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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Serum Human

Classification: Biological fluid -> blood -> serum -> human

Citations 103

"Sensitive Assay For Clavulanic Acid And Sulbactam In Pharmaceuticals And Blood Serum Using A Flow Injection Chemiluminometric Method"
Anal. Chim. Acta 2000 Volume 414, Issue 1-2 Pages 15-23
Fatma A. Aly, Nawal A. Alarfaj and Abdulrahman A. Alwarthan

Abstract: A sensitive, simple and inexpensive chemiluminescence (CL) method using flow injection is described for the determination of two β-lactamase inhibitors sulbactam sodium and clavulanic acid (potassium salt) in their pure form, in pharmaceutical preparations and added to blood serum. The method is based on the enhancing effect of these compounds on the CL generated by the oxidation of luminol with hydrogen peroxide in alkaline medium. The CL intensity is a linear function of sulbactam sodium concentration over the range 0.1-150 µg mL-1 with a detection limit (2 x noise) of 0.05 µg mL-1, and for clavulanic acid over the range 0.01-12 µg mL-1 with a detection limit of 0.01 µg mL-1. The method is applied successfully to the determination of the drugs in their dosage forms without interference from co-formulated drugs. The method is specific for the intact drugs in the presence of their degradation products.
Clavulanic acid Sulbactam Chemiluminescence Indirect Optimization Method comparison

"Flow Injection Chemiluminometric Determination Of Albumin"
Anal. Chim. Acta 2000 Volume 403, Issue 1-2 Pages 137-143
N. T. Deftereos, N. Grekas and A. C. Calokerinos

Abstract: A new, simple analytical procedure for the determination of 5.00-300 µg mL-1 of albumin is presented, based on the chemiluminogenic reaction of the analyte with potassium permanganate in the presence of polyphosphoric acid (PPA). Serum globulins are also chemiluminescent, but their interference was eliminated by precipitation with saturated ammonium sulfate solution. The low limit of detection (4 µg ml-1) allows extensive dilution of the samples after globulin precipitation so that no other interferences are observed. Relative standard deviations were in the range 0.4-3.8% and the method was applied to the determination of albumin in human sera with good agreement with the bromcresol green (BCG) spectrophotometric method.
Albumin Chemiluminescence Interferences Method comparison

"Sulfide Measurements By Flow Injection Analysis And Ion Chromatography With Electrochemical Detection"
Anal. Chim. Acta 2000 Volume 409, Issue 1-2 Pages 27-34
Innocenzo G. Casella, Maria R. Guascito and Elio Desimoni

Abstract: Dissolved sulfide ions are separated and detected by using ion chromatography (IC) and electrochemical detection with a glassy carbon electrode modified by electrochemical deposition of palladium particles. The influence of various experimental factors such as pH, loading of palladium, applied potential and flow rate of mobile phase is evaluated. The effect of some common organic and inorganic compounds on the amperometric signal is also evaluated. Under optimal chromatographic conditions, the detection limit for sulfide (S/N = 3) was 0.3 µM (15 pmol injected) and the calibration plot was linear for 0.5-350 µM sulfide (r=0.9964, n=16). The separation and detection of sulfides in human blood serum is presented as an example of an application to real specimens.
Sulfide HPIC Electrode Amperometry Post-column derivatization

"Flow Injection Catalytic Determination Of Copper In Human Blood Serum"
Anal. Chim. Acta 1981 Volume 127, Issue 1 Pages 39-46
S. M. Ramasamy and Horacio A. Mottola

Abstract: Copper(II) ions catalyze the iron(III)-thiosulfate indicator reaction. This chemical system has been successfully used in a closed-loop apparatus with unsegmented continuous flow for the determination of copper in human blood serum. The sought-for species (the catalyst) is removed from the recirculating reagent by controlled-potential electrolysis after detection has taken place. Such electrolysis is also employed for in situ regeneration of the main reagent, iron(III). The reported method (a fixed-time, variable-signal kinetic determination) has a limit of detection for copper(II) of 0.25 g mL-1 and permits 325 determinations per hour with a relative standard deviation of 2%.
Copper Electrochemical analysis Closed loop Catalysis

"Homogeneous Enzyme-linked Assays Mediated By Enzyme Antibodies; A New Approach To Electrode-based Immunoassays"
Anal. Chim. Acta 1984 Volume 162, Issue 1 Pages 363-367
Susan B. Brontman and M. E. Meyerhoff

Abstract: A non-isotopic competitive-binding immunoassay for the detection of antigens is proposed. In this method, adenosine deaminase is coupled covalently to an analyte protein and catalyses the hydrolysis of adenosine to inosine and NH3. The anti-enzyme antibody inhibits the enzyme, whereas the antibody selective for the antigen reverses the inhibition. In the presence of free antigen in the assay mixture, there is competition for anti-antigen antibody sites, and protection against the anti-enzyme antibodies is reduced. The enzyme reaction is monitored in an automated continuous-flow system by use of an NH4+-selective electrode. Use of the method is exemplified by the determination of human serum albumin.
Albumin Immunoassay Electrode Enzyme

"Further Studies On The Reaction Of Amines And Proteins With 4-fluoro-7-nitrobenzofurazan"
Anal. Chim. Acta 1985 Volume 170, Issue 1 Pages 81-87
Hiroshi Miyano, Toshimasa Toyo'oka and Kazuhiro Imai

Abstract: The reaction of glycine with the title compound (NBD-F) was investigated to establish conditions that provide a high formation rate of the fluorescent adduct NBD-glycine and a low hydrolysis rate of the reagent. The effects of temp., organic solvents and buffer solution are reported. Conditions are given for derivatizing both low-mol.-wt. amines and proteins with NBD-F after HPLC separation. For low-mol.-wt. amines, acetonitrile is preferred to ethanol as reaction solvent, in the presence of borate buffer of pH 8 to 8.5. For proteins (separated by size-exclusion chromatography), water (e.g., 50%) should be present for sensitive reaction. Detection limits for human serum albumin, β-lactoglobulin and myoglobin are 6.6 pmol, 8.4 pmol and 11 pmol, respectively; these are larger than the limits of u.v. detection at 210 nm, but fluorescence detection may have advantages in selectivity.
Albumin β-Lactoglobulin Myoglobin HPLC Spectrophotometry Post-column derivatization

"Flow Injection Extraction And Gas-chromatographic Determination Of Terodiline In Blood Serum"
Anal. Chim. Acta 1985 Volume 175, Issue 1 Pages 281-287
Lage Nord, Stig Johansson and Harald Brötell

Abstract: A flow injection manifold comprising a 500 µL stainless-steel sample loop, a glass segmentor, a 4-m stainless-steel and nickel (2 m each) extraction coil and a poly(methyl methacrylate)-membrane phase separator was designed. Human serum containing terodiline(I) was injected at 30 samples h-1 into heptane - pentanol (49:1) containing N-t-butyl-1-methyl-4,4-diphenylbutylamine as internal standard (1 mL min-1) that was then mixed with 0.3 M NaOH (0.5 mL min-1) and subsequently segmented with more of the organic phase (0.5 mL min-1). After extraction a fraction of the organic phase (collected at 0.4 mL min-1) was injected (2 or 7 µL) on to a 25-m fused-silica capillary column coated with OV-1701 (cross-linked), at 180°C, with He - H (23:2) as carrier gas (4 mL min-1) and N-selective detection. The extraction was quantitative, i.e., none of the contact materials adsorbed I significantly. The coefficient of variation for 7 determinations of 400 ng mL-1 of I in serum was 3%. The GC determination alone gave a coefficient of variation of 4% (n = 10).
Terodiline GC Sample preparation Extraction Membrane Phase separator

"Use Of Holding Coils To Facilitate Long Incubation Times In Unsegmented Flow Analysis. Determination Of Serum Prostatic Acid Phosphatase"
Anal. Chim. Acta 1986 Volume 179, Issue 1 Pages 225-231
B. F. Rocks and R. A. Sherwood, M. M. Hosseinmardi and C. Riley

Abstract: An automated distribution valve is described that directs samples into one of four holding coils where they remain for a pre-determined time. The device is applied to the determination of acid phosphatase activity by a method similar to that described by Fabiny-Byrd and Ertingshausen [Clin. Chem. (Winston-Salem, N.C.), 1972, 18, 841] in which 1-naphthyl phosphate is hydrolyzed and the liberated 1-naphthol is coupled with a diazonium salt for absorbance measurement at 405 nm. The within-batch coefficient of variation (n = 10) at 4.2 and 23.5 iu L-1 are 8.0 and 2.0%, respectively. The results compared well with those obtained by a manual method (r = 0.98).
Acid phosphatase Spectrophotometry Method comparison Holding coil Valve

"Flow Injection Methods For The Determination Of Uracil Derivatives With Voltammetric Detection"
Anal. Chim. Acta 1988 Volume 211, Issue 1-2 Pages 175-193
B. Bouzid and A. M. G. Macdonald

Abstract: Flow injection methods are described for the determination of 18 uracil derivatives by differential pulse amperometry (d.p.a.) and differential pulse cathodic-stripping voltammetry (d.p.c.s.v.). The carrier stream was 0.05 M Na2B4O7 - 0.01 M KNO3 - 0.1 M HNO3 (or NaOH) at pH 7.6 containing 0.001% of Triton X-100 (to avoid the need for deaeration) for the detection of compounds having peak potentials in the range 180 to 70 mV (vs. Ag - AgCl). Calibration graphs were rectilinear for most compounds in the range 1 to 0.1 µM by d.p.a. and about one order of magnitude lower by d.p.c.s.v. The limit of determination for 5-iodouracil was 5 nM (~1.2 ng mL-1). Pre-separation was needed for applications to blood or urine. A deproteinization procedure followed by reversed-phase HPLC is described for the simultaneous determination of 5-fluorouracil, 5'-deoxy-5-fluorouridine and uric acid in human serum, with sequential spectrophotometric (268 nm) and amperometric detection.
Amperometry HPLC Voltammetry Stopped-flow Triton X Surfactant

"Determination Of Copper(II) In A Complex Matrix After Prereduction To Copper(I) In A Flow Injection System With An Amperometric Detector"
Anal. Chim. Acta 1990 Volume 228, Issue 1 Pages 117-122
Mouna Noufi, Ch. Yarnitzky and Magda Ariel

Abstract: A flow injection manifold and an amperometric detector equipped with a carbon-paste electrode in a wall-jet type configuration were used to determine Cu in complex media at +0.050 V vs. Ag - AgCl. Both the reduction current peak of Cu(II) and the oxidation peak of Cu(I) (obtained in the presence of hydroxylamine in the reagent stream) were proportional to the Cu(II) concentration. in the original sample. Acidified Cu(II) samples containing additional Fe(II), Zn, Pb(II) and relatively high concentration. of serum albumin were analyzed by using a chelating column to retain the heavy metal ions, while allowing albumin to run to waste. The retained metals were subsequently eluted with HNO3 into a stream of Na acetate or Na acetate - hydroxylamine. The oxidation (hydroxylamine) system was preferred as it gave the best sensitivity and reproducibility, minimum Fe interference and an electrode surface which remained uncontaminated and stable throughout the analytical run. Sample throughput was 25 h-1. The method was applied to the determination of Cu in standard human blood serum.
Copper(II) Amperometry Electrochemical analysis Electrode Electrode Chelation Column Sensitivity Interferences

"Study Of The Mimetic Peroxidase-catalyzed Chemiluminescence Reaction"
Anal. Chim. Acta 1990 Volume 237, Issue 2 Pages 497-502
Yun-Xiang Ci, Hong-Bin He, Wen-Bao Chang and Jing-Shi Liu

Abstract: The chemiluminescence behavior of the reaction in which the Mn-TPPS-4 complex ( the mimetic enzyme of peroxidase (manganese tetrakis(sulfophenyl)porphine)) acts as a catalyst for the oxidation of luminol by hydrogen peroxide was studied. The reaction product luminescences at 427 nm. Trace amounts of hydrogen peroxide and glucose can be determined with detection limits of 5.5 times 10^-9 and 2.7 times 10^-9 M, respectively. The characteristics of Mn-TPPS-4 were compared with those of horseradish peroxidase.
Hydrogen peroxide Glucose Chemiluminescence Catalysis Redox Immobilized enzyme

"Design And Properties Of A Flow Injection Analysis Cell Using Potassium-selective Ion-sensitive Field-effect Transistors As Detection Elements"
Anal. Chim. Acta 1991 Volume 245, Issue 2 Pages 159-166
Peter D. van der Wal, Ernst J. R. Sudhölter and David N. Reinhoudt

Abstract: The combination of flow injection analysis (FIA) and chemically modified ion-sensitive field-effect transistors (CHEMFETs) is described. In a wall-jet cell, two identical potassium-selective CHEMFETs were used for a differential measurement using a platinum (pseudo-)reference electrode. Silicone-rubber membrane materials, chemically bound to the SiO-2 gate oxide, were used with valinomycin as the ionophore. The optimized FIA system showed a linear response of 56 mV per decade for potassium concentrations above 5 times 10^-5 M. Preliminary results of potassium determinations in human serum and urine samples are presented.
Potassium Field effect transistor Silicone membrane Optimization Apparatus

"Simultaneous Spectrophotometric Determination Of Iron And Copper In Serum With 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N-sulfopropylamino)aniline By Flow Injection Analysis"
Anal. Chim. Acta 1992 Volume 261, Issue 1-2 Pages 197-203
Sam Woo Kang, Tadao Saki* and Noriko Ohno, Kazunori Ida

Abstract: The system described and illustrated incorporates a double-beam spectrophotometer with two flow cells. The sample is injected into 0.1 M HCl - 0.1 mM KIO4 as carrier and mixed with a 0.1 mM solution of the cited reagent in acetate buffer of pH 4.5 in a 15-cm reaction coil. The solution is passed through the first flow cell for measurement of the absorbance due to Cu(II) at 558 nm. A 10 mM solution of Na ascorbate in the buffer is introduced into the stream, which passes through a 700-cm reaction coil heated at 60°C before measurement of the absorbance at 558 nm in the second flow cell; in this instance an inverted peak for Fe(II) and a positive peak for Cu(II) are obtained. The Fe(III) present after adding the sample to the carrier does not interfere. The calibration graphs are rectilinear for 50 to 200 µg L-1 of Cu(II) or Fe(II) and the limit of detection is 2.4 µg l-1. Nickel and Co interfere at concentration. >10 µg L-1 which, however, are not encountered in serum samples. Serum is deproteinized with trichloroacetic acid at 90°C before analysis. Results on human, horse, bovine, chicken and goat serum agreed with those obtained by ICP-AES. A method for the simultaneous spectrophotometric determination of iron and copper by flow injection analysis was developed using 2-(5-bromo-2-pyridylazo)-5-(N-propyl-N- sulfopropylamino)aniline. The flow system utilizes a double-beam spectrophotometric detector with two flow cells. The reagent forms water-sol. chelates with Cu(II) and Fe(II) in an acetate-buffered medium at pH 4.5. The molar absorptivities of the complexes are 65,000 L mol-1 cm-1 at 578 nm for Cu(II) and 87,000 L mol-1 cm-1 at 558 nm for Fe(II). In one reaction coil Fe(II) is oxidized to Fe(III) by KIO4, so the Fe(II) chelate is not formed and only the colored complexes with Cu(II) are monitored in the first flow cell. In a second reaction coil, the solution merges with sodium ascorbate solution to reduce Cu(II) and Fe(III) to Cu(I) and Fe(II). Thus only the Fe(II) complex is measured in the second flow cell. Cu(II) and Fe(II) in the range 50-200 µg L-1 are determined at 558 nm. The sample throughput is 30 h-1 with a precision of 1.2%.
Iron Copper Spectrophotometry Simultaneous analysis Method comparison Buffer Complexation Interferences

"Amperometric Techniques In Flow Injection Analysis: Determination Of Magnesium In Sera And Natural Waters"
Anal. Chim. Acta 1992 Volume 269, Issue 1 Pages 41-48
Alison J. Downard, Joanne B. Hart, H. Kipton J. Powell* and Shuanghua Xu

Abstract: For water analysis, the flow injection analysis system (described) was based on the complexation of Mg by 0.15 mM Eriochrome Black T at pH 11.5 in 15% 1,2-diaminoethane - 0.3 M KCl during flow-through a reaction coil (100 cm x 0.51 mm); water was used as the carrier (0.6 mL min-1) with amperometric detection in a wall-jet microflow cell with a glassy carbon electrode (cf., Downard et al., ibid., 1992, 256, 117) and 0.45 mM EGTA and 0.04 M triethanolamine as masking agents. Rectilinear calibrations from 0.027 to 1.8 mg kg-1 of Mg were achieved with a detection limit of 5.5 µg kg-1 and coefficient of variation of 0.7% for water. Similarly conditions for serum were described (details given). Results were compared with those obtained by AAS. Magnesium was determined in a flow injection system by formation of the magnesium-Eriochrome Black T (EBT) complex at pH 11.5 (5% 1,2-diaminoethane in 0.1 M KCl) and amperometric measurement of the excess of EBT at +0.175 V on a glassy carbon electrode. From modeling calculations, pH and concentrations of EBT (3.75 x 10^-5M) and masking agent EGTA (1.12 x 10^-4M) were chosen so as to minimize interference from calcium. Electrode fouling from EBT oxidation products was countered by use of 0.2% surfactant (Hostapur SA 93) in the carrier stream. Iron(III) was masked with triethanolamine. For anal. of natural waters the linear working range was 0.027-1.8 mg kg-1 Mg and the relative standard deviation was 0.7% at 1.6 mg kg-1 Mg (n=11); the detection limit was 5.5 µg kg-1. Anal. of human blood serum samples was effected by two methods: direct injection of serum after a 20-fold dilution (Hostapur SA 93 countered the association of EBT with serum albumin) and after dialysis (2.5 min) to sep. Mg from acidified serum in the flow system. The linear working range for direct injection was as above and the relative standard deviation was 0.4% (n=9) at 1.0 mg kg-1 Mg. For each method the results were compared with those from flame atomic absorption spectrometry.
Magnesium Amperometry Electrode Method comparison Interferences Linear dynamic range Dialysis

