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

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Blood Plasma

Classification: Biological fluid -> blood -> plasma

Citations 131

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

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

"Comparison Of Spectrophotometric And Chemiluminescence Methods For The Determination Of Blood Glucose By Flow Injection Analysis"
Anal. Chim. Acta 1984 Volume 164, Issue 1 Pages 103-109
P. J. Worsfold, J. Farrelly and M. S. Matharu

Abstract: The sample was injected into a stream of 0.18 M NaCl, which was dialysed against either 0.2 M KH2PO4 - 12.5 mM Na2EDTA - 4.2 mM disodium 3,5-dichloro-2-hydroxybenzenesulfonate (in the spectrophotometric procedure) or 0.2 M phosphate buffer (1.2 mM in Na2EDTA; pH 6.5) (for chemiluminescence). The diffusate was passed through a reactor tube containing immobilized glucose oxidase, and the H2O2 formed was determined either by spectrophotometry at 505 nm, via the peroxidase-catalyzed oxidation of 4-aminoantipyrine, or by chemiluminescence involving the Fe(III)-catalyzed reaction of H2O2 with luminol. Calibration graphs were rectilinear in the range 0 to 25 mM and the coefficient of variation were 0.3 to 2.3% and 1.3 to 3.9% for the spectrophotometric and chemiluminescent procedures, respectively. Results for 10 samples of plasma correlated well (r = 0.972 for spectrophotometry and 0.988 for chemiluminescence) with those obtained with an electrochemical stat analyzer.. The sampling rate was 75 h-1.
Glucose Chemiluminescence Clinical analysis Spectrophotometry Dialysis Immobilized enzyme Reactor

"Determination Of Glucose In Blood By Flow Injection Analysis And An Immobilized Glucose Oxidase Column"
Anal. Chim. Acta 1984 Volume 166, Issue 1 Pages 111-118
M. Masoom and Alan Townshend

Abstract: Glucose oxidase was immobilized on controlled-porosity glass and used in a glass column (25 mm x 2.5 mm). The H2O2 (1 µM) produced by the enzymatic reaction between the glucose oxidase and glucose was detected by the current produced in a flow-through cell with two platinum electrodes having a p.d. of 0.6 V. The carrier stream (2 mL min-1) was 0.1 M phosphate buffer (pH 4.5) and a fixed sample volume (~20 µL) was delivered into the stream by means of a rotary valve. Glucose (0.2 mM) could be determined in plasma with either a dialyser or a column (25 mm x 2.5 mm) of Cu diethyldithiocarbamate on controlled-porosity glass inserted before the enzyme column. Results agreed well with those obtained by a conventional analyzer. system and no deterioration of the enzyme was observed over a 10-month period.
Glucose Amperometry Electrode Dialysis Immobilized enzyme Reactor Controlled pore glass

"Flow Injection Determination Of Adenosine And Inosine In Blood Plasma With Immobilized Enzyme Columns Connected In Series And Fluorimetric Detection"
Anal. Chim. Acta 1986 Volume 186, Issue 1 Pages 131-137
Yohji Hayashi, Kiyoshi Zaitsu and Yosuke Ohkura

Abstract: Samples were mixed with HClO4, and centrifuged, the supernatant solution was treated with K2CO3 followed by centrifugation, and urate oxidase and catalase, were added to the supernatant solution. After 30 min at 37°C, the solution was treated as above, the supernatant solution was diluted with Tris - HCl buffer of pH 8.0 and an aliquot was analyzed on successive columns (4 cm x 0.86 mm) of adenosine deaminase, purine nucleoside phosphorylase, xanthine oxidase, urate oxidase and horse-radish peroxidase, immobilized on amino-derivatized controlled-pore glass AMP-CPG 1400 (120 to 200 mesh; 140 nm pore size). The carrier and reagent solution contained 0.15 M NaCl - 50 mM Na2HPO4 - 10 mM Na2EDTA and 0.1 M Tris - HCl buffer (pH 8.0); the reagent solution also contains 5 mM 3-(4-hydroxyphenyl)propionic acid(I),flow rates were 0.25 mL min-1, and the H2O2 formed was detected fluorimetrically after reaction with I at 405 nm (excitation at 305 nm). Calibration graphs were rectilinear from 0.5 to 500 pmol of adenosine or inosine, with recoveries of ~100% and coefficient of variation of 1% (n = 10).
Adenosine Inosine Clinical analysis Fluorescence Column Controlled pore glass Immobilized enzyme

"Online Electrochemical Derivatization Combined With Diode Array Detection In Flow Injection Analysis. Rapid Determination Of Etoposide And Teniposide In Blood Plasma"
Anal. Chim. Acta 1987 Volume 202, Issue 1 Pages 35-47
M. A. J. Van Opstal, J. S. Blauw, J. J. M. Holthuis, W. P. Van Bennekom and A. Bult

Abstract: The cited antineoplastic agents (I and II, respectively) are oxidized electrochemically in a flow-through cell equipped with two porous-graphite working electrodes, followed by spectrophotometric detection of the respective o-quinone compounds at 365 nm. Plasma (1 ml) is extracted with 2 mL of 1,2-dichloroethane and, after centrifuging for 2 min at 2500 g, 1 mL of the organic layer is evaporated to dryness. The residue is dissolved in 100 to 200 µL of carrier solution [Britton - Robinson buffer solution (pH 4) - methanol (1:1)] and a 25 µL portion of this solution is injected into the carrier stream (0.2 mL min-1). The optimum oxidation potentials for I and II were +500 and +450 mV (vs. a proprietary reference electrode), respectively. Calibration graphs were rectilinear in the range 1 to 50 µg mL-1 of I or II, and the limit of detection was 6 ng for each compound. For 2.5 and 25 µg mL-1 of I or II, the coefficient of variation were 6.5 and 3.2%, respectively. Recovery of 10 µg mL-1 of I or II was 95.7 ± 2.7% (n = 6) and the max. injection frequency was 40 h-1. Potential interference from catecholamines and metabolites is discussed.
Etoposide Teniposide Clinical analysis Spectrophotometry Electrode Electrochemical product conversion Interferences Optimization

"Amperometric Assay Of Glucose And Lactic Acid By Flow Injection Analysis"
Anal. Chim. Acta 1988 Volume 209, Issue 1-2 Pages 231-237
Bo A. Petersson

Abstract: Plasma was injected into a carrier stream of aqueous phosphate buffer (containing Na2EDTA, Na benzoate, NaH2PO4, Na2HPO4 and NaCl) at 1 mL min-1. Immobilized glucose or lactate oxidase membrane electrodes were coupled to an amperometric detector and polarized at +600 mV for detection of H2O2. The response was rectilinear for up to 40 mM glucose(I) and up to 10 mM lactic acid(II), with coefficient of variation of 1.5%. Results for determination of I and II agreed well with those obtained by established methods.
Glucose Lactic acid Amperometry Clinical analysis Electrode Computer Immobilized enzyme Method comparison

"Flow Injection Spectrophotometric Determination Of Glucose In Blood Plasma With Bindshedler's Green Leuco Base As Color Reagent"
Anal. Chim. Acta 1988 Volume 214, Issue 1 Pages 455-461
Masanori Akiba and Shoji Motomizu

Abstract: The hydrogen peroxide produces in the oxidation of glucos in an immobilized glucose oxidase reactor is determined by using Bindschedler's green (leuco base) as color reagent with iron(II) as catalyst; the increase in the absorbance at 725 nm is measured. For 100 µL samples, calibration was almost linear in the range 0-2.5 mg L-1 glucose; the relative standard deviation for 1 mg L-1 glucose was 0.6% (n = 10) and the detection limit (S/N = 2) was 0.02 mg L-1. The injection rate was 20 h-1. Glucose was determined satisfactorily in control sera and in real blood sera.
Glucose Clinical analysis Spectrophotometry Catalysis Enzyme Merging zones

"Effect Of Whole Blood And Plasma On The Permeability Of Glucose Through Different Cellulose And Cellulose Acetate Membranes"
Anal. Chim. Acta 1990 Volume 231, Issue 2 Pages 165-173
Lars Risinger, Thomas Buch-Rasmussen, Gillis Johansson

Abstract: The transfer of glucose (I) from whole blood, plasma and aqueous standards through different membranes for use in biosensors was carried out with use of a flow injection system (diagram given) incorporating a flow-through dialysis cell that contained the membrane being studied. Detection was with use of a glucose dehydrogenase enzyme reactor and a graphite electrode at 0 mV vs. Ag - AgCl. The membranes studied were: cellulose acetate; Spectra/por 4 (mol. wt. cut off = 12,000 to 14,000; Spectrum Medical Industries, Los Angeles, CA); Spectra/por 6 (mol. wt cut off = 1,000) and cellulose acetate Cuprophan (mol. wt cut off = 5,000). Theoretical models are proposed for the effect of plasma viscosity and haematocrit on glucose transfer rate. Lower membrane permeability resulted in less dependence on haematocrit and smaller differences between results in aqueous solution, plasma and whole blood.
Glucose Sensor Cellulose acetate Membrane Viscosity Dialysis

"Flow Injection System With Photodiode Array Detection And Multivariate Data Evaluation For Quantifying Teniposide In Blood Plasma"
Anal. Chim. Acta 1990 Volume 241, Issue 1 Pages 23-30
M. J. P. Gerritsen and G. Kateman, M. A. J. van Opstal and W. P. van Bennekom, B. G. M. Vandeginste

Abstract: The micelle-mediated method described by van Opstal (J. Chromatogr., 1989, 495, 139) was applied. For the calibration a partial least squares model was used. Although prediction is possible within 10% of the LC value, the authors admit that the present application requires further optimization with respect to calibration.
Teniposide Spectrophotometry Multivariate calibration Micelle Optimization Partial least squares

"Flow Injection Analysis With Tetrameric Calixarene-based Potentiometric Detection"
Anal. Chim. Acta 1991 Volume 251, Issue 1-2 Pages 149-155
Martin Telting-Diaz, Dermot Diamond* and Malcolm R. Smyth

Abstract: A thin-film membrane was prepared (described) containing methyl p-t-butylcalix[4]aryl acetate or p-t-butylcalix[4]arene methyl tetraketone, K tetrakis-p-chlorophenylborate, 2-nitrophenyl octyl ether and PVC. The membrane was inserted into a flow-through potentiometric detector for flow injection analysis. For determination of Na, plasma was diluted 10-fold with Tris buffer and a 100 µL portion was injected into a carrier stream containing physiological concentration. of plasma electrolytes. Results compared well with those obtained with a Technicon analyzer..
Sodium Electrode Potentiometry Buffer

"Determination Of Mitozantrone Using Phase-selective A.c. Adsorptive Stripping Voltammetry In A Flow System With Selectivity Enhancement"
Anal. Chim. Acta 1992 Volume 256, Issue 2 Pages 231-236
Juan Carlos Cortina Villar, Agustin Costa García and Paulino Tuñon Blanco*

Abstract: Urine was diluted 1:20 to contain standard additions of 25 nM-, 50 nM- and 75 nM-mitozantrone (final concentration.), and 0.1 mL portions of these solution were injected into a carrier stream of 0.1 M HClO4 (pH 1.12; 1.5 mL min-1). The mitozantrone became adsorbed on a carbon-paste electrode (prep. described), from which it was stripped by a.c. voltammetry. For 1 µM-mitozantrone in urine, the value obtained was 0.985 µM with a coefficient of variation (n = 5) of 3.7%. A flow cell incorporating a stationary carbon paste electrode coupled with a.c. voltammetry was used. Preconcentration. and determination of the cancer chemotherapy drug mitoxanthrone (I) were achieved via a flow injection approach utilizing adsorption of the drug on the electrode, followed by medium exchange and a.c. voltammetry on the adsorbed surface. A linear response was obtained in the concentration. range 5 x 10^-9-1.5 x 10^-7 M with aqueous samples. The method was used for the determination of I in urine without any sample pretreatment; the relative standard deviation obtained was 1.1% (n = 5) with a concentration. of 1 x 10^-6M. The proposed method is more selective than other methods suggested for the determination of I in urine and plasma samples, which usually require some kind of sample pretreatment.
Mitozantrone Voltammetry Selectivity Method comparison Standard additions calibration

"Robust Estimation Of Selectivity Coefficients Using Multivariate Calibration Of Ion-selective Electrode Arrays"
Anal. Chim. Acta 1993 Volume 276, Issue 1 Pages 75-86
Dermot Diamond* and Robert J. Forster

Abstract: Selectivity coefficients for Na, Ca, and K ISE estimated by traditional methods, e.g., separate and mixed solution methods, are compared with values obtained by an approach using multivariate calibration using simplex optimization and non-linear modeling of the array characteristics. The method was used to predict the concentration. of Sa, Ca and K in blood plasma and mineral water samples using dip and FIA measurements. For the array FIA data, the possibility of using an enhanced kinetic-based selectivity for analytical measurements is raised. Further improvement in the three-electrode array performance by using a fourth multiple ionophore electrode is discussed. Results obtained are compared with single electrode dip and FIA measurements of Na in plasma samples.
Sodium Calcium Potassium Electrode Selectivity coefficient Multivariate calibration Simplex Optimization Method comparison

"Catalytic Determination Of Copper In Blood Plasma Using Flow Injection Biamperometry"
Anal. Chim. Acta 1993 Volume 281, Issue 2 Pages 299-304
Jacek Michaowski and Marek Trojanowicz*

Abstract: Blood plasma was pre-treated according to the procedure of Gubler et al., followed by centrifugation at 2300 g for 5 min. The supernatant solution was injected into a carrier solution (2.5 ml/min) of water and merged with streams of 0.02 M iron(III) nitrate solution (2.5 ml/min) and 0.28 M sodium thiosulfate solution (2.1 ml/min), into a flow cell (100 cm long) maintained at 32°C to the amperometric detector (comprising of two polarized Pt wire electrodes). At a polarizing voltage of 80 mV a linear dependence of current intensity on Cu(II) concentration. (0.2-0.8 µg/l) was observed. Tolerance levels are presented for various inorganic species. For all the species tested, the tolerated concentration. were less than or equal to the upper levels normally present in blood serum. The results obtained compared well with those obtained by AAS.
Copper Biamperometry Catalysis

"Needle-type Enzyme-based Lactate Sensor For In Vivo Monitoring"
Anal. Chim. Acta 1993 Volume 281, Issue 3 Pages 503-511
Yibai Hu, Yanan Zhang and George S. Wilson

Abstract: The cited implantable probe (diagram given) was constructed from a PTFE-coated Pt-Ir wire (0.25 mm o.d.) which was stripped to form a 1.5 mm long cavity, 3 mm from the tip. Ag wire (0.05 mm diameter) was wrapped round the sensor and anodized in Cl--containing buffer to form a Ag/AgCl reference electrode. The cavity was dip-coated in 6% cellulose acetate in acetone/ethanol (1:1) to form a membrane to which 0.6 µL of a solution of 1.5% lactate oxidase/2% BSA/0.3% glutaraldehyde was applied. The sensor was soaked in water, dried, coated with 5% polyurethan solution, and stabilized in 0.1 M phosphate buffer of pH 7.4 for 10^-20 days. Measurements were made at +600 V vs. Ag/AgCl in the same buffer. The sensor was applied in a FIA system (described) to determine plasma and blood lactate. The calibration graph was linear from 0.05 mM (detection limit) to 12 mM lactate, the average recovery was 95.8% and the response time was 30 s; no RSD are given. The sensor was applied in vivo in rats with satisfactory results. An error of ~3.1% was caused by endogenous substances. The sensors were O2-independent down to a pO2 of ~10 Torr.
Lactate Electrode Electrode Sensor Interferences In vivo monitoring

"Continuous-flow Assay Of Ammonia In Plasma Using Immobilized Enzymes"
Anal. Chim. Acta 1994 Volume 294, Issue 1 Pages 43-47
R. Quilesa, J. M. Fernández-Romerob, E. Fernándeza and M. D. Luque de Castrob,*

Abstract: Heparinized plasma containing NH3 were diluted sixfold with carrier-buffer solution before injection of a 0.3 mL portion for FIA into the 0.15 M triethanolamine carrier, adjusted to pH 8 with 5 M HCl, (0.8 ml/min) containing 25 mM 2-oxoglutarate, 1 mM ADP and 0.2 mM NADH. The carrier was fed through a reaction tube (5 cm x 1 mm i.d.) of glutamate dehydrogenase [NAD(P)+] immobilized on controlled-pore glass (120-200 mesh) at 37°C to the flow-cell detector of a spectrofluorimeter for measurement of the decrease in fluorescence due to NADH consumption. The calibration graph was linear for 5 µM to 0.5 mM NH3 with RSD 2% and sampling frequency of up to 40/h. Results correlated well with those obtained using a Kodak Ektachem analyzer. and bromophenol blue.
Ammonia Fluorescence Controlled pore glass Immobilized enzyme

"Highly Sensitive Flow Injection Determination Of Glucose In Plasma Using An Immobilized Pyranose Oxidase And A Chemiluminometric Peroxidase Sensor"
Anal. Chim. Acta 1997 Volume 354, Issue 1-3 Pages 205-210
Nobutoshi Kiba*, Akiko Itagaki, Satoru Fukumura, Kazuya Saegusa and Motohisa Furusawa

Abstract: A flow-injection system is proposed for the determination of glucose in plasma. Chemiluminometric measurement of glucose was based on the luminol reaction with hydrogen peroxide produced by immobilized pyranose oxidase within a flow-through cell containing immobilized peroxidase. Pyranose oxidase was immobilized on toresylate-poly(vinyl alcohol) beads and packed into a stainless steel column (20 mm x 4 mm i.d.). Peroxidase is immobilized on toresylate-hydrophilic vinyl polymer beads and packed into transparent PTFE tube (20 cm x 1.0 mm i.d.). Plasma (1 µl) was diluted with water to 25 ml. Sample solution (10 µl) was injected into the carrier stream. The maximum sample throughput was 180 h-1. The calibration graph was linear from 10 nM to 5 µM glucose; the detection limit (signal to noise ratio=3) was 3 nM (5 pg in 10 µl injection). The chemiluminometric peroxidase sensor was stable for 30 days.
Glucose Chemiluminescence Sensor Immobilized enzyme

"Simultaneous Multiple Injection In Monosegmented Flow Analysis"
Anal. Chim. Acta 1998 Volume 371, Issue 2-3 Pages 317-324
Vanessa O. Brito and Ivo M. Raimundo Jr.*

Abstract: A flow approach for simultaneous multiple injection (SMI) in monosegmented flow anal. (MSFA) is described. In this approach, the sample and the reagent (or other solution, such as a diluting fluid) are simultaneously injected into the reaction coil of a monosegmented flow analyzer. The monosegment is homogenized while it is carried towards the detector. The sample dilution procedure is not based on a gradient concentration. pattern and dilution factors up to 150 were obtained by using hydrodynamic sampling. The system was applied to perform sample dilution in the determination of glucose in blood plasma. The results show good correlation with those obtained by the Clinical Hospital of UNICAMP. The SMI approach was also applied to add reagent to the sample in nitrite determination in natural water, providing a methodology that has a sampling frequency of 72/h, a relative standard deviation of <2%, at 60 µg/L N-NO2-, a linear response range up to 260 µg/L and a 2s limit of detection of 5 µg/L N-NO2-. Thus, the sensitivity is close to that of the manual reference method. Recovery tests carried out with sea water samples also showed that MSFA overcomes the Schlieren effect, without needing any special procedure.
Glucose Nitrite Injection technique Segmented flow Dilution

