University of North Florida
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Stuart Chalk, Ph.D.
Department of Chemistry
University of North Florida
Phone: 1-904-620-1938
Fax: 1-904-620-3535
Email: schalk@unf.edu
Website: @unf

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Blood Serum

Classification: Biological fluid -> blood -> serum

Citations 444

"Luminol Electrochemiluminescence-based Fibre Optic Biosensors For Flow Injection Analysis Of Glucose And Lactate In Natural Samples"
Anal. Chim. Acta 1999 Volume 381, Issue 1 Pages 1-10
Christophe A. Marquette and Loïc J. Blum

Abstract: A fiber optic biosensor based on the electrochemiluminescence of luminol has been developed for glucose and lactate flow injection analysis, The electrochemiluminescence of luminol was generated using a glassy carbon electrode polarised at +425 mV vs. a platinum pseudo-reference electrode. After optimization of the reaction conditions and physicochemical parameters influencing the sensor response, the measurement of hydrogen peroxide could be performed in the range 1.5 pmol-30 nmol. Glucose oxidase or lactate oxidase, were immobilized on polyamide and collagen membranes. With collagen as the enzymatic support, the detection limits for glucose and lactate were 60 pmol and 30 pmol, respectively, whereas with the enzymatic polyamide membranes, the corresponding values were 150 pmol and 60 pmol. In each case, glucose and lactate measurements could be performed over four decades. The luminol electrochemiluminescence-based biosensors were used for glucose and lactate measurements in sera and also for lactate measurements in whey solutions. Good agreements were obtained between the present method and reference methods. For glucose analysis in serum, the coefficient of variation for 53 repeated measurements performed over a 10 h period was 4.8% while for lactate analysis, 80 assays performed over a 15 h period gave a coefficient of variation of 6.7%.
Glucose Lactate Sensor Chemiluminescence Electrode Method comparison Standard method Optimization

"Determination Of Acid Phosphatase Activity In A Double Injection Flow System With Electrochemical Detection"
Anal. Chim. Acta 2000 Volume 406, Issue 2 Pages 225-232
J. Castañón-Fernández, M. T. Fernández-Abedul and A. Costa-García

Abstract: An enzyme assay for the measurement of total and prostatic acid phosphatase activity by flow injection analysis with electrochemical detection has been developed. The substrate (α-naphthyl phosphate) and enzyme are simultaneously injected to the system by means of a double injection valve. The α-naphtol produced after stopping the flow is amperometrically quantified. The calibration curve is linear from 0.01 to 1.3 u/L with a limit of detection of 0.006 u/L. Results were compared with that obtained spectrophotometrically.
Acid phosphatase Amperometry Electrode Dual injection Stopped-flow Method comparison

"Kinetic Determination Of Acid Phosphatase Activity By Double Injection Flow Analysis With Electrochemical Detection"
Anal. Chim. Acta 2000 Volume 413, Issue 1-2 Pages 103-108
J. Castañón-Fernández, M. T. Fernández-Abedul and A. Costa-García

Abstract: A two-points kinetic procedure has led to an enzyme assay for the measurement of total and prostatic acid phosphatase (PAP) activity by flow injection analysis with electrochemical detection. A double injection valve is employed for the simultaneous injection of the substrate (α-naphthyl phosphate) and enzyme. Successive injections are directed into different reaction coils for amperometric measurement of the α-naphthol produced at different times. Total and prostatic activity is determined in control sera and results were compared with those obtained spectrophotometrically.
Acid phosphatase Amperometry Electrode Dual injection Stopped-flow Method comparison Kinetic

"Highly-sensitive Flow Injection Determination Of Hydrogen Peroxide With A Peroxidase-immobilized Electrode And Its Application To Clinical Chemistry"
Anal. Chim. Acta 2000 Volume 406, Issue 2 Pages 201-207
Keiko Yamamoto, Takanori Ohgaru, Masaki Torimura, Hideaki Kinoshita, Kenji Kano and Tokuji Ikeda

Abstract: A flow injection system for determination of hydrogen peroxide was constructed using a peroxidase/ferrocene-embedded carbon paste (POD-Fc-CP) electrode covered with a membrane. H2O2 was reduced bioelectrocatalytically at 100 mV (versus Ag/AgCl/sat. KCl). A Nafion(R)-coated cellulose acetate membrane prepared on the surface of water proved to be suitable for the covering membrane. The detection limit (signal :noise = 6) for H2O2 was 200 fmol (10 nM x 20 µl) and the response linearity was retained at least up to 25 µM. The relative standard deviation was 4.4% for 1.0 µM water (n = 7). The flow injection system was successfully applied to the determination of glucose and urate in serum by using the corresponding oxidase-immobilized pre-column. The detection limits were 1.5 pmol for glucose and 2 pmol for urate.
Hydrogen peroxide Glucose Urate Electrode Electrode Amperometry Apparatus

"Clearance Of Increased Serum Advanced Glycosylation End Products In Patients With End Stage Renal Disease By Hemodialysis"
Acta Pharmacol. Sin. 2000 Volume 21, Issue 3 Pages 276-280
Liu NF, Sun ZL, Liu BC

Abstract: To study the effect of removal of hemodialysis using acetate membrane on serum advanced glycosylation end products (AGEP) in 36 patients (59.1±1.6) with end stage renal disease (ESRD). METHODS: Serum AGEP levels were determined with quantitative fluorescence spectrometry, flow injection analysis (FIA), and competitive enzyme-linked immunosorbent assay (ELISA) using a polyclonal antibody directed against AGEP. RESULTS: The serum AGEP levels in patients with ESRD quantified by fluorescence spectrometry, FIA, and ELISA were higher than those in controls [(25±5) vs (7.5±1.5), (6.1±1.8) vs (1.4±0.5), and (37±20) vs (9±10) kU/L, respectively] and markedly reduced to [(22±6), (4.2±1.4), and (19±14) kU/L, respectively] after hemodialysis. CONCLUSION: Increased serum AGEP levels in the circulation of patients with ESRD were reduced effectively by hemodialysis.
Dialysis Membrane

"Determination Of Salicylate In Blood Serum Using An Amperometric Biosensor Based On Salicylate Hydroxylase Immobilized In A Polypyrrole-glutaraldehyde Matrix"
Talanta 2000 Volume 51, Issue 3 Pages 547-557
Laércio Rover Júnior, Graciliano de Oliveira Neto, João Roberto Fernandes and Lauro Tatsuo Kubota

Abstract: The use of an amperometric biosensor for the salicylate determination in blood serum is described. The biosensor is based on salicylate hydroxylase (EC 1.14.13.1) electropolymerized onto a glassy carbon-working electrode with polypyrrole and glutaraldehyde, to improve the biosensor lifetime. The hexacyanoferrate: (II) was also incorporated to work as a redox mediator to minimize possible interferences. The salicylate is enzymatically converted to catechol, which is monitored amperometrically by its electrooxidation at + 0.170 V versus SCE (saturated calomel electrode). Salicylate determination was carried out maintaining the ratio between β-NADH and salicylate at 4:1 (30°C). The amperometric response of the biosensor was linearly proportional to the salicylate concentration between 2.3 x 10^-6 and 1.4 x 10^-5 mol L-1, in 0.1 mol L-1 phosphate buffer (pH 7.8), containing 0.1 mol L-1 KCl and 5.0 x 10^-4 mol L-1 Na(2)H(2)EDTA, as supporting electrolyte. The recovery studies, in the presence of several interfering compounds, showed recoveries between 96.4 and 104.8%. The useful lifetime of the biosensor in the concentration range evaluated was at least 40 days, in continuous use. Blood serum samples analyzed by this biosensor showed a good correlation compared to the spectrophotometric method (Trinder) used as reference, presenting relative deviations lower than 7.0%.
Salicylate Amperometry Sensor Electrode Apparatus Detector Method comparison Interferences

"Disposable Interference-free Glucose Biosensor Based On An Electropolymerised Poly(pyrrole) Permselective Film"
Anal. Chim. Acta 2000 Volume 420, Issue 1 Pages 9-17
M. Quinto, I. Losito, F. Palmisano and C. G. Zambonin

Abstract: Miniaturised disposable amperometric biosensors for glucose determination in serum are described. A commercially available three-electrode system created on a planar corundum ceramic base was used as biosensor substrate and the working Pt electrode was modified by an electrogenerated overoxidized poly(pyrrole) film (PPYox). After the optimization of the PPy layer synthesis, two different approaches were investigated for glucose oxidase (GOD) immobilization: enzyme entrapment into the electropolymerised film by an all-electrochemical procedure (PPYox/GOD) and gel-entrapment over the PPYox modified electrode by co-crosslinking with glutaraldehyde/bovine serum albumin (PPYox/GOD-gel). A comparison of the sensitivities to glucose, performed by dropping 50 µl of the sample solutions onto the relevant electrode systems, showed that both sensors have a linearity range extending up to 10 mM, though Pt/PPYox/GOD-gel sensor is more sensitive (168±15 versus 53±7 nAm/M), The two sensors showed remarkable anti-interferent selectivity, moreover the PPYox/GOD-gel sensor had also a good stability and could be used for flow injection analysis of glucose, with a linearity range extending up to 25 mM.
Glucose Sensor Amperometry Electrode Interferences

"Development Of A Sequential Injection System In The Capillary Format For Determinations Of The IpaC Protein"
Anal. Chim. Acta 2000 Volume 409, Issue 1-2 Pages 3-8
D. M. Spence, E. D. Knoll, B. T. Ruotolo and J. Bjerregaard

Abstract: A sequential injection (SI) system in the capillary format is reported for protein concentration determinations employing sample volumes in the microliter to nanoliter range. By incorporating a programmable syringe pump and computer control over the selection valve and data acquisition system, the invasion plasmid antigen C (IpaC) protein concentration in a purified sample was determined. Results of flow rate, temperature, and injection studies are presented. Replicate measurements of a bovine serum albumin (BSA) standard resulted in a relative standard deviation in peak area of 3.1%. In addition, protein concentrations as low as 5 x 10^-6 M were determined in less than 3 min. This system was subsequently applied to the determination of a real IpaC sample using multiple standard additions. The error in the determination, relative to a spectrophotometric method of determination, was 7.1%. The total sample consumption for the calibration and unknown concentration determination was 60 µl.
Protein, IpaC Sequential injection Capillary Standard additions calibration Computer Method comparison

"In Situ Preparation Of A Cholesterol Biosensor: Entrapment Of Cholesterol Oxidase In An Overoxidized Polypyrrole Film Electrodeposited In A Flow System -Determination Of Total Cholesterol In Serum"
Anal. Chim. Acta 1999 Volume 385, Issue 1-3 Pages 213-222
Juan C. Vidal, Esperanza García and Juan R. Castillo

Abstract: A straightforward method for constructing cholesterol biosensors by entrapment of cholesterol oxidase within a polypyrrole (PPy) film electropolymerized in a flow system is proposed. This method enables adjustment of the biosensor characteristics and features low reagent consumption. Prior to use, the PPy film is overoxidized to provide it with anion-exclusion properties in order to minimize the interference of electroactive species such as ascorbic acid and uric acid. A comprehensive preliminary study of the influence of 2-propanol and Triton X-100 added to the cholesterol solution on the flow injection determination of this substrate, and the effects of different experimental variables on the biosensor response and its selectivity against interfering electroactive species was carried out, The proposed cholesterol oxidase based biosensor, named Pt/PPy-ChOx, was applied to the determination of cholesterol in reference serum samples, and the results were consistent with certified values.
Cholesterol Sensor Electrode Reference material Interferences

"Flow Injection Analysis. 7. Use Of Ion-selective Electrodes For Rapid Analysis Of Soil Extracts And Blood Serum. Determination Of Potassium, Sodium And Nitrate"
Anal. Chim. Acta 1977 Volume 88, Issue 1 Pages 1-16
J. Ruzicka, E. H. Hansen and E. A. Zagatto

Abstract: The flow injection principle is used with novel design of a flow cell, in which the ion-selective an reference electrode are incorporated. The reproducible mixing of the sample with the carrier stream of electrolyte and the precise timing of potentiometric measurements in the continuously moving stream are the most important parameters influencing the quality of direct potentiometric measurements. Methods for the determination of NO3- and K in soil extracts, and Na and K in blood serum are described. At sampling rates of 125 determinations per hour, a standard deviation of only 0.8% is typical. Simultaneous measurements of two ions on one stream are described; advantages and drawbacks of the use of ion-selective electrodes in continuous-flow measurements are discussed; and the new possibilities which the flow injection method offers are contemplated.
Nitrate Potassium Sodium Clinical analysis Electrode Electrode Electrode Sample preparation Flowcell Apparatus Dual detection

"Optimization Of A Sodium Ion-selective Electrode For Use In Serum Measurements"
Anal. Chim. Acta 1977 Volume 89, Issue 1 Pages 101-109
Ulla Fiedler

Abstract: A Na+-selective polymeric membrane electrode was optimized, especially with reference to serum measurements. For a given combination of polymer and ligand, the choice of membrane solvent is critical. Potentiometric measurements and electrodialysis experiments showed that the dielectric constant and solubility parameter of the solvent influence the electrode selectivities, stability, slope, and limit of detection. For use in serum measurements, it is essential to use an appropriate analytical technique, such as flow injection analysis, which permits exposures to serum for only short periods of time.
Sodium Electrode Potentiometry Optimization Apparatus

"Flow Injection Analysis. 8. Determination Of Glucose In Blood Serum With Glucose Dehydrogenase"
Anal. Chim. Acta 1977 Volume 89, Issue 2 Pages 241-254
E. H. Hansen, J. Rika and B. Rietz

Abstract: The flow injection principle is used for enzymatic determination of glucose in blood serum with glucose dehydrogenase. Methods based on single-point determination on deproteinized serum samples, single-point determination comprising continuous-flow dialysis, and 2-point kinetic assay, are described and discussed. Special consideration was given to optimizing the flow parameters in order to minimize reagent consumption, yet to maintain a high sampling rate. Thus, for the single-point determination, a sampling rate of 120 samples/h was achieved at an expenditure of 3.12 g-units of glucose dehydrogenase/sample.
Glucose Clinical analysis Spectrophotometry Sample preparation Optimization Dialysis Kinetic Enzyme

"Flow Injection Analysis. 6. The Determination Of Phosphate And Chloride In Blood Serum By Dialysis And Sample Dilution"
Anal. Chim. Acta 1976 Volume 87, Issue 2 Pages 353-363
Elo H. Hansen and Jaromir Rika

Abstract: The rapid determination of Cl- and inorganic phosphate (Pi) in blood serum based on continuous-flow spectrometry was adapted to flow injection analysis by applying dialysis as well as differential dilution of the injected samples. In addition to the manual injection procedure, an automated microsampling unit was developed and used. By continuous dialysis Cl- could be determined reproducibly at a sampling rate of 125 samples/h at sample volumes of ≤60 µL. A similar sampling rate and high precision were obtained for the determination of Pi by using differential dilution.
Chloride Phosphate Phosphates Clinical analysis Spectrophotometry Dialysis Dilution

"Flow Injection Analysis Of Calcium In Serum, Water And Waste Water By Spectrophotometry And By Ion-selective Electrode"
Anal. Chim. Acta 1978 Volume 100, Issue 1 Pages 151-165
E. H. Hansen, J. Rika and Animesh K. Ghose

Abstract: The flow injection technique is a fast, reliable, and sensitive method for the determination of Ca in various aqueous as well as serum samples; spectrophotometric or potentiometric detection can be used. At sampling rates of 100-10 samples/h, with 30 µL sample injections, high reproducibility of measurement and low reagent consumption are achieved in both methods. In the spectrophotometric method (with o-cresolphthalein complexon), the anal. readout is available within 12 s after sample injection at a total reagent consumption of 0.75 mL/anal. The potentiometric measurement of the Ca activity in serum is placed on a reliable basis by alternating measurements of serum samples and aqueous standards without incurring any nonreproducible changes in potential between aqueous and serum solutions This permits the simultaneous determination of pH and pCa, the anal. readout being available within 3 s of sample injection. The good agreement between the results obtained with the flow injection method and those attained by atomic absorption and EDTA titrns. as well as pCa stat-measurements show that the new methods are potentially suitable for routine anal.
Calcium pH Spectrophotometry Electrode Potentiometry Method comparison

"Flow Injection Analysis With A Fluorimetric Detector For Determinations Of Glycine And Albumin"
Anal. Chim. Acta 1979 Volume 106, Issue 2 Pages 395-399
Joy I. Braithwaite and J. N. Miller

Abstract: Flow injection analysis with fluorometric detection was used in the determination of glycine and albumin, with o-phthalaldehyde being used in determination of the latter. Preliminary variables and applications of flow injection analysis were examined, using quinine sulfate and m-hydroxybenzoic acid which are intrinsically fluorescent. System response was linear in the range of 0-8 µg/mL for glycine and concentrations as low as 2 pg/mL were detectable. The level of glycine in orange juice and dietetic cola was 0.33-0.41 and 0.17 g/dL, respectively. Serum albumin determinations flow injection analysis had a relative standard deviation of 1.8%. The method allowed sampling rates of 180/h for both glycine and albumin.
Glycine Albumin Clinical analysis Fluorescence Optimization

"Stopped-flow And Merging Zones - A New Approach To Enzymatic Assays By Flow Injection Analysis. Determination Of Glucose In Serum"
Anal. Chim. Acta 1979 Volume 106, Issue 2 Pages 207-224
J. Rika and E. H. Hansen

Abstract: Four alternative flow injection methods based on the concept of merging zones were developed for the assay of glucose in serum with glucose dehydrogenase. Special attention was paid to the dispersion and synchronization of the merging zones and to methods of measuring the blank value of serum. The optimal procedure, based on rate measurements, allowed the assay of glucose at a rate of 100 determinations/h, with the anal. readout available 30 s after sample injection. The assay requires <1 unit of enzyme/sample.
Glucose Clinical analysis Spectrophotometry Optimization Dispersion Enzyme Stopped-flow Merging zones

"Development Of A Glucose Analyzer Based On Immobilized Glucose Oxidase"
Anal. Chim. Acta 1979 Volume 106, Issue 2 Pages 233-242
B. Watson, D. N. Stifel and F. E. Semersky

Abstract: A dedicated instrument for the determination of glucose in serum or plasma is described. The flow system includes an amperometric sensor to measure the hydrogen peroxide liberated from the total conversion of a 2-l sample injected into a column of immobilized glucose oxidase covalently coupled to a porous alumina substrate. The instrument digitally displays the glucose concentration and is capable of testing 60 samples/h. No sample or reagent preparation is necessary. The accuracy, precision, and selectivity are discussed.
Glucose Amperometry Clinical analysis Potentiometry Immobilized enzyme Reactor

"Photochemical-reaction Detectors In Continuous-flow Systems: Applications To Pharmaceuticals"
Anal. Chim. Acta 1980 Volume 114, Issue 1 Pages 137-146
A. H. M. T. Scholten, U. A. Th. Brinkman and R. W. Frei

Abstract: Several basic parameters of a photochemical reactor coupled to a high-performance liquid Chromatographie system are discussed. The non-fluorescent clobazam and desmethylclobazam and three phenothiazines, which exhibit native fluorescence, are used as model compounds. On irradiation with ultra-violet light, the reaction products formed display fluorescence (clobazam, desmethylclobazam) or unproved fluorescence characteristics (phenothiazines). The effects of carrier stream (mobile-phase) composition, time of irradiation and band broadening in the reactor on the fluorescence signal are described. The polarity of the organic solvents used (methanol, ethanol, acetonitrile) appears to have an important effect on the fluorescence intensity. For clobazam and desmethylclobazam, detection limits of 70 and 120 pg, respectively, were calculated after an irradiation time of 28 s with methanol-0.01 M (pH 5) acetate buffer (1:1) as mobile phase. The method is applied to the determination of both compounds in serum and urine samples.
Clobazam Desmethylclobazam Phenothiazines Chromatography Fluorescence Photochemistry

"Automation Of An Energy-transfer Immunoassay By Using Stopped-flow Injection Analysis With Merging Zones"
Anal. Chim. Acta 1980 Volume 114, Issue 1 Pages 183-189
C. S. Lim and J. N. Miller, J. W. Bridges

Abstract: A homogeneous fluorescence energy-transfer immunoassay for serum albumin has been automated by using flow-injection analysis. Application of the merging-zone and stopped-flow principles permits low consumption of labelled reagents and samples, and sub-micromolar concentrations of albumin can be rapidly and precisely determined. Studies on individual serum samples show good agreement with other techniques, including a fluorimetric dye-binding assay that has also been automated by using mergingzone flow-injection analysis.
Albumin Clinical analysis Fluorescence Immunoassay Enzyme Merging zones Stopped-flow

"Miniaturization In Analytical Chemistry - A Combination Of Flow Injection Analysis And Ion-sensitive Field-effect Transistors For Determination Of PH, Potassium And Calcium"
Anal. Chim. Acta 1980 Volume 118, Issue 1 Pages 45-53
A. U. Ramsing, J. Janata, J. Rika and M. Levy

Abstract: The combination of flow injection analysis and ion-sensitive field effect transistors is described for determinations of pH, potassium and calcium ions. The reference electrode is placed in the bypass of the sample injector, thereby avoiding the detrimental effect of serum proteins on the liquid junction potential for the reference electrode. The reagent consumption is halved compared to the use of the conventional electrodes without adversely affecting the sampling rate and the standard deviation of the measurement. An attractive feature of this combination is the possibility of multi-ion analysis.
Calcium Potassium pH Field effect transistor Electrode Miniaturization

"Determination Of Ethanol By Air-stream Separation With Flow Injection And Electrochemical Detection At A Nickel Oxide Electrode"
Anal. Chim. Acta 1980 Volume 120, Issue 1 Pages 75-80
T. N. Morrison, K. G. Schick and C. O. Huber

Abstract: Ethanol is separated by an air stream from a 1 mL sample with collection in 1 mL of 0.2 mol L-1 sodium hydroxide. Measurement is by voltammetry at a tubular, catalytic nickel oxide electrode with 30-l sample injected into a continuous flow stream. Relative standard deviation for repetitive measurements is 1.8% for synthetic samples. Serum samples extracted for 100 s and then measured yielded linear results for concentrations of 2 x 10^-4-5 x 10^-2 mol L-1 ethanol (0.001-0.23%), with relative standard deviation of 2.0%. The time per determination was about 2 min.
Ethanol Electrochemical analysis Electrode Potentiometry Voltammetry Air segmentation

"Use Of Immobilized Alcohol Oxidase In The Continuous-flow Determination Of Ethanol With An Oxygen Electrode"
Anal. Chim. Acta 1981 Volume 123, Issue 1 Pages 125-133
E. L. Gulberg and G. D. Christian

Abstract: Immobilized alcohol oxidase was used in the determination of blood alcohol. The alcohol oxidase catalyzed the aerobic oxidation of ethanol and the oxygen concentration was monitored with an oxygen membrane electrode in a flow cell. The enzyme was immobilized either by covalent attachment via glutaraldehyde to the inside walls of nylon tubing, or by adsorption onto three separate controlled-pore glass support materials: TiO2, SiO2, or AL2O3. The supports were packed into 10 cm lengths of 3 mm i.d. glass tubing or 30 cm lengths of 5 mm i.d. nylon tubing. The five methods of immobilization were compared for stability and activity toward ethanol. Immobilization on silanized glass beads results in the highest activity and greatest stability of the reactor.
Ethanol Electrode Electrode Immobilized enzyme

"Direct Spectrophotometric Determination Of Thiocyanate In Serum And Urine With A Continuous-flow Analyser"
Anal. Chim. Acta 1981 Volume 128, Issue 1 Pages 169-175
Gianfranco Giraudi, Caterina Grillo

Abstract: A spectrophotometric method for the rapid measurement of thiocyanate in serum and urine without separation from interfering substances is described. Thiocyanate reacts immediately with chloramine-T in presence of iron(III) chloride catalyst to give cyanogen chloride, which reacts with a mixture of -picoline (4-methylpyridine) and barbituric acid to form a soluble violet-blue product, which is measured at 605 nm. Other components of physiological fluids react more slowly and do not interfere if the reaction time of the chlorinating step is kept very short. The proposed procedure is compared with a highly selective method, based on the oxidation of thiocyanate to cyanide, and good agreement was obtained for both serum and urine. The method is readily adapted to a continuous-flow procedure with a Technicon AutoAnalyzer.
Thiocyanate ion Spectrophotometry

"Flow Injection Analysis For Glucose And Urea With Enzyme Reactors And Online Dialysis"
Anal. Chim. Acta 1981 Volume 130, Issue 1 Pages 45-53
L. Gorton and L. &Ouml;gren

Abstract: A flow injection system for glucose and urea determination is described. The glucose determination uses immobilized glucose oxidase in a reactor designed to give 100% substrate conversion. The hydrogen peroxide formed is converted to a colored complex with 4-aminophenazone and N,N-dimethylaniline. The coupling is catalyzed by a reactor containing immobilized peroxidase. The colored complex is measured in a flow-through spectrophotometric cell. Urea is converted to ammonia in a reactor with immobilized urease and detected with an ammonia gas membrane electrode. Proteins and other interfering species from serum samples are removed in an on-line dialyzer. Calibration curves are linear for glucose in the range 1.6 x 10^-4-1.6 x 10^-2 M and for urea in the range 10^-4-10^-1 M. The samples are 25 µL for glucose determination and 100 µL for urea determination. Linear ranges can be changed by varying the sample sizes. The effects of the dialyser, enzyme reactors and detectors on dispersion are evaluated.
Glucose Urea Clinical analysis Spectrophotometry Electrode Electrode Immobilized enzyme Dialysis Reactor

"Optimal Speed As A Function Of System Performance For Continuous-flow Analysers"
Anal. Chim. Acta 1983 Volume 145, Issue 1 Pages 51-58
Svetla Angelova, H. W. Holy

Abstract: The limitation to high rates of sample throughput with continuous flow automated analyzers is unacceptably high sample-to-sample carry-over. Previous programs for carry-over correction have involved relatively large computer capacities. Here, the simple expression derived for carry-over can be easily programmed in BASIC for a Commodore PET operating in real time. The program includes peak picking and calculation of the calibration graph and sample concentrations. At 240 samples per hour, the results obtained for total protein (linear calibration graph) and haptoglobin (non-linear calibration plot) showed acceptable precision and recovery and correlated well with the same determination conducted at normal operating speeds. Standard Auto Analyzer equipment was used throughout. A parameter which monitors the instrument function is also calculated; this replaces the conventional visual examination of the curve for function monitoring. At high sample rates with degraded curves, visual curve examination is not effective.
Protein, total Haptoglobin Review

"A Flow-through Detector For Simultaneous Determinations Of Glucose And Urea In Serum Samples"
Anal. Chim. Acta 1983 Volume 145, Issue 1 Pages 213-217
Marco Mascini and Giuseppe Palleschi

Abstract: Glucose and urea electrodes are prepared by immobilizing glucose oxidase and urease on nylon net and fixing the nets on oxygen and ammonia gas sensors. Both enzyme electrodes are fixed in a single flow cell (40 µL volume). Serum is diluted tenfold with 0.1 M Tris buffer (pH 8.3) to fit the calibration graphs for both sensors. Samples are pumped for 1 min, with wash periods of 2 min for recovery to the baseline. Results on serum samples are in good agreement with the results obtained by conventional spectrophotometry.
Glucose Urea Clinical analysis Electrode Electrode Immobilized enzyme

"Reduction Of Analytical Variance By Using A Discrete-time Data-weighting Filter To Estimate Abrupt Changes In Batch-type Processes. 2. Applications"
Anal. Chim. Acta 1983 Volume 151, Issue 2 Pages 441-446
Rob T. P. Jansen, Hennie N. J. Poulisse

Abstract: The filter algorithm was applied to the results for 250 batches of blood serum analyzed by a continuous-flow system for inorganic phosphate over a period of 25 days. A reduction in variance of ~50% of the original value was achieved. Similar results were obtained with data from four other routine analytical methods.
Phosphate Computer Signal processing Digital filter

"Repetitive Enzymatic Determination Of Glucose With Regeneration And Recycling Of Coenzyme And Enzymes"
Anal. Chim. Acta 1983 Volume 153, Issue 1 Pages 181-190
P. Roehrig, C. -M. Wolff and J. P. Schwing

Abstract: A closed-loop flow injection system is described. The carrier stream consisted of 40 mL of 0.1 M phosphate buffer of pH 7.4 that was 2.25 mM in 2-oxoglutarate (I), 25 mM in ammonium acetate and 3.9 mM in NAD+ and contained 10 iu mL-1 of glucose dehydrogenase (II) and 0.2 unit of glutamate dehydrogenase (III) (i.e., the activity that transforms 0.2 µmol min-1 of I into glutamate in the presence of 0.1 mM NADH), and was circulated at 0.14 mL s-1 from a reservoir through two identical spectrored because of the low stability of NO2- in H2SO4. When the electrode was pre-treated anodically at +1.75 V for 5 min with phosphate buffer and the electrode was operated at +1.0 V vs. a SCE, response was rectilinear over several orders of magnitude and the detection limit was 3 ppb.
Glucose Clinical analysis Spectrophotometry Closed loop Enzyme Kinetic Standard method Method comparison

"Amperometric Determination Of Phospholipids In Blood Serum With A Lecithin-sensitive Electrode In A Flow Injection System"
Anal. Chim. Acta 1983 Volume 153, Issue 1 Pages 337-340
Toshio Yao, Yoshiaki Kobayashi and Minoru Sato

Abstract: By use of glutaraldehyde as described previously (Ibid., 1983, 148, 27), choline oxidase and phospholipase D were cross-linked with bovine serum albumin and bonded as an inner and an outer layer, respectively, to a platinum electrode (2 cm x 1 cm x 0.1 mm) previously treated with 3-aminopropyltriethoxysilane, and the resulting electrode was used as the anode in a flow-through cell (cf. Ibid., 1983, 153, 175). The sample of serum (10 µL) was injected into a carrier stream (1.5 mL min-1) of 0.1 M phosphate buffer of pH 9.0 that contained 0.1% of Triton X-100 and was 40 µM in CaCl2 and passed through an electrolytic column packed with reticulated vitreous carbon to remove electro-active interfering compounds (e.g., ascorbic acid and uric acid) (cf. Bunseki Kagaku, 1983, 32, 253) and then into the flow-cell. At the electrode surface, lecithin was hydrolyzed by the immobilized phospholipase D to form choline, which was oxidized by the immobilized choline oxidase, and the H2O2 produced was determined amperometrically at +0.8 V. Calibration graphs were rectilinear in the range 0.2 to 3.5 mg mL-1 of lecithin, and the limit of detection was 0.08 mg mL-1. For 1 to 3 mg mL-1 of lecithin the coefficient of variation were 1.5%. Results for two control sera agreed with the manufacturer's data. The enzyme layers retained ~80% of their original activity after one month of regular use. The injection rate was 60 h-1
Phospholipids Lecithin Amperometry Clinical analysis Electrode Electrode Interferences Immobilized enzyme Triton X Surfactant

"Flow Injection Analysis For Cholinesterase In Blood Serum By Use Of A Choline-sensitive Electrode As An Amperometric Detector"
Anal. Chim. Acta 1983 Volume 153, Issue 1 Pages 169-174
Toshio Yao

Abstract: By use of glutaraldehyde as described previously (Ibid., 1983, 148, 27), choline oxidase was cross-linked with bovine serum albumin and bonded to a platinum electrode (2 cm x 1 cm x 0.1 mm) previously treated with 3-aminopropyltriethoxysilane, and the resulting electrode was used as the anode in a flow-through cell (cf. Ibid., 1983, 153, 175). To determine cholinesterase in blood, the sample (5 µL) was injected into a carrier stream (0.9 mL min-1) of 1 mM acetylcholine chloride in 0.1 M phosphate buffer of pH 7.5, and the mixture was passed through a column packed with Cu(II) dithiocarbamate-modified silica gel (cf. Bunseki Kagaku, 1981, 30, 740) to remove electroactive impurities and then through a PTFE zone-dispersion coil. The choline produced was oxidized by the immobilized choline oxidase, and the H2O2 produced was determined amperometrically at +0.8 V. Calibration graphs were rectilinear in the range 0.25 miu to 0.1 iu of cholinesterase injected, and the limit of detection was 40 µiu. For 0.026 iu, the coefficient of variation was 1.8%. The activity of the enzyme layer remained high after two months in repeated use. The injection rate was 40 h-1
Enzyme, cholinesterase Amperometry Electrode Reactor Immobilized reagent Silica gel

"Amperometric Determination Of Glucose In Blood Serum With A Chemically Modified Enzyme-membrane Electrode In A Continuous-flow System"
Anal. Chim. Acta 1983 Volume 153, Issue 1 Pages 175-180
Toshio Yao

Abstract: A flow-cell with a working volume of 12 µL (described and illustrated) has been developed for use with the amperometric glucose sensor described previously (Ibid., 1983, 148, 27). To determine glucose in serum, the sample (5 µL) is injected into a carrier stream (2.9 mL min-1) of 0.1 M phosphate buffer of pH 7.5, and the mixture is passed through a column packed with Cu(II) dithiocarbamate-modified silica gel (cf. Bunseki Kagaku, 1981, 30, 740) to remove electroactive impurities before it enters the flow-cell. Glucose is oxidized by glucose oxidase immobilized on the surface of the anode, and the H2O2 produced is determined amperometrically at +0.8 V. Calibration graphs are rectilinear in the range 0.01 to 8 mg mL-1 of glucose in the sample, and the limit of detection is 2 µg mL-1. The method is specific for β-D-glucose. For 0.5, 1 and 2 mg mL-1 of glucose, the coefficient of variation (n = 7, 10 and 10, respectively) were <1%. Results for two control sera agreed with the manufacturer's data. The activity of the enzyme layer remained high after two months in repeated use. The injection rate is 100 h-1
Glucose Amperometry Electrode Electrode Silica gel Immobilized reagent

"Synthesis Of O,o'-dihydroxyazo Compounds And Their Application To The Determination Of Magnesium And Calcium By Flow Injection Analysis"
Anal. Chim. Acta 1984 Volume 159, Issue 1 Pages 289-298
H. Wada and G. Nakagawa, K. Ohshita

Abstract: Seven such compounds were synthesized, and were found to be highly sensitive reagents for Mg (e = 47,000) and Ca (e = 39,000). Because of its ease of preparation and purification and its stability in alkaline solution, 4-(4-diethylamino-2-hydroxyphenylazo)-3-hydroxynaphthalene-2,7-disulfonic acid was the preferred reagent. By masking Fe(III), Al, Cu(II), Zn, Mn(II) and Cd with 2,3-dimercaptopropanol(I) and triethanolamine(II), the sum of Mg and Ca was determined. Magnesium was determined alone by masking Ca and the other metals with a ligand buffer containing Ba, 3,6-dioxaoctane-1,8-diamine-NNN'N'-tetra-acetic acid, I and II, and Ca could then be determined by difference. Calibration graphs were rectilinear over the ranges 0.1 to 1.2 mg L-1 (Mg) and 0.4 to 4 mg L-1 (Ca). The method was used to determine Mg and Ca in tap-water and a standard serum; the results compared well with those for the waters obtained by AAS or EDTA titration and with the certified value for the serum. A diagram of the flow injection manifold is given; the PTFE tubing was 0.5 mm in i.d. except for the back-pressure coil (0.25 mm i.d., 50 cm long). The chromogenic reagent and buffer solution were each delivered at 0.81 mL min-1 with a peristaltic pump, and the sample was injected into the buffer stream via a rotary valve. Sample and reagent solution were mixed in a 300-cm coil and passed to a flow cell (10 mm light path; 20 µL volume) for measurement of absorbance.
Calcium Magnesium Spectrophotometry Interferences Method comparison Reference material Chromogenic reagent

"Spectrophotometric Determination Of Lithium Ion With The Chromogenic Crown Ether, 2'',4''-dinitro-6''-trifluoromethylphenyl-4'-aminobenzo-14-crown-4"
Anal. Chim. Acta 1984 Volume 162, Issue 1 Pages 285-291
Y. P. Wu and G. E. Pacey

Abstract: The named reagent {16-(ααα-trifluoro-4,6-dinitro-o-tolylamino)-2,6,9,13-tetraoxabicyclo[12.14.0]octadeca-1(14),15,17-triene} reacts with alkali-metal ions, e.g., Li+, in aqueous solution to form complexes that can be extracted into CH2Cl2 for spectrophotometry at 464 nm. The calibration graph was rectilinear for 14 to 560 mg L-1 of Li, and, in the determination of 280 mg L-1 of Li, 3 g L-1 of Na could be tolerated. The method was applied to the determination of Li in serum (doped with LiCO3) by batch or flow injection analysis, and the results obtained agreed with those of AAS The method was more selective and sensitive than an ion-pairing method based on use of benzo-14-crown-4.
Lithium Metals, alkali Clinical analysis Spectrophotometry Sample preparation Crown ether Extraction Method comparison

"Determination Of Urea With An Ammonia Gas-sensitive Semiconductor Device In Combination With Urease"
Anal. Chim. Acta 1984 Volume 163, Issue 1 Pages 143-149
F. Winquist, A. Spetz and I. Lundstr&ouml;m, B. Danielsson

Abstract: An Ir - Pd metal oxide semiconductor capacitor and urease were used in two systems. One comprised essentially a column of immobilized urease in a flow injection system. Urea was determined in whole blood and serum (after 500-fold dilution) by this method; the serum values were in reasonably good agreement with those obtained by a spectrophotometric method, the coefficient of variation (n = 10) for 4.5 mM urea in whole blood was 4.6%, and recoveries of additions of 0.5 to 2 mM urea to whole blood ranged from 85 to 106%. The second system was an enzyme probe based on urease enclosed between a dialysis membrane and a gas-permeable membrane. The dialysis membrane formed part of a flow-cell through which the buffered sample was pumped, and the capacitor was mounted 0.1 mm from the other membrane. Up to 5 mM urea could be determined. For the first method, the limit of detection was 0.2 µM-urea, and the calibration graph was rectilinear up to 50 µM, for 150 µL portions of the diluted samples. For the second method, the calibration graph was non-rectilinear, and the limit of detection was 0.01 mM.
Urea Electrode Dialysis Method comparison Immobilized enzyme Reactor

"Determination Of Ammonia In Air And Aqueous Samples With A Gas-sensitive Semiconductor Capacitor"
Anal. Chim. Acta 1984 Volume 164, Issue 1 Pages 127-138
F. Winquist, A. Spetz and I. Lundstr&ouml;m, B. Danielsson

Abstract: The properties of a new type of ammonia gas-sensitive semiconductor capacitor are described. The sensor is based on a palladium MOS field-effect capacitor with a thin layer (3 nm) of iridium surrounding the palladium gate. The lower limit of detection for ammonia in air is 1 ppm (0.59 mg kg-1). The analytical characteristics and temperature-dependence of the sensor in measurements of ammonia in air are evaluated. Ammonia in aqueous solutions is determined by the use of a continuous flow system utilizing a gaspermeable membrane in combination with the sensor. The calibration plot of the voltage drop of the capacitor vs. ammonia concentration in 150 µL samples is linear in the concentration range 0.2 x 10^-6-5 x 10^-5 M. Ammonia is determined in rain and river water as well as in whole blood and blood serum; 15 samples per hour can be assayed. Analytical recovery studies and the selectivity properties of the system are described and discussed. Finally, the properties of the flow-through system in continuous monitoring are described.
Ammonia Electrode

"Stopped-flow Injection Determination Of Copper(II) At The Ng/ml Level"
Anal. Chim. Acta 1984 Volume 165, Issue 1 Pages 177-185
F. L&aacute;zaro, M. D. Luque de Castro and M. Valc&aacute;rcel

Abstract: The catalytic action of Cu(II) on the di-2-pyridyl ketone hydrazone - H2O2 system is quantitative. The oxidation product shows an intense blue fluorescence at 427 nm (excitation at 350 nm) in a strongly acidic medium. The sampling rate (72 h-1), coefficient of variation (1.4%) and freedom from interference from most foreign ions allow determination of 0.2 to 300 ng mL-1 of Cu in foods (e.g., fruits and rice) and blood serum.
Copper(II) Fluorescence Interferences Catalysis Stopped-flow PPB

"Galactose Determination In An Automated Flow Injection System Containing Enzyme Reactors And An Online Dialyzer"
Anal. Chim. Acta 1985 Volume 167, Issue 1 Pages 123-136
Bo Olsson, Hans Lundb&auml;ck and Gillis Johansson

Abstract: In this method galactose is removed from serum by dialysis, and the diffusate passes through a Bond-Elut NH2 - Cu(II) column to remove interference and then through a catalase reactor to remove H2O2 from the solution The D-galactose is oxidized by galactose oxidase immobilized in a reactor to produce H2O2, which reacts chromogenically with peroxidase. Finally the reaction products are determined spectrophotometrically at 514 nm. The system is controlled from a personal computer, which also collects and analyzes data. The calibration graph is rectilinear in the range 10 µM to 14 mM galactose. Recovery from serum is ~100%.
Galactose Spectrophotometry Interferences Dialysis Enzyme

"Enzymatic Immunoassay Of α-fetoprotein, Insulin And 17-α-hydroxyprogesteron Based On Chemiluminescence In A Flow Injection System"
Anal. Chim. Acta 1985 Volume 167, Issue 1 Pages 241-248
Masako Maeda and Akio Tsuji

Abstract: Optimum conditions have been established for the luminol - H2O2 - K3Fe(CN)6 reaction for use in enzyme immunoassays, with glucose oxidase as the enzyme label. The antibody-coated solid-phase method was used for the separation of bound and free fractions. Chemiluminescence was measured in a flow injection system with a detector equipped with a spiral flow cell. The method was applied to determination of α-foetoprotein by sandwich enzyme immunoassay and of insulin and 17α-hydroxyprogesterone by competitive-binding immunoassay, with detection limits of 1.8 fmol, 1.5 fmol and 0.25 pg, respectively.
α-Fetoprotein Insulin 17-Hydroxyprogesterone Chemiluminescence Clinical analysis Immunoassay Flowcell

"Indirect Atomic Absorption Spectrometric Determination Of Perchlorate By Liquid-liquid Extraction In A Flow Injection System"
Anal. Chim. Acta 1985 Volume 169, Issue 1 Pages 161-169
M. Gallego and M. Valc&aacute;rcel

Abstract: Serum or urine samples were centrifuged and the supernatant solution were subjected to flow injection analysis. The sample was mixed with the carrier solution [acetate buffer solution (pH 4.75) that was 4.8 mM in ascorbic acid and contained 8 µg mL-1 of Cu] and the mixture was extracted with 0.2% 6-methylpicolinaldehyde azine solution in isobutyl methyl ketone. The extract was mixed with water and analyzed by AAS The calibration graph was rectilinear for 0.1 to 5.0 µg mL-1 of ClO4- and the detection limit was 70 ng mL-1. Recovery was 98.5 to 107% and the coefficient of variation was 0.7%.
Perchlorate Spectrophotometry Sample preparation Extraction Tecator Indirect

"Determination Of Cholesterol By Flow Injection Analysis With Immobilized Cholesterol Oxidase"
Anal. Chim. Acta 1985 Volume 174, Issue 1 Pages 293-297
M. Masoom and Alan Townshend

Abstract: The injected sample passes at pH 7 through a column of cholesterol oxidase, immobilized on controlled-porosity glass by cross-linking with glutaraldehyde, and the H2O2 produced is detected with an amperometric flow-cell detector. The calibration graph of peak height vs. concentration. is rectilinear for up to 80 mg dl-1 with a coefficient of variation of 1 to 3%, the detection limit is 0.2 µg, and the achievable sample throughput is 80 h-1. The method was applied to the determination of cholesterol in blood serum, wax-wool alcohol and a butter extract and gave results in good agreement with those obtained by established procedures.
Cholesterol Amperometry Controlled pore glass Immobilized enzyme Method comparison

"Synthesis Of Bidentate Pyridylazo And Thiazolylazo Reagents And The Spectrophotometric Determination Of Copper In A Flow Injection System"
Anal. Chim. Acta 1985 Volume 176, Issue 1 Pages 41-50
K. Ohshita, H. Wada and G. Nakagawa

Abstract: Of four such reagents (syntheses given) investigated as spectrophotometric reagents for Cu(II), 3-[4-(3,5-dibromo-2-pyridylazo)phenyl(ethyl)amino]propanesulfonic acid(I) showed the most sensitive reaction, forming a 1:2 (metal-to-ligand) complex in acetate medium (pH 4.5) in the presence of Na dodecyl sulfate(II). The sample (60 µL) is injected into a carrier stream (1 mL min-1) of 0.1 M acetate buffer (pH 4.5) and then mixed with a reagent stream (1 mL min-1) containing 40 µM-I, 0.1 M acetate buffer and 0.4% of II. The mixture is passed through a reaction coil (2 m x 0.5 mm) and the absorbance is measured at 638 nm (e = 124,000). Calibration graphs are rectilinear for 20 to 200 µg L-1 of Cu(II) and the sampling rate is 60 h-1. Nickel, Pd(II), AgI, Hg(II), Co(II) and Co(III) also form complexes with I but may be tolerated to varying degrees. The method has been applied to the determination of Cu in serum, and the results for two reference samples were in good agreement with certificate values.
Copper(II) Spectrophotometry

"Potentiometric Detection In Flow Analysis"
Anal. Chim. Acta 1986 Volume 179, Issue 1 Pages 359-370
K. T&oacute;th, J. Fucsk&oacute;, E. Lindner, Zs. Feh&eacute;r and E. Pungor

Abstract: Flow-through potentiometric detection in flow injection analysis is discussed and the performance characteristics of ion-selective electrodes with respect to rectilinear response, detection limit, selectivity and stability are studied. A method for the determination of F- with a selective electrode in rain-water is described, and a K+-selective electrode described by Tarcali et al. (Anal. Abstr., 1986, 48, 11J139) was applied to the analysis of blood serum. The results were in good agreement with those obtained by ion-chromatographic methods (for F-) and flame-photometric methods (for K).
Potassium Fluoride Electrode Electrode Potentiometry Method comparison Review

"Membrane-dialyser Injection Loop For Enhancing The Selectivity Of Anion-responsive Liquid-membrane Electrodes In Flow Systems. 2. Selective Sensing System For Salicylate"
Anal. Chim. Acta 1986 Volume 186, Issue 1 Pages 81-90
Qinlin Chang and M. E. Meyerhoff

Abstract: The dialyser assembly contains a vinylmethylsilicone rubber membrane (0.1-mm thick); the salicylate is trapped in a static layer of 50 mM 4-morpholinylethanesulfonic acid - NaOH buffer (pH 6.5). This recipient/carrier solution is then flushed to a tubular polymer membrane electrode cast from a solution of PVC, Mn(III) tetraphenylporphyrin chloride, and dibutyl sebacate in THF. Response to salicylate is almost Nernstian over the range 0.1 to 10 mM with a 2-min trapping time, compared with 2 µM to 10 mM without the dialysis unit; detection limits depend on the trapping time. The system is very selective for salicylate; only benzoic acid interferes significantly. For serum samples, recoveries range from 86 to 105%.
Salicylate Electrode Electrode Electrode Potentiometry Dialysis Injector Interferences Selectivity Silicone membrane