"Catalytic Behaviour Of Iron(II)-oxime Complexes In The Chemiluminescence Reaction Of Luminol With Hydrogen Peroxide"
Anal. Chim. Acta 1993 Volume 277, Issue 1 Pages 67-72
Yun-Xiang Ci*, Jian-Ke Tie, Feng-Ji Yao, Zhao-Lan Liu, Sa Lin and Wei-Qing Zheng

Abstract: Chemiluminescence produced by the catalytic effect of iron(II)-oxime complexes on the oxidation of luminol by hydrogen peroxide was studied. This is a very fast chemiluminescence reaction and the emission spectrum is similar to that of peroxidase-catalyzed chemiluminescence. Different kinds of iron(II)-oxime complexes were compared and it was fund that the symmetrical dioxime complex (four nitrogen ligands) has a higher catalytic activity than the asymmetric monooxime complex (two nitrogen and two oxygen ligands). The presence of EDTA can greatly accelerate the chemiluminescence reaction, which was considered to be a result of the formation of a mixed-ligand complex. Other homologues of EDTA were tested and showed different degrees of enhancement. Optimum conditions for the chemiluminescence reactions were examined. Combined with a flow injection technique, trace amounts of hydrogen peroxide (2 times 10^-7-1 times 10^-4 M) and, using glucose oxidase, glucose (0.79-160 µg mL-1) can be determined by using the iron(II)-dimethylglyoxime complex as a typical catalyst. The detection limit of hydrogen peroxide was 1.3 times 10^-8 M. The relative standard deviation was 1% for the determination of 2 times 10^-7 M H-2O-2 (n=11). The feasibility of utilizing the proposed method for the determination of glucose in human serum was examined.
Glucose Chemiluminescence Indirect Optimization Complexation Catalysis

"Study Of The Europium(III)-tetracycline-thenoyltrifluoroacetone System By Using The Stopped-flow Mixing Technique: Determination Of Tetracycline In Serum"
Anal. Chim. Acta 1994 Volume 292, Issue 1 Pages 133-139
P. Izquierdo, A. Gómez-Hens and D. Pérez-Bendito*

Abstract: The addition of thenoyltrifluoroacetone (I) to the Eu(III)-tetracycline system in the presence of Triton X-100 gave rise to a 30-fold enhancement in fluorescence emission intensity at 612 nm (excitation a 342 nm) compared to that of the Eu(III)-tetracycline alone. In the determination of tetracycline a 0.15 mL portion of 5.4 µM-I/20 µM-Eu(III)/10 mM Tris buffer of pH 7.4 reagent was mixed with 0.15 mL of analyte solution (20-1000 mg/ml of I) in 0.01% Triton X/100-10 mM Tris buffer of pH 7.4 in a Hitachi F-2000 spectrofluorimeter equipped with a stopped-flow module (cf., Ibid., 1987, 199, 29) and the fluorescence intensity was monitored at 612 nm (excitation at 342 nm) for 10^-15 s. The calibration graph was linear for 20-1000 ng/ml of tetracycline, but the range could be extended to 10 µg/ml by increasing the reagent concentrations tenfold. The method was used to determine therapeutic levels of tetracycline in human serum (81-5 µg/ml). For 2 µg/ml of tetracycline the intra- and inter-day RSD (n = 5) were 0.9 and 1.1%, respectively. The mean recovery was 98.8%. No interference was observed.
Tetracycline Fluorescence Interferences Stopped-flow Triton X Surfactant

"Biosensors For Enantioselective Analysis"
Anal. Chim. Acta 1994 Volume 293, Issue 3 Pages 271-276
Thomas Kullicka, Roland Ulbera, Hartmut H. Meyerb, Thomas Scheperc and Karl Schügerla,*

Abstract: Enantioselective enzyme FET for the detection of β-hydroxy acid esters and aromatic amino esters were prepared by immobilizing various enzymes on to the pH-sensitive gate surface of a pH-FET. Esterase was used for the unspecific hydrolysis of the substrate into the respective acid and alcohol and α-chymotrypsin and lipase were used for the enantiomeric specific hydrolysis of S-phenyl-alanine methyl ester and S-3-hydroxy-3-phenylpropanoic acid ethyl ester, respectively. The enzymes were immobilized by crosslinking with glutardialdehyde and human serum albumin. The biosensors and a Ag+/Ag reference electrode were mounted in a flow-through detector cell of a FIA system. The esterase-FET gave a linear response for up to 2 g/l of R,S-phenyl-alanine methyl ester and up to 1 g/l for R,S-3-hydroxy-3-phenylpropanoic acid ethyl ester. The α-chymotrypsin-FET and the lipase-FET gave linear responses for up to 2 g/l for S-phenyl-alanine methyl ester and 5 g/l of S-3-hydroxy-3-phenylpropanoic acid ethyl ester, respectively. The sensors gave a maximum signal at pH 8. The sensor signal exhibited good reproducibility (no details given) and the sensor life-time was >40 days.
L-phenylalanine methyl ester 3-Hydroxy-3-phenylpropanoic acid ethyl ester Sensor Electrode

"Thiophilic Gels: Applications In Flow Injection Immunoassays For Macromolecules And Haptens"
Anal. Chim. Acta 1995 Volume 303, Issue 2-3 Pages 223-230
Derek A. Palmer* and James N. Miller

Abstract: Human serum albumin (630 nM) was labelled with 3.1 µM-fluorescein isothiocyanate (I) in 4 mL of hydrogen carbonate buffer of pH 9 for 4 h at room temperature, and the mixture purified through a Sephadex G-25 PD-10 column (no details given), eluting the product (II) at the void volume with PBS of pH 7.4. The albumin (III) and fluoriphore concentrations were determined at 280 and 495 nm respectively. Portions of 10 µL of II and 100 µL of III were reacted for 2 min with 100 µL of III-antiserum (1+49 in PBS buffer), and 25 µL injected onto a glass T-gel affinity column (10 cm x 6.6 mm i.d.) 50 mM sodium phosphate buffer of pH 8 containing 500 mM K2SO4 as mobile phase (binding buffer) and fluorimetric detection at 525 nm (excitation at 495 nm). The bound complex was eluted with 50 mM sodium phosphate buffer of pH 8 at the same flow rate. Theophylline (IV) was determined by the injection of IV-antiserum (1+49 dilution in PBS of pH 7.4) onto the T-gel column using binding buffer, and reaction on-column with a mixture of 5 µL of theophylline-I and 100 µL of sample, the bound complex being eluted as before. Calibration curves for the determination of III and IV are presented. For III, RSD (n = 8) of 3.9-8.4% were obtained, with a detection limit of 4 µg/ml. For IV recoveries of 114.6-96.3% were obtained with RSD of 3.7-5.7% and a detection limit of 80 ng IV.
Albumin Theophylline Immunoassay Spectrophotometry Fluorescence

"Pulsed Electrochemical Detection Of Proteins Using Conducting Polymer Based Sensors"
Anal. Chim. Acta 1995 Volume 315, Issue 1-2 Pages 27-32
W. Lu, H. Zhao and G. G. Wallace*

Abstract: These studies reveal that sensitive responses for proteins can be generated at selected conducting polymer electrodes using flow-injection analysis (FIA) and pulsed electrochemical detection. The responses observed are dependent on the nature of the polymer, the nature of the electrolyte anion/cation as well as on the electrolyte concentration and pH. These factors can be used to modify selectivity. Detection limits in the order of nanomolar have been estimated.
Albumin Myoglobin Ovalbumin α-Lactalbumin Voltammetry Sensor

"Arsenic Speciation In Serum Of Uraemic Patients Based On Liquid Chromatography With Hydride Generation Atomic Absorption Spectrometry And Online UV Photo-oxidation Digestion"
Anal. Chim. Acta 1996 Volume 319, Issue 1-2 Pages 177-185
X. Zhang*, R. Cornelis, J. De Kimpe and L. Mees

Abstract: An on-line method was developed for the speciation of arsenic species in human serum, including monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB) and arsenocholine (AsC). The method is based on cation-exchange liquid chromatography (LC) separation, UV-photo-oxidation for sample digestion and continuous hydride generation (HG) atomic absorption spectrometry (AAS) for the measurement of arsenic in the LC eluent. By developing the technique of argon segmented flow in the post-column eluent, an substantial improvement in Chromatographic resolution for the separation of these four arsenic species was obtained. The LC separation, photo-oxidation, hydride generation and AAS measurement could be completed on-line within 10 min. The detection limits were 1.0, 1.3, 1.5 and 1.4 µg/l of arsenic for MMA, DMA, AsB and AsC, in serum respectively. The concentration of the 4 species was determined in serum samples of patients with chronic renal insufficiency. Only AsB and DMA were significantly detected by the present method. The main part of arsenic in human serum is AsB. No MMA, AsC and inorganic arsenic were detected in these 6 samples.
Arsenic HPLC Spectrophotometry Online digestion UV reactor Speciation Photochemistry

"Determination Of Trace Elements In Human Serum By Inductively Coupled Plasma Atomic-emission Spectrometry With Flow Injection"
Anal. Chim. Acta 1996 Volume 320, Issue 2-3 Pages 185-189
H. B. Lim*, M. S. Han and K. J. Lee

Abstract: Measurements were made with a Labtam (Australia) P1000 ICP-AES system and a commercially available LC system. Human serum was subjected to 4 cycles of freezing and thawing to remove coagulation factors, then ultrafiltration was performed with 1.2, 0.8, 0.45 and 0.22 µm pore-size prefilters and the samples were lyophilized. They were analyzed for Mg, Cu, Zn, Fe, Sr and Li. The operating parameters are tabulated. Albumin was used for matrix matching. Recoveries for Cu, Fe, Mg and Li were in the range 95-99.8% for a standard reference material; RSD were 1.3%. Calibration graphs were linear.
Magnesium Copper Zinc Iron Strontium Lithium Spectrophotometry Reference material

"Electroluminescent Detection Of Enzymatically Generated Hydrogen-peroxide"
Anal. Chim. Acta 1996 Volume 327, Issue 3 Pages 253-260
MaEsther Fernández Laespada, JoséLuis Pérez Pavón and Bernardo Moreno Cordero*

Abstract: The present work studies the analytical possibilities of the electroluminescent detection of hydrogen peroxide generated in an enzymatic reaction. Glucose oxidation through the enzyme glucose oxidase (GOD) was used as the study system. The hydrogen peroxide generated reacts in situ with the radical obtained by electrochemical oxidation of luminol (5-amino-2,3-dihydrophthalazine-1,4-dione) on a glassy carbon electrode. The detection limit (2 x background noise) was 4.3 x 10^-6 M. The variables affecting the electroluminescent detection of hydrogen peroxide in the enzymatic reaction were studied. A linear calibration range of 5-50 µg mL-1 glucose was achieved. The proposed system was applied to the determination of glucose in different types of fruit juice and human serum. A dialysis membrane was coupled to the flow system in order to avoid electrode fouling, giving a linear calibration range of 50-300 µg mL-1. Glucose solution was merged with glucose oxidase and the flow was stopped for a preset time. The generated H2O2 was injected into a stream of carbonate buffer of pH 10.6 which was merged with a stream of 50 µM luminol dissolved in the same buffer. The mixture was passed through a reaction coil and then to a 30 µL flow cell constructed from Plexiglas and equipped a vitreous C working electrode held at 1.9 V vs. Ag/AgCl reference electrode. A Pt electrode served as the counter electrode. The emitted light was measured by means of a photomultiplier tube connected to a conventional spectrophotometer. The system was used for the determination of glucose in human serum and fruit juice samples. The detection limit was 4.3 µM glucose. Calibration graphs were linear for the range 50-300 µg/ml. Recoveries were >92%.
Glucose Chemiluminescence Electrode Enzyme Stopped-flow Buffer Dialysis

"Chemiluminescent Flow Injection Determination Of Alkaline Phosphatase And Its Applications To Enzyme Immunoassays"
Anal. Chim. Acta 1997 Volume 339, Issue 1-2 Pages 139-146
Jin-Ming Lin, Akio Tsuji and Masako Maeda*

Abstract: The flow injection chemiluminescence method for determining alkaline phosphatase (ALP) was based on the catalyzed reaction of cortisol-21-phosphate to produce cortisol which was detected by its chemiluminescence reaction with lucigenin (NN'-dimethyl-9,9'-biacridinium dinitrate). Sample (100 µL) was injected into a Tris hydrochloride buffer carrier stream (0.5 ml/min) at pH 10 which was merged with the reagent stream (1.5 ml/min) containing 0.1 mg/mL lucigenin and 0.06% cetyltrimethylammonium bromide in 0.1 M NaOH. The chemiluminescence was detected using a photomultiplier tube. The calibration graph for 4-50 pM-ALP was presented. The detection limit was 1 pM-ALP and the RSD (n = 5) was <6.1%. The method was applied to FIA for 17-α-hydroxyprogesterone and human chorionic gonadotropin in urine or serum using ALP as the enzyme label. The results correlated with those obtained by other methods.
Enzyme, alkaline phosphatase 17-Hydroxyprogesterone Gonadotropin chorionic Chemiluminescence Immunoassay Catalysis Method comparison

"Potentiometric Enzyme Electrode In A Flow Injection System For The Determination Of Urea In Human Serum Samples"
Anal. Chim. Acta 1998 Volume 369, Issue 1-2 Pages 129-137
Izabela Wa

Abstract: A urea biosensor with urease covalently bound directly to the membrane of the ammonium ion selective electrode was developed. The enzyme electrode was tested in single and double channel flow injection analysis (FIA) systems. Optimized measurement conditions allowed determination of urea within the linear range of 0.5-8 mM in the single channel and of 1-10 mM in the double channel FIA system. The response time of the biosensor was short enabling frequent sample injections (every 1.5-3 min). Long term stability of the enzyme electrode allows application of the same biosensor in the FIA systems for up to two months. Under optimized conditions the determination of urea is possible at physiol. and pathological levels. The results of urea determination in real, human serum samples were in good agreement with those obtained by routine clinical analytical methods.
Urea Potentiometry Electrode Electrode Optimization Method comparison

"Amperometric Biosensor For Uric Acid Based On Uricase-immobilized Silk Fibroin Membrane"
Anal. Chim. Acta 1998 Volume 369, Issue 1-2 Pages 123-128
Yu-Qing Zhang*, Wei-De Shen, Ren-Ao Gu, Jiang Zhu and Ren-Yu Xue

Abstract: An amperometric urate sensor based on a uricase-immobilized silk fibroin membrane and an O electrode in flow injection analysis are described. The biosensor shows that recoveries of uric acid in human serum and urine are at 94.2-102.6% and 92.5-97.9%, respectively. The relative standard deviations (RSDs) for repeatedly monitoring standard urate solution, human serum and urine are 2.37, 3.72 and 2.95%, respectively., based on 100 measurements. The urate sensor based on the uricase-immobilized membrane is capable of detecting 60-70 human serum samples per h. Also, a piece of uricase-immobilized fibroin membrane used at the sensor could not only be stored for over 2 yr, but also repeatedly monitored >1000 times for biosamples such as human serum or urine.
Uric acid Sensor Amperometry Electrode Immobilized enzyme Silk fibroin membrane

"Characterization And Analytical Use Of A Polypyrrole Electrode Containing Anti-human Serum Albumin"
Anal. Chim. Acta 1998 Volume 371, Issue 1 Pages 39-48
J. N. Barisci, D. Hughes, A. Minett and G. G. Wallace*

Abstract: The direct incorporation of anti-human serum albumin (AHSA) into a polypyrrole (PP) film by galvanostatic polymn. is a simple method for the preparation of an electrochemical sensor. However, the reproducibility of this approach is an area that requires attention. The amt. of antibody incorporated during polymer formation increases with increasing AHSA concentration. and decreasing c.d. Binding of human serum albumin (HSA) to the polymer-confined AHSA occurs readily, but is accompanied by a relatively large proportion of nonspecific interactions. The presence of phosphate ions hinders to a certain extent the PP/AHSA-HSA binding. Detection of HSA by flow injection analysis is possible using the PP/AHSA electrode operated under pulsed amperometric conditions in a NaNO3 carrier solution Pretreatment of the electrode with a bovine serum albumin solution decreases the response due to nonspecific binding.
Albumin Amperometry Sensor Electrode Electrode Apparatus Detector Interferences

"Simultaneous Determination Of Lithium, Sodium And Potassium In Blood Serum By Flame Photometric Flow Injection Analysis"
Talanta 1996 Volume 43, Issue 5 Pages 735-739
George Narh Dokua and Victor P. Y. Gadzekpoa,*