"Chemiluminometric Branched Chain Amino Acids Determination With Immobilized Enzymes By Flow Injection Analysis"
Anal. Chim. Acta 1998 Volume 375, Issue 1-2 Pages 65-70
Nobutoshi Kiba*, Masaki Tachibana, Kazue Tani and Takao Miwa

Abstract: A tri-enzyme sensor was developed for the flow injection determination of branched chain amino acids (L-valine, L-leucine and L-isoleucine). Leucine dehydrogenase, NADH oxidase and peroxidase were coimmobilized covalently on tresylate-hydrophilic vinyl polymer beads and packed into transparent PTFE tube (20 cm x 1.0 internal diameter), which was used as flow cell. The calibration graph was linear for 30 nM-5 µM; the detection limit (signal-to-noise = 3) was 10 nM. The sampling rate was 25 h-1 without carryover. The sensor was stable for two weeks. The sensor system was applied to the determination of branched chain amino acid in plasma.
Amino Acids l-Valine l-Leucine l-Isoleucine Chemiluminescence Immobilized enzyme Vinylpolymer beads

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

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

"Validation Of The Determination Of Copper And Zinc In Blood Plasma And Urine By ICP-MS With Cross-flow And Direct Injection Nebulization"
Talanta 1997 Volume 44, Issue 8 Pages 1389-1396
J. Szpunar*, J. Bettmer, M. Robert, H. Chassaigne, K. Cammann, R. Lobinski and O. F. X. Donard

Abstract: Use of a Perkin-Elmer cross-flow nebulizer and a direct-injection nebulizer (Microneb 200, CETAC, Omaha, Ne, USA) were compared for the ICP-MS determination of Cu and Zn using an Elan 600 instrument (Perkin Elmer SCIEX). Plasma and urine were diluted 20- and 10-fold, respectively, in 0.05% HNO3 and 10 µg/l 89Y was added as internal standard. Isobaric interferences were eliminated for 65Cu and most of the Zn nuclides. Flow injection direct-injection nebulization was optimized for the analysis of 1-2 µL samples (parameters tabulated). Standard additions was used to eliminate matrix effects. Results using the flow injection method on reference materials agreed with the certified values.
Copper Zinc Mass spectrometry Nebulizer Reference material Interferences Standard additions calibration

"Controlled-dispersion Flow Analysis In Clinical Chemistry. Determination Of Albumin, Triglycerides And Theophylline"
Analyst 1984 Volume 109, Issue 7 Pages 847-850
Bernard F. Rocks, Roy A. Sherwood and Clifford Riley

Abstract: The flexibility of the cited technique (cf. Riley et al., Anal. Abstr., 1983, 45, 2J11) is illustrated by its application to the cited assays. In the determination of albumin by the bromocresol green method, only 240 nl of sample was required and reagent consumption was 360 µL per cycle (cycle time 30 s). Calibration graphs were rectilinear for up to 70 g l-1, and at the 42 g L-1 level the coefficient of variation was 1.2%. Triglycerides and theophylline were determined by enzymatic - spectrophotometric methods by application of the merging-zones (cf. Bergamin F. et al., Ibid., 1979, 36, 6J8) and stopped-flow techniques. The triglyceride assay was performed with use of a single-phial reagent, and involved a combination of linked enzyme systems resulting in measurement of the reduced form of 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride at 503 nm. At the 1.83 mM level, the coefficient of variation was 5.2% (n = 20). The determination of theophylline was based on use of the EMIT kit (Syva) and measurement of the change in absorbance at 340 nm due to conversion of NADH into NAD+. Calibration graphs were rectilinear in the range 2.5 to 4 mg L-1 and results obtained on a control serum (15 mg l-1) agreed well with expected values.
Albumin Triglycerides Theophylline Clinical analysis Spectrophotometry Dispersion Merging zones Small sample Stopped-flow Reagent consumption

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

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

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

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

"Characteristics Of Flow Injection Inductively Coupled Plasma Mass Spectrometry For Trace Metal Determination"
J. Anal. At. Spectrom. 1988 Volume 3, Issue 2 Pages 349-354
John R. Dean, Les Ebdon, Helen M. Crews and Robert C. Massey

Abstract: Flow injection sample introduction in ICP-MS alleviates problems arising from high salt content, viscosity and high acid concentration. A commercial ICP-MS instrument (Plasmaquad) was used, sample aerosol generation was achieved with either a fixed cross-flow nebulizer or a high-solids nebulizer, and a manually operated flow injection valve was used. All operating conditions are tabulated. Biological reference materials were analyzed, following digestion by various methods, by ICP-MS with external calibration or isotope dilution. Results agreed well with certified values for both methods. The technique was applied to determine Pb in powdered mixed diet samples and Zn isotope ratios in blood plasma. The main advantage of flow injection over continuous nebulization was the small amount of sample required.
Lead Metals, trace Zinc Mass spectrometry Mass spectrometry Sample preparation Interferences Nebulizer Reference material Viscosity

"Determination Of Creatinine By An Ammonia-sensitive Semiconductor Structure And Immobilized Enzymes"
Anal. Chem. 1986 Volume 58, Issue 1 Pages 145-148
Fredrik Winquist, Ingemar Lundstroem, and Bengt Danielsson

Abstract: The determination is described of creatinine in biological samples, by using an enzymatic flow injection system (diagram presented). A 25-fold dilution was used for whole blood and plasma and a 1000-fold dilution for urine. An NH3-sensitive Ir - metal oxide semiconductor sensor was developed to detect the NH3 produced by immobilized creatinine deiminase. Endogenous NH3 was removed by the action of immobilized glutamate dehydrogenase. The sensitivity for 85 µL aliquots was 0.2 µM-creatinine and the calibration graph was rectilinear for up to 30 µM. The results correlated well with those obtained by conventional spectrophotometry. The recoveries ranged from 93 to 105% and the only significant interferences were from low-mol.-wt. amines.
Creatinine Electrode Interferences Immobilized enzyme Method comparison

"Tubular Microporous Membrane Entrapped Enzyme Reactors For Flow Injection Analysis"
Anal. Chem. 1987 Volume 59, Issue 9 Pages 1356-1360
Hoon Hwang and Purnendu K. Dasgupta

Abstract: The enzyme reactor consisted of a bundle of 15 to 18 microporous hollow fibers (Celgard X-20, 400 µm i.d., 25 µm wall, 0.02 µm pore size), folded in half to form a U with sharp bends, and with the exterior surface of the bundle coated with cured epoxy adhesive. Connections to both ends of the bundle were made with tightly nested PTFE tubes of increasing diameter, connected to PTFE inlet and outlet tubing (0.8 mm i.d.). Schematic diagrams are given. To determine H2O2, flow streams of 4-hydroxyphenylacetic acid solution (derivatization reagent) and sample, injected into water carrier, were mixed and passed through the reactor, which contained entrapped peroxidase. The fluorescent dimer formed by enzyme-catalyzed oxidation with H2O2 was detected fluorimetrically at 370 nm (excitation at 326 nm). The response was rectilinear from the detection limit (600 ng l-1) to >1 mg l-1. The system was applied similarly to determine plasma glucose; a glucose oxidase reactor preceded the peroxidase reactor. The response was rectilinear from the detection limit (13 µg l-1) to 2.5 g l-1.
Hydrogen peroxide Glucose Fluorescence Celgard Enzyme Hollow fiber membrane

"Selective Determination Of Histamine By Flow Injection Analysis"
Anal. Chem. 1990 Volume 62, Issue 18 Pages 1971-1976
James M. Hungerford, Kevin D. Walker, Marleen M. Wekell, Jack E. LaRose, and Harold R. Throm

Abstract: A flow injection analysis (FIA) method for the determination of histamine is described. Control of reaction timing allows exploitation of a transient, chemical-kinetic increase in selectivity that occurs when o-phthalaldehyde reacts with histamine. The molar fluorescence ratio (selectivity) of histamine/histidine reaches a maximum value of 800 in 32 s, precluding the need for separation of histamine from histidine, spermidine, and other potential interferences in biological samples. Online dilution prevents matrix effects and affords a linear response up to approximately 4.45 mM histamine, or 500 mg of histamine free base/100 g. Under these conditions the detection limit (3 times peak-to-peak baseline noise) is 5.5 pg (corresponding to 0.60 mg of histamine free base/100 g of sample) and throughput is 60 injections per hour. The high sensitivity and high selectivity of the method allow the rapid determination of histamine in fish with minimal sample conditioning and will find application in the determination of endogenous histamine as well, such as in blood plasma and brain tissue.
Histamine Fluorescence Kinetic Selectivity Interferences Dilution Detection limit

"Noncompetitive Flow Injection Immunoassay For A Hapten, α-(difluoromethyl)ornithine"
Anal. Chem. 1993 Volume 65, Issue 9 Pages 1152-1157
P. Chandrani Gunaratna and George S. Wilson

Abstract: Plasma containing α-(difluoromethyl)ornithine (eflornithine hydrochloride; I) was diluted 200-fold with phosphate buffer, pH 7.4, and incubated with an affinity-purified antibody, conjugated to horse-radish peroxidase, for 15 min at room temperature Portions were injected into a carrier stream (0.3 ml/min) of the same buffer and the excess antibody was separated on a column (6 cm x 3.8 mm) of Reacti-Gel HW-65F beads to which a conjugate of the I with BSA was covalently attached. The outflow from this column, containing the bound I, was mixed with 10 mM 3-(p-hydroxyphenyl)propionic acid and 5 mM H2O2 in phosphate buffer, pH 8.5 (0.3 ml/min) and the fluorescence was measured at 405 nm (excitation at 320 nm). Calibration graphs were rectilinear for 0.05-2.5 nM-I with a detection limit of 20 pM and within-run RSD (n = 6) of 2.6, 0.5, and 2.4% and between-run (n = 5) of 6.3, 2.2 and 4.3% for 0.21, 0.84 and 1.7 nM-I, respectively. Cross-reaction with ornithine and putrescine was eliminated by the purification of the antibody. The Reacti-Gel column was used for 50 samples before regeneration. The method gave good correlation with the results from an amino-acid analyzer., with post-column detection with o-phthalaldehyde (r = 0.94, n = 76). A noncompetitive flow injection immunoassay method has been developed to assay small haptens. In this assay the sample containing the hapten is incubated with excess enzyme-labeled monovalent antibody for a brief period. The excess antibody is then separated from the bound antibody by eluting through an antigen-immobilized immunoaffinity column. The enzyme label of the eluting antibody-hapten complex is fluorometrically detected. The applicability of the method is demonstrated by assaying α-(difluoromethyl)ornithine (DFMO), an anticancer drug in human plasma samples. The assay is sensitive enough to detect 200 amol of DFMO. Interferences from other similar endogenous amines have been eliminated by selective immunoaffinity purification of the antibodies.
α-Difluoromethylornithine Immunoassay Fluorescence Immobilized antigen Interferences

"Enzyme Electrodes For Lactate, Pyruvate And Lactate Dehydrogenase Activity Determination"
Fresenius J. Anal. Chem. 1987 Volume 328, Issue 3 Pages 259-262
D. Weigelt, F. Schubert und F. Scheller

Abstract: The enzyme was applied in 5% gelatin solution to the external membrane of an O electrode and covered with another membrane. The electrode was then connected to a commercial O meter, e.g., the Glukometer GKM 01. With use of lactate 2-monooxygenase in the electrode, methods are described for the determination of lactate (1 to 40 mM) in plasma (both manual and flow injection) and of L-lactate dehydrogenase. Pyruvate (0.1 to 2 mM) was determined with use of a mixture of lactate monooxygenase and L-lactate dehydrogenase in the electrode. Lactate was determined similarly; after the addition of NADH, pyruvate was reduced to lactate, and, thus, lactate and pyruvate could be determined in the same sample. Results for the determination of lactate and pyruvate in biological fluids agreed well with those obtained spectrophotometrically.
Lactate Enzyme, lactate dehydrogenase Pyruvate Electrode Electrode Method comparison

"Fast Lipoprotein Chromatography (FLPC): Novel Approach To The Analysis Of Plasma Lipoprotein Fractions"
Fresenius J. Anal. Chem. 1990 Volume 337, Issue 1 Pages 126-127
Hans-Wolfgang Schultis, Hans v. Baeyer, Heidemarie Neitzel, Eberhard Riedel, R&uuml;diger Siekmeier, Winfried M&auml;rz, Hubert Scharnagl and Werner Gro&szlig;

Abstract: Plasma (20 µL) was analyzed by gel-permeation chromatography on a 30-cm Superose 6 column with 0.1 M Na2HPO4 - 0.2 M NaCl (pH 7.4) as mobile phase (0.3 mL min-1), post-column derivatization with CHOD-PAP cholesterol reagent (Boehringer Mannheim) and detection at 500 nm. The response was rectilinear for 80 µL of hypercholesterolaemic plasma, and the coefficient of variation (n = 13) for very-low-density-, low-density- and high-density-lipoprotein cholesterol were 5.8, 2.0 and 1.9%, respectively. Results were more reproducible than those by conventional methods, and only a small amount of sample, with no pre-treatment, was required.
Cholesterol GPC Spectrophotometry Column Post-column derivatization Small sample

"Simultaneous Determination Of Ascorbic Acid And Dehydroascorbic Acid By HPLC With Post-column Derivatization And Fluorimetric Detection"
Fresenius J. Anal. Chem. 1992 Volume 342, Issue 4-5 Pages 462-466
Michaela Capellmann Contact Information and Hermann M. Bolt

Abstract: Plasma was solid-phase extracted on anion-exchange columns and ascorbic acid (I) and dehydroascorbic acid (II) were eluted with 0.4 M Na citrate solution A portion of the eluate was analyzed by HPLC on a column (25 cm x 4 mm) of Spherisorb C18-2 equipped with a guard column of similar material and a mobile phase of 5 mM Na acetate - 2.3 mM tetrabutylammonium hydroxide adjusted to pH 3.5 with acetic acid. I and II were detected fluorimetrically at 420 nm (excitation at 340 nm) after post-column derivatization with 1,2-diaminobenzene dihydrochloride containing Cu acetate as oxidizing agent. Detection limits were 16 and 3 ng for I and II, respectively. Results with solid-phase extraction were superior to those obtained with conventional ppt. of plasma proteins by m-phosphoric acid.
Ascorbic acid dehydroascorbic acid HPLC Fluorescence Sample preparation Post-column derivatization

"Flow Injection Analysis With Fourier Transform Infrared Detection For Clinical And Process Analysis"
Fresenius J. Anal. Chem. 1994 Volume 348, Issue 8-9 Pages 530-532
E. Rosenberg and R. Kellner

Abstract: Using glucose and urea as model compounds, 500 µL aqueous solution were pumped (1.1 mL/min) to an FTIR spectrometer fitted with 25 µm pathlength CaF2 windows and background spectra were obtained. A second injection was pumped to an enzyme reactor containing either native or glass-bead immobilized (cf. 'Methods in Enzymology', Vol. 44, Academic Press, NY), glucose oxidase from Aspergillus niger and urease from Canavalia ensiformis. After 10 min, the reaction mixture was pumped to the spectrometer and a second spectrum obtained. The difference in spectra at 1085 and 1125 cm-1 for glucose and 1366 or 1438 cm-1 for urea were correlated to concentrations. Up to 50 mM glucose was quantitatively converted. Applications of the method to fruit juices and simulated plasma containing 40 g/l of BSA are discussed and correlated with reference methods.
Glucose Urea Spectrophotometry Clinical analysis

"Liposome-based Flow Injection Enzyme-immunoassay For Theophylline"
Microchim. Acta 1990 Volume 100, Issue 3-4 Pages 187-195
Tai -Guang Wu and Richard A. Durst

Abstract: A peristaltic pump was used to supply, in 0.1 M Tris buffer (pH 7.2) as carrier, a standard solution of theophylline (I) or plasma sample, in the same buffer, to a column (17.8 cm x 2.5 mm) of glass beads coupled to monoclonal anti-theophylline antibodies. The injector (Rheodyne type 7010) then delivered a solution of liposomes that encapsulated horse-radish peroxidase and had been sensitized with 4-(1,3-dimethylxanthin-8-yl)butyric acid (II). Competition between I and II for the antibodies occured, and unbound liposomes were eluted for post-column reaction with H2O2 and 4-fluoriphenol. This reaction caused release of F-, which was determined with an Orion model 69-09 ion-selective electrode. The column was then washed with glycine - HCl solution to dissociate the antigen - antibody complex and reactivate the column. Calibration graphs are presented for two liposome compositions (10 and 20 miu mL-1 of enzyme activity). I can be detected over the concentration. range 0.2 to 4000 ng mL-1, i.e., a detection limit of 100 fmol in a 0.1 mL sample. For an activity of 10 miu mL-1, the coefficient of variation (n = 6) was 4.6% at the level of 4.3 ng mL-1. The assay takes ~10 min.
Theophylline Immunoassay Electrode Buffer pH Column Enzyme Liposomes Calibration Glass beads Detection limit

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

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

"Determination Of Methylguanidine In Plasma And Urine By High Performance Liquid Chromatography With Fluorescence Detection Following Post-column Derivatization"
Anal. Biochem. 1990 Volume 184, Issue 2 Pages 213-218
Venkata K. Boppana*, Gerald R. Rhodes* and David P. Brooks

Abstract: Plasma (1 ml) was mixed with 0.2 µg of ethylguanidine as internal standard, adjusted to pH 11 with 26 µL of 1 M NaOH and applied to a 1 mL weak-cation-exchange extraction column, which was washed with water before elution with methanolic 1% trifluoroacetic acid. After evaporation under N, the residue was dissolved in 0.2 mL of the mobile phase [0.06 M chloroacetate buffer (pH 4.0) - methanol (17:3)], and a 0.1 mL portion was subjected to HPLC on an Ultrasil cation-exchange silica (10 µm) column (25 cm x 4.6 mm) at 60°C with a mobile phase flow rate of 0.75 mL min-1, post-column mixing at 70°C with NaOH and ninhydrin solution and fluorescence detection with a 470 nm cut-off filter (excitation at 390 nm). Urine was diluted tenfold with water before injection of a 20 µL portion. Calibration graphs were rectilinear for 1 to 1000 ng mL-1 of methylguanidine in plasma and 0.1 to 600 µg mL-1 in urine, and the detection limit was 100 pg. The intra- (n = 5) and inter-day (n = 15) coefficient of variation for 10, 100 and 1000 ng mL-1 of methylguanidine in plasma were 2.49, 1.30 and 1.38% and 7.51, 5.20 and 7.98%, respectively.
Methylguanidine HPLC Fluorescence Post-column derivatization Silica Detection limit

"Automated Spectrophotometric Flow Injection Assay Of Alkaline Phosphatase"
Anal. Lett. 1988 Volume 21, Issue 12 Pages 2381-2388
Masoom, M.

