Characterization of desoxymethyltestosterone main urinary metabolite produced from cultures of human fresh hepatocytes.

Desoxymethyltestosterone (DMT; 17ß-hydroxy-17α-methyl-5α-androst-2-ene) is a designer steroid present in hormonal supplements distributed illegally as such or in combination with other steroids, for self-administration. It figures on the list of substances prohibited in sports and its detection in athlete's urine samples is based upon the presence of the parent compound or the main urinary metabolite, which has not been characterized yet. Following its isolation from cultures of human fresh hepatocytes and S9 fractions of liver homogenates, we were able to identify this metabolite as being 17α-methyl-2ß,3α,17ß-trihydroxy-5α-androstane. Other minor metabolites were also characterized. The production, isolation, NMR, mass spectral analyses and chemical synthesis are presented.

Development of a candidate reference method for the simultaneous quantitation of the boar taint compounds androstenone, 3α-androstenol, 3ß-androstenol, skatole, and indole in pig fat by means of stable isotope dilution analysis-headspace solid-phase microextraction-gas chromatography/mass spectrometry.

The steroidal pig pheromones androstenone (5α-androst-16-en-3-one), 3α-androstenol (5α-androst-16-en-3α-ol), and 3ß-androstenol (5α-androst-16-en-3ß-ol) as well as the heterocyclic aromatic amines skatole and indole, originating from microbial degradation of tryptophan in the intestine of pigs, are frequently recognized as the major compounds responsible for boar taint. A new procedure, applying stable isotope dilution analysis (SIDA) and headspace solid-phase microextraction-gas chromatography/mass spectrometry (HS-SPME-GC/MS) for the simultaneous quantitation of these boar taint compounds in pig fat was developed and validated. The deuterated compounds androstenone-d(3), 3ß-androstenol-d(3), skatole-d(3), and indole-d(6) were synthesized and successfully employed as internal standards for SIDA. The new procedure is characterized by a fast, simple, and economic sample preparation: methanolic extraction of the melted fat followed by a freezing and an evaporation step allows for extraction and enrichment of all five analytes. Additional time-consuming cleanup steps were not necessary, as HS-SPME sampling overcomes fat-associated injector and column contamination. The method has been validated by determining intra- and interday precision and accuracy as well as the limit of detection (LOD) and limit of quantitation (LOQ). Additionally, a cross-validation for androstenone, skatole, and indole was carried out comparing the results of 25 back fat samples obtained simultaneously by the new SIDA-HS-SPME-GC/MS procedure with those obtained in separate GC/MS and high-performance liquid chromatography fluorescence detection (HPLC-FD) measurements. The cross-validation revealed comparable results and confirms the feasibility of the new SIDA-HS-SPME-GC/MS procedure.

Microbial oxidation of anabolic steroids.

Microbial transformation of two anabolic steroids, ethylestrenol (1) and nandrolone (2), were carried out. Ethylestrenol (1), when incubated with Rhizopus stolonifer (TSY 0471), yielded two oxidative metabolites named 17alpha-ethyl-3beta,17beta-dihydroxy-19-norndrost-4-ene (3) and 17alpha-ethyl-17beta-hydroxy-19-norandrost-4-en-3-one (4), while incubation of compound 2 with the same fungus yielded two oxidative metabolites, 19-norandrost-4-en-3,17-dione (5) and 6alpha,17beta-dihydroxy-19-norandrost-1,4-dien-3-one (6).

[The isolation and identification of two new epimer from the mother liquid of testosterone].

AIM: To study the impurity of the drug testosterone. METHODS: Chromatography methods were used to separate the chemical constituents. Their structures were determined by NMR and MS spectral analysis. RESULTS: Two new epimers were isolated from the mother liquid of the drug. CONCLUSION: These new epimers were identified as 3alpha-ethoxyandrost-4-en-17beta-ol, 3beta-ethoxyandrost-4-en-17beta-ol.

Reversed-phase HPLC separation and chromatographic-spectral characterization of 17 beta-(2'-Thiazolyl)androst-5-en-3 beta-ols and their acetates.

Simple isocratic HPLC separation of a series of thiazolyl steroids with the 17 beta-2' linkage is described. The chromatographic and spectral characterization utilizes both on-the-fly UV spectral maxima and absorbances changes.

Formation of 5,16-androstadien-3 beta-ol from pregnenolone in human testicular microsomes.

A microsomal fraction of testicular tissue from a patient with prostatic carcinoma was incubated with [4-14C]pregnenolone in the presence of an NADPH-generating system for different periods of time. The metabolites were separated by Sephadex LH-20 column chromatography and then identified by thin-layer chromatography, radio-gas chromatography, and crystallization studies. Pregnenolone was converted to a major metabolite, 5-androstene-3 beta,17 beta-diol via 17-hydroxypregnenolone and then dehydroepiandrosterone. Another major metabolite was 5,16-androstadien-3 beta-ol, which increased with the time of incubation and accumulated in the incubation medium. After 120 min of incubation, 34.6% of the precursor was converted to 5-androstene-3 beta,17 beta-diol and 15.1% to 5,16-androstadien-3 beta-ol. In addition to the above-mentioned steroids, 16 alpha-hydroxypregnenolone, 5-pregnene-3 beta,20 alpha-diol, and 5-androstene-3 beta,17 alpha-diol were identified as minor metabolites of pregnenolone. From these results it was concluded that human testicular microsomes possess enzymic activities for the synthesis of 5,16-androstadien-3 beta-ol, as well as androgens from pregnenolone.

The sex pheromone precursor androsta-5,16-dien-3 beta-ol is a major early metabolite in in vitro pregnenolone metabolism in human testicular homogenates.

In an earlier report we described the early time sequence of the in vitro metabolism of [4-14C]pregnenolone ([4-14C]P5) to testosterone in homogenates of human and rat testes and demonstrated the appearance of mainly delta 5 (humans)- and delta 4 (rats)-steroids within minutes after starting the incubation. In this study strong evidence is presented for the substantial synthesis from P5 of the sex pheromone precursor androsta-5,16-dien-3 beta-ol (ADL) in human, but not rat, testicular homogenates. The 16-unsaturated C19 steroid ADL appeared after 1 min of incubation, and within 5 min reached values (17-23% of total radioactivity added as [4-14C]P5) comparable to those of the major delta 5-steroids 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Thus, in humans, as in boars, the sex attractant precursor ADL is a major early testicular metabolite of P5.