Abstract: Serum was diluted with 1 mM Li(I) and water and injected into a carrier stream of water (5 ml/min). The stream was split three ways into coils 6 cm x 1 mm i.d., 100 cm x 1 mm i.d. and 210 cm x 1 mm i.d. separating the stream into three samples by time. The emerging stream then passed to the photometer where Li(I) was determined in the sample portion, the filter switched and Na was determined in the second sample portion and the filter was switched again and K was determined. Calibration graphs were linear for 0.01-1 mM Li, 14-140 mM Na and 0.5-5 mM K. Results were compared to those obtained by the batch method.
Lithium Potassium Sodium Spectrophotometry Sample splitting Method comparison

"Enzymic Methods For The Determination Of α-glycerophosphate And α-glycerophosphate Oxidase With An Automated FIA System"
Talanta 1998 Volume 45, Issue 5 Pages 1015-1021
Efstratios R. Kiranas, Miltiades I. Karayannis and Stella M. Tzouwara-Karayanni*

Abstract: A procedure for the enzymatic determination of α-glycerophosphate (α-GP) has been developed, using an automated inhouse FIA (flow injection anal.) system, with immobilized glycerol-3-phosphate oxidase (GPO) on non-porous glass beads, following optimization of the immobilization and anal. parameters. Fabricated single bead string reactors (SBSR) were used in connection with the FIA system, following optimization of its parameters. The half-life of GPO-SBSR regarding reduction of the enzyme activity was found to be 110 days for its use in 20 triplicate measurements daily and storage at 4°C in the appropriate buffer. The regression equation of the calibration graph for the determination of α-GP was: Amax = (10 ± 2) x 10^-4 + (22,134 ± 12) x 10^-4 (mmol L-1 α-GP). The lower limit of quantitation was 0.74 µmol L-1 α-GP and the RSD of the method 0.05% (r = 0.9999). The same FIA system and procedure can be also used for the determination of the GPO activity, with the α-GP as substrate. The regression equation for this calibration graph was: Amax = (23 ± 18) x 10^-4 + (190 ± 1) x 10^-4 (µg mL-1 GPO), the lower limit of quantitation was 0.782 x 10^-3 mg mL-1 (0.782 ppm) GPO and the RSD of the method 0.53% (r = 0.9999). Serum samples obtained from hospitalized patients were de-proteinized by gel filtration and analyzed under pseudo-first order conditions, at various concentrations of α-GP. A kinetic study of the reduction of α-GP in serum vs. time is given and an observed reaction rate constant kob = 106.5 x 10^-4 min-1 was determined.
α-glycerophosphate Non-porous glass beads Immobilized enzyme Optimization Single bead string reactor

"A Model Immunoassay Using Automated Flow Injection Analysis"
Analyst 1984 Volume 109, Issue 3 Pages 339-341
Paul J. Worsfold and Arwel Hughes

Abstract: A model immunoprecipitin reaction between concanavalin A (I; representing the antibody) and yeast mannan (II; antigen) was studied by use of stop - flow merging zones in a microcomputer-controlled system. I in acetate buffer solution (30 µL) was injected into a carrier stream of buffer (Na acetate - NaCl - Brij-35, pH 6.2, at 0.5 mL min-1). Simultaneously, II in acetate buffer (30 µL) was injected into a separate flow at 0.5 mL min-1 of carrier buffer solution (pH 6.2). The two streams (at 25°C) were merged by passage through a mixing coil into the flow-through cell (7.9 µL; 10-mm path) for continuous measurement of turbidity at 400 nm. The rate of reaction could be measured by stopping the flow and performing a two-point kinetic analysis.
Concanavalin A Clinical analysis Immunoassay Spectrophotometry Turbidimetry Computer Kinetic Merging zones Stopped-flow

"Determination Of Human Serum Immunoglobulin G Using Flow Injection Analysis With Rate Turbidimetric Detection"
Analyst 1985 Volume 110, Issue 11 Pages 1303-1305
Paul J. Worsfold, Arwel Hughes and David J. Mowthorpe

Abstract: The reaction between human serum IgG and goat antihuman serum is elaborated and a modified version of the manifold described previously is used with the stopped-flow merging zones technique (cf. Anal. Abstr., 1984, 46, 10203). The rate of the reaction was monitored by turbidimetric detection at 340 nm. The results for a U.S. national reference preparation was 1145 mg dl-1 compared to the quoted value of 1128 mg dl-1; the between-batch coefficient of variation was 9.8% (n = 7) over several weeks and the within-batch coefficient of variation was from 3.6 to 5.2% (n = 10).
Immunoglobulin G Spectrophotometry Turbidimetry Merging zones Stopped-flow

"Enzymatic Determination Of Urea In Water And Serum By Optosensing Flow Injection Analysis"
Analyst 1986 Volume 111, Issue 8 Pages 865-873
Tracy D. Yerian, Gary D. Christian and Jaromir Ruzicka

Abstract: During flow injection analysis, the pH change associated with the enzymatic degradation of urea to NH3 and HCO3- is measured by reflectance spectrophotometry via a fiber-optic sensor. The pH change is detected by monitoring the reflectance spectrum of a ColorpHast (Merck) indicator strip that is situated in an integrated micro-conduit at the tip of a bifurcated fiber-optic bundle. Samples (50 µL) and reagent (25 µL of a solution containing 60 mg of urease per 100 mL of carrier buffer) are injected into the carrier buffer, containing 1 mM Tris - HCl and 140 mM NaCl (pH 7.0 for aqueous solution or 7.4 for serum samples), via a split-loop injector, and the intensity of the reflected light is monitored at 610 nm. To minimize errors due to variations in the background sample pH, a stopped-flow kinetic procedure is used. For human serum, the response is rectilinear up to 2 mM urea. Results for 12 samples of human serum agreed with those obtained by an ASTRA enzymatic method with conductometric detection.
Urea Spectrophotometry Enzyme Kinetic Method comparison Optical fiber Optosensing Stopped-flow

"Determination Of Urea In Serum By Using Naturally Immobilized Urease In A Flow Injection Conductimetric System"
Analyst 1991 Volume 116, Issue 4 Pages 357-360
Lourival C. de Faria, Celio Pasquini and Graciliano de Oliveira Neto

Abstract: A flow injection method was developed, aimed at the determination of urea in human serum. The system makes use of the naturally immobilized urease present in Canavalia ensiformis DC (jack bean). A column is filled with small pieces of this bean, and the sample (50 µL) containing urea passes through it carried by a 1% NaCl solution. On leaving the column the stream is merged with an alkaline reagent (0.5 mol L-1 NaOH; 0.5% disodium dihydrogen ethylenediaminetetraacetate). The ammonium ions, arising from the enzymatic reaction that occurs inside the column, are changed into the molecular form, which permeates a polytetrafluoroethylene membrane and is received in a de-ionized water acceptor stream. The ammonia ionizes causing an increase in the conductance, which is proportional to the urea content of the sample. About 40 samples can be processed in 1 h with negligible carry-over and with a relative standard deviation of 1% or less. The results are in agreement with those obtained by a standard spectrophotometric method. Aqueous 0.1% serum solution (50 µL) was injected into a carrier stream (1 mL min-1) containing aqueous 1% NaCl and passed through a jack bean (Canavalia ensoformis DC) tissue column (3 cm x 2.5 mm). The eluate was merged with a stream (1 mL min-1) of 0.5 M NaOH - 0.5% disodium dihydrogen ethylenediaminetetra-acetate then passed through a PTFE membrane into an water acceptor stream (2 mL min-1), which had been passed through an ion-exchange column. The conductance of the final solution was measured. The calibration graph [conductance vs. urea (I) concentration.] was rectilinear from 1 to 10 µg mL-1 of I; the coefficient of variation (n = 10) for 3 µg mL-1 of I was 0.8%. Sample throughput was 40 h-1. Results agreed (r = 0.987) with those obtained by a standard spectrophotometric method (Sampson and Baird, Clin. Chem., 1979, 25, 1721).
Urea Conductometry Ion exchange Column Immobilized enzyme Teflon membrane Method comparison

"Pyoverdin-doped Sol-gel Glass For The Spectrofluorimetric Determination Of Iron(III)"
Analyst 1995 Volume 120, Issue 2 Pages 431-435
J. M. Barrero, C. C&aacute;mara, M. C. P&eacute;rez-Conde, C. San Jos&eacute; and L. Fern&aacute;ndez

Abstract: Pyoverdin was entrapped in sol-gel glass (details given) and used for the determination of Fe(III). Two methods, viz., continuous-flow and flow injection (FI), were used. In the continuous-flow method, Fe(III) solution in 0.05 M MES buffer of pH 6.5 (buffer A) was pumped (1.8 ml/min) through a flow cell filled with 200 mg of the pyoverdin-doped sol-gel glass (maintained at 4°C). The decrease in the fluorescence intensity was measured at 405 nm (excitation at 330 nm). Regeneration was effected with 1 M HCl. In the FIA method, Fe(III) solution (1 ml) was injected into a stream (1.2 ml/min) of buffer A and passed through the flow cell, and the fluorescence was measured as described above. For the continuous-flow method, the calibration graph was linear from 3 (detection limit) to 300 ng/ml of Fe(III) and the RSD (n = 10) was 3%. For the FIA method, the calibration graph was linear from 20 (detection limit) to 900 ng/ml of Fe(III) and the RSD (n = 10) was 4.5%. The continuous-flow method was applied to tap water and the FIA method to human serum.
Iron(III) Fluorescence Method comparison Indirect Sol-gel

"Development Of A Novel Luminol-related Compound, 3-propyl-7,8-dihydropyridazino-[4,5-g]quinoxaline-2,6,9(1 H)-trione, And Its Application To Hydrogen Peroxide And Serum Glucose Assays"
Analyst 1995 Volume 120, Issue 4 Pages 1083-1086
Junichi Ishida, Hiromi Arakawa, Maki Takada and Masatoshi Yamaguchi

Abstract: Manual and flow injection (FI) methods with chemiluminescence detection are described for H2O2 determination, using the cited luminol-related compound (I). In the manual method, 100 µL 5 µM-microperoxidase in 10 mM phosphate buffer of pH 7 (buffer A) and 100 µL 10 µM-I in buffer A were added to 100 µL of sample solution and the chemiluminescence intensity produced was measured. In the FI method, a 100 µL portion of the sample was injected into a reagent stream (0.5 ml/min) containing 20 µM I and 12 µM microperoxidase in 10 mM phosphate buffer of pH 8. The mixture passed to a flow cell and the chemiluminescence produced was measured. Calibration graphs were linear from 20-5000 and from 3-300 pmol of H2O2 for the manual and FI methods, respectively; the corresponding detection limits were 13 and 1.3 pmol. The RSD (n = 8 and 6, respectively) were 4.5-5.8% and 2% for the manual and FI methods, respectively. The manual method was applied to the determination of serum glucose (details given). The results agreed with those obtained by spectrophotometry. Manual and flow injection methods with chemiluminescence detection were developed for the determination of hydrogen peroxide (H2O2) using a novel luminol-related compound, 3-propyl-7,8-dihydropyridazino[4,5-g]quinoxaline-2,6,9(1H)- trione (PDIQ), having a higher efficiency than luminol. The methods are based on the chemiluminescence produced by the reaction of H2O2 with PDIQ in the presence of microperoxidase in alkaline media. Detection limits for manual and flow injection methods are 13 pmol per 100 µL of test solution and 1.3 pmol per 100 µL injection volume, respectively, at a ratio of chemiluminescence intensities (or peak heights) of test to blank of 2. The manual method was applied to the determination of glucose in human serum. The method correlated well with the conventional spectrophotometric method (4 = 0.998).
Glucose Hydrogen peroxide Chemiluminescence Method comparison

"Direct Microcomputer-controlled Determination Of Zinc In Human Serum By Flow Injection Atomic Absorption Spectrometry"
J. Anal. At. Spectrom. 1986 Volume 1, Issue 6 Pages 453-456
Kirsten Wiese Simonsen, Bent Nielsen, Arne Jensen and Jan Rud Andersen

Abstract: Samples (typically 50 µL) are drawn untreated through a three-way valve from an autosampler into a carrier stream of water, propelled (~6.5 mL min-1) by the negative pressure at the nebulizer, and pass through a polyethylene dispersion tube (32 cm x 0.5 mm); 40 samples can be analyzed in duplicate (with blank injection) per hour. The autosampler and valve are controlled by a Commodore 64 microcomputer, which receives the analogue output of the Perkin-Elmer 460 spectrometer via an analogue integrator, a peak detector (each with its sample-and-hold circuit) and an Intersil DM-4100D digital voltmeter with tri-state binary-coded decimal output. Calibration of peak areas is effected daily with aqueous standard solution; the limit of detection is 0.14 mg L-1 of Zn. Results for serum containing known amounts of Zn were accurate.
Zinc Spectrophotometry Computer

"Liquid Chromatography With An Inductively Coupled Plasma Mass-spectrometric Detector For Simultaneous Determination Of Gold Drug Metabolites And Related Metals In Human Blood"
J. Anal. At. Spectrom. 1989 Volume 4, Issue 8 Pages 767-771
Susan G. Matz, R. C. Elder and Katherine Tepperman

Abstract: Blood plasma or serum (0.5 ml) was digested with 2.5 mL of aqueous 40% HNO3 with heating for 30 s at 700 W. The cooled digest was analyzed by ICP-MS in conjunction with a flow injection system. The mobile phase comprised water, aqueous 5% HNO3, 50 mM NH4 acetate buffer (pH 5.5) or 50 mM Tris buffer (pH 6.5). Matrix effects were significant. Detection limits were 0.2 to 0.7 ppb of Au, Zn and Cu, and calibration graphs were rectilinear up to 1000 ppb. The digests were also determined by ICP-MS after separation by HPLC on a column (15 cm x 4.6 mm) of Alltech WAX 300 anion exchanger with 20 to 200 mM Tris buffer (pH 6.5) as mobile phase or on a column (30 cm x 7.5 mm) of Bio-Sil TSK 250, with 25 mM Tris buffer (pH 7.7) as mobile phase. The HPLC - ICP-MS system was applied in the simultaneous determination of sixteen elements.
Copper Gold Zinc Mass spectrometry Sample preparation Buffer Calibration Column Method comparison Interferences

"Inductively Coupled Plasma-mass Spectrometry For Direct Multielement Analysis Of Diluted Human Blood And Serum"
J. Anal. At. Spectrom. 1997 Volume 12, Issue 9 Pages 1005-1009
EBBA BARANY, INGVAR A. BERGDAHL, ANDREJS SCH&Uuml;TZ, STAFFAN SKERFVING and AGNETA OSKARSSON

Abstract: A method for the inductively coupled plasma mass spectrometry (ICP-MS) multi-element analysis of diluted human blood and serum was used for the following elements: Co, Ni, Cu, Zn, Ga, Se, Rb, Mo, Ph, Pd, Cd, Sn, Sb, W, Pt, Hg, Tl and Pb. Sample pretreatment was a simple dilution (ten times for blood and five times for serum) with a solution containing 5 g L-1 of 25% ammonia, 0.5 g L-1 Triton X-100, and 0.5 g L-1 EDTA in Millipore water. In and Sc were used as internal standards. For sample introduction a flow injection type technique (based on time instead of volume) was used. The determinations mere carried out first in a peak-jumping mode for selected masses, and then in a scanning mode. Each determination of a preparation took 75 s. The results for reference samples agreed with recommended or certified values for Co, Cu, Zn, Rb, Cd, Tl and Pb in blood, and for Rb, Mo and Cd in serum. For Ni and Hg in blood, and Cu and Zn in serum, the results agreed with one of two reference samples. The detection limits for all these elements (except for Tl) were sufficient for analysis of samples from the general population. On the other hand, the results for Se in blood, and for Co, Ni, Se, Sn and Hg in serum did not agree with recommended or certified values. No reference samples are available for Ga, Mo, Ph, Pd, Sn, Sb, W, or Pt in blood, or for Ga, Ph, Pd, W, Pt, or Pb in serum. Generally, the limits of detection for the elements in the latter group (below 0.15 µg l-1) are close to or above the levels present in the general population.
Cobalt Copper Gallium Nickel Zinc Cadmium Molybdenum Palladium pH Rubidium Selenium Antimony Lead Mercury Platinum Thallium Tin Tungsten Mass spectrometry Multielement Interferences Timed injection Reference material Triton X Surfactant

"Repetitive Determinations Of Amylase, Maltose, Sucrose And Lactose By Sample Injection In Closed Flow-through Systems"
Anal. Chem. 1978 Volume 50, Issue 12 Pages 1665-1670
D. P. Nikolelis and Horacio A. Mottola

Abstract: Conditions have been developed for the repetitive determination of amylase and some disaccharides by coupling enzyme-catalyzed reactions yielding glucose as a product with the glucose oxidase catalyzed oxidation of this sugar. Determinations have been performed by sample injection into a continuously circulated reagent mixture and monitoring of oxygen depletion with a three-electrode amperometric system. Maltose, sucrose, and lactose in the range of 50 to 500 mg/100 mL, 10 to 250 mg/100 mL, and 25 to 250 mg/100 mL, respectively, and amylase in the range of 50 to 500 units/100 mL have been determined with relative errors and relative standard deviations (population) of about 2%. The maximum determination rate is 240 injections/h for maltose, 700 injections/h for sucrose and lactose; and 120 injections/h for amylase at room temperature. Applications to real samples (a variety of food products, human blood serum, and serum calibration references and controls) are reported.
Disaccharides α-Amylase Maltose Sucrose Lactose Clinical analysis Electrode Closed loop

