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The microbial transformation of anabolic androgenic steroid mestanolone (1) with Macrophomina phaseolina and Cunninghamella blakesleeana has afforded seven metabolites. The structures of these metabolites were characterized as 17β-hydroxy-17α-methyl-5α-androsta-1-ene-3,11-dione (2), 14α,17β-dihydroxy-17α-methyl-5α-androstan-3,11-dione (3), 17β-hydroxy-17α-methyl-5α-androstan-1,14-diene-3,11-dione (4), 17β-hydroxy-17α-methyl-5α-androstan-3,11-dione (5), 11β,17β-dihydroxy-17α-methyl-5α-androstan-1-ene-3-one (6), 9α,11β,17β-trihydroxy-17α-methyl-5α-androstan-3-one (7), and 1β,11α,17β-trihydroxy-17α-methyl-5α-androstan-3-one (8). All the metabolites, except 5 and 6, were identified as new compounds. Substrate 1 (IC50 = 27.6 ± 1.1 μM), and its metabolites 2 (IC50 = 19.2 ± 2.9 μM) and 6 (IC50 = 12.8 ± 0.6 μM) exhibited moderate cytotoxicity against the HeLa cancer cell line (human cervical carcinoma). All metabolites were noncytotoxic to 3T3 (mouse fibroblast) and H460 (human lung carcinoma) cell lines. The metabolites were also evaluated for immunomodulatory activity, and all were found to be inactive.

Anabolic-androgenic steroids (AAS) are testosterone derivatives developed for steroid-replacement and treatment of debilitating conditions. They are widely used by athletes in elite sports and bodybuilding due to their muscle-building and performance-enhancing properties. Excessive AAS use is associated with cardiovascular diseases, mood changes, endocrine and metabolic disorders; however, the underlying mechanisms remain unknown. Selective androgen receptor modulators (SARMs) aim to reduce adverse androgenic effects, while maximizing anabolic effects. This study assessed potential steroidogenic disturbances of 19 AAS and 3 SARMs in human adrenocortical carcinoma H295R cells, comparing basal and forskolin-activated states by mass spectrometry-based quantification of nine major adrenal steroids. Mesterolone, mestanolone and methenolone increased mineralocorticoid but decreased adrenal androgen production, indicating CYP17A1 dysfunction. Cell-free activity assays failed to detect direct CYP17A1 inhibition, supported by molecular modeling. The mRNA expression levels of 3β-HSD2, CYP17A1, CYP21A2, CYP11B1 and CYP11B2 were unaffected, suggesting indirect inhibition involving post-translational modification and/or impaired protein stability. Clostebol and oxymetholone decreased corticosteroid but increased dehydroepiandrosterone biosynthesis in H295R cells, suggesting CYP21A2 inhibition, sustained by molecular modeling. These AAS did not affect the expression of key steroidogenic genes. None of the SARMs tested interfered with steroidogenesis. The chosen approach allowed the grouping of AAS according to their steroidogenic-disrupting effects and provided initial mechanistic information. Mesterolone, mestanolone and methenolone potentially promote hypertension and cardiovascular diseases via excessive mineralocorticoid biosynthesis. Clostebol and oxymetholone might cause metabolic disturbances by suppressing corticosteroid production, resulting in adrenal hyperplasia. The non-steroidal SARMs exhibit an improved safety profile and represent a preferred therapeutic option.

A new chemically modified electrode was constructed and applied to the electro-oxidation of the oxymetholone. Also, the electrode was applied to the simple, rapid, highly selective and sensitive determination of oxymetholone (OXM) in pharmaceutical and plasma samples using square wave voltammetry (SWV). The multi-walled carbon nanotubes modified glassy carbon electrode (MWCNT/GCE) were prepared by casting of the multi-walled carbon nanotubes (MWCNT) suspension on the glassy carbon electrode surface. The limit of detection and the linear range were found to be 1.36 and 2.00-90.00 ng mL(-1) of OXM, respectively. The effects of potentially interfering substances on the determination of this compound were investigated and found that the electrode is highly selective. The proposed modified electrode was used for the determination of OXM in human plasma and pharmaceutical samples. This reveals that MWCNT/GCE shows excellent analytical performance for the determination of OXM in terms of very low detection limit, high sensitivity, very good repeatability and reproducibility over other methods reported in literature.

Microbial transformation of mestanolone (1) using the plant pathogenic fungus, Rhizopus stolonifer, resulted in the production of two known metabolites, identified as 11α-hydroxymestanolone (3) and 6α-hydroxymestanolone (4). Transformation of 17-methyl-1-testosterone (2) by R. stolonifer yielded two known metabolites, methandrostenolone (5) and 11α,17β- dihydroxy-androsta-1,4-diene-3-one (6). These transformations included α-hydroxylations at C-11 and C-6, dehydrogenation at C-4, androsta and a demethylation at C-17 positions. Structures of transformed products were determined using spectroscopic techniques.

