University of North Florida
Browse the Citations
-OR-

Contact Info

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

View Stuart Chalk's profile on LinkedIn

Plasma Human

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

Citations 45

"Determination Of Selenium Balance In Healthy Children By AAS-hydride Generation And By INAA Technique"
Acta Aliment. 2002 Volume 31, Issue 3 Pages 227-234
Adányi, N., Váradi, M., Sziklai-László, I., Snyder, P., Snyder, R. D., Cser, M. Á.

Abstract: Total daily Se intake was determined by duplicate diet collection, venous blood samples were taken and urine was collected over 24 h in order to measure selenium input and output in healthy, American and Hungarian children aged 8 to 17 living in Budapest. The American children consumed not only locally processed food. Food samples were weighed, mixed, homogenised and the Se content was determined by Instrumental neutron activation analysis (INAA). The Se concentration of blood, plasma and urine samples was determined by atomic absorption spectrometry-hydride generation (AAS-HG) after wet digestion. Se intake calculated for wet weight was 62±18.5 µg/day in American children. In the Hungarian children the mean Se intake was about 35% less than in the Americans. Se concentrations in plasma were 0.84±0.16, in whole blood 1.13±0.17 µmol L-1 in the Americans, higher than those in healthy Hungarian children (0.64±0.10 and 0.83±0.12 µmol L-1, respectively) of similar age and gender. Urinary Se output calculated for creatinin was higher in the children from abroad (27.0±9.5 µg Se/day/g creatinin) compared to Hungarians (11.0±5.0 µg Se/day/g creatinin).
Selenium Spectrophotometry Volatile generation

"Spectrophotometric And Spectrofluorimetric Determination Of Famotidine And Ranitidine Using 1,4-benzoquinone Reagent"
Anal. Lett. 1999 Volume 32, Issue 7 Pages 1403-1419
Abdel Kader S. Ahmad; M. Abdel Kawy; M. Nebsen

Abstract: Spectrophotometric and spectrofluorimetric methods were adopted for the analysis of Famotidine and Ranitidine depending on their reaction with 1,4 Benzoquinone reagent at pH 5.2 and 5.6, respectively. The absorbances of the resulting condensation products were measured at 502 and 508 nm for Famotidine and Ranitidine, respectively. Concentrations adhering to Beers law were from 40-160 µg mL-1 for Famotidine and from 20-100 µg mL-1 for Ranitidine. Furthermore the resulting condensation products exhibited fluorescence at 665 nm when excited at 290 nm and the calibration graphs were rectilinear from 0.4-1.4 µg mL-1 for Famotidine and from 0.21 µg mL-1 for Ranitidine. Different parameters affecting these reactions were thoroughly studied. Also these methods were applied to the pharmaceutical preparations and the results were satisfactory. The validities of the methods were ascertained by the standard addition technique revealing fine results in consideration to the mean recovery percent and standard deviation. The spectrofluorimetric method was a hundred times more sensitive then the spectrophotometric method. The proposed methods were sensitive, accurate, and precise as statistically compared with the official methods of analysis of Famotidine and Ranitidine.
Spectrophotometry Fluorescence Standard additions calibration Standard method Optimization

"Determination Of Flufenamic Acid And Mefenamic Acid In Pharmaceutical Preparations And Biological Fluids Using Flow Injection Analysis With Tris(2,2 -bipyridyl)ruthenium(II) Chemiluminescence Detection"
Anal. Chim. Acta 2000 Volume 416, Issue 1 Pages 87-96
Fatma A. Aly, Salma A. Al-Tamimi and Abdulrahman A. Alwarthan

Abstract: A novel chemiluminescent method using flow injection has been investigated for the rapid and sensitive determination of flufenamic and mefenamic acids. The method is based on a tris(2,2-bipyridyl)ruthenium(III) chemiluminescence reaction. Ru(bipy)(3)(3+) is chemically generated by mixing two streams containing solutions of tris(2,2-bipyridyl)ruthenium(II) and acidic cerium(IV) sulfate. After selecting the best operating parameters calibration graphs were obtained over the concentration ranges 0.07-6.0 and 0.05-6.0 µg mL-1 for flufenamic acid and mefenamic acid, respectively. The Limits of detection (s/n = 3) were 3.6 x 10^-9 M flufenamic acid and 2.1 x 10^-7 M mefenamic acid. The method was successfully applied to the determination of these compounds in pharmaceutical formulations and biological fluids. A proposal for the reaction pathway was given.
Flufenamic acid Mefenamic acid Chemiluminescence Method comparison Optimization

"Chemiluminescence Determination Of Catecholamines In Human Blood Plasma And Urine Using Diphenylethylenediamine As Pre-column Derivatization Reagent In Liquid Chromatography"
Anal. Chim. Acta 1994 Volume 298, Issue 3 Pages 431-438
Gamal H. Ragab, Hitoshi Nohta, Masaaki Kai and Yosuke Ohkura

Abstract: A chemiluminescence detection of the fluorescent derivatives of catecholamines (norepinephrine, epinephrine and dopamine) and isoproterenol as an internal standard is described for the highly sensitive liquid Chromatographic determination of these compounds. The amines were converted by reaction with 1,2-diphenylethylenediamine into the corresponding fluorescent derivatives, which were separated on a reversed-phase column (TSK gel ODS-120T) with isocratic elution. The derivatives in the column eluate were detected by the post-column chemiluminescence reaction system using bis[4-nitro-2-(3,6,9-trioxade-cyloxycarbonyl)phenyl] oxalate and hydrogen peroxide. The method allowed the determination of catecholamines in 50 µL of human blood plasma and 10 µL of 20 times diluted urine. The detection limits for the amines were 150-450 amol per 100 µL injection volume at a signal-to-noise ratio of 3.
Catecholamines Chemiluminescence Pre-column derivatization

"Determination Of Branched-chain L-amino-acids By Flow Injection Analysis With Co-immobilized Leucine Dehydrogenase/NADH Oxidase And Chemiluminescence Detection"
Anal. Chim. Acta 1995 Volume 311, Issue 1 Pages 71-76
Nobutoshi Kiba*, Akira Kato and Motohisa Furusawa

Abstract: An enzyme reactor column (5 cm x 4 mm i.d.) containing leucine dehydrogenase and NADH oxidase co-immobilized on to aminated poly(vinyl alcohol) beads was incorporated into a FIA system for the determination of branched-chain L-amino-acids (BCAA). Sample (20 µL) was injected into a 2 mM NAD+ carrier solution (0.15 ml/min) and merged with a stream of 2 mM luminol in 0.4 M carbonate buffer at pH 10 (0.15 ml/min). The flow passed through the enzyme reactor column at 40°C. The eluate from the reactor column was merged with a 10 mM potassium hexacyanoferrate stream (0.3 ml/min) before passing through a mixing coil (50 cm x 0.5 mm i.d.) to the detector flow cell (100 µL) where the chemiluminescence was measured. The calibration graph was linear for 0.5-600 µM-BCAA (based on L-isoleucine) and the detection limit was 0.3 µM. RSD (n = 7) was 0.78% at the 15 µM level. The method was applied to the determination of BCAA in human plasma. Plasma samples were diluted 10-fold with 0.1 M carbonate buffer at pH 10 prior to analysis. The within day and day-to-day coefficients of variation for the determination of 436 µM-BCAA in plasma were 0.87 and 1.6%, respectively. The recoveries of 546 µM-2.45 mM BCAA from plasma were 98-102%.
Amino acids, L Chemiluminescence Heated reaction Immobilized enzyme Poly vinyl alcohol beads

"Light Scattering-based Determination Of Fibrinogen In Human Plasma Using An Automated Continuous System"
Anal. Chim. Acta 1996 Volume 327, Issue 2 Pages 101-106
M. P. da Silva, J. M. Fernández-Romero and M. D. Luque de Castro*

