Masatoki Katayama

Simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography with fluorescence derivatization

A sensitive method was developed for the simultaneous determination of six adenyl purines in human plasma by high-performance liquid chromatography. The adenyl purines (adenine, adenosine, AMP, ADP, ATP and cyclic AMP) were derivatized using 2-chloroacetaldehyde for fluorescence detection, and the reaction and separation conditions were reinvestigated to improve sensitivity for small volume sample analysis. Each derivatized purine was separated on a Capcell Pack SG120A column with mobile phase consisting of 0.05 M citric acid-0.1 M dipotassium hydrogen phosphate (pH 4.0)-methanol (97+3). The detection limits were 100-1000 fmol/ml by fluorescence detection, some 500 times better than previous reports. The proposed method was applied to determine adenyl purines in human plasma. The purine levels were as follows: ATP (9.2-22.2 pmol/ml), ADP (5.5-22.2 pmol/ml), AMP (0.8-3.2 pmol/ml). Other purines, adenine, adenosine, cAMP were lower than 0.1 pmol/ml.

Determination of amines by flow-injection analysis based on aryl oxalate-Sulphorhodamine 101 chemiluminescence

Abstract A flow-injection method for amines based on aryl oxalate-Sulphorhodamine 101 chemiluminescence is described. Fifty-five aliphatic, aromatic and heterocyclic amines were studied by the proposed method without a derivatization reaction or expensive apparatus. Twenty-eight amines were detected at levels of 1.0 × 10 −10 –4.0 × 10 −6 M (signal-to-noise ratio = 3, 20-μl injection) with linear calibration up to 1.0 × 10 −4 –1.0 × 10 −3 M, respectively. The relative standard deviation ( n = 6) was 4.8% at 1.0 × 10 −6 M triethylamine. The proposed method was applied to the determination fo histamine in fish and compared favourably with a liquid chromatographic method using fluorescence detection after derivatization.

Determination of estrogens in plasma by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole.

2-(4-Carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) was used as a pre-column derivatization reagent for corticosteroids analysed by high-performance liquid chromatography with fluorimetric detection. Eight corticosteroids were derivatized with CDB to their esters in acetonitrile containing 4-piperidinopyridine and 1-isopropyl-3-(3-dimethylaminopropryl)carbodiimide perchlorate. The resulting CDB esters were extracted with a Sep-Pak C18 cartridge and the esters were separated on a reversed-phase column (Zorbax ODS) with water-methanol (25:75, v/v) containing 5 mmol/l tetramethylammonium hydrogensulphate as the mobile phase. The limits of detection for steroids were 0.06-0.3 pg per 100 microliters of plasma (signal-to-noise = 3). The within-day relative standard deviations (n = 6) were 7.8-11.1%, and day-to-day relative standard deviations (n = 6) were 7.0-10.4%.

Determination of progesterone and 17-hydroxyprogesterone by high performance liquid chromatography after pre-column derivatization with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a- diaza-s-indacene-3-propionohydrazide

Progesterone, 17-hydroxyprogesterone and four other 3-keto steroids were determined by high performance liquid chromatography with fluorescence detection. Each steroid was derivatized with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene++ +-3- propionohydrazide (BODIPY FL hydrazide) and separated on a Wakosil 5C4 column with acetonitrile-water (7 + 3) as mobile phase. The limits of detection of progesterone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone and 17-methyltestosterone were 550-3700 fmol per 10 microliters injection (signal-to-noise ratio = 5) serum. The calibration curves were linear up to 1000 ng/ml serum. The proposed method was most sensitive among the available high performance liquid chromatographic methods after fluorescence and chemiluminescence pre-labeling with dansylhydrazine.

Determination of alcohols by high-performance liquid chromatography after pre-column derivatization with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole

Abstract A sensitive method for the determination of fatty alcohols using high-performance liquid chromatography with fluorescence detection has been developed. The alcohols were derivatized with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole to their esters in the presence of 4-piperidinopyridine and 1-isopropyl-3-(3-dimethylaminopropyl)carbodiimide perchlorate. The resulting esters were extracted with a Sep-Pak ODS cartridge, and then the esters were separated on a reversed-phase column (Zorbax ODS) with methanolpropan-2-ol (85:15 v/v) as the mobile phase. The esters were detected by fluorescence spectrophotometry (excitation 338 nm, emission 428 nm). The limits of detections for alcohols were 0.2–0.4 pg per 20 μl (signal-to-noise ratio of 3) in an acetonitrile solution.

Determination of β-blockers by high performance liquid chromatography coupled with solid phase microextraction from urine and plasma samples

The solid phase microextraction (SPME) method was applied to drug monitoring of β-blockers in human urine and plasma. Six β-blockers (alprenolol, atenolol, oxprenolol, pindolol, β-propranolol, and timolol) were extracted from urine and plasma samples by SPME fiber coated with 85-μm polyacrylate for 2 h at 30°C. The β-blockers extracted were separated by Capcell PackTM SG120 (4.6 × 150 mm, 5 μm) with 0.05 M phospholic acid-methanol (55 + 45, v/v) and detected by photometric detection (280 nm) and fluorometric detection (excitation wavelength 220 nm, emission wavelength at 360 nm). The recoveries of β-blockers (500 μg/ml) from urine were 84.3 to 88.4%, and from plasma they were 86.4 to 90.2%. The detection limits were 2 to 25 μg/ml by the photometric detection, 0.4 μg/ml of β-propranolol by fluorescence detection. The relative standard deviations were 10.1 to 15.3% for 500 μg/ml β-blockers in urine, 9.9 to 13.2% for 5 μg/ml of β-blockers in plasma, respectively.

