Yoshihiko Shinohara

Simultaneous determination of methionine and total homocysteine in human plasma by gas chromatography–mass spectrometry

A gas chromatographic-mass spectrometric method for the simultaneous determination of methionine and total homocysteine in human plasma is described. DL-[2H4]Methionine and DL-[2H8]homocystine were used as internal standards. The method involved reduction of the disulfide bond with dithiothreitol, purification by cation-exchange chromatography using a BondElut SCX cartridge and derivatization with isobutyl chlorocarbonate in water-ethanol-pyridine. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of N(O,S)-isobutyloxycarbonyl ethyl ester (IBC-OEt) derivatives for methionine and [2H4]methionine, respectively, and the fragment ions ([M+H-COOisoBu-COOEt]+) for TBC-OEt derivatives for homocysteine and [2H4]homocysteine, respectively. The sensitivity, specificity, accuracy and precision of the method were demonstrated to be satisfactory for measuring concentrations of methionine and total homocysteine in human plasma.

Determination of plasma testosterone by mass fragmentography using testosterone-19-d3 as an internal standard : Comparison with radioimmunoassay

Analytical procedures for the measurement of testosterone by mass fragmentography (MF) using trideuterated testosterone (testosterone-19,19,19-d3) are described. For the calculation of plasma testosterone, peak height ratios were measured by MF performed on the molecular ions of the TFA derivative of testosterone (m/e 480) and testosterone-19,19,19-d3 (m/e 483). The sensitivity of the method was judged from the lower limit of detection of the mass spectrometer which was at 10 pg. For the measurement of the precision, the inter- and intra-assay coefficients of variation (C.V.) were calculated by using a pooled plasma sample; they were 3.15% and 1.79%, respectively. The specificity was investigated by the use of 5 alpha-dihydrotestosterone and the MF method was found to afford a highly selective technique. These results obtained by MF have been compared with the results obtained by a radioimmunoassay method.

Application of radioluminography to off-line counting of radioactivity in high-performance liquid chromatographic eluates

Abstract An off-line counting method for the determination of carbon-14 in HPLC eluates was developed using radioluminography (RLG). A succession of aliquots of the eluate were collected in the flat-bottomed wells of a polystyrene microplate, evaporated to dryness and contacted with an imaging plate, and radioactivity was determined with a Bio-image analyser. The limit of detection was 0.35 Bq per injection. The inter- assay relative standard deviation was less than 3% over the range 2–20 Bq. The RLG off-line counting method was utilized to determine [ 14 C]eicosapentaenoic acid metabolites formed by rat hepatic microsomes. The results were compared with those obtained with an off- line liquid scintillation counting method and an on-line counting method.

Determination of 14C in microplates by radioluminography

A method alternative to liquid scintillation counting for detecting 14C was developed. The new method involves putting an aqueous radioactive sample onto the flat-bottomed wells of a polystyrene microplate, preparing a pellicle by lyophilization, and determining the radioactivity using radioluminography. It provides a simple, inexpensive, sensitive and reliable technique for determining the radioactivity of a few hundred samples simultaneously.

Determination of testosterone propionate in human plasma by gas chromatography— mass spectrometry

A specific, sensitive and accurate quantitative analysis of testosterone propionate in human plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. For the calculation of testosterone propionate in plasma, peak height ratios were measured by selected-ion monitoring performed on the molecular ions of the trifluoroacetyl derivative of testosterone propionate (m/z 440) and testosterone propionate-19,19,19-d3 (m/z 443). The sensitivity of the method was judged from the lower limit of the detection of the mass spectrometer which was at 20 pg. The inter-assay coefficients of variation and relative error at a concentration of 1.31 ng/ml of plasma were 5.47% and -2.3%, respectively. The method described was applied to the determination of plasma concentrations of testosterone propionate-19,19,19-d3 following an intramuscular dose of testosterone propionate-19,19,19-d3 in a healthy male volunteer.

Simultaneous determination of the enantiomers of leucine and [2H7]leucine in plasma by capillary gas chromatography–mass spectrometry

A method for the stereoselective assay of D- and L-enantiomers of both leucine and [2H7]leucine in rat plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. DL-[2H3]leucine was used as an internal standard. The method involved purification by cation-exchange chromatography using BondElut SCX cartridge and derivatization with hydrochloric acid in methanol to form methyl ester followed by subsequent chiral derivatization with (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride to form diastereomeric amide. The derivatization made the separation of the leucine enantiomers possible with good gas chromatographic behavior. Quantitation was performed by selected-ion monitoring of the quasi-molecular ions of the diastereomers on the chemical ionization method. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of leucine enantiomers.