Abstract: The FIA method was based on the precipitation of fibrinogen in the presence of ammonium sulfate and the detection of the suspended solid by light scattering with an incident wavelength of 340 nm. Plasma (180 µL) was injected into a 30 mM guanidine hydrochloride/60 mM titriplex III carrier stream (2.9 ml/min) at pH 4.9 which was merged with 88 mM ammonium sulfate (2.9 ml/min) in the same buffer. The flow was then passed through a reactor coil (50 cm x 0.5 mm i.d.) to the detector. The calibration graph was linear for 1-20 mg/l fibrinogen, the detection limit was 0.5 mg/l and the RSD were 1.33% for 5-12 mg/l fibrinogen. The sampling frequency was 80/h. The method was used to analyze 35 human blood samples and the results were confirmed by a nephelometric method. The recoveries of 5 and 12 mg/l fibrinogen from spiked plasma were 89-108%.
Fibrinogen Turbidimetry Spectrophotometry Precipitation

"Pulsed-amperometric Detection Of Urea In Blood Samples On A Conducting Polypyrrole-urease Biosensor"
Anal. Chim. Acta 1997 Volume 341, Issue 2-3 Pages 155-160
S. B. Adelojua*, S. J. Shaw and G. G. Wallace

Abstract: A biosensor for urea was fabricated by immobilizing urease into a conducting polypyrrole film deposited onto a Pt electrode by constant current polymerization. The biosensor was incorporated into a FIA system and used as a pulsed amperometric detector for determining urea in blood. Plasma samples were diluted 1:100 fold and 50 µL volumes were injected into a 0.05 M phosphate buffer carrier stream (0.1 ml/min, pH 7) and propelled through an anion-exchange column (not specified) to remove undesirable anions. The flow passed through the detection cell equipped with the biosensor, a Ag/AgCl reference electrode and a Pt auxiliary electrode. Urea was determined by applying pulsed potentials of -70 mV and -400 mV for 120 ms each. The calibration graph was linear for 0.1-4.5 mg/l urea, the detection limit was 0.06 mg/l and the RSD was 3% for >=6 mg/l urea. The recoveries of 0.6-6 mg/l urea from spiked plasma were 95-112%.
Urea Amperometry Sensor Ion exchange Immobilized enzyme

"Continuous-flow System For The Evaluation Of The Extrinsic Coagulation Pathway"
Talanta 1996 Volume 43, Issue 9 Pages 1531-1537
J. M. Fernández-Romeroa and M. D. Luque de Castroa,*

Abstract: A flow injection method was developed for monitoring the final step of the coagulation pathway by which human plasma is clotted in the presence of an excess of thromboplastin. Plasma (50 µL) and calcium thromboplastin (50 µL) were simultaneously injected into separate carrier streams (0.8 ml/min) of 10 mM 5,5-diethylbarbiturate buffer (pH 7.3), which were then were combined and passed through a reaction coil at 50°C. The flow was stopped when the reaction mixture entered the flow cell, and the rate of the clotting reaction was monitored spectrophotometrically at 340 nm for 90 s. The calibration graph of prothrombin activity (%) vs. coagulation rate was linear for prothrombin activities in the range 5-100%, and the RSD were 0.9-2.8% (n = 11) at the 20, 50 and 80% prothrombin activity levels. A linear relationship was also established between the coagulation reaction rate and the commonly used prothrombin time parameter. Results for prothrombin activity in 25 samples exhibited good correlation (r = 0.998) with those obtained by a nephelometric procedure (Rousseaux et al., Spectra Biol., 1988, 6, 53).
Spectrophotometry

"Determination Of Albumin With Bromocresol Purple Using Controlled-dispersion Flow Analysis"
Analyst 1985 Volume 110, Issue 6 Pages 669-671
Bernard F. Rocks, Scott M. Wartel, Roy A. Sherwood and Clifford Riley

Abstract: Albumin was determined in human blood plasma by controlled-dispersion analysis in which the consumption of bromocresol purple reagent (which also contained Brij 35 and acetate buffer of pH 4.9) was minimized with use of merging zones. Slugs of sample and reagent were transported by non-segmented water streams to a T-junction for mixing before feeding to a flow-through photometer. Each 30-s cycle consumed 2.4 µL of sample, 16.6 µL of reagent and <2 mL of water. Throughputs of 180 samples h-1 were achieved by introducing the succeeding sample before its predecessor was completely washed out. At fast flow rates, readings taken on the trailing edge of the peak were more reproducible than those taken at the maxima. The coefficient of variation were 0.8 and 1.5% for concentration. of 32 and 48 g l-1, respectively.
Albumin Spectrophotometry Dispersion Merging zones

"Chemiluminescence Determination Of Naltrexone Based On Potassium Permanganate Oxidation"
Analyst 1998 Volume 123, Issue 5 Pages 1053-1056
A. Campiglio

Abstract: A rapid method by chemiluminescence is described for the determination of naltrexone. The method is based on the chemiluminescence reaction with potassium permanganate in sulfuric acid medium. The optimum conditions for the chemiluminescence emission were investigated. With the integrated chemiluminescence intensity for 10 s after KMnO4 injection as a quantitative parameter, naltrexone can be determined over the concentration range 50-1000 ng mL-1 with a detection limit of 2.5 ng mL-1 and with a RSD (n = 10) of 0.9% and 0.5% at levels of 1000 and 100 ng mL-1, respectively. The method was applied to the determination of naltrexone in pharmaceutical preparations.
Naltrexone Chemiluminescence

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

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

"Competitive Heterogeneous Enzyme Immunoassay For Digoxin With Electrochemical Detection"
Anal. Chem. 1986 Volume 58, Issue 1 Pages 135-139
Kenneth R. Wehmeyer, H. Brain Halsall, William R. Heineman, Charlels P. Volle, and I Wen Chen

Abstract: Human plasma (375 µL) was placed simultaneously with 25 µL of an alkaline phosphatase-labelled digoxin solution in digoxin-specific antibody-coated reagent cuvettes. After incubation and washing procedures, 300 µL of enzyme substrate (phenyl phosphate) was added and the solution was further incubated at room temperature The phenol (generated by the enzyme) was detected amperometrically at +670 mV vs. silver - AgCl at a carbon-paste electrode after separation on a 5-cm ODS column. A detection limit of 50 pg mL-1 of digoxin was obtained in plasma samples. The feasibility and limitations of using flow injection analysis with electrochemical detection for determination of the enzyme product were also discussed. Good correlation was obtained between the proposed method and an RIA method.
Digoxin Amperometry HPLC Electrode Immunoassay Method comparison

"Non-linear Calibration Of Ion-selective Electrode Arrays For Flow Injection Analysis"
Anal. Chem. 1992 Volume 64, Issue 15 Pages 1721-1728
Robert J. Forster and Dermot Diamond

Abstract: An ion-selective electrode array, modelled by the Nikolskii - Eisenmann equation, was simplex-optimized for the simultaneous determination of Na, K and Ca by flow injection analysis. The array comprised three highly selective electrodes and a fourth, a multiple ionophore electrode, that responded selectively to the three cations, but to varying degrees. The response surface of each electrode in the array was determined by using mixed calibration solution, and the dependence of the model parameters on the analyte concentration. was studied. The advantages of the combination of selective and non-selective electrodes in the array compared with single-electrode measurements and sparingly selective electrode arrays are discussed. The determination of cations in real samples at low and high concentration. with coefficient of variation of 8 and 2.5%, respectively, is demonstrated by the analysis of mineral water and human plasma. The application of an ion-selective (ISE) array modeled via the Nikolskii-Eisenmann equation for the simultaneous determination of sodium, potassium, and calcium in flow injection analysis system is described. The array consists of three highly selective electrodes and a fourth sensor which responds selectively to the three cations but to differing degrees. The response surface of each electrode within the array is determined using mixed calibration solutions and this response modeled using simplex optimization. The dependence of the model parameters on analyte concentration. has been investigated. The kinetics of the selective response have been examined by modeling the potentiometric response of the array at short times following analyte injection and the application of this response for anal. investigated. The combination of sensors employed offers considerable advantage over existing single electrode measurements and arrays containing sparingly selective electrodes, such as prediction polling resulting in improved precision and accuracy, diagnosis of electrode performance without recalibration and accurate characterization of array response using simple models. The ability to determine sodium, potassium, and calcium in real samples at low and high concentrations. to within 8 and 2.5% error, respectively, is demonstrated using mineral water and human plasma samples.
Potassium Sodium Calcium Electrode Electrode Electrode Electrode Potentiometry Calibration Non-linear regression Kinetic Response surface Optimization Simplex