Improvement of sensitivity and selectivity of high-performance liquid chromatography for anti-retroviral drugs (non-reverse transcriptase inhibitors) by diamond-electrode electrochemical and fluorescence detection

The new non-reversed transcriptase inhibitor (NRTI) drugs for treatment of acquired immunodeficiency syndrome (AIDS) are reported. An improvement in the sensitivity and selectivity of high-performance liquid chromatography was obtained by diamond electrode-electrochemical detector and fluorescence detector owing to different structural information. The four anti-retroviral NRTI drugs (abacavir, didanosine, lamivudine and zidovudine) were separated on a CapcellPak C18 UG120 column (250 mm x 4.6 mm I.D., 5 microm) with an acetonitrile-25 mM potassium dihydrogenphosphate buffer (pH 8.0; 1:9, v/v) as the mobile phase. We applied dual detection (electrochemical detection and florescence detection) for improving the peak identification and also for improved selectivity, which assisted monitoring by trace-volume samples (e.g., plasma). The electrochemical detector, equipped with a diamond electrode, was set at 2000 mV (applied voltage) and the fluorescence detector was set at excitation wavelength 275 nm and emission wavelength 315 nm. The detection limits of the four NRTIs in spiked plasma were 1-100 ng/ml by electrochemical detection and 5-10 pg/ml by fluorescence detection. The calibration graphs were linear up to 20 microg/ml by electrochemical detection and 10 microg/ml by fluorescence detection. This is the first report of LC analysis of NRTIs by electrochemical detection, also combined with fluorescence detection. The detection limits of didanosine, lamivudine and zidovudine were improved 20-fold by electrochemical detection and 500-fold by fluorescence detection compared to previous reports on UV detection. The selectivity was also improved by dual detection. The proposed method was applied to the preliminary monitoring of NRTIs in plasma.

BLOOD COAGULATION FACTOR X (FX) IN HUMAN SEMINAL PLASMA

The active form of human blood coagulation factor X (FXa, EC 3.4.21.6) showing N - f -Benzoyl-L-isoleucyl-L-glutamyl-L-glycyl-L-arginine- p -nitroanilide (S-2222) hydrolyzing activity was first detected in human semen (seminal plasma) by affinity chromatography using anti-human coagulation factor X, and this enzyme activity was inhibited by anti-human FX. This enzyme has been associated with the human coagulation factor X (FX) in human semen (seminal plasma) by Western blot analysis, and the molecular mass of mature FX was also estimated to be 59 KDa by SDS-PAGE and Western blot analysis.

Preliminary Monitoring of Bisphenol A and Nonylphenol in Human Semen by Sensitive High Performance Liquid Chromatography and Capillary Electrophoresis After Proteinase K Digestion

Abstract To investigate human exposure by bisphenol A (BPA) and nonylphenol (NP), BPA and NP in human semen were preliminary monitored by sensitive high performance liquid chromatography (HPLC) by fluorescence pre-labeling with 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole (CDB) and capillary electrophoresis (CE) based on miceller electrokinetic chromatography after proteinase K digestion. The semen samples were collected from 57 patients who attended in vitro embryo fertilization program (IVF-ET). They were classified into nine groups according to their properties by semen character (e.g., normozoospermia) and statistical analysis (e.g., sperm count and sperm mobility). The proteinase K digestion was effective in obtaining high recovery value on the extraction process. In this time, BPA and NP were not detected in nine groups (57 samples, limits of detections = 1 and 7 pg/mL). It was suspected that BPA and NP levels in these semen were less than 1 pg/mL and 7 pg/mL, respectively. There were no significant exposures to BPA and NP on semen samples in this time experiments. Further large scale monitoring was needed for diagnosis effect of endocrine disruptor to human sperm production.

Determination of estrogens by high performance liquid chromatography with pre-label derivatization with 2-(4-carboxy)-5,6-dimethylbenzimidazole (II) : Shortening of analysis time and application to monitoring estrogen in serum from in-vitro fertilization embryo transfer (IVF-ET) patients

Abstract We report a sensitive high performance liquid chromatography (HPLC) method for determination of free and conjugated estrogens (estrone, estradiol and estriol) by a fluorescent pre-labeling regent, 2-(4-carboxyphenyl)-5,6-dimethylbenzimidazole, with modification of previous work. The modified method was also tried, in preliminary work, for diagnosis of the in-vitro fertilization embryo transfer (IVF-ET) process. The reagent volumes were changed to one-tenth, derivatization conditions were changed to mild conditions at 40 C, and a solid-phase extraction process by SEP-PAK could be omitted after restudy of reaction conditions. As a result analysis time could be shorted within 40 min. The proposed HPLC method was applied to monitoring of free and conjugated estrogens in the patients who attend in-vitro fertilization embryo transfer (IVF-ET). The subsequent increase of free and conjugated estrone, estradiol and estriol was observed with the progress of follicle growth following ovulation stage in the ...