"Bipolar Pulse Conductometric Detection Of Enzyme Reactions In Flow Injection Systems. Urea In Serum And Urine"
Anal. Chim. Acta 1986 Volume 186, Issue 1 Pages 91-100
Douglas Taylor and Timothy A. Nieman

Abstract: Urea(I) is determined by enzymatic hydrolysis on a column (5.7 cm x 3.2 mm) of urease bound to controlled-pore glass, with phosphate buffer (pH 7.3) as the carrier stream (20 mM for urine and 2 mM for serum samples) containing NaCl; the reaction is monitored by measuring conductivity before and after hydrolysis, with automatic processing to give a differential signal. The calibration graph is rectilinear for 10 mM I, with detection limits of 0.1 mM I in urine and 0.01 mM I in serum; the coefficient of variation is 3%. The sampling rate is 20 h-1. The accuracy compares well with that of existing methods.
Urea Clinical analysis Conductometry Controlled pore glass Immobilized enzyme

"Assay For Guanase In Blood Serum By Flow Injection Analysis With Fluorescence Detection"
Anal. Chim. Acta 1987 Volume 197, Issue 1 Pages 51-56
Yohji Hayashi, Kiyoshi Zaitsu and Yosuke Ohkura

Abstract: A substrate solution (400 µL) of 35 parts of 0.1 M Tris - HCl buffer (pH 8) mixed with 1 part of 2 mM guanine in 0.01 M NaOH and 2 parts each of urate oxidase solution (0.5 iu mL-1) and catalase solution (40 iu mL-1) was incubated initially for 2 min at 37°C and then for a further 30 min following the addition of serum (10 µL). The reaction was stopped by adding 4 M HClO4 (50 µL) and the product was mixed with 2 M K2CO3 (90 µL) and centrifuged. Aliquots (20 µL) of supernatant solution were injected into the carrier stream (0.25 mL min-1), which consisted of 0.15 M NaCl and 10 mM Na2EDTA and was blended with the reagent stream (0.25 mL min-1) which contained 5 mM 3-(4-hydroxyphenyl)propionic acid in 0.1 M Tris - HCl. The blended stream passed sequentially through a 1-m mixing coil and columns containing immobilized xanthine oxidase, urate oxidase and horse-radish peroxidase, which were all immersed in a water bath at 37°C. The fluorescence of xanthine generated was measured in a 12 µL flow cell at 405 nm (excitation at 305 mm). Calibration graphs were rectilinear for up to 500 pmol of xanthine per injection with a detection limit of 0.5 pmol. The immobilized-enzyme columns were found to be stable for three months.
Enzyme, guanase Fluorescence Heated reaction Immobilized enzyme

"Simultaneous Determination Of Iron And Copper Ions By Flow Injection Analysis With A Multichannel Photodiode-array Detector"
Anal. Chim. Acta 1987 Volume 200, Issue 1 Pages 515-521
Hiroko Wada, Tomoaki Murakawa and Genkichi Nakagawa

Abstract: A single chromogenic reagent, 7-hydroxy-8-(2-pyridylazo)naphthalene-2-sulfonic acid(I), was used in a flow injection manifold for the simultaneous determination of Fe(II) and Cu(II) with photodiode-array detection. The reagent solution comprised 0.1 mM I in 0.4 M acetic acid - Na acetate buffer (pH 5.0) containing 0.2 mM Na nitrilotriacetate (to prevent interference from Zn). The carrier solution was the same buffer but containing 10 mM L-ascorbic acid. Both solution had a flow rate of 0.80 mL min-1. The sample (80 µL) was injected into the carrier stream and mixed with the reagent in a double coil. Iron was determined from the absorbance at 764 nm, and Cu from the difference in absorbance at 550 and 764 nm. Calibration graphs were rectilinear up to 8 µM for each metal, and the detection limit was 0.03 µg mL-1 for each. Application of the method to the analysis of blood serum is described.
Iron(2+) Copper(II) Spectrophotometry Computer Interferences Multicomponent Chromogenic reagent

"Continuous-flow Enzymatic Determination Of Creatinine With Improved Online Removal Of Endogeneous Ammonia"
Anal. Chim. Acta 1987 Volume 200, Issue 1 Pages 61-72
M. E. Collison and M. E. Meyerhoff

Abstract: The endogenous NH3 (1 mM) in a physiological sample (diluted with buffer of pH 9.5) is removed by an online gas pre-dialysis unit at 45°C, comprising a PTFE coil and an acid reservoir trap. The sample is passed to a flow-through enzyme reactor coil containing immobilized creatinine deiminase which converts creatinine(I) into NH3 and N-methylhydantoin. The liberated NH3 is detected with a membrane electrode. The peak potentials are directly proportional to I concentration. The method is precise and selective and I can be determined in serum and urine samples containing abnormally high levels of NH3. Determination of I in serum samples (n = 30) correlated with an existing Technicon AutoAnalyser colorimetric method (r = 0.996).
Creatinine Electrode Electrode Dialysis Enzyme Heated reaction Interferences Method comparison Reactor

"Automated Flow Injection Determination Of Sulfonamides By The Bratton-Marshall Reaction For Clinical Analysis, Assays And Dissolution Studies Of Formulation"
Anal. Chim. Acta 1988 Volume 204, Issue 1-2 Pages 271-283
M. A. Koupparis and P. I. Anagnostopoulou

Abstract: Two flow injection manifolds are described based on the Bratton - Marshall reaction, one for 2 to 20 mg L-1 of sulfonamide and the other for 0.5 to 5 mM. N-(1-Naphthyl)ethylenediammonium chloride is used as chromogenic reagent with detection at 545 nm. The method was used in the determination of sulfonamides in serum, urine, feeds and formulations, and for automated dissolution studies of tablets. The detection limit was 0.6 to 1.1 mg l-1, and the coefficient of variation was <0.5% (n = 10). The analysis rate was 72 samples h-1. The pseudo-titrimetric method gave a 1.3% mean difference from the HPLC technique.
Sulfonamides Clinical analysis Spectrophotometry Dissolution rate Method comparison Chromogenic reagent

"Determination Of Serum Oestradiol By Normal-phase High Performance Liquid Chromatography With Peroxyoxalate Chemiluminescence Detection"
Anal. Chim. Acta 1988 Volume 205, Issue 1-2 Pages 255-260
Osamu Nozaki and Yasuhiro Ohba, Kazuhiro Imai

Abstract: 17α-Oestradiol(I) was extracted from serum (500 µL) with use of an ODS minicolumn (6 mm x 8 mm i.d.). I was eluted with methanol and, after evaporation of the solvent, was derivatized with dansyl chloride. After cleanup on the ODS minicolumn and re-elution with methanol, the eluate was evaporated to dryness and the residue was dissolved in 60 µL of mobile phase. A 40 µL portion of this solution was analyzed by HPLC on two columns (15 and 10 cm x 1.9 mm) of Nucleosil 50-5 in series, with hexane - CHCl3 (7:3) containing 0.1% of ethanol as mobile phase (0.3 mL min-1). The eluate was mixed (0.4 mL min-1) with 8 mM bis-(2,4,6-trichlorophenyl) oxalate - 0.1 M triethylamine in CHCl3 and 0.6 M H2O2 in methanol containing 6% of acetate buffer (pH 4.0), and the chemiluminescence was measured. Overall recovery of I was ~90%, and the limit of detection was 50 pg.
Oestradiol Chemiluminescence HPLC Post-column derivatization

"Bioluminescence Flow System For Determination Of Branched Chain L-amino Acids In Serum And Urine"
Anal. Chim. Acta 1988 Volume 205, Issue 1-2 Pages 229-237
S. Girotti, A. Roda, M. A. Angellotti and S. Ghini, G. Carrea and R. Bovara, S. Piazzi and R. Merighi

Abstract: Deproteinized urine was centrifuged at 1000 g, the supernatant liquid was filtered (0.45 µm), and the filtrate was diluted 10 to 100-fold with 0.11 M Na4P2O7 buffer of pH 9.0. Serum samples were filtered and diluted with the same buffer. A 10 µL portion of the test sample was injected into an air stream which was mixed with a reagent stream containing NAD+ in the buffer solution and fed through a 1-m reaction coil containing immobilized leucine dehydrogenase, whereupon branched-chain L-amino-acids (L-leucine, L-valine and L-isoleucine) were oxidized to the corresponding 2-oxo-acids, with the formation of NADH. The product was mixed with 10 µM-flavine mononucleotide - 27 µM-decanol - 0.5 mM dithiothreitol in 0.1 M potassium phosphate buffer (pH 6.9) and fed into a second reaction coil where NADH was oxidized in the presence of immobilized NADH oxidoreductase (FMN) and alkanal mono-oxygenase (FMN-linked) and the luminescence was measured. Calibration graphs were rectilinear for 20 to 2000 pmol of acid; the limit of detection was 5 pmol. Within- and between-assay coefficient of variation (n = 10) were 7% and 9.7%, respectively, and recovery was 85 to 116%. Other amino-acids and branched-chain 2-oxo-acids did not interfere. Results for 41 serum samples correlated well with those obtained by ion-exchange chromatography.
Amino acids, L l-Isoleucine l-Leucine l-Valine Bioluminescence Immobilized enzyme Indirect Interferences Method comparison

"Flow Injection System For Simultaneous Assay Of Free And Total Cholesterol In Blood Serum By Use Of Immobilized Enzymes"
Anal. Chim. Acta 1988 Volume 207, Issue 1-2 Pages 319-323
Toshio Yao and Tamotsu Wasa

Abstract: Serum (5 µL) was injected into a carrier solution (1.5 mL min-1) of 0.1 M phosphate buffer (pH 7.5) containing 2.0% Triton X-100 and 0.5 mM in K4Fe(CN)6. The stream was split so that part passed through a 10-mm-long immobilized cholesterol esterase column before passing through a 5-mm-long immobilized cholesterol oxidase column; the other part only passed through the latter column. A constant potential of -30 mV vs. Ag - AgCl was applied to a peroxidase electrode and the current was recorded. The max. of the two peaks obtained were rectilinearly related to the concentration. of free and total cholesterol, respectively. The detection limits were 0.8 and 3 µM, respectively. Up to 10 samples could be analyzed in 1 h.
Cholesterol, free Cholesterol, total Amperometry Electrode Immobilized enzyme Triton X Surfactant

"Determination Of Creatinine In Undiluted Blood Serum By Enzymatic Flow Injection Analysis With Optosensing"
Anal. Chim. Acta 1988 Volume 214, Issue 1-2 Pages 147-159
Mette Tranholm Jeppesen and Elo Harald Hansen

Abstract: For the determination of creatinine in serum, a flow injection system was used that included a packed-bed enzyme reactor containing creatinine deiminase bonded to CPG-10 controlled-pore glass to liberate NH4+, which was converted into NH3 and transferred via a gas-permeable membrane (Celgard 2500 polypropylene; 0.04 µm pores) to an indicator stream containing 0.04% bromothymol blue at pH 6.8 for spectrophotometric detection at 620 nm. Endogenous NH3 was either removed by inclusion of a pre-reactor containing CPG-10-bonded glutamate dehydrogenase [NAD(P)+] or was monitored in a parallel system in which the creatinine deiminase reactor was omitted to obtain a reagent blank. The carrier stream in the pre-reactor system was 50 mM Tris containing 5 mM 2-oxoglutaric acid, 2 mM NADH, 20 mM EDTA and 0.5 mM ADP, with a reagent stream of 0.2 M NaOH to provide pH of 11.9. In the reagent-blank system, the carrier stream contained 20 mM Na2B4O7 to give pH of 9.2. The operating range was up to 1 mM for NH3 and for creatinine. Enzymic conversion efficiency was 91 ± 2%, and within-run reproducibility was ±4%.
Creatinine Clinical analysis Spectrophotometry Celgard Controlled pore glass Gas diffusion Immobilized enzyme Optosensing

"Flow Injection Determination Of Branched-chain L-amino-acids With Immobilized Leucine Dehydrogenase"
Anal. Chim. Acta 1989 Volume 218, Issue 1 Pages 161-166
Nobutoshi Kiba, Sachie Hori and Motohisa Furusawa

Abstract: Leucine dehydrogenase was immobilized on polystyrene Bio-Beads in a stainless-steel column (10 cm x 4 mm) and the column was incorporated into a flow system for determination of L-leucine, L-isoleucine and L-valine. A solution of 10 mM NAD+ in 1 mM Na4 EDTA was mixed with buffer solution (glycine - KCl - KOH of pH 11) before injection of the sample. The solution then passed to the enzyme reactor (thermostatted at 40°C) and the NADH produced was monitored fluorimetrically at 465 nm (excitation at 340 nm). The calibration graph was rectilinear from 5 µM to 0.5 mM isoleucine. The detection limit for L-leucine was 1 µM. The method was applied in the analysis of serum.
l-Leucine l-Isoleucine l-Valine Fluorescence Polystyrene beads Column Buffer Immobilized enzyme Heated reaction Detection limit

"Spectrophotometric Flow Injection Determination Of Urea In Body Fluids By Using An Immobilized Urease Reactor"
Anal. Chim. Acta 1989 Volume 218, Issue 1 Pages 151-155
Petr Solich, Miroslav Pol&aacute;ek and Rolf Karl&iacute;ek, Olga Valentov&aacute; and Miroslav Marek

Abstract: Urease was immobilized on poly(glycidyl methacrylate)-coated controlled-porosity glass which was then packed into PTFE tubing. The reactor was incorporated into a flow system for determination of urea. Serum was diluted 1:50 and urine 1:1000 or 1:2000 with water before injection into 0.2 M phosphate buffer (pH 6.9) containing 2 mM Na2 EDTA, 1 mM NaN3 and 5 µM-2-mercaptoethanol. The solution passed to the enzyme reactor before being mixed with 0.37 M Na salicylate - 4 mM Na nitroprusside - 0.25 M NaOH in aqueous 30% ethanol, and then with 30 mM NaClO and passing to a reaction coil (1.5 m x 0.5 mm) at 50°C. The absorbance of the solution was measured at 700 nm. The calibration graph was rectilinear for 25 to 500 µM-urea, and the coefficient of variation (n = 10) for 300 µM-urea was 1.1%.
Urea Spectrophotometry Buffer Immobilized enzyme Controlled pore glass

"Kinetic Determination Of Lactate Dehydrogenase In Blood Serum By Multi-detection With A Cyclic Flow Injection System"
Anal. Chim. Acta 1989 Volume 219, Issue 2 Pages 191-199
Juan M. Fernandez-Romero, M. D. Luque de Castro and M. Valc&aacute;rcel

Abstract: Cyclic flow injection allows the repeated passage of the reacting plug through the detector, resulting in multiple peak recordings. The required sensitivity can be selected by using procedures based on fixed-time measurements (peak maxima or minima) or reaction-rate measurements (signal increment between two successive maxima or minima). The method was applied in the determination of lactate dehydrogenase (I) in serum, based on its catalytic effect on the reduction of pyruvate to lactate by NADH. The reaction was monitored at 340 nm. Under optimum conditions, calibration graphs were rectilinear for 0.1 to 2.0 iu L-1 of I. The average recoveries of I were in the range 99.1 to 102.3%. The coefficient of variation (n = 11) were 0.2 to 3.5%.
Enzyme, lactate dehydrogenase Spectrophotometry Kinetic Multidetection Closed loop Peak analysis Sensitivity Optimization

"Flow Injection Determination Of L-tyrosine In Serum With An Immobilized Tyrosinase Reactor And Fluorescence Detection"
Anal. Chim. Acta 1989 Volume 224, Issue 1 Pages 133-138
Nobutoshi Kiba, Masae Ogi and Motohisa Furusawa

Abstract: Serum (100 µL) was deproteinized with Na2WO4 - H2SO4 and filtered, and 50 µL of the filtrate was injected into a carrier stream of 0.3 M phosphate buffer of pH 7.2 (0.6 mL min-1), which passed through a separation column (5 cm x 4 mm) of Capcell 120 C18 (5 µm) and a reactor column (5 cm x 4 mm) containing the cited enzyme immobilized on controlled-pore glass beads and was then mixed with a stream of 5 M KOH (1 mL min-1). The fluorescence was measured at 490 nm (excitation at 375 nm). The calibration graph was rectilinear in the range 0.1 µM to 0.1 mM tyrosine, the detection limit was 50 nM, and the coefficient of variation (n = 10) for the determination of 5.0 µM-tyrosine was 2.0%.
Tyrosine Fluorescence Buffer Column Immobilized enzyme Controlled pore glass Detection limit

"Determination Of Total 3α-hydroxy-bile Acids In Serum By A Bioluminescent Flow Injection System Using A Hollow-fibre Reactor"
Anal. Chim. Acta 1989 Volume 225, Issue 2 Pages 273-282
Kayoko Oda, Shigeru Yoshida and Shingo Hirose, Tatsumori Takeda

Abstract: A bacterial luciferase and NADH-flavin mononucleotide oxidoreductase were covalently co-immobilized on CNBr-activated Sepharose 4B. A permeable membrane reactor was used to introduce NAD+ and the bioluminescent reagent [comprising flavin mononucleotide, dithiothreitol and decanal in phosphate buffer (pH 6.8)]. The column, filled with the immobilized bioluminescent enzyme, was placed in front of a photomultiplier tube inside a photon counter to give a continuous-flow light-emitting system which was versatile and simple. The membrane reactor flow injection system allowed >20 samples h-1 to be analyzed. The detection limit was 10 fmol of NADH for a 1 µL injection. The calibration graphs for Na cholate were rectilinear up to 10 µM and 5 to100 µM. Excellent reproducibility, precision and sensitivity were achieved. Results agreed with those obtained by fluorimetry.
Bile acids, 3-α hydroxy Cholate Bioluminescence Hollow fiber membrane Buffer Column Detection limit Sensitivity Method comparison Sepharose beads Immobilized enzyme

"Flow Injection And Liquid Chromatographic Determination Of Aluminum Based On Its Fluorimetric Reaction With 8-hydroxyquinoline-5-sulfonic Acid In A Micellar Medium"
Anal. Chim. Acta 1989 Volume 225, Issue 2 Pages 339-350
Jos&eacute; Ignacio Garci&aacute; Alonso, Angeles L&oacute;pez Garci&aacute;, Alfredo Sanzmedel and Elisa Blanco Gonzales, Les Ebdon and Phil Jones

Abstract: Reaction of Al with 8-hydroxyquinoline-5-sulfonic acid (I) in cationic micelles of hexadecyltrimethylammonium bromide (II) formed a strongly fluorescent compound and allowed continuous determination of Al in flowing systems. At pH 6.3, with 0.2 mM I and 2 mM II, the detection limit for Al by flow injection analysis was 0.1 ng mL-1. The calibration graph was rectilinear for 100 ng mL-1, and the peak-height precision was 2% for 10 ng mL-1. Interference was greatly decreased compared with the batch method. The reaction was applied to the post-column detection of Al in fresh and high-salinity waters and dialysis fluid by ion chromatography, and for speciation of Al in serum after separation of proteins by ion-exchange LC.
Aluminum Fluorescence Interferences Buffer Speciation 8-hydroxyquinoline-5-sulfonic acid Dialysis Micelle

"Continuous-flow Bioluminescent Assay Of NADH Using A Fibre-optical Sensor"
Anal. Chim. Acta 1989 Volume 222, Issue 2 Pages 331-336
L. J. Blum, S. M. Gautier and P. R. Coulet

Abstract: The sensor comprises bacterial luciferase - oxidoreductase co-immobilized on pre-activated nylon membranes and placed in close contact with the tip of a glass optical fiber bundle. A light-tight flow cell was adapted to fit the end of the bundle. Sample solution were prepared in 0.1 M phosphate buffer (pH 7.8), and the reagent solution comprised 50 mM phosphate buffer (pH 7) containing 2 mM dithiothreitol, 0.2% of bovine serum albumin, 30 µM-FMN and 9.5 µM-decanal. The calibration graph was rectilinear from 2 pmol (the detection limit) to 1 nmol of NADH, and the coefficient of variation was 3.4% for 0.1 nmol. The sampling rate was 25 h-1. No loss in activity was observed after 150 determinations over 3 days.
Nicotinamide adenine dinucleotide reduced Sensor Immobilized enzyme Nylon Membrane Buffer Buffer Optical fiber

"Determination Of Magnesium In Blood Serum Using A Flow Injection System With A Potential-scanning Electrochemical Detector Equipped With A Thin-film Deaerator"
Anal. Chim. Acta 1990 Volume 234, Issue 1 Pages 161-166
Rivka Goldik, Chaim Yarnitzky and Magda Ariel

Abstract: A flow injection analysis system is described involving formation of the eriochrome black T (I) - Mg complex and its detection via its adsorptive reduction current peak at a potential-scanning electrochemical detector, equipped with a static-Hg-drop electrode vs. Ag - AgCl and a novel thin-film deaerator (illustrated). The method is basically an adaptation of a previous batch method (cf. An et al., Talanta, 1985, 32, 479). Diluted serum (100 µL) is injected into a carrier stream (0.6 mL min-1) of 2% ethylenediamine solution which is merged with the reagent stream (0.4 mL min-1) containing 0.003 to 0.004% of I, and the solution is passed through a 12-cm reaction coil to the deaerator - detector device. The calibration graph is rectilinear for 0.14 µg mL-1 of Mg. Results showed good agreement with those obtained by AAS. Sampling rate is 35 h-1.
Magnesium Electrochemical analysis Electrode Calibration Method comparison

"Utilization Of Adsorption-immobilized Urease In Gas Diffusion Flow Injection"
Anal. Chim. Acta 1990 Volume 237, Issue 2 Pages 503-508
T. L. Spinks and G. E. Pacey

Abstract: The enzyme was immobilized by addition of perfluorialkyl chains to the free amine groups of the enzyme and then adsorption on a PTFE gas-permeable microporous membrane. Injections. of 100 µL of urea were made into a carrier stream of 0.02 M Tris - HCl buffer (pH 8.5) at 0.9 mL min-1. This stream was merged with another stream of the same buffer. When the sample plug reached the membrane there was a 1 min stopped-flow period for conversion of the urea to NH3. The indicator on the acceptor side was Tecator NH3 - N mixed indicator (pH 6.4). When the streams were re-started the change in indicator absorbance was measured at 590 nm. The method was applicable in the range 0.1 to 500 mM urea. The method was used for the determination of urea in whole blood serum, with a sample throughput of 50 samples h-1.
Urea Spectrophotometry Gas diffusion Immobilized enzyme Membrane Buffer pH Stopped-flow Tecator

"Enzyme Co-immobilization For The Sequential Determination Of Lactic Acid And Glucose In Serum"
Anal. Chim. Acta 1990 Volume 238, Issue 2 Pages 411-415
M. T. Morales, P. Linares, M. D. Luque de Castro and M. Valc&aacute;rcel

Abstract: Lactic acid (I) and glucose (II), in a phosphate buffer carrier solution (pH 8.5), were determined by flow injection analysis in a reactor of immobilized glucose-6-phosphate dehydrogenase, hexokinase and L-lactate dehydrogenase at pH 8.0. The products, NADH and NADPH, from I and II, respectively, were monitored at 340 nm. Calibration graphs were rectilinear from 10 to 400 and 2 to 100 µg mL-1 of I and II, respectively, with corresponding coefficient of variation (n = 11) of 1.63 and 2.30%. Recoveries were 88.84 to 118.4% for I and 92.72 to 110.3% for II in serum. Mixtures of the analytes (up to 1:10) could be resolved.
Lactic acid Glucose Immobilized enzyme Buffer pH Calibration

"Determination Of L-alanine In A Flow Injection System With An Immobilized Enzyme Reactor"
Anal. Chim. Acta 1990 Volume 239, Issue 2 Pages 307-310
Nobutoshi Kiba, Hirohisa Tagami and Motohisa Furusawa

Abstract: The sample was injected into a merged stream of 15 mM NAD+ and 0.2 M glycine - NaCl - NaOH buffer, pH 10.3, at 0.8 mL min-1, which flowed into a reactor of alanine dehydrogenase immobilized on poly(vinyl alcohol) beads at 40°C. The fluoresence of the NADH produced was measured at 465 nm (excitation at 340 nm). The response was rectilinear from 0.5 to 500 µM, with a limit of detection of 0.2 µM and a coefficient of variation (n = 9) of 0.9% for 100 µM. The coefficient (n = 5) for serum and beverages were between 1% for 0.02 to 410 µM.
l-Alanine Fluorescence Immobilized enzyme Buffer Detection limit

"Spectrofluorimetric Determination Of Diquat By Manual And Flow Injection Methods"
Anal. Chim. Acta 1991 Volume 244, Issue 1 Pages 99-104
T. P&eacute;rez-Ruiz, C. Mart&iacute;nez-Lozano and V. Tom&aacute;s

Abstract: In the manual method, sample solution, containing 0.03 to 9 µg of diquat (I), was mixed with 2 mL of 0.5 M borax buffer (pH 8), 1 mL of 0.5% sodium dithionite and water to 10 mL and, after 5 min, the resulting stable radical of I was detected fluorimetrically at 497 nm (excitation at 428 nm). The method was also adapted for use in a flow injection procedure (diagram of apparatus and details given. The calibration graph was rectilinear from 3 to 900 and 18 to 4000 µg L-1 of I by the manual and flow injection methods, respectively; the detection limit by the manual method was 0.4 µg L-1 and coefficient of variation were 2%. The method was applied in the determination of I in commercial herbicide formulations, water, potatoes, flowers and soil (sample prep. described) and in serum and urine directly with use of a standard additions method.
Diquat Fluorescence Buffer Standard additions calibration

"Dehydrogenase And Ethanol Assay Based On Visible Semiconductor Laser Spectrometry"
Anal. Chim. Acta 1991 Volume 251, Issue 1-2 Pages 191-195
Totaro Imasaka, Toshiyuki Higashijima and Nobuhiko Ishibashi*

Abstract: Methods are described for the assay of dehydrogenases and the determination of NAD+ and ethanol based on visible semiconductor laser spectrometry of methylene blue. Lactate dehydrogenase (I) was assayed in serum by using a batch system. Serum (20 µL) was incubated at 37°C for 20 min with reagent solution (1 ml) containing lactic acid, NAD+, diaphorase and methylene blue in Tris buffer solution (pH 8.6) and the reaction was stopped with 1 M HCl (5 ml). Fluorescence was measured with a semiconductor laser fluorimeter. Alcohol dehydrogenase (II) and ethanol (III) were determined by using a flow system (described with diagram). II solution (0.5 µL) was injected into a merging zone (5 µL) containing diaphorase (0.6 iu), NADH (5 nmol), III (300 nmol) and 500 mM methylene blue at pH 8.7. III (1 µL) was injected into a merging zone containing II (0.6 iu), diaphorase (0.1 iu), NAD+ (5 nmol) and 500 mM methylene blue at pH 8.7. For NAD+ determination, the merging zone contained diaphorase (0.6 iu) and methylene blue (2.5 pmol) in Tris buffer solution (pH 8.7). Detection limits were 0.1 miu of II and 10 nmol of III. The calibration graph for NAD+ was rectilinear up to 40 nmol.
Ethanol Enzyme, lactate dehydrogenase Fluorescence Sensor Buffer Heated reaction Merging zones

"Simultaneous Determination Of Amitriptyline, Imipramine And Perphenazine In Pharmaceutical Preparations And Blood Serum By Multi-wavelength Spectrophotometry"
Anal. Chim. Acta 1991 Volume 252, Issue 1-2 Pages 107-113
J. M. Garc&iacute;a Fraga, A. I. Jim&eacute;nez Abizanda, F. Jim&eacute;nez Moreno and J. J. Arias Le&oacute;n*

Abstract: Several methods were developed for the simultaneous determination of the binary mixtures amitriptyline-imipramine (1.00-10 µg mL-1), amitriptyline-perphenazine (1.00-30.00 and 1.00-8.00 µg mL-1) and imipramine-perphenazine (1.00-30.00 and 1.00-8.00 µg mL-1) and the ternary mixture amitriptyline-perphenazine-imipramine using a diode-array spectrophotometer and a multi-component analysis program. The methods were applied to the determination of the components of synthetic perphenazine-imipramine mixtures, real amitriptyline-imipramine and amitriptyline-imipramine-perphenazine mixtures in blood serum and real amitriptyline-perphenazine mixtures in pharmaceutical preparations.
Amitriptyline Imipramine Perphenazine Spectrophotometry Multivariate calibration

"Rapid Screening Of Porphyrins Using Flow Injection Analysis And Visible Laser Fluorimetry"
Anal. Chim. Acta 1991 Volume 254, Issue 1-2 Pages 189-196
Carmen W. Huie, Joseph H. Aiken and William R. Williams

Abstract: Urine and serum were diluted with 0.15 M hexadecyltrimethylammonium bromide in 0.01 M NaH2PO4 buffer, pH 7.4, and the solution were injected into a carrier stream of the same buffer. The fluorescence of the compounds was measured at ~620 nm with excitation at 488 nm provided by an Ar laser. Urine spiked with 0.02, 0.04 and 0.06 µmM was clearly distinguishable from the blank, these concentration. representing the lower range for abnormal urine. With serum the limit of detection was ~0.015 µM.
Porphyrins Fluorescence Buffer pH

"Flow Injection Determination Of L-glutamate In Serum With An Immobilized Glutamate Dehydrogenase Reactor"
Anal. Chim. Acta 1992 Volume 256, Issue 2 Pages 221-224
Nobutoshi Kiba*, Takami Moriya and Motohisa Furusawa

Abstract: Serum (10 µL) is treated with 10 µL of 5% Na2WO4 solution and 30 µL of 0.15 M H2SO4 and filtered through an Airpress-30 filter (Tosoh), and 30 µL of the filtrate is injected into a carrier stream (0.4 mL min-1) of 5 mM NAD+ in 0.05 M glycine buffer of pH 9.5. The stream passes through a stainless-steel column (3 cm x 4 mm) containing glutamate dehydrogenase (NAD(P)+) immobilized on poly(vinyl alcohol) beads (method described), and the NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is rectilinear for 0.5 to 500 µM-glutamate, and the detection limit is 0.2 µM. Glutamate dehydrogenase is immobilized on poly(vinyl alcohol) beads and packed into a stainless-steel column (3 cm x 4 mm i.d.). Serum is deproteinized with tungstic acid. Sample solution (30 µL) is injected into the carrier stream [5 mM NAD+ in glycine buffer (pH 9.5)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.5-500 µM glutamate; the detection limit is 0.2 µM.
l-Glutamate Fluorescence Immobilized enzyme Poly vinyl alcohol beads Buffer

"Direct Determination Of Copper In Serum By Flow Injection Analysis"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 87-89
Hiroko Wada*, Xiao Jing Zhou, Tomosuke Ishizuki, Hiromichi Yamada and Genkichi Nakagawa

Abstract: A 30 µL sample of serum was injected into a carrier stream of 0.5 M acetate buffer (pH 4.2) containing 1% of Triton X-100 and 3% of ethanol. This solution was mixed in a coil (25 cm x 0.45 mm i.d.) with 3.5 M guanidinium chloride (pH 4.2) containing 4.5 mM KIO3 and 0.4 mM KI and then in a 15-cm coil with a stream of aqueous 0.3 mM 4-methyl-5-(sulfomethylamino)-2-(2-thiazolylazo)benzoic acid; each flow rate was 0.6 mL min-1. The absorbance of the Cu complex was measured at 600 nm, which is preferred to the λmax of 585 nm because of the lower background and less interference from bilirubin and haemoglobin. The sampling frequency was 60 h-1. The coefficient of variation at 1.20 mg L-1 was 0.7% (n = 6), and results agreed with those by two methods involving sample pre-treatment. Cu in serum was determined with a flow injection system based on spectrophotometry using 2-(2-thiazolylazo)-4-methyl- 5-(sulfomethylamino)benzoic acid (TAMSMB) as chromogenic reagent. A serum sample (30 µL) is directly injected into the carrier containing 0.5 M HOAc-NaOAc buffer (pH 4.2), 1% Triton X 100 and 3% EtOH. The carrier merges into the stream of 3.5 M guanidine HCl containing 4.5 x 10^-3 M KIO3 and 4.0 x 10^-4 M KI, and then Cu(II) reacts with TAMSMB in the flow system. The absorbances are monitored at 600 nm. The sampling frequency is 60/h.
Copper Spectrophotometry Triton X Chromogenic reagent Interferences Surfactant

"Flow Injection Amperometric System For Enzyme Immunoassay"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 45-52
D. M. Ivnitskii*, R. A. Sitdikov and V. E. Kurochkin

Abstract: A mathematical model has been developed for describing and thus optimizing the operation of a flow injection amperometric detector with a Pt-wire working electrode and a Ag - AgCl (saturated KCl) counter electrode, which was used with a continuously circulating carrier solution of 0.1 M acetate buffer of pH 4.2 containing 0.1 M KCl for the detection of enzymatically produced iodine (from enzyme immunoassay of IgE with a commercial kit) in the injected sample. The agreement between calculated and experimental analytical parameters was satisfactory. Simultaneous evaluation by means of a photometric ELISA reader showed that the electrochemical sensor could be used to determine 1.5 to 100 kiu L-1 of IgE in blood. The technique could be used in conjunction with appropriate immunosorbents for the immunoenzymatic determination of hormones, enzymes, drugs, antibodies and viruses. A flow injection amperometric system was developed for monitoring the results of enzyme immunoassay. The operation of the flow-through amperometric detector is described by a limiting current equation for the working wire electrode. The possibility of the optimization of the geometry and hydrodynamic properties of amperometric detectors in flow systems was demonstrated. The flow system was tested in the determination of peroxidase and IgE in blood serum. Peroxidase (marker enzyme) was determined in the concentration. range 5 x 10^-12 - 4 x 10^-10 M. The detection limit for IgE in blood serum was 3.1 ng mL-1. The sample throughput was 400 hr-1.
Immunoglobulin G Enzyme, peroxidase Amperometry Immunoassay Electrode Modeling Optimization

"Flow-through Sensing Device Based On Derivative Synchronous Fluorescence Measurements For The Multi-determination Of B6 Vitamers"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 269-274
Danhua Chen, M. D. Luque de Castro and M. Valc&aacute;rcel*

Abstract: The apparatus incorporated a laboratory-built dual PTFE injection valve and a conventional fluorescence flow cell containing C18 material (60 to 100 µm beads). Portions of the sample (containing 50 nM to 1.5 µM concentration. of the vitamers) were injected, simultaneously with 3 M NH3 buffer containing 0.5 mM Be(NO3)2 (pH 9.9 or 7.9), into water as carrier. After passage through a 3-m reaction coil, the stream entered the flow cell, where the reaction products were trapped. The flow was stopped 2 min after reaction and synchronous-scanning fluorimetry was carried out between 500 and 320 nm (emission) with a 50-nm difference between the excitation and emission wavelengths. The first derivative of the scan was used for quantitation. Pyridoxic acid could be determined from the inverted peak at 377 nm. The peaks of pyridoxal and pyridoxal 5'-phosphate overlapped, but their magnitudes differed at the two pH values (the peak of pyridoxal being decreased more markedly at pH 7.9), allowing separate determination from the peaks at 446 nm (pH 9.9) and 440 nm (pH 7.9). Calibration graphs were rectilinear from 0.05 to 15 µM for pyridoxal and 0.05 to 10 µM for the other vitamers. The sampling rate was 6 h-1. The method was applied to serum samples that had been deproteinized with trichloroacetic acid at room temp.
Vitamin B6 Fluorescence Spectroscopy Valve Stopped-flow

"Comparison Of Electrochemiluminescence And Amperometric Detection Of Lipid Hydroperoxides"
Anal. Chim. Acta 1992 Volume 262, Issue 2 Pages 217-223
S. Sakura*, J. Terao

Abstract: Serum containing uric acid, ascorbic acid and tocopherol interferents was used for comparison of the cited techniques. Cyclic voltammograms were measured, for both methods, at vitreous carbon and Pt electrodes, in a flow injection system with phosphate buffer (pH 7.4) in a 30% acetonitrile solution Optimum conditions for electrochemical oxidation (ECO) were 0.3 to 0.5 mL min-1 flow rate and pH 7 to 8. Pre-treatment to remove uric acid and ascorbic acid is necesssary. Tocopherol does not interfere. Optimum conditions for electrochemiluminescence (ECL) were 0.3 to 0.5 mL min-1 flow rate, 10 to 20 µM luminol and pH of flow solution 7 to 9. Interferents should be removed prior to measurement. Detection limit for ECO at a vitreous carbon electrode was 0.05 nmole, the coefficient of variation (n = 5) was 4.9% and the signal to noise ratio was 1.36. The detection limit for ECL at a vitreous carbon electrode was 0.3 nmole, the coefficient of variation (n = 5) was 1.3% and the signal to noise ratio was 1.5. At a Pt electrode the detection limit for ECL was 0.1 nmole, the coefficient of variation (n = 5) was 5.3% and the signal to noise ratio was 2.5.
Hydroperoxides, lipid Chemiluminescence Amperometry Electrode Electrode Interferences Signal to noise ratio Optimization

"Co-immobilized Enzyme Columns In Determining Serum Creatinine Using Creatininase, Creatinase And Sarcosine Oxidase By Flow Injection Analysis And Chemiluminescence Detection"
Anal. Chim. Acta 1992 Volume 262, Issue 2 Pages 315-321
Masayoshi Tabata*, Masayuki Totani, Jiro Endo

Abstract: The column of co-immobilized creatininase - creatinase - sarcosine oxidase (2:5:3) was selected from six wt. ratios of the enzymes. In the cited determination, detection was based on the chemiluminometric rate assay of H2O2 produced. The flow rate for luminol, hexacyanoferrate(III) and 10 mM K phosphate buffer (pH 8.5) was 0.6 mL min-1. Total chemiluminescence emission was determined from the area under the light - time curve. The method was compared with the modified Jaffe reaction rate method. The calibration graph was rectilinear up to 1.0 mM creatinine. Within-day and day-to-day coefficient of variation (n = 10) were 2.4 and 3.7%, respectively. Creatinine recovery was 97 to 101%. The title method was based on the chemiluminometric rate assay of hydrogen peroxide produced from creatinine by consecutive reactions of three immobilized enzymes. The procedure is independent of the concentration. of endogenous creatine in serum, although creatininase catalyzes the reversible reaction. Further, because of the shifts of pH optima caused by immobilization of the three enzymes, successive reactions could be carried out under almost optimum conditions at pH 8.5. The method was found to give perfect calibration linearity, with accurate recoveries ranging from 97 to 101% for up to 1.0 mM creatinine. The within-day and day-to-day relative standard deviations (n = 10) were 2.4% and 3.7%, respectively, for 0.10 mM creatinine and 1.6% and 2.1%, respectively, for 0.96 mM creatinine. The results correlated satisfactorily with those obtained by the modified Jaffe reaction rate method.
Creatinine Chemiluminescence Immobilized enzyme Method comparison Indirect Optimization

"Total And Individual Determination Of Creatine Kinase Isoenzyme Activities By Flow Injection And Liquid Chromatography"
Anal. Chim. Acta 1992 Volume 263, Issue 1-2 Pages 43-52
M. D. Luque de Castro* and J. M. Fernandez-Romero

Abstract: A flow manifold was devised for online coupling of a cyclic flow injection system to a LC system. Sample was injected simultaneously into the loops of the flow injection and LC systems. By appropriate switching of the selective valve, the combined stream is trapped in a closed circuit consisting of an enzyme reactor containing two auxiliary enzymes co-immobilized on controlled-pore glass (120 to 200 mesh); spectrophotometric or fluorimetric detection was used. The optimum values of the experimental parameters were evaluated. The method was applied to the determination of the total and individual activity of creatine kinase isoenzymes in serum. Recoveries were >95%. Calibration graphs were rectilinear from 0.01 to 2.00 iu L-1 and the coefficient of variation were 2%. A flow injection liquid chromatography method for the simultaneous determination of total and individual creatine kinase isoenzyme activities is proposed. The flow manifold coupled to the liquid chromatograph is a cyclic circuit that allows each eluted isoenzyme to be trapped within. This circuit includes a spectrophotometric detector and a reactor containing co-immobilized auxiliary enzymes that take part in the post-column derivatization reaction. The iterative passage of the reacting zones through the enzymatic reactor provides a series of multiple-peak recordings that can be used to determine total and individual isoenzymes; such recordings supply a wealth of information and enhanced sensitivity for the determinations Linear calibration graphs between 0.01 and 2.00 U L-1 and relative standard deviations <2.0% were obtained in all instances. The application of the method to serum samples provided results consistent with those obtained by the standard method and recoveries within the range 95-105%.
Enzyme, creatine kinase, isoenzymes HPLC Immobilized enzyme Controlled pore glass Post-column derivatization

"Flow Injection Determination Of Glucose In Serum With An Immobilized Pyranose Oxidase Reactor"
Anal. Chim. Acta 1992 Volume 269, Issue 2 Pages 187-191
Nobutoshi Kiba*, Fumito Ueda and Motohisa Furusawa, Takeshi Yamane

Abstract: Pyranose oxidase (I) or glucose oxidase (II) were immobilized on controlled-pore glass beads and packed into a stainless-steel column (5 cm x 4 mm) by the slurry-packing method. Glutaraldehyde solution (2%) in 0.1 M phosphate buffer (pH 7.0) was pumped through the column for 2 h at 0.2 mol min-1 and the column was washed with water. Enzyme solution (1000 iu of I or II in 0.05 M phosphate buffer (pH 7.0) was circulated through the column for 3 h at 0.2 mL min-1. Serum samples (10 µL) were injected into the carrier stream of the flow injection system (diagram given) and the H2O2 produced in the reactor at 40°C was detected by measuring the chemiluminescence emitted on mixing with luminol and K3Fe(CN)6. The calibration graph was rectilinear from 0.2 to 500 and 0.5 to 1000 µM of glucose for immobilized I and II, respectively; the corresponding detection limits were 0.05 and 0.1 µM.
Glucose Chemiluminescence Immobilized enzyme Heated reaction Controlled pore glass Indirect

"Flow Injection Determination Of 1,5-anhydroglucitol In Serum With An Immobilized Pyranose Oxidase Reactor And Chemiluminescence Detection"
Anal. Chim. Acta 1993 Volume 271, Issue 1 Pages 47-51
Nobutoshi Kiba*, Fumito Ueda, Kazuya Saegusa, Yuusuke Toto and Motohisa Furusawa, Takeshi Yamane

Abstract: A stainless-steel enzyme reactor column (5 cm x 4 mm) containing pyranose oxidase bonded via glutaraldehyde onto aminopropyl-bonded controlled-pore glass (200 to 400 mesh; pore diameter 59 nm) was used at 50°C with a pre-column (4 cm x 4 mm) of TSKgel SAX anion-exchange resin (10 µm; Cl- form) for sample cleanup and a flow stream of 0.1 M NaH2PO4 - 0.05 M citric acid (pH 4.5). Post-column reaction with 0.7 mM luminol and 0.2 M K3Fe(CN)6 in a 0.3 M carbonate buffer (pH 10.5) was followed by luminescence detection of H2O2. The rectilinear calibration range was 0.4 µM to 0.2 mM 1,5-anhydroglucitol (I) with a detection limit of 0.2 µM. Human serum was analyzed and recoveries of I were 97 to 104% with coefficient of variation of 2.0% within-day or 2.4% between-day for 144 µM-I.
1,5-Anhydroglucitol Chemiluminescence Controlled pore glass Reactor Immobilized enzyme Heated reaction

"Flow Injection Spectrophotometric Enzymic And Non-enzymic Methods For The Determination Of Direct And Total Bilirubin In Serum"
Anal. Chim. Acta 1993 Volume 276, Issue 2 Pages 271-279
J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro* and M. Valc&aacute;rcel

Abstract: The first method involves an automatic flow injection system for the sequential determination of total and direct bilirubin based on catalytic oxidation (with bilirubin oxidase) of conjugated bilirubin in an acidic medium and the oxidation of unconjugated bilirubin in a basic medium. A stopped-flow system was used and the change in absorbance was measured at 460 nm. The calibration graph was rectilinear from 0.09 to 25 µM and from 0.14 to 13 µM for total and direct bilirubin, respectively. The second method is based on the differential reactivity (in an acidic medium) of conjugated and unconjugated bilirubin with Erlich reagent. Methanol was used as accelerator and the azorubin pigment formed was monitored at 560 nm. The calibration graph was rectilinear from 0.85 to 144 µM and from 3.8 to 125 µM for total and direct bilirubin, respectively. For both methods the coefficient of variation was 3%. No significant interference was found from substances commonly present in serum. Results agreed well with those obtained by an established conventional method.
Bilirubin, total Spectrophotometry Enzyme Stopped-flow Kinetic Method comparison Interferences

"Pulsed Amperometric Detection Of Proteins Using Antibody Containing Conducting Polymers"
Anal. Chim. Acta 1993 Volume 279, Issue 2 Pages 209-212
O. A. Sadik and G. G. Wallace*

Abstract: The antibody containing electrode was prepared by galvanostatically electropolymerising pyrrole monomer from an aqueous solution containing 0.05 M pyrrole and 100 mg/l of antihuman serum albumin on to a Pt disc at a current density of 0.5 mA/cm2. The prepared electrode was used with Pt gauze auxiliary and Ag/AgCl reference electrodes in an FIA system with 0.1 M NaNO3 as eluent (1 ml/min). The amperometric response to human serum albumin was measured using a pulsed potential at an initial potential of +0.4 V, a final potential of 0.0 V and a pulse width of 120 ms. The reproducibility of the response was ± 5% in the range 5-50 ppm of human serum albumin and the detection limit was 1 mg/l.
Proteins Albumin Amperometry Electrode

"Spectrophotometric Determination Of Magnesium In Serum By Using A Flow Injection System With An Immobilized Enzyme Reactor"
Anal. Chim. Acta 1993 Volume 283, Issue 1 Pages 447-452
J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro and M. Valc&aacute;rcel*, R. Quiles-Zafra

Abstract: The method was based on the activating effect of Mg(II) on the hydrolysis of 2-nitrophenyl β-D-galactopyranoside (I) by β-galactosidase with all other reactants present in excess. In a merging-zones configuration (illustrated), portions of the sample and a solution of 4.7 g/l of NaCl and 60.3 mg/l of I in reagent A (aqueous 0.1 M Tris/4 mM dithiothreitol/0.4 mM EGTA of pH 7.5) were simultaneously injected into a stream of reagent A. In a region maintained at 37°C, the solution were mixed in a single-bead string reactor and passed through a reactor containing β-galactosidase immobilized on controlled-pore glass. A flow of 1 M NaOH was introduced and merged with the stream in a reactor coil, and the absorbance was monitored at 405 nm. Optimized operating parameters were established. Forty samples could be analyzed in 1 h, and response was linear for 5-20 µM-Mg. Within-run and between-run RSD (n = 11) were 0.78-2.91% and 1.7-3.21%, respectively. The method showed good selectivity, with no interference from a 20-fold amount of calcium. Recovery of added Mg was 96-113% and results were well correlated with those of AAS.
Magnesium Spectrophotometry Immobilized enzyme Merging zones Optimization Method comparison Controlled pore glass Interferences

"Flow Injection Analysis For Total Cholesterol With Photometric Detection"
Anal. Chim. Acta 1994 Volume 287, Issue 1-2 Pages 59-64
Achim Krug*, Roman G&ouml;bel and Robert Kellner