"Amperometric Determination Of Hydrogen Peroxide With A Manganese Dioxide Film-modified Screen Printed Carbon Electrode"
Fresenius J. Anal. Chem. 1998 Volume 362, Issue 2 Pages 194-200
Klemens Schachl, Hailemichael Alemu, K. Kalcher, Helmut Moderegger, Ivan Svancara, Karel Vytras

Abstract: A carbon thick film electrode modified with an MnO2-film was investigated as an amperometric detector for H2O2 in flow injection analysis (FIA). At an operating potential of +0.48 V vs. Ag/AgCl catalytic oxidation of the analyte was exploited for amperometric monitoring. Experimental parameters, such as pH of the carrier, working potential, flow rate, and injection volume were optimized. The amperometric signals were linearly proportional to the concentration. of H2O2 in the range from 0.005-10 mg/L, showing a detection limit (3s) of 2.3 µg/L. The method was applied to the determination of H2O2 in rainwater and to a simple assay to quantify glucose in human plasma.
Hydrogen peroxide Glucose Amperometry Electrode Electrode Electrode Sensor Apparatus Detector Optimization

"High Performance Liquid Chromatographic Determination Of Catecholamines And Their Precursors And Metabolites In Human Urine And Plasma By Post-column Derivatization Involving Chemical Oxidation Followed By Fluorescence Reaction"
Anal. Biochem. 1992 Volume 200, Issue 2 Pages 332-338
Hee-Kyoung Jeon, Hitoshi Nohta and Yosuke Ohkura

Abstract: Urine or plasma was mixed with isoproterenol and 3,4-dihydroxyphenylpropionic acid (internal standards) and HClO4. After centrifugation, the supernatant solution was adjusted to pH 1.5 to 2.0 with 2 M K carbonate and centrifuged. The supernatant solution was applied to a Toyopak IC-SP S cartridge, eluted with 1.5 M KCl in 100 mM HCl - methanol (93:7; for urine) or 2 M NaClO4 - methanol (93:7; for plasma). Eluates were analyzed by HPLC (cf. Anal. Sci., 1991, 7, 257). The calibration graphs were rectilinear for 2 to 1000 pmol of L-dopa and 0.2 to 200 pmol for other catecholamines. The coefficient of variation were 1.9 to 5.4 and 3.2 to 7.3% for urine and plasma, respectively. Detection limits were from 0.5 to 95 pmol mL-1.
Catecholamines HPLC Fluorescence Post-column derivatization

"Quantitation Of Toremifene And Its Major Metabolites In Human Plasma By High Performance Liquid Chromatography Following Fluorescent Activation"
Anal. Lett. 1987 Volume 20, Issue 6 Pages 871-879
Holleran, W.M.;Gharbo, S.A.;De Gregorio, M.W.

Abstract: Toremifene 2-[4-(4-chloro-1,2-diphenylbut-1-enyl)phenoxy]-NN-dimethylethylamine; I , and its major metabolites, N-demethyl-I and the 2,.yza.4-[4-chloro-1-(4-hydroxyphenyl)-2-phenylbut-1-enyl]phenoxy-analogue of I, were extracted from plasma containing nafoxidine (as internal standard) with butanol - hexane (1:49). The extract was evaporated, the residue was dissolved in methanol and the solution was irradiated for 2 min by using a 15-W Hg vapor lamp (254 nm) to form fluorescent phenanthrene forms of the analytes. A portion of the solution was applied to an Ultrasphere ODS column with aqueous 93% methanol containing 0.1% of triethylamine as mobile phase and fluorescence detection (excitation at 266 nm). Calibration graphs were rectilinear for up to 500 ng mL-1 for all analytes. The detection limits were 8, 15 and 5 ng mL-1 for I, N-demethyl-I and the other metabolite, respectively. The extraction efficiency for I was 88%.
Toremifene HPLC Fluorescence Post-column derivatization

"An Aromatic Phosphine Reagent For The HPLC - Fluorescence Determination Of Hydroperoxides. Determination Of Phosphatidylcholine Hydroperoxides In Human Plasma"
Anal. Lett. 1988 Volume 21, Issue 6 Pages 965-975
Akasaka, K.;Ohrui, H.;Meguro, H.

Abstract: A mixture of plasma (0.5 ml), methanol (0.5 ml) and CHCl3 (1 ml) containing 0.003% of 2,6-di-t-butyl-p-cresol (solvent A) was shaken vigorously and centrifuged at 1000 g for 10 min. The CHCl3 layer was separated, and the aqueous phase was extracted with solvent A (2 x 1 ml). The combined CHCl3 solution were evaporated, the residue was dissolved in solvent A (15 µL), and a 10 µL portion was analyzed by HPLC. A column (25 cm x 4.6 mm) of TSK-gel silica 60 was used, with CHCl3 - methanol - water (9:21:1) as mobile phase (0.6 mL min-1), and detection at 235 nm. The eluate was mixed (0.3 mL min-1) with a solution of 3 mg of diphenyl-1-pyrenylphosphine(I) in 400 mL of methanol - acetone (3:1), in a stainless-steel coil (10 m x 0.25 mm) at 70°C. The mixture was then passed through a 0.5-mm i.d. coil in a water bath at 20°C, and the resulting I oxide was detected fluorimetrically at 380 nm (excitation at 352 nm). The peak height ratio of phosphatidylcholine hydroperoxides to phosphatidylcholine (internal standard) was rectilinearly related to concentration. up to 60 pmol in 0.5 mL of plasma; the coefficient of variation (n = 5) was 5%.
Phosphatidylcholine HPLC Fluorescence Heated reaction Post-column derivatization

"Determination Of Codeine In Human Plasma And Drug Formulation Using A Chemically Modified Electrode"
Electroanalysis 1998 Volume 10, Issue 8 Pages 536-540
Jyh-Myng Zen*, Ming-Ren Chang, Hsieh-Hsun Chung, Ying Shih

Abstract: Both flow injection methodology and square-wave voltammetry were developed and evaluated for determining codeine in human blood plasma and pharmaceutical formulations using a Nafion/Ru oxide pyrochlore chemical modified electrode. Combining the electrocatalytic function of the Ru oxide pyrochlore with charge-exclusion and the pre-concentration features of Nafion perform well in codeine detection. Compared to a bare glassy C electrode, the chemical modified electrode exhibits a shift of the oxidation potential in cathodic direction and a marked enhancement of the current response. A linear calibration plot is obtained over the 0-32 µM range in 0.05 M HClO4 solution with a detection limit (3s) of 10 nM in the square-wave voltammetric method. While, in flow injection anal., a linear calibration plot is obtained over the 0.5-40 µM range with a detection limit of 0.86 ng. Quant. anal. was performed by the standard addition method for codeine content in human plasma and a commercial available drug.
Codeine Electrode Electrode Voltammetry Method comparison Apparatus Detector

"Online Electrochemical Reagent Production For Fluorescence Detection Of Phenothiazines In Liquid Chromatography"
J. Chromatogr. A 1986 Volume 354, Issue 1 Pages 249-257
W. Th. Kok, W. H. Voogt, U. A. Th. Brinkman and R. W. Frei