Abstract: Enzyme standard solution (0 to 250 iu l-1), sample (20 µL) and substrate solution (6 mM 4-nitrophenyl phosphate; 20 µL) were simultaneously injected into separate streams of 0.1 M diethanolamine buffer (pH 9.8), each pumped equally to give an overall flow rate of 2.5 mL min-1. Flow-line tube diameter was 0.5 mm and the distance from the confluence point to the detector was 50 cm, inclusive of a packed reactor (2.5 cm x 2.5 mm) containing glass beads (0.5 to 0.75 mm). Absorbance was monitored at 405 nm (based on the rate of formation of 4-nitrophenol from the substrate). Calibration graphs were rectilinear over the range studied. Within-batch precision was 1.8%; total analysis time was 85 s per sample.
Enzyme, alkaline phosphatase Spectrophotometry Computer Glass beads Indirect

"Determination Of N-acetylcysteine, Intact And Oxidized, In Plasma By Column Liquid Chromatography And Post-column Derivatization"
J. Chromatogr. A 1987 Volume 385, Issue 1 Pages 343-356
Margareta Johansson, Douglas Westerlund

Abstract: Plasma was deproteinized with 2.3 M HClO4 and a portion of the supernatant solution was injected on to a column (10 cm x 4.4 mm) packed with Microspher C18 (3 µm) with a Waters nitrile guard column. Oxidized forms of acetylcysteine(I) (coupled to small sulfides) in the supernatant solution were subjected to reductive cleavage of the disulfides with dithiothreitol before injection. The total concentration. of I (including oxidized forms and the fraction coupled to proteins) was determined by treatment of the plasma with dithiothreitol in alkaline solution before protein precipitation The mobile phase was phosphate buffer (pH 2) - acetonitrile (99:1) containing 0.1 mM EDTA with post-column derivatization by the addition of 0.05 M borate buffer (pH 11) and 50 µM-pyrenemaleimide in acetonitrile, followed by fluorimetric detection at 389 nm (excitation at 342 nm). The coefficient of variation for I and total I were 14% (within-run) and 6.8% (between-run), respectively. The determination limit was 240 nM-I.
N-acetylcysteine HPLC Fluorescence Post-column derivatization

"Automated Analysis Of Mitomycin C In Body Fluids By High Performance Liquid Chromatography With Online Sample Pretreatment"
J. Chromatogr. A 1987 Volume 420, Issue 1 Pages 53-62
U. R. Tjaden, E. A. De Bruijn, R. A. M. Van Der Hoeven, C. Jol, J. Van Der Greef and H. Lingeman

Abstract: Plasma, ascites fluid and urine were pre-treated by dialysis across a cellulose membrane, followed by concentration. of the mitomycin(I)-containing acceptor stream on pre-columns (3 cm x 2 mm) of Polygosil C8 (40 to 63 µm). Pretreatment was fully automated by using continuous-flow and column-switching techniques. HPLC was carried out on a column (10 cm x 3 mm) of Nucleosil C8 (5 µm) with, as mobile phase, aqueous 15% acetonitrile, and detection at 360 nm. Up to 100 samples a day could be analyzed if two alternating pre-columns were used. The calibration graph was rectilinear from 5 to 200 ng mL-1 and was independent of the biological matrix. Within- and between-day coefficient of variation ranged from 4.4 to 2.9 and 5 to 11.3%, respectively, for plasma. Recoveries were 25% and the limit of determination was 0.5 to 1 ng mL-1. Floxuridine, fluorouracil, doxorubicin, vincristine and bleomycin did not interfere.
Mitomycin C HPLC Sample preparation Spectrophotometry Interferences Membrane Preconcentration Dialysis

"Comparison Of Flow Injection Analysis With High Performance Liquid Chromatography For The Determination Of Etoposide In Plasma"
J. Chromatogr. B 1988 Volume 432, Issue 1 Pages 395-400
M. A. J. Van Opstal and P. Krabbenborg, J. J. M. Holthuis, W. P. Van Bennekom and A. Bult

Abstract: HPLC was performed with use of a guard column (2 cm x 3.9 mm) of LiChrosorb RP-18 (5 to 10 µm), an analytical column (7.5 cm x 3.9 mm) of Novapak phenyl and an electrochemical detector; the mobile phase (1 mL min-1) was 10 mM phosphate buffer (pH 7) - methanol (9:11). The apparatus and conditions for flow injection analysis were as described previously (Anal. Abstr., 1988, 50, 8D97). Both methods produced rectilinear calibration graphs but flow injection analysis had high blank responses due to interfering plasma components. Detection limits were 1.5 and 0.15 µg mL-1 by flow injection analysis and HPLC, respectively. Flow injection analysis is a good alternative to HPLC for determination of I in plasma for >1.5 µg mL-1.
Etoposide HPLC Interferences Method comparison

"Chromatography, Flow Injection Analysis And Electrophoresis In Computer Assisted Comparative Biochemistry: Its Application And Possibilities In Clinical Research. Preliminary Studies On Crohns Disease"
J. Chromatogr. A 1988 Volume 440, Issue 1 Pages 261-273
V. R. Villanueva and M. Mardon

Abstract: A computer-assisted multicomponent analytical system, developed for comparative biochemical studies, was used for a clinical study of Crohn's disease in which 73 subjects, of comparable age and sex distribution, were considered: 40 with Crohn's disease, 16 with ulcerative colitis and 17 healthy volunteers as controls. Blood samples (5 ml) were taken to recover plasma and red cells. After extraction and fractionation of low- and high-molecular weight substances, the samples were analyzed by ion-exchange chromatography and electrophoresis. The contents of amino acids, sugars, polyamines and proteins in the plasma and the red cells from the three groups of individuals were compared using statistical (means, variance, principal components analysis) and graphical profile methods. The first results indicate that the content of red cells, in comparison with plasma, allows the best differentiation of the three groups of subjects considered. In particular, the amino acids (Asp, Thr, Ser, Glu, Gly, Ala and Leu), the polyamines (spermidine and spermine) and glucose, show the most significant differences. The methodology followed and the results obtained, together with possible uses of this computer-assisted multicomponent analytical system in problems concerning clinical research, are discussed.
Amino Acids Glucose Amines, poly Clinical analysis HPIC Electrophoresis Computer Chemometrics Multicomponent

"High Performance Liquid Chromatographic Analysis Of Arginine-containing Peptides In Biological Fluids By Means Of A Selective Post-column Reaction With Fluorescence Detection"
J. Chromatogr. A 1988 Volume 444, Issue 1 Pages 123-131
Gerald R. Rhodes and Venkata K. Boppaa

Abstract: Plasma (1 ml) containing SKF 104146 (1 µg mL-1) as internal standard was cleaned up on a 1 mL cation-exchange solid-phase extraction cartridge. The cartridge was washed with water (1 ml), and the washings were applied to the cartridge which was then washed with aqueous 1% trifluoroacetic acid (I; 1 ml), water (2 ml) and methanol (2 ml). The peptide was eluted with methanolic 2% I (2 ml). Following evaporation of the solvent, the residue was dissolved in chloroacetate buffer of pH 3.2 (prep. described) - methanol (1:1; 0.1 ml), and the solution was analyzed by HPLC on a column (25 cm x 2 mm) of Ultrasphere C8 (5 µm), at 60°C, with a mobile phase (0.3 mL min-1) of chloroacetate buffer - methanol. For some samples, Na octanesulfonate was used as ion-pair reagent. The eluate was mixed with ninhydrin and NaOH, and the product was determined fluorimetrically at 470 nm (excitation at 390 nm). The range of application of the method was 0.5 to 100 nM; the detection limit was 50 fmol.
Peptides, arginine containing HPLC Fluorescence Heated reaction Post-column derivatization

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

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

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

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

"High Performance Liquid Chromatographic Determination Of Monohydroxy-compounds By A Combination Of Pre-column Derivatization And Post-column Reaction Detection"
J. Chromatogr. A 1992 Volume 593, Issue 1-2 Pages 29-36
Venkata K. Boppana*, Richard C. Simpson, Kathleen Anderson, Cynthia Miller-Stein, Timothy J. A. Blake, Bruce Y. H. Hwang and Gerald R. Rhodes

Abstract: Oxiracetam (I) was determined in plasma after derivatization with propyl isocyanate in pyridine at 50°C for 1 h to form the corresponding propyl carbamate ester. The solvent was evaporated to dryness, the residue was dissolved in solvent A [0.05 M acetate buffer (pH 6) - methanol (9:1)] and the solution was analyzed by HPLC. A column (25 cm x 2 mm) of Ultrasphere ODS (5 µm) was used with gradient elution with solvent A (9:1 for 5 min, to 4:1 over 4 min, held for 1 min, then to 1:1 in 1 min, held for 5 min). Post-column derivatization of the ester was carried out at 90°C with methanolic phthalaldehyde solution (2 mg mL-1) and 3-mercaptopropionic acid. The resulting fluorescence was detected at 455 nm (excitation at 340 nm). The calibration graph was rectilinear for 2 to 2000 ng of I with coefficient of variation of 3% (n = 6). The limit of detection was 0.5 ng of I.
Oxiracetam HPLC Fluorescence Post-column derivatization Heated reaction Pre-column derivatization

"Online Continuous-flow Dialysis Thermospray Tandem Mass Spectrometry For Quantitative Screening Of Drugs In Plasma: Rogletimide"
J. Chromatogr. A 1992 Volume 598, Issue 2 Pages 189-194
E. van Bakergem, R. A. M. van der Hoeven, W. M. A. Niessen*, U. R. Tjaden and J. van der Greef, G. K. Poon and R. McCague

Abstract: Plasma (1.26 ml) was introduced into a water air-segmented donor stream (0.42 mL min-1) and passed through a dialysis membrane into a non-segmented water stream (0.6 mL min-1). The acceptor stream was concentrated on a column (12 x 2 mm) of Polysil C8 (63 µm). The trapping column was subsequently desorbed for online separation of rogletimide (I) by HPLC on a column (10 cm x 4.6 mm) of Nucleosil C8 (5 µm) with a mobile phase of 30% methanol in 100 mM ammonium acetate and MS or MS - MS detection. Gluthetimide was used as an internal standard. The calibration graph was rectilinear from 5 (determination limit) to 360 ng mL-1 of I and the coefficient of variation was 5.2% (n = 5) at the 100 ng mg-1 level. The application of a continuous-flow dialysis system, consisting of a membrane dialyzer and a trace enrichment column, in online combination with tandem mass spectrometry via a thermospray interface is described. The method is applied to the quantitation of drugs in complex biological matrixes containing macromol. interferences. The potential of the method is demonstrated by the quant. anal. of the anti-cancer drug rogletimide in the plasma of patients after treatment.
Rogletimide Mass spectrometry Dialysis Interface Interferences Internal standard Preconcentration

"Determination Of BAY Y 3118, A Novel 4-quinolone, In Biological Fluids Using High Performance Liquid Chromatography And Photothermal Post-column Derivatization"
J. Chromatogr. B 1993 Volume 616, Issue 1 Pages 87-93
Hans Guenter Schaefer

Abstract: Plasma was diluted with 0.05 M H3PO4 and centrifuged; saliva and urine were diluted with 0.1 M phosphate buffer of pH 7.5. Portions of the resulting solution were injected on to a column (25 cm x 4.6 mm) of Nucleosil 100 C18 (5 µm) operated at 50°C and protected by a similarly packed guard column with acetonitrile/[0.1 M tetrabutylammonium bromide/0.05 M H3PO4 (1:1)] (19:81; pH 2) as mobile phase (1 ml/min). Post-column derivatization of the eluate was performed, utilizing either photolysis at 254 nm in a Beam Boost reaction unit or combined thermolysis and photolysis in a laboratory-made reactor (details given). Fluorimetric detection was performed, employing excitation at 277 nm and an emission cut-off filter (418 nm). Calibration graphs were rectilinear from 0.01-2.0 mg/l of BAY y 3118 (I) in plasma and 1-500 mg/l of I in urine. Intra- and inter-day RSD were from 3.3-5.1% and 2.6-9.8%, respectively, for plasma and from 0.5-3.8% and 2.5-6.9%, respectively, for urine. The method was used to determine the pharmacokinetic behavior of I in a healthy human volunteer. A reversed-phase high performance liquid chromatographic (HPLC) method that allows the sensitive and selective quantification of a novel 4-quinolone (BAY y 3118, I) in biological fluids is described. After sample dilution with 0.05 M phosphoric acid (plasma) or 0.1 M phosphate buffer pH 7.5 (urine), samples can be directly injected into the HPLC system. Prior to fluorescence detection, I is decomposed to fluorescence compound(s) by post-column derivatization utilizing either photolysis (Beam Boost reaction unit) or a combination of thermolysis and photolysis (laboratory-made post-column reactor). Compared with fluorescence detection alone, derivatization increases the signal intensity (about 80-fold) and the selectivity of the detection significantly. Concentrations down to 0.01 mg/l could be quantified in biological fluids. Only thermolysis was not able to decompose I to fluorescence products. Investigations on the stability of I in plasma and urine demonstrate good stability under the different conditions tested. The method was applied to human plasma and urine samples from a subject after a single oral dose of 100 mg of I.
4-Quinolone HPLC Fluorescence Post-column derivatization

"Determination Of Oxiracetam In Human Plasma By Reversed-phase High Performance Liquid Chromatography With Fluorimetric Detection"
J. Chromatogr. A 1993 Volume 631, Issue 1-2 Pages 227-232
Richard C. Simpson, Venkata K. Boppana, Bruce Y. -H. Hwang and Gerald R. Rhodes

Abstract: Reversed-phase HPLC methodology utilizing pre-column derivatization and post-column reaction fluorimetric detection has been developed and applied to the determination of oxiracetam in human plasma. The method involves preliminary isolation of oxiracetam and internal standard from plasma by solid-phase extraction prior to the formation of their n-propyl carbamate derivatives. The carbamate derivatives were subsequently isolated by solid-phase extraction and subjected to a gradient liquid chromatographic separation on an octadecylsilica column prior to online post-column alkaline hydrolysis to produce the corresponding primary amine, which was in turn derivatized with o-phthalaldehyde and 3-mercaptopropionic acid to yield a fluorescent isoindole. The isoindole was then quantified using a fluorescence detector. The method provided an on-column detection limit of 0.5 ng of oxiracetam and was sufficiently sensitive, accurate, and precise to support pre-clinical or clinical pharmacokinetic studies.
Oxiracetam HPLC Fluorescence Pre-column derivatization Post-column derivatization

"Determination Of Panomifene In Human Plasma By High Performance Liquid Chromatography"
J. Chromatogr. A 1994 Volume 668, Issue 2 Pages 419-425
V. Erd&eacute;lyi-T&oacute;tha,*, E. Papa, J. Kralov&aacute;nszkya, E. Bojtib and I. Klebovichb

Abstract: Plasma containing 2 µL/ml of tamoxifen citrate (TMX, internal standard), aqueous 50% methanol and acetonitrile were vortex mixed for 1 min and centrifuged at 5000 rpm for 1 h, the supernatant solution was diluted with 0.05 M phosphate buffer of pH 3 containing 2% heptanesulfonic acid. The sample was transferred to a phenyl liquid-solid extraction micro-column and PAN and TMX were eluted with 50 mM phosphate buffer of pH 3 containing 5 mM heptanesulfonic acid (eluent A)/acetonitrile (1:4). The eluate was evaporated to dryness under N2. The residue was dissolved in eluent A/acetonitrile (3:7) and analyzed by HPLC on a Si-100-S 10 Phenyl column (25 cm x 4.6 mm i.d.) with a Si-100-S Phenyl pre-column (2 cm x 4.6 mm i.d.), eluent A containing 0.3 ml/l of triethylamine at 80°C/acetonitrile at 60°C (1:3) as the mobile phase (1.2 ml/min), post-column photochemical derivatization by irradiation with a Hg lamp (254 nm) and fluorescence detection at 378 nm (excitation at 257 nm). The calibration graph was linear for 1-100 ng/ml of PAN with a detection limit of 20 pg/ml and within-day RSD (n = 6) of 2.89-8.9%. The recoveries of 5-100 ng/ml of PAN and 20 ng/ml of TMX were 67-74% and 76%, respectively.
Panomifene HPLC

"Determination Of Free Trimethyllysine In Plasma And Tissue Specimens By High Performance Liquid Chromatography"
J. Chromatogr. B 1984 Volume 306, Issue 1 Pages 79-87
Alan T. Davis, Stephen T. Ingalls and Charles L. Hoppel

Abstract: Trimethyl-lysine and triethyl-lysine (internal standard) are extracted from acid-deproteinized plasma or the acid-soluble fractions of tissue homogenates by sorption on a mixed-bed ion-exchange column [Dowex 1-X8 (OH-) and 50W-X8 (NH4+)] and elution with aqueous 1 M NH3 and water. The compounds are transferred into HCl for HPLC on a Radial-Pak C18 cartridge (10 cm x 5 mm) with a guard column of Co:Pell ODS and with aqueous 30% acetonitrile, containing 50 mM Na dodecyl sulfate and 50 mM NaH2PO4, as mobile phase; fluorimetric detection involves post-column derivatization with phthalaldehyde - 2-mercaptoethanol. The calibration graph is rectilinear from 0.5 to 4 µM; the detection limit is 25 pmol injected.
6-N,N,N-Trimethyllysine HPLC Ion exchange Fluorescence Post-column derivatization

"Determination Of The Antibiotic Fludalanine {3-fluoro-D-[2-2H]alanine} In Plasma And Urine By High Performance Liquid Chromatography Using A Packed-bed, Post-column Reactor With Phthalaldehyde And 2-mercaptoethanol"
J. Chromatogr. B 1985 Volume 338, Issue 1 Pages 357-367
D. G. Musson, S. M. Maglietto and W. F. Bayne

Abstract: Plasma was mixed with 3,3-difluoro-D-alanine(I) as internal standard, and transferred to an ultra-filter and centrifuged; the filtrate was then analyzed. Urine plus I was mixed with acetonitrile and applied to a Sep-Pak silica column and the fraction eluted with water was analyzed. Analysis was by HPLC on a Radial-Pak C18 cartridge (10 cm x 8 mm; 5 µm) with a Guard-Pak C18 cartridge and a mobile phase (2 mL min-1) of Na dodecyl sulfate (50 mg mL-1) in aqueous 10% methanol containing 2 mL L-1 of 85% H3PO4, at pH 2.5. Post-column derivatization was achieved with phthalaldehyde and 2-mercaptoethanol in borate buffer solution in a glass-bead-packed reactor at 40°C with fluorimetric detection at 455 nm (excitation at 340 nm). The calibration graphs were rectilinear for 0.25 to 20 µg mL-1 (plasma) and 0.5 to 200 µg mL-1 ( urine). The intra- and inter-assay coefficient of variation were 6.14 and 8.7%, and 5.18 and 8.3%, for plasma and urine, respectively; the respective recoveries were 48.9 and 66.5%.
Fludalanine HPLC Fluorescence Heated reaction Post-column derivatization Glass beads

"High Performance Liquid Chromatographic Determination Of Plasma And Brain Histamine Without Previous Purification Of Biological Samples: Cation-exchange Chromatography Coupled With Post-column Derivatization Fluorimetry"
J. Chromatogr. B 1985 Volume 344, Issue 1 Pages 115-123
Atsushi Yamatodani, Hiroshi Fukuda and Hiroshi Wada, Toshinao Iwaeda, Takehiko Watanabe

Abstract: Plasma and homogenized brain were extracted with HClO4 and the extracts were analyzed directly by HPLC on a column (10 cm x 6 mm) of TSK gel SP-2SW (5 µm) with 0.25 M KH2PO4 as mobile phase (0.6 mL min-1). After the elution of histamine(I), spermidine and other strongly basic compounds were eluted with 0.5 M KH2PO4. I was determined fluorimetrically at 450 nm (360-nm excitation) after online post-column derivatization with phthalaldehyde. The detection limit was 0.05 pmol of I, and the calibration graph was rectilinear for 10 pmol. Recovery was 98.5% and the within- and between-day coefficient of variation were <3%.
Histamine Spermidine HPLC Fluorescence Post-column derivatization