Abstract: Serum (70 µL) was injected into a carrier stream (3 ml/min) of 0.1 M phosphate buffer of pH 7/2-propanol/Triton X-100 (83:13:4) and the stream was passed through a glass column enzyme reactor (10.5 cm x 1.5 mm i.d.) containing a 45 mm section filled with cholesterol esterase immobilized on to controlled pore glass (CPG) beads (120-200 mesh; 127 nm pore diameter) and a 60 mm section filled with cholesterol oxidase immobilized on to CPG. The sample stream passed through the cholesterol esterase section first. Hydrogen peroxide generated was determined by post-column reaction with 3.5 mM 2,2-azinobis(3-ethylbenzthiazoline-6-sulfonate) catalyzed by horse-radish peroxidase (5 U/ml) and with detection at 423 nm. The calibration graph was linear for 0.1-0.86 mM cholesterol and the RSD (n = 8) for 5.1 mM cholesterol was 2.5%. The sample throughput was 50 samples/h. Reducing species do not interfere at concentrations >10 times their normal levels in serum.
Cholesterol, total Spectrophotometry LC Interferences Controlled pore glass Triton X Surfactant

"Novel Mercury-coated Carbon Fibre Voltammetric Detector For Use In Adsorptive Stripping Flow Analysis"
Anal. Chim. Acta 1994 Volume 289, Issue 2 Pages 169-176
Jos&eacute; Amez del Pozo, Agust'in Costa-Garc&iacute;a and Paulino Tu&ntilde;&oacute;n-Blanco

Abstract: A mercury-coated carbon fiber micro-electrode was used in an FIA system for the determination of mitozantrone (MZT) by a.c. stripping voltammetry. The FIA manifold was constructed by connecting the injection loop to the detection cell via a PTFE tube (50 cm x 1 mm i.d.). The flow passed over the carbon fiber working electrode (7.5 µm diameter) and then over the Ag/AgCl reference electrode. The stainless-steel outlet from the detector cell acted as the auxiliary electrode. The carrier stream was 0.1 M HClO4. The Hg film was formed on the working electrode by injecting 0.5 mL of 10 mM Hg(II) nitrate and applying a potential of -1.2 V for 180 s. A 0.5 mL volume of the MZT solution was injected into the carrier stream (0.2 ml/min) and, after a delay of 270 s, the voltammogram was recorded by scanning from 0.0 to -1.5 V at 10 mV/s. The optimum a.c. monitoring conditions were a superimposed potential of 30 mV, a phase angle of 90°C and a fixed frequency of 75 Hz. After each recording the electrode was activated at 0.4 V for 60 s. The method was applied to the determination MZT in blood serum (details given). The calibration graph was linear for 0.45 (detection limit) to 4.5 µM-MZT.
Mitozantrone Voltammetry Electrode Electrode Electrode

"Heterogeneous Fluorescence Immunoassays Using Flow Injection Analysis With Protein G Solid-phase Reactors"
Anal. Chim. Acta 1994 Volume 290, Issue 1 Pages 154-158
Muhammad Y. Khokhar, James N. Miller and Nichola J. Seare

Abstract: An FIA heterogeneous fluorescence immunoassay for insulin was developed using a protein G immunoreactor and rhodamine isothiocyanate (RITC) as a pH resistant fluorescent label. Wild-type protein G was immobilized on agarose beads which packed in a glass micro-column (5 cm x 3 mm i.d.) to form the immunoreactor. The immunoassay proceeded by incubating the insulin samples (0.05-2.25 ng/ml in 1 ml) with 30 mL of a 1:25 dilution of guinea-pig anti-bovine insulin antibodies for 45 s. RITC-insulin conjugate (1.3 µg in 5 mL of PBS of pH 7.4) was added and, after a further 120 s incubation, a 125 µL portion was injected into 0.05 M Tris buffer of pH 8.8 carrier stream (flow rates not given) of the FIA manifold. After 300-350 s, the carrier stream was changed to 0.1 M citrate buffer of pH 2.5 containing 0.1% Triton X-100 to elute the antibody-bound RITC-insulin conjugate which had adhered to the protein reactor. Fluorescence detection was at 585 nm (excitation at 546 nm). Serum was diluted before analysis by the proposed method and the recovery of 0.12 ng/ml of insulin from serum averaged 120.1%.
Insulin Fluorescence Immunoassay Triton X Surfactant

"Flow Injection Determination Of 3-hydroxybutyrate In Serum With An Immobilized 3-hydroxybutyrate Dehydrogenase Reactor And Chemiluminescence Detection"
Anal. Chim. Acta 1994 Volume 290, Issue 3 Pages 357-361
Nobutoshi Kiba*, Hidekazu Koemado and Motoshisa Furusawa

Abstract: Serum (10 µL) was diluted 10-fold with 0.1 M glycine/0.1 M NaCl/0.1 M NaOH buffer of pH 9.3 (buffer A), ultrafiltered through an Advantec Q0100 membrane (mol. wt. cut-off 10 000) and injected into pre-merged streams (0.25 ml/min) of 5 mM NAD+ in 0.01 M phosphate buffer of pH 7 and buffer A. The stream passed through an enzyme reactor (4 cm x 4 mm i.d.; 40°C) containing 3-hydroxybutyrate (I) dehydrogenase immobilized on poly(vinyl alcohol) beads (13 µm diameter) and then merged with a stream (0.5 ml/min) of 600 µM-1-methoxy-5-methylphenazinium methylsulfate before passing through a reaction coil (10 m x 0.5 mm i.d.; 40°C). It was then mixed with pre-merged streams (0.5 ml/min) of 3 mM luminol in 0.4 M carbonate buffer of pH 10.5 and 20 mM potassium hexacyanoferrate(III), for chemiluminescent detection of the generated H2O2. The calibration graph was linear for 0.5-300 µM-I, the detection limit in serum was 1 µM, the within-day and day-to-day RSD were 0.87% and 1.5%, respectively, and the recoveries from spiked serum were >98%. Sample throughput was 20/h and the reactor was stable for 1 month.
3-hydroxybutyrate Chemiluminescence Immobilized enzyme

"Determination Of 3-hydroxybutyrate In Serum By Flow Injection Analysis Using A Co-immobilized 3-hydroxybutyrate Dehydrogenase/NADH Oxidase Reactor And A Chemiluminometer"
Anal. Chim. Acta 1994 Volume 298, Issue 1 Pages 129-133
Nobutoshi Kiba*, Hidekazu Koemado, Junko Inagaki and Motohisa Furusawa

Abstract: A flow-injection system with a co-immobilized enzyme reactor is described for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase and NADH oxidase were immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (5 cm x 4 mm i.d.). Serum was diluted 10-fold with borate buffer (pH 9.4) and ultrafiltered. Filtrate (20 µL) was injected into the carrier stream. The hydrogen peroxide produced was detected chemiluminometrically via a luminol-hexacyanoferrate(III) reaction. The calibration graph was linear for 2 x 10^-7 M-5 x 10^-4 M; the detection limit was 8 x 10^-8 M and the sample throughput was 30 h-1 without carryover.
3-hydroxybutyrate Chemiluminescence Immobilized enzyme Reactor

"Spectrophotometric Cell Comprising Parallel Rotating And Stationary Bioreactors: Application To The Determination Of Glucose In Serum Samples"
Anal. Chim. Acta 1995 Volume 300, Issue 1-3 Pages 299-305
Julio Rabaa, Shaofeng Lib and Horacio A. Mottolab,*

Abstract: A spectrophotometric cell comprising parallel bioreactors facing each other and containing immobilized enzyme preparations is described. The lower reactor rotates to minimize diffusional constraints, and the upper reactor is fixed to provide an integrated design for the realization of coupled enzyme-catalyzed reactions. The operating characteristics of the cell are illustrated with the determination of glucose using glucose oxidase [EC 1.1.3.4] and horseradish peroxidase [EC 1.11.1.7] as immobilized enzymes (horseradish peroxidase on the rotating reactor and glucose oxidase on the stationary one). The H2O2 produced in the dissolved-oxygen oxidation of β-D-glucose enters into oxidative coupling in a reaction with N,N-dimethylaniline and 4-aminophenazone which is catalyzed by horseradish peroxidase; the absorbance of the colored complex formed provides the basis for monitoring. The cell was incorporated into a continuous-flow/stopped-flow/continuous-flow operation, and the determination was based on the rate of response under stopped-flow conditions. The overall approach was applied to the determination of glucose in standards of human serum and samples of bovine blood serum.
Glucose Spectrophotometry Immobilized enzyme

"Stopped-flow Kinetic Determination Of Nalidixic Acid And Norfloxacin Based On Lanthanide-sensitized Fluorescence"
Anal. Chim. Acta 1995 Volume 303, Issue 1 Pages 39-45
S. Panadero, A. G&oacute;mez-Hens and D. P&eacute;rez-Bendito*

Abstract: Nalidixic acid and norfloxacin were determined in this work for the first time by applying kinetic methodology to complex formation reactions involving energy-transfer processes. By using initial-rate measurements, a fluorescence-based method based on the formation of the complexes of these compounds with terbium(III) in the presence of Tris buffer was developed. The formation rate of these complexes is very high, so the stopped-flow mixing technique is required in order to make kinetic measurements, which, in addition, allows the method to be automated. A second ligand, EDTA, is also used to decrease the reaction rate of both systems via ligand-exchange, which is necessary in order to obtain appropriate kinetic data. The reaction rate is obtained within only 0.1-0.2 s after the reactants are mixed, which allows ready application of the proposed method to routine analyzes. The calibration graph is linear over the range 0.02-7.0 µg mL-1 nalidixic acid and 0.4-9.0 µg mL-1 norfloxacin. The detection limit for nalidixic acid and norfloxacin is 0.006 and 0.13 µg mL-1, respectively. The selectivity and precision of the method were also studied. The proposed kinetic method was satisfactorily applied to the direct determination of these compounds in serum samples.
Nalidixic acid Norfloxacin Fluorescence Indirect Stopped-flow Kinetic

"Air-segmented Flow Injection: A Hybrid Technique For Automated, Low Dispersion Determinations"
Anal. Chim. Acta 1995 Volume 303, Issue 2-3 Pages 231-239
Yunsheng Hsieh and S. R. Crouch*

Abstract: The hardware requirements of the title technique are discussed, and the degree of instrumental sample dispersion critically evaluated using 10 µM-phenol red in a borate buffer of pH 9.5. Applications of the technique to the analysis of glucose in wine and serum at 510 nm as previously described (Ibid., 1993, 284, 159) are presented, with a calibration graph linear over the range 10^-80 ppm glucose. Over the range 0.0093-0.42% glucose in wine, RSD (n = 5) of 0.46-0.78% were obtained. At 79 mg/dl glucose in serum an RSD (n = 5) of 3% was obtained.
Glucose Spectrophotometry Air segmentation Dispersion

"Inductively Coupled Plasma Mass-spectrometric Determination Of Gold In Serum: Comparison With Flame And Furnace Atomic Absorption Spectrometry"
Anal. Chim. Acta 1995 Volume 304, Issue 3 Pages 317-321
Masahito Higashiura*, Hiroshi Uchida, Tetsuo Uchida and Hiroko Wada

Abstract: Three analytical methods for the determination of gold in the serum of rheumatoid patients have been compared, i.e., flame and graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry (ICP-MS) with the respective detection limits of 30, 1.0 and 0.01 ng mL-1. The stability of gold solutions was also investigated. With 0.1% Triton X-100 and 0.1 M hydrochloric acid the gold concentration at ng mL-1 levels in standards was constant for 4 days, and the levels in 2000-fold diluted serum were the same for at least 7 days. The analytical results obtained with the above three methods correlated quite well, however, ICP-MS was the best technique, because of its specificity, sensitivity, wide dynamic range, speedy and ease of measurement.
Gold Mass spectrometry Spectrophotometry Spectrophotometry Method comparison

"Spectrofluorimetric Flow Injection Determination Of Potassium In Serum Based On Enzyme Activation"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 178-186
J. M. Fern&aacute;ndez-Romeroa, M. D. Luque De Castroa,* and R. Quiles-Zafrab

Abstract: The activation of the pyruvate kinase (PK) catalyzed reaction between phospho(enol)pyruvate (PEP) and ADP by K formed the basis of the FIA method. The product, pyruvate, was treated with NADH in the presence of lactate dehydrogenase (LDH) and the decrease in concentration of NADH was monitored at 460 nm. The decrease in NADH concentration was proportional to K+ concentration. Na+ interference was eliminated by a constant concentration of Li+ in the samples. The interference due to ammonium was eliminated by its conversion to glutamate via glutamate dehydrogenase (GDH). Three enzyme reactors containing immobilized GDH, PK and LDH were connected in series to form the FIA manifold. A carrier solution containing 200 mM triethanolamine, 10 mM Mg2SO4 and 40 mM LiCl of pH 7.2 was used at 0.3 ml/min. Serum was diluted 100-fold with 3 mM Kriptofix-221 in carrier buffer and injected into the carrier stream. The carrier stream was merged with a reagent stream (0.3 ml/min) containing 6 mM ADP/6 mM PEP/0.5 mM NADH in carrier buffer and passed through the enzyme reactors at 37°C to the spectrophotometric detector. The calibration graph was linear for 1-10 µM-K. Recoveries of 1-3 mM K were 95-105%. RSD (n = 8 or 11) for 2-8 mM K.
Potassium Fluorescence Heated reaction Immobilized enzyme Interferences

"Flow Injection Chemiluminometric Determination Of Ascorbic Acid Based On Its Sensitized Photo-oxidation"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 299-307
Tom&aacute;s P&eacute;rez-Ru&iacute;z*, Carmen Mart&iacute;nez-Lozano and Antonio Sanz

Abstract: The chemiluminescent reaction between lucigenin and the photo-oxidation products of ascorbic acid was utilized for the determination of ascorbic acid. The FIA system allowed 275 µL sample solution and 215 µL 1 mM toluidine blue (as photosensitizer) to be injected simultaneously into two phosphate buffer streams (both at 1.1 ml/min) at pH 3. The streams were merged before passing through a photoreactor equipped with a reaction coil (2 m x 0.5 mm i.d.) and a W halogen lamp. The stream was merged with streams of 0.1 mM lucigenin (1.1 ml/min) and 0.7 M KOH (4.1 ml/min). The three merged zones travelled 6 cm (0.5 mm i.d.) to the detector cell were the chemiluminescence was measured with a photomultiplier tube. The calibration graph was linear for 1.1 nM-0.3 mM ascorbic acid and the detection limit was 0.2 nM. The RSD (n = 11) for the determination of 0.2 mM, 20 µM and 0.7 µM-ascorbic acid were 0.43, 1.65 and 1.22%, respectively. The sampling frequency was 80 samples/h. The method can be applied to the determination of ascorbic acid in pharmaceutical preparations, fruit juices, soft drinks and blood serum.
Ascorbic acid Chemiluminescence Photochemistry

"Fluorimetric Flow Injection Determination Of Theophylline Based On Its Inhibitory Effect On Immobilized Alkaline Phosphatase"
Anal. Chim. Acta 1995 Volume 308, Issue 1-3 Pages 159-163
M. S&aacute;nchez-Cabezudo, J. M. Fern&aacute;ndez-Romero and M. D. Luque de Castro*

Abstract: The dual channel flow injection manifold for the determination of theophylline consisted of a dual injection valve, a reactor at 40°C containing bovine alkaline phosphatase immobilized on controlled pore glass and a fluorimetric detector. Sample (500 µL) and 225 µL 2 mM 4-methylumbelliferone phosphate solution (substrate) were simultaneously injected into two carrier streams (both at 1.2 ml/min) of 0.5 M Tris hydrochloride buffer of pH 10. The streams were merged and the flow was passed through the reactor (50 cm x 0.5 mm i.d.) to the detector. The released 4-methylumbelliferone was detected at 445 nm (excitation at 365 nm). The presence of theophylline inhibited the enzymatic reaction and reduced the fluorimetric signal. The calibration graph was linear for 10^-200 µM-theophylline and the RSD (n = 3) at the 20 µM level was 3.5%. The sampling frequency was 40 samples/h. The recovery of 20-60 µM-theophylline from spiked blood serum was >93%.
Theophylline Fluorescence Immobilized enzyme Heated reaction Indirect Controlled pore glass

"A Stopped-flow/continuous-flow Method For Kinetic Determinations"
Anal. Chim. Acta 1995 Volume 309, Issue 1-3 Pages 277-282
Yun-Sheng Hsieh and S. R. Crouch*

Abstract: Using the kinetics of glucose (I) oxidation in wine as a model system, wine samples were diluted in phosphate buffer as described previously (Ibid., 1993, 284, 159), injected into carrier streams (0.16-0.98 ml/min) of air and buffer and detected at 510 nm with the detector placed within the sample loop. Calibration graphs were linear for 10^-80 ppm I. At ~0.44% and 0.019% of I in wine and using air- and buffer-carriers, RSD (n = 4 and 3) of 1.2% and 3.9%, and 1.5% and 1% were obtained, respectively. Method accuracy was assessed with the determination of I in standard sera, with an error of 8% (no details given). The use of acetone, ethanol, methanol and IBMK as carrier streams resulted in no significant system improvement.
Glucose Spectrophotometry Kinetic Optimization Stopped-flow MIBK Reference material

"Evaluation Of Multicomponent Flow Injection Analysis Data By Use Of A Partial Least Squares Calibration Method"
Anal. Chim. Acta 1995 Volume 310, Issue 1 Pages 53-61
O. Hern&aacute;ndez, A. I. Jim&eacute;nez, F. Jim&eacute;nez* and J. J. Arias

Abstract: A 132 µL sample of 0.454-1.59 µg/ml Co(II), 0.536-1.879 µg/ml Cu(II) or 0.322-1.612 µg/ml Zn(II) as nitrates was injected into a water carrier stream which was mixed sequentially with 0.5 M borate buffer of pH 9 and 0.1 mM 2-carboxy-2-hydroxy-5-sulfoformazylbenzene (Zincon). Formation of Co-Zincon (λmax = 656 nm, ε = 13 200), Cu-Zincon (λmax = 600 nm, ε = 27 300) and Zn-Zincon (λmax = 618 nm, ε = 50 600) complexes was completed in a reactor coil (160 cm x 0.5 mm i.d.) and spectra were recorded with a diode array spectrometer using an 18 µL flow cell. The overlapping spectra were resolved by multivariate calibration (details given). The RSD for Co, Cu and Zn were 0.018-0.048%, 0.02-0.052% and 0.004-0.011%, respectively. Up to a 100-fold excess of K, Li, Ba, Ca and Mg did not interfere; Mn and Ni did not interfere in 5-fold excess. Fe(III) was tolerated up to a 10:1 metal/interferent ratio in the presence of 0.1 M phosphate or 0.1 M NaF. Levels of Zn determined in blood serum were in good agreement with values determined by AAS; agreement for Cu was less satisfactory. The levels of Co, Cu and Zn found in veterinary products agreed with those determined by AAS.
Cobalt Copper Zinc Spectrophotometry Chemometrics Complexation Simultaneous analysis Multivariate calibration Interferences Partial least squares

"Direct And Simultaneous Determination Of Uric Acid And Glucose In Serum With Electrochemical Filter/biosensor Flow Injection Analysis System"
Anal. Chim. Acta 1995 Volume 315, Issue 1-2 Pages 137-143
Yusuke Okawa*, Hiroyuki Kobayashi and Takashi Ohno

Abstract: A flow-through electrolysis cell, named an electrochemical filter (ECF), with a three-electrode configuration was developed for the elimination and determination of electrochemically active components in biofluids, which give generally a nonspecific response to electrochemical biosensors. The use of a reference electrode ensured the precise control of the working electrode potential of the ECF and thus the quantitative current efficiency of the electrolysis. A flow-injection analysis setup was constructed by connecting two ECFs and a flow-through amperometric biosensor for glucose in series for the direct and simultaneous determination of glucose and uric acid in serum. The first ECF coulometrically determined uric acid, and the second one eliminated interferents with the biosensor: the sample was thus treated on-the-fly and then delivered to the biosensor, so that it determined glucose amperometrically without interference. The concentrations of glucose and uric acid measured in human control sera agreed well with the values determined with conventional methods. The system exhibited the wide dynamic range in the high glucose concentration region, thus can be applied to the analysis of serum of hyperglycaemic patients.
Glucose Uric acid Amperometry Sensor

"Flow Injection Method For Determination Of Uranium In Urine And Serum By Inductively Coupled Plasma Mass Spectrometry"
Anal. Chim. Acta 1996 Volume 334, Issue 3 Pages 295-301
Avraham Lorber, Zeev Karpas* and Ludwik Halicz

Abstract: Urine acidified with one-hundredth its volume of HNO3 or untreated serum was injected from a 100 µL loop into a carrier stream comprising 0.6% HNO3 containing 1 g/l of Triton X-100. The stream was passed to the ICP nebulizer and the intensity of the 238U+ signal was measured during a dwell time of 250 ms in the mass spectrometer. The total analysis time was 60 s and a throughput of 50 samples per h was achieved. The detection limit was 1.5 ng/l of U. Memory and matrix effects were reduced by adding the Triton to the carrier. The method was applied to the determination of U in samples taken from a population not occupationally exposed to U or its compounds. The technique was validated by analysis of standard reference materials, by dilution experiments and by laser-induced fluorescence.
Uranium Mass spectrometry Reference material Triton X Surfactant

"The Kinetics-based Electrochemical Determination Of Serum Glutamate Pyruvate Transaminase Activity With A Gold Microelectrode"
Anal. Chim. Acta 1997 Volume 353, Issue 2-3 Pages 319-323
Ya-Nan He and Hong-Yuan Chen*

Abstract: A kinetics-based differential pulse voltammetric (DPV) method was reported for the determination of glutamate pyruvate transaminase (GPT) activity in human serum with a gold microelectrode. The production rate of NAD+ via GPT enzymatic reaction was measured electrochemically by following the increase in the DPV peak current of the NAD+ reduction peak appearing at - 1.07 versus Ag/AgCl. A linear response over a range of GPT activity from 0-200 U1-1 was obtained under the optimal conditions. The detection limit was 0.3 U1-1. Satisfactory results were obtained for the determination of GPT in patient serum samples.
Enzyme, activity Enzyme, glutamate pyruvate transaminase Polarography Electrode Electrode Kinetic

"Magnesium Ion-selective Electrode Optimization And Flow Injection Analysis Application"
Anal. Chim. Acta 1997 Volume 356, Issue 1 Pages 105-111
Nikolas A. Chaniotakisa,*, John K. Tsagatakisa, Elizabeth A. Moschoua, Steven J. Westb and Xiaowen Wenb

Abstract: The development of manual and Flow Injection Analysis (FIA) methods for the determination of magnesium in aqueous samples by means of a magnesium ion-selective electrode (ISE) is described. A sample pre-treatment solution containing pH buffering compounds and alkaline earth metal complexing agents was developed to minimize interferences and optimize the limits of detection. For the achievement of the desired potential stability of the electrode in the FIA mode, a pulseless syringe pump was employed to eliminate the possibility of a variable streaming potential. This, in conjunction with a laminar flow cell with low and well-controlled dead volume allowed for the simple, semi-automatic, direct, inexpensive, and accurate determination of magnesium in water samples. Results from both the manual and FIA method were corroborated by capillary electrophoresis (CE) and Atomic Absorption Spectroscopy (AAS). 16 References
Magnesium(II) Electrode Potentiometry Optimization Buffer Complexation Interferences Method comparison

"Glucose Oxidase Polyion Complex-bilayer Membrane For Elimination Of Electroactive Interferents In Amperometric Glucose Sensor"
Anal. Chim. Acta 1998 Volume 364, Issue 1-3 Pages 173-179
Fumio Mizutani*, Yukari Sato, Yoshiki Hirata, Takahiro Sawaguchi and Soichi Yabuki

Abstract: An amperometric glucose-sensing electrode was prepared by immobilizing glucose oxidase (GOx) on a polyion complex membrane. First, a monolayer of 3-mercaptopropionic acid (MPA) was made on the surface of a gold electrode by immersing it in an ethanol solution containing MPA. Aqueous solutions of poly-L-lysine and poly-4-styrenesulfonate were successively placed on the electrode surface and allowed to dry. A GOx layer was then formed on the poly-L-lysine/poly-4-styrenesulfonate-complex layer by crosslinking the enzyme by the addition of a glutaraldehyde solution. The polyion complex layer was effective for eliminating electrochemical interferents such as L-ascorbic acid, uric acid and acetaminophen. whereas the hydrogen peroxide produced through the GOx-catalyzed reaction permeated rapidly through the layer. This resulted in a rapid response (100% response in <5 s) with a low interference level (e.g., the ratio of response for L-ascorbic acid to that for the same concentration of glucose was 0.07). The electrode was applied to the assay of glucose in beverages and sera, and could be used for more than two months,
Glucose Amperometry Electrode Electrode Electrode Interferences Apparatus Detector

"Chemiluminescence Flow-sensing System For Hydrogen Peroxide With Immobilized Reagents"
Anal. Chim. Acta 1998 Volume 372, Issue 3 Pages 357-363
Wei Qin, Zhujun Zhang*, Baoxin Li and Shuna Liu

Abstract: A novel chemiluminescence (CL) sensing system for hydrogen peroxide combined with flow injection analysis (FIA) is described. It is prepared by electrostatically immobilizing the anal. reagents, luminol and cobalt(II), on a strongly basic anion-exchange resin and a weakly acid cation-exchange resin, respectively. Hydrogen peroxide is sensed by the CL reaction with luminol and cobalt(II) bleeding from the ion-exchange column with immobilized reagents by hydrolysis. The calibration graph is linear in the range 4 x 10^-8 to 1 x 10^-5 mol L-1, and the detection limit is 1.2 x 10^-8 mol L-1 hydrogen peroxide. A complete anal. could be performed in 1 min with a relative standard deviation of <5%. The system could be used for 50 h and was applied successfully to the determination of hydrogen peroxide in rainwater and glucose in serum by measuring the formation of hydrogen peroxide from a packed bed reactor with immobilized glucose oxidase.
Hydrogen peroxide Glucose Ion exchange Chemiluminescence Immobilized reagent Immobilized enzyme Resin

"Determination Of Choline-containing Phospholipids In Serum With A Fiber-optic Biosensor"
Anal. Chim. Acta 1998 Volume 374, Issue 1 Pages 19-29
M. D. Marazuela and M. C. Moreno-Bondi*

Abstract: An optical fiber biosensor for choline-containing phospholipids monitoring in serum samples is described. Phospholipids are hydrolyzed by the enzyme phospholipase-D to choline which is analyzed with a fiber-optic biosensor based on choline oxidase and oxygen transduction. Several supports have been evaluated for enzyme immobilization. The optimum biosensing layer consists of the enzyme immobilized on a nylon membrane and placed onto an oxygen sensitive layer formed by a luminescent Ru(II) complex, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), dispersed in a silicone membrane. The performance of the biosensor has been optimized using a flow injection system. The linear dynamic range of the biosensing membranes is found to be 0.08-3.00 mg mL-1 phosphatidylcholine. Studies of the reproducibility, stability and interferences of the device, as well as the application of the sensor to measurements in serum samples, are reported.
Choline Phospholipids Sensor Sensor Optimization Apparatus Detector Interferences

"Alternative Use Of Flow Injection Analysis And The Combination Of Liquid Chromatography And Flow Injection Analysis For The Determination Of Total And Individual Bile Acid Concentrations In Serum"
Anal. Chim. Acta 1991 Volume 249, Issue 2 Pages 461-467
A. Membiela, F. L&aacute;zaro, M. D. Luque de Castro and M. Valc&aacute;rcel

Abstract: The sample was passed through a Sep-Pak C18 cartridge and the bile acids were eluted with methanol. The total acid content was determined by flow injection analysis and was based on the oxidation of the acids, catalyzed by 3-α-hydroxysteroid dehydrogenase and NAD+, the reduced forms of which being monitored fluorimetrically. Samples with abnormal acid concentration. were analyzed for individual analytes on a column (20 cm x 4 mm) of Nucleosil C18 (10 µm) with methanol - 20 mM KH2PO4 (9:1) as mobile phase, pH 4.5, with detection at 460 nm (excitation at 340 nm). Recoveries by both methods were between 87.3 and 115.7%. The calibration graphs were rectilinear from 1 to 10 µg mL-1, and the coefficient of variation (n = 11) were between 2.7 and 3.0% for 4 µg mL-1 of each acid.
Bile acid LC Fluorescence Catalysis Column pH

"Sensitive Flow-Injection Amperometric Detection Of Iodide Using Mn3+ And As3+"
Anal. Sci. 2005 Volume 21, Issue 5 Pages 525-529
Snezana D. NIKOLIC, Jelena J. MUTIC, Aleksandar D. LOLIC, and Dragan D. MANOJLOVIC

Abstract: A rapid, selective, and sensitive kinetic flow-injection method for iodide content determination with amperometric detection on a platinum electrode was developed. The method is based on the catalytic effect of iodide on the Mn3+ reaction with As3+ in the presence of sulfuric acid. The calibration curve was linear in the concentration range from 5.0 x 10^-7 to 1.0 x 10^-4 mol/L iodide. The limit of detection (LOD) was found to be 5.0 x 10^-9 mol/L iodide. The relative standard deviations (RSD) were 1.68% and 3.03% for 1.0 x 10^-3 mol/L standard and 1.0 x 10^-6 mol/L iodide solution (n = 6), respectively. The method has been successfully applied for determination of iodide in waters, table salts, fodder, organic substances and human blood sera. The results were compared with those obtained by a standard AOAC (Association of Official Analytical Chemists) method, as well as with those obtained by a kinetic spectrophotometric procedure for determination of iodide.
Iodide Amperometry Electrode Method comparison Interferences Catalysis

"Spectrometric Rapid Sampling - Mixing System For Analytical - Clinical Methods"
Talanta 1979 Volume 26, Issue 7 Pages 549-561
D. L. Krottinger, M. S. McCracken and H. V. Malmstadt*

Abstract: A rapid sampling/mixing system has been designed which conforms to certain criteria necessary for its use as a clinical analyzer.. These include low solution volume (<100 µL) for each determination on a sample, rapid cycle time (<2 sec to take an aliquot, mix and transfer reactants to an observation cell), good precision (reproducibility better than 0.2%), and ready automation (requiring only two electronic signals to perform a complete cycle). This device has been incorporated into an automated spectrophotometer to be used for a variety of clinical methods. Equilibrium methods for the determination of calcium and albumin are presented that require measurement times of only 6 and 7 sec, respectively. A reaction-rate procedure for total protein is presented that requires only 3 sec per sample. Precisions obtained with these procedures are typically better than 1% in the normal serum range. Results on samples from hospital patients are compared with values obtained on an SMA 12/60 instrument.
Calcium Albumin Protein, total Clinical analysis Spectrophotometry Method comparison

"Homogeneous Enzymatic Fluorescence Immunoassay Of Serum IgG By Continuous-flow Injection Analysis"
Talanta 1982 Volume 29, Issue 12 Pages 1109-1112
Tim A. Kelly and Gary D. Christian*

Abstract: A new technique for automated homogeneous immunoassay has been developed and applied to the determination of serum IgG. An enzyme label, horseradish peroxidase (HRP), conjugated to the antibody (anti-human IgG) was inhibited on immunochemical association. The inhibition of activity was monitored as a decrease in the laser-induced fluorescence of dichlorofluorescein, produced by the HRP-catalyzed oxidation of leuco-diacetyldichlorofluorescein by hydrogen peroxide. The entire procedure was performed by flow-injection analysis at a rate of 60 samples per hour. Serum IgG concentrations from 1.4 to 25 mg/ml could be determined after a 1:700 dilution, with a within-run precision of ±9.8%.
Immunoglobulin G Clinical analysis Fluorescence Immunoassay

"Mixed Reagents In Multi-component Flow Injection Analysis. Simultaneous Determination Of Iron And Copper In Blood Serum With Mixed Bathocuproinedisulfonate And Bathophenanthrolinedisulfonate Or Ferrozine"
Talanta 1989 Volume 36, Issue 4 Pages 463-467
V. Kub&aacute;, D. B. Gladilovich and L. Sommer, P. Popov

Abstract: Deproteinized serum (0.5 ml) was mixed with 0.1 mL of 0.1 M ascorbic acid, 0.1 mL of 3 M trichloroacetic acid and 0.3 mL of 0.33 M formate buffer (pH 3.5) and, after 15 min, the solution was centrifuged and a 0.4 mL portion of supernatant solution was diluted with 0.1 mL of water. A 20 µL portion of solution and 20 µL of reagent solution [0.6 mM bathocuproinedisulfonate (I) - 0.2 mM bathophenanthrolinedisulfonate or 2 mM I - 0.2 mM ferrozine] were synchronously injected into the carrier stream (1 mM HNO3) of the flow injection system. A PTFE reaction coil (27 cm x 0.6 mm) was used and the flow rate was 0.5 mL min-1. Spectra were recorded with a photodiode-array detector over the range 390 to 590 nm. A computer program, ORTHO, was used to evaluate results. The merging zones multi-component method was suitable for determination of 0.7 to 33 µM Fe(II) and 0.4 to 35 µM Cu(I), and for Cu-to-Fe ratios of 10:1 to 1:10. The coefficient of variation for Fe and Cu were, respectively, 3 and 6% in artificial mixtures and 2 and 5% in serum (n = 5 or 7).
Iron Copper Spectrophotometry Buffer Computer Multicomponent Merging zones

"Flow-reversal Flow Injection Analysis. 2. Determination Of Glucose With A Double-pump System"
Talanta 1989 Volume 36, Issue 12 Pages 1233-1236
Jun'ichi Toei

Abstract: A procedure in which the sample and reagents are mixed in the sample loop of a 6-way valve [cf. Part I, Analyst (London), 1988, 113, 475] has been extended with the use of a second pump. A sample is injected into a stream of reagent, which is pumped slowly into the loop, then flow is reversed and the sample - reagent plug is pumped, in a rapid flow of carrier solution, to the detector by the second pump. Advantages of the system (diagram given) are low consumption of reagents and formation of sharp peaks. The system is demonstrated by the determination of glucose in serum by using glucose oxidase (Wako glucose test C kit); response is rectilinear up to 20 mg mL-1, and reagent consumption is 0.4 mL per sample compared with 3.0 mL per sample in the normal test.
Glucose Flow reversal Reagent consumption

"Simultaneous Flow Injection Analysis Of Three Components With Online Dialysers In Series. Determination Of Sodium, Potassium And Chloride In Blood Serum"
Talanta 1991 Volume 38, Issue 9 Pages 1033-1039
Jacobus F. van Staden,

Abstract: A procedure was developed for the simultaneous flow injection determination of Na, K and Cl- in blood serum from a single sample injection. Two flow-through dialysers are incorporated in the system, which separates (i) Na and K for determination by flame photometry in a 15 mM Li+ medium and (ii) Cl- for determination by UV spectrophotometry at 485 nm with Hg(SCN)2 as color reagent. A coefficient of variation of 1% was obtained with 100 µL samples at a sampling rate of 106 h-1.
Chloride Potassium Sodium Spectrophotometry Dialysis

"Potassium Sodium Chloride Integrated Micro-conduits In A Potentiometric Analytical System"
Talanta 1991 Volume 38, Issue 9 Pages 989-993
Cui Hongbo and Sun Junyan

Abstract: An integrated micro-conduit flow injection analysis manifold has been developed that incorporates flow-through K+-, Na+- and Cl--selective electrodes. The characteristics of the electrodes were evaluated, showing a rapid response of 10 s, a reproducibility of ±0.1 mV, and stability over 8 h. Samples of water, soil extracts and serum were analyzed, giving results in agreement with those obtained by flame-photometric and AgNO3 titrimetric methods.
Chlorine Potassium Sodium Electrode Potentiometry Sample preparation

"Flow Injection Analysis Of Serum Urea Using O-phthalaldehyde And Naphthylethylenediamine"
Talanta 1992 Volume 39, Issue 10 Pages 1233-1238
D. Narinesingh and A. Pope, T. T. Ngo

Abstract: The colorimetric reaction, modified by Lequang et al. (cf. Spectra Biol., 1987, 87, 44) was applied to the cited determination . Calibration graphs (at 517 nm) were rectilinear up to 51 mg L-1 of urea-N, when the flow injection manifold was operated at 42°C and 0.44 mL min-1. The recoveries were 95 to 99%. The within-day coefficient of variation were 0.5 to 2.2% and the between-day coefficient of variation were 2.1 to 3.9%. Some sulfur compounds and amino-acids interfered at concentration. >0.76 and >21 mg l-1, respectively. The results agreed well with those obtained by the free urease/Berthlot and the hospital (Abbott Bichromatic Analyser) method. An anal. procedure was developed for the determination of urea by flow injection analysis (FIA). The methodology is based on the color that develops (λmax, 517 nm) when urea reacts with o-phthalaldehyde in the presence of naphthylethylenediamine under acidic conditions. Calibration curves are linear up to 51 mg urea N/dL when the FIA manifold is operated at 42°C, utilizing 90 µL samples and a flow-rate of 0.44 mL/min. By manual injection up to 40 samples can be analyzed per h. Recovery yields ranged 95-99%. The within-day CV was 0.5-2.2% whereas the day-to-day CV was 2.1-3.9%. When applied to the anal. of urea in serum samples, excellent correlations (>0.998) are obtained when the FIA results are compared with those obtained for the same serum samples analyzed by the free urease/Berthelot's and the hospital method (employing the Abbot Bichromatic Analyzer). Interferences are observed with S compounds such as sulfanilamide (>0.76 mg/dL) as well as with many amino acids but at relatively high concentrations. (>21 mg/dL).
Urea Spectrophotometry Heated reaction Interferences Method comparison

"Bioluminescent Flow Sensor For L-phenylalanine Determination In Serum"
Talanta 1993 Volume 40, Issue 3 Pages 425-430
S. Girotti*, E. Ferri, S. Ghini and R. BudiniGiacomo Carrea and Roberto BovaraSandro Piazzi and Roberto MerighiAldo Roda,

Abstract: A bioluminescent flow sensor for the determination of L-phenylalamine in serum is described. The sensor monitors the reduced form of NAD produced by the dehydrogenase with use of a bioluminescent detection coil. Analyte and enzyme are immobilized on to separate nylon coils and mounted in series in the flow injection system. The response is rectilinear for 1 to 100 µM, and the detection limit is 0.5 µM.
l-Phenylalanine Bioluminescence Sensor

"Simultaneous Determination Of Multi-components By Flow Injection Analysis. Determination Of Copper And Zinc In Serum Using Zincon As Coloring Reagent"
Talanta 1993 Volume 40, Issue 4 Pages 511-514
Ren-Min Liu, Dao-Jie Liu and Ai-Ling Sun

Abstract: Serum (1 ml) was shaken with 2 M HC1 (1 ml), set aside for 10 min then heated with 20% trichoroacetic (0.5 ml) for 10 min at 90°C to 95°C. After centrifugation at 3000 rpm, supernatant solution was adjusted to pH 7 and two portions of the solution were injected into a carrier stream comprising boric acid (12.4 g), KCl (13 g), sodium citrate (3.1 g), sodium triphosphate (1.5 g), 1,10-phenanthroline (0.13 g) dissolved in water (800 ml), adjusted to pH 9 and diluted to 1 l. To determine Cu and Zn, one sample plug was mixed with aqueous 0.05% Zincon solution before the absorbance was measured at 606 nm. To determine Zn, a Cu masking reagent (5% thiourea) was merged with the second plug before mixing with Zincon solution as above The cailbration graphs were rectilinear up to 5 µg mL-1 of Cu and Zn. Recoveries were quantitative. The coefficient of variation for a synthetic mixture of 0.5 µg mL-1 of Cu and Zn were 0.9 and 0.7% respectively. Interferences caused by Fe(III), Mn(II), Ni(II), Co(II), Al(III) and Cd(II) are masked by the compounds in the carrier solution Results were in good agreement with those obtained by an AAS. Sample throughput was ~45 h-1.
Copper Zinc Spectrophotometry Multicomponent Dual detection Interferences

"Flow Injection Determination Of L-tyrosine In Serum With Immobilized Tyrosinase"
Talanta 1993 Volume 40, Issue 7 Pages 995-998
Nobutoshi Kiba, Hiroshi Suzuki and Motohisa Furusawa

Abstract: Serum was deproteinized by 5% sodium tungstate solution and 0.15 M H2SO4, filtered and a portion was injected into 0.2 M phosphate buffer solution of pH 7.2 (0.4 ml/min). The mixture passed through a separation column (4 cm x 4 mm) of Capcell 120 C18 and then through the enzymatic reactor (5 cm x 4 mm) containing monophenol monooxygenase immobilized on controlled pore glass (cf. Kiba et al., Anal. Chim. Acta, 1989, 224, 133). The effluent from the reactor was mixed with 30 mM 1,2-diphenylethylenediamine in 0.6 M HCl/ethanol (3:7) and the mixture passed through a reaction coil (1 m) held at 105°C and then through a cooling coil (10 cm) at 15°C, before measurement of the fluorescence at 480 nm (excitation at 350 nm) in a flow-through cell. The calibration graph was linear for 1-200 µM-L-tyrosine with a detection limit of 0.2 µM. Interference from cysteine or ascorbate was avoided. Recoveries of 70-985 µM-L-tyrosine from serum were 98-105%. Within- and between-day RSD were 1 and 1.8%, respectively, for 64 µM-L-tyrosine. Results agreed well with those obtained by an amino-acid analyzer..
Tyrosine Fluorescence Immobilized enzyme Controlled pore glass Heated reaction Interferences

"Enzymic Flow Injection Determination Of Urea In Blood Serum Using Potentiometric Gas Sensor With Internal Nonactin-based ISE"
Talanta 1994 Volume 41, Issue 8 Pages 1229-1236
Tadeusz Krawczyski velKrawczyk and Marek Trojanowicz*Andrzej Lewenstam,

Abstract: Cellulose triacetate (66 mg) was dissolved in 2 mL CHCl3/CH2Cl2 (1:1), 120 mL dipentyl phthalate [or di(2-ethylhexyl) sebacate] and 4 mg nonactin added. A 6 mm diameter membrane was formed and mounted on an electrode body with a hydrophobic gas-permeable membrane, an internal filling solution of 0.1 M Tris buffer of pH 8-8.2 and containing a Ag/AgCl reference electrode. Serum was diluted 1:9 (no details given) and 200 mL injected into a 3.2 ml/min flow of water, and passed through a column (6 cm x 4 mm i.d.) of glass-immobilized urease. The column effluent was mixed with a 4.8 ml/min flow of NaOH and passed through a second enzyme reactor. Calibration graphs were linear from 10 µM to 10 mM NH4Cl. At 0.1, 1 and 10 mM NH4Cl, RSD (n = 8) were 0.62, 0.24 and 0.65%, respectively. At 0.1 mM and 1 mM NH4Cl, electrode recovery times were 3-4 and 7-8 min, respectively.
Urea Potentiometry Electrode Electrode Sensor

"Flow Injection Determination Of 3-hydroxybutyrate In Serum With An Immobilized 3-hydroxybutyrate Dehydrogenase Reactor And Fluorescence Detection"
Talanta 1994 Volume 41, Issue 9 Pages 1583-1586
Nobutoshi Kiba, Hidekazu Koemado and Motohisa Furusawa,

Abstract: A flow-injection system with an immobilized enzyme reactor is proposed for the determination of 3-hydroxybutyrate. 3-Hydroxybutyrate dehydrogenase is immobilized on aminated poly(vinyl alcohol) beads and packed into a stainless-steel column (4 cm x 4 mm I.D.). Serum is diluted and filtered. Sample solution (20 µL) is injected into the carrier stream [4 mM NAD+ in glycine buffer (pH 9.3)]. The NADH formed is detected at 465 nm (excitation at 340 nm). The calibration graph is linear for 0.7-500 µM 3-hydroxybutyrate; the detection limit is 0.5 µM.
3-hydroxybutyrate Fluorescence Immobilized enzyme Reactor

"Flow Injection Fluorimetric Analysis Of Sulfamethoxazole In Pharmaceutical Preparations And Biological Fluids"
Talanta 1994 Volume 41, Issue 12 Pages 2159-2164
C. Lopez Erroz, P. Vi&ntilde;as and M. Hern&aacute;ndez C&oacute;rdoba*,

Abstract: Pharmaceuticals were dissolved in ethanol and filtered, and urine and serum were diluted with water and filtered. The filtrate was injected into a stream of 0.01 M o-phthaldialdehyde in 0.7 M H3PO4 and merged with a stream of 0.01 M β-mercaptoethanol in 0.7 M H3PO4. The resulting solution flowed through a reactor coil (2.5 m x 0.5 mm i.d.) thermostatted at 40°C and into a flow cell for fluorimetric detection at 412 nm (excitation at 302 nm). The system flow rate was 1 ml/min. The calibration graph was linear for 0.01-2.5 µg/ml of sulfamethoxazole with a detection limit of 7 ng/ml. The RSD (n = 10) were 3.5, 2.2 and 2.1% for 0.01, 0.05 and 1.5 µg/ml, respectively. Interferences from tablet fillers and other compounds are discussed.
Sulfamethoxazole Fluorescence Interferences

"Electrothermal Atomic Absorption Spectrometric Determination Of Ultratrace Amounts Of Tin By In Situ Preconcentration In A Graphite Tube Using Flow Injection Hydride Generation With Online Ion-exchange Separation"
Talanta 1995 Volume 42, Issue 3 Pages 375-383
Guanhong Tao and Zhaolun Fang*

Abstract: Geological samples (0.5 g) were treated with 5 mL HNO3/HF (7:3) at 40°C. After boiling nearly to dryness a further 2 mL acid was added. After cooling, the digests were diluted to 50 mL and 1 mL was diluted further to 100 mL with 2 M HCl. Hair (0.2 g) or 1 mL serum was acid-treated as above and then the digests were diluted to 50 mL with 2 M HCl. Tap water samples were filtered and acidified to 2 M HCl with concentrated HCl. The samples were passed through a microcolumn (3 cm x 3 mm i.d.) packed with anion-exchanger D-201 (Shenyang Organic Chemicals Co.). The Sn was retained as its chlorostannate complex and subsequently eluted with water into the hydride generation system. The hydride and H2 evolved were separated from the liquid phase in a gas-liquid separator and transferred into a Pd-coated graphite tube pre-heated to 300°C to collect the analyte which was later atomized at 2300°C, followed by AAS. Tin was determined at a sampling frequency of 30/hr with a detection limit of 0.01 µg/l. RSD were ~1.5%.
Tin Sample preparation Spectrophotometry Phase separator Preconcentration Reference material Volatile generation Ultratrace Volatile generation

"Development Of A High Performance Liquid Chromatographic System With Enzyme Reactors For The Determination Of N-acetyl Branched-chain Amino-acids"
Talanta 1995 Volume 42, Issue 3 Pages 449-453
Nobutoshi Kiba, Yukio Oyama and Motohisa Furusawa