Abstract: Thioridazine(I) in human plasma was determined after deproteinization, but without an extraction step, on a column (25 cm x 4.6 mm) packed with LiChrosorb RP-8, with a mobile phase comprising aqueous 70% methanol containing 0.1 M LiNO3, 0.05 M tetraethylammonium perchlorate, 1 mM KBr and 0.1 mM EDTA, followed by post-column derivatization in which electrolytically generated Br was applied as an oxidant to enhance the sensitivity of fluorescence (excitation at 345 nm, detection at 425 nm). The detection limit was 0.5 ng or 5 ppb in plasma; reproducibility (n = 10) was 4.6% at 100 ppb of I. Recoveries of I at 20, 100 and 200-ppb levels were ~93, 101 and 95%. The described detection system was compared with other HPLC detection methods; its detection limits are no better than those obtained with other modes, but its selectivity is such that sample extraction is not necessary. The proposed system is compatible with sample concentration methods, e.g., pre-column techniques, and was applied in the determination of other phenothiazines.
Phenothiazines HPLC Fluorescence Electrochemical reagent generation Post-column derivatization

"Determination Of Citrulline And Homocitrulline By High Performance Liquid Chromatography With Post-column Derivatization"
J. Chromatogr. B 1990 Volume 497, Issue 1 Pages 37-43
Ichiro Koshiishi, Yumiko Kobori and Toshio Imanari

Abstract: For determination of citrulline (I) in plasma, samples were extracted with trichloroacetic acid and subjected to HPLC on a column (15 cm x 4 mm) of TSK gel SCX with 50 mM citrate buffer containing 0.3 mM NaCl as mobile phase (0.4 mL min-1). The post-column colorimetric reaction was carried out with phthalaldehyde and N-(1-naphthyl)ethylenediamine; detection was at 520 nm. For homocitrulline (I) in urine, acidified samples were applied to a column of Amberlite CG-120 (H+ form) cation exchange resin; II was eluted with 0.1 M Tris - HCl buffer and subjected to HPLC as described. Both I and II were well separated with no interference from protein amino-acids or urea. Calibration graphs were rectilinear in the ranges 2.6 to 500 and 4.3 to 500 µM, respectively. Results indicated that the level of I in the plasma of uremia patients is higher than that in healthy plasma, and that II is excreted into healthy human urine but not into plasma.
Citrulline Homocitrulline HPLC Post-column derivatization Buffer Calibration Interferences Amberlite

"Determination Of Thiamine And Its Phosphate Esters In Human And Rat Blood By High Performance Liquid Chromatography With Post-column Derivatization"
J. Chromatogr. A 1985 Volume 332, Issue 1 Pages 181-188
Mieko Kimura and Yoshinori Itokawa

Abstract: Blood, erythrocytes and plasma were deproteinized with aqueous 10% trichloroacetic acid. After centrifugation, the supernatant solution were analyzed by HPLC on a µBondapak C18 column (25 cm x 4 mm) with 0.2 M NaH2PO4 in aqueous 0.3% acetonitrile as the mobile phase (1.0 mL min-1); the eluates were subjected to post-column derivatization with 0.1% of K3Fe(CN)6 in aqueous 15% NaOH and spectrofluorimetric detection at 450 nm (excitation at 375 nm). The detection limit for thiamine and its phosphate esters was 30 fmol and recoveries were 95.0 to 100.2%. The calibration graphs were rectilinear for 50 to 200 fmol.
Thiamine Thiamine triphosphate Thiamine monophosphate HPLC Fluorescence Post-column derivatization

"Quantification Of Physiological Amino Acids By Gradient Ion-exchange High Performance Liquid Chromatography"
J. Chromatogr. B 1993 Volume 613, Issue 2 Pages 223-230
Svend Erik M&oslash;ller

Abstract: Human or rat plasma, human cerebrospinal fluid or 0.4 M HClO4 extracts of rat brain were mixed with sulfosalicylic acid and α-aminoadipic acid (internal standard) at 4°C, then filtered before analysis on a column (15 cm x 3 mm) of 5 µm Li cation-exchange material (sulfonated divinylbenzene-styrene copolymer) equipped with a guard column (2 cm x 3 mm) and maintained at 50°C. The mobile phase (0.2 and 0.3 ml/min) comprised a gradient (details given) of 0.64 M lithium citrate buffer (pH 7.50) in 0.24 M lithium citrate buffer (pH 2.27) and detection was by post-column phthalaldehyde derivatization and fluorimetry at 448 nm (excitation at 340 nm). Intra-assay RSD for 28 amino-acids present in human plasma were 3.4-17.6% and the inter-assay RSD were 2.7-39.0%. The response was rectilinear for 50-5000 pmol for 32 amino-acids, and up to 40 compounds could be determined in a 3-h run. A single-column gradient lithium ion-exchange chromatographic method with post-column derivatization and fluorimetric detection for the quantification of physiological amino acids is described. The method runs automatically, requires a minimum of sample preparation, separates all amino acids in plasma and cerebrospinal fluid, and most compounds in brain extract, in addition to some amino acids used therapeutically and in pharmacological studies. About 40 compounds can be quantitated within a run time of 3 h. The within-assay and between-assay coefficients of variations for principal amino acids in plasma samples are satisfactory. The system has performed conveniently and with high stability in the daily routine work and is cost-saving based on laboratory-prepared buffers.
Amino Acids HPLC Fluorescence Post-column derivatization

"Simple And Rapid Determination Of Clomiphene Cis And Trans Isomers In Human Plasma By High Performance Liquid Chromatography Using Online Post-column Photochemical Derivatization And Fluorescence Detection"
J. Chromatogr. B 1993 Volume 617, Issue 1 Pages 168-172
I. &Uuml;rm&ouml;s, S. M. Benk&ouml; and I. Klebovich

Abstract: A validated, sensitive and rapid high performance high performance liquid chromatographic method has been developed for the analysis of both cis and trans isomers of clomiphene in human plasma. The method involves a new off-line pre-column solid-phase extraction for sample preparation of clomiphene from human plasma in the presence of an internal standard. Analysis was performed by isocratic elution on a LiChrospher 100 RP-18 5-microns column using online post-column photochemical derivatization, with fluorescence detection at 247 nm (excitation) and 378 nm (emission). The limit of quantitation was 0.75 ng/ml for the cis isomer and 1.25 ng/ml for the trans isomer. The precision and accuracy of method were between good laboratory practice (GLP) required limits. Plasma containing 1,4-diethylamino-ethyoxy)phenyl-1,2-diphenylethanol (internal standard) was treated with 3 M NaCl and water and applied to a Bond Elut C18 column previously activated with methanol and water. Elution was effected with methanol, the eluate was evaporated to dryness under N at 50°C and the residue was dissolved in the mobile phase. A portion (20 µL) of the resulting solution was analyzed on a column (25 cm x 4 mm) of LiChrospher 100 RP-18 (5 µm) with acetonitrile/methanol/H2O/1% NH4Cl/1% K2CO3 (475:15:10:2:4) as mobile phase (1 ml/min) with post-column photochemical derivatization and fluorimetric detection at 378 nm (excitation at 247 nm). The calibration graph was linear for 2-2000 ng/ml of clomiphene with a limit of quantitation of 0.75 and 1.25 ng/ml for the cis and trans isomers, respectively. The limit of detection was 0.4 ng/ml. Recoveries were 75 and 62% for the cis and trans isomer, respectively. Inter- and intra-day RSD were 1.8-5% and 4.9-18.9% for the cis isomer; the corresponding values for the trans isomer were 2.6-4.7% and 3.5-9.4%
cis-Clomiphene trans-Clomiphene HPLC Fluorescence Photochemistry Post-column derivatization

"Determination Of Alpidem, An Imidazopyridine Anxiolytic, And Its Metabolites By Column-switching High Performance Liquid Chromatography With Fluorescence Detection"
J. Chromatogr. A 1994 Volume 668, Issue 2 Pages 403-411
L. Flaminio, M. Ripamonti and V. Ascalone*

