Kuniko Mitamura

Gas chromatography and high-performance liquid chromatography of natural steroids

This review article underlines the importance of gas chromatography (GC), high-performance liquid chromatography (HPLC) and their hyphenated techniques using mass spectrometry (MS) for the determination of natural steroids, especially in human biological fluids. Steroids are divided into eight categories based on their structures and functions, and recent references using the above methodologies for the analysis of these steroids are cited. GC and GC-MS are commonly used for the determination of volatile steroids. Although HPLC is a widely used analytical method for the determination of steroids including the conjugated type in biological fluids, LC-MS is considered to be the most promising one for this purpose because of its sensitivity, specificity and versatility.

Derivatization of thiol-containing compounds

The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography-mass spectrometry is also described.

Oxysterols are substrates for cholesterol sulfotransferase

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7α/7β-hydroxycholesterol and 5α,6α/5β,6β-epoxycholesterol as well as the 7α-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.

Identification of dehydroepiandrosterone metabolites formed from human prostate homogenate using liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry

The identification of the in vitro metabolites of dehydroepiandrosterone formed from human prostate homogenate was investigated by hyphenated techniques using the stable-isotope dilution method. A mixture of dehydroepiandrosterone and [2H4]dehydroepiandrosterone was incubated with hypertrophied human prostate tissue homogenate in the presence of NAD, NADH and NADPH. The metabolites were extracted with AcOEt-hexane, purified by solid-phase extraction, and then analyzed by LC-atmospheric pressure chemical ionization MS and/or GC-MS. Androst-5-ene-3beta,17beta-diol (major product), androst-4-ene-3,17-dione, testosterone, 5alpha-dihydrotestosterone, androsterone, and 7alpha-hydroxydehydroepiandrosterone were identified in comparison with authentic samples based on their chromatographic behavior and mass spectra.

Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection.

A method for the determination of 25-hydroxyvitamin D3, the major metabolite of vitamin D3 in human plasma, using a non-radioactive internal standard and reversed-phase high-performance liquid chromatography with UV detection (265 nm) has been developed. The method was applied to the determination of the metabolite in plasma from healthy subjects (n = 25) and from patients with chronic renal failure (n = 12). 25-Hydroxyvitamin D3 3-sulfate, a major conjugated metabolite of 25-hydroxyvitamin D3, was also determined and the correlation between the concentrations of these metabolites was examined. The study showed that almost equal amounts of both compounds were detected in the plasma of healthy subjects, however, in two subjects, the amount of sulfate in the free form was found to be about twice as high as normally detected. In contrast, the free form was predominant in the plasma of patients with chronic renal failure and the sulfate was not detected in four patients.

Studies on neurosteroids: IX. Characterization of estrogens in rat brains using gas chromatography–tandem mass spectrometry

The characterization of the classical estrogens (estrone, estradiol, estriol) and guaiacol estrogens (2-hydroxyestrone 3-methyl ether, 4-hydroxyestrone 3-methyl ether) in rat brains was performed using gas chromatography-tandem mass spectrometry (GC-MS-MS). Estrogens were purified from Wistar strain rat brains by some chromatographic pre-treatments, such as solid-phase extraction, preparative thin-layer chromatography or preparative high-performance liquid chromatography. After the derivatization with O-methylhydroxylamine and/or N,O-bis(trimethylsilyl)trifluoroacetamide, estrogens were identified by comparison of their chromatographic behavior during GC-MS-MS operating in the product ion scan mode and comparison with the product ion MS spectra of an authentic sample. These evidences suggested that estrogens exist in rat brains as neurosteroids or neuroactive steroids.

Studies on neurosteroids. Part XIII. Characterization of catechol estrogens in rat brains using liquid chromatography-mass spectrometry-mass spectrometry

The existence of catechol estrogens in rat brains was clarified using liquid chromatography-atmospheric pressure chemical ionization-ion trap-mass spectrometry-mass spectrometry (LC-APCI-MS2). The catechol estrogens were extracted in the presence of ascorbic acid and then derivatized to acetates with acetic anhydride and pyridine. After a successive purification with silica gel mini-column chromatography, reversed-phase solid-phase extraction and preparative HPLC, the obtained fractions containing the catechol estrogen acetates were subjected to LC-APCI-MS2. 2-Hydroxyestrone, 2-hydroxyestradiol and their 4-hydroxy isomers were identified as acetates by comparison with authentic samples based on their chromatographic behavior and mass spectral data. The derivatization to acetate was useful for the treatment of labile catechol estrogens.

Characterization of monoglucuronides of vitamin D2 and 25-hydroxyvitamin D2 in rat bile using high-performance liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

The characterization of vitamin D2 3-glucuronide, 25-hydroxyvitamin D2 3-glucuronide and 25-hydroxyvitamin D2 25-glucuronide, biliary metabolites obtained from rats dosed with vitamin D2 and 25-hydroxyvitamin D2 per os, was carried out using HPLC-atmospheric pressure chemical ionization (APCI)-MS. The glucuronide obtained from bile specimens was identified by comparison of its chromatographic behaviour with an authentic sample using HPLC-APCI-MS operating in the negative-ion mode. Methylation of the respective fraction with diazomethane gave the methyl ester, which was also confirmed by HPLC-APCI-MS operating in the positive-ion mode. The (M-H)- and (M + NH4)+ ions were monitored in the selected-ion monitoring mode.

High-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase.

The high-performance liquid chromatographic separation of bile acid pyrenacyl esters with cyclodextrin-containing mobile phase is presented. Compared with conventional methods, inclusion chromatography gives much more satisfactory separation of derivatized bile acids in a short time. The application of this method to the separation of glycine-conjugated bile acids in human bile is also described.

Retention behavior of vitamin D and related compounds during high-performance liquid chromatography

The retention behavior of vitamin D 2 -D 5 and provitamin D 2 -D 5 are examined using high-performance liquid chromatography. Inclusion chromatography using cyclodextrin as the mobile phase additive in reversed-phase high-performance liquid chromatography is also used for this purpose. Reversed-phase high-performance liquid chromatography is more effective than normal-phase high-performance liquid chromatography in separating these analogs. The addition of methyl- β-cyclodextrin to the mobile phase is effective in separating the pair of vitamin-D 2 and - D 3 or provitamin-D 2 and -D 3 The separation of the pair of stereoisomeric Cookson-type derivatives of vitamin-D 2 or -D 3 was also examined and found that normal-phase high-performance liquid chromatography is effective for this purpose.