"Automated Determination Of Amoxycillin In Biological Fluids By Column Switching In Ion-pair Reversed-phase Liquid Chromatographic Systems With Post-column Derivatization"
J. Chromatogr. B 1985 Volume 344, Issue 1 Pages 285-296
Jan Carlqvist and Douglas Westerlund

Abstract: Plasma was deproteinized with HClO4 and buffered with citrate - phosphate buffer solution (pH 5.4) - 1 M NaOH (11:4) before analysis. Urine was adjusted to pH 4.85 with citrate - phosphate buffer solution before analysis. The chromatographic system comprised a guard column (5 mm x 4 mm) of Spherisorb S5 ODS (5 µm) and two analytical columns (10 cm x 4.6 mm) of CP Microspher C18 (3 µm) separated by a switching valve. Amoxycillin(I) was isolated on the first column with a mobile phase (0.75 mL min-1) of 10 to 11% of methanol in phosphate buffer solution (pH 7.4) containing 1 mM Na hexyl sulfate, and the eluate fraction containing I was transferred to the second column; I was eluted with a mobile phase (1 mL min-1) of 30 to 35% of methanol in phosphate buffer solution (pH 7.4) containing 1 mM tetrahexylammonium hydrogen sulfate. The eluate was mixed with fluorescein in acetonitrile before fluorimetric detection at 470 nm (372.5-nm excitation). The detection limits for I in plasma and urine were 10 and 25 ng mL-1, respectively, and the calibration graph was rectilinear for 0.05 to 75.2 µg mL-1 of I in urine.
Amoxycillin HPLC Fluorescence Post-column derivatization

"Development Of A High Performance Liquid Chromatographic Assay For Digoxin Using Post-column Fluorogenic Derivatization"
J. Chromatogr. B 1986 Volume 377, Issue 1 Pages 233-242
Elizabeth Kwong and Keith M. McErlane

Abstract: Digoxin(I) and its metabolites were separated by HPLC on a column (15 cm x 4.6 mm) of Spherisorb ODS II (3 µm) with a mobile phase of methanol - water - ethanol - propan-2-ol (52:45:3:1) at 0.3 mL min-1. Post-column derivatization was achieved by reaction with dehydroascorbic acid formed in situ from H2O2 - ascorbic acid in HCl, and fluorimetric detection was at 435 nm (excitation at 360 nm). Rectilinear calibration graphs were obtained for 1.5 to 10 ng of I. The method should be applicable to plasma.
Digoxin HPLC Fluorescence Post-column derivatization

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

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

"Analysis Of Digoxin At Therapeutic Concentrations Using High Performance Liquid Chromatography With Post-column Derivatization"
J. Chromatogr. B 1986 Volume 381, Issue 1 Pages 357-363
E. Kwong and K. M. McErlane

Abstract: Plasma samples (3 ml) were deproteinized with acetone, then extracted with CH2Cl2 containing 2% of propanol; after evaporation of the extract to dryness and dissolving the residue in aqueous 50% methanol the solution was analyzed by HPLC. A column (15 cm x 4.6 mm) of ODS material (3 µm) was used with a mobile phase of methanol - ethanol - propan-2-ol - water (52:3:1:45) at 0.3 mL min-1 and the eluate was subjected to post-column fluorogenic derivatization with HCl and dehydroascorbic acid before fluorimetric detection at 425 nm (excitation at 360 nm). The calibration graph was rectilinear from 0.5 to 4.0 ng mL-1 and the detection limit for digoxin was 0.5 ng mL-1. Results correlated well (r = 0.999) with those of a RIA and there was no interference from digoxin metabolites.
Digoxin HPLC Fluorescence Interferences Post-column derivatization

"Simultaneous Quantification Of Cycloserine And Its Prodrug Acetylacetonylcycloserine In Plasma And Urine By High Performance Liquid Chromatography Using Ultra-violet Absorbance And Fluorescence After Post-column Derivatization"
J. Chromatogr. B 1987 Volume 414, Issue 1 Pages 121-129
D. G. Musson, S. M. Maglietto, S. S. Hwang, D. Gravellese and W. F. Bayne

Abstract: Plasma was mixed with 0.2 M sodium borate buffer (pH 9.75), 6-aminohexanoic acid(I) (internal standard) and 5-methoxyindol-3-ylacetic acid (internal standard) and centrifuged. The supernatant solution was analyzed by HPLC. Urine was mixed with I, α-aminobutylhistidine and NaHCO3 and extracted with propan-2-ol. The organic phase was analyzed by HPLC. The HPLC was achieved on a column (24 cm x 5 mm) of ODS-Hypersil (10 µm) with a mobile phase (2.3 mL min-1) of propan-2-ol - water - concentrated acetic acid - decanesulfonate (pH 4.4) and post-column derivatization with phthalaldehyde - 2-mercaptoethanol. Detection was at 313 nm or fluorescent at 455 nm (excitation at 340 nm). The calibration graphs were rectilinear for 0.3 to 7.5 and 0.3 to 15.0 µg mL-1 of acetylacetonylcycloserine(II) and D-cycloserine(III), respectively, in plasma, and for 2.0 to 100.0 µg mL-1 of II or III in urine.
Pentizidone sodium Cycloserine HPLC Fluorescence Spectrophotometry Post-column derivatization

"Rapid And Sensitive Method For The Determination Of Arterial - Venous Differences And Leg Efflux Of 3-methylhistidine Using Ion-pair High Performance Liquid Chromatography And Post-column Fluorescence Derivatization"
J. Chromatogr. B 1987 Volume 414, Issue 1 Pages 174-179
Eva Andersson, Erik H&aring;kanson, L&ouml;rgen Larsson, Johannes M&aring;rtensson

Abstract: Plasma, deproteinized with trichloroacetic acid, was diluted with the mobile phase [5 mM Na heptanesulfonate - concentrated acetic acid (250:1) (pH 4) containing 4% of methanol]. The solution was subjected to HPLC on a column (25 cm x 4.6 mm) of Nucleosil C18 (7 µm) with a mobile-phase flow rate of 0.8 mL min-1, post-column derivatization with phthalaldehyde reagent and fluorescence detection at 420 nm (excitation at 350 nm). The calibration graph was rectilinear for 25 pmol to 10 nmol of 3-methylhistidine and the limit of detection was 3.2 pmol. Recovery was 96% and the coefficient of variation was 2.95%.
3-Methylhistidine HPLC Fluorescence Post-column derivatization

"Body Fluid Analysis Of A Phosphonic Acid Angiotensin-converting Enzyme Inhibitor Using High Performance Liquid Chromatography And Post-column Derivatization With Phthalaldehyde"
J. Chromatogr. B 1989 Volume 487, Issue 1 Pages 135-141
Harold Kadin, Harry G. Brittain, Eugene Ivashkiv and Allen I. Cohen

Abstract: Plasma or urine (1 ml) was mixed with 0.1 M Na acetate and a fluorinated analogue of the cited drug, i.e., 1- 6-amino-2-[hydroxy-(4-phenylbutyl)phosphinoyl]hexanoyl proline, as internal standard. The solution was applied to a column of C8 and elution was effected with methanol. After evaporation, the residue was dissolved in the mobile phase (200 µL). The solution was analyzed by HPLC on a column (15 cm x 4.1 mm) of PRP-1 (5 µm) with dilute H3PO4 as mobile phase (0.85 mL min-1). Post-column derivatization with phthalaldehyde was followed by fluorimetric detection at 418 nm (excitation at 229 nm). The calibration graphs were rectilinear from 0.2 to 10 µg mL-1 and 2 to 500 ng mL-1 for urine and plasma, respectively, with corresponding detection limits of 0.1 µg mL-1 and 1 ng mL-1.
Phosphoric acid HPLC Fluorescence Post-column derivatization

"High Performance Liquid Chromatographic Analysis Of Unchanged Cis-diamminedichloroplatinum (cisplatin) In Plasma And Urine With Post-column Derivatization"
J. Chromatogr. B 1990 Volume 529, Issue 1 Pages 462-467
Masafumi Kinoshita, Naomi Yoshimura and Hiroyasu Ogata, Daijiro Tsujino, Toshiaki Takahashi, Satoru Takahashi, Yuji Wada and Kazuhiko Someya, Tetsuro Ohno, Keisou Masuhara and Yoshio Tanaka

Abstract: Plasma was ultra-filtered and analyzed directly. Urine was centrifuged and diluted 1:10 before analysis. HPLC was carried out on a column (15 cm x 4.6 mm) of Hitachi No. 3013-N anion-exchange resin (5 µm), with a Guard-Pak CN pre-column and acetonitrile - 10 mM NaCl (17:3) as mobile phase (0.7 mL min-1). The eluate was mixed with 26 µM-K2Cr2O7 (0.14 mL min-1) and 6.6 mM NaHSO3 (0.7 mL min-1), and cisplatin was determined as Pt by AAS at 265.9 nm (heating program given). The limit of detection was 80 ng mL-1 and the calibration graph was rectilinear for 30 µg mL-1. Recovery was 95.0 to 104.0% from plasma and 98.0 to 103.8% from urine, with corresponding coefficient of variation of 4.9 to 7.9% and 3.0 to 5.5%. The method is applicable to pharmacokinetic studies.
HPLC Post-column derivatization Dilution Column Detection limit Calibration

"Quantitation Of Busulfan In Plasma By High Performance Liquid Chromatography Using Post-column Photolysis"
J. Chromatogr. B 1990 Volume 532, Issue 1 Pages 429-437
Joachim Blanz, Cornelius Rosenfeld, Barbara Proksch and Gerhard Ehninger, Klaus-Peter Zeller

Abstract: Plasma was applied to a Bond Elut C8 cartridge and busulfan (I) was eluted with methanol by centrifugation. The eluate was heated at 70°C for 40 min with 4 M NaI and heptane and, after cooling, the organic phase was mixed with 2-methoxyethanol. The heptane phase was evaporated and the remaining extract was analyzed by HPLC on a column (25 cm x 4.6 mm) of LiChrosorb CN (5 µm) with a mobile phase (1 mL min-1) of aqueous 20% methanol, post-column derivatization in a photochemical reaction unit and detection at 226 nm. The calibration graph was rectilinear from 0.05 to 5 µg mL-1 of I and the limit of detection was 20 ng mL-1. Recovery was 93% and the intra- and inter-day coefficient of variation were 2.7 and 7.6 to 11.5%, respectively.
Busulfan HPLC Spectrophotometry Post-column derivatization Detection limit C8 Photochemistry

"Determination Of 2-mercaptopropionylglycine And Its Metabolite, 2-mercaptopropionic Acid, In Plasma By Ion-pair Reversed-phase High Performance Liquid Chromatography With Post-column Derivatization"
J. Chromatogr. B 1991 Volume 564, Issue 1 Pages 258-265
Pierre Leroy, Alain Nicolas*, Catherine Gavriloff and Muriel Matt, Patrick Netter and Bernard Bannwarth, Bernard Hercelin and Michel Mazza

Abstract: Plasma (1 ml) was heated at 50°C for 30 min with 0.2 M phosphate buffer of pH 8 (0.2 ml) and 10% tributylphosphine in CHCl3 (0.2 ml), and proteins were precipitated by addition of ethanol to the mixture and cooling in an ice-bath. The filtrate was centrifuged, and a 50 µL portion of the supernatant was analyzed on a column (12.5 cm x 4 mm) of LiChrospher RP-18e (5 µm), fitted with a guard column (4 mm x 4 mm) of the same material, with acetonitrile - 0.01 M phosphate buffer of pH 7.0 (1:3) containing 5 mM cetrimonium bromide. After derivatization with pyrene maleimide, the fluorescence of the derivatives was measured at 370 nm (excitation at 260 nm). The calibration graphs were rectilinear from 0.2 to 10 and from 0.2 to 5 µg mL-1 of 2-mercaptopropionylglycine (tiopronin; I) and 2-mercaptopropionic acid (II), with coefficient of variation (n = 5) of 6.2 and 9.9% for 0.2 µg mL-1 of I and II, respectively. Recoveries were 95% and the limit of detection were 0.05 µg mL-1 of analyte. Sampling rate was 5 to 6 h-1.
2-Mercaptopropionylglycine 2-Mercaptopropionic acid HPLC Fluorescence pH Post-column derivatization Precipitation

"High Performance Liquid Chromatographic Determination Of Clenbuterol And Cimaterol Using Post-column Derivatization"
J. Chromatogr. B 1991 Volume 564, Issue 2 Pages 537-549
Dirk Courtheyn*, Carlo Desaever and Roland Verhe

Abstract: Animal tissues, faeces and feeding-stuffs were extracted with dilute 0.5 M HCl saturated with ethyl acetate; the resulting extracts or liquid samples, e.g., urine, plasma, blood and bile were purified on Chem Elut CE 120 columns and eluted with toluene - CH2Cl2. The eluate was mixed with 0.1 M HCl, ultrasonicated and centrifuged before a portion of the mixture was analyzed by HPLC on a column (15 cm x 4.6 mm) of Nova-Pak C18 (4 µm) with 25 mM sodium dodecyl sulfate and 0.02 M anhydrous acetic acid buffer of pH 3.5 containing 1 M NaOH - acetonitrile (53:47) as mobile phase (1.3 mL min-1). The eluate was derivatized with use of three reagents (details given) before the absorbance was measured at 537 and 493 nm for cimaterol and clenbuterol, respectively. Detection limits for liquid and solid samples were 0.1 ng mL-1 and 0.2 ng g-1, respectively. Results agreed well with those obtained by high performance TLC and GC - MS.
Cimaterol Clenbuterol HPLC Spectrophotometry Buffer Column Detection limit pH Post-column derivatization

"Determination Of Ampicillin In Biological Fluids By Coupled-column Liquid Chromatography And Post-column Derivatization"
J. Chromatogr. B 1991 Volume 567, Issue 1 Pages 121-128
K. Lanbeck-Vall&eacute;n*, J. Carlqvist and T. Nordgren

Abstract: Plasma (0.5 ml) was mixed vigorously with 70% trichloracetic acid in Na2PO4 - citric acid buffer solution, pH 5.4 (200 µL) and centrifuged at 2400 g for 10 min. A portion (400 µL) of the supernatant liquid was mixed with 1 M NaOH (85 µL) to adjust the pH to ~5. Paediatric plasma (100 µL) was treated in a similar manner. Urine was diluted with 0.5 M Na2PO4 - citric acid buffer solution of pH 4.85. Portions (20 to 100 µL) of the solution were analyzed by HPLC on a column of Perkin Elmer 3 x 3 (3 µm) connected in series with a column (10 cm x 4.6 mm) of Microspher C18 (3 µm) with elution (1 mL min-1) with 17% methanol in phosphate buffer solution of pH 7.4 containing 1 mM Na hexylsulfate for the first column and 35% methanol (for plasma) or 30% methanol (for urine) in phosphate buffer solution of pH 7.4 for the second column. The eluate from the second column was reacted with fluorescamine (0.16 mg mL-1) in acetonitrile and the fluorescence was measured at 470 nm (excitation at 372 nm). Detection limits were 14 nM and 570 nM-of ampicillin in plasma and urine, respectively.
Ampicillin HPLC Fluorescence Buffer Column pH Post-column derivatization

"Determination Of Neomycin In Plasma And Urine By High Performance Liquid Chromatography. Application To A Preliminary Pharmacokinetic Study"
J. Chromatogr. B 1991 Volume 571, Issue 1-2 Pages 189-198
Badar Shaikh*, Jean Jackson and Greg Guyer, William R. Ravis

Abstract: Plasma (1 ml) was deproteinized with 20% trichloroacetic acid solution and the solution was centrifuged. The urine sample was also centrifuged and both supernatant solution were analyzed on a column (15 cm x 4.6 mm) of Supelcosil LC-8-DB (5 µm). A guard column (2 cm x 4.6 mm) of LC-8-DB Supelguard was also used. The mobile phase consisted of 11 mM 1-pentanesulfonate, 56 mM Na2SO4, 7 mM acetic acid and 1.5% of methanol. Neomycin (I) was determined following post-column derivatization with phthalaldehyde at 33°C and fluorescence detection at 455 nm (excitation at 340 nm). Calibration graphs were rectilinear from 0.3 to 20 µg mL-1 of I. The average intra- and inter-assay coefficient of variation for I were 4.4 and 9.8%, respectively.
Neomycin B HPLC Fluorescence Column Kinetic Post-column derivatization

"Determination Of Cisplatin And Cis-diammineaquachloroplatinum(II) Ion By Liquid Chromatography Using Post-column Derivatization With Diethyldithiocarbamate"
J. Chromatogr. B 1994 Volume 652, Issue 2 Pages 203-210
Anita Andersson* and Hans Ehrsson

Abstract: Flash frozen plasma samples were thawed and ultrafiltered at 4000 g for 30 min using filters with a 10 000 mol. wt. cut off. The ultrafiltrate was stored at -25°C before use. Portions (20 or 50 µL) were analyzed on an anion-exchange 5 µm Nucleosil SB column (7 cm x 3.2 mm i.d. or 15 cm x 4.6 mm i.d.) for cisplatin and on a cation-exchange 5 µm Nucleosil SA column (15 cm x 3.2 mm i.d.) for the monohydrated complex, with 0.125 M succinic acid of pH 5.2/methanol (2:3) as mobile phase (0.5 ml/min). Post-column reaction was performed using a packed bed reactor steel column (50 cm x 0.35 cm i.d.) of glass beads (details given), operated at 115°C with a mobile phase (0.17 ml/min) of sodium diethyldithiocarbamate in aqueous methanol. Detection was at 344 nm. The RSD of the determination was 11.5% for 20 ng of the monohydrate and 8% for 9 ng of cisplatin.
Cis-platin Cis-diamminemonoaquachloro-Platinum(II) ion HPLC Glass beads Post-column derivatization

"Assay Of The Antiangiogenic Compound TNP-470, And One Of Its Metabolites, AGM-1883, By Reversed-phase High Performance Liquid Chromatography In Plasma"
J. Chromatogr. B 1994 Volume 652, Issue 2 Pages 187-194
William D. Figga,*, Herman J. C. Yehb, Alain Thibaulta, James M. Pludac, Fumio Itohd, Robert Yarchoane and Michael R. Coopera

Abstract: Plasma (1 ml) was acidified with 100 µL of 2% H2SO4 and a 500 µL portion was mixed with 1 mL of acetonitrile/0.01 M acetic acid and centrifuged at 5643 g for 15 min at 4°C. A portion (1 ml) of the supernatant solution was evaporated at 40°C under N2 and the residue was dissolved in 100 µL of acetonitrile to which 200 µL of water was added. The mixture was subjected to centrifugal microfiltration (0.46 µm pore size) at 3705 g for 15 min and a portion of the filtrate was analyzed on a Nova-Pak C18 column (15 cm x 3.9 mm i.d.) operated at 40°C and protected with a Nova-Pak C18 Guard-Pak. Gradient elution (1 ml/min) was effected with aqueous 30-95% acetonitrile over 18 min. Post-column derivatization was carried out with sodium 8-quinolinethiolate, followed by further separation of the derivatives by HPLC as above except with fluorimetric detection at 426 nm (excitation at 250 nm). The average recoveries of TNP-470 and AGM-1883 were 88% and 85%, respectively, and the calibration curves were linear from 2.5-160 ng/ml. The inter-assay RSD was 18%.
Antiangiogenic, TNP-470 Antiangiogenic, AGM-1883 HPLC