Abstract: Alpidem, 6-chloro-2-(4-chlorophenyl)-N,N-dipropylimidazo<1,2-a>pyridine- 3-acetamide, is an anxiolytic imidazopyridine that undergoes a first-pass elimination after oral administration to humans; it is actively metabolized and three circulating metabolites have been identified in plasma due to N-dealkylation, oxidation or a combination of both processes. For the determination of the unchanged drug and its metabolites in human plasma, a column-switching HPLC method was developed. The method, based on solid-phase extraction (performed online), involves the automatic injection of plasma samples (200 µL) on to a pre-column filled with C18 material, clean-up of the sample with water in order to remove protein and salts and transfer of the analytes to the analytical column (after valve switching) by means of the mobile phase. All the processes were performed in the presence of an internal standard, a compound chemically related to alpidem. During the analytical chromatography, the pre-column was flushed with different solvents and after regeneration with water, it was ready for further injections. The analytical column was a C8 type and the mobile phase was acetonitrile-methanol-phosphate buffer solution (45:15:45, v/v/v) at a flow-rate of 1.5 mL min-1. The column was connected to a fluorimetric detector operating at excitation and emission wavelengths of 255 and 423 nm, respectively. The limits of quantitation of alpidem and three metabolites were 2.5 and 1.5 ng mL-1, respectively, in human plasma.
Alpidem HPLC Fluorescence

"Analysis Of 6-mercaptopurine In Human Plasma With A High Performance Liquid Chromatographic Method Including Post-column Derivatization And Fluorimetric Detection"
J. Chromatogr. B 1982 Volume 233, Issue 1 Pages 249-255
R. E. Jonkers, B. Oosterhuis, R. J. M. Ten Berge, C. J. Van Boxtel

Abstract: A relatively simple assay with improved reliability and sensitivity for measuring levels of 6-mercaptopurine in human plasma is presented. After extraction of the compound and the added internal standard with phenyl mercury acetate, samples were separated by ion-pair reversed-phase high-performance liquid chromatography. On-line the analytes were oxidized to fluorescent products and detected in a flow-fluorimeter. The within-day coefficient of variation was 3.8% at a concentration of 25 ng/ml. The lower detection limit was 2 ng/ml when 1.0 mL of plasma was used. Mercaptopurine concentration versus time curves of two subjects after a single oral dose of azathioprine are shown.
6-Mercaptopurine HPLC Fluorescence Post-column derivatization

"Improved High Performance Liquid Chromatographic Method For The Determination Of 6-N,N,N-trimethyl-lysine In Plasma And Urine: Biomedical Application Of Chromatographic Figures Of Merit And Amine Mobile Phase Modifiers"
J. Chromatogr. B 1986 Volume 380, Issue 2 Pages 285-299
Paul E. Minkler, Elizabeth A. Erdos, Stephen T. Ingalls, Ronda L. Griffin and Charles L. Hoppel

Abstract: Samples of blood and urine were collected from rats and human subjects; the urine samples were hydrolyzed with concentrated HCl at 105°C for 18 h. The hydrolysate or blood plasma samples were purified on successive columns (3.5 cm x 5 mm) of Dowex 50-X8 (NH4+ form) and Dowex 1-X8 (OH- form), respectively; elution was effected with aqueous 2.2 M NH3. The eluate was analyzed by HPLC on a cartridge (10 cm x 5 mm) of Radial-Pak C18 (10 µm) equipped with a pre-column (5 cm x 4 mm) of Co:Pell ODS; the mobile phase consisted of 2.5 mM Na dodecyl sulfate - 30 mM NaH2PO4 - 20 mM 3-(dimethylamino)propane-1,2-diol in acetonitrile. Post-column derivatization was carried out by treating the eluate with phthalaldehyde - 2-mercaptoethanol in 0.5 M H3BO3. Detection was by fluorimetry at 418 nm (excitation at 240 nm). The detection limit was 0.2 nmol mL-1 of analyte. The effect of pH of the mobile phase on the performance of the system was investigated.
6-N,N,N-Trimethyllysine HPLC Fluorescence Optimization Post-column derivatization

"Method For Determination Of Free And Total Glutathione And γ-glutamylcysteine Concentrations In Human Leucocytes And Plasma"
J. Chromatogr. B 1987 Volume 420, Issue 1 Pages 152-157
Johannes M&aring;rtensson

Abstract: Leucocytes and plasma were prepared as previously described for glutathione (Metabolism, 1986, 35, 118) except that cell lysis was performed for total glutathione(I) and γ-glutamylcysteine(II) analysis before incubation with dithiothreitol. After incubation with sulfosalicylic acid, dithiothreitol was removed from total I samples by ethyl acetate extraction. All solution were applied to a p-acetoxymercurianiline-Sepharose 4-B affinity column before elution with 25 mM mercaptoacetic acid in 50 mM HNO3 containing 1 mM Na2EDTA. HPLC was performed on a column (25 cm x 4.6 mm) of Spherisorb C18 (5 µm) with a guard column (25 cm x 4.6 mm) of Polygosil 60 C12 (25 to 40 µm). The mobile phase (1.2 mL min-1) was 5 mM Na heptanesulfonate (pH adjusted to 1.8 with H3PO4) containing 11% of methanol. Post-column derivatization with phthalaldehyde was performed before detection by measuring fluorescence at 420 nm (excitation at 350 nm). EDTA was added to all solution to prevent oxidation of the compounds. Recoveries were 98 and 96% for reduced and total I, respectively, and 96 and 92% for reduced and total II, respectively. The calibration graph was rectilinear from 20 pmol to 2 nmol of I and the detection limit was 4.5 pmol. The inter-assay coefficient of variation (n = 5) were 6.4 and 10.1% for I and II, respectively.
Glutathione γ-Glutamylcysteine HPLC Fluorescence Post-column derivatization

"Direct Determination Of Tamoxifen And Its Four Major Metabolites In Plasma Using Coupled-column High Performance Liquid Chromatography"
J. Chromatogr. B 1994 Volume 655, Issue 2 Pages 261-268
Karen M. Fried and Irving W. Wainer*

Abstract: Human plasma (150 µL) was mixed with 150 µL of acetonitrile, centrifuged and portions (50 µL) of the supernatant were loaded automatically onto a semi-permeable surface (SPS) CN guard column (1 cm x 4.6 mm i.d.). After washing with water, tamoxifen (I) and its metabolites were eluted (1 ml/min) with 35% acetonitrile in 20 mM potasium phosphate buffer of pH 3.1 (buffer A) and the eluate was analyzed on a Regis Rexchrom CN (5 µm) column (25 cm x 4.6 mm i.d.) with a similar guard column operated with buffer A as mobile phase (1 ml/min). Post-column derivatization using an ICT Beam Boost photochemical reactor with a 5 m reaction coil (Astec, Whippany, NJ, USA) was performed, giving phenanthrene derivatives which were detected fluorimetrically using a 370 nm cut-off filter (excitation at 250 nm). The calibration graphs for I, its N-desdimethyl- and N-desmethyl- derivatives and tamoxifen-ol were linear from 50-2000 ng/ml, and the calibration graph for 4-hydroxy-I was linear from 5-200 ng/ml; the corresponding detection limits were 5-8 ng/ml and 2 ng/ml. The RSD were 7% and the recoveries were 89% (84-88% for tamoxifen-ol).
Tamoxifen HPLC

"Determination Of The Renin Inhibitor Ro 42-5892 In Human Plasma By Automated Pre-column Derivatization, Reversed-phase High Performance Liquid Chromatographic Separation And Electrochemical Detection After Post-column Irradiation"
J. Chromatogr. B 1995 Volume 665, Issue 2 Pages 373-381
J. Leube* and G. Fischer