Simultaneous determination of androstenedione, 11β-hydroxyandrostenedione, and testosterone in human plasma by stable isotope dilution mass spectrometry

This study describes a GC-MS method for the simultaneous determination of androstenedione (AD), 11beta-hydroxyandrostenedione (11beta-OHAD), and testosterone (TS) in human plasma. [19,19,19-(2)H(3)]Androstenedione (AD-(2)H(3)), 11beta-hydroxy-[1,2,4,19-(13)C(4)]androstenedione (11beta-OHAD-(13)C(4)), and [1,16,16,17-(2)H(4)]testosterone (TS-(2)H(4)) were used as internal standards. Pentafluoropropionic (PFP) derivatization with good GC behavior was employed for the GC-MS analysis of the three steroids. The detection limit of the present GC-MS-SIM method was found to be 1 pg per injection for AD (S/N ratio=4.5), 5 pg for 11beta-OHAD (S/N ratio=5.0), and 1 pg for TS (S/N ratio=4.4), respectively. Calibration curves were linear from 0.22 to 2.80 ng/mL (r=0.9998) for AD, from 0.56 to 3.19 ng/mL (r=0.9996) for 11beta-OHAD, and from 2.05 to 10.3 ng/mL (r=0.9996) for TS. The intra- and inter-day assay reproducibilities in the amounts of the three androgens determined were in good agreement with the actual amounts added, the relative errors (R.E.) were -3.1 to 2.4%. The inter-assay relative standard deviation (R.S.D.) was less than 5.3%. The present method provides a sensitive and reliable technique for the simultaneous determination of AD, 11beta-OHAD, and TS in plasma. The method can be applied to pharmacokinetic and metabolic studies of androgens with a particular interest in evaluating the conversion of AD to 11beta-OHAD and the interconversion of AD and TS in humans.

Synthesis of deuterium labeled 17-methyl-testosterone

The synthesis of two forms of selectively deuterated 17-methyl-testosterone is described. 17-Methyl-d3-testosterone was prepared by the Grignard reaction of dehydroepiandrosterone with deuterium labeled methyl magnesium iodide followed by an Oppenauer oxidation. 17-Methyl-d3-testosterone-19,19,19-d3 was prepared by treating 3,3-ethylenedioxy-5,10-epoxy-5 alpha, 10 alpha-estran-17-one with deuterium labeled methyl magnesium bromide followed by hydrolysis and dehydration of the 5 alpha-hydroxyandrostane derivative.

Stable isotope dilution analysis of human urinary metabolites of 17α-methyltestosterone

A method based on gas chromatography-mass spectrometry-selected-ion monitoring was developed to measure the main metabolites of 17alpha-methyltestosterone, 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, in human urine. 17alpha-Methyl-[(2)H3]-5alpha-androstan-3alpha,1 7beta-diol and 17alpha-methyl-[(2)H3]-5beta-androstan-3alpha,17 beta-diol were used as internal standards. The methods involved purification using a Sep-Pak C(18) cartridge, hydrolysis by beta-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammon ium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)-2xTMSOH]+) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, and can be applied to pharmacokinetic studies of 17alpha-methyltestosterone.

Determination of the enantiomers of suprofen and [2H3]suprofen in plasma by capillary gas chromatography-mass spectrometry

A method for the stereoselective assay of the (+)- and (-)-enantiomers of suprofen and [2H3]suprofen in human plasma was developed using gas chromatography-mass spectrometry-selected-ion monitoring. (+/-)-[2H7]Suprofen was used as an internal standard. The method involved diethyl ether extraction and chiral derivatization with S-(-)-1-(naphthyl)ethylamine to form diastereomeric amide. The diastereoisomers were separated on a capillary gas chromatograph-mass spectrometer. Quantitation was achieved by selected-ion monitoring of the quasi-molecular ions of the diastereoisomers. The sensitivity, specificity, accuracy and reproducibility of the method were demonstrated to be satisfactory for application to pharmacokinetic studies of suprofen enantiomers.