"Quinoxalinone Derivatization Of Biological Carboxylic Acids For Detection By Peroxyoxalate Chemiluminescence With High Performance Liquid Chromatography"
J. Chromatogr. B 1994 Volume 653, Issue 2 Pages 123-130
Bryan W. Sandmann and Mary Lynn Grayeski*

Abstract: A quinoxalinone fluorescent tag is evaluated as a carboxylic acid derivatizing reagent for detection by peroxyoxalate chemiluminescence. The synthetic procedure for the quinoxalinone was modified to give a yield that is significantly increased over that reported previously. The new conditions use less hazardous reagents, and produce a final product greater than 97% pure, without the need for intermediate clean-up steps. The derivatization reaction is also modified to give increased yields of greater than 85% compared to 74% obtained previously. The post-column chemiluminescence reaction conditions are optimized to give detection limits of 500 attomole/injection-10 times lower than the fluorescence previously obtained. The reagent is used for the first time on a plasma sample extract. Typical method precision is 4%.
Quinoxalinone HPLC Chemiluminescence Post-column derivatization

"Determination Of Zuclopenthixol And Its Main N-dealkylated Metabolite In Biological Fluids Using High Performance Liquid Chromatography With Post-column Photochemical Derivatization And Fluorescence Detection"
J. Chromatogr. B 1994 Volume 658, Issue 2 Pages 319-325
Bodil Brandt Hansen and Steen Honor&eacute; Hansen

Abstract: The cis-isomer of clopenthixol (I; zuclopenthixol) and its main dealkyl metabolite (II) were extracted from plasma and urine by adsorption on to a Bond-Elut CN cartridge and eluted therefrom with acetonitrile/butylamine (9:1). Analysis was by HPLC on a Spherisorb S5 CN column (12 cm x 4.6 mm i.d.), operated at 40°C with 6 mM dodecyltrimethylammonium bromide in H2O/acetonitrile/0.2 M phosphate buffer of pH 6.5 (59:36:5) as mobile phase. Eluate was fed through a PTFE photochemical reactor coil (5 m x 0.5 mm i.d.) irradiated by a low-pressure 8 W mercury UV lamp and fluorimetric detection at 435 nm (excitation at 260 nm). The calibration graph was linear from 0.1-50 ng of I injected. Recoveries of 0.1-50 ng/ml of I and II were 62-79% and 82-89%, respectively and corresponding RSD (n = 6) were 7.2-16.6% and 5.8-12.5%. The limit of detection was 6 pg of I.
Zuclopenthixol N-dealkylzuclopenthixol HPLC Fluorescence Post-column derivatization UV reactor Photochemistry

"Determination Of Oxalate In Urine And Plasma Using Reversed-phase Ion-pair High Performance Liquid Chromatography With Tris(2,2'-bipyridyl)ruthenium(II)-electrogenerated Chemiluminescence Detection"
J. Chromatogr. B 1995 Volume 665, Issue 1 Pages 27-36
David R. Skotty and Timothy A. Nieman*

Abstract: Oxalate is quantitated in both urine and plasma samples using reversed- phase ion-pair high performance liquid chromatography (HPLC) with tris(2,2'-bipyridyl)ruthenium(II) [Ru(bpy)2+(3)]-electrogenerated chemiluminescent (ECL) detection. Underivatized oxalate was separated on a reversed-phase column (Zorbax ODS) using a mobile phase of 10% methanol in 100 mM phosphate buffer at pH 7.0. The eluted compounds were combined with a stream of 2 mM Ru(bpy)2+(3) at a mixing tee before the ECL flow-cell. In the flow-cell, Ru(bpy)2+(3) is oxidized to Ru(bpy)3+(3) at a platinum electrode, and reacts with oxalate to produce chemiluminescence (CL). Urine samples were filtered and diluted prior to injection. Plasma samples were deproteinized before injection. A 25 µL aliquot of sample was injected for analysis. Possible interferants, including amino acids and indole-based compounds, present in biological samples were investigated. Without the separation, amino acids interfere by increasing the total observed CL intensity; this is expected because they give rise to CL emission on their own in reaction with Ru(bpy)3+(3). Indole compounds exhibit a unique interference by decreasing the CL signal when present with oxalate. Indoles inhibit their own CL emission at high concentrations. By use of the indicated HPLC separation, oxalate was adequately separated from both types of interferants, which thus had no effect on the oxalate signal. Urine samples were assayed by both HPLC and enzymatic tests, the two techniques giving similar results, differing only by 1%. Detection limits were determined to be below 1 µM (1 nmol/ml) or 25 pmol injected. The working curve for oxalate was linear throughout the entire clinical range in both urine and plasma.
Oxalate HPLC Chemiluminescence Interferences Post-column derivatization Indirect

"Validation Of The Determination Of Amino Acids In Plasma By High Performance Liquid Chromatography Using Automated Pre-column Derivatization With O-phthaldialdehyde"
J. Chromatogr. B 1995 Volume 669, Issue 2 Pages 177-186
Durk Fekkes*, Astrid van Dalen, Margriet Edelman and Ans Voskuilen

Abstract: A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5 µm Spherisorb ODS 2 column (125 x 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).
Amino Acids HPLC Pre-column derivatization

"Automated Analysis Of Free And Total Concentrations Of Three Antiepileptic Drugs In Plasma With Online Dialysis And High Performance Liquid Chromatography"
J. Chromatogr. B 1995 Volume 669, Issue 2 Pages 281-288
Karianne Johansen*, Mette Krogh, Alf Terje Andresen, Asbj&oslash;rg S. Christophersen, Gustav Lehne and Knut E. Rasmussen

Abstract: A fully automated method for determination of the free and total concentration of drugs with a varying degree of protein binding is described. The antiepileptic drugs phenytoin, carbamazepine and phenobarbitone were chosen to demonstrate the utility of this technique. The method was based on the ASTED system and combined on-line equilibrium dialysis at 37°C with concentration of the dialysate on a trace enrichment column and HPLC determination with UV detection. The dialysis cell was a modification of the ASTED dialysis cell and 22% of the free concentration of the drugs were recovered in the recipient channel of the dialyser after 10 min of dialysis at 37°C. The free concentration, the total concentration as well as the drugs protein binding could be determined. The method was shown to be well suited for routine monitoring of the free and the total concentrations of the drugs in plasma from epileptic patients.
Phenytoin Carbamazepine Phenobarbitone HPLC Spectrophotometry Dialysis

"Voltammetric Determination Of Cyadox Using Adsorptive Accumulation In A Flow-through System"
J. Electroanal. Chem. 1986 Volume 214, Issue 1-2 Pages 115-123
Miloslav Kopanica and Vra Star&aacute;

Abstract: Cyadox in de-gassed (with He) 0.05 M NaClO4 - DMF (19:1) as carrier (0.4 mL min-1) was adsorbed from the sample plug on a hanging-mercury-drop electrode at +0.1 V and, when the plug had passed, was subjected to differential pulse stripping to -1.0 V (scan rate 10 or 20 mV s-1; pulse amplitude -50 mV; pulse duration and interval between pulses 0.1 s). For a l mL injection of sample solution the peak current at -0.68 V varied rectilinearly with the Cyadox concentration. in the range 3 to 150 ng mL-1. The method was applied successfully to porcine plasma diluted 1:3 with the carrier, although the presence of plasma components necessitated a flow rate of 0.2 mL min-1, an accumulation potential of -0.1 V, a 'washing' period of 5 min after the plug has passed, and a scan rate of 10 mV s-1. The limit of determination in plasma was 10 ng mL-1.
Cyadox Electrode Voltammetry

"Direct Injection Of Plasma And Urine In Automated Analysis Of Catecholamines By Coupled-column Liquid Chromatography With Post-column Derivatization"
J. Pharm. Biomed. Anal. 1984 Volume 2, Issue 2 Pages 315-333
Per Olof Edlund* and Douglas Westerlund

Abstract: Adrenaline(I), noradrenaline(II) and dopamine(III) were selectively adsorbed from plasma on a column (1 cm x 4.6 mm) of benzeneboronic acid gel (Affi-Gel 601) or from urine on a column (2 cm x 3.8 mm) of dihydroxyboryl-silica or Aba-silica, and were eluted with aqueous H3PO4 containing 2 mM Na decyl sulfate(IV). The eluted catecholamines were enriched as ion pairs on a column (2 cm x 4.6 mm) of Supelcosil LC-18-DB (5 µm), before elution with methanol - buffer solution (1:4) and separation on a column (7.5 cm x 4.6 mm) of Supelcosil LC-18-DB (3 µm), with phosphate buffer (pH 6.65) - citrate buffer (pH 6.65) - methanol (3:3:2) (containing 2 mM (IV) and 0.3 mM EDTA) as the mobile phase (0.9 mL min-1). III was detected by coulometry at +0.3 V, and I and II by fluorimetry, as the trihydroxyindoles after post-column derivatization, at 510 nm (excitation at 400 nm). The limits of detection were 0.05, 0.04 and 1.6 pmol for I, II, and III, respectively, with coefficient of variation between 2 and 4%.
Adrenaline Noradrenaline Dopamine HPLC Fluorescence Coulometry Post-column derivatization

"Validation Of A Liquid Chromatography Post-column Derivatization Assay For The Determination Of Cisplatin In Plasma"
J. Pharm. Biomed. Anal. 1994 Volume 12, Issue 2 Pages 265-271
H. H. Farrish*, P. -H. Hsyu, J. F. Pritchard, K. R. Brouwer and J. Jarrett

Abstract: Plasma (400 µL) was mixed with acetonitrile (400 µL), centrifuged and portions (200 µL) of the supernatant were mixed with 100 µL of 0.01 M citric acid/0.1 mM hexadecyltrimethylammonium bromide (CTAB) buffer (buffer A) of pH ~2.5 and 700 µL of CH2Cl2. After centrifugation, portions (150 µL) of the supernatant were analyzed by HPLC on a Shandon BDS-Hypersil C18 column (10 cm x 4.6 mm i.d.; 5 µm) coated with CTAB, operated at 25°C with a Brownlee Polymer RP guard column (1.5 cm x 3.2 mm i.d.; 7 µm), buffer A of pH 5 as mobile phase (0.7 ml/min) and post-column reaction (reagent flow rate 0.2 ml/min) with 0.117 mM potassium dichromate in an OPA reactor (0.2 ml), then at 30°C with 28.16 mM NaHSO3 in a CRX-390 reactor (1 ml) for detection at 290 nm. The calibration graph was linear from 0.06-30 µg/ml of cisplatin (I) and the inter-run RSD were 4-8.2% (n = 8); the recoveries of I from plasma and plasma/acetonitrile (1:1) supernatant were 105.6% and 103%, respectively. After 18 h at 4°C, 17% of I was lost in the plasma extract. The method was applied to drug interaction studies in dogs.
Cis-platin HPLC Spectrophotometry Post-column derivatization

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

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

"Microfabricated Glucose, Lactate, Glutamate And Glutamine Thin-film Biosensors"
Sens. Actuat. B 1994 Volume 19, Issue 1-3 Pages 587-591
Th. Schalkhammer, Ch. Lobmaier, B. Ecker, W. Wakolbinger, E. Kynclova, G. Hawa and F. Pittner

Abstract: Glutaminase, glucose, lactate and glutamate oxidase-bonded sensors were fabricated on Al2O3 ceramics by immobilization in a polyimine gel or co-crosslinking with polyvinylpyrrolidone and nitrene (details given). The sensors contained integrated Pt and Ag/AgCl counter and reference electrodes. Sensors were mounted in a flow cell and test solution were pumped through the cell at 3 ml/min. Cyclovoltammetry was performed by scanning from -500 to +700 mV at 50 mV/s. Calibration graphs were linear for 0-20 mM glucose and 0-120 mM lactate, glutamate and glutamine. After use, the cell assembly was washed with 0.12 M PBS of pH 7. Applications to the FIA analysis of glucose, lactate, glutamate and glutamine in plasma, blood and fermentation broths are discussed.
Glucose Lactate Glutamate Glutamine Voltammetry Electrode Electrode Sensor

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

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

"Sensitive Determination Of Nitroprusside In Blood By High Performance Liquid Chromatography"
Anal. Sci. 1988 Volume 4, Issue 2 Pages 203-206
T. WATANABE, Y. NAGAMINE, T. TOIDA, I. KOSHIISHI and T. IMANARI

Abstract: Blood or plasma (100 µL) was mixed with 3 M HClO4 (100 µL) in a light-shielded container on an ice - water bath. After centrifugation, a portion (150 µL) of the supernatant solution was mixed with 1 M K2HPO4 to ppt. unused HClO4 and the mixture was centrifuged. A 20 µL portion of the supernatant solution was analyzed by HPLC on a column (15 cm x 4 mm) of Asahipak BEST 502Q with 0.1 M acetate buffer (pH 6.0) containing 0.3 M NaClO4 as mobile phase (0.5 mL min-1). The eluate was allowed to react successively with 5 mM dithiothreitol in 0.05 M Tris - HCl buffer (pH 9.0) containing 5 mM EDTA, aqueous 0.5% chloramine T and barbituric acid - pyridine - concentrated HCl - water (3:30:6:164, w/v/v/v). The product was measured by fluorimetry at 607 nm (excitation at 583 nm). The calibration graph was rectilinear for 0.4 to 400 pmol of nitroprusside.
Nitroprusside HPLC Fluorescence Post-column derivatization

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

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

"Analysis Of Penicillins In Biological Material By Reversed-phase Liquid Chromatography And Post-column Derivatization"
Acta Pharm. Suec. 1979 Volume 16, Issue 3 Pages 187-214
Westerlund D, Carlqvist J, Theodorsen A.

Abstract: The procedure comprises injection of the biological fluid or homogenate after protein precipitation, separation by reversed phase liquid chromatography (LiChrosorb RP 8), post-column derivatization with air-segmentation and finally detection of the UV-absorbance at 310 nm. The derivatization involves formation of the mercuric mercaptide of penicillenic acid and is specific for penicillins with an intact ring system. The conditions for liquid chromatography and derivatization are optimized for the determination of ampicillin [69-53-4] and mecillinam [32887-01-7], but the method is general and may be adapted to any penicillin [1406-05-9] or cephalosporin [11111-12-9]. The influence of pH and MeOH content of mobile phase was systematically studied. By the injection of 100 mL plasma, ampicillin and mecillinam can be quant. determined at 100 ng/mL with a precision of 8%, but the sensitivity increases in direct proportion to the injected volume up to 2 mL. The method was applied to determinations in plasma, whole blood, urine and lymph.
Penicillins HPLC Spectrophotometry Post-column derivatization Optimization

"Determination Of Artesunate And Dihydroartemisinine In Plasma By Liquid Chromatography With Post-column Derivatization And U.v. Detection"
Acta Pharm. Suec. 1984 Volume 21, Issue 4 Pages 223-234
Edlund PO, Westerlund D, Carlqvist J, Wu BL, Jin YH

Abstract: The sample (1 ml), 1 mL of sulfate buffer solution (pH 1.2) and 0.1 mL of aqueous 30% H2O2 [to dissociate the complex formed between artesunate(I) and haemoglobin] were mixed at room temperature After 15 min, 4 mL of hexane - butanol - heptafluorobutanol (72:5:3) was added and, after mixing and centrifugation, the organic phase was removed and evaporated at 40°C under N. The residue was dissolved in acetonitrile (100 µL) and 200 µL of 0.1 M acetic acid was added. A portion (100 µL) of this solution was analyzed by HPLC on a column (10 cm x 4.6 mm) of Microsphere C18 (3 µm), with acetonitrile - acetate buffer of pH 5.1 (11:9) as mobile phase (0.45 mL min-1). Post-column derivatization with 1 M KOH in aqueous 90% methanol produced u.v.-absorbing compounds, which could be detected at 289 nm. Detection limits for I and dihydroartemisinine were ~50 nM, and calibration graphs were rectilinear up to 5 and 3.5 nmol, respectively. The chemistry of the post-column reaction is discussed.
Artesunate Dihydroartemisinine HPLC Spectrophotometry Post-column derivatization

"Automated Determination Of Vitamin C In Food Stuffs And Biological Materials"
Analusis 1989 Volume 17, Issue 9 Pages 519-525
Bourgeois, C.F.;Chartois, H.R.;Coustans, M.F.;George, P.R.

Abstract: The sample (e.g., organ tissue, blood plasma, foodstuff) is extracted with aqueous 5% metaphosphoric acid and the extract is placed in an automated apparatus. Ascorbic acid is oxidized by iodine to dehyroascorbic acid, which is coupled with 4,5-dimethyl-1,2-phenylenediamine to give a fluorescent quinoxaline derivative. This compound is extracted into isoamyl alcohol, back-extracted into a basic aqueous solution where it forms a hydrophilic salt, and the fluorescence is measured against that of a borate buffer. Compared with conventional fluorimetric methods the measurement is carried out in an almost pure solution, thus minimizing matrix interferences. The mean recovery was 98.7%, the coefficient of variation was 1.4% and the sensitivity was 20 ng mL-1.
Ascorbic acid Fluorescence Sample preparation Automation Redox Extraction Buffer Sensitivity Apparatus Interferences

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

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

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

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

"The Measurement Of Oxalate In Biological Fluids Using An Immobilized Enzyme - Bioluminescent Assay"
Ann. Clin. Biochem. 1987 Volume 24, Issue S1 Pages 227-228
Parkinson, I.S.;Adonai, L.R.;Channon, S.M.;Tomson, C.R.V.;Ward, M.K.;Laker, M.F.