Abstract: The renin inhibitor Ro 42-5892 has been found to be very potent, thereby necessitating a sensitive assay method for the evaluation of its pharmacokinetics in man. We report here the development of a very sensitive and selective HPLC assay for the analysis of this compound in human plasma. Ro 42-5892 was extracted from plasma with dichloromethane, derivatized with 2,4-dinitrofluorobenzene and then chromatographed on a Novapak C18 column (150 x 3.9 mm I.D.) with acetic acid buffer (pH 7)-acetonitrile (100:85). Detection was performed by irradiation at 254 nm, followed by electrochemical oxidation at 550 mV. The extraction recovery of Ro 42-5892 from human plasma (mean 102%) was quantitative. With this method a limit of quantitation of 0.3 ng/ml was achieved. The assay was linear up to 5 ng/ml, had acceptable inter-assay precision (12.2%) and accuracy (9.3%) and was successfully tested for selectivity. This assay was successfully applied to over 250 samples from a pharmacokinetic study in hypertensive patients.
Renin, inhibitor HPLC Electrode Pre-column derivatization Post-column derivatization

"Determination Of Nifedipine In Human Plasma By Flow Injection Tandem Mass Spectrometry"
J. Chromatogr. B 1998 Volume 710, Issue 1-2 Pages 115-120
Jan Dankers*, Jos van den Elshout, Gertrude Ahr, Erich Brendel and Cees van der Heiden

Abstract: For use in clinical studies, a fast and sensitive assay method was developed for the determination of nifedipine in human plasma samples. The assay method is based on tandem mass spectrometry detection (HPLC-MS-MS). The effect of flow injection as well as HPLC separation on the results of the nifedipine determination were evaluated. The limit of quantification is 0.5 ng/mL and the accuracy (as determined by spiking recovery) was found to be good.
Nifedipine Mass spectrometry HPLC Method comparison

"Determination Of Drugs In Biosamples At Picomolar Concentrations Using Competitive ELISA With Electrochemical Detection: Application To Steroids"
J. Pharm. Biomed. Anal. 1993 Volume 11, Issue 6 Pages 459-467
Karin Kronkvist, Ulf L&ouml;vgren, Lars-Erik Edholm and Gillis Johansson

Abstract: Plasma (2.5 ml) containing budesonide was centrifuged and pre-treated on a solid-phase extraction C18 Bond-Elut column with 35% ethyl acetate in heptane as mobile phase. After drying, sample was dissolved in 350 µL of 5 mM formic acid containing 25% ethanol and 300 µL of the solution was injected onto a column (5 cm x 2.1 mm) of C18 Nucleosil (3 µm) fitted with a Guard-Pak Resolve silica column, with ammonium acetate buffer solution containing 35% methanol as mobile phase (0.5 ml/min). Microtitre plates were coated with 100 µL of purified sheep anti-budesonide IgG (diluted 1:5000 in acetate buffer, pH 5) and incubated overnight at room temperature After blocking with BSA/Tween 20, 200 µL of enzyme-conjugated steroid (diluted 1:10 000) and sample were added and incubated overnight at room temp., then 150 µL of 2 mM p-aminophenyl phosphate was added with incubation for 1 h in the dark. p-Aminophenol was determined by injection into a FIA system with ECD at a vitreous carbon electrode at 250 mV vs. Ag/AgCl. The linear range for plasma spiked with budesonide was 10 pM (detection limit) to 100 pM with a RSD (n = 6) of 27.8-43.4%. A competitive ELISA with electrochemical detection in a flow injection system (FIA) has been developed for determinations of the steroid drug budesonide in biological samples. Plasma samples were cleaned from interfering and cross-reacting compounds by two pretreatment steps consisting of a solid-phase extraction and a liquid chromatography fractionation. The enzyme label was alkaline phosphatase, which was used with p-aminophenyl phosphate (PAPP) as a substrate. The product, p- aminophenol, was detected electrochemically at a glassy carbon electrode at 250 mV (vs Ag/AgCl). The limited stability of both the substrate and the product influenced the performance of the method and had to be taken into account in the procedure by a normalization with time. Budesonide could be quantified in plasma samples down to 10 pM. The major sensitivity-limiting factor was the amperometric background response, probably due to spontaneous hydrolysis of PAPP to p- aminophenol.
Drugs Steroids 4-Aminophenol Budesonide Amperometry Electrode Interferences

"Determination Of Glucose By Flow Injection Analysis With Merging-zone Technique"
Anal. Sci. 1987 Volume 3, Issue 2 Pages 181-183
K. UCHIDA, D. YOSHIZAWA, M. TOMODA and S. SAITO

Abstract: The sample is injected into a flow stream of phosphate buffer (pH 6.95) containing 0.15 M NaCl and 6% of Triton X-100. An Iatrochrome GLU-Lq enzyme kit (Iatron Laboratories) reagent containing aldose 1-epimerase (1.6 iu mL-1), glucose oxidase (17 µiu mL-1), peroxidase (5.6 miu mL-1) and 1 mM aminoantipyrine is injected into a reagent stream containing the same liquid carrier. The flow rates for the sample and reagent streams are 0.5 and 2.0 mL min-1, respectively. The flow streams are mixed in a reaction coil at 45°C and the eluate is monitored at 580 nm. The calibration graph is rectilinear for up to 180 mg L-1 of glucose (r = 0.9998); recovery is >95% and the coefficient of variation (n = 3) is <1%. The method is applicable for the determination of glucose in human plasma. Interference caused by ascorbic acid is eliminated by addition of L-ascorbate oxidase.
Glucose Clinical analysis Spectrophotometry Merging zones Heated reaction Interferences Triton X Surfactant

"A Sensitive And Rapid FIA With An Immobilized Enzyme Column Reactor And Peroxyoxalate Chemiluminescence Detection For The Determination Of Total D-amino Acids In Human Plasma"
Anal. Sci. 1997 Volume 13, Issue 6 Pages 945-950
M. WADA, N. KURODA, S. AKIYAMA and K. NAKASHIMA

Abstract: The immobilized enzyme column reactor (IMER) (7 cm x 2 mm i.d.) was prepared by the glutaraldehyde method and featured aminopropyl-controlled pore-glass. The analysis is based on a specific enzyme reaction with D-amino acid oxidase and peroxyoxalate chemiluminescence detection with bis(2,4,6-trichlorophenyl) oxalate and 2,4,6,8-tetrathiomorpholinopyrimido[5,4-d]pyrimidine in acetonitrile as chemiluminescence (CL) reagent. Plasma samples (20 l) spiked with 5 l of a known concentration of standard D-amino acids were added to ethanol (1 ml). After centrifugation at 2000 g for 6 min, 800 l of supernatant was transferred to a phial and evaporated to dryness. The residue was dissolved in 200 l carrier solution (20 mM imidazole-HNO3 buffer) and a 10 l portion was injected into the FIA system. Flow rates were 0.5 ml/min for carrier and 1 ml/min for CL reagent. Eleven types of D-amino acids could be detected quantitatively, with detection limits in the range 0.4-30 pmol/10 l injection. Results were in satisfactory agreement with those obtained by a colorimetric method. Five samples could be analyzed within 10 min.
Amino acids, D Chemiluminescence Immobilized enzyme Reactor

"Galactose Biosensors Using Composite Polymers To Prevent Interferences"
Biosens. Bioelectron. 1995 Volume 10, Issue 3-4 Pages 359-370
Paul Manowitz, Paul W. Stoecker and Alexander M. Yacynych

Abstract: A biosensor using a composite polymer to prevent interferences was used in a flow injection analysis system for the detection of galactose in human plasma. The biosensor consisted of galactose oxidase immobilized on a platinized carbon electrode that had been modified with a composite polymer. The composite polymer showed improved selectivity to hydrogen peroxide compared with either of its individual polymeric components, Nafion and a copolymer of diaminobenzene and resorcinol. The composite polymer minimized the effect of possible interference from urate, ascorbate, and acetaminophen. This analytical system had a minimum detection limit of 50 µM, linearity to 6 mM, a storage stability of greater than 30 days, and a high sample throughput (~120 samples/h).
Galactose Electrode Electrode Sensor Interferences

