Preconcentration and spectrophotometric determination of oxymetholone in the presence of its main metabolite (mestanolone) using modified maghemite nanoparticles in urine sample.

A novel and sensitive extraction procedure using maghemite nanoparticles (γ-Fe2O3) modified with sodium dodecyl sulfate (SDS), as an efficient solid phase, was developed for removal, preconcentration and spectrophotometric determination of trace amounts of oxymetholone (OXM), in the presence of mestanolone (MSL). Combination of nanoparticle adsorption and easily magnetic separation was used for the extraction and desorption of OXM. The preparation of γ-Fe2O3 nanoparticles were obtained by co-precipitation method and their surfaces were modified by SDS. The size and properties of the produced γ-Fe2O3 nanoparticles were determined by X-ray diffraction analysis, FT-IR and scanning electron microscopy measurements. OXM and MSL became adsorbed at pH 3.0. The adsorbed drugs were then desorbed and determined spectrophotometrically using a selective complexation reaction for OXM. The calibration graph was linear in the range 15.0-3300.0 ng mL(-1) of OXM with a correlation coefficient of 0.9948. The detection limit of the method for determination of OXM was 4.0 ng mL(-1). The method was applied for the determination of OXM in human urine samples.

Mass spectrometric description of novel oxymetholone and desoxymethyltestosterone metabolites identified in human urine and their importance for doping control.

The metabolism of two anabolic steroids - oxymetholone and desoxymethyltestosterone - was reinvestigated to identify new targets potentially valuable for the antidoping analysis. Excretion urine samples from the laboratory reference collection were used in this study. Following fractionation of the urinary extract by means of high performance liquid chromatography (HPLC), each fraction was subjected to gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) analysis after trimethylsilylation. About 20 metabolites were found for desoxymethyltestosterone and more than 40 for oxymetholone, with many of them being isomeric compounds. In addition to the well-known reduced and hydroxylated metabolites, 18-nor-17,17-dimethyl and 18-nor-17-hydroxymethyl-17-methyl steroids were also identified. Having evaluated all the metabolites in terms of how long they could be detected, we suggest that 18-nor-2ξ,17ß-hydroxymethyl-17α-methyl-5α-androst-13-en-3α-ol is an important marker of oxymetholone abuse. In case of desoxymethyltestosterone, better detectability could be achieved if 18-nor-17,17-dimethyl-5α-androst-13-en-2ξ,3α-diol is monitored. These novel metabolites could be detected using GC-MS/MS at least for 14 days after administration of these anabolic steroids compared to 5-7 days for previously reported metabolites.

Detection of urinary metabolites common to structurally related 17alpha-alkyl anabolic steroids in horses and application to doping tests in racehorses: methandienone, methandriol, and oxymetholone.

Methandienone, methandriol, and oxymetholone, which are anabolic steroids possessing 17alpha-methyl and 17beta-hydroxy groups, were developed as oral formulations for therapeutic purposes. However, they have been used in racehorses to enhance racing performance. In humans, it has been reported that structurally related anabolic steroids having the 17alpha-methyl and 17beta-hydroxy groups, including 17alpha-methyltestosterone, mestanolone, methandienone, methandriol, and oxymetholone, have metabolites in common. In this study, we found that metabolites common to those of 17alpha-methyltestosterone and mestanolone were detected in horse urine after the administration of oxymetholone, methandienone, and methandriol. Based on analytical data, we confirmed these to be the common metabolites of five structurally related steroids, 17alpha-methyltestosterone, mestanolone, oxymetholone, methandienone, and methandriol. Furthermore, we detected hitherto unknown urinary metabolites of methandriol and oxymetholone in horses. The parent steroid itself was detected in horse urine after the administration of methandriol, other than metabolites common to 17alpha-methyltestosterone and mestanolone. On the other hand, the major metabolite of oxymetholone was mestanolone, aside from metabolites presumed to be the stereoisomers of 2-hydroxymethyl-17alpha-methyl-5alpha-androstan-3,17beta-diol and 2,17alpha-di(hydroxymethyl)-5alpha-androstan-3,17beta-diol. The simultaneous detection of common metabolites and other main metabolites would help us narrow down the candidate-administered steroid for the doping tests in racehorses.

Studies on anabolic steroids. 10. Synthesis and identification of acidic urinary metabolites of oxymetholone in a human.

Two major unconjugated acidic metabolites of oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one, 1), namely, 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,3-dioic acid (2) and 3 alpha,17 beta-dihydroxy-17 alpha-methyl-5 alpha-androstane-2 beta-carboxylic acid (6a), were detected by gas chromatography/mass spectrometry in urine samples collected after oral administration of 1 to a human volunteer. Reference steroid 2 was synthesized and identified. The identification of urinary metabolite 6a was based on the synthesis of its stereoisomers and the isomerization of the methyl ester 6b to its 2-epimer, 3 alpha,17 beta-dihydroxy-17 alpha-methyl-5 alpha-androstane-2 alpha-carboxylic acid methyl ester (9b). The mechanisms accounting for the formation of these acidic metabolites are discussed.