Abstract: An adaptation of the enzymatic determination of plasma oxalate (Parkinson et al., Clin. Chim. Acta, 1985, 152, 335) is described in which the bioluminescent enzymes NAD(P)H dehydrogenase (FMN) and alkanal monooxygenase (FMN-linked) (luciferase) are immobilized on CNBr-activated Sepharose (details given). The method involves adjusting plasma pH to 3.0 and incubating it with oxalate decarboxylase for 30 min at room temperature The formate formed acts as a substrate for formate dehydrogenase, generating NADH, which is determined by bioluminescence with a continuous-flow system containing the immobilized enzymes. Recovery was >99%, and levels of 1 µM-oxalate could be determined. The inter-batch coefficient of variation was 8.4% (13 determinations of 17 µM).
Oxalate Clinical analysis Bioluminescence Immobilized enzyme

"Cyanide Dissociation From The Hemoglobin Of Parascaris Equorum"
Biochim. Biophys. Acta 1994 Volume 1205, Issue 2 Pages 252-257
Giovanni Antonini, Andrea Bellelli, Antonio Concetti, Giancarlo Falcioni and Maurizio Brunori*

Abstract: The reduction of cyanomethemoglobin by dithionite leads to the appearance of an intermediate, the complex of cyanide with ferrous hemoglobin, whose dissociation is easily followed in a stopped-flow apparatus. This reaction was studied in the hemoglobin from the parasitic nematode Parascaris equorum, whose extremely high oxygen affinity is due to a very low dissociation rate. The rate of cyanide dissociation from ferrous Parascaris hemoglobin is not so dramatically different from that of other hemoglobins and myoglobins. Other features of the reaction are: (i) the rate constant of cyanide release is pH independent, an observation which is agreement with the possible absence of the distal histidine, given the mechanism suggested in a previous study (Bellelli, A., Antonini, G., Brunori, M, Springer, B.A. and Sligar, S.G. (1990) J. Biol. Chem. 265, 18898-18901), and (ii) the time-course shows no kinetic cooperativity. The structural basis of the extremely high oxygen affinity of Parascaris hemoglobin cannot be explained on the basis of the results here reported. This study also confirms that, even though cyanide binding to ferrous hemoglobins is controlled by distal interactions, the functional behavior of this ligand is characteristic and differs from the behavior of oxygen.
Cyanide, complexes, metal Spectrophotometry Stopped-flow Kinetic

"Endogenous Plasma N-acetylcysteine And Single Dose Oral Bioavailability From Two Different Formulations As Determined By A New Analytical Method"
Biopharm. Drug Dispos. 1991 Volume 12, Issue 5 Pages 343-353
Bernard Gabard, Hermann Mascher

Abstract: Plasma (0.5 ml) was mixed with 0.5 mL of water and 30 µL of 10% tributyl phosphine in methanol. After 30 min at 37°C, 0.15 mL of 20% HClO4 was added and the solution was centrifuged at 2500 g. A portion (100 muwl) of the supernatant solution was analyzed by HPLC on a column (8 cm x 4 mm) of Nucleosil 120 C18 (3 µm) with ethanol - 0.1 M H3PO4 - triethylamine (40:960:1), containing glycine at 100 µg mL-1, as mobile phase (1 mL min-1). The column eluate was mixed with 0.5 M NaOH - 0.1 M H3BO3 (41:59), containing o-phthalaldehyde (10 µg mL-1), at 1 mL min-1 in a reaction tube (described) and detected at 475 nm (excitation at 325 nm). The calibration graph was rectilinear from 0.06 to 8 µM-acetylcysteine; the detection limit was 6 nM. The coefficient of variation (n = 3) were 4.4% at 60 nM and 0.1% at 8 µm. The method is specific for thiols, e.g., cysteine, glutathione, thioglycolic acid and homocysteine.
Cysteine Glutathione Homocysteine Thioglycolic acid Thiols HPLC Fluorescence Column Post-column derivatization

"An FIA Biosensor System For The Determination Of Phosphate"
Biosens. Bioelectron. 1991 Volume 6, Issue 7 Pages 581-587
K. B. Male and J. H. T. Luong*

Abstract: A flow injection analysis (FIA) biosensor system for the determination of phosphate was constructed using immobilized nucleoside phosphorylase and xanthine oxidase and an amperometric electrode (platinum vs silver/silver chloride, polarized at 0.7 V). When a phosphate- containing sample was injected into the detection cell, phosphate reacted with inosine in the carrier buffer to produce hypoxanthine and ribose-1-phosphate in the presence of nucleoside phosphorylase. Hypoxanthine was then oxidized by xanthine oxidase to uric acid and hydrogen peroxide, which were both detected by the amperometric electrode. The response of the FIA biosensor system was linear up to 100 µM phosphate, with a minimum detectable concentration of 1.25 µM phosphate. Each assay could be performed in 5-6 min and the system could be used for about 160 repeated analyzes. This system was applicable for the determination of phosphate in various food products and plasma, and the results obtained agreed well with those of the enzymatic assay. The development and performance of the cited flow injection system for phosphate determination are described. The biosensor incorporates purine-nucleoside phosphorylase (I) and xanthine oxidase (II), co-immobilized on a pre-activated nylon membrane and attached to the tip of a amperometric electrode (Pt vs. Ag - AgCl, polarized at 0.7 V). On injection of the sample solution into the flow cell, phosphate reacts with 1 mM inosine in the carrier buffer solution [100 mM imidazole - 100 mM NaCl (pH 7)] in the presence of I to produce hypoxanthine and ribose 1-phosphate. The hypoxanthine is then oxidized by II to uric acid and H2O2, which are detected by the amperometric electrode. The system response was rectilinear up to 100 µM-phosphate, with a detection limit of 1.25 µM. The response time was 2 min, the assay time was 5 to 6 min, and the system could be used for ~160 analyzes. The method was applied to phosphate determination in plasma and various food products. The results agreed well with those obtained by enzyme assay.
Phosphate Amperometry Electrode Electrode Sensor Biotechnology Buffer Immobilized enzyme Method comparison Sensitivity

"Improvement Of The Selectivity Of An Flow Injection Analysis Amperometric Biosensor System For Glucose"
Biosens. Bioelectron. 1993 Volume 8, Issue 5 Pages 239-247
K. B. Male and J. H. T. Luong*

Abstract: In the cited flow injection analysis system, samples (75 µL) were injected into a stream (31 ml/h) of 1 mM acetate buffer of pH 4-7.2 and carried to a column (12 cm x 2.54 mm i.d.) of acetate AG 1-X8 anion-exchange resin (200-400 mesh) to remove interfering substances including uric acid and ascorbic acid. The stream then merged with a stream (31 ml/h) of 100 mM acetate buffer of pH 5.5 containing 1 M NaCl and passed through an enzyme reactor column (6 cm x 2.54 mm i.d.) prepared by immobilizing glucose oxidase onto aminopropyl glass beads using glutaraldehyde (details given) and packing the beads into Tygon tubing. H2O2 was determined using an amperometric flow cell with an Immunodyne membrane (3 µm) and a Pt electrode held at +700 mV vs. Ag/AgCl. The optimum pH for ascorbic acid and uric acid removal were 4 and >6, respectively; the anion-exchange column was less efficient at removing paracetamol. The calibration graph was linear for up to 1 mM glucose and the detection limit was 10 µM. Reproducibility was good and the enzyme reactor was stable for >2000 analyzes; sample throughput was 17/h. The method was applied to urine, plasma, fruit juices and pea and bean seed extracts (details given). A flow injection analysis (flow injection analysis) biosensor system has been developed for the determination of glucose from urine, blood plasma and foodstuffs. Glucose oxidase was immobilized onto porous aminopropyl glass beads via glutaraldehyde activation to form an enzyme column. The hydrogen peroxide released from the conversion of glucose to gluconic acid was monitored by a platinum electrode vs. silver/silver chloride poised at +700 mV. As a novel aspect to the improvement of the selectivity of the biosensor system, an anion exchange column was placed upstream to remove uric acid, ascorbic acid or acetaminophen, three major electroactive interfering substances which usually occur in urine and blood plasma. Among several resins tested, the effective adsorption of uric and ascorbic acids could be accomplished using an acetate anion exchanger, and the selectivity coefficient was pH dependent. The binding of acetaminophen to the resin was much less efficient and, in all cases, the selectivity coefficient was independent of the operating temperature up to 37°C. When applied to real samples, the data obtained by the biosensor system compared well with those of the standard hexokinase assay. The immobilized glucose oxidase could be reused for at least 2000 repeated analyzes without loss of its original activity.
Glucose Amperometry HPIC Electrode Sensor Biotechnology Selectivity Immobilized enzyme Interferences Glass beads

"Determination Of Tetracycline In Plasma By Flow Injection Method With Chemiluminescence Detection"
Bunseki Kagaku 1984 Volume 33, Issue 10 Pages 568-570
Owa, T.;Masujima, T.;Yoshida, H.;Imai, H.

Abstract: Tetracycline(I) was extracted from the plasma sample into ethyl acetate as the Ca chelate. The organic phase was separated by centrifugation and evaporated to dryness, and the residue was dissolved in 0.2 M NaOH for flow injection analysis. I was oxidized with 40 mM K2S2O8 and then mixed with 10 mM 1,3-dibromo-5,5-dimethylhydantoin to produce chemiluminescence, which was measured at 440 nm. Calibration graphs were rectilinear for 1.2 to 19 µg mL-1 of I in plasma.
Tetracycline Chemiluminescence Clinical analysis Chelation

"Flow Injection Analysis With Membrane Separation. Determination Of Ammonia In Blood And Urine"
Bunseki Kagaku 1984 Volume 33, Issue 10 Pages 505-509
Aoki, T.;Uemura, S.;Munemori, M.

Abstract: The sample, made alkaline with 1 M NaOH, was injected into the outer tube of a separation unit (shown diagrammatically), which comprised an inner micro-porous PTFE tube containing phthalaldehyde - 2-mercaptoethanol reagent, and an outer tube containing water. Ammonia permeated into the inner tube and the reaction product flowed into a flow cell for fluorimetric determination at 486 nm (370-nm excitation). Calibration graphs were rectilinear for 1 µM to ~1 mM NH3. Various compounds commonly found in blood and urine did not interfere. Recovery of NH3 was 95 to 108, 104 to 110 and 97 to 101% from whole blood, plasma and urine, respectively.
Ammonia Clinical analysis Fluorescence Gas diffusion Interferences Teflon membrane

"A Sensitive Post-column Derivatization/UV Detection System For HPLC Determination Of Antitumour Divalent And Quadrivalent Platinum Complexes"
Chem. Pharm. Bull. 1995 Volume 43, Issue 1 Pages 108-114
Kizu, R.;Yamamoto, T.;Yokoyama, T.;Tanaka, M.;Miyazaki, M.

Abstract: Methods for the determination of cisplatin (I), oxoplatin (II), carboplatin (III), oxaliplatin (IV) and tetraplatin (IV), in human plasma ultrafiltrate and urine (100 l samples) are described). For I, an MCI gel CDR10 column (8 cm x 4.6 mm i.d.) was used with 10 mM acetate buffer of pH 5.5 containing 0.1 M Na2SO4 and 30% acetonitrile as mobile phase. For II a MCI gel CPK08 column of similar dimensions was used with 50 mM K2SO4 as mobile phase. For III, IV and V and Inertsil ODS-2 column (25 cm x 4.6 mm i.d.) was used with 10 mM acetate buffer of pH 5.5 containing 5% acetonitrile as mobile phase. The flow rate was 1 ml/min and the columns were operated at 40°C. Post-column reaction at 60°C and 0.3 ml/min was performed with 40 mM NaHSO3 in 10 mM acetate buffer of pH 5.5 in a reaction coil (10 m x 0.5 mm i.d.) for detection at 290 nm. The calibration graphs were linear for 0.05-20 M-I, 0.1-20 M II, and 0.2-20 M III, -IV and -V and the corresponding detection limits were 20, 40, 60, 100 and 60 nM; the intra-day RSD were <5%. The recoveries were >>95%. The method was used to study the pharmacokinetics of I and II in rabbits.
Cis-platin Carboplatin Oxaliplatin Tetraplatin Oxoplatin HPLC Spectrophotometry Post-column derivatization

"Development And Performance Of An Automated HPLC Analyser For Catecholamines"
Chromatographia 1987 Volume 24, Issue 5 Pages 363-370
K. -S. Boos, B. Wilmers, R. Sauerbrey, E. Schlimme

Abstract: An automated dual-column HPLC system is described for the determination of catecholamines in body fluids, based on the use of a bonded-phase material with both size-exclusion and affinity-chromatographic properties (cf. Boos et al., '8. Koenigsteiner Chromatographie Tage', Waters GmbH, Koenigstein, 1985, p. 219), a microprocessor-controlled column-switching technique and an optional reaction system for post-column derivatization for fluorimetric detection. For a standard mixture of noradrenaline(I) and adrenaline(II), the detection limits were 300 and 2 pg of I or II with measurement of natural and trihydroxyindole fluorescence, respectively. From 20 fmol mL-1 to 3 nmol mL-1 of I and II can be determined. Coefficients of variation are tabulated for catecholamines in urine and plasma or serum.
Catecholamines Noradrenaline Adrenaline SEC LC Fluorescence Post-column derivatization

"Catecholamines:"
Chromatographia 1988 Volume 25, Issue 3 Pages 199-204
Boos, K.S.;Wilmers, B.;Sauerbrey, R.;Schlimme, E.

Abstract: Noradrenaline(I) and adrenaline(II) were determined in plasma (serum) and urine by using a pre-column (3 cm x 4 mm) of hydrophilic vinyl polymer and an analytical column (12.5 cm x 4 mm) of LiChrospher RP-18 (5 µm) linked by an automatic switching valve. The mobile phase was phosphate buffer of pH 8.7 for the pre-column and phosphate buffer of pH 3.0 - methanol - water in the analytical column. Post-column derivatization was effected by sequential treatment of the eluate with buffered K3Fe(CN)6, ascorbic acid and NaOH. The fluorescent trihydroxyindole derivatives formed were detected at 520 nm (excitation at 410 nm). The limit of detection was 2 pg. In urine, the within-batch coefficient of variation were 5.66 and 6.67% for I and II, respectively. Recoveries (n = 20) from urine were 93.61 ± 4.14 and 95.61 ± 3.25% for I and II, respectively; the corresponding figures for plasma (n = 15) were 103.54 ± 3.89 and 103.18 ± 4.11%.
Adrenaline Noradrenaline HPLC Fluorescence Post-column derivatization

"Aminopropyl-silica As An Advantageous Alternative To Nonpolar Sorbents For Continuous Cleanup/preconcentration Of Vitamin D-3 Metabolites"
Chromatographia 1998 Volume 47, Issue 7-8 Pages 367-372
F. Ortiz Boyer, J. M. Fern&aacute;ndez Romero, M. D. Luque de Castro and J. M. Quesada

Abstract: A new procedure for continuous cleanup and concentration of hydroxyvitamin D3 metabolites prior to their separation by HPLC and UV-detection is reported. The process is based on the use of aminopropyl-silica as solid-phase sorbent as an alternative to the use of nonpolar sorbents. The improvement thus achieved has been tested by comparing the results with those obtained using octadecyl-C18 as non-polar sorbent. The comparison has been based on the calibration graphs (linear range, detection and quantitation limits), precision and multiple standard addition method.
Previtamin D(3) 7-Hydrocholesterol 25-Hydroxyvitamin D3 Calcitriol 24,25-Dihydroxyvitamin D3 HPLC Silica Preconcentration Pre-column extraction

"Determination Of Total Carbon Dioxide In Plasma By Automated Flow Injection Analysis"
Clin. Chem. 1979 Volume 25, Issue 3 Pages 443-445
H Baadenhuijsen and HE Seuren-Jacobs

Abstract: A procedure for measuring total CO2 in plasma is described. It is based on the principles of the flow injection analysis technique which makes use of unsegmented fast-flowing reagent streams as developed by Ruzicka et al. The further methodological design resembles the silicone-rubber membrane technique of Kenny and Cheng. CO2 in the sample is released by reaction with H2SO4. Appropriate amounts of CO2 permeate through the membrane that separates the acid reagent stream and a buffered cresol-red indicator stream. The experimental set-up and functioning of this system are described. A procedure is described for measuring total CO2 in blood plasma by the flow injection analysis technique, which uses unsegmented fast-flowing reagent streams, as developed by J. Ruzicka, et al. (1975). CO2 in the sample is released by reaction with H2SO4. Appropriate amounts of CO2 permeate the membrane that seps. the acid reagent stream and a buffered cresol red indicator stream. The experimental setup and functioning of this system are described.
Carbon dioxide Clinical analysis Spectrophotometry Dialysis Gas diffusion Method comparison Silicone membrane

"Continuous-flow Fluorimetry Of Low Galactose Concentrations In Blood Or Plasma"
Clin. Chem. 1980 Volume 26, Issue 2 Pages 282-285
JM Henderson and FW Fales

Abstract: Clearance of 0-100 mg/L concentrations of galactose from the blood depends on nutrient hepatic blood flow. We can measure such concentrations, which was not previously possible, by a continuous-flow method involving the use of galactose oxidase and peroxidase, the latter being coupled to a fluorogenic substrate, p-hydroxyphenylacetic acid. Interfering substances in the peroxidase reaction are removed by zinc/alkali precipitation. Sensitivity is maximized by using saturating concentrations of the enzymes and substrate. In prepared plasma test samples with galactose concentrations of 10, 40, 70, and 100 mg/L, the within-run CV's ranged from 2.1 to 8.6%, and day-to-day CV's from 2.2 to 17.2%, the largest CV's being for the 10 mg/L concentration. Normal subjects are shown to clear galactose more efficiently than subjects with moderate cirrhosis.
Galactose Clinical analysis Fluorescence Air segmentation Technicon

"Continuous-flow System For Automation Of Latex Immunoassay By Particle Counting"
Clin. Chem. 1983 Volume 29, Issue 6 Pages 1007-1011
AM Bernard and RR Lauwerys

Abstract: The method is based on the agglutination, by protein, of calibrated latex particles coated with a specific antibody. The automated system consists of a modified sampler, a peristaltic pump, a thermostatic bath, a manifold, an optical cell counter equipped with a double-threshold system, and a recorder with a continuously adjustable scale. The reaction mixture (sample plus antibody-coated latex beads) is incubated in a heated mixing coil for 25 min, and the extent of agglutination is measured with the cell counter. No external shaking is required. A wide variety of proteins in plasma and urine, including human ferritin, β2-microglobulin, retinol-binding protein and albumin, have been determined. The detection limits range from 1 pM to 0.1 nM. Within- and between-assay coefficient of variation are <10%. In the assay of ferritin, sera are pre-treated to prevent interference from chylomicrons, complement and rheumatoid factor.
Proteins Ferritins β-2-Microglobulin Albumin, protein Clinical analysis Biochemical analysis Immunoassay Interferences Heated reaction Latex

"Determination Of Thiamine And Thiamine Phosphate Esters In Blood By Liquid Chromatography With Post-column Derivatization"
Clin. Chem. 1983 Volume 29, Issue 12 Pages 2073-2075
M Kimura and Y Itokawa

Abstract: A modification to the method of Kimura et al. (cf. Anal. Abstr., 1983, 44, 2E36) is described.Blood, erythrocytes or plasma (0.2 ml) and 0.2 mL of aqueous 10% trichloroacetic acid were mixed, and the mixture was centrifuged at 35,000 g.A 100 µL portion of supernatant solution was analyzed by HPLC on a column (25 cm x 4 mm) of µBondapak C18, with 0.2 M NaH2PO4 in aqueous 0.3% acetonitrile as mobile phase (1.0 mL min-1).The eluate was mixed with 0.01% K3Fe(CN)6 in 15% NaOH solution to form thiochrome phosphate esters, and the fluorescence was measured at 450 nm (excitation at 375 nm).Thiamine(I), I mono-, I tri-, and I poly-phosphates were eluted and measured as single peaks.The limit of determination of I or its esters was 30 fmol.
Thiamine Thiamine triphosphate Thiamine monophosphate Clinical analysis HPLC Fluorescence Post-column derivatization

"Alternative Methods Evaluated For Assaying Low Concentrations Of Galactose In Plasma"
Clin. Chem. 1987 Volume 33, Issue 3 Pages 420-421
B Noe, JM Henderson, and MH Kutner

Abstract: The continuous-flow fluorimetric assay previously described (cf. Henderson and Fales, Ibid., 1980, 26, 282) was compared with a manual adaptation and with an electrochemical method performed with a YSI Model 27 Analyser (Yellow Springs Instrument Co.). In both fluorimetric assays the fluorescence was measured at 414 nm with 317-nm excitation. In the electrochemical method a galactose oxidase membrane electrode was used. There was good correlation between results by the fluorimetric methods (r = 0.987) but not between those and results by the electrochemical method (r = 0.524). The electrochemical method also overestimated galactose and is not considered suitable for use in determining low concentration. (<100 mg l-1) of galactose.
Galactose Clinical analysis Electrode Electrode Fluorescence Method comparison

"More-sensitive Enzyme-multiplied Immunoassay Technique For Procainamide And N-acetylprocainamide In Plasma, Serum And Urine"
Clin. Chem. 1988 Volume 34, Issue 5 Pages 957-960
PR Henry and RA Dhruv