"Simultaneous Determination Of Pyridoxal And Pyridoxal-S-phosphate In Human Serum By Flow Injection Analysis"
Anal. Chem. 1985 Volume 57, Issue 11 Pages 2101-2106
Pilar Linares, M. D. Luque de Castro, and Miguel Valcarcel

Abstract: Methods are described for the sequential, simultaneous and differential kinetic determination of pyridoxal(I) and pyridoxal 5'-phosphate(II). They are based on oxidation by CN-, yielding fluorescent derivatives, which are determined at 425 nm (excitation at 345 nm). The sequential method involves the use of a diverting valve to provide the carrier for each of I and II, while the other methods use the fluorescence of I and of the oxidation product of II (simultaneous method) or the fluorescence of both oxidation products (differential kinetic). Serum is deproteinized with trichloroacetic acid and treated with 0.6 M phosphate buffer (pH 7.4) before analysis. The calibration range is 10 nM to 1 mM, with coefficient of variation of <±1%. At the 10 nM level in serum, the average error was ±10 to 15%. Optimum conditions are presented for each method. The sampling rate is 30 h-1.
Pyridoxal Pyridoxal phosphate Fluorescence Kinetic Tecator

"Electropolymerized 1,2-diaminobenzene As A Means To Prevent Interferences And Fouling And To Stabilize Immobilized Enzyme In Electrochemical Biosensors"
Anal. Chem. 1990 Volume 62, Issue 11 Pages 1111-1117
Sylvia V. Sasso, Raymond J. Pierce, Robert Walla, and Alexander M. Yacynych

Abstract: A reticulated vitreous-carbon electrode was partially platinized and then coated with glucose oxidase as previously described (see preceding abstract). For use in the flow injection determination of glucose in human serum, the electrode was further coated with a protective film by electropolymerization of o-phenylenediamine. This coating prevented the adsorption of serum proteins on the electrode and decreased interference due to oxidation at the electrode of such constituents as ascorbic acid, uric acid and cysteine; it also improved the thermal stability of the enzyme. The working life of such a biosensor was 1.5 to 2 months and the shelf life was >3 months.
Glucose Electrode Sensor Interferences Immobilized enzyme

"Asymmetric Carbonate Ion-selective Cellulose Acetate Membrane Electrodes With Reduced Salicylate Interference"
Anal. Chem. 1993 Volume 65, Issue 21 Pages 3151-3155
Kang Shin Lee, Jae Ho Shin, Sang Hyun Han, Geun Sig Cha, Doo Soon Shin, and Hai Dong Kim

Abstract: The asymmetric electrodes were prepared as described by Cha and Meyerhoff (Talanta, 1989, 36, 271). The hydrophilic layer, formed by direct base hydrolysis of one side of an unplasticized cellulose triacetate membrane, faced the sample solution, and the selective layer, incorporating 1-butyl-4-(trifluoroacetyl)benzene, tridodecylmethylammonium chloride and plasticizer [bis-(2-ethylhexyl) sebacate or adipate], was fused to the other side of the membrane. The internal filling solution was aqueous 0.1 M NaH2PO4/0.1 M Na2HPO4/0.01 M NaCl, and a double-junction Ag/AgCl electrode was used as the external reference electrode. The response of the asymmetric membranes, used in wall-jet configuration for detection in FIA, to small anions, including carbonate, was virtually unchanged compared with that of unmodified carbonate-selective cellulose triacetate-matrix or PVC-matrix membranes, but the response to salicylate and other larger anions was lower and also very slow. When the asymmetric membrane electrode was used to determine 25 mM CO2, no interference was caused by up to 1 mM salicylate. The electrode was also used in a static arrangement to determine total CO2 in blood serum.
Carbon dioxide Electrode Electrode Interferences Cellulose acetate

"Automatic Determination Of Malate Dehydrogenase Activity By Two Flow Injection Modes"
Fresenius J. Anal. Chem. 1990 Volume 336, Issue 8 Pages 676-678
Juan Manuel Fernandez-Romero and M. D. Luque de Castro

Abstract: Malate dehydrogenase activity was determined by normal (A) and stopped-flow (B) flow injection analysis by monitoring the decrease in absorbance due to NADH uptake. Injected volumes of 100 µL for A and 160 µL for B were mixed with 1 mM NADH and phosphate buffer solution (pH 7.0) in reactors 1.5 m and 20 cm long, respectively. For B, a delay of 28 s with a 60 s stop time were used. Both methods are suitable from 0.02 to 1.5 U l-1, and recoveries from human serum samples were >96%. Sample throughput was 60 h-1 and 36 h-1 for A and B, respectively.
Enzyme, malate dehydrogenase Spectrophotometry Automation Stopped-flow Buffer

"Fluorimetric Enzymic Flow Injection Determination Of Bile Acids In Human Serum"
Fresenius J. Anal. Chem. 1990 Volume 338, Issue 6 Pages 749-751
Antonio Membiela, Fernando L&aacute;zaro, M. D. Luque de Castro and Miguel Valc&aacute;rcel

Abstract: Bile acids were determined in serum based on the enzymatic oxidation of the analytes in a stream of 0.1 M phosphate buffer (pH 8.0) and 2.25 mM NAD+ and fluorimetric detection. Tauro-, cheno-, glyco- and chenodeoxy-cholic acids were determined from 0.3 to 10 µg mL-1. The coefficient of variation was 3%. Recoveries of bile acids were 93.6 to 104.4%. The method was fast, simple and convenient.
Bile acid Fluorescence Enzyme Buffer pH

"Development Of An Automated Flow Injection Chemiluminescence Immunoassay For Human Immunoglobulin G"
Fresenius J. Anal. Chem. 1995 Volume 352, Issue 7-8 Pages 793-796
Andrea Hacker, Martin Hinterleitner, Curt Shellum and Gerald G&uuml;bitz

Abstract: An immunoreactor flow cell was constructed comprising a transparent PTFE tube (2 cm x 1.5 mm i.d.) packed with anti-human IgG immobilized on Affi-prep 10 support (details given). The immunoreactor was equilibrated with assay buffer of 0.01 mM sodium phosphate buffer/0.14 M NaCl/0.05% NaN3 of pH 7 at 0.3 ml/min, serum sample was injected at 0.03 ml/min and, after 1 min, the buffer was changed to the elution buffer of 32 mM 85% phosphoric acid of pH 1.8 (0.3 ml/min) and H2O2/NaOH solution was injected. Detection was by chemiluminescence. The detection limit was 7 fmol/injection of IgG in serum with intra-assay RSD (n = 7) of 1-3% and inter-assay RSD (n = 21) of 1.4%. Recoveries of 15.6 and 31.2 µM-IgG from serum were 102.2 and 103.4%, respectively.
Immunoglobulin G Chemiluminescence Immunoassay Column Immobilized antibody

"Determination Of Alkaline Phosphatase Activity In Human Sera By Mid-FTIR Spectroscopy"
Fresenius J. Anal. Chem. 1998 Volume 360, Issue 6 Pages 717-720
B. Lendl A, P. Krieg A, R. Kellner

Abstract: Fourier transform infrared spectroscopy is applied to the determination of alkaline phosphatase (ALP) activity in human sera. 4-nitrophenylphosphate was found to be an excellent ALP substrate for FT-IR spectroscopic detection. The developed method is based on the acquisition of two FT-IR spectra: one recorded immediately after mixing the sample and assay solution and the other after an incubation time of 10 min. Spectral changes in the mid IR range due to the enzymatic reaction could be correlated to the ALP activity in the sample. Experimental conditions were established such that the clinically relevant range for determination of ALP activity in human sera (50 to 900 U/L) was covered. The method was applied to the analysis of ALP activities in standard solutions as well as in human sera yielding results which agreed well with those obtained by a standard reference method.
Enzyme, alkaline phosphatase Spectrophotometry

"Flow Injection Potentiometry For The Assay Of Potassium In Biological Fluids"
Microchem. J. 1992 Volume 45, Issue 2 Pages 232-247
Judit Jeney, Kl&aacute;ra T&oacute;th*, Brno Lindner and Ern Pungor

Abstract: Potassium-selective solvent polymeric membranes (PVC and modified PVC) were made with the use of various ionophores, and discs of the membranes (7 mm diameter) were mounted in a commercially available electrode body. The performance characteristics were evaluated by the use of KCl solution, blood serum, and urine-like electrolytes, and a macro wall-jet potentiometric detector was designed (description given) for the determination of K in undiluted urine samples (using a flow injection technique). A carrier solution of 1 mM KCl, 130 mM MgCl2 and 24.8 mM Tris (adjusted to pH 8) was selected. Within-run precision of the analytical data (relevant to urine samples) in comparison with data obtained by the batch operation mode and by a commercial continuous-flow ion-analyzer (SERA 210) are presented in diagrammatic form. A correlation study is also presented relating to the potassium concentration. of undiluted urine obtained by the flow injection technique, the continuous-flow method (SERA 210) and flame AES. The performance characteristics (selectivity, slope of the electrode response, response time) of poly(vinyl chloride and modified poly(vinyl chloride)-based solvent polymeric, K-selective membrane electrodes were evaluated in relevance to the anal. of ionic constituents in biological samples. A flow injection system incorporating a macro wall-jet potentiometric detector was designed and its utility for the K assay in undiluted urine samples was evaluated. Anal. data obtained by flow methods for K content of patients' blood serum and urine samples had been correlated with flame atomic emission spectrometric data.
Potassium Potentiometry Electrode Method comparison

"Determination Of Vanadium By Its Catalytic Effect On The Oxidation Of Gallocyanine With Spectrophotometric Flow Injection Analysis"
Microchem. J. 1996 Volume 53, Issue 2 Pages 139-146
Ali A. Ensafi and A. Kazemzadeh

Abstract: Portions (200 µL) of standard V solutions were injected into a carrier stream of water at 0.5 ml/min delivered by a 12-channel peristaltic pump in a flow injection system (schematic shown). The sample-containing carrier stream was mixed sequentially with streams of 3.5 µM-gallocyanine (I) in a citrate buffer of pH 4 and 0.9 M NaBrO4 solution (both at 0.5 ml/min) in a 250 cm reaction coil. The decrease in the absorbance of I resulting from its V-catalyzed oxidation by bromate was measured at 620 nm. The calibration graph was linear from 0.01-100 ng/ml of V(V) [0.002-20 ng/200 µL solution] and the detection limit was 0.001 ng/200 µL. RSD (n = 10) were 3.2, 2, 1.7 and 1.1%, respectively, for 0.01, 0.1, 5 and 20 ng V(V). Tolerance levels for forty foreign ions and cations are listed. A throughput of 40 samples/h was achieved. The method was applied to the analysis of V(V) in human serum, crude petroleum and river water with recoveries of 100-105%.
Vanadium Spectrophotometry

"Microdetermination Of Proteins With The 1,10-phenanthroline-H2O2-cetyltrimethylammonium Bromide-Cu(II) Chemiluminescence System"
Microchem. J. 1998 Volume 60, Issue 3 Pages 217-223
Li Zheng Ping, Li Ke An and Tong Shen Yang

Abstract: Proteins can enhance the chemiluminescence (CL) intensity of the 1,10-phenanthroline-H2O2- cetyltrimethylammonium bromide (CTMAB)-Cu(II) system because unsaturated complexes of Cu(II) with protein have a much stronger catalytic effect on the CL reaction than does Cu(II). On this basis, a new flow injection analysis method for detection of some proteins was established. The method gives Linear responses over two orders of magnitude and detection limits at the 0.02-0.05 µg mL-1 level for bovine serum albumin, human serum albumin, γ-globulin, and egg albumin. The method was used for determination of proteins in human serum with satisfactory results.
Proteins Albumin Chemiluminescence

"Amperometric Assays Of Total And Free Cholesterols In Serum By The Combined Use Of Immobilized Cholesterol Esterase And Cholesterol Oxidase Reactors And Peroxidase Electrode"
Anal. Biochem. 1985 Volume 149, Issue 2 Pages 387-391
Toshio Yao, Minoru Sato, Yoshiaki Kobayashi and Tamotsu Wasa

Abstract: A flow injection system for assays of total cholesterol and free cholesterol was described. The total cholesterol assay system included an amperometric peroxidase electrode to measure hexacyanoferrate(III) converted from hydrogen peroxide, which was generated by injecting a 2 µL sample into the packed-bed reactors of immobilized cholesterol esterase and cholesterol oxidase covalently bound to silica. The free cholesterol was assayed with the same system without the cholesterol esterase reactor. The peak current was linearly related to cholesterol in the range 2-160 mg/dl and to total cholesterol in the range 3-300 mg/dl; the assay speed was about 80 samples/h for free cholesterol and 40 samples/h for total cholesterol. Reliable results were obtained in the assays of free cholesterol and total cholesterol in human sera. Both the reactors and the peroxidase electrode retained over 90% of their original activities, even after repetitive use for 4 and 2 months, respectively. Cholesterol esterase and cholesterol oxidase were separately immobilized on silica packed into columns (length 10 and 30 cm, respectively). The injection of a sample (2 µL) containing cholesterol (free and/or esterified) resulted in generation of H2O2 by the immobilized enzymes, and the H2O2 was used to generate Fe(CN)63- from Fe(CN)64- in an immobilized peroxidase electrode. The Fe(CN)63- could be measured amperometrically at low potential (-50 mV vs. silver - Ag+), so that there was little interference from other normal constituents of serum. Free cholesterol was assayed by omission of the cholesterol esterase reactor. The peak current was rectilinearly related to cholesterol concentration. in the range 2 to 160 mg dl-1 or cholesteryl palmitate concentration. in the range 3 to 300 mg dl-1. About 80 samples h-1 could be assayed for free cholesterol or 40 h-1 for total cholesterol. Within-assay coefficient of variation were <1.5%. Results agreed well with those from a chemical method. The reactors and the electrode retained >90% of their original activities after repetitive use for 4 and 2 months, respectively.
Cholesterol, free Amperometry Electrode Interferences Immobilized enzyme Silica

"Flow Injection Determination Of Proteins Using Enhanced Peroxyoxalate Chemiluminescence Applied To The Determination Of Immunoglobin G And Albumin In Serum"
Anal. Biochem. 1991 Volume 197, Issue 2 Pages 340-346
R. deLavalle and M. L. Grayeski

Abstract: The intensity of the chemiluminescence (CL) signal from an aqueous peroxyoxalate CL reaction can be significantly enhanced in the presence of various proteins with hydrophobic sites. A flow injection measurement for various hydrophobic proteins based on this CL enhancement was developed. The enhancement is due to the inclusion of the CL species in the favorable environment provided by the protein's hydrophobicity, which results in efficient light production. Various protein structures were evaluated; the degree of enhancement depends on the protein structure and CL reaction conditions. The CL enhancement measurement in the flow injection system is made after the introduction of the protein solution to the main phosphate buffer stream followed by the addition of the CL reagent streams: (1) hydrogen peroxide in water and (2) 8-anilino-1-naphthalene sulfonic acid and 4,4'-oxalylbis-(trifluoromethylsulfonylimino)ethylene bis(4-methyl morpholinium trifluoromethane sulfonate) in acetonitrile. Although prior separation of proteins is required before the measurement, the advantage of this approach is increased sensitivity without derivatization of the protein. The enhancement was demonstrated for several proteins, including antibodies, which suggests that this approach may be generally applicable to a variety of measurements, including immunoassay determinations. This CL enhancement was used to develop a simple and accurate flow injection measurement for the determination of albumin and IgG in human serum. A flow injection system (diagram presented) is presented for the determination of hydrophobic proteins, based on enhanced peroxyoxalate chemiluminescence. The protein sample was carried in the main phosphate buffer stream (pH 7.5) to mix with 4,4'-oxalyldisulfonylbis(trifluoromethylimino)diethylenebis-(4-methylmorpholinium trifluoromethanesulfonate) - 8-anilinonaphthalene-1-sulfonic acid reagent followed by H2O2. The resulting light emission was detected by a photomultiplier tube. Separation of proteins before measurement was achieved on open protein G - Sepharose 4 fast-flow columns. The method was applied to the development of an automated method to determine albumin and IgG in human serum.
Proteins Albumin Immunoglobulin G Chemiluminescence Buffer Hydrophobic membrane

"An Amperometric Flow Injection Analysis Biosensor For Glucose Based On Graphite Paste Modified With Tetracyanoquinodimethane"
Anal. Biochem. 1993 Volume 214, Issue 1 Pages 233-237
Pandey P. C., Glazier S. and Weetall H. H.