Abstract: Immobilized enzymes were used as column reactors in a high-performance liquid chromatographic system for the specific detection of N-acetyl branched-chain amino acids (AcBCAs) such as N-acetyl-L-valine (AcVal), N-acetyl-L-leucine (AcLeu) and N-acetyl-L-isoleucine (AcIle). Aminoacylase and leucine dehydrogenase were immobilized onto poly(vinyl alcohol) beads. The AcBCAs were separated as three peaks on a Capcell C1 SG120 column with 0.03 M phosphate buffer (pH 8.0). Aminoacylase was capable of hydrolysing the AcBCAs to amino acids, which react with β-nicotinamide adenine dinucleotide (NAD+) in the presence of leucine dehydrogenase. The reduced nicotinamide adenine dinucleotide (NADH) produced was monitored fluorimetrically. The calibration graphs were linear from 4 to 200 µM for AcVal and AcLeu, and from 5 to 300 µM for AcIle; detection limits for AcVal, AcLeu and AcIle were 2, 2 and 3 µM, respectively. The immobilized aminoacylase reactor should be renewed every five days owing to a poor stability of aminoacylase.
Amino acids, N-acetyl N-acetylleucine N-Acetylvaline N-Acetylisoleucine HPLC Immobilized enzyme Post-column derivatization Reactor

"Determination Of Hydrazine Derivatives By Flow Injection Analysis With Spectrophotometric Detection"
Talanta 1995 Volume 42, Issue 10 Pages 1465-1469
M. I. Evgen'ev, S. Y. Garmonova, I. I. Evgen'eva and H. C. Budnikov

Abstract: A diagram is presented of a flow injection manifold used in the spectrophotometric determination of hydrazine-based drugs. The method was based on the reaction of 4-chloro-5,7-dinitrobenzofurazan (DNBF) with the hydrazine derivatives. The 0.02 M DNBF acetonitrile solution was injected directly into a carrier solution of the hydrazine derivatives (concentration range 0.15-4 µg/ml) and the absorbance at 510 nm was measured. For the determination of 1-hydrazinophthalazine hydrochloride in apressin drug, powdered tablets containing ~10 mg of the drug were diluted to 100 mL with water. The mixture was shaken, filtered and a 5 mL portion was diluted to obtain a final solution containing ~1 µg/ml drug. For the determination of hydrazine drugs in plasma, urine and albumin, 1 mL TCA was added to 4 mL sample followed by centrifugation at 6000 rpm. The supernatant was neutralised with 0.5 mL acetate buffer of pH 5.5 and analyzed by the above procedure. The calibration graphs were linear from 0.15-4 µg/ml, with sampling rates of 28-32/h. Interferences from other compounds are tabulated. Flow injection analysis for the determination of hydrazine derivatives based on their nucleophilic substitution reaction with 4-chloro-5,7-dinitrobenzofurazan in aqueous medium, and spectrophotometric detection has been described. The calibration graphs were linear in the range from 0.15 to 4.0 µg mL-1 of hydrazine derivatives, with sampling rates of up to 28-32 samples h-1. Interferences from amino compounds, benzoic acids, aliphatic amines and ammonia have been evaluated. The procedure has been applied to the determination of hydrazine derivatives in serum, urine, apressin drugs and artificial mixtures. (16 references)
Drugs Hydrazine Spectrophotometry Interface Interferences

"Chemiluminescence Investigation Of NH2OH-fluorescein-copper(II) System And Its Application To Copper Analysis In Serum"
Talanta 1995 Volume 42, Issue 11 Pages 1619-1623
Jinming Lin*, and Toshiyuki Hobo

Abstract: A novel chemiluminescent system, fluorescein-NH2OH-OH-, was developed for the determination of copper(II) in serum. A weak light emission arises from hydroxylamine in the presence of the organic reagent fluorescein in basic aqueous solution. Under the conditions of 1.2 x 10^-3 mol L-1 NH2OH and 5 x 10^-3 mol l(-3) fluorescein, the light intensity is linearly dependent upon the concentration of copper(II) within the range 1-20 ppb. The relative standard deviation of the determination of copper(II) is 4.2% (n = 13) and the detection limit is 0.5 ppb. The system is highly selective for copper except in the presence of iron(II,III) and cobalt(II). In conjunction with potassium fluoride as masking agent, the method was successfully applied to the determination of microamounts of copper(II) in serum. A mechanistic study of the chemiluminescence reaction is also discussed. (19 References)
Copper(II) Chemiluminescence Interferences Indirect

"Poly(o-aminophenol)-modified Bienzyme Carbon Paste Electrode For The Detection Of Uric Acid"
Talanta 1996 Volume 43, Issue 5 Pages 785-796
E. Miland, A. J. Miranda Ordieres, P. Tu&ntilde;&oacute;n Blanco*, M. R. Smyth and C. &Oacute; F&aacute;g&aacute;in

Abstract: A biosensor was fabricated by packing uricase and horseradish peroxidase mixed with unmodified carbon paste into the well of a working electrode. The monomer o-aminophenol was electropolymerized at the surface area of the electrode in 0.1 M acetic acid of pH 5 at 0-0.7 V for 10 min. Cyclic voltammetry and amperometry were performed using the above electrode with a stainless steel contact, a Ag/AgCl/saturated KCl reference electrode and a Pt wire auxiliary electrode. Calibration graphs were linear up to 0.1 mM uric acid with a detection limit of 3.14 µM and an RSD (n = 4) of 2.2% for 20 µM-uric acid. The sensor reponse time was 37 s and the response was stable for 2 days. The effects of interferences are discussed. The use of the sensor to determine uric acid in serum using a FIA system is described. A carrier stream of 0.1 M phosphate buffer of pH 7.5 (1 ml/min) was used with an applied potential of 0.05 V vs. Ag/AgCl. Recoveries of uric acid from serum were 90-95%.
Uric acid Electrode Electrode Sensor Amperometry Voltammetry Interferences

"Flow Injection Analysis Of L-lactic Acid Using An Enzyme-polyion Complex - Coated Electrode As The Detector"
Talanta 1996 Volume 43, Issue 10 Pages 1815-1820
Fumio Mizutania,*, Soichi Yabukia and Yoshiki Hirataa

Abstract: An enzyme electrode for L-lactic acid was fabricated by applying 0.04 M potassium phosphate buffer of pH 7 containing 10 µM-lactate oxidase and 10 mM poly-L-lysine and the same buffer containing 20 mM poly(4-styrene-sulfonate) successively on a polished glassy carbon electrode. The electrode was then used in a FIA system. Serum or sour milk was injected into a carrier stream (0.5 ml/min) of 0.1 M potassium phosphate buffer of pH 7.7 which passed to a flow-through cell comprising the enzyme electrode a Ag/AgCl reference electrode, a stainless-steel auxiliary electrode and a PTFE gasket. The potential was set at 1 V. Calibration graphs were linear up to 2.5 mM lactic acid with a determinable concentration range up to 6 mM. The detection limit was 20 µM and the RSD (n = 100) was 1.4% for 2 mM lactic acid.
l-Lactic acid Electrode Electrode Electrode Buffer

"Determination Of L-phenylalanine In Serum By Flow Injection Analysis Using Immobilized Phenylalanine Dehydrogenase And Fluorimetric Detection"
Talanta 1996 Volume 44, Issue 1 Pages 131-134
Nobutoshi Kiba*, Akiko Itagaki and Motohisa Furusawa

Abstract: A flow injection system with an immobilized enzyme reactor is proposed for the determination of L-phenylalanine. Phenylalanine dehydrogenase from Rhodoccus sp. M4 was immobilized on tresylated poly (vinyl alcohol) beads (13 µm) and packed into a stainless-steel column (5 cm times 4 mm i.d.). Serum sample was deproteinized with tungstic acid and filtered through an ultrafiltration membrane. The sample solution (30 µl) was injected into the carrier stream (water). The NADH formed was detected at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 µm L-phenylalanine; the detection limit was 0.3 µm, The sample throughout was 25 h-1 without carryover. The half-life period of the immobilized enzyme was 23 days. Serum (50 µL) was deproteinized by addition of 100 µL 10% sodium tungstate solution and 100 µL 0.06 M H2SO4 and filtration through an ultrafiltration membrane (mol. wt. cut off 10 000). A portion (30 µL) of the filtrate was injected into a carrier stream (0.2 ml/min) of water which merged with streams (0.2 ml/min) of 8 mM NAD+ and 2% glycerol in 0.2 M glycine buffer of pH 10 that had been pre-mixed in a mixing coil (100 cm x 0.5 mm i.d.). The flow passed through an enzyme column reactor (5 cm x 4 mm i.d.) at 20°C prepared by immobilization (details given) of phenylalanine dehydrogenase on poly(vinyl alcohol) beads (10 µm diameter). The NADH produced was detected fluorimetrically at 465 nm (excitation at 340 nm). The calibration graph was linear for 0.9-600 µM-L-phenylalanine and the detection limit was 0.3 µM. The within-day and day-to-day RSD were 0.88% and 1.2%, respectively. Recoveries were 97-103%. Results agreed with those obtained by LC.
l-Phenylalanine Sample preparation Fluorescence Filter Immobilized enzyme Reactor Method comparison Poly vinyl alcohol beads Buffer

"Determination Of Ion-selective Electrode Characteristics By Non-linear Curve Fitting"
Talanta 1997 Volume 44, Issue 10 Pages 1847-1858
Paddy Kane and Dermot Diamond*

Abstract: A simple practical method of determining potentiometric selectivity coefficients of ion-selective electrodes (ISEs) is described in which electrode characteristics (slope, potentiometric selectivity coefficients and cell constant) can be determined by fitting the experimental data obtained using the fixed interference (FI) method to an appropriate model by non-linear least-squares regression. The proposed method is simple to implement practically, and data processing can be easily achieved through use of the optimization add-on, Solver, bundled with Microsoft Excel. The flexibility of the method is demonstrated by modelling the response of a valinomycin potassium-selective electrode with the Nikolskii-Eisenman equation and a recently proposed alternative to the Nikolskii-Eisenman equation for cases where the ionic charges on the primary and interfering ions are unequal.
Lithium Electrode Electrode Membrane Calibration Signal processing

"Flow Injection Analysis Of Potassium Using An All-solid-state Potassium-selective Electrode As A Detector"
Talanta 1998 Volume 46, Issue 6 Pages 1293-1297
Shinichi Komaba, Junko Arakawa, Michiko Seyama, Tetsuya Osaka*, Ikuo Satoh and Sadako Nakamura

Abstract: The concentration. of potassium was determined by a combination of flow injection analysis (FIA) with an all-solid-state potassium sensor detection. The all-solid-state potassium-selective electrode possessing long-term potential stability was fabricated by coating an electroactive polypyrrole/poly(4-styrenesulfonate) film electrode with a plasticized poly(vinyl chloride) membrane containing valinomycin. The simple FIA system developed in this lab. demonstrated sensitivity identical to that in the batch system and achieved considerably rapid assay (150 samples h-1). Analyses of soy sauce and control serum samples by this FIA system yielded results in good agreement with those obtained by conventional measurements.
Potassium Electrode Electrode Method comparison Apparatus Detector Reference material

"Rapid Flow Analysis With Inductively Coupled Plasma Atomic Emission Spectroscopy Using A Micro-injection Technique"
Analyst 1982 Volume 107, Issue 1280 Pages 1335-1342
P. W. Alexander, R. J. Finlayson, L. E. Smythe and A. Thalib

Abstract: An introduction system for liquid micro-samples in inductively coupled plasma atomic-emission spectroscopy is described that allows the injection of 5-500 µL volumes into a rapidly flowing carrier reagent stream leading to the nebuliser. The effect on analyte signal was studied as a function of flow-rate, injection volume and sample concentration. It is shown that the carrier flow-rate determines the response time, sensitivity, precision and sample carry-over in the nebuliser. By the use of relatively rapid flow-rates of up to 7.5 mL min-1, fast injection of 10 µL samples is achieved at an injection rate of 240 h-1 with a relative standard deviation of 1.5% for a single-element analogue readout. Digital readout is used for multi-element determinations with similar or better precision. Detection limits of the order of 0.1 mg l-1 are obtained for 10 µL injections, limited by the volume injected, with a proportionate decrease in detection limit for increasing volumes.
Calcium Magnesium Iron Potassium Sodium Clinical analysis Spectrophotometry Injection technique

"Simultaneous Multi-element Analysis Of Blood Serum By Flow Injection Inductively Coupled Plasma Atomic Emission Spectrometry"
Analyst 1984 Volume 109, Issue 3 Pages 327-332
Cameron W. McLeod, Paul J. Worsfold and Allan G. Cox

Abstract: For the determination of Na, K, Ca, Mg, Li, Cu, Fe and Zn, the sample (2 µL to 2 ml) of serum or standard aqueous solution was injected into a carrier (aqueous solution of non-ionic surfactant at 2.2 mL min-1) for nebulization and ICP-AES The plasma emission was examined in a 30-channel direct-reading spectrometer interfaced to a microcomputer for data acquisition and analysis (peak areas were measured). Detection limits depended on the volume injected; the method was sufficiently sensitive to determine all the elements except Li in 20 µL of human serum. Matrix effects occurred with undiluted serum, but were decreased by use of relatively high r.f. power and low injection volume; dilution (1:1) of the serum eliminated the interference.
Calcium Copper Iron Lithium Magnesium Potassium Sodium Zinc Spectrophotometry Interferences Multielement

"Simultaneous Determinations In Flow Injection Analysis. A Review"
Analyst 1984 Volume 109, Issue 4 Pages 413-419
Maria Dolores Luque de Castro and Miguel Valc&aacute;rcel Cases

Abstract: In flow injection analysis (f.i.a.), the different characteristics resulting from the relative location of several detectors allows a sub-classification into either series or parallel configuration. Detection by potentiometric techniques is common in the series mode, and sequential ion-selective electrodes permit the simultaneous determination of several species, such as alkali or alkaline-earth metals, in serum. With the detectors in parallel, consideration is given to single-injection operation, with splitting of the sample, simultaneous multiple injection and zone sampling. Simultaneous determinations with use of a single detector are discussed in terms of sequential injection, stream-splitting with two cells aligned in the same optical path, with use of a pH gradient or f.i.a., and ion-exchange association. Examples of determinations are given; thus, with ion exchange, Zn and Cd in the range 10 to 100 and 20 to 200 ng mL-1, respectively, can be determined at a rate of 3 min per sample. Methods of f.i.a. based on differential kinetics are fully described, and difficulties inherent in this methodology are mentioned.
Cadmium Zinc Ion exchange Electrode Potentiometry Simultaneous analysis General Gradient technique Kinetic Merging zones Reactor Review Stopped-flow Zone sampling pH gradient

"Controlled-dispersion Flow Analysis With Atomic Absorption Detection For The Determination Of Clinically Relevant Elements"
Analyst 1985 Volume 110, Issue 5 Pages 493-496
Roy A. Sherwood, Bernard F. Rocks and Clifford Riley

Abstract: The determination of Li, Mg, Ca, Cu and Zn in plasma or serum is carried out by AAS, with use of the controlled-dispersion analysis instrument described previously (Anal. Abstr., 1983, 45, 2J11). The calibration graphs are rectilinear for 40 µM for Cu and Zn, 2 mM for Li, 3.5 mM for Ca and 1.5 mM for Mg. Within-batch coefficient of variation are 0.9 to 2.0%, and the results correlate well (r 0.96) with those of conventional flow injection analysis. A 'sandwich - slug' technique for economizing on reagent is described.
Lithium Magnesium Calcium Copper Zinc Clinical analysis Spectrophotometry Dispersion

"Problems In The Application Of Ion-selective Electrodes To Serum Lithium Analysis"
Analyst 1986 Volume 111, Issue 5 Pages 567-570
V. P. Y. Gadzekpo, G. J. Moody and J. D. R. Thomas

Abstract: Three types of Li+-selective electrodes were studied in microconduit flow injection analysis for their response to 0.21 to 2 mM Li in serum samples. Possible interference (e.g., from proteins or NaCl) was examined. Two of the electrodes were based on PVC matrix membranes of a commercial Li electrode (Philips) and dodecylmethyl-14-crown-4. The third was based on PVC containing the tetraphenylborate of a Ba polypropoxylate and dioctylphenyl phosphonate (solvent mediator). Adjustment of the serum NaCl level to 153.9 mM improved the data to match more closely those obtained by flame photometry. However, even the most effective electrodes studied, namely that based on Ba polypropoxylate, only produced results which matched the flame photometric data for 5 out of the 10 samples examined.
Lithium Clinical analysis Electrode Electrode Potentiometry Interferences

"Lithium Ion-selective Electrodes Containing TOPO. Determination Of Serum Lithium By Flow Injection Analysis"
Analyst 1987 Volume 112, Issue 1 Pages 61-64
Robert Y. Xie and Gary D. Christian

Abstract: Incorporation of 1% of trioctylphosphine oxide (I) and 1% of K tetrakis-(4-chlorophenyl)borate in a membrane of PVC and 2-nitrophenyl octyl ether (33:65) containing 3% of 14-crown-4 markedly increased selectivity to Li+ relative to Na+; response to Li+ was rectilinear from 2 to 200 mM. This electrode was used in a flow injection system with 8.25 mM Na2B4O7 as carrier, a silver - AgCl reference electrode and a 3 M KCl flow salt bridge. For ten serum samples of 0.41 to 1.24 mM Li+ and 150 mM Na+, the average relative error compared with the Li+ concentration. determined by AAS was -2.9% and the difference between the two methods was not statistically significant. Precision for six replicates at 0.5 mM Li+ was ±4.9 µM. Addition of I to ETH 1810 and UWXC 10 electrodes reduced their selectivity to Li+.
Lithium Electrode Electrode Potentiometry Method comparison Selectivity

"Chemically Immobilized Bi-enzyme Electrodes In The Redox Mediated Mode For The Flow Injection Analysis Of Glucose And Hypoxanthine"
Analyst 1987 Volume 112, Issue 1 Pages 65-70
G. J. Moody, G. S. Sanghera and J. D. R. Thomas

Abstract: Working electrodes were prepared by immobilizing glucose oxidase and peroxidase, or xanthine oxidase and peroxidase, on activated nylon mesh stretched over a smooth platinum electrode (oxidase - peroxidase ratio 2:1). The electrodes were used in a wall-jet type of chamber in a Stelte micro-cell with a vitreous-carbon auxiliary electrode and a silver - AgCl reference electrode. For flow injection analysis, samples were introduced into 100 mM NaH2PO4 buffer solution containing 2.5 mM K4Fe(CN)6 at pH 7.0 and 2.75 mL min-1. The Fe(II) - Fe(III) couple was utilized for amperometric determination of H2O2 generated by the enzyme reactions, allowing a low applied potential without the need for sample pretreatment. Calibration was rectilinear from 0.02 to 3 mM for glucose and from 2 to 100 µM for xanthine and hypoxanthine. Glucose was determined in blood serum and hypoxanthine in extracts of fish. The results agreed well with those obtained with an enzyme test kit and by a recommended spectrophotometric method, respectively.
Glucose Hypoxanthine Xanthine Electrode Electrode Electrode Sample preparation Method comparison

"Flow Injection Analysis Use Of Immobilized Enzymes For The Determination Of Ethanol In Serum"
Analyst 1987 Volume 112, Issue 3 Pages 259-261
Juan Ruz, Mar&iacute;a Dolores Luque de Castro and Miguel Valc&aacute;rcel

Abstract: Alcohol dehydrogenase was immobilized on silanized controlled-pore glass with glutaraldehyde as the coupling agent (details given). A single-channel system included a reactor (20 cm x 1.2 mm o.d.) containing the immobilized enzyme. Normal-flow and stopped-flow methods were used. Serum (50 µL) was diluted to 5 mL with carrier solution, aliquots (30 to 280 µL) were injected into the carrier stream (1 mL min-1) and the NADH formed from NAD+ was detected at 340 nm. For normal flow injection analysis the calibration graphs were rectilinear for 1 to 24 µg mL-1 with a detection limit of 0.3 µg mL-1 and a sampling frequency of 40 hr-1. For stopped-flow analysis the calibration graph was rectilinear for 0.5 to 28 µg mL-1, with a detection limit of 0.1 µg mL-1 and a sampling frequency of 30 hr-1.
Ethanol Spectrophotometry Controlled pore glass Immobilized enzyme Stopped-flow

"Rapid, Miniaturized Procedure For The Determination Of Glucose Based On Flow Injection Principles"
Analyst 1987 Volume 112, Issue 11 Pages 1565-1568
Jun'-ichi Toei

Abstract: The proposed flow injection method is based on the Wako glucose C enzymatic test kit procedure (Wako Pure Chemicals, Osaka, Japan). The reagent (70 µL) is introduced into a variable-volume mixing device (described and illustrated) that is placed within the injection loop of a six-port valve. The whole unit is heated to 40°C in a water bath. The sample solution (1 µL) is injected into the mixing device and, after 1 min, the reaction mixture is transferred to the water carrier stream (1 mL min-1) and the absorbance is monitored at 535 nm in a flow-through spectrophotometer. The calibration graph is rectilinear up to 5 g L-1 of glucose and the coefficient of variation at 2 and 5 g L-1 are 1.01 and 3.34%, respectively (n = 7). Results for two samples of serum were in excellent agreement with those obtained with the manual Wako test kit.
Glucose Clinical analysis Spectrophotometry Microfluidic Mixing chamber Heated reaction Method comparison

"High Performance Liquid Chromatography Using A Color-forming Agent As A Component Of The Mobile Phase. Separation And Determination Of Magnesium And Calcium In Clinical Samples With O-cresolphthalein Complexan"
Analyst 1988 Volume 113, Issue 2 Pages 247-250
Jun'ichi Toei

Abstract: The cited determination was carried out on a column (5 cm x 4 mm) of TSK-GEL SP-2 SW. The optimum mobile phase (1 mL min-1) contained 0.2 M KCl, 0.02 M acetic acid - Na acetate buffer (pH 4.0) and 0.1 mM o-cresolphthalein complexan. Detection was at 575 nm after mixing the column eluate with 0.4 M NH3 - NH4Cl buffer (pH 10.4). Pre-treatment of samples (milk, urine, serum or saliva) removed protein-bound Ca; pre-treatment on Toyo-Pack SP cartridges to remove free Ca was unsuccessful. Transition metals and Na did not interfere at physiological concentration. The coefficient of variation (n = 7) for 2.1 ppm of Mg and 28.9 ppm of Ca in milk were 1.41 and 1.26%, respectively.
Calcium Magnesium Clinical analysis Spectrophotometry Interferences Post-column derivatization

"Determination Of Glucose In Clinical Samples By Flow Reversal Flow Injection Analysis"
Analyst 1988 Volume 113, Issue 3 Pages 475-478
Jun'ichi Toei

Abstract: A multiple detection system for flow injection analysis is described and illustrated; it consists of an open flow stream arrangement and only one pump and one detector. Samples pass through the detector and are then pumped back to the same detector in the reverse direction (by rotation of a six-way valve). The effects are studied of flow changeover time on peak height and peak area for detection of acetone in an aqueous 0.1% solution at 254 nm. Results agreed well with those obtained by normal flow injection analysis. The technique was applied to determine glucose in serum by a two-point rate assay by using an enzyme test kit. Analysis was carried out at a flow rate of 1 mL min-1, with a sample volume of 1 µL, first and second flow changeover times of 0.5 and 1.22 min, respectively, and with detection at 535 nm. Results agreed well with those from the batch method and the coefficient of variation (n = 7) was 2.1%.
Glucose Clinical analysis Spectrophotometry Flow reversal Kinetic Method comparison Multidetection

"Factors Concerning The Design And Calibration Of An Amperometric Enzyme Electrode System For The Flow Injection Analysis Of Cholesterol"
Analyst 1988 Volume 113, Issue 9 Pages 1419-1422
G. J. Moody, G. S. Sanghera and J. D. R. Thomas

Abstract: Cholesterol oxidase (5 mg mL-1 in phosphate buffer) is immobilized on a nylon mesh, which is then attached to a smooth Pt electrode. A serum sample (0.5 ml) is injected into a stream (2.3 mL min-1) of 100 µM-NaH2PO4 (pH 7.0) containing 1% of Triton X-100 in a flow injection system. A three-electrode detector system (Metrohm EA1102) is used. The electrodes are operated at +600 mV vs. Ag - AgCl. Factors affecting the enzyme electrode lifetime were studied. The calibration graph was rectilinear from 10 µM to 1 mM cholesterol.
Cholesterol Amperometry Clinical analysis Electrode Electrode Calibration Surfactant Triton X

"Determination Of Selenium In Blood Plasma And Serum By Flow Injection Hydride Generation Atomic Absorption Spectrometry"
Analyst 1990 Volume 115, Issue 3 Pages 275-278
Kieran McLaughlin, Darioush Dadgar, Malcolm R. Smyth and Dorothy McMaster

Abstract: A flow injection hydride generation atomic absorption spectrometric (AAS) method has been used to determine the selenium concentrations of human serum and plasma samples following digestion with nitric, sulfuric and perchloric acids. In the hydride generation process, reduction was carried out by sodium tetrahydroborate to produce a hydride that was atomized in a flame-heated atomisation cell. The method had a detection limit of 1.2 ng mL-1 and a sensitivity of 2.1 ng mL-1. Within-run precisions of 5.8% at 20 ng mL-1 and 4.5% at 80 ng mL-1, and between-run precisions of 4.8% at 69 ng mL-1 and 3.4% at 80 ng mL-1 were obtained. An inter-laboratory comparison study with a graphite furnace AAS method was carried out and the results showed excellent agreement. The flow injection method of sample introduction allowed the use of a sample volume of 330 µL with an injection rate of 90 injections per hour. Plasma or serum (2 ml) was digested at 140°C for 20 min (temp. raised to 140°C over 20 min) with 5 mL of 16 M HNO3 and cooled; 2.5 mL of 18 M H2SO4 and 1 mL of 11.6 M HClO4 were added and the temperature was increased to 140°C (held for 15 min), then to 205°C over 15 min (held until white fumes appeared). The residue was cooled, then heated at 95°C for 30 min with 5 mL of 5 M HCl. After cooling and dilution to 25 mL with water, a 330 µL portion was injected into the flow injection hydride generator with 1% (w/v) NaBH4 (3.9 mL min-1) - 1 M HCl (5.4 mL min-1). The H2Se produced was carried by the Ar stream to the heated silica cell of the AAS instrument and atomized at 900°C. Absorbance was measured at 196 nm. The detection limit of Se was 1.2 ng mL-1; sensitivity was 2.1 ng mL-1. Within- and between-run precisions were 4.5 and 3.4%, respectively, at 80 ng mL-1 of Se. Recoveries were 95 to 109%. Results from an inter-laboratory comparison study with graphite-furnace AAS agreed well.
Selenium Spectrophotometry Sample preparation Detection limit Graphite Dilution Sensitivity

"Flow Injection Of Lithium Ion Using Chromogenic 14-crown-4 Derivatives As Extraction-spectrophotometric Reagents"
Analyst 1990 Volume 115, Issue 9 Pages 1251-1255
Keiichi Kimura, Shin-ichi Iketani, Hidefumi Sakamoto and Toshiyuki Shono

Abstract: A system for flow injection of Li+ has been designed, with use of proton-dissociable chromogenic 14-crown-4 derivatives as the extraction-spectrophotometric reagents, and the analytical conditions have been optimized. This flow injection system showed high selectivity for Li+ reflecting the cation-complexing property of the chromogenic crown ethers. The determination of Li+ in the clinical range in blood under a high Na+ background of 130-160 mM was feasible, with a small sample size (50 µL) and high sampling rate (more than 100 injections per hour), with this method. The proposed extraction-spectrophotometric flow injection system was, therefore, found to be promising for the efficient determination of Li+ in biological samples, such as blood sera, with a high Na+ background. Conditions for the use of six 14-crown-4 derivatives were determined. The best results were obtained with 14-crown-4-dinitrophenol at pH 9.5 for a carrier stream of H3BO3 - KOH and a solvent stream of CHCl3, both at 0.75 mL min-1. Detection was at 410 nm and the coil length was 1 m. The samples e.g., blood sera, and the crown ether were injected into the aqueous and organic streams respectively and the Li+ was extracted in the coil. The response was rectilinear up to 2 mM with a limit of detection of 0.1 µM. High backgrounds caused by the presence of Na+ were compensated for, by preparing calibration standards containing 145 mM Na+.
Lithium Spectrophotometry Sample preparation Crown ether Selectivity Small sample Detection limit Calibration Extraction

"Determination Of Creatine Kinase Activity Using A Co-immobilized Auxiliary Enzyme Reactor Coupled Online With A Flow Injection System"
Analyst 1991 Volume 116, Issue 2 Pages 167-169
J. M. Fern&aacute;ndez-Romero and M. D. Luque de Castro

Abstract: Two flow injection methods (based on spectrophotometric and spectrofluorimetric detection) were developed for the determination of over-all creatine kinase activity. Despite the complexity of the reactions involved (both include three enzyme-catalyzed steps), the manifold is very simple because the two auxiliary enzymes which catalyse the two-step indicator reaction are co-immobilized on controlled-pore glass. The features of the proposed methods (calibration ranges between 0.1 and 2.0 and 0.01 and 1.0 U l-1, relative standard deviation 0.93 and 0.53% for the spectrophotometric and spectrofluorimetric methods, respectively) allow the successful determination of the analyte activity in serum samples (recoveries better than 95-105% for both methods). In the flow injection method described, a sample of serum is injected into a stream of 100 mM Tris - acetate buffer (pH 7.00) and mixed with the reagent stream containing 20 mM Mg acetate, 1 mM EDTA, 10 µM-p'p5-di(adenosine-5')pentaphosphate, 1 or 0.5 mM AMP, 10 mM N-acetyl-L-cysteine, 2 or 1.5 mM ADP, 15 mM creatine phoshate, 1.5 mM D-glucose and 1.5 or 2 mM NADP in the same buffer. The mixture was passed through a reaction coil and an enzymatic reactor containing hexokinase and glucose-6-phosphate dehydrogenase immobilized on controlled pore glass (cf. Masoom and Townshend, Anal. Chim. Acta., 1984, 166, 111). Detection was by spectrophotometry or fluorimetry (where two concentration. are given, they refer to the two methods, respectively). Calibration graphs were rectilinear for 0.1 to 2 and 0.01 to 1 iu l-1, respectively, and coefficient of variation were 1% (n = 11). Recoveries were 95 to 105%.
Enzyme, creatine kinase Fluorescence Buffer Controlled pore glass Immobilized enzyme

"Flow Injection Electrochemical Enzyme Immunoassay For Theophylline Using A Protein A Immunoreceptor And P-aminophenyl Phosphate P-aminophenol As The Detection System"
Analyst 1992 Volume 117, Issue 11 Pages 1679-1682
Derek A. Palmer, Tony E. Edmonds and Nichola J. Seare

Abstract: An online immobilization of the antibody - antigen complex on controlled-pore glass - protein. A competitive electrochemical enzyme immunoassay was developed (details given). The p-aminophenol phosphate hydrolysis product was determined at ± 0.2 V vs. SCE. In the analysis of serum, the coefficient of variation were 6.8 to 8.7%; the detection limit was 25 ng mL-1 of theophylline. Recoveries were 95.5%. A competitive electrochemical enzyme immunoassay has been developed for the antiasthmatic drug theophylline, utilizing a controlled-pore glass-protein A immunoreactor and flow injection techniques. p-Aminophenyl phosphate, a substrate for alkaline phosphatase, has been used in this assay, and its hydrolysis product p-aminophenol was determined at +0.2 V vs. the SCE. For each sample the antibody-protein A reaction takes place at near-neutral pH, and the complexes are eluted at acid pH. Serum theophylline has been determined by this method, and good relative standard deviations and percentage recoveries have been achieved.
Theophylline Electrode Immunoassay Controlled pore glass Immobilized protein

"Bioluminescent Flow-sensing Device For The Determination Of Magnesium(II)"
Analyst 1993 Volume 118, Issue 7 Pages 849-853
Stefano Girotti, Elida Ferri, Severino Ghini, Pavel Rauch, Giacomo Carrea, Roberto Bovara, Aldo Roda, Maria Antonietta Giosu&eagrave;, Piero Masotti and Gianni Gangemi

Abstract: A continuous-flow system was developed for the determination of magnesium in serum, drugs and beverages. The system used firefly luciferase (LUC) or recombinant luciferase (r-LUC) from Escherichia coli (1 mg/ml) immobilized on a nylon coil activated with triethyloxonium tetrafluoroborate, 1,6-diaminohexane and glutaraldehyde or immobilized on a column of epoxy methacrylate (Eupergit) placed inside a luminometer. The bioluminescence continuous-flow assay manifold for the nylon coil system involved two streams. The first supplied the coil with the working bioluminescent solution and the second was a continuous-flow of air into which a known volume (5-20 µL) of sample was injected. The same system was used for the Eupergit column except that the air was replaced with Tris acetate buffer. The optimal conditions were 0.05 mM luciferin and 0.3 mM ATP at a flow rate of 0.4 ml/min for an LUC-Eupergit column or an r-LUC-nylon coil; the corresponding responses were linear for 0.05-6.7 and for 0.01-6.7 mM Mg. Both the intra- and inter-assay RSD were 7%. There was no interference from Ca(II), Mn(II), Fe(II), Cu(II), Zn(II), or Co(II).
Magnesium(II) Bioluminescence Interferences

"Rapid Fluorescence Flow Injection Immunoassay Using A Novel Perfusion Chromatographic Material"
Analyst 1994 Volume 119, Issue 5 Pages 943-947
Derek A. Palmer, Mark Evans, James N. Miller and Martin T. French

Abstract: Texas Red-labelled testosterone (100 µL) was mixed with 100 µL of testosterone antiserum diluted 1:19 in PBS of pH 7.4. After 2 min, 25 µL of the solution were injected into a carrier stream of PBS of pH 7.4 (2.6 ml/min). The stream was passed through a POROS IIA immunoreactor (protein A immobilized on a perfusion matrix). The bound complex was eluted with citrate buffer of pH 2.5 (2.6 ml/min) and detected fluorimetrically at 625 nm (excitation at 595 nm). The detection limit was 0.5 µg/ml of testosterone. The intra- and inter-assay RSD (n = 5) for 1 µg/ml of testosterone were 1.9 and 10.7%, respectively. The recovery was 94.7%. The method was applied to the analysis of serum. An online assay was also carried out in which the testosterone antiserum, Texas Red-labelled testosterone and testosterone sample were sequentially irreversibly immobilized on to the POROS IIA immunoreactor (details given).
Testosterone Immunoassay Fluorescence Perfusion

"Determination Of Substrates Using Poly(ethylene Glycol)-stabilized Dehydrogenase Enzymes By Microlitre Per Minute Flow Injection"
Analyst 1995 Volume 120, Issue 4 Pages 1091-1096
James R. Marsh and Neil D. Danielson

Abstract: Several enzymatic substrates, viz., pyruvate (I), lactate (II) and cortisone (III), were determined using the cited method. For I determination, the sample solution (0.5 µL) was injected into a carrier stream (25 µL/min) of 0.067 mM NADH, 2.6 iu/ml lactate dehydrogenase (LDH) and 15% PEG-8000 in 0.2 M Tris buffer of pH 7.3 at 30°C and the decrease in NADH absorbance was measured at 340 nm. For II, the sample solution (0.5 µL) was injected into a carrier stream (50 µL/min) of 3.6 mM NAD+, 50 iu/ml LDH and 15% PEG-8000 in a hydrazine buffer of pH 9 at 30°C and the formation of NADH was measured at 425 nm (excitation at 338 nm). For III determination, the sample solution (0.5 µL) was injected into a carrier stream (254 µL/min) of 0.5 iu/ml hydroxysteroid dehydrogenase, 0.067 mM NADH and 15% PEG-8000 in 0.03-M-Tris buffer of pH 7.6 at 30°C and the decrease in NADH absorbance was measured at 340 nm. Calibration graphs were linear from 0.01-0.5, from 0.3-20 and from 0.05-2 mM of I, II and III, respectively. Detection limits were 8 µM I and 0.28 mM II (no value given for III). The RSD were 2% for I and 1% for II (no value given for III). The method was used to determine II in serum. Flow injection (FI), at a flow rate of µL min-1, is an effective method for enzymatic substrate determination using low concentrations of poly(ethylene glycol) (PEG)-stabilized soluble enzymes. PEG stabilizes dehydrogenase enzymes for at least several days by promoting sub-unit association. Band broadening of knitted open tubular reactors is reduced as flow rate decreases below 300 µL min-1 and a small tubing diameter is important for a faster rate of absorbance signal increase with residence time. Small (0.5 µL) sample injections also ensure narrow FI peaks. The determination of several substrates such as pyruvate, lactate, and cortisone using appropriate PEG-stabilized enzymes is demonstrated with this FI instrument at 25 or 50 µL min-1 with sample throughputs of the order of 2-3 min per sample. The determination of lactate in serum samples is also possible. The advantage of this method, sample throughput, is not sacrificed but enzyme consumption is considerably less, compared to standard mL min-1 FI.
Cortisone Lactate Pyruvate Fluorescence Spectrophotometry Knotted reactor Low flow rate Open tubular reactor

"Flow Injection Spectrofluorimetric Method For The Determination Of Magnesium In Blood Serum"
Analyst 1995 Volume 120, Issue 8 Pages 2115-2118
Loukas N. Zachilas, Pinelopi C. Ioannou, Christoforos Polydorou and Constantinos E. Efstathiou

Abstract: Serum was diluted 1000-fold and a portion (80 µL) of the resulting solution was injected into a carrier stream (2.42 ml/min) of aqueous 50% ethanol. Streams (0.85 ml/min each) of ethanolic 0.1 mM 2-hydroxy-l-naphthaldehyde salicyloylhydrazone and aqueous ethanolic 0.1 M ammonia buffer of pH 11.6 were mixed before being merged with the sample zone. The reactants passed through a 50 cm reaction coil and the fluorescence intensity was measured at 460 nm (excitation at 420 nm). The calibration graph was linear from 4.8-120 µg/l of Mg and the detection limit was 2 µg/l (2 mg/l for serum). Recoveries of Mg from spiked serum were 96.2-102% and the RSD (n = 4) was 0.7-3.5%. The throughput was 120 samples/l. Results were in good agreement with those obtained by spectrophotometry. A flow injection configuration for the spectrofluorimetric determination of magnesium is proposed. The method is based on the formation of a strongly fluorescent complex of magnesium ion with 2- hydroxy-1-naphthaldehyde salicyloylhydrazone in alkaline, aqueous- ethanolic (50% v/v) solutions. The optimal chemical conditions for complex formation in the mixed solvent were studied and the flow injection manifold was optimized and used for the determination of magnesium in 1000-fold diluted serum samples without any other pre- treatment. The measurement throughput was 120 h-1. The detection limit of the method was 2 µg L-1 (2 mg L-1 for serum). The concentration range of application is 4.8-120 µg L-1 (0.2-5.0 µmol l-1). Within-run relative standard deviations for the method at 4.8, 24 and 120 µg L-1 of magnesium were 3.5, 1.2 and 0.7%, respectively. Analytical recoveries range from 96.2 to 102% (mean 98.8%).
Magnesium Fluorescence Method comparison Complexation

"Development Of A Flow Fluoriimmunosensor For Determination Of Theophylline"
Analyst 1995 Volume 120, Issue 10 Pages 2589-2591
Carlos M. Rico, M. del Pilar Fernandez, Ana M. Gutierrez, M. Concepcion Perez Conde and Carmen Camara

Abstract: A 30 µL portion of theophylline (I) polyclonal antibody diluted 100-fold was injected into a carrier stream (0.18 ml/min) of 50 mM Tris buffer of pH 8.8 in 0.5 M NaCl (buffer A). The antibodies were carried to a flow cell packed with protein A immobilized on controlled-pore glass. After allowing 80 s for binding to occur, a portion (30 µL) of a mixture of fluorescein isothiocyanate-labelled I (200 µg/l) and standard I solution was injected into a carrier stream (0.18 ml/min) of buffer A. The two antigens were carried to the flow cell containing the bound antibodies and, after 200 s had elapsed, the fluorescence intensity was measured at 520 nm (excitation at 495 nm). The calibration graph was linear up to 10 µg/l of I and the detection limit was 0.3 µg/l. The within-day RSD (n = 6) was 3.3-4.4%. The through-put was 10^-12 samples/h. The method was applied to the analysis of I in serum. Results ageed well with those obtained by an HPLC reference method.
Theophylline Immunoassay Fluorescence Sensor Immobilized enzyme Controlled pore glass Method comparison

"Determination Of Inorganic Phosphate By Flow Injection With Fluorescence Quenching"
Analyst 1995 Volume 120, Issue 10 Pages 2613-2616
Elena Diacu, Pinelopi C. Ioannou, Christoforos K. Polydorou and Constantinos E. Efstathiou

Abstract: Portions (80 µL) of standard phosphate (I) solution containing 4 µM of Sc was injected into a stream aqueous 50% ethanol containing 0.01 M glycine buffer and 0.1 M NaCl ajusted to pH 4 (2.62 ml/min; solution A) of a flow injection system and merged with a stream of 3- or 30 µ-M-2-hydroxy-1-naphthaldehyde p-methoxybenzoylhydrazone (HNAMBH) reagent in solution A (0.92 ml/min). The mixture was passed through a 50 cm PTFE reaction coil and the fluorescence intensity was measured at 490 nm (excitation at 430 nm). The calibration graphs were linear from 3-15 from 15-150 µg/l, respectively, of I for HBAMBH concentrations of 3 and 30 µM and the corresponding detection limits were 0.8 and 3 µg/l. The within-run RSD (value of n not given) were 0.9-5.7%. The throughput was 120 samples/h. The method was applied to the analysis of I in serum after a 1000-fold dilution of the sample. Recoveries were 95.5-103.9% for 29.1-82.6 mg/l of added I.
Phosphate Fluorescence Indirect Quenching

"Multicomponent Analysis By Flow Injection Using A Partial Least-squares Model. Determination Of Copper And Zinc In Serum And Metal Alloys"
Analyst 1996 Volume 121, Issue 2 Pages 169-172
Oscar Hern&aacute;ndez, Francisco Jim&eacute;nez, Ana Isabel Jim&eacute;nez and Juan Jos&eacute; Arias

Abstract: Sample (192 µL) was injected into a carrier stream (1.75 ml/min) which had been previously merged with a stream of 0.1 M borate buffer of pH 9 and also with a reagent stream of aqueous 10% methanolic 0.1 mM 4-(4'-methyl-2'-thiazolylazo)-2-methylresorcinol. The mixture was passed through a 80 cm reactor after which the absorption spectra were recorded from 520-620 nm at 1 s intervals for 40 s on a Hewlett-Packard 8452 A spectrophotometer, equipped with a diode-array detector, using an integration time of 0.4 s. A partial least-squares multivariate calibration method, employing the computer program Unscrambler, was used to process the data obtained (details given), thus enabling the simultaneous determination of Cu and Zn to be achieved. The results obtained agreed with those obtained by AAS.
Copper Zinc Spectrophotometry Partial least squares Method comparison Multivariate calibration

"Colloidal Gold Supported Onto Glassy Carbon Substrates As An Amperometric Sensor For Carbohydrates In Flow Injection And Liquid Chromatography"
Analyst 1996 Volume 121, Issue 2 Pages 249-254
Innocenzo G. Casella, Angelo Destradis and Elio Desimoni

Abstract: The cited sensor was prepared by depositing 10 µL 30 mM Au(III) on the surface of a vitreous C electrode. The electrode was dried and conditioned in 0.2 M NaOH by cycling the potential between -0.6 and 0.4 V vs. SCE for 30 min. The feasibility of using the Au-modified electrode as an amperometric sensor for carbohydrates was evaluated with a flow injection system, and the following LC method was developed for carbohydrate determination. The sample was analyzed on a Carbopac PA1 anion-exchange column (25 cm x 4 mm i.d.), with 0.2 M NaOH/1 µM-Au(III) as mobile phase (0.6 ml/min) and pulsed amperometric detection at dual Au-modified electrode, in serial configuration, held at 0.65 V vs. Ag/AgCl for 0.2 s, then at -0.7 V for 0.2 s and finally at 0.2 V for 0.4 s. Calibration graphs were linear over 2-3 orders of magnitude of concentration for the carbohydrates studied. Detection limits were 4-12 ng. RSD (n = 6) were 0.8-3.5%. The LC method was used to determine glucose in serum and urine. Recoveries of glucose were 97.7-102.1%. The results obtained agreed with those obtained by an enzymatic method.
Carbohydrates Glucose Amperometry HPIC Sensor Electrode Method comparison

"Precipitation Flow Injection Immunoassay For Human Immunoglobulin G"
Analyst 1996 Volume 121, Issue 11 Pages 1565-1568
Andrea Hacker, Gerald G&uuml;bitz, Juan-Manuel Fernandez-Romero, Maria D. Luque de Castro and Miguel Valcarcel

Abstract: The sample was diluted with 50 mM Na2HPO4 buffer of pH 7 containing 0.9% NaCl (buffer A) and a portion (130 µL) of the resulting solution was mixed 195 µL fluorescein isothiocyanate-labelled anti-human IgG diluted buffer A such that there was a 10-fold molar excess of the antibodies. A portion (75.4 µL) of the mixture was injected into a carrier stream (0.5 ml/min) of 50 mM Na2HPO4 buffer of pH 7 containing 6% PEG 6000 and 0.9% NaCl of a flow injection manifold (schematic shown). The solution was passed through a PTFE reaction coil (2 m x 0.76 mm i.d.) maintained at 37°C and the precipitate formed was collected on a 5 µm metallic filter. The precipitate was subsequently dissolved with a stream (0.7 ml/min) of 1 M NaOH and the fluorescence intensity of the resulting solution was measured at 521 nm (excitation at 485 nm). The calibration graph was linear from 250-2000 fmol human IgG and the detection limit was 140 fmol. Intra- and inter-assay RSD (n = 7 and 21, respectively), were 1-3 and 1.2-5%, respectively. The method was applied to serum: the recoveries of added human IgG were 96-102%.
Immunoglobulin G Immunoassay Precipitation

"Miniaturized Graphite Sensors Doped With Metal-bathophenanthroline Complexes For The Selective Potentiometric Determination Of Uric Acid In Biological Fluids"
Analyst 1997 Volume 122, Issue 8 Pages 815-819
Saad S. M. Hassan and Nashwa M. H. Rizk

Abstract: Miniaturized poly(vinyl chloride) matrix membrane sensors in an all- solid-state graphite support, responsive to urate anion, were developed. The membranes incorporate lipophilic ion-pair complexes of urate anion with ruthenium(III), iron(II), nickel(II) and copper(I) bathophenanthroline (4,7-diphenyl-1,10-phenanthroline) counter cations. The sensors demonstrate a near-Nernstian response to urate over the concentration range 1 x 10^-2-1 x 10^-5 mol L-1 and have micromolar detection limits and good selectivity properties. The response is virtually unaffected by pH changes in the range 7-10 and the response times are 5-10 s in aqueous solutions and in human serum and urine samples. A flow injection detector incorporating an iron(II) bathophenanthroline-urate graphite sensor was used for continuous monitoring of uric acid. The minimum detectable concentration was approximately 8 µg mL-1 and the sample throughput was approximately 120 h-1. Direct potentiometric determination of uric acid in the static and hydrodynamic modes of operation over the range 15 µg mL-1-1.5 mg mL-1 showed average recoveries of 98.7 and 97.8% with RSDs of 0.6 and 0.7%, respectively. Application of the method to the determination of uric acid in human serum and urine gave results that compared favourably with those obtained by the standard spectrophotometric method.
Uric acid Potentiometry Electrode Electrode Electrode Sample preparation Sensor Detection limit Method comparison Process monitoring Ion pair extraction

