Kazuhiro Yamamoto

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.

Determination of Eicosapentaenoic, Docosahexaenoic, and Arachidonic Acids in Human Plasma by High-Performance Liquid Chromatography with Electrochemical Detection

A high-performance liquid chromatography with electrochemical detection (HPLC-ECD) system was developed for the simultaneous determination of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and arachidonic acid (AA) in human plasma. In the present HPLC-ECD system, EPA, DHA, and AA were separated using a reverse-phase C30 column and detected based on the voltammetric reduction of 3,5-di-tert-butyl-1,2-benzoquinone (DBBQ). Chromatographic peak areas were proportional to the concentration of EPA, DHA, and AA from 0.75 μM to 0.1 mM (r > 0.998). The concentrations of EPA, DHA, and AA in plasma from healthy human subjects after overnight fasting were determined, and the ratio of EPA to AA was obtained by the present HPLC-ECD method, which required 40 μL of human plasma and a simple procedure of sample preparation using diethyl ether extraction. Moreover, changes in EPA, DHA, and AA concentrations in a human subject were monitored before and after fish oil supplement administration by the present HPLC-ECD system.

Electrochemical detection of tocopherols in vegetable oils by supercritical fluid chromatography equipped with carbon fiber electrodes

A flow-through column electrolytic cell for supercritical fluid chromatography has recently been developed. To examine the applicability of the cell to real samples, the determination of tocopherols in edible vegetable oils was carried out. Tocopherols (α, β, γ and δ) and 2,2,5,7,8-pentamethyl-6-hydroxychroman (internal standard) were eluted and fully separated within 10 min using a mixture of supercritical carbon dioxide and methanol containing 1.0 mol L−1 ammonium acetate (98 : 2, v/v) as a mobile phase and a conventional silica gel column as a stationary phase. The current peak area detected in the electrochemical cell was found to be linear with the amount of samples injected in the range from 5 μmol L−1 to 200 μmol L−1 (r ≧ 0.997). The repeatability was not more than 1.82% RSD (n = 6) and the intermediate precision was also not more than 3.55% RSD (n = 3). The limit of detection (S/N = 3) on the column was less than 3.43 pmol. By the present internal standard method, tocopherols in various vegetable oils were determined with a simple sample preparation procedure.