Abstract: A biosensor system using flow injection analysis (FIA) has been developed for the analysis of glucose in human serum. The system consists of the enzyme glucose oxidase incorporated into graphite paste modified with the electroactive material tetracyanoquinodimethane (TCNQ). TCNQ acts as an efficient mediator for oxidation of the reduced enzyme at 200 mV vs Ag/AgCl. The flow injection assay described has detection limits of 2 mM glucose using a 100 µL sample injection through a 250 µL sample loop. Data are presented to show the effect of sample injection volume and flow rate on the response of the FIA sensor. The biosensor exhibited excellent reproducibility for 800 injections. The loss of response after 800 injections was due to leaching of TCNQ from the graphite paste. Each assay takes 3 min giving a sample throughput of 20 per hour at a flow rate of 30 ml/h. The sensor was applied to the determination of glucose in human serum. The glucose measurements are in good agreement with those of a commercially available spectrophotometric method. Data showing the effect of interfering substances, ascorbic acid and acetaminophen, on the response of the sensor are also reported. A biosensor system was developed comprising glucose oxidase incorporated into a graphite paste modified with tetracyanoquinodimethane which acted as an efficient mediator for the oxidation of the reduced enzyme at 200 mV vs. Ag/AgCl. Serum (100 µL) was injected into a stream of 0.1 M phosphate buffer of pH 7 (30 ml/h) in a flow cell and the enzyme electrode was maintained at 200 mV vs. Ag/AgCl. The calibration graph was linear up to 200 mM glucose with a detection limit of 2 mM glucose. Total time for the procedure was 3 h with a sample throughput of 20/h.
Glucose Amperometry Sensor Electrode Electrode Method comparison Redox Interferences

"Flow Injection Analysis Of Glucose In Human Serum By Chemiluminescence"
Anal. Lett. 1982 Volume 15, Issue 21-22 Pages 1751-1766
C. Ridder; E. H. Hansen; J. Ruzicka

Abstract: Combining the rigidly controllable conditions of Flow Injection Analysis with detection by chemiluminescence, a method for determination of glucose in serum via enzymatic degradation by glucose in serum via enzymatic degradation by glucose oxidase is described. With a Standard deviation of less than 2% the sampling frequency is 75 samples per hour. Results obtained by the proposed procedure are compared with those acquired by a routine AutoAnalyzer method used at a local hospital. It is shown that any influence on the yield of chemiluminescence due to difference in viscosity of the injected samples effectively can be eliminated.
Glucose Chemiluminescence Clinical analysis Enzyme

"Combining Flow Injection Analysis And Immobilized Enzymes For Rapid And Accurate Determination Of Serum Glucose"
Anal. Lett. 1991 Volume 24, Issue 5 Pages 727-747
Narinesingh, D.;Stoute, V.A.;Davis, G.;Shaama, F.;Ngo, T.T.

Abstract: Glucose oxidase and peroxidase in an optimum activity ratio of 1.8:1 were co-immobilized on 2-fluoro-1-methylpyridinium-activated Fractogel (BioProbe International, Tustin, CA, USA) in 0.05 M phosphate coupling buffer of pH 8.0; the optimum loading was 0.72 mg mL-1 of protein. The resulting material was packed in Tygon tubing (2 cm x 2 mm) to provide a bioreactor, which was incorporated into a flow injection manifold (illustrated). The centrifuged serum sample was mixed (1:1) with 0.1 M phosphate buffer of pH 6.0 containing 0.02 M 3-methylbenzothiazolin-2-one hydrazone and 1.0 mM 3-dimethylaminobenzoic acid, and a 25 µL portion was injected into a flow (0.6 mL min-1) of 0.1 M phosphate buffer (pH 6.0). To compensate for background effects, the mixture was allowed to flow alternately through an enzyme reactor and a blank reactor, then passed through a detector operated at 590 nm. The optimum temperature and reaction time were 30°C and 50 s, respectively. The calibration graph of the difference signal vs. glucose concentration. was rectilinear up to 7 mg mL-1. The within-day coefficient of variation at 555 µg mL-1 was 2.0% (n = 15). The bioreactor retained 95% of its activity after storage in the buffer solution for 60 days. Results on a range of patients` samples correlated well with those obtained by a hospital method.
Glucose Buffer Immobilized enzyme Reactor

"Synthetic IgG Receptor, Avid AL: Applications In Quantitative IgG Determination And In Immunoprecipitation"
Anal. Lett. 1991 Volume 24, Issue 11 Pages 2005-2015
Narinesingh, D.;Pope, A.;Khatter, N.;Ngo, T.T.

Abstract: Flow injection analysis and low-pressure liquid affinity chromatography are combined to give a method for the determination of IgG using the affinity gel Avid AL as IgG receptor. The cartridge was conditioned with PBS binding buffer (0.01 M Na2HPO4 - 0.15 M NaCl - 0.01% NaN3, pH 7.3) at 1.5 mL min-1 and human IgG standards were injected into the buffer stream at 0.5 mL min-1. The absorbance of unbound protein in the effluent was monitored at 280 nm. When the value returned to baseline, the column was eluted with 0.1 M acetic acid at 2.9 mL min-1 and the absorbance was monitored as before. Between each sample injection the column was conditioned with regeneration and binding buffer. At an optimized sample loop size of 150 µL, the calibration graphs were rectilinear up to 2.5 mg mL-1 (375 µg applied to column), the recovery was 99 to 101% and the coefficient of variation were 4.1 and 1.9% for 0.36 and 1.32 mg mL-1 of IgG, respectively (n = 12). The determination of IgG in human serum had a throughput rate of 10 min and a detection limit of 0.06 mg mL-1. The quantitative immuno-precipitation of soluble antibodies using Avid AL is also described.
Immunoglobulin G LC Buffer Column Precipitation Synthesis

"Flow Injection Analysis Of Human Serum Albumin Using A Stabilized, Immobilized Cibacron Blue Support"
Anal. Lett. 1992 Volume 25, Issue 9 Pages 1721-1739
Narinesingh, D.;Pope, A.;Ngo, T.T.

Abstract: A diagram of the flow injection manifold is given. The affinity column, operated at 25°C, consisted of a 1 mL cartridge packed with Blue Avid Gel P previously conditioned with 20 mM Tris containing 150 mM NaCl of pH 7.5 (TBS). Portions (160 µL) of human serum albumin (HSA) were injected into the system, and unbound material was washed out with the TBS. Once the absorbance (280 nm) had returned to the baseline, the albumin bound to the gel was eluted with TBS containing 2 M KCl (1 mL min-1), and the absorbance at 280 nm was measured. The method was applied to standard solution of HSA containing 2.5 mg mL-1, serum samples treated with HSA standard solution, and patients' serum. The calibration graph was rectilinear, with a mean recovery of 98% and a coefficient of variation of 4.3%. This paper reports on the development of a simple, accurate method which combines low pressure liquid affinity chromatography and flow injection analysis for the quantitation of albumin in human serum. The pseudo-affinity matrix consists of Cibacron Blue covalently linked to a 2-fluoro-1-methylpyridinium salt activated Fractogel support. The albumin can be readily eluted at pH 7.5 using a Tris buffer (20 mM) containing 2 M KCl. Linear calibration curves are obtained (r = 0.999) at albumin concentrations up to at least 250 mg dL-1. Recovery yields in the range 95% to 103% (mean = 98%) are obtained. The within day as well as the day to day relative standard deviation is less than 4.3%. Comparison of the albumin concentrations in serum samples analyzed for by the title method with those obtained for the samples analyzed for by the hospital method (bromocresol green method) gave good correlations (r = 0.999).
Albumin Spectrophotometry Sample preparation Solid phase extraction Immobilized reagent

"Direct Determination Of Potassium In Human Blood Serum By Flow Injection Flame Photometry With Automatic Dilution"
Anal. Lett. 1996 Volume 29, Issue 10 Pages 1719-1727
Ana Cl&aacute;udia de Paiva; Graciliano de Oliveira Neto; Matthieu Tubino; Nelci F. Hoehr

Abstract: Serum (100 ml) was injected into the flow system via a flow injection manifold (schematic given) and diluted fourfold with water. The flow was diluted into two parts by a splitter and mixed with water in a ratio of 1:8 with a final dilution to ~1.32 after passing through a second splitter. Readings were taken with use of a flame photometer. The sampling rate was ~60 samples/h. The sampling flow rate was 3 ml/min (other flow rates are fixed by the tube diameter and are given). Calibration was linear up to 12.5 mM K+. The experimental error was ±5%. The results compared well with those obtained by a manual photometric method.
Potassium Spectrophotometry Automation Dilution Method comparison Sample splitting

"Testosterone Determination Using Rapid Heterogeneous Competitive-binding For Enzyme-linked Immunosorbent Assay In Flow Injection"
Anal. Lett. 1996 Volume 29, Issue 12 Pages 2125-2139
Di Benedetto, L.T.;Dimitrakopoulos, T.;Davy, R.M.;Iles, P.J.

Abstract: Heparinized blood or serum (50 µL) and 200 µL horseradish peroxidase-testosterone conjugate were incubated in polystyrene wells coated with testosterone antibody. After washing the plates with Tween 20, 200 µL H2O2/tetramethylbenzidine in acetate/citrate buffer was added and the plates re-incubated. The reaction was terminated by the addition of 50 µL 1 M H2SO4. The tetramethylbenzidine species were measured spectroscopically at 450 nm via flow injection. The ELISA kit used was a FERTIGENIX TESTO-EASIA (Medgenix Diagnostics, Australia) testosterone ELISA kit. The analysis time was reduced to 1 h compared with the conventional 150 min by reducing the antibody incubation time. The results agreed well with those obtained by an IRMA. The calibration range extended up to 12.2 ng/ml of testosterone and the assay was applied to male and female patients. The RSD was 0.5% for peak height precision.
Testosterone Immunoassay Spectrophotometry Method comparison

"Differential-pulse Voltammetric Determination Of Serum Aspartate-aminotransferase Activity Using DCIP As Redox Mediator At A Gold Micro Electrode"
Anal. Lett. 1997 Volume 30, Issue 7 Pages 1279-1291
Ya-Nan He; Hong-Yuan Chen; Xun-Yang Luo

Abstract: Human serum aspartate amino- transferase(AST) activity, was measured voltammetrically at a gold µelectrode. 2,6-dichloroindophenol(DCIP) was used as the redox mediator and electroactive species. Sensitive measurements free from spectrophotometric interferences can be made by a differential pulse voltammetric detection technique. The instrumentation is simple and inexpensive. The method yields reproducible results that were in excellent agreement with those obtained using tile standard spectrophotometric method for determining serum AST activity in the clinical laboratory. 14 References
Enzyme, aspartate aminotransferase Voltammetry Electrode Electrode Interferences Redox Method comparison

"Electrooxidation Of Aliphatic Amines And Their Amperometric Detection In Flow Injection And Liquid Chromatography At A Nickel-based Glassy Carbon Electrode"
Electroanalysis 1998 Volume 10, Issue 15 Pages 1005-1009
Innocenzo G. Casella *, Simona Rosa, Elio Desimoni

Abstract: The electrocatalytic oxidation of mono- and poly-amines was investigated in alkaline solutions at a nickel-based chemically modified glassy carbon electrode. -NiOOH exhibited electrocatalytic activity towards amines oxidation. FIA experiments were performed to test the electrode as an amperometric sensor. Under constant-potential amperometric mode (at 0.55 V vs. 4,0 M Ag/AgCl/Cl-), detection limits of all investigated analytes ranged within 1.2-3.8 pmol (S/N = 3), and the linear dynamic range extended over three orders of magnitude above the detection limits. Chromatographic separations, performed by an ion moderated partition column in alkaline solutions equipped with the Ni-CME detector, allowed very simple quantitation of aliphatic amines, avoiding tedious and time-consuming derivatization processes or post-column addition procedures. The Ni-CME showed good temporal stability and appreciable sensitivity.
Amines, aliphatic Amperometry Electrode HPIC Catalysis

"Determination Of Pseudouridine In Human Urine And Serum By High Performance Liquid Chromatography With Post-column Fluorescence Derivatization"
J. Chromatogr. A 1990 Volume 515, Issue 1 Pages 495-501
Yoshihiko Umegae, Hitoshi Nohta and Yosuke Ohkura

Abstract: Urine (100 µL) was mixed with 100 µL of 0.5 mM 5-fluorouridine (I; internal standard) and 800 µL of water. Serum (0.5 ml) was mixed with 0.5 mL of 0.05 mM I and 0.5 mL of 2 M HClO4, and the mixture was centrifuged at 1000 g and 4°C for 10 min. The supernatant solution (0.5 ml) was mixed with 65 µL of 2 M K2CO3 and re-centrifuged. An aliquot (100 µL) of either solution was analyzed by HPLC on a column (15 cm x 4.6 mm) of TSK gel ODS-80 (5 µm), with 2 or 4% of methanol in 10 mM phosphate (pH 5.0) as mobile phase (0.5 mL min-1). Detection was at 254 nm, and, after mixing the eluate with 2 mM NaIO4 and 20 mM 1,2-bis-(4-methoxyphenyl)ethylenediamine (each at 0.25 mL min-1), by fluorimetry at 470 nm (excitation at 340 nm). Calibration graphs were rectilinear for 0.5 to 50 nmol of pseudouridine in 100 µL of urine or 0.1 to 4 nmol of I in 0.5 mL of serum. Limits of detection were 40 and 8 nM, respectively.
Pseudouridine HPLC Fluorescence Post-column derivatization Column pH Calibration Detection limit

"Determination Of Ochratoxin A At The Ppt Level In Human Blood, Serum, Milk And Some Foodstuffs By High Performance Liquid Chromatography With Enhanced Fluorescence Detection And Immunoaffinity Column Cleanup: Methodology And Swiss Data"
J. Chromatogr. B 1995 Volume 666, Issue 1 Pages 85-99
Bernhard Zimmerli* and Rudolf Dick

Abstract: An improved specific analytical method for ochratoxin A (OA) is presented, combining HPLC separation with enhanced fluorescence detection by post-column addition of ammonia. Commercial immunoaffinity columns (Biocode) were for the first time applied to the cleanup of extracts of body fluids; they could be used up to 20 times for blood serum. The extraction efficiency of OA from human serum and milk as well as its derivatization to esters were studied and improved. The quantitation limit for OA was improved and estimated at 5-10 pg/g for human milk and serum. The mean recovery of OA from serum and milk was estimated at 85%. The overall coefficient of variation for OA determinations in serum, milk and selected foodstuffs was estimated at 10% (concentration range 0.01-5 ng/g). The method was applied to sera of 368 blood donors, 10 pairs of maternal and fetal sera, as well as to 40 human milk samples and selected foodstuffs; the results are discussed.
Ochratoxin A HPLC Fluorescence Post-column derivatization

"Kinetic Determination Of Aspartate Aminotransferase In Human Serum With A Flow Injection/multidetection System"
J. Pharm. Biomed. Anal. 1991 Volume 9, Issue 9 Pages 679-684
J. M. Fern&aacute;ndez-Romero* and M. D. Luque De Castro

Abstract: Photometric-kinetic methods for the determination of activity of aspartate aminotransferase are proposed. The flow injection manifold used for this purpose includes a selecting valve which allows the sample to be trapped in a closed circuit where a solid reactor housing an auxiliary enzyme and a conventional single detector allows a multipeak recording to be obtained for each sample. This record represents a typical kinetic curve from which much information can be obtained to develop fixed-time and reaction-rate methods for the determination of the analyte based on its catalytic action on the L- aspartic acid-2 oxoglutarate system. The linear range is found to be between 1 and 500 U l-1, with relative standard deviation less than 1%. The utility of the methods is illustrated by the determination of the analyte in human serum from healthy and sick individuals. Sample was injected into a stream of 0.1 M Tris - HNO3 buffer of pH 7.5 which merged successively with 6 mM 2-oxoglutarate in Tris buffer and 200 mM aspartic acid and 1 mM NADH also in Tris buffer. The mixture passed through the manifold (illustrated) to a selecting valve which trapped the reacting plug in a closed circuit. The plug passed through an enzymatic reactor (5 cm long) containing malate dehydrogenase immobilized on controlled-pore glass and the decrease in absorbance was monitored at 340 nm. The calibration graph was rectilinear for 1 to 500 U L-1 of aspartate aminotransferase; coefficient of variation was 1% for 30 U l-1.
Enzyme, aspartate aminotransferase Buffer Catalysis Controlled pore glass Kinetic Immobilized enzyme pH

"Ultra-sensitive Electrochemical Enzyme Immunoassay For Thyroid Stimulating Hormone In Human Serum"
J. Pharm. Biomed. Anal. 1994 Volume 12, Issue 6 Pages 787-793
Zhengrong Yu, Yan Xu* and Michael P. C. Ip

Abstract: The Tandem-E TSH HS immunoenzymometric assay kit (Hybritech Inc., San Diego, CA, USA) for thyroid stimulating hormone (TSH) was adapted for this assay. Serum containing TSH and mouse monoclonal anti-human TSH antibody conjugated with bovine alkaline phosphatase were placed in a test-tube, then a single monoclonal anti-human TSH IgG coated bead was added. After incubation at 32°C for 2 h, substrate solution, 4-aminophenyl phosphate, was added. The product, 4-aminophenol, was detected by oxidative amperometry in a flow injection system with a vitreous carbon working electrode operated at 325 mV vs. Ag/AgCl, with a stainless-steel auxiliary electrode. The calibration graph was linear from 50 fmol to 100 pmol of 4-aminophenol and the limit of detection was 10.9 fmol. Intra-assay RSD were 8.0% for 0.02-60 miu/l of TSH. The detection limit was 0.01 piu/l of TSH. Results correlated well with those by Bio-Rad immunoradiometric assay (r = 0.992) and an immunochemiluminometric assay (r = 0.986).
4-Aminophenol Sample preparation Immunoassay Amperometry Electrode Electrode

"Detection And Quantitation Of Gadolinium Chelates In Human Serum And Urine By High Performance Liquid Chromatography And Post-column Derivatization Of Gadolinium With Arsenazo III"
J. Pharm. Biomed. Anal. 1995 Volume 13, Issue 7 Pages 927-932
Erlend Hvattum*, Per Trygve Normann, Gene C. Jamieson, Jan-Ji Lai and Tore Skotland