"Flow Injection Online Photochemical Reaction Coupled To Spectrofluorometry For The Determination Of Thiamine In Pharmaceuticals And Serum"
Analyst 1998 Volume 123, Issue 5 Pages 1017-1021
Hengwu Chen, Jingping Zhu, Xiaoxia Cao and Qiongjun Fang

Abstract: The photochemical reaction of thiamine was studied with a photochemical reactor made by coiling a knotted PTFE reactor around a low-pressure Hg lamp. Acetone, which was previously reported to be a sensitizer for the photochemical reaction that took place in situ in a flow-through cell, severely depressed the fluorescence signal of the photochemical reaction that took place online in the knotted PTFE reactor when Na sulfite was involved in the photochemical reaction. Experiments revealed that the effect of acetone on the photochem. reaction was dependent on the intensity of the irradiation. that was used to induce the photochemical reaction, and that acetone might impair the photochemically induced fluorescence if strong UV irradiation. was applied to induce the photochemical reaction and Na sulfite was used to enhance the fluorescence signal. Based on these observations, a flow injection online photochemical-spectrofluorometric method for the determination of thiamine was developed without using acetone. With the proposed method, a detection limit of 0.11 µg L-1 thiamine, a relative standard deviation of 0.36% for 11 determinations of 1 mg L-1 thiamine and a sampling frequency of 100 h-1 were achieved. The developed method was successfully applied to the determination of the thiamine content in various pharmaceutical preparations and serum.
Thiamine Fluorescence Knotted reactor UV reactor Photochemistry

"Development Of A Biparametric Bioanalyzer For Creatinine And Urea. Validation Of The Determination Of Biochemical Parameters Associated With Hemodialysis"
Analyst 1998 Volume 123, Issue 6 Pages 1321-1327
M. Jurkiewicz, S. Alegret, J. Almirall, M. Garc&iacute;a and E. F&agrave;bregas

Abstract: The construction and evaluation of an automated urea and creatinine biparametric biosystem using flow injection analysis (FIA) are described. The biosystem uses enzyme reactions that hydrolyze urea and creatinine producing ammonium ions. The enzymes used were creatinine deiminase and urease, which are immobilized covalently in flow reactors. The reactor with creatinine deiminase has the enzyme immobilized on controlled-pore glass beads, whereas urease is immobilized on a nylon open tubular reactor. Detection is realized with a flow-through ammonium ion-selective electrode with an inner solid-state contact (graphite-epoxy composite). Ammonium ions are separated from alkali ion interferents through a gas-diffusion cell. The bioanalyzer is fully automated using software and electronics developed ex profeso in our labs. The analyzer was validated off-line by measuring urea and creatinine from discrete effluent samples from hemodialysis equipment. Results agreed with concurrent analyzes realized using hospital lab. methods. There were no significant differences between the two sets of results at the 95% confidence level. Finally, the biparametric bioanalyzer was validated online by measuring creatinine and urea levels in artificial kidney effluents. These measurements were useful in the determination of key biochemical parameters of clinical interest such as the mass of urea and creatinine extd. from the patient as well as the initial concentration. of creatinine and urea in blood plasma. When the results of the bioanalyzer were compared with those yielded by the usual methods, they showed no significant differences at the 95% confidence level when determining the mass of the analytes extd. by the hemodialyzer or when determining the urea concentration. in blood plasma. However, when measuring the creatinine concentration. in blood plasma using the developed bioanalyzer, significant differences appeared.
Creatinine Urea Sensor Electrode Gas diffusion Interferences Automation Method comparison

"Highly Sensitive Flow Detection Of Uric Acid Based On An Intermediate Regeneration Of Uricase"
Analyst 1998 Volume 123, Issue 8 Pages 1775-1780
Yasushi Hasebe, Kimihisa Nawa, Shinya Ujita and Shunichi Uchiyama

Abstract: The principle of the signal amplification of a uric acid sensor based on dithiothreitol (DTT)-mediated intermediate regeneration of uricase was applied to a flow injection system with an immobilized uricase reactor and a DTT-containing carrier. Highly sensitive detection for nM to µM order of uric acid was achieved when 10 mM TRIS-HCl buffer (pH 10.0) containing 20 mM DTT was used as a carrier at 0.6 mL min-1 and 37°C. The sensitivity of the uric acid was much improved over a batch method using a uricase membrane-coupling electrode, and the detection limit (ca. peak current 8 nA) of uric acid was found to be down to 3 x 10^-10 M (amplification factor; more than 10000). This chemical amplified flow-system is very useful for the direct assay of uric acid in highly diluted biological fluids (urine and serum) without complicated pretreatment of the samples, because this sensor has the potential to detect trace amounts (nM to µM) of uric acid in highly diluted body fluids in which the concentration. of interfering constituents was decreased to negligible levels. Good correlation was observed between this system and conventional spectrophotometry. The immobilized uricase reactor could be re-used for at least 4 months of repeated analysis without loss of activity and was stable if stored at 4°C in 10 mM TRIS-HCl buffer, pH 9.0.
Uric acid Electrode Electrode Immobilized enzyme Reagent recycling Heated reaction

"Optimization Of Flow Injection Hydride Generation Inductively Coupled Plasma Mass Spectrometry For The Determination Of Selenium In Water And Serum Samples"
J. Anal. At. Spectrom. 1995 Volume 10, Issue 10 Pages 871-874
M. Angeles Quijano, Ana Maria Guti&eacute;rrez, M. Concepcion P&eacute;rez Conde and Carmen C&aacute;mara

Abstract: Water was acidified with concentrated HCl (3 mL per 5 mL sample), boiled to 100°C for 10 min and after cooling diluted to 10 mL with water. Serum (1 ml) was diluted to 50 mL with water and treated similarly to the water samples (loc. cit.); Si was used as anti-foaming agent. The effects of NaBH4 concentration, HCl concentration, flow rates, sample volume, forward power and carrier gas flow rates were studied and optimized. Optimum conditions were: 0.5% (m/v) NaBH4; 0.6 M HCl; HCl and NaBH4 flow rates of 3.5 and 1.7 ml/min, respectively; sample volume = 100 µL with 30 cm coil lengths, an r.f. power of 1350 W; and an Ar flow-rate = 1.01 ml/min. The interference from 31 elements was investigated; serious interference was caused by some transition elements. The calibration graph was linear up to 30 µg/l and the detection limit was 35 ng/l of Se which is 100 times better than that possible with traditional pneumatic nebulization. RSD (n not stated) were 5%. Recoveries were 92-104%.
Selenium Mass spectrometry Volatile generation Optimization Speciation Interferences Volatile generation

"Enzymatic Determination Of Urea In Serum Based On PH Measurement With The Flow Injection Method"
Anal. Chem. 1979 Volume 51, Issue 2 Pages 199-203
J. Ruzicka, E. H. Hansen, Animesh K. Ghose, and H. A. Mottola

Abstract: A method based on flow injection analysis incorporating a flow-through capillary pH electrode is described for the enzymatic determination of urea in aqueous and serum samples. By maintaining a constant buffering capacity of the carrier stream solution, a linear relation between the recorded pH signal and the urea content was obtained. Anaytical readouts were obtained within 30 s of sample introduction, and the sampling rate was 60 samples/h. The reproducibility of measurement was ±.0.0029 pH unit, corresponding to ±0.52%. When solution urease was used in the carrier stream, its consumption was 25 units per analysis. The possibility of further reducing the enzyme consumption by using the merging zone principle in the flow injection stop-flow system is discussed.
Urea Clinical analysis Electrochemical analysis Electrode Enzyme Stopped-flow Buffer Merging zones

"Automated Continuous-flow Determination Of Serum Albumin By Differential Pulse Polarography"
Anal. Chem. 1980 Volume 52, Issue 12 Pages 1896-1900
P. W. Alexander and M. H. Shah

Abstract: A polarographic study of the interaction of serum albumin with potassium titanium(1V) oxalate is reported over a buffer range of pH 4-5. The shift in the half-wave potential of the titanium reagent after reaction with albumin is used to develop a novel automated method for determination of albumin in blood serum. Polarographic analysis in a continuous-flow system is operated in the differential pulse mode at a fixed potential of -0.68 V. Under controlled solution conditions at pH 4.89, albumin is shown to selectively react with the Ti(1V) reagent without serious interference from other serum components including the major globulin and glycoprotein fractions unless they are present in excess of the albumin concentration. The polarographic method for analysis of 15 human serum samples is compared to the bromocresol green spectrophotometric dye-binding method for albumin determination, giving a correlation coefficient of 0.9934.
Albumin Polarography

"Continuous-flow Analysis For Uric Acid In Biological Fluids, With Immobilized Uricase In A Closed-loop System"
Anal. Chem. 1980 Volume 52, Issue 14 Pages 2332-2338
Assaha Iob and Horacio A. Mottola

Abstract: Relatively inexpensive and/or stable enzymes can be directly used in solution for repetitive determinations in closed-flow systems. Important clinical determinations (e.g., uric acid in biological fluids), however, require enzymes that are not sufficiently stable and/or inexpensive to be used in solution for relatively long periods of time and at the high activity levels needed for success in unsegmented closed-flow systems. This paper reports on the chemical immobilization of uricase on controlled pore glass, certain characteristics of the immobilized enzyme preparation, and application of it as packing in mixing- delay coils for the determination of uric acid In biological fluids. Determinations can be performed at a 100 samples/h rate with satisfactory precision (24% RSD). The method was compared with a regularly used colorimetric procedure in the SMA 18/90 analyzer [correlation coefficient = 0.98 for 22 samples of human blood serum]. The immobilized enzyme preparation retains over 70 % of its initial activity after repetitive use for more than 10 months.
Uric acid Clinical analysis Spectrophotometry Closed loop Enzyme Immobilized enzyme

"Evaluation Of Dual-wavelength Spectrophotometry For Drug Level Monitoring"
Anal. Chem. 1984 Volume 56, Issue 13 Pages 2352-2354
Susumu Honda, Tadao Konishi, and Hirokazu Chiba

Abstract: Serum sulphanilamide was converted into an azo-dye by treatment with a solution of HCl, NaNO2 and sulphamic acid and coupling with N-1-naphthylethylenediamine. Detection was at 423 and 538 nm for the simple elimination mode and at 660 and 538 nm for the k-factor mode. All three modes studied (viz, simple elimination, k-factor and difference) were less subject to interference than was the single-wavelength technique, and they afforded coefficient of variation (n = 10) of <1.2%. Automation of the method based on the simple elimination and k-factor modes was achieved.
Sulfanilamide Spectrophotometry Detector

"Automated Flow Injection Phenol Red Method For Determination Of Bromide And Bromide Salts In Drugs"
Anal. Chem. 1986 Volume 58, Issue 2 Pages 322-326
Paraskevi I. Anagnostopoulou and Michael A. Koupparis

Abstract: The chloramine T - phenol red method was optimized by the simplex technique. The calibration graph was rectilinear from 1 to 10 mg l-1, with a coefficient of variation (n = 10) from 0.2 to 2.5% at the highest and lowest levels, respectively, and a detection limit of 90 µg l-1. Interference from Cl- and NaHCO3 was eliminated (up to 40 and 10 g l-1, respectively), and that of NH3 was decreased (tolerance limit 25 mg l-1). Glucose, starch, talc (each at 10 g l-1), Carbowax 4000, Mg stearate, Na dodecyl sulfate (each at 1 g l-1), and gelatin (0.5 g l-1) also do not interfere. Ions that react with Chloramine T, Br- or the Br2 produced during the reaction interfere, as do sulfonamides, isoniazid, salicylates, imipramine and propranolone hydrochlorides, and almost all organic substances with a nucleophilic substituent on the phenyl group. Interfering compounds having an acidic or basic functional group can be removed online by Amberlite XAD-2 resin. Recoveries of 2.5 to 5 mg L-1 of Br- added to seawater were from 98.6 to 103.3% and from serum samples spiked with 19.6 to 103 mg L-1 they were from 97.2 to 103%. In the analysis of eight commercial drugs, recoveries by the proposed method agreed well with those by the USP XX 1980 method. Up to 120 measurements h-1 can be performed.
Drugs Bromide Clinical analysis Spectrophotometry Amberlite Interferences Simplex

"Liquid Chromatographic Determination Of Guanidines With An Anion-exchange Column Used Simultaneously As Separator And Post-column Reagent Generator"
Anal. Chem. 1986 Volume 58, Issue 7 Pages 1380-1383
Hans Jansen, Elisabeth G. Van der Velde, Udo A. T. Brinkman, Roland W. Frei, and Hans Veening

Abstract: Samples were injected on to a stainless-steel column (10 cm x 4.6 mm) packed with Annex A-28 anion-exchange resin (OH- form). The mobile phase (0.26 mL min-1) was aqueous 25 mM acetate - methanol (9:1) at pH 7.0. The components were separated and OH- was generated for post-column derivatization with phenanthrenequinone. Fluorescence detection was at 460 nm (excitation at 375 nm). Detection limits ranged from 3 to 40 ng and the method was used to separate guanidines added to serum and haemodialysate.
Guanidines HPLC Fluorescence Ion exchange Post-column derivatization

"Serum Lithium Analysis By Coated Wire Lithium Ion-selective Electrodes In A Flow Injection Analysis System"
Anal. Chem. 1986 Volume 58, Issue 8 Pages 1806-1810
Robert Y. Xie and Gary D. Christian

Abstract: Blood serum samples (200 µL) were injected into the donor stream (H2O flowing at 0.4 mL min-1) which flowed through a dialyser. The dialysed ions were carried by the acceptor stream (7 mM Na2B4O7 at 0.3 mL min-1) to the coated-wire electrode detector consisting of platinum, copper or silver wire coated with a dialkyl 14-crown-4 ether (3-dodecyl-3-methyl-1,5,8,12-tetraoxacyclotetradecane). The effect of sample size and flow rate, concentration. and pH of the acceptor stream on the electrode response is discussed. Results agreed well with those obtained by AAS.
Lithium Electrode Dialysis Method comparison Optimization

"Reversibly Immobilized Glucose Oxidase In The Amperometric Flow Injection Determination Of Glucose"
Anal. Chem. 1987 Volume 59, Issue 22 Pages 2688-2691
W. Uditha De Alwis, Brian S. Hill, Bruce I. Meiklejohn, and George S. Wilson

Abstract: Glucose oxidase(I) is reversibly immobilized by utilizing reaction sequences involving antibody - antigen reactions. In method A, I - anti-human IgG (goat) conjugate is passed through a packed-bed reactor in which human IgG is covalently immobilized. In method B, polyclonal goat anti-mouse IgG Fab' fragment immobilized on a support is used to reversibly immobilize a mouse monoclonal anti-I antibody that is used to immobilize the I. The reactors are coupled to a flow injection analysis system. After the immunological reaction, glucose is determined in biological fluids by amperometric monitoring of the H2O2 produced by the I-catalyzed reaction with O. The calibration graphs were rectilinear from 1.1 nmol to 11 µmol and from 0.11 nmol to 1.1 µmol for reactors A and B, respectively. The detection limits were 50 pmol (A) and 12 pmol (B). The precision of the H2O2 determination was >1%. On exhaustion, the reactors can be reloaded to within 3% of the original activity.
Glucose Amperometry Electrode Immobilized enzyme Reactor

"Electrochemical Platinization Of Reticulated Vitreous-carbon Electrodes To Increase Biosensor Response"
Anal. Chem. 1990 Volume 62, Issue 11 Pages 1106-1110
George H. Heider, Sylvia V. Sasso, Keming Huang, Alexander M. Yacynych, and Henry J. Wieck

Abstract: A method is given for the partial plating of a reticulated vitreous-carbon electrode (2 cm x 3 mm diameter) with Pt. The electrode was then coated with glucose oxidase with use of the carbodi-imide immobilization technique. The resulting electrode was used for the determination of glucose in serum by flow injection analysis (max. sampling rate 118 h-1). Samples (5 or 100 µL) were injected into the carrier stream (0.1 M phosphate buffer of pH 7.4; 2 mL min-1). The H2O2 formed in the enzymatic reaction was detected amperometrically at +0.6 V vs. the SCE. Calibration graphs were rectilinear for up to 50 and 10 mM glucose, respectively, for the two sample sizes, with corresponding detection limits of 0.25 and 0.05 mM. These responses make the electrode applicable in the clinical range (3.5 to 6.5 mM).
Glucose Electrode Sensor Immobilized enzyme Buffer Calibration Detection limit

"Use Of Ionomer Membranes To Enhance The Selectivity Of Electrode-based Biosensors In Flow Injection Analysis"
Anal. Chem. 1990 Volume 62, Issue 22 Pages 2418-2424
Sara A. Rosario, Geun Sig Cha, Mark E. Meyerhoff, and Marek Trojanowicz

Abstract: The use of ionomer membranes to enhance the selectivity of potentiometric enzyme electrodes in flow injection measurement arrangements is examined. The ionomer membranes employed are permeable to analyte substrates but relatively impermeable to detectable ions that would normally interfere with the measurement of the substrates if the enzyme electrodes were in direct contact with the sample. As a model system, the selectivity of enzyme electrodes prepared with nonactin-based ammonium-sensitive polymeric membranes is evaluated. In the preferred configuration, a thin hydrophilic anion-exchange membrane is incorporated within a flow-through dialysis unit upstream from the enzyme-electrode detector. As the sample passes through the dialysis unit, neutral or anionic analyte molecules (urea or glutamine) move through the membrane while the permeation of endogenous ammonium ions and other cations in the sample is retarded. A flowing recipient buffer on the other side of the membrane carries the analyte substrate to the enzyme-electrode detector. Enhancements in selectivity for analyte substrates over endogenous ammonium and potassium ions are greater than or equal to 9-fold when compared to enzyme-electrode flow injection analysis (FIA) systems assembled without the ionomer membrane unit. The analytical utility of the proposed system is demonstrated by the accurate measurements of urea in blood serum and L-glutamine in hybridoma bioreactor media.
l-Glutamine Urea Potentiometry Ion exchange Sensor Electrode Membrane Selectivity Interferences Dialysis Detector Buffer

"Continuous-flow/stopped-flow System Incorporating Two Rotating Bioreactors In Tandem. Application To The Determination Of Alkaline Phosphatase Activity In Serum"
Anal. Chem. 1994 Volume 66, Issue 9 Pages 1485-1489
Julio Raba and Horacio A. Mottola

Abstract: Sample was mixed with aqueous 4-nitrophenyl dihydrogen phosphate in the presence of 1 mM diethanolamine/10 mM Mg2+ and reaction was allowed to proceed for a fixed time (e.g., 5 min at 30°C) to hydrolyse a proportion of the substrate before quenching with 0.4 M HCl, adjustment of the pH to 7, addition of methanol and dilution with carrier buffer A. Streams of carrier buffer A (1 mM diethanolamine/1 mM MgCl2; pH 9-10) and the sample solution were mixed in a rotary four-way valve injector and passed through a rotating bioreactor containing immobilized alkaline phosphatase (cf. Matsumoto et al., Ibid., 1993, 65, 636). The resulting solution was mixed with 0.1 M phosphate buffer of pH 7.5 before entering a second rotating bioreactor containing immobilized alcohol oxidase, where unchanged methanol was oxidized to formaldehyde and H2O2. Within this bioreactor, the H2O2 produced was detected at a Pt ring electrode operated at +0.6 V vs. an Ag/AgCl reference electrode in conjunction with a stainless-steel tubular auxiliary electrode. The recorded response was directly related to the alkaline phosphatase activity in the sample. The method was used to assay alkaline phosphatase in serum.
Enzyme, alkaline phosphatase Electrode Electrode Stopped-flow

"Miniaturized Biosensors Employing Electropolymerized Permselective Films And Their Use For Creatinine Assays In Human Serum"
Anal. Chem. 1996 Volume 68, Issue 21 Pages 3832-3839
Marcel B. Madaras and Richard P. Buck

Abstract: Miniaturized disposable amperometric biosensors for creatinine and creatine were fabricated by depositing a multilayer structure on to a polyimide foil. A Pt electrode was galvanostatically deposited on to the foil and coated with an inner permselective layer formed by the electropolymerization of 1,3-diaminobenzene. The enzyme layers were then deposited by crosslinking with glutaraldehyde in the presence of BSA. Creatine amidinohydrolase (CI)/sarcosine oxidase (SO) were immobilized for the creatine biosensor and C/creatinine amidohydrolase (CA) for the creatine + creatinine biosensor. Finally an outer polymeric membrane of Nafion/poly(2-hydroxyethyl methacrylate) was deposited. The performances of the biosensors were evaluated in batch and flow injection systems. Calibration graphs were linear from 0.9-1.2 mM for the creatine biosensor in the batch system and up to 2 mM in the flow injection system; the detection limits were 20 and 10 µM, respectively. Similar responses were obtained for the creatinine + creatine biosensor. A differential approach using both biosensors was used to determine creatinine in blood serum; the results were verified by a classical spectrophotometric method.
Creatine Sensor

"Automation Of Solvent Extraction Of Drugs In Continuous-flow Systems"
Fresenius J. Anal. Chem. 1981 Volume 305, Issue 4 Pages 277-284
Peter Neubert und Klaus Reiff

Abstract: The performance of liquid/liquid extractions in continuous flow-systems is described by means of two different fluorescent compounds. The conventional AutoAnalyzer-System (using air-segmentation) is compared with the Flow Injection Analysis (FIA)-method. The extractability of compounds from biological fluids is evaluated with regard to sampling rate, solvent and reagent requirements and to recovery depending on the kind of extraction used and on the protein content of the sample. An automated system for the extraction of drugs from serum and urine is described. It is intended to be a part of a fully automated system for sample preparation (extraction, change of solvent) for chromatographic analysis.
Drugs Clinical analysis Fluorescence Sample preparation Solvent extraction

"Determination Of Creatinine In Urine And Serum By Flow Injection Analysis Using The Jaffe Reaction"
Fresenius J. Anal. Chem. 1983 Volume 315, Issue 2 Pages 141-144
Jacobus F. van Staden

Abstract: A simple, modified, flow-injection procedure for the determination of creatinine in urine and deproteinized serum using the Jaffe reaction is described. The method is suitable for the flow-injection analysis of creatinine at a rate of up to 120 samples per hour with a coefficient of variation of better than 2.9%. The baseline drift is eliminated by the addition of 20% methanol to the picric acid solution.
Creatinine Clinical analysis Spectrophotometry

"Determination Of Aluminum In Hemodialysis Fluids By A Flow Injection System With Preconcentration On A Synthetic Chelate-forming Resin And Flame Atomic Absorption Spectrophotometry"
Fresenius J. Anal. Chem. 1986 Volume 325, Issue 3 Pages 300-302
Pedro Hernandez, Lucas Hernandez and Jose Losada

Abstract: In a single-line flow injection system, a serum sample (from uraemic patients) containing 25 to 200 ng of Al is injected into a flow of buffer solution (aqueous 0.5 M NH3 - ammonium acetate, pH 8) and passed through a micro-column (2 cm x 2 mm) of anion-exchange resin modified with Chrome Azurol S (C. I. Mordant Blue 29), giving 100% retention of Al at a flow rate <2 mL min-1. The Al is eluted with 100 µL of 1 M NaOH at 1.5 mL min-1 into the N2O - acetylene flame of the spectrometer. Calibration is rectilinear in the range 25 to 200 µg l-1. The detection limit is 15 µg L-1 (<10 µg L-1 with standard additions) and the coefficient of variation is 0.88 to 1.48%.
Aluminum Clinical analysis Spectrophotometry Chelation Resin Preconcentration Standard additions calibration Dialysis

"Rapid, Miniaturized Procedure For The Stopped-flow Determination Of Glucose Based On Flow Injection Analysis"
Fresenius J. Anal. Chem. 1988 Volume 332, Issue 2 Pages 167-170
Jun'ichi Toei

Abstract: Glucose(I) was determined in serum by a commercial enzyme test kit (Glucose Test C Wako; Wako Pure Chemicals, Osaka, Japan) with use of a flow injection system (described) in which the detector is situated in the sample loop. Absorbance was measured at 254 and 505 nm. The coefficient of variation was 2.17% for glucose in serum.
Glucose Spectrophotometry Closed loop Enzyme Stopped-flow

"Spectrophotometric Determination Of Phosphorus As Phosphate In Organic Compounds And Materials Of Biological Origin Using A Flow Injection Manifold With A Mixing Chamber"
Fresenius J. Anal. Chem. 1992 Volume 342, Issue 9 Pages 734-736
D. Thorburn Burns, N. Chimpalee and M. Harriott

Abstract: Serum was treated with 10% trichloroacetic acid and the mixture was centrifuged. A portion of the supernatant solution was diluted with water and injected into a carrier stream (2.6 mL min-1) of aqueous 1% PVC which merged sequentially with streams (all at 1.3 mL min-1) of 20% HNO3, 0.5 mM Crystal violet (C. I. Basic Violet 3) and 0.1 M sodium molybdate. After passing through a reaction coil, the absorbance of the mixture was measured at 560 nm. The calibration graph was rectilinear up to 1.25 µg mL-1 of phosphate; the detection limit was 0.01 µg mL-1. The coefficient of variation (n = 10) was 0.6% for 0.5 µg mL-1. Sample throughput was 100 h-1. The method was also applied to the determination of P in organic compounds and plants. A flow injection manifold with mixing chamber has been developed for the determination of phosphate by reaction with molybdate and Crystal Violet. The insoluble blue dye salt is kept in colloidal solution with poly(vinyl alcohol) and the absorbance is measured at 560 nm. The injection rate is 100 h-1. The detection limit is 0.01 µg of PO43-/mL based on 250 µL injection volumes The system has been applied to the determination of phosphate in serum samples and after appropriate mineralization to organic compounds and to plant materials.
Phosphorus Sample preparation Spectrophotometry Mixing chamber

"Determination Of Serum Alkaline Phosphatase Activity By Electrochemical Detection With Flow Injection Analysis"
Fresenius J. Anal. Chem. 1993 Volume 346, Issue 6-9 Pages 859-862
Samuel D. Jackson, H. Brian Halsall, Amadeo J. Pesce and William R. Heineman

Abstract: Serum was added to 15 mM phenyl phosphate and 0.1 mM Mg(II) in 0.84 M aminomethylpropanol buffer of pH 10.3 pre-heated to 37°C and two portions of the mixture were subjected to analysis, one 2 min after addition of the serum and one 6 min after the addition. FIA was performed on a BAS LCEC analyzer. (BioAnalytical Systems, IN, USA) with a flow rate of 0.6 ml/min. The phenol produced was detected at +0.7 V using a carbon paste electrode vs. Ag/AgCl. The calibration graph for phenol was rectilinear for 0.1-10 µM, corresponding to 40-1000 iu/l of the enzyme. The RSD was 5%. The method was not affected by haemolysis, icteric or lipaemia or by the presence of such electroactive substances as urate, ascorbate and paracetamol. Results compared well with those obtained by a reference spectrophotometric method.
Enzyme, alkaline phosphatase Electrochemical analysis

"Advances In Photolithographically Fabricated ENFET Membranes"
Fresenius J. Anal. Chem. 1994 Volume 349, Issue 8-9 Pages 666-669
Astrid Z&uuml;rn, Beatrice Rabolt, Manfred Gr&auml;fe and Helmut M&uuml;ller

Abstract: ENFET (enzyme FET) biosensors were constructed by coating an n-channel depletion mode ISFET (1.2 x 4.2 mm) with a solution of acrylamide, Michler's ketone, benzophenone, bisacrylamide and urease in aqueous 50% ethanol. The coated chip, which contained two ISFET gates (16 x 400 µm) with SiO2/Si3N4 gate insulator, was exposed to UV radiation through a mask and the crosslinked membrane was developed by washing with water. When used in a flow injection system a biosensor obtained from a coating solution containing 40 mg/ml of urease showed a high sensitivity to urea with a linear calibration range from 1-500 mM and a response time (95%) of 20 s. The response was not inhibited by exposure for 5 min to 10 mg/l of Cu(II) at 1.8 ml/min. The biosensors were stable for 30 days either dry or immersed in buffer solution. Compounds present in serum or waste water from urea production did not interfere. Biosensors obtained from coating solution containing 7 mg/ml of urease were used to detect pesticides, e.g., down to 0.1 µg/l of Carbofuran, by monitoring the inhibition by the pesticides of the biosensor urea response. These biosensors should be useful in screening for pesticides in drinking water.
Urea Pesticides Sensor Spectrophotometry Field effect transistor Interferences

"Determination Of Lipase Activity In Porcine Pancreas And Clinical Analysis Of Lipase In Human Serum With Surface Acoustic Wave Enzyme Sensor System"
Fresenius J. Anal. Chem. 1996 Volume 354, Issue 1 Pages 118-121
Kai Ge, Dezhong Liu, Kang Chen, Lihua Nie, Shouzhuo Yao

Abstract: The 61 MHz surface acoustic wave generator was made from a Y,Z-cut lithium niobate piezoelectric crystal connected to a pair of parallel platinum electrodes mounted in the 6 mL detection cell. Changes in the conductivity of the solution caused by enzyme reactions produce a change in the frequency response of the sensor detected by a frequency counter and recorded. For the assay of lipase (I), a 6 mL portion of 41.3 mM triacetin in 5 mM Tris buffer of pH 8 was transferred to the cell and the temperature was maintained at 30°C by magnetic stirring. Portions of standard solutions of I were added and the change in frequency was recorded as a function of time. For calibration graphs the initial rate of change of frequency was plotted against the concentration of I. The calibration graph was linear up to 500 U/l of I and the detection limit was 0.3 U/l. This is more sensitive than colorimetric, turbidimetric or flow injection methods, but less sensitive than immunoassay. Results from the injection of serum samples, including those of pancreatitis patients, correlated well with those from direct conductivity measurements, but conductivity was less precise and more restricted by ionic strength. The method was also applied to study the effects of activators and inhibitors of I.
Enzyme, lipase Clinical analysis Sensor Sensor

"Activated Platinum Electrodes As Transducer For A Glucose Sensor Using Glucose Oxidase In A Photopolymer Membrane"
Fresenius J. Anal. Chem. 1997 Volume 357, Issue 5 Pages 534-538
D. Wilke, H. M&uuml;ller, N. Kolytsheva

Abstract: Lyophilized glucose oxidase and 5 mg dodecyltrimethylammonium bromide (surfactant) were mixed with 121 mg poly(methyl methacrylate)/bisphenol A-bis (2-hydroxypropyl methacrylate/Michler's ketone/benzophenone (70:30:1:20) dissolved in 1 mL CHCl3 (photopolymer system). Dry planar Pt electrode surfaces (preparation details given) were spin-coated with this cocktail. After solvent evaporation for ~2 min, the electrodes were exposed to UV light for 60 s then conditioned in phosphate buffer for 12 h. Three- and two- electrode techniques were used for cyclic voltammetric and amperometric flow injection measurements, respectively, with a SCE reference and a Pt wire auxiliary electrode. Amperometric measurements were made in an H-form wall-jet cell where one part was the SCE. Glucose measurements were accelerated using a FIA system with a flow rate of 1 ml/min. Calibration graphs were linear from 0.05-10 mM glucose. Electrodes were maintainable for >20 days after coating with the enzyme membrane. The sensor was applied to determination of glucose in diluted serum; calibration graphs were linear from 3-15 mM and results agreed with those obtained using standard photometry.
Glucose Amperometry Electrode Electrode Sensor Voltammetry Method comparison

"Monosegmented Flow-analysis Of Slow Enzymatic Reactions: Determination Of Triglycerides In Serum"
Fresenius J. Anal. Chem. 1998 Volume 360, Issue 1 Pages 100-103
A. N. Ara&uacute;jo A, Jos&eacute; A. M. Catita A, Jos&eacute; L. F. C. Lima A, Elias A. G. Zagatto

Abstract: A monosegmented flow system (MCFA) is proposed to achieve slow enzymatic spectrophotometric determinations, here applied to the determination of triglycerides in blood serum. The sample (4.5 µL), enzymatic reagent (150 µL) and an air plug (100 µL) are simultaneously inserted into a carrier stream buffered to pH 7.9 (Tris HCl). In order to avoid the cumbersome step of air removal, a relocating detector was used. This system handles about 60 samples/h, yielding precise results (RSD usually <2.5%). Sensitivity is 56 mAU L/mmol up to 6 mmol/L triglycerides. Accuracy was assessed by running 50 samples already analyzed by a conventional procedure yielding the equation CMCFA(mmol/L) = 1.00(±0.04) CRef(mmol/L) -0.03(±0.08); r=0.990.
Triglycerides Kinetic

"Determination Of Chromium(III) In Urine, Blood Serum, And Hair Using Flow Injection Chemiluminescence Analysis"
Fresenius J. Anal. Chem. 1998 Volume 361, Issue 5 Pages 509-511
R. Escobar, M. S. Garc&iacute;a-Dom&iacute;nguez, A. Guira&uacute;m, F. F. de la Rosa

Abstract: A flow injection (FI) chemiluminescence method for the determination of Cr(III) in blood serum, urine, and hair samples is reported. It is based on the chromium-catalyzed light emission from the luminol oxidation by H2O2. The app. consists of an FI system with a flow cell formed by a coiled transparent tube suitable for chemiluminescence detection. The specificity of the method is achieved in presence of EDTA. The detection limit under optimum conditions is 0.01 µg/L of Cr(III). Precision and accuracy were evaluated by determining Cr(III) concentrations in urine standards from the National Institute of Standard and Technol. (NIST).
Chromium(III) Chemiluminescence Indirect EDTA Optimization Reference material

"Synthesis Of 4-methyl-5-(3-sulfopropylamino)-2-(2-thiazolylazo)benzoic Acid And The Application To The Flow Injection Analysis Of Copper(II)"
Microchim. Acta 1983 Volume 81, Issue 3-4 Pages 235-244
H. Wada, T. Ishizuki and G. Nakagawa

Abstract: The cited reagent (I) and its 5-(2-sulfoethylamino)-analogue (II) were synthesized, and their acidity constants and the formation constants of their Cu(II) and Ni(II) complexes were determined. The effect of the size of the sulfoalkyl group on the properties of I and II as reagents was evaluated by comparison with the 5-(sulfomethylamino)-analogue (III); the sensitivity towards Cu decreased in the order I, II, III. Use of I in the spectrophotometric determination of 25 ppb to 25 ppm of Cu by a flow injection procedure is described. The e value of the complex was 58,000, and max. absorption was at 610 nm. The method was used to determine Cu in blood serum.
Copper Spectrophotometry

"Spectral And Non-spectral Interferences In Inductively Coupled Plasma Mass Spectrometry"
Microchim. Acta 1995 Volume 119, Issue 3-4 Pages 277-286
Richard F. J. Dams, Jan Goossens and Luc Moens

Abstract: An overview is presented on the identification and elimination of interferences in ICP-MS. Elimination of spectral interferences by anion exchange, matrix modification and mathematical correction are discussed. Calibration methods, adapted sample preparations and flow injection techniques for the elimination of nonspectral interferences are then considered. Applications of the correction methods to the analysis of water, soil, sewage, urine, serum and wine are described. (50 references).
Mass spectrometry Sample preparation Interferences Matrix removal Review

"Fiber Optic Fluorimetric Enzyme Sensors For Hydrogen-peroxide And Lactate, Based On Horseradish-peroxidase And Lactate Oxidase"
Microchim. Acta 1995 Volume 121, Issue 1-4 Pages 237-247
Florian Schubert, Fang Wang, and Herbert Rinneberg

Abstract: An optical biosensor for the determination of hydrogen peroxide based on immobilized horseradish peroxidase is described. The fluorescence of the dimeric product of the enzyme catalyzed oxidation of homovanillic acid is utilized to determine the concentration of H2O2. The membrane-bound enzyme is attached to a bifurcated fiber bundle permitting excitation and detection of the fluorescence by a fluorimeter. The response of the sensor is linear from 1 to 130 µM hydrogen peroxide; the coefficient of variation is 3 %. The sensor is stable for more than 10 weeks. The operating pH for maximal sensor response is 8.15. This allows the sensor to be used in combination with oxidase reactions producing hydrogen peroxide, as is demonstrated with a co-immobilized lactate oxidase-horseradish peroxidase optode for the determination of L-lactate. The fluorescence intensity of this sensor depends linearly on the concentration of lactate between 3 and 200 µM and a throughput of 10 samples per hour is possible. The precision is in the same range as that of the monoenzyme optode. The lifetime of the bienzyme sensor for lactate is considerably shorter than that of the peroxidase sensor; it is limited by the stability of the immobilized lactate oxidase enzyme. The sensor has been applied to the determination of lactate in control serum. (32 references)
Hydrogen peroxide Lactate Sensor Fluorescence Sensor Immobilized enzyme Optical fiber Reference material

"Metal-ion Incorporation Into N-methyl-5,10,15,20-tetrakis(4-sulfonatophenyl)porphine And Its Differential Rates As Applied To The Kinetic Determination Of Copper(II) And Zinc(II) In Serum"
Microchim. Acta 1986 Volume 88, Issue 1-2 Pages 33-47
S. Funahashi, Y. Ito, H. Kakito, M. Inamo, Y. Hamada and M. Tanaka

Abstract: Metal-ion complexes with the named reagent(I) are formed by mixing I with metal-ion solution and buffer solution The reaction was applied to the determination of Cu(II) and Zn in acidified (1 M HCl) serum. Thus, the acidified serum was heated on a water bath for 2 min before addition of aqueous 20% trichloroacetic acid. The mixture was centrifuged, the pH of the supernatant solution was adjusted to 7.0 for Cu or 8.0 for Zn, and I was added to the diluted solution. The rate of complex formation with Cu or Zn was measured spectrophotometrically at 439 or 430 nm, respectively. Results agreed well with those by AAS. A flow injection method for determination of Cu in serum is also given.
Copper Zinc Spectrophotometry Complexation Kinetic Method comparison

"New Continuous-flow Micro-method For Determination Of Creatine In Serum Or Plasma"
Microchem. J. 1980 Volume 25, Issue 4 Pages 500-506
E. D. Ryan and W. H. C. Walker

Abstract: We report here the development of an assay in which the major barrier to achieving increased sensitivity in a continuous flow system, i.e., the dialysis step, is eliminated. The specificity of the assay is enhanced by removal of the plasma proteins by prior trichloroacetic acid (TCA) precipitation and by controlling the pH during color development with a phosphate buffer system. Only 7.5 ,ul of plasma is required for this assay.
Creatinine Spectrophotometry Method comparison

"Continuous-flow Determination Of Reducing Sugars In Serum By Reaction With Periodate Ions, With Use Of A Flow-through Periodate-sensitive Electrode"
Microchem. J. 1983 Volume 28, Issue 2 Pages 227-234
Laboratory of Analytical Chemistry, University of Athens, Athens, Greece

Abstract: A continuous-flow method for the determination of reducing sugars in serum is described. The sample reacts with an excess of periodate in a flow system and the decrease in periodate activity is monitored with a periodate-sensitive flow-through electrode. The recorded potential peak heights are indirectly linearly related to the reducing sugar concentration expressed as glucose, in the range 50-290 mg/100 mL. The analysis is completely automated and requires no sample pretreatment, and samples can be analyzed at the rate of 30 per hour with average errors and relative standard deviation of about 1-2%. Comparison with an enzymatic method for serum gave satisfactory results.
Carbohydrates Sugars, reducing Electrode

"Study Of Continuous-flow Automation For Serum Iron On Comparing Several Sensitive Reagents"
Microchem. J. 1983 Volume 28, Issue 2 Pages 275-284
Joseph D. Artiss, Donald R. Strandbergh and Bennie Zak

Abstract: Three reagents, for the determination of serum iron concentrations, are compared for their use on an AutoAnalyzer. Under identical conditions, all three reagents ferrozine, 2,4-bis (5,6-diphenyl-1,2,4-triazin-3-yl)pyridinetetrasulfonate, and ferene S, behave in a similar manner. The interference of copper is studied as well as the possibility of using various masking reagents to reduce this interference. It is demonstrated that, contrary to a previously reported manual procedure, thioglycolic acid, thiourea, and neocuproine, the masking reagents, do not all result in exactly the same statistics. As well, it is shown that the time of addition of the masking reagent (i.e., pre- or postdialysis) is not particularly important. Comparison of a manual procedure, involving the preparation of a protein-free filtrate with one of the reagents on the AutoAnalyzer, shows very good correlation. Comparison of the various iron reagents with the AutoAnalyzer suggests that the prime advantage of one over the others is sensitivity.
Iron

"Continuous-flow Potentiometric Determination Of α-amylase Activity In Serum And Urine"
Microchem. J. 1985 Volume 32, Issue 2 Pages 183-190
E. P. Diamandis, A. Papanastasiou-Diamandi, T. K. Christopoulos and T. P. Hadjiioannou

Abstract: α-Amylase was caused to react with a buffered starch solution (pH 6.9) at 40°C for 30 min under controlled continuous-flow conditions, after which the reducing sugars produced were oxidized by IO4-. The consumption of IO4- was monitored continuously with a IO4--sensitive flow-through electrode (details given). The endogenous reducing substances of serum or urine were measured with the same system by incubation of the sample with starch solution (pH 4.7) in the presence of NaF as an α-amylase inhibitor. The difference in the reducing power (as glucose) was used to calculate the α-amylase activity of the samples. Values obtained correlated well with those from an amyloclastic method (r = 0.90 to 0.96).
α-Amylase Electrode Electrode Potentiometry Heated reaction

"Multicomponent Flow Injection Analysis Using Spectrophotometric Detection With Reagent Spectral Overlap. Application To Determination Of Calcium And Magnesium In Blood Serum Using Eriochrome Black T"
Microchem. J. 1986 Volume 34, Issue 1 Pages 67-75
Amin T. Haj-Hussein and Gary D. Christian

Abstract: Proteins were removed from the serum by precipitation with 0.2% HClO4 and the filtrate was buffered before injection via a 20 µL sample loop into the reagent carrier stream of 0.32 mM Eriochrome Black T (C. I. Mordant Black 11) in aqueous 20% ethanol buffered with NH3 - HClO4 to pH 10.1 or 10.5. After passage through a 43-cm mixing coil the absorbance of the complexes formed with Ca and Mg was measured at 520 nm (10-mm flow cell). Sequential measurements were made at pH 10.1 and 10.5 to obtain different relative absorbances of the two complexes. An iterative procedure (graphic or computational) is presented for blank absorbance correction. In simultaneous measurement of Ca and Mg, direct data was required for determination of one metal so that the other could be estimated; for 1 ppm of Mg and 2 ppm of Ca the coefficient of variation was ~2% for each metal.
Calcium Magnesium Clinical analysis Spectrophotometry Multicomponent

"A Microsampling System For Flame Atomic Absorption Spectrometry Using Hydrodynamic Injection"
Microchem. J. 1995 Volume 51, Issue 3 Pages 360-366
Xu S. K. and Fang Z. L.

Abstract: A flow injection microsampling system for AAS was developed. The system (manifold shown) was based on air-transported sample loading and hydrodynamic injection. A diagram of the injector is given. An augmenting water stream (6 ml/min) between the sample loop and nebulizer prevented interferences from nebulizer suction during sample loading. A Perkin-Elmer AS-90 autosampler was used to obtain aqueous Cu samples at 1.2 ml/min. After loading, samples in the sample loop were transported by a water carrier at 5.2 ml/min into the nebulizer. Cu was determined at 432.8 nm using an air-acetylene flame. A total of 0.8 mL sample was required for triplicate determinations (80 µL sampling loop). The calibration graph for Cu was linear for 0.5-2 mg/l and the RSD was 1.7%. The method was applied to serum diluted 1:1 using a sampling frequency of 100/h. The results for a bovine serum referenced material agreed well with the certified values and the RSD were 1.7-2.7%.
Copper Spectrophotometry Hydrodynamic injection Reference material Interferences

"Application Of Photochemical Inhibition To The Fluorimetric Determination Of Nitroprusside In A Flow Injection System"
Microchem. J. 1995 Volume 52, Issue 1 Pages 33-40
Perezruiz T., Martinezlozano C., Tomas V. and Val O.

Abstract: Sample was mixed with 60 µM-phloxin (tetrachlorotetrabromofluorescein) in 0.1 M phosphate buffer of pH 6 and portions (185 µL) were injected into a stream (1.5 ml/min) of 0.1 M EDTA adjusted to pH 6 with 0.2 M NaOH. The sample and reagent streams were deaerated with Ar. The sample plug passed to a reactor coil (200 cm x 0.5 mm i.d.) where it was stopped and irradiated for 30 s using a halogen lamp. Optical fibers guided the light from the lamp to a 100 cm length of the reactor coiled around a glass tube (1 cm diameter) and placed over a mirror. The fluorescence was measured at 543 nm (excitation at 530 nm). The calibration graph was linear for 6-70 µM-sodium nitroprusside and the detection limit was 0.29 µg/ml. The RSD (n = 10) at 5 µg/ml was 1.18%. Catecholamines interfered. The method was applied to a pharmaceutical preparation with recoveries of 99.3-101.8%; the results agreed well with those obtained by polarography. The method was also applied to thiosalicylic acid-treated human serum with recoveries of 98.8-101.7%.
Nitroprusside Fluorescence Photochemistry Method comparison Interferences Optical fiber

"A Chemiluminescence Flow Injection Analysis Of Serum 3-hydroxybutyrate Using A Bioreactor Consisting Of 3-hydroxybutyrate Dehydrogenase And NADH Oxidase"
Anal. Biochem. 1995 Volume 229, Issue 1 Pages 133-138
Tabata M. and Totani M.