"Enzyme-epoxy Membrane Based Glucose Analysing System And Medical Applications"
Biosens. Bioelectron. 1996 Volume 11, Issue 8 Pages 735-742
Mana Sriyudthsak, Tara Cholapranee, Montri SawadsaringkarnNapaporn Yupongchaey and Penprapa Jaiwang

Abstract: A semi-automated FIA system for glucose determinations employed a thin-film glucose sensor fabricated by coating an epoxy resin containing glucose oxidase and ferrocene (electron mediator) on to a Ti/Pt electrode (area 7.2 mm2, thickness ~1000 angstrom). The sensor was operated at a potential bias of 200 mV with 1-10 mM phosphate buffer of pH 7.2 as the carrier stream (0.8-3 ml/min) and an injection volume of 20 µL. Glucose concentrations of 50-4000 mg/dl were measured and a linear response was obtained up to 400 mg/dl. The RSD (n = 30) for 500 mg/dl at 3 ml/min was ~3%. The sampling frequency was 60 samples/h. No interference was observed from sucrose, lactose, maltose, ascorbic acid and uric acid. The system was used to determine glucose in blood plasma and the results were correlated with those obtained by a commercial automatic machine (Olympus model Reply).
Glucose Sensor Interferences

"Serotonin And 5-hydroxyindole-3-acetic Acid In Human Plasma And Rat Brain Determined By Liquid Chromatography With Post-column Derivatization And Fluorescence Detection"
Clin. Chem. 1993 Volume 39, Issue 11 Pages 2355-2356
Junichi Ishida, Ryuji Iizuka and Masatoshi Yamaguchi

Abstract: Plasma or rat brain homogenate (5-hydroxyindole-3-acetamide as internal standard) was deproteinized with HClO4 and analyzed by HPLC [cf. Analyst (London), 1993, 118, 165] with post-column derivatization with benzylamine in the presence of K3Fe(CN)6 in weakly alkaline medium and fluorescence detection of the oxazole derivatives at 480 nm (excitation at 345 nm). Response was linear up to 500 pmol injected. Detection limits were 1.6 nM-5-hydroxytryptamine (serotonin) and 3.3 nM-5-hydroxyindole-3-acetic acid. Recovery was 67.7-95.0% and within-day RSD (n = 10) were 2.2-3.9%.
Serotonin 5-Hydroxyindole-3-acetic acid HPLC Fluorescence Clinical analysis Post-column derivatization Letter

"The Utilization Of Intravenously Infused Pyridoxine In Humans"
Clin. Chim. Acta 1994 Volume 229, Issue 1-2 Pages 27-36
Janos Zempleni* and Werner K&uuml;bler

Abstract: Plasma (70 µL) was mixed with 15 µL of 10 M HCl and 500 µL was transferred onto a membrane and centrifuged at 4°C for 35 min at 17 000 g. Before analysis the sample was mixed with 0.1 M sodium acetate of pH 3.8 (1:1). Washed erythrocytes (300 µL) were mixed with 300 µL of 0.1 M sodium acetate buffer of pH 3.8. Trichloroacetic acid (200 µL, 20%) was added with centrifugation for 5 min at 12 000 g then 350 µL of the supernatant was mixed with 700 µL of sodium acetate buffer. A 100 µL portion was analyzed on a 5 µm RP 18 column (12.5 cm x 4 mm i.d.) equipped with a pre-column (4 x 4 min i.d.) with gradient elution (1.5 ml/min) with 0.03 M KH2PO4/4 mM octanesulfonic acid, pH 2.7/methanol (details given) and post-column derivatization with 0.5 M K2HPO4 and 10 µL/ml of 37% NaHSO3 (pH 7.5, flow rate 0.07 ml/min). Detection was at 400 nm (excitation at 330 nm). The method allowed the determination of 4-pyridoxic acid, pyridoxal, pyridoxal 5'-phosphate, pyridoxamine, pyridoxamine 5'-phosphate and pyridoxine. Intra- and inter-run RSD (n = 10) were 2.8-7.2 and 6.2-10.3%, respectively. Detection limits were 10 nM and recoveries were 94.2-96.5%.
Pyridoxal Pyridoxal phosphate Pyridoxamine 4-Pyridoxic acid Pyridoxine HPLC Clinical analysis

"Quantitation Of Circulating Hydroxyvitamin D-3 In Human Plasma By A Continuous Cleanup/concentration Procedure Prior To HPLC-UV Detection"
Clin. Chim. Acta 1998 Volume 274, Issue 2 Pages 139-149
F. Ortiz-Boyer, J. M. Fern&aacute;ndez-Romero, M. D. Luque de Castro* and J. M. Quesada

Abstract: A method for the determination of hydroxyvitamin D-3 metabolites (25-hydroxyvitamin D-3, 24,25-dihydroxyvitamin D-3 and 1,25-didydroxyvitamin D-3) based on a continuous cleanup/pre-concentration procedure coupled with HPLC and UV-detection is reported here. The method exhibits a linear range between 0.05 and 100 ng/mL (r2 = 0.9917) with CV values lower than 6.5%, and has been checked by applying it to plasma samples from a hospital with acceptable recoveries. The results compare well with those obtained by routine radioimmunoassay (y = 2.784±1.37 + 0.333±0.05 σ(yx), r = 0.8233, n = 19 for 25-hydroxyvitamin D-3). The sampling frequency was 4 h-1; 12 analytes h-1.
25-Hydroxyvitamin D3 HPLC Spectrophotometry Preconcentration Method comparison Sample pretreatment

"Determination Of Vitamin B6 In Foods And Other Biological Materials By Paired-ion High Performance Liquid Chromatography"
J. Agric. Food Chem. 1985 Volume 33, Issue 3 Pages 359-363
Jesse F. Gregory and Debra Feldstein

Abstract: Food, human plasma or milk, or rat liver or muscle samples, were homogenized in sulfosalicylic acid with 4'-deoxypyridoxine (as internal standard) and CH2Cl2. After centrifugation, the aqueous phase was applied to a column of Bio-Rad AG2-X8 anion-exchange resin (200 to 400 mesh; Cl- form) with 0.1 M HCl as mobile phase. The fraction containing pyridoxine was subjected to HPLC on a column (3 cm x 4.6 mm) of octadecylsilica (3 µm) with gradient elution with 0 to 2.5% of propan-2-ol in 33 mM potassium phosphate - 8 mM octanesulfonic acid (pH 2.2). Post-column derivatization was with NaHSO3 in 1 M sodium phosphate buffer (pH 7.5) and fluorescence detection was at 400 nm (excitation at 330 nm). The coefficient of variation were <2%; recoveries were 78.7 to 103.2%. Good correlation with published data was achieved.
Vitamin B6 Pyridoxine HPLC Ion exchange Fluorescence Post-column derivatization

"Determination Of Some β-endorphin Fragments In Human Plasma By High Performance Liquid Chromatography With Laser-induced Fluorescence Detection"
J. Controlled Release 1990 Volume 13, Issue 2 Pages 129-139
C. M. B. van den Beld**, U. R. Tjaden*, N. J. Reinhoud, D. S. Stegehuis and J. van der Greef

Abstract: Plasma was deproteinized with trichloroacetic acid and applied to a column of Sephadex G-50. The appropriate fraction was concentrated on to a pre-column of Amberlite XAD-2 before analysis by HPLC on a column (10 cm x 3 mm) of C18 material with a mobile phase (0.75 mL min-1) of 0.01 M Na phosphate buffer (pH 2.4) - acetonitrile - 21.5 mM Na 1-octanesulfonate (763:232:5). The eluate was derivatized with 1.65 mM o-phthaldialdehyde - 9.25 mM 2-mercaptoethanol in 0.05 M borate buffer solution (pH 9.4) for 10 s before laser-induced fluorescence detection at 450 or 515 nm (excitation at 351.1, 363.8 or 488 nm). The calibration graph was rectilinear for 1 to 100 ng of des-enkephaline-γ-endorphin; the limit of detection was 0.4 ng mL-1. The method was more senstive than a method involving pre-column derviatization with fluorescein-5-isothiocyanate (described).
β-Endorphin (6-17) HPLC Fluorescence Buffer pH Post-column derivatization Detection limit Calibration