Studies on anabolic steroids--8. GC/MS characterization of unusual seco acidic metabolites of oxymetholone in human urine.

One of the biotransformation routes of oxymetholone (17 beta-hydroxy-2-hydroxymethylene-17 alpha-methyl-5 alpha-androstan-3-one) in man leads to the formation of 17 beta-hydroxy-17 alpha-methyl-5 alpha-androstan-3-one (mestanolone). To demonstrate that this latter steroid may be formed by decarboxylation of an intermediate metabolite of oxymetholone bearing a 2-carboxylic group, we studied the urinary excretion of oxymetholone acidic metabolites. Five new acidic metabolites are reported here for the first time, among which four are unusual seco steroids resulting from the oxidative cleavage of the A-ring. The most abundant compound is 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,3-dioic acid 1, the cumulative excretion of which accounted for 1.52% of the dose. Three other seco diacids were produced in smaller amounts, namely 17 beta-hydroxy-17 alpha-methyl-2,3-seco-5 alpha-androstane-2,4- dicarboxylic acid 3, 17 beta-hydroxy-17 alpha-methyl-1,3-seco-5 alpha-androstane-1,3-dioic acid 4 and 17 beta-hydroxy-17 alpha-methyl-2,4-seco-5 alpha-androstane-2,4-dioic acid 5. The fifth acidic metabolite was identified as 3 alpha, 17 beta-dihydroxy-17 alpha-methyl-5 alpha-androstane-2 beta-carboxylic acid 2. The excretion in urine of these acidic metabolites suggests that the 2-hydroxymethylene group in oxymetholone is readily oxidized to yield the corresponding beta-keto acid which can be (1) decarboxylated to form mestanolone; (2) reduced at C-3 to give compound 2; and (3) further oxidized to afford the unexpected seco diacids 1, 3, 4 and 5. The identity of compounds 1 and 2 was ascertained by GC/MS and 1H and 13C-NMR analysis of reference compounds. The other metabolites were characterized by GC/MS analysis.

[Identification of oxymetholone metabolites in the urine using gas chromatography-mass spectrometry]. / Identifikacija metabolita oksimetolona u urinu metodom gasna hromatografija-spektrometrija masa.

Metabolite of 17-methyl-17-hydroxy-2-(hydroxymethylen)-androstan-3-one (oxymetholane) in urine after a single oral administration was monitored by gas chromatography+mass spectrometry. During the investigation prepared TMS-ethers and TMS-enol-ethers of conjugated steroid fraction two new metabolites of oxymetholone have been identified: tetrahydrooxymetholone and tetrahydro-6-hydroxy-oxymetholone.

Studies on anabolic steroids. V. Sequential reduction of methandienone and structurally related steroid A-ring substituents in humans: gas chromatographic-mass spectrometric study of the corresponding urinary metabolites.

The biotransformation of methandienone (17 beta-hydroxy-17 alpha-methylandrosta-1,4-dien-3-one) in human adults, more particularly the sequential reduction of its A-ring substituents, was investigated by gas chromatography-mass spectrometry. Two pairs of 17-epimeric tetrahydro diols resulting from the stereoselective reduction of the delta 4- and 3-oxo groups and of the delta 1-function were characterized. The major diols were 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol and 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol, which were both excreted in the conjugate fraction in a 1:3.8 ratio. The immediate metabolic precursors of the 5 beta-diol, namely 17 beta-hydroxy-17 alpha-methyl-5 beta-androsta-1-en-3-one and 17 alpha-methyl-5 beta-androsta-1-en-3 alpha,17 beta diol and their corresponding 17-epimers, were also identified in post-administration urine samples. These data indicated that reduction of methandienone A-ring substituents proceeds according to the sequence. delta 4-, 3-oxo- and delta 1-. The A-ring reduction products of the structurally related steroids mestanolone, 17 alpha-methyltestosterone and oxymethone were also characterized and provided further analytical and metabolic evidence supporting the proposed route of methandienone A-ring reduction. It was also demonstrated using synthetic 17 beta-sulfate conjugates of methandienone and 17 alpha-methyltestosterone that their corresponding 17-epimers are formed by nucleophilic substitution by water of the labile sulfate moiety. The steroidal metabolites were identified on the basis of their characteristic mass spectral features and by comparison with authentic reference standards. Metabolic pathways accounting for the occurrence of the metabolites of interest in post-administration urine samples are proposed.