Abstract: Human serum (1 µL) was injected into a stream (0.6 ml/min) of 10 mM potassium phosphate buffer of pH 8.5 containing 0.7 µM FAD and 0.1 mM NAD. The stream passed through a column (2 cm x 2 mm i.d.) packed with ~102 mg (wet wt.) of 3-hydroxybutyrate dehydrogenase/NADH oxidase (5:1) co-immobilized on to porous alkylamine glass beads (80-120 mesh) using glutaraldehyde (preparation described). The H2O2 produced was reacted with luminol/hexacyanoferrate by mixing with streams (0.6 ml/min) of 0.7 mM luminol and 20 mM potassium hexacyanoferrate(III) and the chemiluminescence was measured. The calibration graph was linear for 1 pmol (detection limit) to 0.2 mM 3-hydroxybutyrate and the within- and day-to-day RSD were 1.2-2% and 1.7-2.7%, respectively (n = 10). The recoveries were 98-102%. The results agreed well with those obtained by the spectrophotometric method of Williamson et al. Ascorbic and uric acids interfered. The column activity decreased by 20% over five weeks (1500 runs). We describe a simple method for the highly sensitive chemiluminescence--flow injection analysis of 3-hydroxybutyrate in serum using a bioreactor column consisting of the two immobilized enzymes, 3-hydroxybutyrate dehydrogenase and NADH oxidase. The method was based on measuring the level of chemiluminescence formed by the reaction of a luminol-hexacyanoferrate mixture with hydrogen peroxide. The hydrogen peroxide was produced by the NADH oxidase reaction from NADH which was formed in the conversion of 3-hydroxybutyrate to acetoacetate by the 3-hydroxybutyrate dehydrogenase reaction. Among three immobilized enzyme columns, a co-immobilized, small 3-hydroxybutyrate dehydrogenase/NADH oxidase bioreactor alone (2 x 20 mm i.d.) readily hydrolyzed all of the injected 3-hydroxybutyrate into acetoacetate, although 3-hydroxybutyrate dehydrogenase catalyzed the reversible reaction. The present method generated linearity of the data up to 1.5 mM 3-hydroxybutyrate with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from 3-hydroxybutyrate correlated satisfactorily with those obtained by other well-established methods. The co-immobilized 3-hydroxybutyrate dehydrogenase/NADH oxidase reactor unit showed good operational stability over a 5-week period, during which it was repeatedly used for 1500 analyzes.
3-hydroxybutyrate Chemiluminescence Immobilized enzyme Glass beads Interferences Method comparison

"The Peroxidase-catalyzed Oxidation Of NADH As A Indicator Reaction For Repetitive Determinations By Sample Injection In Closed Flow-through Systems: The Determination Of LDH In Blood Serum"
Anal. Biochem. 1979 Volume 97, Issue 2 Pages 255-263
D. P. Nikolelis, Chih-duen C. Painton and Horacio A. Mottola*

Abstract: The air oxidation of reduced nicotinamide adenine dinucleotide (NADH), catalyzed by peroxidase, provides a useful 'indicator reaction' for the determination of both NADH and serum lactate dehydrogenase (LDH). Application of this indicator reaction for repetitive determinations by sample injection into a continuously circulated reagent mixture, and by monitoring of oxygen depletion with an amperometric sensor, is described. At determination rates of 260 determinations/h, NADH and LDH have been determined in the range of 3.8 x 10^-4 to 7.6 x 10^-3 M and 70 to 700 U/mL, respectively, and with relative errors and standard deviations (population) of about 2%. LDH has been determined in human blood serum and the injection procedure compared with a modified Wroblewski-Ladue procedure; the Pearson correlation coefficient was found to be 0.999 (40 samples). Recovery studies are also reported for LDH in serum calibration references and controls.
Enzyme, lactate dehydrogenase Amperometry Clinical analysis Closed loop Enzyme

"Fluorimetric Determination Of Guanidino-compounds By New Post-column Derivatization System Using Reversed-phase Ion-pair High Performance Liquid Chromatography"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 392-398
Yoshie Kobayashi, Hiroaki Kubo and Toshio Kinoshita

Abstract: Nine guanidino-compounds and creatinine in serum were separated in 25 min by reversed-phase ion-pair HPLC with post-column derivatization on a column (15 cm x 4.6 mm) of Nucleosil C8 (5 µm). The mobile phase (1 mL min-1) was 1 mM Na 1,2-naphthoquinone-4-sulfonate (fluorogenic reagent) and 15 mM Na octanesulfonate in water - acetonitrile - methanol (92:3:5) adjusted to pH 4.0 or 3.5 with acetic acid; 1 M NaOH was delivered at 0.5 mL min-1 to a reaction coil (5 m x 0.5 mm) and the fluorescent products formed were detected at 505 nm (excitation at 355 nm). The detection limit was <20 ng and recovery from serum was 93 to 108%. The method was accurate and reproducible and could be used for routine determinations in haemodialysis patients. Guanidino-drugs could also be determined by using this system.
Drugs Guanidino compounds HPLC Fluorescence Heated reaction Post-column derivatization Dialysis

"Selective Determination Of Lithium In Biological Fluids Using Flow Injection Analysis"
Anal. Biochem. 1987 Volume 160, Issue 2 Pages 243-250
G. E. Pacey, Y. P. Wu and K. Sasaki

Abstract: The batch extraction of the cryptand 211 - Li - resazurin complex into 1,2-dichloroethane was carried out at 24°C for 10 min and the absorbance of the organic phase was measured at 630 nm. The chromogenic aza-12-crown-4 batch extraction of Li into CHCl3 was carried out at 24°C for 10 min and the absorbance of the organic phase was measured at 400 nm. Both systems were successfully automated by using a PTFE flow injection system (diagram presented). For the cryptand 211 - resazurin system with 1000 ppm of Na present the calibration graph was rectilinear for 0.7 to 2.1 ppm of Li for batch extraction (0.28 to 1.12 ppm for flow injection) and for the chromogenic aza-12-crown-4 system it was rectilinear for 0.3 to 2 ppm of Li (both methods). The methods were used in the determination of Li in serum and urine, with flow injection giving the better precision and accuracy. Good agreement was observed with known values and AAS results.
Lithium Spectrophotometry Method comparison Theory

"Continuous-flow Automated Assay For Iodimetric Estimation Of Hydroperoxides"
Anal. Biochem. 1989 Volume 176, Issue 2 Pages 353-359
Sian M. Thomas*, Wendy Jessup, Janusz M. Gebicki

Abstract: Lipid hydroperoxides in aqueous samples or heptane extracts of biological materials were determined by an automated continuous-flow iodimetric method. The reagent was 1% KI in methanol - acetic acid (2:1) (made 1 mM with respect to desferal, used to inactivate contaminating Fe) and the wash solution was 2% acetic acid. The formation of tri-iodide ions was measured at 360 nm. All operations were protected from light. The detection limit was 60 pmol of hydroperoxide; coefficient of variation were 3, 3.6 and 2.5% (n = 10) for serum, aqueous lipid and heptane extracts of biological material, respectively. Results correlated well with those by the manual iodimetric assay (r = 0.999).
Hydroperoxides, lipid Spectrophotometry Detection limit Method comparison

"Flow Injection Analysis Of Serum Urea Using Urease Covalently Immobilized On 2-fluoro-1-methylpyridinium Salt-activated Fractogel And Fluorescence Detection"
Anal. Biochem. 1990 Volume 188, Issue 2 Pages 325-329
D. Narinesingh, R. Mungal and T. T. Ngo

Abstract: Serum samples were analyzed for their urea content using fluorescence flow injection analysis incorporating an immobilized urease bioreactor and a gas permeable separator. The urease was immobilized under mild and facile conditions to a hydrophilic 2-fluoro-1-methylpyridinium-activated support. The ammonia released as a result of urease-catalyzed urea hydrolysis diffused through a gas permeable membrane into a constant stream of o-phthaldehyde solution to form a highly fluorescent product with lambda ex at 340 nm and lambda em at 455 nm. Up to 25 serum samples can be analyzed per hour. The within-day coefficient of variation (CV) was 1.12% and the day-to-day CV was 1.25% for serum containing 10.50 mg urea nitrogen dl-1. The bioreactor shows excellent storage (at 4°C) and operational stabilities (at 37°C). Urea was determined in serum by flow injection analysis (illustrated) with urease immobilized on 2-fluori-1-methylpyridinium toluene-4-sulfonate-activated Fractogel. The ammonia released diffuses through a gas-permeable membrane into a stream of phthalaldehyde. The fluorescence of the resulting product is measured at 455 nm (excitation at 340 nm). Calibration graphs were rectilinear up to 1400 mg dl-1 and the detection limit was 0.1 mg dl-1. Recovery was 98 to 103% and coefficient of variation were 1.5%.
Urea Fluorescence Enzyme Immobilized enzyme Gas diffusion Membrane Calibration Detection limit

"Flow Injection Analysis With Electrochemical Detection Of Reduced Nicotinamide Adenine Dinucleotide Using 2,6-dichloroindophenol As A Redox Coupling Agent"
Anal. Biochem. 1991 Volume 192, Issue 1 Pages 243-250
Hua T. Tang, Kiamars Hajizadeh, H. Brian Halsall* and William R. Heineman*

Abstract: The determination of reduced nicotinamide adenine dinucleotide (NADH) by electrochemical oxidation requires a more positive potential than is predicted by the formal reduction potential for the NAD+/NADH couple. This problem is alleviated by use of 2,6-dichloroindophenol (DCIP) as a redox coupling agent for NADH. The electrochemical characteristics of DCIP at the glassy carbon electrode are examined by cyclic voltammetry and hydrodynamic voltammetry. NADH is determined by reaction with DCIP to form NAD+ and DCIPH2. DCIPH2 is then quantitated by flow injection analysis with electrochemical detection by oxidation at a detector potential of +0.25 V at pH 7. NADH is determined over a linear range of 0.5 to 200 µM and with a detection limit of 0.38 µM. The lower detection potential for DCIPH2 compared to NADH helps to minimize interference from oxidizable components in serum samples.
Nicotinamide adenine dinucleotide oxidized Electrochemical analysis Electrode Voltammetry Electrode Voltammetry Interferences Redox

"Determinations Of Lactate And Lactate Dehydrogenase Activity In Serum With The Flow Injection Analysis System Involving Immobilized Enzyme Column And Chemiluminescence"
Anal. Biochem. 1991 Volume 193, Issue 1 Pages 112-117
Masayoshi Tabata, Masayuki Totani* and Takashi Murachi

Abstract: The methods for the highly sensitive flow injection analysis of lactate and lactate dehydrogenase (LDH) activity in serum using immobilized enzymes in column form and chemiluminescence detection which does not require a blank correction are described. The methods were based on the determination of chemiluminescence formed by the reaction of a luminol-ferricyanide mixture with hydrogen peroxide. This hydrogen peroxide was produced by the lactate oxidase (LOD) reaction from lactate, which was in serum or was produced by the action of LDH in serum. The action of LDH in a flow injection analysis system was performed for 2 min in an incubation coil placed parallel to the substrate-buffer line between the LOD column and the LOD/catalase column. Endogenous lactate in serum was removed by an immobilized LOD/catalase column prior to the action of LDH. The present method gave perfect linearity of the data up to 5.6 mmol/liter for lactate and 1840 IU/liter for LDH activity with satisfactory precision, reproducibility, and accurate reaction recoveries. The results from the lactate and LDH activity correlated satisfactorily with those obtained by other well-established methods. Serum was applied to a column of lactate oxidase - catalase immobilized on alkylamine glass beads. The eluate was incubated at 37°C for 2 min with phosphate buffer solution (pH 7.5) containing pyruvate and NADH. The mixture was applied to a lactate oxidase column and the eluate was subjected to chemiluminometric determination (J. Appl. Biochem., 1984, 6, 251). Lactate in serum was determined as above but the sample was applied directly on to the lactate oxidase column. The calibration graph was rectilinear for 5.6 mM lactate and the detection limit was 10 µM. For lactate dehydrogenase, the calibration graph was rectilinear for 1840 IU L-1 and the within- and day-to-day coefficient of variation were 3.0 and 3.9%, respectively. The results correlated well (r = 0.974) with those obtained on a Hitachi Model 726 discrete automatic analyzer. with use of pyruvate as substrate.
Lactate Enzyme, lactate dehydrogenase Chemiluminescence Buffer Column Glass beads Heated reaction Immobilized enzyme

"Determination Of Serum Cholinesterase Activity By Liquid Chromatography With Electrochemical Detection"
Anal. Biochem. 1991 Volume 196, Issue 2 Pages 377-384
R. Brent Miller and C. LeRoy Blank

Abstract: Serum was incubated at room temperature for 10 min in phosphate buffer solution (pH 7.2) containing ethylhomocholine, choline and acetylcholine. After addition of HClO4 and centrifugation, the supernatant solution was subjected to HPLC on a C18 (3 µm ODS) column (10 cm x 3.2 mm). The mobile phase (0.9 mL min-1) was Tris buffer solution (pH 7.5) containing acetic acid, tetramethylammonium chloride, Na octyl sulfate, NaN3, EDTA and acetonitrile. Post-column derivatization was effected with choline oxidase and acetylcholinesterase with electrochemical detection at 0.5 V vs. Ag - AgCl. The limit of detection was 3 pmol of choline or 0.1 µmol mL-1 h-1 of cholinesterase. The calibration graph was rectilinear for up to 269 µmol mL-1 h-1, with a coefficient of variation of 1.7%.
Enzyme, cholinesterase HPLC Electrochemical analysis Buffer C18 Column Heated reaction Post-column derivatization

"Semi-automated Flow Injection Procedure For Acetylcholinesterase And Cholinesterase Activities"
Anal. Biochem. 1992 Volume 200, Issue 1 Pages 176-179
Ignacio L&oacute;pez Garc&iacute;a, Fernando Ruiz Abell&aacute;n and Manuel Hern&aacute;ndez C&oacute;rdoba

Abstract: A stopped-flow flow injection method is described (details and diagram of apparatus given) for the assay of cholinesterase (I) and acetylcholinesterase (II) based on the Ellman colorimetric method. For determination of I, serum (100 µL) was diluted to 10 mL with phosphate buffer solution For determination of II, 25 µL of blood was mixed with 1 mL of isotonic saline. The erythrocytes, pelleted by centrifugation, were haemolysed in 1 mL of water and the haemolysate was analyzed. Sample solution (50 µL) was injected into a carrier stream (0.1 mL min-1) comprising 2 mM acetylthiocholine and 0.2 mM 5,5'-dithiobis-(2-nitrobenzoic acid) in 0.1 M phosphate buffer solution (pH 7.2) and the resulting stream was passed through a 46 cm reaction coil at 37°C to a flow cell where the flow was stopped to allow detection at 410 nm. The calibration graphs for I and II were rectilinear and the FIA was significantly more sensitive than the manual procedure. A stopped-flow FIA adaptation of the classical Ellman's colorimetric procedure for the measurement of acetylcholinesterase or cholinesterase activity is described. The samples are injected into a flow analytical system which is provided with an electronic timer and an electrovalve in order to stop the flow when the sample is inside the measurement cell. In this way, the absorbance-time relationship is obtained with a savings of sample, time, and reagents.
Acetylcholinesterase Enzyme, cholinesterase Spectrophotometry Heated reaction Stopped-flow Buffer Valve Method comparison

"Simultaneous Determination Of Nitrate And Nitrite In Biological Samples By Multichannel Flow Injection Analysis"
Anal. Biochem. 1995 Volume 231, Issue 2 Pages 383-386
Phillip F. Pratt, Kasem Nithipatikom and William B. Campbell

Abstract: In a multichannel flow injection analyzer. (a modified Automated QuikChem Ion Analyzer, Lachat Instruments Inc., Milwaukee, WI, USA; modifications described), the sample was divided into two channels, one representing total nitrite (obtained by Cd reduction of nitrate to nitrite) and the second representing only nitrite. The absorbance of the product of the reaction of nitrite with Greiss reagent was measured at 540 nm. The detection limit was 25 nM-nitrite or -nitrate. The calibration graph was linear from 25 nM to 20 µM nitrite. Recovery from bovine coronary endothelial cells was 95% for both ions. The method was applied to the determination of the ions in serum and bovine coronary artery endothelial cells and rat cerebellar granule cells. An automated method for the simultaneous determination of nitrite and nitrate in biological samples by using a multichannel flow injection analyzer has been developed. The method was based on the reaction of nitrite with Greiss reagent. The sample solution was injected and equally divided into two channels; channel one (1) represented total nitrite obtained by cadmium reduction of nitrate to nitrite while channel two (2) represented only nitrite. The absorbance of the color product was measured by photometric detectors with 540-nm filters. This method combines high reproducibility of sample introduction via flow injection and sensitivity of spectrophotometric detection. The detection limit is 25 nM for both nitrite and nitrate. The chemistry manifolds are constructed of Teflon tubing which, along with a low- pressure Flowfit connector system, provides for low maintenance, ease of use, and high sample throughput. We demonstrated that the system can be used for the determination of both nitrate and nitrite in a variety of biological samples as well as a comparison of the results from this system and the HPLC system.
Nitrate Nitrite Spectrophotometry Method comparison Lachat Multichannel

"A Stopped-flow, Reaction-rate Method For Total Serum Protein"
Anal. Lett. 1980 Volume 13, Issue 13 Pages 1115-1128
Wai-Tak Law; S. R. Crouch

Abstract: A reaction-rate method for total serum protein is described. The method uses a stopped-flow analyzer to obtain information on the rate of formation of the biuret reaction product. Measurement times can be as low as 100 ms after initiation of the reaction in the stopped-flow system. However, reaction times of 10 s are shown to give higher precision with relative standard deviations in the range of 0.1 to 1.4%. The results for total serum protein determinations are also accurate as shown by comparison with values obtained by equilibrium-based biuret procedures.
Protein Clinical analysis Spectrophotometry Rate constants Stopped-flow Method comparison

"A Simple Electrochemical Method For Quantitative Determination Of Acetaminophen In Serum"
Anal. Lett. 1981 Volume 14, Issue 13 Pages 1003-1012
Andrew Falkowski; Robert Wei

Abstract: A simple electrochemical method for the determination of acetaminophen in serum is described. The eleotrode and associated electronics are simple, reliable, and inexpensive to build. The apparatus can be operated at a rate of 2-3 determinations per minute using only 10 µl serum per determination. The procedure includes extraction of acetaminophen in ethyl acetate and subsequent oxidative amperometric detection of the drug by a form of flow-injection analysis. The system parameters of buffer, pH, and redox potential have been optimized to permit measurement of less than 10 µg/ml of acetaminophen. The determination is linear over the range of 10^-300 µg/ml with a C.V. of less than 3% for replicate analysis of the same sample.
Acetaminophen Acetaminophen Electrochemical analysis Small sample

"Direct Determination Of Glucose In Blood Serum Using Trinder's Reaction"
Anal. Lett. 1984 Volume 17, Issue 5 Pages 385-396
M. J. Medina; J. Bartroli; J. Alonso; M. Blanco; J. Fuentes

Abstract: A flow injection analysis system (illustrated schematically) is described; the method is based on Trinder's reaction, which is a two-stage process with oxidation of β-D-glucose by glucose oxidase to produce H2O2 followed by peroxidase-catalyzed oxidative coupling to form a colored quinonimine dye. Serum (15 µL) is injected directly into water as carrier (0.5 mL min-1) without deproteinization or dialysis and dispersed in a 1-m coil before mixing with a stream of enzyme solution (12 iu mL-1 of glucose oxidase, 1.2 iu mL-1 of peroxidase, 1.5 mM 4-aminoantipyrine and 100 mM phosphate buffer of pH 7.0) and one of 9.3 mM phenol (each at 0.5 mL min-1). Reaction occurs in a single bead string reactor (length 1 m) at 37°C, and the solution then passes through a 1-m coil at 37°C before detection at 505 nm. Calibration graphs are rectilinear in the range 50 to 400 mg dl-1 and sampling frequency is 60 h-1.
Glucose Spectrophotometry Heated reaction Immobilized enzyme Single bead string reactor Dialysis

"Determination Of Lactate Dehydrogenase Isoenzyme (LD-1) Using Flow Injection Analysis With Electrochemical Detection After Immunochemical Separation"
Anal. Lett. 1985 Volume 18, Issue 3 Pages 345-355
Takashi Toyoda; Shia S. Kuan; George G. Guilbault

Abstract: The lactate dehydrogenase isoenzyme was separated from serum with use of an Isomune-LD kit (Roche Diagnostics), which incorporates a double antibody system. The supernatant solution containing the enzyme was incubated for 5 min at 37°C with a reagent solution comprising Li lactate, dihydrolipoamide reductase (NAD+) and K3Fe(CN)6 in phosphate - KCl buffer solution (pH 9). To the solution was added NAD+ (1 mg mL-1) to initiate the reaction, and the products were sampled after 2, 5 and 8 min. Amperometric detection was effected with a thin-layer transducer connected to a polarographic analyzer.; calibration graphs were rectilinear for up to 202 iu L-1 of LD-1 and 385 iu L-1 of total enzyme.
Enzyme, lactate dehydrogenase Amperometry

"An Electrochemical Determination Of Uric Acid In Sera By A Flow-through Equipment With And Without Uricase"
Anal. Lett. 1985 Volume 18, Issue 15 Pages 1799-1820
Jachen; G. Grunig; K. Bertermann

Abstract: The enzyme electrode was prepared by sandwiching a urate oxidase - gelatin layer (2 to 3 iu cm-2 and 25 to 30 m thick) between two cellulose membranes (Nephrophan, 20 m, VEB Filmfabrik Wolfen, E. Germany) and stretching this film over a platinum electrode. The enzyme-free electrode comprised a Nephrophan film stretched over the platinum electrode. Diluted serum samples were analyzed by using an apparatus comprising an automatic sampler, a peristaltic pump, either of the electrodes, an amperometric measuring device and a recorder. Both electrodes gave rectilinear responses up to ~6 mM uric acid (0.282 mM in the diluted samples) but results with the enzyme electrode showed greater accuracy and precision, both within runs and between days.
Uric acid Electrochemical analysis Enzyme

"Bioluminescent Flow Sensors: L-alanine Determination In Serum And Urine"
Anal. Lett. 1987 Volume 20, Issue 8 Pages 1315-1330
Girotti, S.;Roda, A.;Piazzi, S.;Carrea, G.;Piacentini, A.L.;Angellotti, M.A.;Bovara, R.;Ghini, S.

Abstract: L-Alanine is determined by a bioluminescence continuous-flow technique based on its reaction with NAD+ catalyzed by L-alanine dehydrogenase(I). Urine samples are deproteinized and centrifuged and the supernatant solution are filtered. The filtrates are diluted with sodium pyrophosphate buffer (pH 9) before passage of portions, plus the buffer and 2 mM NAD+, through a nylon coil containing immobilized I. The bioluminescent solution [10 µM-flavine mononucleotide in 0.1 M potassium phosphate buffer (pH 6.9), 27 µM-decanal and 0.5 mM dithiothreitol] is added to the emergent solution, which then passes through a coil on which alkanal monooxygenase (FMN-linked) and NAD(P)H dehydrogenase (FMN) are immobilized. The bioluminescence is measured at 340 nm. The detection limit was 0.5 µM-L-alanine and response was rectlinear from 5 to 1500 pmol. Recovery was 94 to 108%. Serum samples are analyzed similarly but require only membrane filtration as pretreatment.
l-Alanine Bioluminescence Sensor Immobilized enzyme Nylon

"An Improved Zone Sampling Method For Flow Injection Analysis"
Anal. Lett. 1988 Volume 21, Issue 9 Pages 1633-1651
Toei, J.

Abstract: An improved zone sampling method where a small volume mixing device is placed before the fraction loop has been developed. In this method the samples are dispersed by the gradient chamber (device) and its dispersion profile of the injected samples is exponential. Therefore, the fraction difference caused by a lug of the fraction timing is small and consequently the optimization of system is very easy. The method was applied to the predilution of the samples in clinical flow injection analysis.
Glucose Spectrophotometry Dilution Enzyme Optimization Zone sampling

"The Determination Of Total Serum Cholesterol By Flow Injection Analysis With Amperometric Detection"
Anal. Lett. 1990 Volume 23, Issue 3 Pages 425-435
Alexis Carpenter; William C. Purdy

Abstract: As a cleanup step, serum (0.25 ml) was passed through a column of Amberlite IRA-400 (Cl- form), the column was washed with 5 mL of 0.1 M phosphate buffer (pH 7.4), and the eluate was diluted to 25 mL with the carrier solution (phosphate buffer containing 0.1% of Triton X-100). This solution was analyzed in a flow injection system involving immobilized cholesterol (I) esterase and I oxidase, and detection of the H2O2 produced with a Pt electrode at +600 mV vs. Ag - AgCl. The thin-layer flow cell used is illustrated. The limit of detection was ~2.6 µM, and the calibration graph was rectilinear up to 31 mM. In analysis of three standard reference materials, the bias was -1.4 to -2.4%, and the coefficient of variation were 1.5% (n = 16).
Cholesterol Amperometry Electrode Buffer Immobilized enzyme Amberlite Reference material Triton X Surfactant

"Photometric And Fluorimetric Determination Of Creatine Kinase Activity By Using Co-immobilized Auxiliary Enzymes And An Open - Closed Flow Injection Manifold"
Anal. Lett. 1991 Volume 24, Issue 5 Pages 749-765
M. D. Luque de Castro; J. M. Fern&aacute;ndez-Romero

Abstract: The method involves reaction between creatine phosphate (I) and ADP catalyzed by creatine kinase (II), phosphorylation of D-glucose by the produced ATP catalyzed by hexokinase (III), and oxidation of the resulting D-glucose 6-phosphate by NADP+ catalyzed by glucose-6-phosphate dehydrogenase (IV); the NADPH formed is monitored photometrically at 340 nm or fluorimetrically at 470 nm (excitation at 340 nm). The serum sample is diluted with 0.1 M Tris - acetate buffer of pH 7.0 before injection into a stream of the same buffer, which is then merged with a stream containing ADP and I and passed through a reaction coil. Subsequently, the sample plug is passed repeatedly through an enzyme reactor containing III and IV co-immobilized on controlled-pore glass, the flow-through detector and two reaction coils; the series of peaks of increasing height thus obtained is used to provide fixed-time or reaction-rate measurements of II activity. Optimum values of the reaction variables are tabulated. Typical rectilinear ranges of calibration were 0.01 to 1.00 or 2.00 iu L-1 of II, and recoveries of added II (0.1 or 0.2 iu l-1) were quantitative.
Enzyme, creatine kinase Fluorescence Buffer Catalysis Controlled pore glass Immobilized enzyme pH

"Bioluminescent Flow Sensor For L-glutamate"
Anal. Lett. 1992 Volume 25, Issue 4 Pages 637-652
Girotti, S.;Ghini, S.;Budini, R.;Pistillo, A.;Carrea, G.;Bovara, R.;Piazzi, S.;Merighi, R.;Roda, A.

Abstract: The method for the analysis of serum or meat, fish or vegetable extract is based on the reaction of the analyte with NAD+ and glutamate dehydrogenase (NAD(P)+). The NADH produced was monitored by the luminescence generated by its reaction with NAD(P)H dehydrogenase (FMN) and luciferase (both from Photobacterium fischeri). The reductant enzyme and the luminescent enzymes were immobilized on separate nylon coils in a continuous-flow system similar to that described previously [Analyst (London), 1990, 115, 889]. The calibration graph was rectilinear from 50 to 1000 µM with coefficient of variation (n = 7) of 7%. The recoveries were between 88 and 105%. The results agreed well with those obtained by a spectrophotometric method for stock cubes and by a chromatographic method for serum.
l-Glutamate Bioluminescence Sensor Immobilized enzyme Nylon Method comparison

"Flow Injection Fluoroimmunoassay For Human Transferrin Using A Protein A Immunoreactor"
Anal. Lett. 1994 Volume 27, Issue 6 Pages 1067-1074
Ren, X.Z.;Miller, J.N.

Abstract: Transferrin (I) was determined in serum by flow injection fluoroimmunoassay using Lucifer Yellow VS as the label, with detection at 525 nm (excitation at 433 nm). A controlled-pore glass micro-column (5 cm x 3 mm i.d.) of PROSEP-A with two injection valves was used. Goat anti-human transferrin (50 µL) was injected into binding buffer (PBS, pH 7.4) through one valve and sample (50 µL) through the other. When washing of tracer was complete, the flow was switched to elution buffer (0.1 M citrate, pH 3) for 6 min, then switched back to binding buffer for 4 min. Flow rates were 0.25 ml/min throughout. The calibration graph was linear for up to 500 µg/ml of I. The RSD (n = 8) for 102 and 208 µg/ml of I were 10.6 and 9.4%, respectively.
Transferrin Immunoassay Fluorescence Controlled pore glass

"Turbidimetric Determination Of Potassium By Flow Injection Analysis"
Anal. Lett. 1994 Volume 27, Issue 8 Pages 1625-1636
De Oliveira Torres, J.R.;Tubino, M.

Abstract: The FIA system comprised two aqueous 2% glycerol carrier solution (1.2 ml/min) into which 400 µL of a 3% sodium tetraphenylboron solution (prep. described) and 400 µL of sample solution containing 2% glycerol were separately introduced. The carrier streams were mixed in a reaction bobbin (1.5 m x 1 mm i.d.) of polyethylene tubing and the absorbance was read at 420 nm. To enhance sensitivity to 1 µg/ml, a stream of aqueous KCl solution (1.5-3 µg/ml; 0.36 ml/min) was introduced into the aqueous glycerol carrier solution The calibration graphs were linear for up to 20 µg/ml of K, the detection limit was ~1 µg/ml and the RSD were ~1%. The sampling rate was 60/h. The method was applied to plant leaves (chicory, cabbage, kale), mineral water and serum rehydration solution; the results agreed well with those obtained by flame photometry (plants) or with the expected values.
Potassium Turbidimetry

"Simultaneous Stopped-flow Determination Of Tetracycline And Doxycycline In Serum Based On Lanthanide-sensitized Luminescence"
Anal. Lett. 1994 Volume 27, Issue 12 Pages 2303-2316
Izquierdo, P.;Gomez Hens, A.;Perez Bendito, D.

Abstract: The intramolecular energy transfer from tetracycline (I) and doxycycline (II) to Eu ions was used for the rapid, simultaneous resolution of mixtures of these antibiotics in serum. The method is based on the formation of the corresponding ternary chelates in the presence of thenoyltrifluoroacetone (III). A syringe drive of the stopped-flow module was filled with a solution containing 54 µM-III, 0.2 mM Eu(III) and 10 mM Tris buffer. The other syringe was filled with a solution containing I and II of 0.1-10 and 0.15-5 µg/ml, respectively (or serum), 0.01% Triton X-100 and 10 mM Tris buffer. In each run, each solution was mixed in the mixing chamber and the variation of luminescence intensity with time monitored at 612 nm (excitation at 342 nm) over 30 s. Optimization of variables is discussed. Calibration graphs were linear for 0.1-10 and 0.15-5 µg/ml for I and II, respectively. Mean recoveries for I and II from serum were 98.7 and 96.4%, respectively, the corresponding within-assay RSD (n = 10) were 1.8 and 2.4% and the between-assay RSD (n = 5) 2.2 and 2.5%.
Tetracycline Doxycycline Fluorescence Stopped-flow Triton X Optimization Surfactant

"Chemiluminescent Determination Of Cholesterol By Flow Injection Analysis With Immobilized Cholesterol Oxidase"
Anal. Lett. 1996 Volume 29, Issue 13 Pages 2281-2288
A. Nabi; A. Rashid; M. Yaqoob

Abstract: The method was based on the chemiluminescence produced from the oxidation of luminol in the presence of Co(II) as catalyst. A schematic diagram of the FIA manifold is given. Sample (30 µL) was injected into 0.1 M phosphate buffer of pH 7 (0.6 ml/min) and passed through an immobilized cholesterol oxidase column (2 cm x 2.5 mm i.d.). The resulting H2O2 was merged 1.8 cm downstream in a glass coil (8 cm x 1 mm i.d.) with 0.1 M carbonate buffer of pH 10.5 containing 0.01 mM luminol and Co(II). The chemiluminescence produced was detected by a photom The detection limit was 0.1 mg/dl of cholesterol and the RSD over the range 10^-60 mg/dl was The method was applied to the determination of I in serum. The results compared well with those obtained by spectrophotometry.
Cholesterol, total Chemiluminescence Immobilized enzyme Method comparison

"An Enzymatic Method For The Determination Of ATP And Glycerol With An Automated FIA System"
Anal. Lett. 1997 Volume 30, Issue 3 Pages 537-552
E. R. Kiranas; M. I. Karayannis; S. M. Tzouwara-Karayanni

Abstract: The flow manifold was equipped with two enzyme reactors coupled in series containing glycerol kinase (GK) and glycerol-3-phosphate oxidase (GPO) immobilized on to non-porous glass beads. The glycerol assays were performed by injecting 85 µL sample solution into a carrier stream of Tris/HCl buffer of pH 8.2 at a flow rate of 0.45 ml/min. The carrier stream was merged with a reagent stream, at a flow rate of 0.06 ml/min, containing ATP/MgCl2 (1:17). The mixture was passed through the GK reactor where glycerol was phosphorylated to produce α-glycerophosphate (α-GP). The flow was then passed through the GPO reactor where α-GP was oxidized to dihydroxyacetone phosphate producing H2O2. Detection was at 510 nm by merging the flow with a colorimetric reagent at a flow rate of 0.06 ml/min containing horseradish peroxidase/4-aminoantipyrine/3.5-dichloro-2-hydroxymethyl sulfonic acid. To determine ATP the first reagent was changed to glycerol/MgCl2. Calibration graphs were linear from 4-70 and 2-160 µM ATP and glycerol, respectively. The method was applied to the determination of glycerol in wines following a 1:5100 dilution and in human serum following deproteinization by gel filtration.
Adenosine-5'-triphosphate Glycerol Spectrophotometry Immobilized enzyme Glass beads

"Flow Injection Determination Of Glucose By A Photoinduced Chemiluminescent Reaction"
Anal. Lett. 1998 Volume 31, Issue 11 Pages 1837-1855
Tomas Parez-R&uacute;z; Carmen Martinez-Lozano; Virginia Tomas; Antonio Sanz

Abstract: A flow injection procedure for the photochem. determination of glucose was developed. The method is based on the photooxidn. of glucose sensitized by 9,10-anthraquinone-2,6-disulfonate (disodium salt). The hydrogen peroxide formed in the photochem. reaction was measured by the chemiluminescent reaction with luminol and hematin. A linear calibration graph was obtained over the range 2.0 x 10^-6-8.5 x 10^-5 mol L-1. The method was applied to determining glucose in blood serum, urine and fruit juices.
Glucose Chemiluminescence Photochemistry

"Evaluation Of Electrodes Coated With Metal Hexacyanoferrate As Amperometric Sensors For Nonelectroactive Cations In Flow Systems"
Electroanalysis 1990 Volume 2, Issue 4 Pages 263-272
Karsten N. Thomsen, Richard P. Baldwin*

Abstract: Two electrodes modified with either nickel or cupric hexacyanoferrate films were evaluated and compared as sensors for nonelectroactive cations in a flow injection system. Both gave responses for group 1A and ammonium ions, but only the electrode modified with cupric hexacyanoferrate was sufficiently stable for use in flowing solutions. This electrode responded to K+, NH-4+, Rb+, and Cs+ ions rather selectively. Within this group, the selectivity could be controlled from general to almost specific toward Cs+ by the potential at which the electrode was poised. The electrode was compatible with a mobile phase of dilute nitric acid commonly used in ion chromatography, and chromatographic detection limits of 2 times 10^-7 M and linear responses over two decades were obtained. The electrode was applied to the ion chromatographic analysis of K+ and NH-4+ in urine and K+ in blood serum samples.
Potassium Ammonium Rubidium Cesium Amperometry Sensor HPIC

"Elimination Of Interferences In Flow Injection Amperometric Determination Of Glucose In Blood Serum Using Immobilized Glucose Oxidase"
Electroanalysis 1990 Volume 2, Issue 8 Pages 607-616
Wojciech Matuszewski, Marek Trojanowicz*, Andrzej Lewenstam

Abstract: A flow-injection system with anodic amperometric detection for glucose determination in human blood serum samples was optimized by a comparison of various arrangements of enzyme immobilization and the elimination of interferences. The best results were obtained using a compact membrane biosensor in a wall-jet, flow-through detector with a platinum disk electrode covered with an evaporated Nafion layer, a polyester membrane with immobilized glucose oxidase, and another protective polyester membrane. In the optimized conditions, a linear response was observed up to 25 mM glucose with a detection limit of 200 M glucose and a sampling rate of 120 samples/h for a 20 µL sample volume. Results of glucose determination in human serurn were compared with a Beckman Glucose Analyzer 2 and a Kone Dynamic analyzer.
Glucose Amperometry Sensor Interferences Immobilized enzyme

"Potentiometric Anion Selectivities Of Polymer Membranes Doped With Indium(III) Porphyrins"
Electroanalysis 1991 Volume 3, Issue 9 Pages 909-916
S. B. Park, W. Matuszewski, M. E. Meyerhoff, Y. H. Liu, K. M. Kadish

Abstract: The cited membranes were prepared to contain 33% PVC, 66% o-nitrophenyl octyl ether and 1% of, e.g., chloro(octaethylporphyrinato)indium(III). Discs of the membrane were cut and placed in ion-selective electrode bodies, with an internal filling of 0.1 M KCl. Potentials were measured vs. Ag - AgCl. The response to 10 µm to 500 mM solution of 7 anions was measured and selectivity coefficient were calculated. The electrode showed enhanced and partly selective response to Cl-; the calibration graph was rectilinear for 1 to 500 mM Cl-. The electrode was applied in the flow injection determination of Cl- in serum (details given). The results obtained compared well with those obtained by the Ektachem analyzer..
Chlorine Electrode Potentiometry

"Simultaneous Assays Of Glucose, Urate And Cholesterol In Blood Serum By Amperometric Flow Injection Analysis"
Electroanalysis 1995 Volume 7, Issue 2 Pages 143-146
T. Yao, M. Satomura, T. Nakahara

Abstract: The flow system featured a 16-way switching valve and a parallel configuration of three immobilized enzyme reactors. The enzymes; namely glucose oxidase, uricase and cholesterol oxidase were immobilized on controlled-pore glass packed into glass columns (1.8 cm x 3 mm i.d). Nafion film-coated and Nafion/cellulose acetate-coated Pt electrodes were used to selectively detect the H2O2 generated without any interference from electroactive species like L-ascorbate and proteins present in serum. The FIA system was similar to one described earlier (J. Biotechnol., 190, 14, 115). Serum was injected into two carrier streams of 0.1 M phosphate buffer of pH 7.5 and a stream of 0.1 M phosphate buffer of pH 7.5 containing 0.75% Triton X-100 (1 ml/min) which then passed through the enzyme reactors to the electrode detectors. The electrochemical flow cell had an Ag/AgCl reference electrode and a stainless steel tube as auxiliary electrode. Signal current was linear for 5-700 mg/dl glucose, 1-80 mg/dl uric acid and 5-100 mg/dl cholesterol. The RSD was better than 2.8% for the simultaneous assay of the three species in human control serum. Up to 32 samples per hour could be assayed.
Cholesterol Glucose Uric acid Amperometry Electrode Electrode Surfactant Interferences Controlled pore glass Immobilized enzyme Triton X

"Flow Injection Amperometric Detection Of Ammonia Using A Polypyrrole-modified Electrode And Its Application In Urea And Creatinine Biosensors"
Electroanalysis 1996 Volume 8, Issue 3 Pages 233-243
Marek Trojanowicz *, Andrzej Lewenstam, Tadeusz Krawczyski Vel Krawczyk, Ilkka L&auml;hdesm&auml;ki, Wojciech Szczepek

Abstract: Pt disc electrodes were immersed in a solution of 100 mM pyrrole in 100 mM NaCl, and polypyrrole formed on the surface over a period of 3 min at 800 mV vs. Ag/AgCl. Using the electrodes in 100 mM NaCl, calibration graphs were linear up to 100 µM-NH4Cl, and an RSD of 0.9-2% at 100 µM-NH4Cl. A mechanism of response of ammonia with the sensor is discussed. Enzyme immobilized sensors were prepared by adding a 5 µL of aqueous 1.5% glutaraldehyde to a suspension of either 0.5-2 mg urease or 1 mg creatinine (I) iminohydrolase in 15 µL water. After mixing, the mixture was spotted on to a wetted Cuprophan membrane, mounted on to the electrode and dried at 4°C for 3-4 h. Serum was diluted tenfold with water and 100 µL injected into a carrier stream (500 µL/min) of water, and merged with a stream (500 µL/min) of 50 mM borate buffer of pH 9.2 containing 140 mM NaCl. Urea was determined using a 1.6 mm diameter Pt electrode at 300 mV vs. Ag/AgCl. Calibration graphs were linear up to 3 mM urea, with an RSD (n = 24) of 0.8%. I was determined similarly using a I-iminohydrolase immobilized sensor, with a calibration graph linear up to 2 mM I and a detection limit of 0.1 mM I.
Ammonia Creatinine Urea Amperometry Sensor Electrode

"Quantitative Liquid Chromatographic Determination Of Cefatrizine In Serum And Urine By Fluorescence Detection After Post-column Derivatization"
J. Chromatogr. A 1979 Volume 177, Issue 2 Pages 323-332
E. Crombez, G. van der Weken, W. van den Bossche and P. de Moerloose

Abstract: A fast, specific and sensitive high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. The drug is determined by the internal standard method, using cephradine as the internal standard. The separation is carried out on a reversed-phase column, filled with octadecylsilane chemically bonded microparticles. The eluent is a mixture of acetonitrile with 0.025 M sodium phosphate buffer (pH 7). Quantitation is effected by fluorescence detection of the fluorophores formed after post-column derivatization with fluorescamine in a packed-bed reactor. The chromatographic conditions and the conditions for the post-column derivatization are discussed. The method has been applied to serum and urine samples, which were analyzed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by the determination of the cefatrizine content in spiked serum and urine samples.
Cefatrizine Fluorescence Post-column derivatization

"Post-column Derivatization System For The Fluorimetric Determination Of Guanidino-compounds With Ninhydrin By Reversed-phase Ion-pair High Performance Liquid Chromatography"
J. Chromatogr. A 1987 Volume 400, Issue 1 Pages 113-121
Yoshie Kobayashi, Hiroaki Kubo and Toshio Kinoshita

Abstract: Serum was mixed with 20% trichloroacetic acid and centrifuged. The supernatant solution was mixed with 0.4 M NaOH and adjusted to pH 2.5 to 3.0. The solution was subjected to HPLC on a column (15 cm x 4.6 mm) of Nucleosil C8 (5 µm) with a mobile phase of 15 mM Na octanesulfonate and 5 mM ninhydrin in water - acetonitrile - methanol (92:3:5; pH 3.5 or 4.0) with post-column derivatization at 75°C, and detection at 500 nm (excitation at 395 nm). Recoveries were 96.1 to 107.0% and the within-run coefficient of variation were 1.59 to 3.00%.
Guanidino compounds HPIC Fluorescence Heated reaction Post-column derivatization

"Liquid Chromatographic And Flow Injection Analysis Of Tetracycline Using Sensitized Europium(III) Luminescence Detection"
J. Chromatogr. B 1988 Volume 433, Issue 1 Pages 149-158
Thomas J. Wenzel, Lisa M. Collette, Deirdre T. Dahlen, Susan M. Hendrickson and Lawrence W. Yarmaloff

Abstract: Europium was used as a fluorophore for the detection of tetracycline(I) in HPLC and flow injection analysis. Gingival fluid was absorbed on filter-paper strips and tetracycline(I) was extracted with 1 mM Na2EDTA in 0.12 M KNO3 - acetonitrile (9:1). HPLC was performed on a column (25 cm x 4.6 mm) of C8 silica (10 µm). Various mobile phases and post-column reagent solution containing Eu(III) were evaluated; the fluorescence was detected at 616 nm (excitation at 392 nm). A 30 µs delay was incorporated in fluorescence measurement to avoid interference from fluorescent impurities. Detection limits were better than those obtained with UV detection. Europium (III) can be used as a luminescent chromophore for detection in the liquid chromatographic and flow injection analysis of tetracycline. Detection is dependent upon an intramolecular energy transfer from the tetracycline to Eu (III). In liquid chromatography, the Eu (III) is added post-column as a complex with ethylenediaminetetraacetic acid. The post-column phase also serves to adjust the pH for optimum sensitivity. The method is highly selective for tetracycline since few compounds are capable of transferring energy to Eu (III). Fluorescent impurities that would otherwise interfere in flow injection analysis can be eliminated through the use of a delay time between the source pulse and the start of data acquisition. The detection limits for tetracycline using sensitized Eu (III) luminescence are better than those obtained using ultraviolet detection. The method is applied to the analysis of tetracycline in urine, blood serum, and gingival crevice fluid.
Tetracycline HPLC Fluorescence Luminescence Complexation Interferences Post-column derivatization

"Online Sample Processing And Analysis Of Diol Compounds In Biological Fluids"
J. Chromatogr. A 1988 Volume 456, Issue 1 Pages 93-104
Karl-Siegfried Boos* and Bernd Wilmers, Eckhard Schlimme, Richard Sauerbrey

Abstract: A coupled dual-column system with optional post-column derivatization (illustrated) was used for online sample processing, trace enrichment and analysis for aromatic 1,2-diol and aliphatic cis-diol biomolecules. Catecholamines were determined in human urine acidified to pH 3.0 to 3.5 with 10 M HCl; noradrenaline and adrenaline were determined in plasma and serum treated with 10 M NaOH to pH 7 and ribonucleosides were determined in urine, serum and milk acidified to pH 4 with concentrated formic acid. Aliquots of the sample solution were applied to the HPLC system consisting of a pre-column and an analytical column. Catecholamines and ribonucleosides were detected by fluorescence and UV spectrometry, respectively. The pre-column packing, phenylboronic acid-modified silica (Hagemeier et al., Ibid., 1983, 268, 291), allowed simultaneous covalent affinity and size-exclusion chromatography.
Catecholamines Diols Ribonucleosides LC SEC Fluorescence Spectrophotometry Post-column derivatization

"Improved High Performance Liquid Chromatographic Method With Fluorimetric Detection For The Determination Of Glycerol Using An Immobilized Enzyme Column Reactor"
J. Chromatogr. A 1988 Volume 456, Issue 2 Pages 398-404
Nobutoshi Kiba*, Haruhiko Maruyama and Motohisa Furusawa

Abstract: In the improved, one-pump system described glycerol was determined by HPLC on a column (30 cm x 7.8 mm) packed with TSK gel SCX (5 µm), fitted with a guard column (5 cm x 6 mm) of Shodex Ionpak KS800P and operated at 40°C ± 0.2°C with a mobile phase (0.7 mL min-1) comprising a mixture (1:1) of carbonate buffer (NaHCO3 - K2CO3, 0.4M, pH 10.0) and 10 mM NAD solution in 0.05 M phosphate buffer (pH 7.0). Post-column derivatization was effected on a stainless-steel column reactor (15 cm x 4.0 mm) packed with glycerol dehydrogenase immobilized on aminobutylpolystyrene beads (prep. described). Fluorescence detection was at 465 nm (excitation at 340 nm). The calibration graph was rectilinear over the range 5 µM to 0.5 mM and the detection limit was 1 µM-glycerol. The method was applied in the determination of triglycerides in serum. Within- and between-day coefficient of variation (n = 10) were 3.2 and 3.6% for 1.54 mM triglycerides and 3.0 and 3.6% for 2.10 mM.
Glycerol Triglycerides HPLC Fluorescence Immobilized enzyme Post-column derivatization Polystyrene beads

"High Performance Liquid Chromatographic Determination Of Some Polar Phospholipids In Serum"
J. Chromatogr. B 1989 Volume 495, Issue 1 Pages 61-70
Jir&iacute; B&ouml;swart, Thomas Schmidt, Pavel Kostiuk, Vera Pac&aacute;kov&aacute; and Karel Stul&iacute;k

Abstract: The experimental conditions have been optimized for high performance liquid chromatographic determination of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and sphingomyelin (SM) in serum. The phospholipids are separated on a silica gel column, using a mobile phase of acetonitrile-methanol-water (100:10:18, v/v), with ultraviolet photometric detection at 200 nm. The limit of detection was 0.2 µg (in 20 µL) for natural phospholipids and 2.5 µg for synthetic phospholipids; the relative standard deviation was ~5%. An alternative detection is tensammetry at a mercury electrode, at a potential of -1.8 V, with an a.c. current frequency of 60 Hz and an amplitude of 20 mV. The tensammetric detection has an advantage in its independence of the structure of the phospholipids. In measurements without a column (flow injection analysis), the tensammetric detection also yields a somewhat lower limit of detection than photometry (0.15 µg per 20 µL), but this value increases more than ten times in chromatographic detection. The precision is poorer and is more susceptible to interferences. The method was applied to the determination of the above substances in the blood of obese children, as a function of physical stress and spa treatment. It was shown that physical exercise causes a decrease in the contents of PE and PC in the patients. On the other hand, the spa treatment has no pronounced effect on the phospholipid content in the blood.
Phosphatidylethanolamine Phosphatidylcholine Sphingomyelin HPLC Electrode Sensor Optimization Method comparison Detection limit Precision Interferences