"Fluorimetric Flow Injection Analysis For Inosine In Human Plasma Using Multi-connected Immobilized Enzyme Columns"
J. Flow Injection Anal. 1985 Volume 2, Issue 1 Pages 50-59
Kiyoshi ZAITSU, Yohji AYASHI and Yosuke OHKURA

Abstract: Flow injection analysis for inosine in human plasma with fluorescence detection was detected. Multi-connected immobilized enzyme columns (purine nucleoside phosphorylase, xanthine oxidase, uricase and horseradish peroxidase were chemically bonded on aminopropy1 glass beads (120-200 mesh), respectively, and these immobilized enzymes were packed in Teflon tubes (40 X 0.86 mm ID), then connected (in the above order) were placed on a flow line. Hydrogen peroxide formed in the enzymatic degradation of inosine through allantoin was measured fluorimetricaly by using the peroxidase substrate, 3-(p-hydroxyphenyl) propionic acid. The limit of determination for inosine was 0.5 pmol/20 µL injection volume.
Inosine Fluorescence Immobilized enzyme

"Fluorescence Detection Of Mexiletine And Its P-hydroxylated And Hydroxymethylated Metabolites In Human Plasma And Urine By High Performance Liquid Chromatography Using Post-column Derivatization With O-phthalaldehyde"
J. Liq. Chromatogr. Relat. Technol. 1994 Volume 17, Issue 3 Pages 659-671
T. Tateishi; K. Harada; A. Ebihara

Abstract: The cited determination was conducted on a Wakosil ODS column (25 cm x 4.6 mm i.d.) with a guard column (1 cm x 4.6 mm i.d.) of the same material, gradient elution (1 ml/min) with 10 mM H3PO4/methanol/1.4 mM 1-octanesulfonic acid (details given) and post-column reaction with o-phthalaldehyde for fluorimetric detection at 445 nm (excitation at 345 nm). The limits of detection were 2 ng/ml for mexiletine and p-hydroxymexiletine and 5 ng/ml for hydroxymethylmexiletine and calibration graphs were linear for 0.01-1 µg/ml for the three analytes. Inter- and intra-assay RSD are tabulated. The method was used to determine mexiletine and the two metabolites in plasma and urine after extraction with ethyl ether; urine was subjected to enzymatic hydrolysis before extraction. 4-Methylmexiletine was used as internal standard.
Mexiletine p-Hydroxymexiletine 6-Hydroxymethylmexiletine Fluorescence HPLC Post-column derivatization

"A Simplified High Performance Liquid Chromatographic Method For Direct Determination Of Warfarin Enantiomers And Their Protein Binding In Stroke Patients"
Ther. Drug Monit. 1994 Volume 16, Issue 5 Pages 509-512
Cai, W.M.;Hatton, J.;Pettigrew, L.C.;Dempsey, R.J.;Chandler, M.H.

Abstract: A simplified method for direct determination of warfarin enantiomers by high-pressure liquid chromatography with fluorescence detection has been developed. This method involves solid phase extraction of warfarin in plasma, pre-column derivatization to form diastereoisomeric esters, and post-column reaction to discriminate each enantiomer separately. Ultrafiltration was employed in the separation of unbound warfarin enantiomers. Twelve plasma samples from six stroke patients taking warfarin regularly were analyzed. The average concentration of total warfarin was 0.47±0.17 mg/L for the S-isomer and 0.69±0.18 mg/L for the R-isomer. The average protein binding was 99.67±0.33% for S-warfarin and 99.44±0.33% for R-warfarin. This methodology provides a quick and reliable technique for determining enantiomeric protein binding of warfarin in clinical settings.
Warfarin Protein HPLC Fluorescence Pre-column derivatization Post-column derivatization

"Chromium (VI) Reducing Capacity Of Ascorbic Acid And Of Human Plasma In Vitro"
Arch. Toxicol. 1992 Volume 66, Issue 1 Pages 45-50
Michaela Capellmann and Hermann M. Bolt

Abstract: In the metabolism of chromium(VI) its reduction in human plasma is of importance; an extracellular reduction of Cr(VI) is regarded as a detoxification step. Ascorbic acid has been suggested to represent the majority of the Cr(VI)-reducing capacity of human plasma. Therefore the kinetics of the reaction of Cr(VI) with ascorbic acid, at biologically realistic concentrations were studied. Ascorbic acid, in 0.2 M HEPES buffer and at concentrations ranging from 14.2 to 113.6 nmol mL-1 (2.5-20.0 µg mL-1), was mixed with Cr(VI) (0.4-1.5 nmol mL-1) and incubated at pH 7.4 and 37°C. In addition, chromate solutions at different concentrations [1.5-100 nmol mL-1 Cr(VI)], were incubated at 37°C with freshly drawn blood. From these incubates, ascorbic acid and its oxidized form, dehydroascorbic acid, were simultaneously analyzed by HPLC and post-column derivatization. Chromate was determined by flow injection analysis. The reaction kinetics of ascorbic acid in HEPES buffer with Cr(VI) is of pseudo-first order at higher concentrations, whilst apparently at lower concentrations kinetics are consistent with an autocatalyzed reaction. Results obtained after spiking human plasma are similar. However, when Cr(VI) was reacted with human plasma, no changes in the intrinsic contents of ascorbic acid of the plasma samples occurred. Also, comparing different plasma samples the intrinsic plasma contents of ascorbic acid and the reduction capacities for Cr(VI) [ranging between 0.48 and 0.63 nmol mL-1 Cr(VI) to be reduced] did not correlate. This shows that the reduction of Cr(VI) in native human plasma is complex and is not only determined by the plasma ascorbic acid levels. This is in contrast to the situation in lung lavage fluids (Suzuki 1988; Suzuki and Fukuda 1990) where the concentrations of ascorbic acid are much higher than in blood.
Chromium(VI)

"Rapid Tandem Mass Spectrometric Method For Determination Of Gabapentin In Human Plasma"
Chromatographia 2005 Volume 61, Issue 9-10 Pages 499-504
K. M. Matar, M. E. Abdel-Hamid

Abstract: A rapid tandem mass spectrometric (MS-MS) method for the quantification of gabapentin (GBP) in human plasma using 4-phenyl-4-aminobutanoic acid as an internal standard (IS) has been developed and validated. The drug and the internal standard were analyzed, by flow injection analysis without chromatographic separation, using a mobile phase of acetonitrile-water-formic acid (50:50:0.025, v/v/v) at a flow rate of 0.1 mL min-1. The run-cycle time was <3 min injection-to injection. Quantitation was achieved using multiple reaction monitoring (MRM) scan at MRM transitions m/z 172 > 154 and m/z 180 > 117 for GBP and the IS, respectively. Ion suppression study indicated practically no suppressive effect of plasma constituents on the mass ions detection of GBP and IS, when measured in MRM scanning mode. Calibration curves were linear over the concentration range of 0.1-10 µg mL-1 (r > 0.999) with a limit of quantification of 0.1 µg mL-1 (RSD%; 7.6 and % DEVs; -3.0 to +17.0%). Validation data showed that the RSD% values were in the range of 1.85 to 13.06%, whereas, the % DEVs values ranged from -1.4 to +10.0% indicating good precision and accuracy. Analytical recoveries of GBP from spiked human plasma were in the range of 98.9 to 101.3%. On the other hand, recoveries of GBP from stored human plasma samples were in the range of 100.0 to 107.5% indicating that GBP was stable in plasma, with no appreciable degradation, when stored at -20°C. The developed method was applied for GBP monitoring in plasma samples of patients treated with GBP.
Gabapentin Mass spectrometry Interferences