Abstract: A narrow-bore high performance liquid chromatography method was developed for simultaneous separation of gadolinium diethylenetriaminepentaacetic acid (GdDTPA), the monomethylamide (GdDTPA-MMA) and the bis-methylamide (GdDTPA-BMA) in human serum and urine. The Gd complexes were detected at 658 nm after post-column derivatization with Arsenazo III. The serum samples were ultrafiltrated, whereas the urine samples were centrifuged and diluted before analysis. With an injection volume of 10 µL on a 2.1 mm ID reversed-phase column, the limit of detection of GdDTPA-BMA was calculated as 0.3 µM and 1.1 µM in serum and urine, respectively. The method was validated with respect to GdDTPA-BMA with a limit of quantification set to 2 µM and 10 µM in serum and urine, respectively. The best fit of the calibration curve was obtained using non-linear regression according to the equation Y = A+BX+CX2 in the concentration ranges 2-800 µM and 10^-2000 µM of GdDTPA-BMA in serum and urine, respectively. The precision of the method was found to range from 1 to 4% RSD. The recoveries of GdDTPA-BMA spiked in serum and urine were higher than 95% with an RSD equal to or less than 4%. The serum samples were stable for at least 5 months when stored at -70°C, and the urine samples were stable for a least 6 months when stored at -20°C.
Gadolinium diethylenetriamine pentaacetic acid Gadolinium monomethylamide Gadolinium bis-methylamide HPLC Spectrophotometry Post-column derivatization

"Potentiometric Flow Injection Determination Of Serum Bromide In Patients With Epilepsy"
J. Pharm. Biomed. Anal. 1997 Volume 15, Issue 12 Pages 1829-1832
Takashi Katsu*, Yuki Moria, Naoyuki Matsukab and Yutaka Gomitab

Abstract: A flow injection system was constructed using a bromide-selective electrode and used to determine serum bromide in patients with epilepsy. A 10 µL serum sample was injected into a carrier stream flowing at 0.12 mL min-1. Potential changes and bromide concentrations were linearly related in the range 3-50 mM. The lower limit of detection for serum bromide was 1 mM and this electrode sensitivity spanned the entire concentration range required for bromide therapy (9-24 mM). The results compared favourably with those obtained by colorimetry.
Bromide Potentiometry Electrode Method comparison

"Simultaneous Determination Of Glycosylated Albumin And D-glucose In Human Serum By High Performance Liquid Chromatography With Post-column Fluorescence Derivatization"
Anal. Sci. 1993 Volume 9, Issue 1 Pages 9-14
G.-Q. ZHANG, M. KAI, H. NOHTA, Y. UMEGAE and Y. OHKURA

Abstract: Serum is diluted 100-fold with 10 mM NaH2PO4 and set aside for 5 min, and a 100 µL portion of the solution is injected on to a TSKgel G2000SWXL column (30 cm x 7.8 mm) operated with 0.1 M phosphate buffer (pH 6.7) - 0.1 M Na2SO4 - 80 mM NaN3 as mobile phase (0.7 mL min-1). The eluate is mixed with 15 mM 4-methoxybenzamidine (I) - 0.55 M NaOH or 5 mM meso-1,2-bis-(4-methoxyphenyl)ethylenediamine (II) - 0.25 M NaOH (0.3 mL min-1) and heated in a reaction coil (7 or 20 m x 0.5 mm, respectively) at 95°C or 140°C, respectively. After passage of the eluate through a cooling coil, the fluorescence is monitored at 470 or 460 nm for I or II, respectively (excitation at 310 or 340 nm, respectively), in a 15 µL flow cell, the peak heights being measured. Glycosylated albumin and glucose are well separated; the coefficient of variation at 2.4 and 5.2 µmol mL-1 of the respective analytes were 3.5 and 2.5% (detection limits 100 and 3 pmol) after reaction with I and 2.6 and 3.5% (150 and 5 pmol) after reaction with II, respectively. A method is also described for determination of the reducing carbohydrates liberated from human serum albumin by acid hydrolysis.
Albumin, glycosylated d-Glucose HPLC Fluorescence Post-column derivatization Heated reaction

"Effect Of Anesthesia With Halothane On The Serum Concentration Of Iron, Copper, Manganese, Zinc And Cobalt In Humans Determined By Flow Injection Analysis And Atomic Absorption Spectrophotometry"
Acta Cient. Venez. 1988 Volume 39, Issue 2 Pages 130-134
Alarcon OM, Cuicas H, Rivas E, Burguera JL, Burguera M.

Abstract: n this study serum concentrations of Zn, Cu, Fe, Mn and Co were evaluated in 40 patients 15 to 70 years old: 25 men (62.5%) and 15 women (37.5%), who underwent surgical treatment and were anesthetized with halothane. The flow injection analysis-atomic absorption spectrophotometry technique was used for each element determination. The concentration of these elements in the preoperative samples were within the "normal" range when compared with previously published values. 60 minutes and 24 hours after exposure to halothane, a remarkable trend to increase serum Zn and Cu concentrations was observed. Theses increases in the concentrations of serum Zn and Cu could be attributed to an hepatotoxic effect of halothane and to surgical trauma, respectively.
Cobalt Copper Iron Manganese Zinc Clinical analysis Spectrophotometry

"Kinetic And Equilibrium Studies Of Porphyrin Interactions With Unilamellar Lipidic Vesicles"
Biochemistry 1994 Volume 33, Issue 32 Pages 9447-9459
Katerina Kuzelova and Daniel Brault

Abstract: The interaction of deuteroporphyrin with dimyristoylphosphatidylcholine unilamellar vesicles of various sizes (ranging from 38 to 222 nm) has been studied using a stopped-flow with fluorescence detection. Beside the kinetics of porphyrin incorporation into vesicles, the transfer of porphyrin from vesicles to human serum albumin has been investigated both experimentally and theoretically. The effects of both vesicle and albumin concentrations indicate that the transfer proceeds through the aqueous phase. It is governed by the rate of incorporation of porphyrin into the outer vesicle hemileaflet (kon), by the exit to the bulk aqueous medium (koff), and by the association (kas) and dissociation (kdis) constants relative to albumin. In both systems studied, a slower transbilayer flip-flop accounts for the biphasic character of the kinetics. This model is strongly supported by the effects of vesicle size, temperature, and cholesterol. The dependence of kon on the vesicle size indicates that the incorporation is diffusion controlled. The constant koff is found to be closely coupled to the phase state of the bilayer. The transbilayer flip-flop rate constant is approximately the same in both directions (approximately 0.4 s-1 at 32°C and pH 7.4). It is strongly affected by the presence of cholesterol in vesicles and by the temperature, with a sharp enhancement around the phase transition. With the exception of very small vesicles obtained by sonication, no influence of the vesicle size on the flip-flop rate was observed. An accelerating effect of tetrahydrofuran, used to improve the solubility of porphyrin, has been noted. Steady-state measurements and kinetics results were in excellent agreement. The interest of systems involving albumin as a scavenger to extract important rate constants, is emphasized.
Porphyrin Fluorescence Kinetic Liposomes Stopped-flow

"Electrosynthesized Non-conducting Polymers As Permselective Membranes In Amperometric Enzyme Electrodes: A Glucose Biosensor Based On A Co-crosslinked Glucose Oxidase/overoxidized Polypyrrole Bilayer"
Biosens. Bioelectron. 1998 Volume 13, Issue 1 Pages 103-112
A. Guerrieri,*, G. E. De Benedetto*, F. Palmisano

Abstract: A glucose amperometric biosensor based on glucose oxidase immobilized on an overoxidized polypyrrole (PPyox) platinum modified electrode, by glutaraldehyde co-crosslinking with bovine serum albumine, is described. The advantages of covalent immobilization techniques (e.g. high loading and long-term stability of the enzyme) are coupled with the excellent interferent rejection of electrosynthesized non-conducting polymers. The sensor showed an apparent Michaelis-Menten constant of 16±0.8 mM, a maximum current density of 490 µA/cm2 and a shelf lifetime of at least three months. Ascorbate, urate, cysteine and acetaminophen at their maximum physiological concentrations produced a glucose bias in the low micromolar range. Flow injection response was linear up to 20 mM glucose with typical sensitivity of 84.0±1.5 nA/mM. The sensor was tested for glucose determination of untreated serum samples from both normal and diabetic subjects; results of amperometric assay compared well with those obtained by a standard enzymatic-colorimetric method.
Glucose Amperometry Electrode Electrode Electrode Method comparison Interferences

"Amperometric Biosensor With A Composite Membrane Of Sol-gel Derived Enzyme Film And Electrochemically Generated Poly(1,2-diaminobenzene) Film"
Biosens. Bioelectron. 1998 Volume 13, Issue 1 Pages 67-73
Toshio Yao*,* and Kazuyoshi Takashima

Abstract: A sol-gel silicate-based biosensor for glucose was made by utilizing a composite membrane of sol-gel enzyme film and electrochemically generated poly(1,2-diaminobenzene) film to improve the selectivity of the sol-gel enzyme sensors. The stability of the sensor was improved by exposing the enzyme layer to glutaraldehyde vapor. The glucose sensor responded rapidly (ca. 15 s) to glucose at 0.6 V (versus Ag/AgCl), without any interferences from electroactive species such as L-ascorbate and urate below 0.2 mM. Reliable results were obtained in the assays of glucose in controlled human sera, with both the steady-state and flow injection measurements. Subsequently, the same sol-gel procedure was applied to the preparation of the sensors for galactose and cholesterol. The galactose and cholesterol sensors responded rapidly to galactose and cholesterol, respectively, although the sensitivity of these sensors was inferior to that of glucose sensor.
Glucose Galactose Cholesterol Amperometry Sensor Electrode Electrode Apparatus Detector

"Natural Silk Fibroin As A Support For Enzyme Immobilization"
Biotechnol. Adv. 1998 Volume 16, Issue 5-6 Pages 961-971
Yu-Qing Zhang

Abstract: A review with 40 references. Silk fibroin derived from Bombyx mori cocoon is being developed and utilized for purposes besides traditional textile material. Fibroin can be easily made up into various forms, several of which can serve as enzyme-immobilized supports. There are numerous reports on immobilized enzymes using these forms of silk fibroin as supports in which the enzyme-immobilized fibroin membranes were characterized in detail by means of spectrophotometry, IR spectra, NMR, ESR. Enzyme-immobilized fibroin membranes have been successfully used in several biosensors for the determinations of glucose, hydrogen peroxide and uric acid in which glucose and urate biosensors in a flow injection system were able rapidly to analyze various biosamples including human whole blood or serum.
Glucose Hydrogen peroxide Uric acid Sensor Review Silk fibroin membrane Immobilized enzyme

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 1. The Flow Injection Analysis Of Protein"
Bull. Chem. Soc. Jpn. 1983 Volume 56, Issue 5 Pages 1382-1387
Tadashi Hara,Motohiro Toriyama and Kazuhiko Tsukagoshi

Abstract: A new method in which luminol-hydrogen peroxide luminescent system is used has been proposed for the determination of the presence of protein. Since the catalytic activity of copper(II) for the chemiluminescent reaction between luminol and hydrogen peroxide decreased when copper(II) interacted with polypeptide linkage, this phenomenon was applied to the determination of protein. Determination of protein was carried out by a flow-injection method. The effects of reagent concentration, flow-rate, and reaction time on the analytical value were examined and the conditions for the determination of protein were established. Similar calibration curves were obtained for human serum albumin, bovine serum albumin, bovine serum α-globulin, and bovine serum γ-globulin. According to the present flow-injection method using chemiluminescent reaction, a small amount of protein could be conveniently and economically determined over a wide range of concentration, 7 x 10^-4 - 7 x 10^-2 g dm-;3, with the detection limit of 0.2 µg and at the rate of about 30 samples per hour. The present method was applicable to the determination of protein in serum.
Albumin Proteins γ-Globulin Clinical analysis Chemiluminescence Catalysis Optimization

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 2. Determination Of Albumin As A Model Protein By Means Of The Flow Injection Analysis Using A Cobalt(III) Complex Compound As A Catalyst"
Bull. Chem. Soc. Jpn. 1984 Volume 57, Issue 1 Pages 289-290
Tadashi Hara,Motohiro Toriyama and Kazuhiko Tsukagoshi

Abstract: A new procedure for the determination of 8 x 10^-9 - 3 x 10^-7 mol L-1 bovine serum albumin as a model protein has been established on the basis of the fact that the catalytic activity of cis-tetraarnminediaquacobalt(III) sulfate for the chemiluminescent reaction between luminol and hydrogen peroxide decreases in the presence of bovine serum albumin.
Albumin γ-Globulin Chemiluminescence Clinical analysis Catalysis Heated reaction

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 3. Flow Injection Analysis Of Protein By Direct Injection"
Bull. Chem. Soc. Jpn. 1984 Volume 57, Issue 6 Pages 1551-1555
Tadashi Hara,Motohiro Toriyama and Kazuhiko Tsukagoshi

Abstract: A flow-injection analysis method of protein by direct injection has been established by use of the luminol-hydrogen peroxide luminescence system. The determination of protein is based on the measurement of the decreasing catalytic activity of copper(II) for the chemiluminescent reaction between luminol and hydrogen peroxide. The present flow-injection analysis in which a protein solution is directly injected is quite different from the previous one in which a protein solution is injected after having been previously allowed to react with copper(II) outside the flow-through system. The optimum conditions For the determination of protein were determined with regard to reagent concentration, flow rate, reaction time, and reaction temperature. The present method is simple, rapid, and applicable to the determination of 2 x 10^-4 - 1 x 10^-1 g L-1 of protein with 40 ng of detection limit at a rate of about 30 samples per hour. The present method was also applied to the determination of the ratio of albumin to globulin in a sample. The present flow-injection system is expected to be a sensitive detector for the determination of small amounts of protein.
Proteins Albumin γ-Globulin Chemiluminescence Heated reaction Optimization

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 5. An Iron - Dyestuff Complex As A Catalyst"
Bull. Chem. Soc. Jpn. 1985 Volume 58, Issue 7 Pages 2135-2136
Tadashi Hara,Motohiro Toriyama,Kouichi Kitamura and Masakatsu Imaki

Abstract: The catalytic activity of the iron(III)-α,β,γ,δ-tetraphenylporphinetrisulfonic acid complex for the chemiluminescence reaction between luminol an H2O2 has been found to decrease in the presence of protein. On the basis of this phenomenon, 2 x 10^-5 - 2 x 10^-3 g L-1 bovine serum albumin was determined with the detection limit of 4 ng, lower than that in the previous paper.
Proteins Albumin Chemiluminescence Catalysis

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 8. Effect Of Heating On The Determination Of Protein"
Bull. Chem. Soc. Jpn. 1986 Volume 59, Issue 11 Pages 3681-3683
Tadashi Hara,Kazuhiko Tsukagoshi,Akihiro Arai and Takeshi Iharada

Abstract: To the sample solution, containing bovine or human serum albumin, was added Cu(II) solution and, after heating at 95°C, the solution was injected into a flow injection apparatus and mixed with buffer solution, H2O2 solution and 1,10-phenanthroline solution The chemiluminescence intensity was measured by a photon counter. The calibration graph was rectilinear from 1 to 100 nM-Cu(II). The apparent coupling constants of the Cu(II) - protein complexes suggest that heating stabilizes the complex and improves the concentration. range for the determination of the protein.
Proteins Albumin Chemiluminescence Catalysis Heated reaction

"Determination Of A Small Amount Of A Biological Constituent By The Use Of Chemiluminescence. 11. Determination Of Protein Using A 1,10-phenanthroline - Hydrogen Peroxide - Osmium(VIII) System"
Bull. Chem. Soc. Jpn. 1987 Volume 60, Issue 6 Pages 2031-2035
Tadashi Hara and Kazuhiko Tsukagoshi

Abstract: Formation of a complex between the analyte protein and Os(VIII) present in known excess was used to lessen the amount of Os available to catalyse the formation of chemiluminescence in the 1,10-phenanthroline - H2O2 system. Reagent solution were 0.12 mM 1,10-phenanthroline, 5% H2O2 and 1.3 mM Os(VIII) and the flow injection system was as previously described (Ibid., 1986, 59, 3681). Calibration graphs were established for the determination of bovine and human serum albumins and human serum γ-globulin under optimum conditions. From 5 µg L-1 to 1 mg L-1 of protein could be determined with a detection limit of 0.25 ng and a coefficient of variation of 2.9% at 0.1 mg L-1 (n = 10).
Albumin γ-Globulin Proteins Chemiluminescence Optimization

"Flow Injection Analysis For Glucose With Chemically Modified Enzyme Membrane Electrode"
Bunseki Kagaku 1984 Volume 33, Issue 4 Pages 213-217
Yao, T.;Nakanishi, K.;Wasa, T.

Abstract: The electrode was constructed by cross-linking glucose oxidase with catalase (electrode A) or with peroxidase (electrode B) and immobilizing the enzymes on a platinum sheet treated with (3-aminopropyl)triethoxysilane. The method involved the enzymatically catalyzed oxidation of β-D-glucose, with amperometric detection of the dissolved O consumed (electrode A) or the Fe(CN)63- generated from Fe(CN)64- (electrode B); measurements were made (vs. a silver - AgCl reference electrode) at -0.7 V (electrode A) and 0.0 V (electrode B). Peak currents were rectilinearly related to glucose concentration. of 20 to 800 mg dl-1 for electrode A (5 µL injections) and of 30 to 400 mg dl-1 for electrode B (1 µL injections). The technique was applied to the determination of glucose in human serum and urine with satisfactory precision (coefficient of variation 2 to 4%). The electrodes retained most of their original activity after repetitive use for two months. Diagrams of the amperometric-detector arrangement and of the flow injection system are presented.
Glucose Amperometry Electrode Electrode Electrode Immobilized enzyme

"Application Of An Immobilized-enzyme Column Reactor To Measurement Of Creatine Kinase Isoenzyme Activity By High Performance Liquid Chromatography"
Bunseki Kagaku 1984 Volume 33, Issue 7 Pages 381-385
Hayashi, M.;Mikami, H.;Ishida, Y.