"Online Electrochemical Reagent Generation For Liquid Chromatography With Luminol-based Chemiluminescence Detection"
J. Chromatogr. A 1991 Volume 557, Issue 1 Pages 13-21
O. M. Steijger, G. J. De Jong, J. J. M. Holthuis, and U. A. Th. Brinkman

Abstract: An on-line method for the generation of electrochemical reagent for liquid chromatography, with luminol-based chemiluminescence detection, has been developed. An ESA Coulochem guard cell, equipped with a porous graphite working electrode, operated at -600 mV and inserted after the column, produces an oxidative reagent for the luminol-based reaction. This method has been compared with the conventional method with post-column addition of hydrogen peroxide as the oxidative reagent. With this novel method a detection limit of 0.15 pmol of ibuprofen (labelled with an isoluminol derivative) can be obtained, and a good alternative for post-column addition of hydrogen peroxide is presented.
Amino Acids Hydroperoxides Lipids Chemiluminescence HPLC Electrochemical reagent generation Post-column derivatization

"Use Of Various Types Of Column Reactors For Flow Injection Analysis"
J. Chromatogr. A 1992 Volume 597, Issue 1-2 Pages 435-442
Masayoshi Tabata*, Takashi Murachi, Jiro Endo and Masayuki Totani

Abstract: Several enzymes, immobilized singly or co-immobilized on glass beads, were aligned in a mini-reactor so that sequential reactions could be carried out before chemiluminescence detection. Conditions were derived for the determination of glucose, NH3, urea and creatinine in one manifold and serum lactate dehydrogenase activity, after removal of lactate, in another. Calibration graphs were rectilinear in ranges up to 20 to 40 mM except for creatinine (0.1 to 1 mM). Two or three different kinds of immobilized enzymes can be aligned in a minireactor so that sequential enzymatic reactions are carried out from upstream to downstream during flow injection analysis A lactate oxidase-catalase reactor, used as a pre-column for removing pre-existing lactate in serum before the lactate dehydrogenase (LDH) reaction, was useful for the determination of serum LDH activity, which did not require any blank correction. A sequential glutamate dehydrogenase-glutamate oxidase reactor was also useful for a novel chemiluminometric determination of ammonia. On the other hand, a co-immobilized creatininase-creatinase-sarcosine oxidase reactor, in spite of containing creatininase which catalyzes the reversible reaction, was the most efficient for the determination of serum creatinine.
Glucose Ammonia Urea Creatinine Enzyme, lactate dehydrogenase Chemiluminescence Immobilized enzyme Glass beads

"Chemiluminescent Detection Of Artemisinin. Novel Endoperoxide Analysis Using Luminol Without Hydrogen Peroxide"
J. Chromatogr. A 1995 Volume 695, Issue 2 Pages 237-242
Michael D. Green*, Dwight L. Mount, G. Daniel Todd and Anthony C. Capomacchia

Abstract: A novel method for artemisinin quantitation employing high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection in the absence of hydrogen peroxide (H2O2), is reported. After elution from the HPLC column, artemisinin is combined with an alkaline solution of hematin and luminol. The resulting CL signal is detected by use of a spectrofluorometer with the excitation lamp disabled, and is proportional to artemisinin concentration. The CL method was optimized and applied to the analysis of artemisinin in spiked human serum. CL in the absence of H2O2 or other known as oxidizing species is remarkable since such oxidizers are usually required to produce CL from luminol under alkaline conditions. Artemisinin, a naturally occurring sesquiterpene, is one of several natural products that contain an endoperoxide functional group. Since H2O2 is not needed in the analysis, the endoperoxide moiety on artemisinin is implicated as a contributing source of superoxide radicals required for the light-producing reaction with luminol.
Artemisinin HPLC Post-column derivatization

"Determination Of Sissomicin, Netilmicin, Astromicin And Micronomicin In Serum By High Performance Liquid Chromatography"
J. Chromatogr. B 1984 Volume 305, Issue 1 Pages 373-379
Takeshi Kawamoto, Ikuo Mashimo and Shigeki Yamauchi, Makoto Watanabe

Abstract: The method was adapted from that described previously for tobramycin (cf. Watanabe et al., Chemotherapy, 1982, 30, 21). Before reversed-phase ion-pair HPLC separation on Zorbax C8 (Zorbax ODS for micronomicin) at 55°C with Na toluene-p-sulfonate as counter-ion in NaH2PO4 solution (pH 2) also containing NaClO4, the aminoglycosides were separated from serum amino-acids by sorption on CM-Sephadex, thus facilitating their determination by post-column derivatization with phthalaldehyde. Internal standards were chosen from within this group of aminoglycosides. Calibration graphs were rectilinear in the ranges 0.32 to 22.8, 0.17 to 11.6, 0.1 to 6.3 and 1.0 to 30 µg mL-1 of sissomicin, netilmicin, astromicin and micronomicin, respectively, and coefficient of variation (n = 6) were 1.9 to 3.1%.
Aminoglycosides Sissomicin Netilmicin Astromicin Micronomicin HPLC Fluorescence Post-column derivatization

"High Performance Liquid Chromatographic Analysis Of Guanidino-compounds Using Ninhydrin Reagent. 2. Guanidino-compounds In Blood Of Patients On Haemodialysis Therapy"
J. Chromatogr. B 1985 Volume 342, Issue 1 Pages 269-275
Yayoi Hiraga and Toshio Kinoshita

Abstract: Serum was deproteinized with 60% trichloroacetic acid and centrifuged. The supernatant solution was adjusted to pH 2.0 to 2.5 and analyzed by HPLC at 50°C on an ISC-05 ion-exchange column (3.8 x 4.6 mm) with gradient elution with 0.05 M trisodium citrate (pH 3.5) and 0.12 M trisodium citrate containing 0.5 M NaCl and 0.1 M H3BO3 (pH 11.4). The eluate was treated with aqueous 0.6% ninhydrin and 1.5 M NaOH at 50°C, and the fluorescence was measured at 500 nm (excitation at 395 nm). Ten guanidino-compounds were separated in 37 min. The day-to-day coefficient of variation (n = 10) were 0.7 to 3.0% and recoveries were 94 to 108%.
Guanidino compounds HPLC Ion exchange Fluorescence Heated reaction Post-column derivatization Dialysis

"High Performance Liquid Chromatographic Determination Of β-aminoisobutyric Acid In The Picomole Range"
J. Chromatogr. B 1986 Volume 374, Issue 1 Pages 129-136
Frank L. Buschman, Gerald Apell and Opendra K. Sharma

Abstract: 3-Aminoisobutyric acid(I) was determined in serum and urine with or without hydrolysis of the sample (for total or free I, respectively). The HPLC was performed on a column (10 cm x 8 mm) of Radial-Pak C18 (5 µm) with 0.4 M Na citrate buffer of pH 4.2 as mobile phase (0.33 mL min-1) and fluorimetric detection at 410 nm (365-nm excitation) after post-column derivatization with phthalaldehyde. For the determination of total I in serum there was a preliminary clean-up by cation exchange on a column (30 cm x 3.9 mm) of W-3H resin to remove phenylalanine and tyrosine; I was eluted with 0.1 M NaH2PO4 buffer of pH 2.5. A rectilinear calibration graph was obtained for 5 pmol to 2 nmol of I.
3-Aminoisobutyric acid HPLC Fluorescence Post-column derivatization Sample pretreatment

"Simultaneous Determination Of Serum Uremic Toxins, Cations And Anions And Urea Determination By Post-column Colorimetry Using Immobilized Enzyme"
J. Chromatogr. B 1986 Volume 378, Issue 1 Pages 95-108
Hideharu Shintani

Abstract: A column-switching method for the simultaneous determination of serum uraemic toxins (e.g., methylguanidine, creatinine and uric acid), urea, cations (e.g., Ca(II), Mg(II), Na+, NH4+ and K+) and anions (e.g., Cl-, ClO4- and HCO3-) is described. Uraemic toxins are determined by ion-pair reversed-phase HPLC on a column (20 cm x 4.6 mm) of Senshupak ODS-SSC-L-1202, with 1 mM Ba hexanesulfonate containing 30 mM BaClO4 as the mobile phase (1 mL min-1) and 210-nm detection. Urea is determined on an immobilized-urease column operated at 55°C with post-column derivatization with indophenol; detection is at 580 nm. Serum cations are determined on a Oyobunko pellicular cation-exchange resin (ASC-4000) column (25 cm x 4.6 mm) with, e.g., 0.37 mM m-phenylenediamine as the mobile phase and conductometric detection. Serum anions are determined on a Wescan anion-exchange column (24 cm x 4.6 mm) with 4 mM K H phthalate (pH 4.5) as the mobile phase. No matrix effects are observed.
Methylguanidine Creatinine Uric acid Urea Ammonium Calcium(2+) Magnesium Potassium Sodium chlorate Chloride Bicarbonate HPLC Conductometry Ion exchange Spectrophotometry Heated reaction Interferences Post-column derivatization Immobilized enzyme

"Reversed-phase High Performance Liquid Chromatographic Method For The Determination Of Warfarin From Biological Fluids In The Low Nanogram Range"
J. Chromatogr. B 1986 Volume 378, Issue 1 Pages 254-260
J. M. Steyn* and H. M. Van Der Merwe, M. J. De Kock

Abstract: Samples of plasma or serum containing warfarin(I) were acidified with 4 M HCl and extracted with ethyl ether; naproxen was added as internal standard. The extract was evaporated to dryness under N and the residue was dissolved in a mobile phase of acetonitrile - 0.1 M NH4H2PO4 (63:37). An aliquot (100 µL) was then analyzed by HPLC on a µBondapak C18 column. Post-column derivatization was effected by treating the eluate with 12% triethanolamine solution; the resulting derivative was detected fluorimetrically at 390 nm (excitation at 320 nm). The detection limit was 0.3 ng mL-1 and calibration graphs were rectilinear for up to 10 µg mL-1 of total I and up to 100 ng mL-1 for the unbound drug. No interference was observed in the presence of some acidic and neutral drugs.
Warfarin HPLC Fluorescence Interferences Post-column derivatization

"Determination Of Oxidizable Inorganic Anions By High Performance Liquid Chromatography With Fluorescence Detection And Application To The Determination Of Salivary Nitrite And Thiocyanate And Serum Thiocyanate"
J. Chromatogr. B 1988 Volume 424, Issue 1 Pages 29-37
Shinzo Tanabe, Michie Kitahara, Masashi Nawata and Kouji Kawanabe

Abstract: Sulfide, SCN-, S2O32- and NO2- were determined by HPLC on a column (15 cm x 4 mm) of TSK gel QAE-2SW (5 µm) with a mobile phase (0.8 mL min-1) of 0.05 M NaNO3 and post-column derivatization, at 80°C for 15 s, with 0.75 M H2SO4 containing 0.25 mM Ce(SO4)2 and 0.003% of sodium bismuthate. Fluorescence detection was at 360 nm (excitation at 260 nm). The limits of detection were 0.1, 0.1, 0.3 and 0.8 nmol for NO2-, SCN-, S2O32- and S2-, respectively. The calibration graphs were rectilinear for 10 nmol per 10 µL injected and the coefficient of variation was <8%. The method was applied in the determination of NO2- and SCN- in saliva and SCN- in serum. Recoveries of NO2- and SCN- were 99.3 and 99.1%, respectively, from saliva and recovery of SCN- from serum was 63%.
Anions, inorganic Nitrite Sulfide Sulfite Thiocyanide HPLC Fluorescence Heated reaction Post-column derivatization

"Development Of A High Performance Liquid Chromatographic - Post-column Fluorogenic Assay For Digoxin In Serum"
J. Chromatogr. B 1989 Volume 496, Issue 1 Pages 321-334
Leanne Embree and Keith M. McErlane

Abstract: Digoxin (I) was extracted from serum, treated with digitoxigenin (internal standard), by a modification of the method of Kwong and McErlane (J. Chromatogr., Biomed. Appl., 1986, 50, 233) and separated from its metabolites on a column (15 cm x 4.6 mm) of Spherisorb ODS II (3 µm) with a Brownlee ODS guard column (1.5 cm x 3.2 mm) and a mobile phase (0.5 mL min-1) of methanol - dehydroascorbic acid - ethananol - propan-2-ol (52:45:3:1). Concentrated HCl was used as the post-column derivatization agent and was delivered by hexane displacement from a PVC pressure vessel. The resulting derivatives were detected fluorimetrically at 425 nm (excitation at 360 nm). The assay was specific for I in the presence of its metabolites. An extraction procedure for determining I in serum without interference from endogenous compounds was also develped. The calibration graphs were rectilinear from 0.5 to 3.3 ng of I and the mean coefficient of variation was 5.6%.
Digoxin HPLC Fluorescence Post-column derivatization Interferences

"High Performance Liquid Chromatographic Determination Of Branched-chain 2-oxo-acids In Serum Using Immobilized Leucine Dehydrogenase As Post-column Reactor"
J. Chromatogr. B 1989 Volume 497, Issue 1 Pages 236-242
Nobutoshi Kiba, Masakazu Muto and Motohisa Furusawa

Abstract: Serum (0.2 ml), containing 20 µL of 0.3 mM 2-oxohexanoic acid as internal standard, was passed through a membrane filter (mol. wt. 30,000), and a 30 µL portion of filtrate was injected on to a column (15 cm x 4 mm) and guard column (1 cm x 4 mm) of Capecell 120 C18 (5 µm), at 40°C. The mobile phase (0.8 mL min-1) was 0.5 M NH4Cl - NH3 buffer of pH 8.5 containing 5 µM-NADH. The eluate was passed through a column (5 cm x 4 mm), also at 40°C, of immobilized leucine dehydrogenase before fluorimetric detection of 2-oxo-acids, e.g., 2-oxo-butyric acid and 2-oxo-pentanoic acid. Calibration graphs were rectilinear in the range 4 to 55 µM. Detection limits were 1 µM. Recoveries were 53.5 to 56.5%, and coefficient of variation were 1.7 to 2.4% (n = 5). Results are compared with those of a direct HPLC method.
Acids HPLC Fluorescence Post-column derivatization Buffer Heated reaction Method comparison Immobilized enzyme

"Determination Of Sulindac And Its Metabolites In Human Serum By Reversed-phase High Performance Liquid-chromatography Using Online Post-column Ultraviolet-irradiation And Fluorescence Detection"
J. Chromatogr. B 1995 Volume 673, Issue 1 Pages 91-96
Madhusudhan Siluveru and James T. Stewart*

Abstract: On irradiation with ultraviolet light, the antiinflammatory agent sulindac and its two metabolites sulindac sulfone and sulindac sulfide form highly fluorescent derivatives. This reaction was exploited for the sensitive and selective detection of these compounds in serum using reversed-phase high performance liquid chromatography on a Ultrasphere octylsilane column (150 x 4.6 mm LD.) at ambient temperature with a flow-rate of 0.5 ml/min. The analytes of interest were isolated from serum using a Bond-Elut C-2 column with satisfactory recovery and selectivity. The detection limits were 10 ng/ml for each of the three analytes using 1 mL of serum and the limit of quantitation was 50 ng/ml. Linear calibration curves from 50 to 1000 ng/ml for all three analytes show coefficients of determination of 0.9999. The post-column ultraviolet irradiation was optimized and the effect of irradiation time on the fluorescence response was determined for all three analytes. Precision and accuracy of the method were 0.4-5.6 and 1.6-4.5% for sulindac, 2.3-5.6 and 1.4-5.3% for sulindac sulfone and 2.5-4.3 and 0.8-2.8% for sulindac sulfide, respectively. (18 references)
Sulindac Sulindac, metabolites HPLC Fluorescence Post-column derivatization Photochemistry UV reactor Optimization

"Sensitive Flow Injection Method With Peroxyoxalate Chemiluminescence Detection Combined With Preparative High Performance Liquid Chromatography For Determination Of Choline-containing Phospholipids In Human Serum"
J. Chromatogr. B 1996 Volume 678, Issue 2 Pages 129-136
Mitsuhiro Wada, Kenichiro Nakashima*, Naotaka Kuroda, Shuzo Akiyama and Kazuhiro Imai

Abstract: Serum samples (5 µL) were diluted to 2 mL with 0.1% aqueous Triton X-100. A 20 µL portion was analyzed for total choline-containing phospholipids (PL) on an immobilized enzyme reactor column (7 cm x 2 mm i.d.) containing phospholipase D and choline oxidase on aminopropyl controlled-pore glass, linked with glutaraldehyde. The carrier solution was 10 mM imidazole buffer of pH 8 containing 0.1% Triton X-100 (1 ml/min) and the post-column chemiluminescent reagent contained 0.4 mM bis-(2,4,6-trichlorophenyl)oxalate and 0.5 µM-2,4,6,8-tetrathiomorpholinopyrimido[5,4,-d] pyrimidine in acetonitrile (1.2 ml/min). Separation of choline-containing PL was performed by adding 100 µL of CHCl3/methanol (1:1) to 5 µL of serum, filtering, then analyzing on a 5 µm Daisopak-SP-120-5-APS aminopropyl column (15 cm x 6.0 mm i.d.) with acetonitrile/methanol/10 mM ammonium phosphate buffer of pH 5.8 (615:264:150) as mobile phase and detection at 205 nm. Fractions were analyzed on the FIA system described above. Calibration graphs were linear for up to 1 nmol PL and the detection limits were 1.3-1.6 pmol; RSD were 1.6-3.3%. RSD for serum were 1.7-7.7%.
Phospholipids HPLC Chemiluminescence Controlled pore glass Immobilized enzyme Triton X Surfactant

"Investigation Of Ion Transfer Across The Membrane-stabilized Interface Of Two Immiscible Electrolyte Solutions. 3. Facilitated Ion Transfer"
J. Electroanal. Chem. 1994 Volume 371, Issue 1-2 Pages 1-11
Bernd Hundhammer, Theodros Solomon, Tadesse Zerihuna, Mulat Abegaz, Aklilu Bekele and Maus Graichenb

Abstract: The transfer of Na+, K+, Cs+ and Tl+ across PT 325 hydrophilic dialysis membrane (Kleinfeld and Co., Hannover, Germany) facilitated by the presence of dibenzo-18-crown-6 has been studied by concentration-pulse FIA. The FIA apparatus was as described in Part II (J. Electroanal. Chem. Interfacial Electrochem., 1989, 266, 133), the membrane-stabilized interface was similar to that described previously (Ibid., 1987, 44, 253), and a four-electrode potentiostat with IR compensation was used to control the potential difference across the interface. The organic receiving phase was 10 mM µ-nitridobis(triphenylphosphorus)-3,3-comobis(undecahydro-1,2-dicarba-3-cobalta-closo-dodecaborate) or µ-nitridobis(triphenylphosphorus)tetraphenylborate in nitrobenzene, and the aqueous supporting electrolyte and carrier solution were both 0.1 M MgSO4. The continuously monitored steady-state current was used to determine Na+ and K+ in standard sera and serum samples diluted 1:50 with water. Results agreed well with those by flame photometry and with the certified values. (For Part II see Anal. Abstr., 1990, 52, 2J6).
Potassium Sodium Spectrophotometry Electrode Reference material Dialysis

"Electrochemical Detection In Flow Injection Analysis For Determining Serum Lipase Activity"
J. Electroanal. Chem. 1995 Volume 396, Issue 1-2 Pages 507-510
K. Takamura, T. Fuse and F. Kusu

Abstract: Serum (15 µL) was incubated with 15 µL of olive oil, 40 µL of water and 35 µL of 0.067 M KH2PO4/0.067 M Na2HPO4 (pH 7) at 37°C for 10 min; for samples of other than serum, the buffer solution of pH 7 was replaced by 35 µL of 0.1 M KH2PO4/0.05 M Na2B4O7 of pH 9. The mixture was then extracted with three 3 mL portions of diethyl ether, the extract was evaporated, and the residue was dissolved in 0.3 mL of the FIA carrier, viz, ethanolic 3 mM 2-methyl-1,4-naphthaquinone/38 mM LiClO4. A 5 µL portion was injected into the carrier stream (0.6 ml/min) and the current at a vitreous-carbon electrode at -0.33 V was monitored in a 2.4 µL wall-jet cell (diagrams presented). The calibration graph of peak heights was linear for 5 µM-0.3 mM linoleic acid, for which the RSD (n = 10) at 0.2 mM was 1.4%. The response was linearly related to lipase activities from 10 (detection limit) to 1000 iu/l, and the RSD (n = 5) at 70 iu/l was 1.2%. Results showed good correlation with those obtained by titration with NaOH and by use of the Nescauto lipase kit-VE. As a simple and rapid method for serum lipase determination, assessment was made of electrochemical detection in flow injection analysis (FIA), by measuring the voltammetric prepeak accompanied by the reduction of 2-methyl-1,4-naphthoquinone (vitamin K-3, VK3) due to the effect of free fatty acids. A sample containing lipase was incubated with olive oil as the substrate at 37°C for 10 min to bring about the release of fatty acids. The sample solution was prepared by dissolving the oily residue extracted by ether in ethanol solution containing 3 mM VK3 and 38 mM LiClO4. A 5 µl aliquot of the sample solution was injected into the flow injection (FI) system. The peak height of the flow signal was linear to lipase activity between 10 and 1000 U L-1 and RSD was 1.2% for 70 U L-1 (n = 5). By this method, as many as 30 samples h-1 can be processed, thus showing this method to be useful and highly efficient. (13 References)
Triacylglycerol lipase Amperometry Electrode Method comparison

"Dibekacin Assay In Serum By Automated Fluorescence Polarization Immunoassay (Abbott Tdx): Comparison With High Performance Liquid Chromatography, Substrate-labelled Fluorescent Immunoassay And Radio-immunoassay"
J. Pharm. Biomed. Anal. 1986 Volume 4, Issue 1 Pages 53-61
B. Rollman, P. Van Der Auwera and P. M. Tulkens

Abstract: Dibekacin(I) was determined in serum by fluorescence polarization immunoassay with reagents from Abbott. The fluorescent label was fluorescein and the wavelengths of the polarized exciting radiation and emission radiation were 484 and 530 nm, respectively. Rectilinear calibration graphs were obtained for up to 9 µg mL-1. I was also determined by HPLC on a 25-cm column of LiChrosorb RP-18 (10 µm) with a mobile phase of 0.02 M p-toluenesulfonic acid - 0.02 M acetate - 0.2 M Na2SO4 - 0.04 M acetic acid - 5% of acetonitrile (1 mL min-1) and fluorimetric detection after post-column derivatization. Determinations were also performed by fluorescence immunoassay and RIA.
Dibekacin HPLC Fluorescence Immunoassay Post-column derivatization

"Enzymic Determination Of Total Cholesterol In Serum By Flow Injection Analysis"
J. Pharm. Biomed. Anal. 1987 Volume 5, Issue 4 Pages 333-340
Juan M. Fernandez-Romero, M. D. Luque De Castro and Miguel Valcarcel*

Abstract: Serum was mixed with aqueous 10% propan-2-ol containing 4% Triton X-100 or 1.5% Brij-35 in buffer solution The solution was subjected to flow injection analysis by mixing with a solution containing cholesterol esterase, cholesterol oxidase, peroxidase and a reagent solution for photometric detection at 500 nm; the reagent solution contained 0.5 mM 4-aminoantipyrine, 10 mM phenol and 2 mM Na cholate in 0.5 M Tris - HCl buffer. For fluorimetric detection at 415 nm (excitation at 325 nm), the reagent solution contained 0.5 mM 4-hydroxyphenylacetic acid in 0.5 M phosphate buffer. The calibration graphs were rectilinear for 0.005 to 0.05 and 0.01 to 0.20 g L-1 of cholesterol and the detection limits were 0.4 to 2.0 mg l-1. The coefficient of variation (n = 33) were 1.28 to 2.08% and recoveries were 96 to 108%.
Cholesterol, total Fluorescence Enzyme Surfactant Triton X

"Automated Flow Injection Determination Of Salicylates Using Trinder Reaction For Clinical Analysis, Assays And Dissolution Studies Of Formulations"
J. Pharm. Biomed. Anal. 1988 Volume 6, Issue 1 Pages 35-46
M. A. Koupparis* and P. I. Anagnostopoulou

Abstract: The construction and use of a flow injection analyzer. with absorbance measurement at 540 nm is described. Salicylate(I), salicylamide(II) and methyl salicylate(III), as well as aspirin after alkaline hydrolysis, were determined by reaction with Fe(III) in weakly acidic medium (0.02 M HNO3). The method has been applied to tablets (and lotions) for determination of 25 to 250 or 80 to 800 mg L-1 of I, 30 to 300 mg L-1 of II and 100 to 1000 mg L-1 of III. It was suitable for dissolution tests. The method was also applied to determine I in serum. Recovery was 96.4 to 103.0% and coefficient of variation were <1%. Interference was much reduced compared with that affecting the manual Trinder method.
Salicylate Methylsalicylate Salicylamide Aspirin Clinical analysis Spectrophotometry Dissolution rate Interferences Method comparison

"Chloride Determination In Serum By A Flow Injection Analysis Precipitation Pseudo-titration Technique Using A Flow-through All-solid-state Silver Electrode"
J. Pharm. Biomed. Anal. 1988 Volume 6, Issue 6-8 Pages 749-755
S. Alegret*, J. Alonso, J. Bartroli, J. Garcia-Raurich and E. Martinez-F&agrave;bregas

Abstract: Details are given of the flow injection system and of the design and construction of the tubular Ag electrode. A 50 µL portion of serum (diluted 1:10) was injected into the system, with 0.1 M KNO3 containing 0.5 mM AgNO3 as supporting electrolyte; a concentration. gradient was achieved by dispersion of the sample in a 0.55 mL mixing chamber, and peak width was measured. The system was applied in determination of Cl- over the range normally found in serum, viz, 96 to 104 mM. The coefficient of variation (n = 12) was 1.8%. Sixty samples could be analyzed in 1 h.
Chloride Electrode Gradient technique Titrations Well stirred mixing chamber Peak width Precipitation

"Determination Of Medroxyprogesterone Acetate In Serum By HPLC With Peroxyoxalate Chemiluminescence Detection Using A Fluorogenic Reagent, 4-(N,N-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole"
J. Pharm. Biomed. Anal. 1992 Volume 10, Issue 10-12 Pages 979-984
S. Uzu, K. Imai*, K. Nakashima and S. Akiyama

Abstract: Serum was centrifuged with acetonitrile at 10,000 g for 1 min and the supernatant solution was mixed with water and applied to a Bond-Elut C18 column. After washing with aqueous 40 and 45% acetonitrile, elution was effected with aqueous 70% acetonitrile. The eluate was evaporated to dryness, dissolved in 0.25 mM 4-(NN-dimethylaminosulfonyl)-7-hydrazino-2,1,3-benzoxadiazole and 0.25% trifluoroacetic acid in acetonitrile and set aside at 4°C for 5 h or overnight. A portion of the reaction mixture was analyzed by HPLC on a column (25 cm x 4.6 mm) of TSKgel ODS 80 (5 µm) in series with a guard column of the same material maintained at 30°C with a mobile phase (0.5 mL min-1) of THF - 100 mM imidazole nitrate buffer of pH 6 (31:19) and post-column derivatization with 0.5 mM bis[4-nitro-2-(3,6,9-trioxadecyloxycarbonyl)-phenyl]oxalate and 2.5 mM H2O2 in acetonitrile - ethyl acetate (1:1) followed by chemiluminescence detection. The calibration graph was rectilinear from 15.6 to 96.6 ng mL-1 of medroxyprogesterone acetate (I) with a detection limit of 9 ng mL-1. Coefficients of variation were 7.4 and 1.7% for 19.3 and 77.3 ng mL-1 of I, respectively.
Medroxyprogesterone HPLC Chemiluminescence Post-column derivatization Heated reaction

"Liquid Chromatographic Separation Of Phenolic Drugs Using Catalytic Detection: Comparison Of An Enzyme Reactor And Enzyme Electrode"
J. Pharm. Biomed. Anal. 1992 Volume 10, Issue 10-12 Pages 789-796
F. Ortega, J. L. Cuevas, J. I. Centenera and E. Dom&iacute;nguez*

Abstract: Tyrosinase (I) catalyses the oxidation - dehydrogenation of phenolic compounds to give quinones, which are readily polymerized to give highly colored species and are electroactive species. Sensitivity of three catalytic detection systems was studied in a flow injection system: (i) an immobilized I reactor (IMER) with photometric detection at 475 nm; (ii) IMER with electrochemical detection with an unmodified graphite electrode (-50 mV) vs. SCE; (iii) I-graphite electrode (-50 mV) vs. SCE. Sample [25 µL, 1 mM dopamine for (i) and (ii), 50 µM-dopamine for (iii)] was injected into 0.1 M phosphate buffer (pH 6.0) carrier stream [0.7 mL min-1 for (i) and (iii), 1 mL min-1 for (ii)]. Sensitivities were 30 mA µg-1, 174 nA µg-1 and 1054 nA µg-1 of dopamine for (i), (ii) and (iii), respectively. A detection limit of 4.5 ng of dopamine was obtained for (iii), the most sensitive method. A catalytic detection system based on the use of immobilized tyrosinase and two different transducers (photometric and electrochemical) is described. Comparison between tyrosinase immobilized in a packed-bed reactor and at the surface of a graphite electrode is discussed in terms of sensitivity in a flow injection system. The enzyme electrode configuration gives the highest sensitivity for the quantitation of dopamine. For the immobilized tyrosinase reactor with photometric detection the range for dopamine is linear up to 0.75 mM (136 µg mL-1) and the immobilized tyrosinase reactor with electrochemical detection and the tyrosinase electrode extends this dynamic range to 1 mM (181 µg mL-1). Liquid chromatographic separation and post-column detection using the tyrosinase electrode is shown for spiked samples of serum.
Phenols HPLC Spectrophotometry Electrode Immobilized enzyme Post-column derivatization Reactor Catalysis

"Flow Injection Extraction-spectrophotometric Determination Of Bromhexine With Orange IV"
J. Pharm. Biomed. Anal. 1995 Volume 13, Issue 9 Pages 1101-1106
Tomas P&eacute;rez-Ruiz*, Carmen Mart&iacute;nez-Lozano, Antonio Sanz and Santos Mondejar

Abstract: Sample (60 µL) containing bromhexine (I) was injected into a stream (0.9 ml/min) of 0.5 M chloroacetate buffer of pH 3 which merged with a stream (0.9 ml/min) of aqueous 0.3 mM orange IV solution [4-(p-anilinophenyl-azo)benzene sulfonic acid sodium salt]. After passing through a reaction coil (30 cm x 0.5 mm i.d.) the resulting stream merged with a stream (1.3 ml/min) of 1,2-dichloroethane and passed through an extractor coil (100 cm x 0.5 mm i.d.), then through a phase separator incorporating a PTFE membrane permeable only to the organic phase. The absorbance of the organic phase was measured at 421 nm. The calibration graph was linear for 5-160 µM-I and the detection limit was 0.5 µM. The RSD were 0.32-0.88% (n = 10). Sample throughout was 40/h. Tolerance limits of foreign compounds are tabulated. The method was applied to TCA-treated human serum with recoveries of 98.5% and to a pharmaceutical preparation with recoveries of 100.5-102%. An automatic flow injection photometric method for the determination of bromhexine is proposed. The drug was determined by formation of an ion- pair with orange IV, extraction into 1,2-dichloroethane and measurement of the absorbance at 412 nm of the organic phase. A linear calibration graph was obtained at concentrations of 5 x 10^-6-1.6 x 10^-4 M of bromhexine. Up to 40 samples h-1 can be processed with an RSD of 0.32- 0.88%. The method was applied to the determination of bromhexine in blood serum and a pharmaceutical preparation.
Bromhexine Spectrophotometry Sample preparation Extraction Organic phase detection Phase separator Teflon membrane

"Flow Injection Determination Of Total Ammonia And Total Carbon Dioxide In Blood Based On Gas-diffusion Separation And With A Bulk Acoustic Wave Impedance Sensor"
J. Pharm. Biomed. Anal. 1998 Volume 16, Issue 5 Pages 759-769
Xiao-Li Su, Bing-sheng Yu, Hu-wei Tan, Xiao-rong Yang, Li-hua Nie and Shou-zhuo Yao*

Abstract: A novel flow injection (FIA) system, for the rapid and direct determination of both total NH3 (TNH3) and total CO2 (TCO2) in clinical blood samples, was developed. Samples were injected into a carrier stream of water, then emerged with a reagent stream, where the analyte was converted into a gaseous species and diffused across a PTFE gas-permeable membrane into an acceptor stream. The trapped NH3/CO2 in the acceptor was determined on line by a bulk acoustic wave (BAW) impedance sensor. At a through-put of 20 and 65 h-1, the proposed system exhibited a linear frequency response up to 200 µmol L-1 ammonium and 20 mmol L-1 bicarbonate with a detection limit of 1.0 and 10 µmol L-1, respectively. Results obtained for TNH3 in serum and TCO2 in plasma were in agreement with those obtained by the conventional glutamate dehydrogenase (GDH) method and gas-sensing electrode method, respectively. The effects of composition of acceptor stream, cell constant of conductivity electrode, sample volume, flow rate and potential interferents on the FIA signals are also discussed.
Ammonia, total Carbon dioxide Conductometry Sensor Gas diffusion Teflon membrane Method comparison Interferences

"Flow-through Biosensor For Sequential Determination Of Total And Prostatic Acid Phosphatase Activity"
Sens. Actuat. B 1995 Volume 23, Issue 1 Pages 9-15
M. S&aacute;nchez-Cabezudo, J. M. Fern&aacute;ndez-Romero and M. D. Luque De Castro

Abstract: Sample (900 µL) diluted 1:5 in 100 mM trisodium citrate dihydrate of pH 4.8 (buffer A) was injected into a carrier stream (1.3 ml/min) of buffer A which merged with a stream of 0.27 mM p-nitrophenylphosphate in buffer A and passed through a reactor coil (250 cm x 0.5 mm i.d.) for hydrolysis. The p-nitrophenol produced was derivatized with 150 mM NaOH in a reactor coil (100 cm x 0.5 mm i.d.). The reaction product was absorbed on Dowex-1 anion-exchange resin (100-200 mesh; chloride form) contained in a biosensor constructed from a 1 mm path length flow cell (details given) for enhancement of detection at 405 nm. The product was eluted from the biosensor with 100 mM EDTA (disodium salt). The system was operated at 40°C. Inhibition of the prostatic acid phosphatase fraction was achieved with 50 mM L-(+)-tartaric acid. The calibration graph was linear for 0.2-20 iu/l of total acid phosphatase (TAP) and the RSD were 0.6-1.5%. The calibration graph for a conventional FIA method without use of the biosensor (details given) was linear for 5-300 iu/l of TAP and the RSD were 1.6-3.3%. Recoveries of TAP and prostatic acid phosphatase from serum were 95-102% and the results agreed well with those using the conventional method.
Acid phosphatase Sensor Spectrophotometry Method comparison Heated reaction Dowex

"Analysis Of Cortisol With A Flow Displacement Immunoassay"
Sens. Actuat. B 1997 Volume 45, Issue 1 Pages 63-69
Wilhelmina A. Kaptein, Jacco J. Zwaagstra, Kor Venema, Marcel H. J. Ruiters and Jakob Korf

Abstract: This study investigates a flow immunoassay for cortisol based on displacement of labelled antigen analogues from immobilized antibodies. On a displacement column, two antibodies with different affinities were immobilized on protein-A Sepharose, and a construct of cortisol and horseradish peroxidase (cort-HRP) was associated to the antibodies. Perfusion of the column with cortisol displaced a proportional amount of cort-HRP. Online detection was performed with an electrochemical detector measuring cort-HRP directly in a flow injection system. For investigating some characteristics of the displacement system, cort-HRP was also detected off-line, with a substrate for HRP, which changes OD492. We tested our system in buffer as well in serum for 2 days at a flow of 4-6 µl min-1. Analysis of the data show a detectable displacement signal at physiological cortisol concentrations. The specificity, stability of the columns and the influence of the antibody affinity were studied. (C) 1997 Elsevier Science S.A. 23 References
Cortisol Radioimmunoassay Immobilized antibody Kinetic Sepharose beads Perfusion

"Miniaturised Ion-selective Sensor Chip For Potassium Measurement In A Biomedical Application"
Sens. Actuat. B 1996 Volume 34, Issue 1-3 Pages 252-257
Albrecht Uhlig*, Frank Dietrich, Ern&ouml; Lindner, Uwe Schnakenberg and Rainer Hintsche

Abstract: A K-sensitive ISE was integrated onto a Si chip (1 x 5 mm i.d.) with a Ag/AgCl reference electrode coated with a poly(hydroxyethyl methacrylate) layer. The ISE incorporated a silicon rubber membrane containing valinomycin and a Ag/AgCl contact. Potentiometric response of the resulting sensor towards K was measured in Ringer's solution. The response time was ~10 s, the calibration graph was linear for up to 1 mM K and RSD were ~2% for continuous measurement in a flow system over 4 days. Response times for determination of K in blood or serum in a FIA system were longer; RSD were 2%. The sensors dimension allow it to be mounted in a catheter or cannular making it suitable for in vivo measurements.
Potassium Sensor Potentiometry

"Electrochemical Filter/biosensor Flow Injection Analysis System For The Direct Analysis Of Practical Samples"
Sens. Actuat. B 1993 Volume 14, Issue 1-3 Pages 541-542
Yusuke Okawa, Hiroyuki Kobayashi and Takashi Ohno

Abstract: Serum (10 µL) was injected into a flow-through electrolytic cell as a electrochemical filter with carrier buffer (1 ml/min) of phosphate-buffered 0.1 M NaCl of pH 6.4. The cell had an Ag/AgCl reference electrode, a carbon felt working electrode, a glucose oxidase monolayer-modified SnO2 electrode as an amperometric biosensor for glucose and was operated at 0.75 V vs. Ag/AgCl (0.3 V for uric acid). The FIA system was described previously (Okawa et al., Chem. Lett., 1991, 849). Glucose and uric acid were determined in control serum samples; results obtained agreed well with the nominal values. It was possible to determine up to 200 mM glucose.
Glucose Uric acid Electrochemical analysis Amperometry Electrode Sensor

"Thick-film Multichannel Biosensors For Simultaneous Amperometric And Potentiometric Measurements"
Sens. Actuat. B 1996 Volume 30, Issue 2 Pages 127-132
A. Silber, M. Bisenberger, C. Br&auml;uchle and N. Hampp*

Abstract: Miniaturized multichannel transducers were combined with FIA to determine several parameters at speed in a single sample. The transducers were fabricated using screen-printing and thick-film technology (details given); they contained 14 Au electrodes as working and counter electrodes and one Ag/AgCl reference electrode. Ion-selective membranes were made based on a PVC-poly(vinyl acetate) copolymer; enzyme membranes were made from organic-solvent-free aqueous polymer dispersions and were applied to the sensor electrodes by screen-printing or solder dispenser systems. The sensor was integrated in an FIA system for analysis of serum. The use of amperometric as well as potentiometric sensor channels on a single transducer successfully enabled control of the metabolite and electrolyte status of the serum.
Sensor Electrode Electrode Electrode Potentiometry Amperometry

"Optical Chemo- And Biosensors For Use In Clinical Applications"
Sens. Actuat. B 1997 Volume 40, Issue 1 Pages 71-77
C. M&uuml;llera, B. Hitzmanna, T. Schepera,* and F. Schubertb

Abstract: Biosensors based on a pH optode were fabricated for penicillin, urea, glucose and creatinine. They incorporated aminofluorescein which was excited at 480 nm and the emission was measured at 520 nm. A layer of the appropriate enzyme was immobilized directly on the optode tip carrying the fluorescent dye. For application in real processes, multichannel measurement was used and the biosensor was integrated into a FIA system. The complex signal was transformed and analyzed by computer using an ANN. Evaluation of data by the trained neural network allowed measurement of buffer ion and analyte concentrations. Results are reported for urea determination in serum and penicillin-G in buffer.
Urea Penicillin G Sensor Fluorescence Clinical analysis Neural network Optosensing Process monitoring

"Development Of Transient Data-acquisition System For Hyphenated Techniques Coupled With Inductively Coupled Plasma Atomic-emission Spectrometry"
Spectrochim. Acta B 1996 Volume 51, Issue 14 Pages 1753-1759
Xiaoru Wang*, Zhixia Zhuang, Chenglong Yang, Pengyuan Yang, Xiaomei Yan and Jianming Lin

Abstract: The apparatus described is suitable for use with FIA, HPLC, ET vaporization and laser ablation coupled with multielement ICP-AES. The internal computer of the spectrophotometer scans ten preselected photomultiplier channels at a maximum rate of 100 data points per s with a minimum integration time of 0.01 s per channel, and the data are transferred at 4800 baud through an RS-232C port to a Compaq 486 computer, which both collects and manipulates the data according to QuickBASIC routines. Good results are demonstrated for the determination of metals in serum by flow injection ICP-AES and in hair by ET-vaporization ICP-AES and for the speciation of metals in tea by HPLC-ICP-AES.
Metals Spectrophotometry HPLC Speciation Computer

"One-Shot Flow Injection Analysis With Immobilized Enzyme Columns: Clinical Applications"
Anal. Sci. 1987 Volume 3, Issue 3 Pages 277-278
M. OHYABU, M. FUJIMURA, K. TANIMIZU, Y. OKUNO, M. TABATA, M. TOTANI and T. MURACH

Abstract: A flow injection system is described (with diagrams) for the determination of glucose, lactic acid, uric acid and urea-N in serum, plasma or urine. Detection is by chemiluminescence at 425 nm after reaction of enzymatically formed H2O2 with luminol.
Glucose Lactic acid Uric acid Nitrogen, urea Clinical analysis Chemiluminescence Apparatus Immobilized enzyme Review Column

"Fluorimetric Determination Of Guanidino-compounds By High Performance Liquid Chromatography Using Water-soluble 9,10-dioxophenanthrene-3-sulfonate"
Anal. Sci. 1987 Volume 3, Issue 4 Pages 363-367
Y. KOBAYASHI, H. KUBO and T. KINOSHITA

Abstract: Serum samples (100 µL) were treated with 20% trichloroacetic acid (20 µL) and the mixture was centrifuged. A 60 µL portion of the supernatant solution was treated with 0.4 M NaOH (15 µL) and the solution (adjusted to pH 2.5 to 3.0) was analyzed by HPLC on a column (7.5 cm x 4.6 mm) of Nucleosil C8 (3 µm). The mobile phase (1 mL min-1) consisted of 5 mM Na octanesulfonate and 2 mM K 9,10-dioxophenanthrene-3-sulfonate in water - acetonitrile - methanol (93:2:5). Post-column derivatization was effected by heating the eluate to 80°C in a reaction coil (10 m x 0.5 mm) with 0.6 M NaOH as carrier solution. Fluorimetric detection was at 520 nm (excitation at 370 nm). The detection limits of nine guanidino-compounds investigated ranged from 6 pmol to 0.1 nmol.
Guanidino compounds HPLC Fluorescence Heated reaction Post-column derivatization

"Data Processing For Flow Injection Analysis Using Personal Computer Of MSX Standard System"
Anal. Sci. 1987 Volume 3, Issue 5 Pages 467-468
D. YOSHIZAWA, M. TOMODA, K. UCHIDA, K. FUKUSHIMA and S. SAITOH

Abstract: A data-processing system compatible with the MSX personal computer was developed. The detector signal from the flow injection analysis system was recorded by an analogue recorder, converted into a digital signal and processed by the computer (program flow-chart presented). The system was successfully applied in the determination of glucose in sera with a data processing time of <1 s.
Glucose Computer Signal processing

"Simultaneous Determination Of Triglycerides And β-D-glucose By Flow Injection Analysis Using Immobilized Enzyme Open Tubular Reactors"
Anal. Sci. 1987 Volume 3, Issue 6 Pages 553-556
S. YONEZAWA, F. MORISHITA and T. KOJIMA

Abstract: Carrier solution, viz, 0.05 M phosphate buffer (pH 8.0) containing 2 mM NAD+, is mixed via a 2- or 5 µL sample loop with serum sample solution containing triglyceride and β-D-glucose. The flow is split: one part passes sequentially through lipoprotein lipase and glycerol dehydrogenase reactors, and the remainder passes through a glucose dehydrogenase reactor, the enzymes being immobilized on the inner surface of whisker-walled glass capillary tubes. The effluent is adjusted to pH 4 to 5 with 0.15 M acetic acid for deproteinization on a Shimpack SPC-RPl column (Shimadzu), and the NADH is detected fluorimetrically (excitation at 340 nm, emission at 460 nm). The hydrolysis of triglycerides takes >10 min, so a stopped-flow stage is incorporated. The calibration graphs were rectilinear for 1 to 5 mM triglyceride and for up to 10 mM β-D-glucose. The coefficient of variation (n = 3) were 1% and 5% for 2 mM triglyceride and 4.0 mM β-D-glucose, respectively.
d-Glucose Triglycerides Fluorescence Immobilized enzyme Open tubular reactor Reactor Stopped-flow

"Micro-injection System For Flame Atomic Absorption Spectrometry Using Carrier Solution"
Anal. Sci. 1987 Volume 3, Issue 4 Pages 291-296
T. MAEDA, Y. TANIMOTO and N. OKAZAKI

Abstract: The nebulizer of the burner system was connected via a PTFE tube (0.5 mm diameter) to a reservoir of carrier solution which was continuously fed into the system during sample injection. Sample solution (20 to 100 l) were injected with a micro-syringe into the carrier stream via a silicone rubber septum. Maximum sensitivity was obtained with a carrier flow rate of 7.5 mL min-1. The effect of sample volume on rectilinearity of response was investigated and reported. The coefficient of variation was <3% for Mg and Ca and <5% for Cu and Zn. With use of La as carrier, the interference of Al and PO43- in the determination of Ca and Mg was suppressed significantly. The method was successfully applied in the determination of Mg, Ca, Cu and Zn in serum.
Magnesium Calcium Copper Zinc Spectrophotometry Injector

"Applicability Of Chromogenic 14-crown-4 Derivative To Extraction-spectrophotometric Flow Injection Analysis For Lithium Ion In Blood Serum"
Anal. Sci. 1988 Volume 4, Issue 2 Pages 221-222
K. KIMURA, S. IKETANI, H. SAKAMOTO and T. SHONO

Abstract: Synthetic serum (50 µL) and 0.3 mM 6-dodecyl-6-(2-hydroxy-3,5-dinitrophenyl)-14-crown-4 solution in CHCl3 (50 µL) were injected simultaneously into 0.125 mM H3BO3 - KOH buffer (pH 9.50) and CHCl3 carrier streams, respectively. The streams were merged at a T-shaped segmentor and passed through a 1-m extraction coil before phase separation with a micro-porous PTFE membrane filter. The absorbance of the organic phase was measured at 410 nm. The calibration graph was rectilinear from 0.4 to 2 mM Li+. There was no interference by Na+.
Lithium Clinical analysis Spectrophotometry Sample preparation Crown ether Extraction Interferences Organic phase detection Phase separator Teflon membrane