Abstract: Procainamide(I) and N-acetylprocainamide(II) were determined by a commercial EMIT method (Syva Co.), modified to permit automated analysis of ~100 samples per day in a working range of 0.1 to 2.0 µg mL-1. A Gilford 203S continuous-flow system was used, with a Beckman Model DU spectrophotometer. The calibration graph was prepared by using I or II concentration. of 0, 0.1, 0.2, 1.6, 1.0 and 2.0 µg mL-1 and had a standard deviation of 5%. In a study in which 0.1 to 10 µg mL-1 of I and II were determined in duplicate, average recoveries, calculated from data obtained on two separate days were 99.5% (coefficient of variation 2.2%) and 100.4% (coefficient of variation 3.0%) for I and II, respectively. At extreme ratios of I to II the accuracy of determining the lower concentration. component became unsatisfactory.
Procainamide N-Acetylprocainamide Clinical analysis Spectrophotometry Immunoassay Simultaneous analysis

"Automated Determination Of Cholinesterase Activity In Plasma And Erythrocytes By Flow Injection Analysis, And Application To Identify Subjects Sensitive To Succinylcholine"
Clin. Chem. 1989 Volume 35, Issue 1 Pages 77-80
P Laine-Cessac, A Turcant and P Allain

Abstract: The cited determination was carried out with use of a flow injection system incorporating a robotic sample preparation and injection unit, a spectrophotometer and an Apple computer. Plasma or haemolysate (5 µL) was mixed with 0.1 M phosphate buffer (pH 7.5) and 10 µL of substrate solution (succinylcholine, butyrylcholine or acetylcholine) and the mixture was heated at 30°C for 20 min. The reaction was stopped by the addition of 10 µL of physostigmine. The choline formed was determined by addition of a color reagent, containing choline oxidase (forming H2O2), peroxidase, phenol and 4-aminoantipyrine and, after further heating, the absorbance was measured at 500 nm. The calibration graph was rectilinear for 0.5 to 4 mM choline. The results using the three substrates correlated well (r 0.94), and the between-day coefficient of variation for plasma analysis was 7% (n = 12). The preferred substrate is succinylcholine. The use of the flow injection system decreases the volume of reagent and sample solution required by a factor of ten.
Enzyme, cholinesterase Succinylcholine Spectrophotometry Clinical analysis Automation Computer Buffer Calibration Robot

"Flow Injection Analysis For Malondialdehyde In Plasma With The Thiobarbituric Acid Reaction"
Clin. Chem. 1992 Volume 38, Issue 10 Pages 2061-2065
H Ikatsu, T Nakajima, N Murayama and T Korenaga

Abstract: Plasma was introduced into the PTFE reaction coil (10 mm x 0.5 mm) of the flow injection apparatus (diagram given) and mixed, at 95°C, with thiobarbituric acid (5 g l-1) - H3PO4 (100 mL l-1). This stream was then mixed with the extracting solvent, methylisobutylketone. The organic phase was continuously separated by a successive phase-separation system, and the absorbance was measured at 532 nm. The calibration graph was rectilinear for up to 10 µM of 1,1,3,3-tetraethoxypropane with a detection limit of 0.27 µM. Recovery of malondialdehyde was 84.5 to 90.2%. A simple, precise, and rapid method to measure plasma malondialdehyde (MDA) was developed by use of solvent extraction- flow injection analysis The reagent solution, containing thiobarbituric acid (TBA), 5 g/L in 100 mL/L phosphoric acid, and extraction solvent (methylisobutyl ketone, MIBK) were propelled with a double-plunger micro-pump at a flow rate of 0.3 mL/min, and 20 µL of sample was introduced into the reagent stream. After TBA-MDA reactant was extd. into MIBK, the organic phase was continuously separated by a successive phase-separation system equipped with two phase separators, and the absorbance of the TBA-MDA reactant was measured at 532 nm. This approach resulted in excellent sensitivity, a CV of <1.5%, a good correlation with the conventional manual method, and a sampling frequency of 7 samples/h, suggesting that this semiautomated method is suitable for measuring plasma MDA.
Malondialdehyde Spectrophotometry Clinical analysis Heated reaction MIBK Phase separator Organic phase detection Method comparison

"Homocysteine And Other Thiols Determined In Plasma By HPLC And Thiol-specific Post-column Derivatization"
Clin. Chem. 1993 Volume 39, Issue 8 Pages 1590-1597
Anders Andersson, Anders Isaksson, Lars Brattstrom and Bjorn Hultberg

Abstract: For determination of total sulfhydryls, plasma or urine was incubated with dithiothreitol (I) for 15 min at 37°C, mixed with sulfosalicylic acid and centrifuged. For determination of free sulfhydryls, acid-pptd. plasma supernatant solution was mixed with boric acid, NaOH solution and I and incubated at 37°C for 15 min. Analysis was by HPLC on a column (10 cm x 3.2 mm) of Brownlee Velosep RP-18 (3 µm) with a mobile phase (0.8 ml/min) of 16 mM NaH2PO4/19 mM H3PO4/8 mM octyl sulfate/4.3% methanol (pH 2.4). Post-column derivatization was with 10 mM 4,4'-dithiodipyridine in 0.01 M HCl/methanol (97:3) and 300 mL of 0.3 M Tris/1 mM EDTA (pH 8.5) followed by detection at 324 nm. The limit of detection was 50 nM-homocysteine and the calibration graph was linear for 0.2-100 µm-homocysteine. The intra- and inter-assay RSD for plasma were 1.1-5.5% (n = 7) and 2.5% (n = 22), respectively. Recoveries were 94.0, 97.1, 86.5 and 112.3% for cysteine, homocysteine, glutathione and cysteinylglycine, respectively.
Cysteinylglycine Glutathione Cysteine Homocysteine HPLC Clinical analysis Post-column derivatization

"Fast Lipoprotein Chromatography: New Method Of Analysis For Plasma Lipoproteins"
Clin. Chem. 1993 Volume 39, Issue 11 Pages 2276-2281
Winfried Marz,Rudlger Seierkmeler, Hubert Scharnagi, Ullrlch B. Selffert, and Werner Gross

Abstract: To separate plasma proteins, 20 µL of plasma was applied directly to a column (30 cm) of Superose 6 with 100 mM Na2HPO4 (pH 7.4)/200 mM NaCl as mobile phase (300 µL/min). Lipoproteins were detected online at 500 nm after post-column derivatization with enzymatic cholesterol reagent (CHOD-PAP; Monotest, Boehringer Mannheim, Mannheim, Germany). Results correlated well with those obtained from an ultracentrifugation/precipitation method (r = 0.933-0.979). Response was linear for up to, e.g., 7.74 g/l of low-density-lipoprotein cholesterol. Inter-assay RSD (n = 13) were 1.9-5.8%. Fast lipoprotein chromatography (FLPC) is a novel method for quantifying lipoproteins. Plasma proteins are separated by fast-flow gel filtration. Lipoproteins are detected by post-column derivatization with an enzymatic cholesterol reagent. FLPC resolves very-low-, low-, and high-density lipoproteins (VLDL, LDL, and HDL, respectively) and completely separates apolipoprotein Al- and apolipoprotein B-containing lipoproteins. CVs for VLDL-cholesterol, LDL-cholesterol, and HDL-cholesterol are 5.8%, 2.0%, and 1.9%, respectively. We compared FLPC with a combined ultracentrifugation and precipitation method and obtained correlation of r = 0.979, 0.978, and 0.933 for VLDL-cholesterol, LDL-cholesterol, and HDL-cholesterol, respectively. Triglyceride concentrations up to 9.00 g/L did not interfere with the quantification of lipoproteins by FLPC. We conclude that FLPC is a precise and reliable method for the analysis of plasma lipoproteins that complements conventional techniques.
Proteins Chromatography Clinical analysis Post-column derivatization Interferences

"Rapid Determination Of Ammonia In Whole Blood And Plasma Using Flow Injection Analysis"
Clin. Chim. Acta 1982 Volume 119, Issue 1-2 Pages 7-14
Gunilla Svensson* and Torbj&ouml;rn Anf&auml;lt

Abstract: A flow injection method for the determination of ammonia in whole blood and plasma is described. The method utilizes diffusion of ammonia into a stream of a pH-sensitive indicator, which is monitored by a photometer. A volume of 90 µL is needed and the result is obtained within 1 min. Ammonia concentrations increase during storage due to deamination. It is proposed to use plasma samples, which could be stored frozen at -20°C without significant change.
Ammonia Clinical analysis Spectrophotometry Gas diffusion

"Sensitization And Visualization Of Biochemical Measurements Using The NAD+ - NADH System By Means Of Meldola Blue. 2. Application To The Continuous-flow Determination Of Plasma Glucose And Urea"
Clin. Chim. Acta 1982 Volume 125, Issue 2 Pages 185-192
J. L. Orsonneau*, K. Meflah, P. Lustenberger, G. Cornu and S. Bernard

Abstract: This paper describes the application of the Meldola blue method to the determination in serum of glucose with glucose dehydrogenase and of urea with urease and glutamate dehydrogenase with a continuous flow analyzer.. It discusses the easy application, reliable results and inexpensiveness of the method.
Glucose Urea Clinical analysis Indirect

"Measurement Of Plasma Oxalate In Healthy Subjects And In Patients With Chronic Renal Failure Using Immobilized Oxalate Oxidase"
Clin. Chim. Acta 1986 Volume 154, Issue 1 Pages 49-58
G. P. Kasidas and G. A. Rose*

Abstract: Plasma, or plasma ultra-filtrate, was incubated with L-ascorbic acid, and the reaction was stopped by addition of 50 mM NaNO2. The mixture was analyzed by continuous-flow-through a column containing immobilized oxalate oxidase; the H2O2 produced was determined by spectrophotometry at 580 nm after reaction with horse-radish peroxidase, 3-dimethylaminobenzoic acid and 3-methylbenzothiazolin-2-one hydrazone. Recovery of oxalate was 84 to 112%.
Oxalate Clinical analysis Spectrophotometry Immobilized enzyme

"An Oxygen-based Enzyme Electrode For Whole Blood Lactate Measurement Under Continuous-flow Conditions"
Clin. Chim. Acta 1986 Volume 155, Issue 3 Pages 295-308
Mark R. Weaver and Pankaj M. Vadgama

Abstract: The enzyme electrode was prepared by immobilizing lactate 2-monooxygenase from Mycobacterium smegmatis on a Millipore ultra-filtration membrane which was applied to the gas-permeable membrane of an O sensor (Radiometer E5046 pO2 electrode). The electrode was housed in a measuring chamber (Radiometer D616) for continuous-flow analysis and an AutoAnalyzer II proportioning pump was used to construct a flow system designed to achieve a blood dilution of 1:40. Response was rectilinear up to 0.8 mM. Results for serum and plasma correlated well with those by a routine fluorimetric method (r = 0.959 and 0.952, respectively). This system permits reagentless continuous in vivo extracorporeal lactate monitoring.
Lactate Clinical analysis Electrode Method comparison Millipore

"Determination Of Plasma Oxalate Concentrations Using An Enzyme - Bioluminescent Assay. 2. Co-immobilization Of Bioluminescent Enzymes And Studies Of In Vitro Oxalogenesis"
Clin. Chim. Acta 1989 Volume 179, Issue 1 Pages 97-108
I. S. Parkinson, S. M. Channon, C. R. V. Tomsona, L. R. Adonai, M. K. Ward*,* and M. F. Laker*

Abstract: Sample or standard solution (4 µL) was incubated with oxalate decarboxylase solution for 30 min at room temperature Formate dehydrogenase was added and incubation was continued for 60 min. A portion of the reaction mixture was transferred to a continuous-flow system for determination of NADH by bioluminescence. The reaction mixture was mixed with a bioluminescent solution of 80 mM NaH2PO4 - trisodium citrate (pH 7.0) containing 30 mM dithiothreitol, 120 µM-flavine mononucleotide and 300 µM-decanal, and this mixture was then passed through a flow-cell containing gel-immobilized NAD(P)H dehydrogenase (FMN) and luciferase enzymes. Luminescence was monitored with a luminometer. The limit of detection was 0.8 µM-oxalate (I). The intra-batch coefficient of variation were 5.2 and 3.8% at 18 and 60 µM-I, respectively. Recovery was 100.7%. There was no interference by 1 mM ascorbate.
Oxalate Bioluminescence Clinical analysis Enzyme Interferences Immobilized enzyme In vitro monitoring

"Determination Of Carbon Dioxide In Blood Plasma By Flow Injection Spectrophotometry"
Fenxi Shiyanshi 1991 Volume 10, Issue 2 Pages 39-40
Fan Shihua, Fang Zhaolun and Li Junxiang

Abstract: Carbon dioxide was determined in plasma (10 µL) by flow injection analysis with separation of CO2 with use of a microporous PTFE membrane, 0.2 M H2SO4 as donor stream (1.6 mL min-1), 0.2 mM NaHCO3 - 0.2 mM Na2CO3 - cresol red in NaOH solution (pH 7.5 to 7.7) as acceptor stream (1.6 mL min-1) and detection at 580 nm. The coefficient of variation (n = 11) was 0.9% and recoveries were 94 to 106%.
Carbon dioxide Spectrophotometry Microporous membrane Teflon membrane

"Detection Of Lipid Hydroperoxides And Hydrogen Peroxide At Picomole Levels By An HPLC And Isoluminol Chemiluminesce Assay"
Free Radical Biol. Med. 1987 Volume 3, Issue 5 Pages 359-361
Yamamoto, Y.;Ames, B.N.

Abstract: Water-soluble and fat-soluble antioxidants were separated by partitioning between aqueous methanol - hexane. The hexane fraction was analyzed by HPLC on a reversed-phase octadecyldimethylsilyl column with methanol - t-butyl alcohol (1:1) as mobile phase. The aqueous methanol fraction was analyzed on a weak anion-exchange aminopropylsilyl column with methanol - 40 mM NaH2PO4 (19:1) as mobile phase. Post-column derivatization was with isoluminol in the presence of microperoxidase and detection was at 430 nm. Interference by antioxidants is avoided and characterization of hydroperoxides at picomole levels in, e.g., blood plasma is possible by this method.
Hydrogen peroxide Hydroperoxides, lipid HPLC Spectrophotometry Interferences Post-column derivatization

"Continuous-flow Quantification Of Total Mercury In Whole Blood, Plasma, Erythrocytes And Urine By Inductively Coupled Plasma Atomic-emission Spectroscopy"
J. Anal. Toxicol. 1992 Volume 16, Issue 2 Pages 99-101
Buneaux F, Buisine A, Bourdon S, Bourdon R.

Abstract: For the determination of Hg, blood and urine underwent sample preparation (details given), before dilution with aqueous NH3 buffer and reduction by sodium borohydride at pH 9.5. The reduced mixture was analyzed by ICP-AES; the test material sample was not nebulized into the torch, but the Hg vapor was collected in a reactor vial and swept into the plasma by the carrier gas (Ar) using a glass apparatus (described). An oxidative mineralization was not required. Optimization of the experimental conditions is discussed. The separation of liquid and gaseous phases decreased background noise and increased the sensitivity of the method; the detection limit was 4 µg l-1. The coefficient of variation (n = 9) was 9% for 35 µg L-1 of Hg. Recoveries were quantitative. The method can be used for routine determinations but it is recommended that a specific standardization is first performed. Mercury determination in blood and urine can be performed by inductively coupled plasma atomic emission spectroscopy after dilution in an ammonia buffer and reduction by sodium borohydride. The proposed method does not need an oxidative mineralization. The sample is not nebulized into the torch, but the mercury vapor, after collection in a reactor vial, is swept into the plasma by the argon carrier gas using the described glass app.
Mercury Spectrophotometry Optimization Volatile generation Phase separator Volatile generation

"Determination Of Specific Proteins By The FIA Principle"
J. Autom. Methods Manag. Chem. 1990 Volume 12, Issue 2 Pages 53-59
IB ANDERSEN

Abstract: Transferrin, haptoglobin, IgG, IgA, IgM and orosomucoid in plasma, and albumin in urine were determined by flow injection analysis on a FIA Star (Tecator) system. Samples were diluted up to 200-fold with carrier solution and a 40 µL portion was injected into carrier solution of 0.05 M phosphate - 0.1 M NaCl buffer (pH 7.4) containing 4 or 7% of polyethylene glycol and mixed with antibody solution; detection was at 405 nm. The results are presented for each protein; the method is not suitable for IgG, IgA and IgM and can be used for orosomucoid if a kinetic setup is applied.
Transferrin Haptoglobin Immunoglobulin G Immunoglobulin A Immunoglobulin M Orosomucoid Albumin Tecator Kinetic

"Continuous-flow Bioluminescent Determination Of ATP In Platelets Using Firefly Luciferase Immobilized On Epoxy Methacrylate"
J. Biolumin. Chemilumin. 1989 Volume 3, Issue 1 Pages 7-11
Giacomo Carrea, Roberto Bovara, Stefano Girotti, Elida Ferri, Severino Ghini, Aldo Roda

Abstract: Plasma was centrifuged, and the supernatant solution was diluted to give 105 platelets mL-1, and then diluted (1:200) with 0.1 mM Tris buffer of pH 8.5 containing 4 mM EDTA and 0.2% of Triton X-100 to release the ATP. Glass columns (3 cm x 2 mm) containing Eupergit C(Sigma)-immobilized firefly luciferase were placed in a luminometer (LKB 1250) in front of the photomultiplier window and Affi-Sep cartridges were inserted directly into the instrument. The manifold was similar to that described earlier (Anal. Chem., 1986, 58, 331), and involved two reagent streams. The first consisted of a bioluminescent solution of 0.02 M Tris - acetate buffer solution of pH 7.75, containing 0.12 mM luciferin, 5 mM Mg2+, 0.1 mM EDTA and 1 mM dithiothreitol; the second stream was a continuous-flow of air, to which a known volume (5 to 50 µL) of sample was intermittently added. The limit of detection was 0.3 pmol of ATP and response was rectilinear between 1 and 500 pmol of ATP. Intra- and inter-assay coefficient of variation were 7.1 (n = 10) and 9.6% (n = 7), respectively. Results correlated well with those obtained by using soluble luciferase (r = 0.99).
Bioluminescence Buffer Glass Column Triton X Detection limit Immobilized enzyme Surfactant

"Enzymatic Determination Of Electrophoretically Separated LDL-cholesterol From Sera Of Cardiac Patients"
J. Chem. Soc. Pak. 1995 Volume 17, Issue 1 Pages 40-42
Anwar, M.;Hashim, M.;Yaqoob, M.;Masoom, M.

Abstract: Plasma was applied to cellulose acetate membrane strips (2 µL/cm) and electrophoresis was performed in 0.05 M barbital buffer of pH 8.6 at a constant current of 0.5 mA/cm over 40 min. The strips were removed and floated on the surface of the fixation solution (5% TCA in 2.5% formaldehyde) for several seconds before soaking completely for 20 min. Staining was performed for 15 min by immersing the strips in Fat Red 7B. The LDL fraction was cut out and the cholesterol was extracted. Cholesterol was determined by an FIA system comprising an immobilized cholesterol esterase/oxidase column as previously described (cf. Atta-ur-Rahman (Ed.) 'Biochemistry for Development', 1988, p. 55).
Cholesterol Column Immobilized enzyme Cellulose acetate

"Determination Of Uric Acid And Xanthine/hypoxanthine In Blood Using Immobilized Enzymes In Flow Injection System"
J. Chem. Soc. Pak. 1995 Volume 17, Issue 2 Pages 109-112
Hussain, G.;Yaqoob, M.;Nabi, A.;Masoom, M.