Abstract: Creatine kinase isoenzymes (MM and MB) from human sera were separated (within 20 min), in a continuous-flow system, on a column (15 cm x 4 mm) of Synchropak AX-300 by gradient elution with Na acetate in Tris acetate buffer solution (pH 8). The individual isoenzymes eluted were then mixed with a substrate (creatine phosphate plus ADP) in the flow line, and the ATP formed was passed (with glucose) through a stainless-steel column (5 cm x 2.1 mm) containing hexokinase - glucose-6-phosphate dehydrogenase immobilized, with glutaraldehyde, on gel-like glass beads (prepared from tetraethoxysilane and HF, and aminopropylsilanized). The NADPH produced was monitored by fluorescence detection at 460 nm (excitation at 340 nm). The detector response was rectilinear for up to 25 nmol of ATP and the detection limit was 15 pmol; this was equivalent to isoenzyme activity from 1.0 to 500 iu l-1. The immobilized enzymes maintained their reactivity even after 100 h of use.
Enzyme, creatine kinase HPLC Fluorescence Catalysis Immobilized enzyme Post-column derivatization Glass beads

"Sensitive Determination Of Proteins By FIA With Coulometric Detection"
Bunseki Kagaku 1989 Volume 38, Issue 9 Pages 454-457
Kubo, H.;Huang, Y.S.;Kinoshita, T.;Nakazawa, H.

Abstract: Sample solution (5 ml) was injected into carrier/reagent solution comprising 0.3 M Na2HPO4 - 1 mM CuSO4 - 20 mM NH3 (pH 12) and passed to a reaction coil (5 m x 0.5 mm) operated at 95°C, and then to a cooling coil (1 m x 0.5 mm) operated at 10°C. Coulometric detection was carried out at 0.7 V. Calibration graphs were rectilinear for 10 to 100 ng of bovine serum albumin (I), human serum albumin, human γ-globulins and ovalbumin. The coefficient of variation (n = 10) at 10 and 25 ng of I were 3.5 and 3.1%, respectively. The detection limit for I was 2.5 ng. Histidine, cysteine, tryptophan and tyrosine were sensitively detected.
Albumin γ-Globulin Ovalbumin Cysteine Histidine Tryptophan Tyrosine Coulometry Heated reaction

"Determination Of Aluminum In Dialyzates And Human Serum By Flow Injection Analysis And Liquid Chromatography With Fluorimetric Detection"
Chem. Anal. 1998 Volume 43, Issue 2 Pages 215-224
Kokot, Z.;Pawlaczyk, J.

Abstract: A flow injection analysis and ion chromatography - fluorimetric method is described for the determination of aluminum in dialyzates and body fluids, based on its complexation with 8-hydroxyquinoline-5-sulfonic acid in the presence of hexadecyltrimethylammonium bromide. In the flow injection analysis the fluorimetric reagent consisted of HQS (0.003 mol/L) and CTAB (0.004 mol/L) in acetate buffer pH = 5.15, at a flow rate 0.5 mL/min. Ion chromatography was performed using Dionex CG2 cation exchange guard column and potassium sulfate (0.1 mol/L, pH = 3.0) as a mobile phase. Serum protein separation was achieved using Superdex 200 HR 10/30 column and phosphate buffer as an eluent. There was a good agreement between the values obtained by this method and graphite furnace atomic absorption spectrometry.
Aluminum HPIC Sample preparation Fluorescence Complexation 8-hydroxyquinoline-5-sulfonic acid Method comparison

"A New Method For Assay Of Ferroxidase Activity And Its Application To Human And Rabbit Serums"
Chem. Pharm. Bull. 1984 Volume 32, Issue 10 Pages 4029-4035
Tanabe, S.;Shioiri, T.;Murakami, K.;Imanari, T.

Abstract: The sample (10 µL) was treated with aqueous 0.3 mM Fe(III) solution (90 µL) and acetate buffer (200 µL) and the mixture was incubated at 30°C. Proteins were pptd. by addition of aqueous 1.25 M HClO4 (200 µL). After centrifuging, the supernatant solution was analyzed by flow injection analysis with use of aqueous 0.5 M NaSCN as the detection reagent; the complex formed was monitored at 480 nm. Tests carried out on human and rabbit serum revealed the presence of a ferroxidase-like substance other than ceruloplasmin. The coefficient of variation (n = 6) was 3.4%. No interferences were observed in the presence of Zn(II), Ni(II), Cd(II), Pb(II), Ca(II), Mg(II), Cl-, PO43-, SO42-, CO32-, CO32-, ClO4-, NO2- or S2O3-, whereas Cu(II), Cr(III), N3-, NO3- and SCN- interfered.
Enzyme, ferroxidase Clinical analysis Spectrophotometry Interferences Enzyme

"Automated Multiple Flow Injection Analysis In Clinical Chemistry. Determination Of Albumin With Bromocresol Green"
Clin. Chem. 1980 Volume 26, Issue 2 Pages 331-334
BW Renoe, KK Stewart, GR Beecher, MR Wills and J Savory

Abstract: We describe an adaptation of automated multiple flow injection analysis instrumentation to an analysis for albumin in serum. The bromcresol green reaction was used to test the utility of the system. The approach yielded albumin results with excellent sensitivity, no measurable carryover, a relative standard deviation of less than 1%, good correlations with published procedures, and no measurable interferences. The simplicity and flexibility of the instrumentation and its performance integrity, as indicated by the analytical results, make this a viable clinical chemical tool. An adaptation of automated multiple flow injection analysis instrumentation to an analysis for albumin in (human) serum is described. The bromcresol green reaction was used to test the utility of the system. The approach yielded albumin results with excellent sensitivity, no measurable carryover, a relative SD of less than 1%, good correlations with published procedures and no measurable interferences. The simplicity and flexibility of the instrumentation and its performance integrity, as indicated by the analytical results, make this a viable clinical chemical tool.
Albumin Clinical analysis Spectrophotometry Interferences Method comparison

"Competitive Heterogeneous Enzyme Immunoassay For Theophylline By Flow Injection Analysis With Electrochemical Detection Of P-aminophenol"
Clin. Chem. 1990 Volume 36, Issue 4 Pages 662-665
EP Gil, HT Tang, HB Halsall, WR Heineman and AS Misiego

Abstract: A competitive enzyme-linked immunoabsorbent assay based on the flow injection amperometric detection of p-aminophenol has been investigated with use of the materials and general procedure of a commercial kit for the determination of theophylline in human serum. The antibody is immobilized on glass beads, and the enzyme label is alkaline phosphatase (EC 3.1.3.1). The high currents generated during the electrochemical detection allowed a rapid (35 min) and simple determination of theophylline throughout its therapeutic range (10-20 mg/L) and also in the subtherapeutic range (detection limit of about 80 µg/L).
Theophylline 4-Aminophenol Immunoassay Amperometry Electrode Clinical analysis Glass beads Immobilized enzyme Detection limit

"Flow Injection Analysis Atomic Absorption Determination Of Serum Zinc"
Clin. Chim. Acta 1984 Volume 137, Issue 2 Pages 151-157
Abdulrahman S. Attiyat and Gary D. Christian*

Abstract: Flow injection analysis-atomic absorption (FIA-AA) determination of zinc in human serum samples is demonstrated. The same samples were analyzed for zinc by conventional atomic absorption spectrometry and no significant difference was found between the two methods. Proteins were precipitated from samples by adding equal volumes of 25% (w/v) trichloroacetic acid and 100 µL aliquots of the protein-free-filtrate were injected. Precision was±3.47% relative standard deviation. The frequency of measurements can be as high as 180 per hour. No pump was used, only the negative pressure from the nebulizer was utilized to aspirate the carrier stream. Simple 1:5 dilution of sample with water, followed by FIA-AA measurements, gave higher zinc levels. The effect of sample volume and the length of dispersion coil on the FIA signal was studied. Serum, deproteinized with trichloroacetic acid, was injected into an AAS instrument via 0.5-mm-i.d. PTFE tubing. The carrier stream (H2O) was introduced by the effect of the negative pressure of the nebulizer. The AAS was carried out with an air - acetylene flame, with measurement at 213.8 nm. Results agreed well with those by conventional AAS; the coefficient of variation was 3.47%. Up to 180 measurements could be made in 1 h. The effects of sample dilution and volume and the length of dispersion coil (between nebulizer and injector) are discussed.
Zinc Clinical analysis Spectrophotometry Method comparison Optimization

"Flow Injection System Based On The Sandwich Technique For Saving Expensive Reagents"
Clin. Chim. Acta 1991 Volume 203, Issue 1 Pages 67-76
Alberto N. Araujo, Jos&eacute; L. F. C. Lima, Juli&aacute;n Alonso-Chamarro*, Jordi Bartrol&iacute; and Manel Poch

Abstract: We report the application of a sandwich technique in flow injection systems which afford low consumption of expensive reagents and two reagent recirculation systems. The potential applicability of the technique thus developed was assessed by determining glucose in serum samples by the enzymatic glucosidase/peroxidase method. It was possible to perform up to 450 determinations with the same amount of reagent used to perform 50 determinations by batch procedures. The sampling rate was 80 determinations per hour with a 0.9% relative standard deviation. Serum was subjected to flow injection analysis with mixing with phosphate buffer solution (pH 7.4) and 11 mM phenol - 0.8 mM 4-aminoantipyrine - peroxidase (900 iu l-1) - glucose oxidase (15,000 iu l-1) and detection at 520 nm. An eight-port injection valve (cf. Alonso et al., Anal. Chim. Acta, 1987, 199, 191) was used to reduce reagent consumption along with a recycling system for the enzyme reagent solution The within-batch coefficient of variation (n = 10) was 0.9%. The method was used to perform 450 determinations with the same amount of reagent used to perform 50 determinations by the batch procedure. The sampling rate was 80 determinations h-1. The results compared well with those obtained using a Hitachi 735 batch analyzer..
Glucose Spectrophotometry Clinical analysis Buffer Calibration Low cost Sandwich technique Reagent consumption Enzyme

"Enzymic Determination Of Bicarbonate In Serum By Flow Injection Analysis"
Clin. Chim. Acta 1995 Volume 235, Issue 2 Pages 169-177
R. Quilesa, J. M. Fern&aacute;ndez-Romerob and M. D. Luque de Castrob,*

Abstract: An automated method for the determination of bicarbonate in human serum is presented. The method is based on the enzymatic reaction between bicarbonate and phosphoenolpyruvate (PEP) in the presence of PEP carboxylase. A diagram is shown of the hydrodynamic flow injection system. The analytical reaction was coupled with a derivatization reaction in which the NADH consumed was monitored at 460 nm (excitation at 340 nm). The enzymes PEP carboxylase and malate dehyrogenase were immobilized on controlled-pore glass. The calibration graph was linear from 25-300 mM. Within- and between-run RSD (n = 11) were 1-2.8 and 1.32-3.58%, respectively. An automated method for the determination of bicarbonate in human serum based on the enzymatic reaction between the analyte and phospho(enol)pyruvate (PEP) in the presence of PEP carboxylase is proposed. The analytical reaction is coupled with a derivatization reaction in which the NADH consumed is fluorimetrically monitored (lambda ex = 340 nm, lambda em = 460 nm). A stopped-flow/flow injection approach is used in which the enzymes (PEP carboxylase and malate dehydrogenase) are immobilized on controlled-pore glass. The linear determination range is between 25 and 300 mmol/l (r2 = 0.9973). The %C.V. for the within- and between-run studies, performed at three concentration levels, ranges between 1.0 and 3.6% and the sampling frequency is 20 per h.
Bicarbonate Fluorescence Clinical analysis Immobilized enzyme Controlled pore glass Stopped-flow

"Bi-enzyme Reactor For Electrochemical Detection Of Low Concentrations Of Uric Acid And Glucose"
Clin. Chim. Acta 1995 Volume 239, Issue 2 Pages 153-165
Ottilia Elekes, Danila Moscone, Kor Venema and Jakob Korf*

Abstract: Human serum (4 µL) was diluted 50- to 100-fold with PBS of pH 7.4 containing 2 mM EDTA and 0.5 mM ferrocenemonocarboxylic acid (buffer A), then left for >6 min before analysis. Portions (2.5 µL) were injected into buffer A and carried at 150 µL/min to a sandwich-type enzyme reactor kept at 30°C containing either uricase and horseradish peroxidase or horseradish peroxidase and glucose oxidase physically co-immobilized between cellulose nitrate filters. The reduction current was measured using a vitreous C electrode held at 0 mV vs. Ag/AgCl and a PTFE/C counter electrode. The calibration graphs were linear for up to 200 µM-uric acid and 1 mM glucose; the detection limits were 30 and 60 nM, respectively. The recoveries of uric acid and glucose were 98-102% and 98-109%, respectively, the within-run RSD were 2% and 3%, respectively (n = 10), and the between-day RSD were 4% and 3%, respectively (n = 10). The results agreed with those obtained using conventional clinical laboratory methods. Sample throughput was 60/h. An enzyme-based flow injection amperometric analysis system for monitoring of uric acid and glucose is described. The oxidase and peroxidase enzymes are physically coimmobilized in a sandwich-type reactor and ferrocene serves as a mediator. The assays are based on the measurement of a reduction current resulting from the enzymatic reactions, at a glassy carbon electrode held at 0.00 mV (vs. Ag/AgCl). The high selectivity (ascorbic acid did not interfere) is coupled to high sensitivity (a detection limit of 30 and 60 nmol/l for uric acid and glucose, respectively; signal/noise = 3) and good stability (the enzymes remained active for more than 6 weeks at 30°C). The usefulness of the assay in clinical chemistry is illustrated by the measurement of human serum uric acid and glucose concentration. The results obtained were in fairly good agreement with those obtained using conventional hospital laboratory methods.
Glucose Uric acid Amperometry Electrode Clinical analysis Calibration Immobilized enzyme Interferences Method comparison

"Flow Injection Determination Of Glutamate In Human Serum And Rat Brain Samples With Immobilized Glutamate Oxidase And Glutamate Dehydrogenase Reactors"
Clin. Chem. Lab. Med. 1994 Volume 32, Issue 10 Pages 767-772
C. D. Stalikas / M. I. Karayannis / Stella M. Tzouwara-Karayanni

Abstract: Two methods are proposed for the determination of regional concentrations of glutamate in the rat brain as well as in human serum. Glutamate oxidase was immobilized on non-porous glass beads and glutamate dehydrogenase was immobilized on glass derivatives. These supports were employed for the construction of Single Bead String Reactors and Packed Bed Reactors, respectively, which in turn were linked to Flow Injection Analysis systems with either photometric or fluorometric detection. Analytical working curves are linear in the range 1-200 µmol/L for packed bed reactors and 10^-500 mmol/l for single bead string reactors. The samples were pretreated depending on their origin and the applied measuring system. Optimal dilution factors were established for the two techniques. Optimal dilution ratios were established and the influence of several added substances was investigated. Recovery and method comparison studies including high performance liquid chromatography verified the accuracy of the proposed methods. Results from within-day and between-day measurements gave relative standard deviations of 4.7 and 5.9% for serum samples and 2.5 and 4.0% for brain samples, respectively.
Glutamate Fluorescence HPLC Clinical analysis Immobilized enzyme Glass beads Single bead string reactor Method comparison

"Fast Screening For Drugs Of Abuse By Solid Phase Extraction Combined With Flow Injection Ionspray-tandem Mass Spectrometry"
J. Anal. Toxicol. 1998 Volume 22, Issue 4 Pages 319-328
W. Weinmann (Wolfgang) and M. Svoboda

Abstract: A fast anal. approach for the simultaneous quant. screening for illicit drugs in serum and urine without tedious chromatography separation steps was developed by combining solid phase extraction (SPE) followed by flow injection analysis (FIA) with ion spray-ionization and tandem mass spectrometry (MS-MS) detection using a PE Sciex API 300 triple-quadrupole MS. MS-MS anal. was performed by sequentially isolating the precursor ions of the analytes and their deuterated standards with subsequent fragmentation and monitoring of one fragment ion for each substance. A multiple-reaction monitoring experiment was set up for morphine (MO), codeine (COD), amphetamine (AMP), benzoylecgonine (BZE), and their deuterated analogs. For method evaluation, serum samples spiked with 2-1000 ng of each drug and deuterated standards were extd. by mixed-mode SPE, redissolved in MeCN-NH4OAc-buffer, and directly injected by flow injection into the ion spray source. The specificity of this new method was demonstrated by testing compounds with similar chemical structure for interferences from the analytes of interest (e.g., hydromorphone, morphine glucuronide, and 6-monoacetylmorphine with MO; dihydrocodeine and hydrocodone with COD; cocaine [COC] and ecgonine methylester with BZE; methamphetamine with AMP). The possibility of interferences of such compounds with the FIA-ion spray-MS-MS screening method is discussed. Spiked serum samples and serum and urine samples from drug addicts and victims of drug abuse were analyzed with FIA-MS-MS and, after derivatization, with gas chromatography-mass spectrometry (GC-MS). Comparable quant. results were obtained with both methods; no interferences with metabolites or other compounds were found. The FIA-ionspray-MS-MS method is a fast, quant., sensitive, and highly specific alternative method to drug-screening by immunoassays, high-performance liquid chromatography, and GC-MS. It can be used for the simultaneous detection of different drugs and metabolites such as opiates, COC, AMP derivatives, and many other drugs.
Drugs Morphine Codeine Amphetamine Benzoylecgonine Mass spectrometry Mass spectrometry Solid phase extraction Interferences Method comparison

"Screening For Ochratoxin A In Blood By Flow Injection Analysis"
J. Appl. Toxicol. 1984 Volume 4, Issue 6 Pages 326-329
Hult K, Fuchs R, Peraica M, Plestina R, Ceovic S.