"Simultaneous Assay Of Aspartate Aminotransferase And Alanine Aminotransferase By Flow Injection Method Using Immobilized Enzyme Reactors"
Anal. Sci. 1988 Volume 4, Issue 6 Pages 579-582
T. SUGAYA, S. NAITO, S. YONEZAWA, F. MORISHITA and T. KOJIMA

Abstract: Flow injection systems for determination of the cited enzymes (I and II, respectively) are described and illustrated. For determination of I, the sample is injected into a carrier stream of 0.3 mM aspartate, 0.05 M α-ketoglutarate, 0.5 mM NADH and 0.1 M Tris buffer (pH 7.5) before passing to a malate dehydrogenase-immobilized reactor maintained at 36°C. After removal of proteins in a column of Toyopearl AF-blue 650MH, I was determined by measuring the decrease in NADH concentration at 470 nm (excitation at 340 nm). For II, sample was separately injected into two carrier streams, one containing 0.3 mM alanine, 0.05 M α-ketoglutarate, 0.5 mM NADH and 0.1 M Tris buffer and the other being the same but without alanine. Each solution passed through a lactate dehydrogenase-immobilized reactor before merging of the solution and proceeding as before. A combined system for simultaneous determination of I and II is also described. The method was applied to determine I and II in serum and results agreed with those obtained by a UV method.
Enzyme, aspartate aminotransferase Alanine aminotransferase Clinical analysis Fluorescence Heated reaction Immobilized enzyme Method comparison

"Glucose Sensor Carrying Monomolecular Layer Of Glucose Oxidase Covalently Bound To Tin(IV) Oxide Electrode"
Anal. Sci. 1989 Volume 5, Issue 5 Pages 507-512
Y. OKAWA, H. TSUZUKI, S. YOSHIDA and T. WATANABE

Abstract: The enzyme-immobilized SnO2 electrode (Watanabe et al., Chem. Lett., 1988, 1183) was immersed in 67 mM phosphate buffer (pH 6.4), to which a portion of glucose solution was added. The mixture was stirred for 10 s and the change in current was recorded; the steady-state current was taken as the output of the sensor, in which Ag - AgCl and Pt reference and counter-electrodes, respectively, were incorporated. The system was used in a flow injection analysis system; the detection limit was 10 µM. Calibration graphs were rectilinear up to 10 mM glucose. The flow injection system was applied for the determination of glucose in diluted serum; anion-exchange HPLC was used for preliminary removal of interfering matrix components.
Glucose Electrode Electrode Sensor Immobilized enzyme Buffer Interferences

"Determination Of Reducing Sugars By High Performance Liquid Chromatography With Post-column Fluorescence Derivatization Using 1,2-bis-(4-methoxyphenyl)ethylenediamine"
Anal. Sci. 1989 Volume 5, Issue 6 Pages 675-680
Y. UMEGAE, H. NOHTA and Y. OHKURA

Abstract: Reducing sugars were separated as their borate complexes by HPLC on a column (15 cm x 4.6 mm) of TSK gel Sugar AXG at 60°C, with 0.5 M borate buffer of pH 8.7 as mobile phase (0.4 mL min-1). Derivatization with the cited reagent was then carried out at 140°C in the presence of NaOH, and the derivatives were detected by fluorimetry at 460 nm (excitation at 330 nm). Serum or urine was deproteinized with acetonitrile in the presence of α-melibiose as internal standard, and borate buffer was added to the centrifuged solution before HPLC. Lactose in urine was hydrolyzed with β-galactosidase at pH 7.3 (phosphate buffer) before the analysis. Response was rectilinear for 0.1 to 10 nmol of reducing sugar injected, and detection limits ranged from 30 to 160 pmol.
Sugars, reducing HPLC Fluorescence Post-column derivatization Heated reaction

"Flow Injection Analysis With Chemiluminescence Detection Of Glucose And Uric Acid Using Immobilized Enzyme Reactor"
Anal. Sci. 1991 Volume 7, Issue 5 Pages 715-718
K. NAKASHIMA, N. HAYASHIDA, S. KAWAGUCHI, S. AKIYAMA, Y. TSUKAMOTO and K. IMAI

Abstract: A rapid and sensitive flow injection method with peroxyoxalate chemiluminescence detection for the determination of glucose (I) and uric acid (II) in serum with use of an immobilized enzyme reactor is described. Chemiluminescence produced in a flow cell by the reaction of H2O2 with bis(2,4,6-trichlorophenyl)oxalate and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine, was monitored. Calibration graphs were rectilinear up to 0.3 mM I and 10 µM II; both detection limits were 0.1 µM. The coefficient of variation was 1.1% for 20 µM I and 2.2% for 5 µM II. The results compared well with those obtained by colorimetric methods.
Glucose Uric acid Chemiluminescence Immobilized enzyme Reactor Sensitivity

"Flow Injection Method For The Polarographic Determination Of L-lactate Using An Immobilized-enzyme Reactor"
Anal. Sci. 1992 Volume 8, Issue 2 Pages 165-168
S. HIKIMA, K. HASEBE and T. KAKIZAKI

Abstract: Any O not consumed in reaction with lactate catalyzed by lactate oxidase is retarded by an ODS column to separate it from the H2O2 and pyruvate products, which are then detected amperometrically. An Inertsil ODS-2 (1 cm x 4 mm) column is used with a static-Hg-drop electrode, a Pt-wire counter electrode and an Ag - AgCl reference electrode. The lactate oxidase was immobilized on Chitopearl. The detection limit was 10 µM and the coefficient of variation was 2.8% at 0.2 mM lactate. The method was applied to serum. A sensitive flow injection system for L-lactate with polarog. detection is described. The system mainly utilizes saturated dissolved oxygen in samples for enzymatic reactions to convert L-lactate and oxygen to pyruvate and hydrogen peroxide; and then, any excess amt. of dissolved, electroreducible oxygen which is not consumed in the enzymatic reaction can be retarded by an ODS-column to sep. it from the pyruvate and hydrogen peroxide. After that, both the pyruvate and hydrogen peroxide are measured by the polarog. method using a static mercury drop electrode. The enzyme, lactate oxidase, was covalently immobilized on Chitopearl composed of chitosan, and packed into a reactor. The detection limit under this system was 9.9 x 10^-6 M and the relative standard deviation at 2.0 x 10^-4 M L-lactate was 2.8%. The method was used to determination of lactate in serum samples with satisfactory results.
l-Lactate Polarography Electrode Indirect Chitosan Immobilized enzyme C18 Interferences

"Quantification Of Histamine By Post-column Fluorescence Detection High Performance Liquid Chromatography Using Orthophthalaldehyde In Tetrahydrofuran And Reaction Mechanism"
Anal. Sci. 1994 Volume 10, Issue 2 Pages 259-265
K. KURUMA, E. HIRAI, K. UCHIDA, J. KIKUCHI and Y. TERUI

Abstract: Serum (1 ml) was mixed with 0.6 mL of 10% trichloroacetic acid and centrifuged for 2 min at 10 500 g and 0.1 mL of the supernatant solution was analyzed by HPLC. Urine was diluted tenfold with water and a 0.1 mL portion was analyzed by HPLC. Both were analyzed on a column (15 cm x 6 mm i.d.) of TSKgel Catecholpak at 40°C with a mobile phase (1 ml/min) of 0.2 M propionate buffer of pH 4.5 containing 86 mM NaCl. For post-column derivatization the eluate was mixed with a 0.1% orthophthalaldehyde solution in THF (0.6 ml/min) at 40°C with fluorescence detection at 430 nm (excitation at 360 nm). The calibration graph was linear for 10^-1280 pg of histamine with a detection limit of 5 pg and RSD (n = 9) of 3.2-4.5%. Recoveries of 7-110 nM-histamine in serum were 104-114% with intra-day and inter-day RSD (n = 5) of 0.8 and 3.3%, respectively, for 27.8 nM-histamine. In urine recoveries were 99-102% for 170-2780 nM-histamine with intra- and inter-day RSD (n =5) of 0.9 and 2%, respectively, for 694 nM-histamine. Reaction mechanism studies are also presented.
Histamine HPLC Fluorescence Post-column derivatization

"Flow Injection Method For The Simultaneous Determination Of D-glucose And 3-hydroxybutyrate With Co-immobilized Enzyme Reactors And A Chemiluminometer"
Anal. Sci. 1995 Volume 11, Issue 4 Pages 605-609
N. KIBA, H. KOEMADO and M. FURUSAWA

Abstract: Plasma or serum (20 µL) was diluted 10-fold with borate buffer of pH 9.4 and filtered (details given) and 80 µL portions of filtrate were injected into a mixed stream of 0.1 M borate buffer of pH 9.4 (0.7 ml/min) and 2 mM NAD+ in 0.01 M-phosphate buffer of pH 7 (0.7 ml/min). The stream was split into two with half pumped through a co-immobilized glucose dehydrogenase/NAPH oxidase on aminated poly(vinyl alcohol) [PVA] beads reactor (10 x 4 mm i.d.) for detection of D-glucose (glucose; I) and the other was pumped through a 3-hydroxybutyrate (II) dehydrogenase/NADH oxidase immobilized on PVA reactor (50 x 4 mm i.d.) for the detection of II. The streams emerging from each reactor were mixed with a stream of 3 mM luminol in 0.4 M carbonate buffer of pH 10.5 (0.5 ml/min) and of 20 mM potassium hexacyanoferrate solution (0.5 ml/mm) and the chemiluminescence intensity produced was measured. The calibration graph was linear from 10 µM to 1 mM and 1 µM to 0.5 mM, respectively, for I and II and the corresponding detection limits were 20 and 0.8 µM. Inter- and intra-day RSD were 0.88 and 1.1 and 2.3 and 2.9%, respectively, for I and II at 5.52 mM and 49 µM, respectively.
Glucose 3-hydroxybutyrate Chemiluminescence Immobilized enzyme Poly vinyl alcohol beads

"Determination Of The Inorganic Phosphorus Content In Serum By The Use Of Immobilized Enzyme Reactions In An FIA System"
Anal. Sci. 1996 Volume 12, Issue 1 Pages 137-139
H. MORI, M. KOGURE, H. TAKEI and H. YAMAMOTO

Abstract: Serum was diluted 1:100 with carrier solution (see below) and a 50 µL portion was injected into a carrier stream of 100 mM PIPES buffer of pH 7.5 containing 100 mM sucrose, 3 mM MgCl2, 1 mM NADP+ and 50 µM-glucose diphosphate (0.4 ml/min) of a flow injection system (schematic shown). The mixture was transferred via an ODS column (4 cm) to an enzyme reactor column (8 cm x 2 mm i.d.) operated at 30°C and packed with 40 iu each of sucrose phosphorylase, phosphoglucomutase and glucose-6-phosphate dehydrogenase immobilized on aminopropyl-bonded glass beads by the method of Mori et al. (Anal. Lett., 1994, 24, 309). The NAPDH formed by the enzyme reactions was fluorimetrically detected at 460 nm (excitation at 340 nm) on a Hitachi F-1000 spectrofluorimeter with a 12 µL flow cell. The calibration graph was linear from 1-100 µM-phosphate (2-6 mg P/100 ml). The RSD (n = 10) was 2.8% for 12.6 µM-phosphate. Results agreed well with those obtained by the method of Berti et al. (J. Clin. Chem. Clin. Biochem., 1988, 26, 399) using an immobilized purine nucleoside phosphorylase-xanthine oxidase-peroxidase enzyme mixture and the Molybdenum Blue method.
Phosphorus Fluorescence Glass beads Immobilized enzyme

"Determination Of Esterase Activity By Flow Injection Analysis With Amperometric Detection"
Anal. Sci. 1996 Volume 12, Issue 2 Pages 343-347
K. TAKAMURA, T. FUSE and F. KUSU

Abstract: For the analysis of lipase (I), sample (15 µL) was incubated with 15 µL olive oil, 40 µL water and 35 µL 0.1 M KH2PO4 and 0.05 M Na2B4O7 buffer of pH 9 for 10 min at 37°C. The mixture was extracted with 3 x 3 mL diethyl ether, the ether layers were combined and evaporated to dryness. The oily precipitate was dissolved in 300 µL ethanol containing 3 mM vitamin K3 (VK3) and 38 mM LiClO4 for test solution preparation. A blank test solution was prepared by omitting incubation. For cholinesterase (II), sample (15 µL) was incubated with 10 µL 0.1 M benzoylcholine and 10 µL 0.01 M phosphate buffer of pH 7.5 for 15 min at 37°C. Ethanol (5 ml) containing 3 mM VK3 and 38 mM LiClO4 was added for test solution. A blank test solution was prepared as above. Portions (5 µL) of test and blank test solutions for both I and II were injected into the carrier solution (0.6 ml/min) of a FIA system and the acids detected at -0.3 V vs. AgCl and the enzyme activities calculated (equation given). The calibration graphs were linear from 10^-1000 and 10^-2000 U/l, respectively, for I and II.
Enzyme, cholinesterase Triacylglycerol lipase Amperometry

"Voltammetric Determination Of Phosphate In Blood"
Anal. Proc. 1982 Volume 19, Issue 9 Pages 443-444
M. A. Abdalla, N. K. Bsebsu and A. G. Fogg

Abstract: Orthophosphate has been determined as 12-molybdophosphate in aqueous acidic molybdate solution by linear-sweep and differential-pulse voltammetry at a glassy carbon electrode in a static system.l The procedure recommended is essentially an anodic-stripping procedure as a positive-going potential scan is used to re-oxidise molybdophosphate reduced at the electrode surface earlier in the scan. These techniques were also applied to the determination of phosphate, silicate, arsenate and germanate in the solution conditions in which the fl-heteropolyacids are stabilised by the addition of acetone or ethanol, and, to the determination of phosphate as phosphovanadomolybdate. All of these methods have been adapted to the voltammetric flow-injection determination of the determinands by injection of the pre-formed heteropolyacids into eluents with the composition of the reagent blanks3 By this means 0.01 and 0.1 pg mL-1 of silica and phosphate, respectively, were determined. The application of these static and flow-injection voltammetric methods to the determination of phosphate in blood serum is described in this paper.
Phosphate Voltammetry

"Monitoring Of Immunoprecipitin Reactions Using Flow Injection Analysis"
Anal. Proc. 1985 Volume 22, Issue 1 Pages 16-17
Arwel Hughes, Paul J. Worsfold

Abstract: An immunoprecipitation method, involving flow injection analysis with automated merging zones (cf. Worsfold and Hughes, Anal. Abstr., 1984, 46, 10D203) is described for the determination of human serum IgG with use of goat anti-human IgG antiserum, and measurement of turbidity (at 340 nm) 44 and 74 s after mixing. The calibration graph is rectilinear for 284.4 mg L-1 with coefficient of variation of 2 to 6%.
Immunoglobulin G Immunoassay Turbidimetry Merging zones

"Applications Of Immobilized Enzymes In Flow Injection Analysis"
Anal. Proc. 1985 Volume 22, Issue 1 Pages 6-8
Stephen, M. Masoom, Alan Townshend

Abstract: Samples of blood serum and soft drinks were injected into a stream of phosphate buffer solution (pH 7) which then passed through a glass column (2.5 cm x 2.5 mm) containing glucose oxidase (immobilized on controlled-pore glass) to a flow-through amperometric detector for the determination of H2O2. Sucrose in soft drinks was determined by use of immobilized β-D-fructofuranosidase, aldose 1-epimerase and glucose oxidase. The limit of detection was 1 µM-H2O2.
Glucose Sucrose Amperometry Immobilized enzyme Controlled pore glass

"Lithium Ion-selective Electrode In Flow Injection Analysis"
Anal. Proc. 1986 Volume 23, Issue 2 Pages 62-64
Gadzekpo, G. J. Moody and J. D. R. Thomas

Abstract: An electrode, based on the Ba adduct of a polyoxypropene glycool, as its tetraphenylborate, with PVC as matrix and, e.g., dioctyl phenylphosphonate as plasticizer, responded to Li+ rectilinearly (slope 57 mV per decade) down to 0.1 mM. In the presence of 0.14 M NaCl, the calibration graph was rectilinear down to 10 mM. The response was good in artificial serum electrolyte. The selectivity of the electrode, which was 27 times more sensitive for Li+ than for Na+, compared favourably with that of other neutral-carrier-type Li+ sensors.
Lithium Electrode Electrode Potentiometry Sensitivity

"Nylon Mesh And Platinum Wire Enzyme Amperometric Electrodes"
Anal. Proc. 1988 Volume 25, Issue 4 Pages 108-110
G. S. Sanghera

Abstract: Three enzyme amperometric electrodes are described for use in flow injection analysis. (I) Single-enzyme electrodes were used for determination of glucose and cholesterol and were based on glucose oxidase and cholesterol oxidase, respectively, immobilized on nylon mesh and held over a Pt electrode. The anodic decomposition of H2O2 (produced in the enzymatic reaction) was monitored at a Pt working electrode. (II) For determination of glucose and xanthine - hypoxanthine, electrodes comprised immobilized (on nylon mesh) glucose oxidase - peroxidase and xanthine oxidase - peroxidase, respectively, and the enzymatically generated H2O2 was monitored amperometrically after its peroxidase-catalyzed reaction with Fe(CN)64-; the generated Fe(CN)63- is reduced at a Pt electrode at -0.1 V vs. Ag - AgCl. (iii) The direct covalent attachment of glucose oxidase to an activated Pt surface was used to form a 'micro' glucose enzyme electrode. The electrodes in(II) were tested for the flow injection determination of glucose in serum and hypoxanthine in fish.
Glucose Cholesterol Hypoxanthine Amperometry Electrode Electrode Immobilized enzyme Method comparison

"Lithium Ion-selective Electrodes: Optimization Studies For Blood Serum Analysis"
Anal. Proc. 1989 Volume 26, Issue 1 Pages 2-4
C. W. Beswick, G. J. Moody, J. D. R. Thomas

Abstract: The selectivity over Na was studied for five Li-selective electrodes, and the best performance was obtained with a membrane consisting of 1% of (dicyclohexylamino)[2-(NN-di-isobutylcarbamoyl)-5-methylcyclohexyl]methanone as sensor, 66% of 2-nitrophenyl octyl ether as mediator, and 33% of PVC. The sensor was incorporated into a flow injection analysis system (cf. Alegret et al., Anal. Abstr., 1986, 48, 9J11), with use of an artificial electrolyte, serum A, as carrier solution (Gadzekpo et al., Analyst (London), 1986, 111, 567). No interference was observed between pH 5.5 and 9.0.
Lithium Electrode Electrode Interferences Optimization

"Flow Injection Analysis Of Phospholipids"
Anal. Proc. 1989 Volume 26, Issue 2 Pages 64-65
F. F. Barretto, J. M. Slater

Abstract: The flow injection system was based on a Stelte micro-cell detector, modified by machining a wall-jet electrode chamber for the working electrode, viz, a Pt electrode covered with nylon mesh on which were immobilized phospholipase D and choline oxidase (1:1). Optimum flow rate was 2.33 mL min-1, and the H202 produced was detected at +650 mV vs. Ag - AgCl. The carrier solution was 0.2 M glycine buffer of pH 10. The calibration graph was rectilinear from 0.2 to 0.6 g L-1 of phospholipids in serum, and the detection limit was 0.01 g L-1 of phosphatidylcholine. The enzyme electrode can be used for >8 weeks.
Phospholipids Palmitoyloleoylphosphatidylcholine Electrode Electrode Electrode Immobilized enzyme Buffer pH Detection limit Detector Optimization

"Flow Injection Fluorescence Immunoassay For Serum Phenytoin Using Perfusion Chromatography"
Anal. Proc. 1994 Volume 31, Issue 1 Pages 7-8
Mark Evans, Derek A. Palmer, James N. Miller and Martin T. French

Abstract: Rhodamine-labelled phenytoin (50 µL) and standard phenytoin (50 µL) were mixed with 100 µL of phenytoin antiserum diluted with PBS of pH 7.4 (details given). The mixture was incubated for 2 min and a 25 µL portion injected into a carrier stream of PBS of pH 7.4 (2.7 ml/min). The stream was passed through a Poros IIA immunoreactor (protein A immobilized on a perfusion matrix) and, after the unbound species had been eluted, the carrier stream was switched to citrate buffer of pH 2.5 (2.7 ml/min) to elute the bound complex which was detected fluorimetrically at 595 nm (excitation at 579 nm). A diagram of the manifold used is given. The detection limit was 0.8 ng/ml of phenytoin, the RSD (n = 10) were 3.2-6% and recoveries were 115-124%. The total assay time was 3.5 min. The method was applied to the analysis of serum.
Phenytoin Chromatography Immunoassay Fluorescence Perfusion

"Thiophilic Gels: An Alternative To Protein A And G For Use In Flow Injection Immunoassays"
Anal. Proc. 1994 Volume 31, Issue 4 Pages 123-125
Derek A. Palmer, Mark Evans and James N. Miller

Abstract: Flow injection immunoassays were developed for theophylline (I) using a thiophilic affinity gel (T-gel). For the pre-incubation assay, fluorescein isothiocyanate labelled I was incubated with sheep anti-I serum for 2 min then injected onto a T-gel immunoreactor using 50 mM sodium phosphate containing 0.5 M potassium sulfate of pH 8 as binding buffer. The bound fraction was eluted with 50 mM sodium phosphate of pH 8 and the elution peak area was measured fluorimetrically at 525 nm (excitation at 495 nm). The assay was completed in just over 5 min; this could be reduced to 5 min using the thiophilic equivalent to POROS affinity matrix [cf. Regnier, Nature (London), 1991, 350, 634]. For the online assay, the antibody and the labelled-sample were sequentially injected onto the T-gel immunoreactor and the bound fraction was measured as above. The assay could be performed in 4 min. Limits of detection were 0.05 and 0.08 µg/ml of I for pre-incubation and online assays, respectively. The corresponding recoveries and RSD were 82.7-92.1% and 95.2-114.6%, and 4.7-9.1% and 3.2-5.7%. The advantages of T-gel over proteins A and G are discussed.
Theophylline Immunoassay

"New Possibilities For Open-closed Flow Injection Systems: An Approach To Interference Removal"
Anal. Proc. 1994 Volume 31, Issue 8 Pages 233-235
R. Quiles-Zafra, J. M. Fern&aacute;ndez-Romero and M. D. Luque De Castro

Abstract: A new use of open-closed systems for the removal of interference in the enzymatic-fluorimetric determination of K in serum is proposed. The method is based on monitoring the signal from the interferent before development of the derivatization reaction. Serum was diluted 1:100 and a 150 µL sample was injected into a carrier stream (1.5 ml/min) of buffer solution of 200 mM triethanolamine/10 mM MgSO4/40 mM LiCl/ 5 M HCl of pH 7.2. this was mixed with a reagent stream of 10 mM ADP/10 mM phospho(enol)pyruvate/0.35 mM NAD H and passed through a reactor containing immobilized glutamate dehydrogenase (to remove ammonium interference) and on to a detector for monitoring NADH consumption. The stream then passed through a reactor containing immobilized pyruvate kinase and lactate dehydrogenase for derivatization. After the first passage of the stream through the detector the system was closed and the derivatization reaction developed and monitored as many times as the reactant plug was recirculated. The linear range was 25-500 µM-K.
Potassium Fluorescence Interferences

"Immobilization Of Glucose Oxidase And Peroxidase To A Urea Derivative Of Granulated Microcrystallized Cellulose"
Acta Biotechnol. 1996 Volume 16, Issue 1 Pages 81-88
I. P. Ivanov

Abstract: Glucose oxidase and peroxidase were immobilized individually to a urea derivative of granulated microcrystallized cellulose activated by formaldehyde. The catalytic properties of the immobilized enzymes were studied and compared to those of the soluble enzymes. The immobilized glucose oxidase and peroxidase were used for the manual determination of the concentration of glucose in sera. The developed method is characterized by high analytical reliability and comparatively low cost. The results correlated well with those obtained by using a BECKMAN glucoanalyzer, utilizing soluble glucose oxidase.
Urea Clinical analysis Reactor Enzyme Immobilized enzyme Method comparison

"Determination Of Calcium And Magnesium In Blood Serum By Flow Injection Analysis And Atomic Absorption Spectrometry"
Acta Cient. Venez. 1982 Volume 33, Issue 4 Pages 371-271
Gallignani, M.;Burguera, J.L.;Burguera, M.

Abstract: NA
Calcium Magnesium Spectrophotometry

"A Simple Flow Injection Analysis For Studies Of Drug-protein Binding Interactions In Serum"
Acta Pharm. Nord. 1989 Volume 1, Issue 5 Pages 291-294
Gizurarson S, Jensen A, Foss S.

Abstract: Studies of drug-protein binding constants (ka) and of the degree of binding (%B) in biological fluids have been carried out by means of flow injection analysis (FIA). The association constants found for clinically relevant concentrations agree with the literature data. The degree of binding found for discoumarol, phenytoin and sulfisoxazole is also in agreement with the literature whereas that of warfarin is slightly lower.
Protein, binding Discoumarol Phenytoin Sulfisoxazole Warfarin Drugs Fluorescence

"Direct Determination Of Sodium And Potassium In Blood Serum By Flow Injection And Atomic Absorption Spectrophotometry"
An. Acad. Bras. Cienc. 1983 Volume 55, Issue 2 Pages 209-211
Burguera, J.L.;Burguera, M.;Gallignani, M.

Abstract: A simple and reliable method for the measurement of sodium and potassium in blood serum without any sample dilution by using flow injection and atomic absorption spectrophotometry is described. A sample throughout of 100 measurements per hour is possible. Matrix problems have not been encountered. The coefficient of variation for within-run determinations is about 1,14 and 2,36% for sodium and potassium, respectively, in serum samples (n = 10). The method is easily adaptable to pediatric research, because of the low required sample volume of 5 µL. Undiluted serum samples are injected into a stream of water, and the Na and K are determined by AAS (air - acetylene flame). Optimum conditions include injection of a 5 µL sample and flow rates of carrier solution of 6 and 4 mL min-1 for Na and K, respectively.
Sodium Potassium Clinical analysis Spectrophotometry Interferences Small sample

"Determination Of Aluminum In Serum By A Flow Injection System With Preconcentration On Dowex A-1 Resin And Flame Atomic Absorption Spectrophotometry"
An. Quim. 1987 Volume 83, Issue 1B Pages 95-100
Hernandez, P.;Rodriguez, J.;Sevilla, M.T.;Hernandez, L.

Abstract: Serum (2 ml) is treated with 0.3 mL of HClO4 and 0.7 mL of concentrated HNO3 in a platinum capsule, the solution is evaporated just to dryness, and the residue is dissolved in 2 mL of 0.4 M acetic acid - ammonium acetate buffer of pH 4. A 1 mL portion of this solution is injected into a stream of the same buffer which passes through a 5.4-cm bed of Dowex A-1 resin, on which the Al is adsorbed. The Al is directly eluted by 6.0 M HNO3 into the nebulizer of a flame AAS instrument for determination at 390 nm. The lower limit of 50 µg L-1 of Al can be improved by using the standard-additions method, which affords a coefficient of variation of 2.0% at 15 µg l-1.
Aluminum Ion exchange Spectrophotometry Dowex Preconcentration Standard additions calibration Resin

"An In-situ Electrosynthesized Poly-o-phenylenediamine/glucose Oxidase Amperometric Biosensor For Flow Injection Determination Of Glucose In Serum"
Ann. Chim. 1992 Volume 82, Issue 5-6 Pages 219-234
Centonze, D.;Guerrieri, A.;Malitesta, C.;Palmisano, F.;Zambonin, P.G.

Abstract: Poly-o-phenylenediamine films were electrochemically grown on Pt electrodes at a constant potential using a 5 mM phenylenediamine (I) solution in acetate buffer, pH 5.2 (I = 0.2). For enzyme immobilization, the I solution contained, e.g., glucose oxidase solution (500 iu mL-1). Glucose in serum (20 µL) was determined by flow injection amperometric detection with the prepared electrode. The detection potential was +0.65 V vs. Ag - AgCl standard electrode, and a flow rate of 0.1 mL min-1 was used at room temperature The results compared well with a colorimetric enzymatic kit method using 4-aminophenazone - phenol as the chromogen. The method was rectilinear up to 15 mM glucose; no electrode fouling was observed during the lifetime of the sensor (1 to 2 weeks). The immobilization of glucose oxidase (GOD) in electrochemically synthesized poly-o-phenylenediamine (PPD) film permits the realization of an amperometric biosensor for the determination of glucose in serum by flow injection analysis (FIA). The biosensor was prepared in-situ (i.e., in the FIA system) by injecting a solution containing the monomer and the enzyme. The influence of some experimental parameters (e.g., deposition potential and charge, monomer and enzyme concentration, pH and flow rate) on the sensor's performance was studied to optimize glucose response minimizing, at the same time, interference effects caused by urate, ascorbate, cysteine, and acetaminophen. Linearity was observed up to about 15 mM and the lifetime of the sensor ranged within 1-2 wk. No electrode fouling was observed and undild. serum could be directly injected. Results for glucose determination in serum compared well (paired t-test at 95% confidence level) with those obtained by a standard enzymatic-colorimetric method.
Glucose Amperometry Sensor Electrode Electrode Immobilized enzyme Method comparison Optimization Interferences

"Improved Continuous Flow Method For Serum Creatinine Using The Jaffe Reaction"
Ann. Clin. Biochem. 1980 Volume 17, Issue 3 Pages 153-154
White WA, Attwood EC.

Abstract: A method for the measurement of creatinine is described in which serum is dialysed directly into a combined alkaline picrate reagent. Baseline noise and drift are eliminated, and precision is improved by avoiding the need to add further reagents after dialysis. This may be of particular interest to laboratories that perform this assay on first-generation AutoAnalyzer equipment.
Creatinine Clinical analysis Technicon

"Improved Continuous-flow Analysis For Serum Bilirubin"
Ann. Clin. Biochem. 1980 Volume 17, Issue 5 Pages 262-271
Porntip H Lolekha

Abstract: A simple and improved method of continuous-flow analysis for total and direct bilirubin in serum is described. Advantages of the method are: small serum and reagent volumes; the use of less concentrated caffeine solution in the accelerator; the substitution of distilled water for hydrochloric acid in the direct bilirubin channel; and the close agreement of results obtained by the present and Jendrassik-Grof reference method when purified bilirubin is used as the calibrating standard.
Bilirubin Clinical analysis Method comparison

"Zinc And Copper Determination In Microsamples Of Serum By Flow Injection And Atomic Absorption Spectroscopy"
Ann. Clin. Biochem. 1982 Volume 19, Issue 5 Pages 338-344
Bernard F Rocks, Roy A Sherwood, Linda M Bayford, Clifford Riley

Abstract: A new approach to the direct determination of copper and zinc in serum and plasma is described. The sample is injected into a continuously pumped stream of water which is fed into the nebuliser of an atomic absorption spectrophotometer. Analytical results are obtained as a series of sharp peaks on a chart recorder. Analytical variables have been investigated, and the proposed method gave results comparable to those obtained using a conventional method based on precipitation of serum proteins with trichloroacetic acid. The proposed method takes less time to perform and was found to give more precise results than the conventional method. In addition, the flow injection analysis method can be performed using microsamples (10-100 µL) and is thus ideally suited for use on children.
Copper Zinc Clinical analysis Spectrophotometry Spectrophotometry Optimization Small sample

"Serum Iron And Total Iron-binding Capacity Determination By Flow Injection Analysis With Atomic Absorption Detection"
Ann. Clin. Biochem. 1983 Volume 20, Issue 2 Pages 72-76
Bernard F Rocks, Roy A Sherwood, Zoe J Turner, Clifford Riley

Abstract: The deproteinised sample (150 µL) is 'injected' into a continuously flowing stream of deionised water which is pumped, via a capillary tube, to the nebuliser of an atomic absorption spectrophotometer. Analytical readout is obtained, in the form of transient peaks, 6 s after sample injection. Before injection traces of haemoglobin are removed from the serum by treatment with trichloroacetic acid-ascorbate solution. This protein precipitant facilitates rapid removal of haemoglobin bound iron without the need for heating. After centrifugation the supernatant solution is introduced into the flowing stream by use of a novel inexpensive 'injector'. Analytical recovery and precision are good, and results compare well with those obtained by a standard AutoAnalyzer procedure.
Iron Clinical analysis Spectrophotometry Method comparison Segmented flow

"Use Of Lucifer Yellow VS As A Label In Fluorescent Immunoassays Illustrated By The Determination Of Albumin In Serum"
Ann. Clin. Biochem. 1983 Volume 20, Issue 4 Pages 213-216
Bailey MP, Rocks BF, Riley C.

Abstract: The properties of Lucifer yellow VS (I; Aldrich Chemical Co.) are described; the advantages of this dye over the more commonly used fluorescent labels are discussed. I was used in a heterogeneous fluoro-immunoassay for human serum albumin, in which the bound fractions were pptd. by polyoxyethylene glycol and the fluorescence of the supernatant solution was measured, with use of a continuous-flow system, at 520 nm (excitation at 430 nm). Results agreed well with those of a dye-binding assay (r = 0.97). The detection limit was ~4 mg l-1. Other potential uses of I are discussed.
Albumin Clinical analysis Immunoassay Fluorescence

"Direct Determination Of Calcium And Magnesium In Serum Using Flow Injection Analysis And Atomic Absorption Spectroscopy"
Ann. Clin. Biochem. 1984 Volume 21, Issue 1 Pages 51-56
Rocks BF, Sherwood RA, Riley C.

Abstract: The use of flow injection analysis for the direct determination of calcium and magnesium in blood serum and plasma is described. An inexpensive rotary valve is used to inject the serum sample (4 µL) into a flowing non-segmented stream of reagent which carries the sample slug through a long narrow-bore coil--where it gradually disperses--and into the nebuliser of an atomic absorption spectrometer. This on-stream sample dilution removes the need for predilution of the sample. The resulting absorbance signals are recorded as peaks less than 40 seconds after sample injection. Analytical recoveries and precision are good for both elements and the results by flow injection analysis compare well with established routine methods. In the system described, for use with serum or plasma, a rotary valve was used to inject a 4 µL sample into a flowing non-segmented reagent stream, which carried the sample 'slug' through a long narrow-bore coil, in which it gradually dispersed, into the nebulizer of the spectrometer. This method of on-stream dilution overcame the need for pre-dilution of the sample, and the resulting absorbance peaks were recorded <40 s after sample injection. Analytical recovery and precision were as good as or better than those of other methods, and the results correlated well with those obtained by using manual dilution.
Calcium Magnesium Clinical analysis Spectrophotometry Dilution Method comparison Small sample

"Examination Of The Hexokinase Method For Serum Glucose Assay Using External Quality Assessment Data"
Ann. Clin. Biochem. 1986 Volume 23, Issue 1 Pages 92-96
Westwood A, Bullock DG, Whitehead TP.

Abstract: Glucose was determined in serum, for the UK External Quality Assessment Scheme, by using hexokinase, by a continuous-flow glucose oxidase (GOD) method, with a Beckman or an ASTRA analyzer. and GOD, or with a manual/discrete analyzer.. The hexokinase method gave results ~0.03 mM below the over-all mean and the inter-laboratory coefficient of variation was ~5.5% (compared with 5.1% generally by the other methods). The bias is unlikely to be of clinical significance and the greater variability may be related to the variety of instruments used with this method. The lowest variability was obtained with the Beckman method, which also gave the lowest mean value. All methods have optimum performance in the glucose concentration. range 10 to 15 mM.
Glucose Clinical analysis Method comparison

"Influence Of Polymer Pre-adsorption On The Performance Of An Electrochemical Serum Biosensor"
Bioelectrochem. Bioenerg. 1993 Volume 31, Issue 3 Pages 311-322
Ana M. Oliveira Brett and Jorge L. C. Pereira, Philippe D&eacute;jardin

Abstract: The biosensor was for the determination of glucose in serum. A nylon membrane was hydrolyzed under reflux in 3 M HCl for 10 min and activated with 12.5% glutaraldehyde in 0.1 M phosphate buffer of pH 8 for 3 h at 40°C. The BSA in the same buffer was coupled to the membrane by incubation at 40°C for 4 h, the membrane was refluxed for 10 min and dried under vacuum for 4 h at 40°C. The membrane was again activated with glutaraldehyde solution and finally, glucose oxidase in 0.1 M phosphate buffer of pH 6 was immobilized for 24 h at 20°C on the nylon. The cleaned C or vitreous electrodes were coated by immersion in one of three aqueous polymer solution of trimethylaminoethylchloride methacrylate, methoxy-poly(ethyleneglycol)methacrylate or acrylnitrile, at 30°C for 3.25 h with KClO3 and NaHSO3 as initiators. FIA was performed (diagram given) with serum injections using the working electrode, Ag/AgCl as reference electrode and a Pt-tube outlet on the cell as auxiliary electrode. The signal on starting sequences of serum injections was less on the polymer-treated electrodes than on the treated ones, but the loss of signal on increasing the number of injections was lower, and there was a more stable response after 20 injections.
Electrochemical analysis Electrode Sensor Membrane

"A Chemiluminometric Method For NADPH And NADH Using A Two-enzyme Bioreactor And Its Application To The Determination Of Magnesium In Serum"
Biomed. Chromatogr. 1990 Volume 4, Issue 3 Pages 123-127
Masayoshi Tabata, Masayuki Totani, Takashi Murachi

Abstract: Chemiluminometric methods are described for the automated flow injection analysis of NADPH and NADH using an immobilized enzyme column reactor and serum magnesium. This application is for the clinical analysis of NADPH and NADH. The reactor for NADPH and NADH contains immobilized L-glutamate dehydrogenase and L-glutamate oxidase, and that for serum magnesium immobilized hexokinase, glucose-6-phosphate dehydrogenase, L-glutamate dehydrogenase and L-glutamate oxidase. Whtained from standard solutions and 0.5% from complex samples (at 1000 mg/L).
Magnesium Nicotinamide adenine dinucleotide oxidized Nicotinamide adenine dinucleotide phosphate oxidized Chemiluminescence Clinical analysis Automation Immobilized enzyme Column Detection limit Method comparison

"Flow Injection Glucose Determination With Long-wavelength Luminescent Oxygen Probes"
Biomed. Sci. 1991 Volume 2, Issue 1 Pages 63-67
Papkovskii DB, Savitskii AP, Yaropolov AI, Ponomarev GV, Rumyantseva VD, Mironov AF.

Abstract: A flow injection method for the determination of glucose in serum is presented. It is based on the enzymatic measurement of oxygen consumption detected via oxygen quenching of the luminescence of certain metalloporphyrins. Phosphorescent water-soluble Pt2+ and Pd(2+)-porphyrins have been characterized by luminescence spectroscopy and decay-time measurements in various buffers, and found to be suitable for oxygen detection in biological systems. A new method for the flow injection analysis of glucose has been developed based on the use of a column of immobilized glucose oxidase and the indicators Pt(2+)-coproporphyrin III and Pd(2+)-coproporphyrin I. The system has been optimized for glucose determination in aqueous samples and in whole serum with the 0.5-200 mM glucose range. Twenty assays can be performed in an hour, and the system has potential for commercial development with biotechnological and medical applications.
Glucose Biotechnology Luminescence Phosphorescence Buffer Column Immobilized enzyme Optimization Quenching

"Enzymatic Regeneration And Automatic Recycling Of NAD+. Application To The Determination Of Glucose"
Biosciences 1982 Volume 1, Issue 6 Pages 93-97
Roehrig, P.;Wolff, C.M.;Schwing, J.P.

Abstract: Glucose was determined enzymatically in human blood serum by using glucose dehydrogenase and regeneration of NAD from the NADH produced by using the glutamate-glutamate dehydrogenase reaction and automated recycling of NAD. The sample (20 mL) was injected into a flow-injection anal. system, and 120 samples/h could be analyzed. The results were compared to those obtained by the Technicon hexokinase method, and the correlation coefficient was 0.98. Compared to a commercial enzymatic equilibrium method, the present kinetic method reduced enzyme consumption by a factor of 20, NAD consumption by a factor of 10, and analysis time by >5 min.
Glucose Spectrophotometry Reagent recycling Enzyme

"Electropolymerized Films To Prevent Interferences And Electrode Fouling In Biosensors"
Biosens. Bioelectron. 1991 Volume 6, Issue 2 Pages 151-160
Robert J. Geise, John M. Adams, Neil J. Barone and Alexander M. Yacynych*

Abstract: Serum glucose was determined by flow injection analysis using electrochemical sensors constructed by all-chemical means. A variety of electrode materials were used; platinization of reticulated vitreous-carbon electrodes was carried out in a solution of 25 mM hexachloroplatinate in 0.1 M phosphate buffer. Glucose oxidase was immobilized onto the surface of the electrode by cross-linking with glutaraldehyde. Isomers of diaminobenzene and dihydroxybenzene were applied by electropolymerization and their effectiveness was evaluated. A film of 1,3-diaminobenzene - resorcinol gave the best protection and stability. The thermal stability of the enzyme was also investigated. The biosensor with the poly(1,3-diaminobenzene - resorcinol) film was effective for 5 months, limited by the lifetime of the enzyme and not the stability of the polymer film. The calibration graph was rectilinear from 2.4 to 10 mM and standard serum samples were accurately determined within ±3%. The electropolymerized films protected the sensors from interferences and fouling due to absorption of proteins. The all-chemical construction could be used for any size or shape of sensor.
Glucose Electrode Sensor Buffer Immobilized enzyme Interferences

"Flow Injection Analysis For Glucose Using An Amperometric Enzyme Electrode Based On Lipid-modified Glucose Oxidase As The Detector"
Biosens. Bioelectron. 1994 Volume 9, Issue 6 Pages 411-414
Fumio Mizutani* and Soichi Yabuki

Abstract: The enzyme sensor was prepared by coating a vitreous-carbon electrode with lipid-modified glucose oxidase applied in benzene solution, and then coating with Nafion. The electrode was used in a flow injection system with 0.1 M potassium phosphate buffer of pH 7 as carrier solution and was operated at a potential of 0.9 V vs. Ag/AgCl. The response to glucose was rapid (2 s) and the calibration graphs were linear up to 10 mM glucose. The electrode was stable for 12 weeks after 2000 glucose injections. The detection limit was 50 µM-glucose. Glucose could be determined in serum and fruit juices or drinks with 1% interference from ascorbic acid; uric acid interfered in the analysis of serum.
Glucose Electrode Electrode Sensor Amperometry Interferences

"An In Situ Electrosynthesized Amperometric Biosensor Based On Lactate Oxidase Immobilized In A Poly-o-phenylenediamine Film: Determination Of Lactate In Serum By Flow Injection Analysis"
Biosens. Bioelectron. 1994 Volume 9, Issue 7 Pages 471-479
F. Palmisano, D. Centonze and P. G. Zambonin*

Abstract: A lactate amperometric biosensor was constructed by electrochemically immobilizing lactate oxidase (lactate 2-monooxygenase) in a poly-o-phenylenediamine film. The sensor was prepared in a FIA system by injecting a plug of solution containing 5 mM o-phenylenediamine and 200 iu of lactate oxidase into a phosphate buffer carrier stream at a flow rate of 50 µL/min. The Pt electrode of the thin-layer amperometric cell was maintained at +0.85 V vs. Ag/AgCl. The calibration graph was linear up to 0.2 mM lactate with a detection limit of 2 µM. Faradaic interferences by ascorbate, urate, cysteine and acetaminophen were minimal. The electrochemical immobilization of lactate oxidase in a poly-o- phenylenediamine film permits the one-step and all-chemical construction of a lactate amperometric biosensor. The sensor was prepared in situ i.e. in the flow injection analysis (FIA) system by simply injecting a plug of a solution containing the monomer and the enzyme. At a flow rate of 50 µL/min linearity was observed up to 0.2 mM lactate and detection limits of about 2 µM could be easily achieved. Faradaic interferences caused by ascorbate, urate, cysteine and acetaminophen were sufficiently minimized to permit lactate determination in diluted serum by FIA. Results obtained by FIA-amperometric detection compared well (according to a proper t-test at a 95% confidence level) with those obtained by a standard enzymatic colorimetric assay. At a flow rate of 1 ml/min a sample throughput higher than 70 sample hr-1 was achieved. After one week of continuous use in the FIA system a 75% decrease in biosensor sensitivity was observed.
Lactate Electrode Electrode Sensor Amperometry Method comparison Interferences

"Lactate Solid-state Biosensor With Multilayer Of Electrodeposited Polymers For Flow Injection Clinical Analysis"
Biosens. Bioelectron. 1996 Volume 11, Issue 11 Pages 1155-1165
Tadeusz Krawczy&ntilde;ski vel Krawczyk, Marek Trojanowicz, Andrzej Lewenstam, Anna Moszczynska

Abstract: The biosensor was produced by electrodepositing the following three polymer layers onto a Pt disc electrode at +0.8 V vs. Ag/AgCl: (i) a polypyrrole layer from 0.4 M monomer solution in 0.05 M phosphate buffer of pH 7.3, deposition time 1 min; (ii) a polyphenol layer from 5 mM monomer solution in the same buffer, deposition time 1 min; and (iii) a poly(o-phenylenediamine) layer containing immobilized lactate oxidase (LOD) from 5 mM monomer/200 iu/ml LOD solution in 0.2 M acetate buffer of pH 5.2, deposition time 30 s. The performance of the biosensor was evaluated in a dual-channel FIA system in which 100 µL of test solution was injected into a water carrier stream (0.4 ml/min). The carrier stream was merged with 0.05 M phosphate buffer (0.4 ml/min) and propelled through a large volume wall-jet detector cell equipped with the lactate biosensor maintained at +0.7 V vs. Ag/AgCl (Pt foil auxiliary electrode). Under these conditions the biosensor did not respond to 1 mM urate, 0.2 mM paracetamol and 0.2 M NaCl. The FIA system was used to determine 2 µM- (detection limit) to 5 mM lactate in both undiluted and diluted serum samples. The RSD (n = 10) for the analysis of 1:10 diluted serum was 0.6%.
Lactate Sensor Clinical analysis

"Free-cholesterol Fiberoptic Biosensor For Serum Samples With Simplex Optimization"
Biosens. Bioelectron. 1997 Volume 12, Issue 3 Pages 233-240
M. D. Marazuela, B. Cuesta, M. C. Moreno-Bondi* and A. Quejido

Abstract: An optical fiber biosensor for free cholesterol monitoring in serum samples is described. Silicone-entrapped tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) complex, the luminescence of which is sensitive to oxygen changes, is used as an optical transducer of the oxidation of cholesterol by cholesterol oxidase. The biocatalyst is entrapped in a graphite powder layer deposited onto the dyed silicone film. Optimization of some interdependent chemical variables which affect the performance of the biosensor has been achieved by application of a super-modified simplex method. The dynamic range of the biosensing membranes is found to be 0.15-3.0 mM of free cholesterol. Studies of the reproducibility, stability and interferences of the device, as well as the application of the sensor to measurements in serum samples, are reported. Simplex optimization has proven to be a very useful tool in the search for the optimal conditions for performing analyzes with the optical fiber biosensor. (C) 1997 Elsevier Science Limited. 31 References
Cholesterol, free Sensor Chemiluminescence Optical fiber Simplex Immobilized enzyme Optimization Interferences