Abstract: Plasma samples were injected without pretreatment but were passed through a glass column (9 x 9 mm i.d.) packed with CPG prior to the uricase column. Plasma (20 µL) was injected into a 0.1 M Tris hydrochloride buffer pH 8 carrier stream (2.5 ml/min) before passing through online immobilized enzyme mini-columns of urate oxidase and xanthine oxidase/urate oxidase followed by amperometric detection for the determination of uric acid and xanthine/hypoxanthine. A diagram of the enzymatic flow system is given. The results agreed well (r = 0.99) with those obtained using a commercial kit. Calibration ranges were 0-10 mg/l (linear) and 0-25 µM (linearity not stated) for uric acid and xanthine/hypoxanthine, respectively. Results are discussed. A flow injection system containing online immobilized enzyme mini-columns of urate oxidase and xanthine oxidase/urate oxidase for the determination of uric acid and xanthine/hypoxanthine is described. For the determination of xanthine and hypoxanthine, a xanthine oxidase column was incorporated online prior to the mate oxidase column. The results of the uric acid analysis in serum were compared with the commercial kit method in use in hospitals with a correlation coefficient of 0.99 and RSD of 1% (n = 6). The method is fast and the cost per analysis is greatly reduced. (10 References)
Uric acid Xanthine Hypoxanthine Amperometry Column 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

"Continuous-flow Analysis Of Ammonia In Perchloric Acid Supernate Of Blood Or Plasma Using An Ammonia-selective Electrode"
J. Clin. Pathol. 1978 Volume 31, Issue 12 Pages 1207-1211
GC Moses, RJ Thibert, and TF Draisey

Abstract: A simple automated method for the estimation of ammonia in perchloric acid supernate of blood or plasma using an ion-selective electrode (Orion Ammonia-selective electrode, Model 95-10) is described. The reliability of the proposed method has been checked against an ion-exchange resin procedure, which has been chosen as a standard procedure. Regression equation and correlation coefficient for the proposed method are y = 0.7 X + 10 and 0.945, respectively, as compared with the chosen standard method. Within-run and between-run precision are 2.1% and 3.5% respectively. The average percent recovery is 97.5% and a tentative range is 13-73 µg/L (9-52 µmol/L) ammonia nitrogen.
Ammonia Clinical analysis Electrode Sample preparation Extraction

"Fluorescence Derivatization Approaches For Quantitative High Performance Liquid Chromatography Of Peptides"
J. Controlled Release 1990 Volume 13, Issue 2 Pages 121-128
Gerald R. Rhodes*, Venkata K. Boppana and Marc J. Rubenfield

Abstract: Peptides were isolated from plasma by solid-phase extraction before analysis. For determination of disulfide-containing peptides, the sample was treated with 100 mM borate buffer (pH 8) containing 10 mM dithiothreitol at 50°C for 30 min, followed by 40 mM ammonium 4-chloro-7-sulfobenzofurazan at 60°C for 2 h. After solid-phase extraction, the sample was subjected to HPLC on a 2-mm i.d. column with gradient elution (described) and fluorescence detection at 470 nm (excitation at 380 nm). For determination of arginine-containing peptides, the sample was subjected to HPLC, at 60°C, with gradient elution (described), followed by post-column derivatization with 0.4 M NaOH and 0.05% ninhydrin at 70°C and fluorescence detection at 470 nm (excitation at 390 nm). The method was applied in the analysis of disulfide- and arginine-containing drugs; the calibration graphs were rectilinear for 0.46 to 185 pmol mL-1 of SKF 101926 and for 0.5 to 100 pmol mL-1 of SKF 105494.
Drugs Peptides HPLC Fluorescence Buffer pH Post-column derivatization Calibration

"Ion-pair Liquid Chromatographic Analysis Of Phenylpropanolamine In Plasma And Urine By Post-column Derivatization With Phthalaldehyde"
J. Liq. Chromatogr. Relat. Technol. 1985 Volume 8, Issue 8 Pages 1489-1500
Robert J. Y. Shi; Winnie L. Gee; Roger L. Williams; Leslie Z. Benet; Emil T. Lin

Abstract: Plasma (0.5 ml) was extracted with 5 mL of CH2Cl2 containing α-methylbenzylamine(I) as internal standard and 0.5 M K2HPO4 solution at pH 11. After centrifuging the mixture, the organic layer was added to a 0.1% solution of HCl in acetonitrile. The solution was evaporated to dryness under N and the residue was dissolved in 200 µL of mobile phase prepared from 2400 mL of acetonitrile, 1600 mL of water, 0.8 g of Na heptane-1-sulfonate and 12 mL of anhydrous acetic acid. Aliquots (40 to 100 µL) were analyzed by HPLC on a column (15 cm x 4.6 mm) containing Ultrasphere ODS (5 µm) with mobile phase at 1.0 mL min-1 and post-column derivatization with phthalaldehyde (details given) and detection by fluorescence measurement at 418 nm (excitation at 340 nm). Urine samples (0.2 ml) were analyzed similarly but without extraction with CH2Cl2, and the mobile phase and column dimensions differed slightly. Calibration graphs based on peak height ratio were rectilinear for 2 to 200 ng mL-1 of phenylpropanolamine in plasma and for 0.5 to 150 µg mL-1 in urine. The detection limits in plasma and urine were 2 ng mL-1 and 0.5 µg mL-1, respectively. The within-day and day-to-day coefficient of variation were <10%.
Phenylpropanolamine HPLC Fluorescence Post-column derivatization

"Liquid Chromatographic Determination Of A Substituted Benzamide In Biological Fluids Using Preconcentration And Post-column Extraction"
J. Liq. Chromatogr. Relat. Technol. 1987 Volume 10, Issue 8-9 Pages 1903-1916
C. de Ruiter; U. A. Th. Brinkman; R. W. Frei

Abstract: Plasma or urine (diluted 1:1 and 1:9, respectively, with 10 mM phosphate buffer of pH 3.0), containing S-(-)-3-bromo-2,6-dimethoxy-N-(1-propylpyrrolidin-2-ylmethyl)benzamide as internal standard, was applied to a pre-column (2 cm x 4.6 mm) of Baker C8 or C18 material or to a disposable cartridge packed with C8 material. The analytical column (10 cm x 4.6 mm) was packed with Spherisorb ODS-2 (3 µm). All columns were pre-treated with hexadecyltrimethylammonium bromide. The mobile phase (1 mL min-1) was aqueous 10% acetonitrile adjusted to pH 3.0 with H3PO4. Post-column derivatization was carried out online with aqueous 0.1 mM Na 9,10-dimethoxyanthracene-2-sulfonate (pH 3.0) and derivatives were extracted into 1,2-dichloroethane. Detection was by fluorescence measurement at 452 nm (excitation at 383 nm). Detection limits were 1 and 15 ng mL-1 of remoxipride [S-(-)-3-bromo-N-(1-ethylpyrrolidin-2-ylmethyl)-2,6-dimethoxybenzamide;(I)] in plasma and urine, respectively. Recoveries were 88 ± 4 and 76 ± 4% of I from plasma and urine, respectively, and corresponding coefficient of variation were 3.5 and 2.2% (n = 5).
S-(-)-3-bromo-N-(1-ethylpyrrolidin-2-ylmethyl)-2,6-dimethoxybenzamide HPLC Fluorescence Sample preparation C8 C18 Preconcentration Post-column derivatization Post-column extraction

"Fluorescamine Post-column Derivatization For The HPLC Determination Of Cephalosporins In Plasma And Urine"
J. Liq. Chromatogr. Relat. Technol. 1988 Volume 11, Issue 14 Pages 2993-3010
M. D. Blanchin; H. Fabra; B. Mandrou

Abstract: Plasma or urine (10 µL) was applied to a column (25 cm x 4 mm) of LiChrosorb RP-18 (7 µm), fitted with a guard column (1 cm x 4 mm) of the same material, with 25 mM phosphate buffer of pH 7.0 - acetonitrile (19:1, 9:1 or 17:3) as mobile phase (1 mL min-1). Post-column derivatization was performed in a PTFE coil (4.5 m x 0.25 mm) with fluorescamine solution (0.2 mg mL-1) in acetonitrile at 0.25 mL min-1. Fluorimetric detection was at 485 nm (excitation at 385 nm). Calibration graphs were rectilinear from 10 to 500 ng of cefaclor, cephalexin, cephradine, cefroxadine, cephaloglycin and cefadroxil. Detection limits ranged from 0.3 to 1.8 ng, and coefficient of variation (n = 12) were 4%. Recoveries were generally 90%.
Cefaclor Cephalexin Cephradine Cefroxadin Cephaloglycin Cefadroxil HPLC Fluorescence Post-column derivatization

"Determination Of Total Pyridoxal In Human Plasma Following Oral Administration Of Vitamin B6 By High Performance Liquid Chromatography With Post-column Derivatization"
J. Pharm. Sci. 1993 Volume 82, Issue 9 Pages 972-974
Hermann Mascher

Abstract: An HPLC method for determining total pyridoxal from plasma was developed for a relative bioavailability comparison of two oral vitamin B6 (pyridoxine HCl) preparations. After cleavage of the pyridoxal-5-phosphate with the acid phosphatase enzyme, the total pyridoxal was determined by HPLC. Pyridoxal was separated on a reversed-phase column, post-column derivatized to pyridoxal-semicarbazide, and then detected by fluorescence and quantitated. The limit of detection was 2 ng/mL and interday variation (3 days) over the whole concentration range (13-215 ng/mL spiked) was < 4.1%. In the relative bioavailability study, 16 human subjects were put on a low vitamin B6 diet for a period of 3 days. On the 2nd and 3rd days, 14 blood samples were taken per subject at the same times each day. The drug was administered on the 3rd day. Total endogenous pyridoxal detected on the 2nd day varied in plasma between 13 and 17 ng/mL. Pharmacokinetic parameters corrected for background are reported for two vitamin B6 (40 mg) preparations. Briefly, the pharmacokinetic results for the Ratiopharm preparation compared with the Hoffmann-La Roche preparation are, respectively: AUC0-24, 369.2 and 352.6 ng.h/mL; AUC24-48, 1638.2 and 1662.3 ng.h/mL; net Cmax, 193.0 and 197.1 ng/mL; tmax, 1.25 and 1.44 h; and relative bioavailability, 97.9% (Westlake, 88-112%) Plasma (1 ml) was vortex-mixed with 0.15 mL of 3 M perchloric acid and the mixture was centrifuged at >1500 g. A 0.5 mL portion of the clear supernatant solution was incubated for 16 h at 40°C with 0.3 mL of 1 M acetate buffer (pH 4.6) and 0.1 mL of aqueous acid phosphatase solution (10 mg mL-1) before vortex-mixing with 0.15 mL of 3 M perchloric acid. After centrifugation, a 20 µL portion of the supernatant solution was subjected to HPLC on a column (12.5 cm x 4 mm) of Nucleosil 120 5 C18 with a mobile phase (2 mL min-1) of 0.05 M perchloric acid and 0.02 M triethylamine in water. The eluate was mixed with semicarbazide hydrochloride in 1.5 M NaOH (3.35 g l-1) in a PTFE tube (10 m x 0.3 mm) at 70°C before fluorimetric detection at 480 nm (excitation at 365 nm). The detection limit was 2 ng mL-1 of pyridoxal and inter- and intra-day coefficient of variation were 1.7 to 4.1% and 0.1 to 9.1%, respectively. The method was used to study the pharmacokinetics of pyridoxal in human plasma..
Pyridoxal HPLC Fluorescence Post-column derivatization

"Liquid Chromatography - Luminescence Methods"
Life Sci. 1987 Volume 41, Issue 7 Pages 901-904
Kazuko Mori

Abstract: Catecholamines in alumina extracts of urine or plasma or in tissue homogenates were separated by HPLC on TSK-gel ODS-1207 with KH2PO4 - acetonitrile - EDTA as mobile phase, pre- or post-column derivatization and fluorimetric detection. After post-column reaction based on trihydroxyindole formation with Fe(CN)63- as oxidant, the fluorescence of adrenaline and noradrenaline derivatives was measured at 520 nm (excitation at 410 nm). Dopamine could not, however, be detected in plasma by this method or by an alternative technique with electrochemical detection. The method could easily be automated, including a pre-column system. By pre-column derivatization with 1,2-diphenylethylenediamine (after cleanup on a cation-exchange column), dopamine and the other catecholamines could be determined in plasma; the detection limit was ~2 fmol. Fluorescence was measured at 480 nm (excitation at 350 nm).
Catecholamines Adrenaline Noradrenaline HPLC Fluorescence Sample preparation Post-column derivatization Pre-column derivatization

"Quantitation Of Glycerophosphorylcholine By Flow Injection Analysis Using Immobilized Enzymes"
Mol. Cell. Biochem. 1996 Volume 162, Issue 2 Pages 83-87
Alessandra Mancini, Francesca Del Rosso, Rita Roberti, Patrizio Caligiana, Alba Vecchini and Luciano Binaglia

Abstract: A method for quantitating glycerophosphorylcholine by flow injection analysis is reported in the present paper. Glycerophosphorylcholine phosphodiesterase and choline oxidase, immobilized on controlled porosity glass beads, are packed in a small reactor inserted in a flow injection manifold. When samples containing glycerophosphorylcholine are injected, glycerophosphorylcholine is hydrolyzed into choline and sn-glycerol-3-phosphate. The free choline produced in this reaction is oxidized to betain and hydrogen peroxide. Hydrogen peroxide is detected amperometrically. Quantitation of glycerophosphorylcholine in samples containing choline and phosphorylcholine is obtained inserting ahead of the reactor a small column packed with a mixed bed ion exchange resin. The time needed for each determination does not exceed one minute. The present method, applied to quantitate glycerophosphorylcholine in samples of seminal plasma, gave results comparable with those obtained using the standard enzymatic-spectrophotometric procedure. An alternative procedure, making use of co-immobilized glycerophosphorylcholine phosphodiesterase and glycerol-3-phosphate oxidase for quantitating glycerophosphorylcholine, glycerophosphorylethanolamine and glycerophosphorylserine is also described.
glycerophosphorylcholine Amperometry Ion exchange Immobilized enzyme Glass beads Method comparison

"Quantification Of Imipenem's Primary Metabolite In Plasma By Post-column Chemical Rearrangement And UV Detection"
Pharm. Res. 1991 Volume 8, Issue 1 Pages 33-39
Donald G. Musson, Richard Hajdu, William F. Bayne and John D. Rogers

Abstract: Pre-treated samples (prep. described) were subjected to HPLC on a column (10 cm x 8 mm) of Resolve C18 Radial-PAK equipped with a column of RCSS Guard PAK C18 with a mobile phase of tetrabutylammonium hydrogen sulfate - H3PO4 - water, adjusted to pH 6.85 with 1 M KOH. The eluate was diluted with 0.426 M H3PO4 (0.6 mL min-1, passed through a column (25 cm x 4.6 mm) packed with 40 µm glass beads and the absorbance was measured at 295 and 320 nm. Calibration graphs were rectilinear for 1 to 100 µg mL-1 of the cited metabolite (I) in plasma and dialysate and for 5 to 100 µg mL-1 in urine . Coefficients of variation were 10%.
Imipenem, N-Formimidoyl thienamycin HPLC Spectrophotometry Column Glass beads pH Post-column derivatization

"Analysis Of Neuropeptides By Perfusion Liquid Chromatography/electrospray Ion-trap Mass Spectrometry"
Rapid Commun. Mass Spectrom. 1994 Volume 8, Issue 4 Pages 333-338
Hung-Yu Lin, Robert D. Voyksner

Abstract: Perfusion high performance liquid chromatography (HPLC) combined with electrospray ion trap mass spectrometry (ITMS) was evaluated for the determination of neuropeptides in plasma. Perfusion HPLC offers the capability of resolving neuropeptides spiked into plasma in 5 min compared to the 30-60 min separations performed on packed capillary C18 columns. Electrospray combined with the ITMS provides the ability to ionize these neuropeptides and mass analyze them with high sensitivity and specificity. Sub-picomole quantities of neuropeptides injected on- column could be specifically detected in a plasma matrix. The electrospray-ITMS mass spectrum of each neuropeptide showed multiply charged ions which could be used to determine or confirm their molecular weights.
Peptides, neuro Mass spectrometry HPLC Perfusion

"Ultra-micro-analysis Of Bioactive Substances: An Approach Based On The Development Of Highly Selective Fluorogenic Reagents"
J. Pharm. Soc. Jpn. 1988 Volume 108, Issue 1 Pages 22-38
OHKURA YOSUKE

Abstract: A series of mono- and di-functional fluorogenic reagents has been developed for the selective derivatization of various bioactive substances and as enzyme substrates. These have been used for fluorimetric determination and for HPLC with pre- or post-column derivatization of the substances in biological materials. Methods are described for HPLC of neuraminic acids in serum, of catecholamines in plasma and urine, of peptides in, e.g., rat brain, of reducing sugars in serum and urine, of fatty acids in serum, of prostaglandins in seminal fluid and of fluorouracil in serum.
Acids, fatty Catecholamines Fluorouracil Neuraminic acids Peptides Prostaglandins Sugars, reducing HPLC Fluorescence Post-column derivatization

"Use Of 4-chloro-5,7-dinitrobenz-2,1,3-oxadiazole In Flow Injection Analysis Of Substituted Hydrazines"
J. Anal. Chem. 1996 Volume 51, Issue 7 Pages 657-662
M. I. Evgen'ev, S. Yu. Garmonov, I. I. Evgen'eva, and G. K. Budnikov

Abstract: A 1:4 mixture of 30% TCA and the sample (blood plasma, urine or an aqueous solution of a drug) was centrifuged and the supernatant liquid was mixed with 0.5 mL of buffer solution (pH 5.5) and DMSO. The hydrazine derivative concentration was found by FIA with 20 mM 4-chloro-5,7-dinitrobenz-2,1,3-oxadiazole in acetonitrile as the reagent and detection at 510 nm. Beer's law was obeyed for ~0.1-2.5 µg/ml of common hydrazine derivatives. Ammonia, amines, amino-acids, NaCl, sodium phosphate and KCl did not interfere.
Hydrazine Spectrophotometry Interferences

"Improved FIA-ABTS Method For Antioxidant Capacity Determination In Different Biological Samples"
Free Rad. Res. 2004 Volume 38, Issue 8 Pages 831-838
Stefano Bompadre, Luciana Leone, Alessia Politi, Maurizio Battino

Abstract: In order to evaluate the actual antioxidant features of foods, beverages and also plasma from patients, a number of assays have been developed in the last few years to determine the so called total antioxidant activity (TAA), intended as the cumulative capacity of a biological sample to scavenge free radicals. Most of the assays partially failed in obtaining a good reproducibility when using plasma because it is composed of a large number of substances, some of which are present at very high concentrations and possess masking features. For these reasons we have improved the widely known ABTS method by means of a FIA system where both temperature and dispersion of sample and reagent were strictly controlled. We found that temperature may be a critical aspect in the measurement of plasma TAA whilst its influence may be less important in the assay of non-complex biological samples. We demonstrated that also the reaction time may be critical, depending on the nature of the substance employed. Data confirmed the high TAA of a methylsalicylate-containing mouthrinse as well as the negligible TAA offered by the chlorhexidine containing one. White wines (Verdicchio) also displayed interesting TAA values. The improved method was useful to screen rapidly, without dilution, with very limited handling of the sample and with high repeatability the TAA of plasma in addition to chemical products, beverages and non-complex biological mixtures.
Antioxidants