Abstract: A micromethod for ochratoxin A detection in human sera by flow injection technique is described. The method requires 50 µL of sera, and it is designed to distinguish samples containing less than 10 ng ochratoxin A per mL. The method is based on fluorescence measurement following a simple extraction procedure for which very small amounts of chemicals are needed. Since the method is not confirmatory, all samples showing fluorescence above a certain intensity have to be reanalyzed with some other method where a confirmation step in included. Because of the small amount of serum needed and the rapid procedure (less than 15 min), a large number of samples can be analyzed very quickly. The method may therefore be applicable for large screening campaigns conducted to determine the presence of ochratoxin A in blood. This conclusion is based on 1675 samples and 147 standards analyzed concurrently by the flow injection technique and an earlier published enzymatic method. The method is also suitable for monitoring ochratoxin A levels in the blood of experimental animals.
Ochratoxin A Clinical analysis Fluorescence Sample preparation Extraction Method comparison

"Automatic-determination Of Malate Dehydrogenase Activity With A Flow Injection Multidetection System"
J. Autom. Methods Manag. Chem. 1990 Volume 12, Issue 2 Pages 49-52
J. M. FERN&Aacute;NDEZ-ROMERO and M. D. LUQUE DE CASTRO

Abstract: Serum was subjected to flow injection analysis with mixing with 0.1 M phosphate buffer solution (pH 7), 50 mM oxaloacetate in buffer solution and 10 mM NADH in 30% ethylene glycol, followed by multipeak detection at 340 nm. The manifold includes a valve for closing the circuit along which the reacting plug is continuously circulated, and the absorbance is measured at a conventional spectrophotometer included in the circuit, each peak corresponding to one passage of the plug through the flow cell of the instrument. The calibration graph was rectilinear from 0.02 to 2.00 iu L-1 of malate dehydrogenase and the coefficient of variation (n = 11) was 0.1 to 0.9%. Recovery was 96 to 103%.
Enzyme, malate dehydrogenase Spectrophotometry Multidetection Buffer Flowcell Calibration Closed loop

"Application And Automation Of Flow Injection Analysis (FIA) Using Fast Responding Enzyme Glass Electrodes To Detect Penicillin In Fermentation Broth And Urea In Human Serum"
J. Autom. Methods Manag. Chem. 1992 Volume 14, Issue 4 Pages 137-143
HELMUT MEIER, FRAN&Ccedil;OIS LANTREIBECQ, and CANH TRAN-MINH

Abstract: Penicillinase and urease were cross-linked as very fine films directly onto the sensitive ends of pH glass electrodes and incorporated into a flow injection analysis system (diagram given). Samples were injected into a carrier stream (4 mL min-1) of buffer solution and passed to the detection cell containing the electrodes. Calibration graphs were rectilinear for 100 mM penicillin and for 10 mM urea; corresponding coefficient of variation were 0.94 and 1.94%. An enzyme immobilization technique was developed to determine the concentration. of biological compounds This technique was applied to penicillinase and urease, which are crosslinked as very fine films directly onto the sensitive ends of pH glass electrodes, thereby dispensing with the need of an online enzyme reactor. The biosensor is incorporated in a FIA system within a magnetically stirred detection cell. Penicillin-V in fermentation broth and urea in human serum samples was detected and the results were compared with HPLC and spectrophotometric methods. Online measurement is achieved through the automation of this FIA system.
Penicillin Urea Electrode Electrode Sensor Method comparison

"A Flow Injection Analysis System Involving Immobilized NADH Oxidase In Column Form For Clinical Analysis"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 33-41
Takashi Murachi*, Masayuki Totani, Masaki Ikemoto and Masayoshi Tabata

Abstract: A highly sensitive FIA system for chemiluminometric determination of reduced coenzyme, NADH, was developed, using immobilized NADH oxidase from Brevibacterium ammoniagenes. The enzyme catalyzed the oxidation of NADH generating hydrogen peroxide which emitted chemiluminescence when mixed with luminol and potassium ferricyanide. The immobilized enzyme reactor was a mini-column, measuring 1 or 2 mm in inner diameter and 20 mm in length, and the sample volume was only 1 µL per assay, with a feeding speed of one sample per min and a lowest detection limit of 10 pmol NADH. A FIA system was also developed for the determination of magnesium in human serum, using an enzyme column reactor with simultaneously coimmobilized hexokinase, D-glucose-6-phosphate dehydrogenase, and NADH oxidase. The performance of the system was as satisfactory as a routine colorimetric assay, but with much higher sensitivity. A flow injection analysis (FIA) system is described for the determination of NADH with a detection limit of 10 pmol. Samples (1 µL) were passed through a mini-column (2 cm x 2 mm) containing immobilized NADH oxidase and the H2O2 generated was determined by a chemiluminometric method using luminol and potassium ferricyanide. A FIA system was also developed for the determination of Mg in human serum using a reactor with co-immobilized hexokinase, D-glucose-6-phosphate dehydrogenase and NADH oxidase. The sensitivity was higher than that for the colorimetric assay using Xylidyl Blue.
Magnesium Nicotinamide adenine dinucleotide oxidized Chemiluminescence Clinical analysis Immobilized enzyme Enzyme Column Catalysis Detection limit Sensitivity Method comparison

"Determination Of Alanine Aminotransferase In Human Serum In An Open-closed Flow Injection Configuration"
J. Biotechnol. 1990 Volume 14, Issue 1 Pages 43-52
J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro and M. Valc&aacute;rcel*

Abstract: Open-closed flow injection systems allow the development of methods based on multidetection by using conventional detectors. The methods proposed here for the determination of alanine aminotransferase (ALT) utilize a photometric detector included in the closed circuit, which allows one to perform fixed-time and reaction-rate measurements whose sensitivity can be increased by using the sum of the analytical signals. The methods, with determination ranges between 1 and 500 U L-1 of ALT, feature sampling frequencies up to 60 h-1. Their application to serum samples provided excellent results, with recoveries between 95 and 106% and averaging at 99.9%. The determination of alanine aminotransferase (I) is based on the transfer of the amino group from L-alanine to 2-oxoglutarate, yielding glutamate and pyruvate within a reactor. A selector valve switched the flow into a closed circuit where, in another reactor, pyruvate was reduced with lactate dehydrogenase and NADH. This indicator reaction was subsequently monitored by the decrease in absorbance at 340 nm in a spectrophotometer flow cell. Repeated passage of the reactive plug through the closed circuit allowed an increase in the sensitivity of the measurements. Recoveries were quantitative for 1 to 500 U L-1 of I. Sample throughput was up to 60 h-1.
Alanine aminotransferase Spectrophotometry Multidetection Detector Sensitivity Flowcell Closed loop

"Use Of A Reversibly Immobilized Enzyme In The Flow Injection Amperometric Determination Of Picomole Glucose Levels"
J. Chem. Soc. Faraday Trans. 1986 Volume 82, Issue 4 Pages 1265-1270
Catherine E. Lomen, Uditha de Alwis and George S. Wilson

Abstract: Glucose oxidase is covalently attached to anti-human IgG by using p-benzoquinone as coupling agent. The resulting conjugate is introduced into a carrier stream of 0.1 M phosphate buffer (pH 6.8) and passed through a µreactor (cf. Anal. Chem., 1985, 57, 2754) containing human IgG covalently bound to controlled pore glass (Lichrospher SI-300). The subsequent immunological reaction within the reactor tube results in the immobilization of enzyme. In the event of a loss of enzyme activity, the enzyme can be removed by dissociating the antibody - antigen complex by elution with 0.1 M phosphate buffer (pH 2.0). The microreactor can then be re-loaded with fresh enzyme. The antigen remains active in binding for >1 year. Results obtained with such a reactor for determination of glucose in four samples of human serum agreed closely with those given by a Beckman Astra instrument.
Glucose Clinical analysis Amperometry Activity Controlled pore glass Immobilized enzyme Method comparison

"Measurement Of Free And Total Hydroxyproline By Automated Flow Injection Of Serum Or Urine Samples From Maintenance Hemodialysis Patients With Renal Osteodystrophy"
J. Clin. Lab. Anal. 1994 Volume 8, Issue 5 Pages 267-272
Uji Y, Karmen A, Okabe H, Hata K, Miura M, Ozaki K, Minamizaki M, Shibata T, Inayama S

Abstract: For the determination of total hydroxyproline, 100 µL of plasma or 1 mL of urine was mixed with 200 µL of 12 M HCl and autoclaved for 3 h at 120°C. The hydrolysate was neutralized with 1 mL of 12 M KOH and 1 mL of 1 M L-cysteine. A 100 µL portion was injected into a carrier stream in a FIA system of chloramine T in borate buffer and KCl adjusted to pH 8.7 with 1 M KOH. The mixture was heated at 120°C in a mixing coil (24 m x 1 mm i.d.) in an Al block. The reaction mixture was merged with a stream of Ehrlich's reagent (1:1), the resulting solution passed through a reaction coil (10 m x 1 mm i.d.) to a double beam photometer for detection at 560 nm. Free hydroxyproline was determined as above but omitting the hydrolysis step and filtering the serum. Calibration graphs were linear up to 1.22 mM with a detection limit of 3.8 µM. The within-run RSD was 2.34, 2.25 and 2.53% for 76, 38 and 19 µM, respectively. The recovery of 10^-50 µM of hydroxyproline in urine was 92-104%. Results agreed well with those obtained by HPLC.
Hydroxyproline Spectrophotometry HPLC Clinical analysis Dialysis

"A Variable Volume Injector Applied To The Atomic Absorption Determination Of Sodium, Potassium, Calcium And Magnesium In Blood Serum By Flow Injection Analysis"
J. Flow Injection Anal. 1990 Volume 7, Issue 1 Pages 11-18
J.L. BURGUERA*, M. BURGUERA and C. Rivas, M. DE LA GUARDIA, A. SALVADOR and V. CARBONELL

Abstract: Different volume of a single standard solution containing Na, K, Ca and Mg were used to determine the cited metals in serum samples. A simple variable-volume injector (Anal. Chim. Acta, 1990, 234, 253) was used to inject the different volume in order to construct a calibration graph. Diagrams are presented of the injector. The coefficient of variation (n = 8) for the determination of 75, 4, 2 and 0.5 µg of Na, K, Ca and Mg, respectively, were 1.5, 1.2, 0.8 and 1.2%, respectively. Results for 22 µL of standard human serum agreed well with the known values.
Sodium Potassium Calcium Magnesium Spectrophotometry Injector Calibration

"Highly Sensitive Amperometric Enzyme Immunoassay For α-fetoprotein In Human Serum"
J. Immunol. Methods 1996 Volume 193, Issue 1 Pages 51-62
Leopoldo Della Ciana*, Giuliana Bernacca, Concetta De Nitti and Anilla Massaglia

Abstract: A sandwich amperometric enzyme immunoassay with flow injection for α-fetoprotein in human serum has been developed with alkaline phosphatase as the enzyme label. p-Hydroxyphenyl phosphate was used as the substrate for alkaline phosphatase. The hydrolysis product, hydroquinone, was detected by oxidative amperometry in a flow injection system. The amperometric wall jet detector was fitted with a glassy carbon working electrode held at 350 mV vs. Ag/AgCl. The detection limit of hydroquinone in 30 mM borate buffer pH 9.5 was 1.2 x 10^-10 M (linearity range: 10^-9 - 5.12 x 10^-6 M. A detection limit for free alkaline phosphatase of 1.2 x 10^-15 M (linearity range: 10^-15 - 10^-13 M), or about 36 000 molecules, was observed (same borate buffer and incubations of 10 min at 25°C). These conditions were maintained for the amperometric α-fetoprotein immunoassay. For comparison purposes, a photometric detection system was set up, with p-nitrophenyl phosphate as enzyme substrate and the same pair of antibodies and incubation conditions. The detection limit for α-fetoprotein obtained by amperometry, 0.07 ng/ml (linearity range = 5-500 ng/ml), was 14 times lower than by photometry. The amperometric enzyme immunoassay correlates well with a commercial colorimetric immunoassay (r = 0.986, slope = 0.967, n = 240).
α-Fetoprotein Immunoassay Amperometry Electrode Method comparison Enzyme

"Additional Advantages Of The Use Of Immobilized Enzymes: Immobilization Of β-D-galactosidase For Magnesium Determination"
Quim. Anal. 1995 Volume 14, Issue 1 Pages 31-35
Quiles Zafra, R.;Fernandez Romero, J.M.;Luque De Castro, M.D.

Abstract: The method was based on Mg activation of the β-D-galactosidase-catalyzed hydrolysis of o-nitrophenyl-β-D-galactopyranoside (I). The enzyme was immobilized on to controlled-pore glass (120-200 mesh) and packed into a glass column (100 cm x 0.5 mm i.d.). The resulting enzyme reactor was incorporated into a FIA system. Sample (50 µL) and I reagent [40 µL; 100 mM Tris hydrochloride buffer of pH 7.5 containing 4 mM DTT and 0.4 mM EGTA (buffer A) with 800 mM NaCl and 2 mM I] were injected into different streams (1.2 ml/min) of buffer A. After merging, the streams passed through a single bead string reactor, then through the enzyme reactor before merging with a stream of 1N-NaOH for detection at 405 nm. Analysis was performed at 37°C. The calibration graph was linear for 5-20 µM-Mg and the within- and between-run RSD were 0.7-2.9% and 1.7-3.2%, respectively. Sample throughput was 40/h. No significant interference from cations was observed. The method was applied to human serum with recoveries of 96-113 %.
Magnesium Spectrophotometry Immobilized enzyme Controlled pore glass Single bead string reactor Interferences Heated reaction

"Application Of Immobilized Protein A In Flow Injection Fluoriimmunoassay"
Shengwu Huaxue Yu Shengwu Wuli Jinzhan 1995 Volume 22, Issue 1 Pages 81-84
Ren Xuezhen, J. N. Miller

Abstract: A convenient, semi-automated flow injection fluoroimmunoassay method has been developed incorporating the flexibility of sample hand1ing of flow injection system containing a immobilized protein A immunoreactor. Protein A can binds the Fc region of most mammlian antibodies at near neutral pH, and the bound antibodies can be eluted from protein A at acid pH. This peculiar property of protein was exploited to separate the antigen bound to antibody and the unbound ones. Experimental variables have been studied and the method has been used to determine the transferrin contents in human serum.
Transferrin Immunoassay Fluorescence Immobilized enzyme

"Comparison Of Digoxin Analysis By High Performance Liquid Chromatography - Post-column Derivatization And Fluorescence-polarization Immunoassay"
Xenobiotica 1990 Volume 20, Issue 6 Pages 635-643
L. Embree and K. M. McErlane

Abstract: The method described previously (J. Chromatogr., 1989, 496, 321) was compared with the Abbott TDx method. The r value between the two methods for digoxin added to drug-free serum was 0.9897 (n = 7). However, serum from digitalized patients showed higher digoxin levels by the TDx method, probably owing to cross-reactivity of metabolites. Cross-reactivity of the TDx method towards endogenous material in the serum of certain patient groups was an even greater problem. The HPLC method (loc. cit.) gave complete resolution of digoxin. The calibration graph for this method was rectilinear for 0.5 to 3.3 ng mL-1 of digoxin in a 3 mL serum sample, with a mean coefficient of variation of 5.6% and a detection limit of 0.5 ng injected.
Digoxin HPLC Immunoassay Fluorescence Post-column derivatization Calibration Detection limit

"A Rapid And Specific Method For The Determination Of Oxytocinase (cystine Aminopeptidase) Activity Using Continuous Flow Analysis And S-benzyl-L-cysteine-P-nitroanilide As Substrate*"
Clin. Biochem. 1975 Volume 8, Issue 1-6 Pages 124-132
C.W. Small and W.B. Watkins

Abstract: 1. An automated method is described for the assay of pregnancy serum cystine aminopeptidase (Oxytocinase, E.C. 3.4.1.10) using continuous flow analysis. S-Benzyl-L-cysteine-p-nitroanilide, the most highly specific substrate so far available for oxytocinase is employed in this assay. Comparison with the manual method from which it has been developed, gave a correlation coefficient of 0.98. Reaction of oxytocinase on the substrate liberated p-nitroaniline, the absorption of which is measured at 410 nm. 2. Blank values are eliminated by the incorporation of a dialyzer in the manifold design and thereby effectively increasing the rate of assay sample to 40 per hour. The lag phase (a) of the continuous flow analysis is 0.17 min while the exponential phase (b) is 0.31 min. Using a 2:1 sample to wash ratio the carry-over is 3% at a 95% of steady state.
Enzyme, cysteine aminopeptidase Spectrophotometry Method comparison Dialysis