A slurry crystallization technique was used in cocrystal screening of two nonionizable pharmaceutical host compounds, stanolone and mestanolone, with 11 pharmaceutically acceptable guest acids. Crystallization was performed simply by adding crystallization solvents to solid mixtures of a host and a guest, which had been prepared using lyophilization of their dimethyl sulfoxide solution. Powder X-ray diffraction and thermogravimetric/differential thermal analysis were used to identify new solid forms. Two resultant new forms, stanolone l-tartaric acid 1:1 cocrystal and mestanolone salicylic acid 1:1 cocrystal, were characterized using single-crystal X-ray diffraction. The hosts, despite having the same steroidal skeleton and the same functional groups that form strong hydrogen bonds, each formed a cocrystal with a different guest molecule. All functional groups of the host and guest molecules that form strong hydrogen bonds were engaged in hydrogen bonding, but, despite the highly analogous molecular struc...

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.

An easily available hypervalent iodine(V) reagent, 2-iodoxybenzoic acid (IBX) immobilized in the ionic liquid [bmim][Br] was found to be an efficient and eco-friendly protocol for the oxidation of 17α-methylandrostan-3β,17β-diol (1). At ambient temperature oxidation of 1 with IBX gave mestanolone (2) in good yield and with an increased stoichiometric amount of IBX, oxidation adjacent to the carbonyl functionality (α,β-unsaturation) occurred to give dehydrogenated 17β-hydroxy-17α-methyl-Δ1-androsten-3-one (3) as the major product in a one-pot reaction. The product is easily obtained by extraction with diethyl ether and evaporation of the solvent.

In the course of investigations on equestrian supplemental products for the presence of doping substances, two products were found to contain forbidden substances. As reported earlier a plant extract (Mexican cactus extract) named “Energy 5” contained the anabolic androgenic steroids (AAS) stanozolol, 17β-hydroxy-17α-methyl-5α-androstane-3β-ol (3β,5α-THMT) as well as mestanolone not declared on the label. In the present study, a product called “Super Kalm Paste” was tested. Analysis by gas chromatography - mass spectrometry (GC-MS) revealed that the preparation contained the class I anti-arrhythmics quinine (trade names KinidinTM, Durules) and cinchonine. The samples were prepared according to a sample preparation procedure established for anabolic steroids in nutritional supplements for humans. The sample treatment comprised the extraction and purification of the analytes as well as the chemical conversion with N-methyl-N-trimethylsilyl-trifluoracetamide (MSTFA) to yield the trimethylsilyl (TMS)-derivatives. To verify whether the administration of such products could lead to positive doping tests, a pilot excretion study on “Energy 5” was conducted with two geldings, and urine samples were collected. Gas chromatography - high resolution mass spectrometry (GC-HRMS) after solid phase extraction and mixed derivatisation has demonstrated the presence of the stanozolol metabolite 16β-hydroxy-stanozolol in urine samples after “Energy 5” application.

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.

This study was aimed to investigate the effects of different doses of two hormones and an anti-aromatase, i.e. 17a methyl testosterone (MT), methyl di hydrotestosterone (MDHT) or mestanolone and letozole in masculinization of Nile tilapia (Oreochromis niloticus) under the condition of brackish water in Bafgh station situated in Yazd province in center of Iran. Each experiment in this study was consisted of 5 treaments with 3 replicates each. A number of 1725 larvaes was distributed randomly among 15 replicates at the beginning of each experiment. Each experiment lasted 45 days and the larvaes were reared in aerated flow-through pots and fiberglass tanks filled with brackish water. The averages for temperature, salinity, pH and dissolved oxygen of water were 26.9 ê, 8 g/l, 7.6 and 5.78% respectively during this study. In experiment 1, three different doses of 40, 60 and 100 mg MT/k of feed were fed to different groups of 7 day post fertilization (dpf) larvaes for 45 days from the beginning of the experiment. The results showed that the larvaes in 40 mg group were 100 percent masculinized based on squash test performed at the end of the experiment but masculinization rates of those in 60 and 100 mg groups were 99.7 and 96.2 perecent respectively. Based on Dunkan test, total body length and weight averages measured in biometry 3 (at the end of the experiment) were not significantly different among groups but in biometry 2 (30 days after the beginning of experiment), they were significantly lesser only in 40 mg group (P

Mild, efficient and eco-friendly oxidation of 17alpha-methylandrostan-3beta-17beta-diol (1) has been studied with three different reagents viz. pentavalent iodine reagent 2-iodoxy benzoic acid (IBX) in DMSO at 65 degrees C, sodium hypochlorite and H2O2/Na2WO4 under phase transfer conditions to give 17beta-hydroxy-17alpha-methylandrostan-3-one (mestanolone 2), a drug intermediate as oxidized product. The H2O2/Na2WO4/PTC gave mestanolone in high yield and purity whereas sodium hypochlorite/PTC system yielded some chlorinated material along with the mestanolone. However, 1 with 2.5 equivalent of IBX gave 17beta-hydroxy-17alpha-methyl-Delta1-androsten-3-one (3) under the similar reaction conditions in good yield and single step reaction.

The use of anabolic steroids was banned by the International Olympic Committee for the first time at the Olympic Games in Montreal in 1976. Since that time the misuse of anabolic steroids by athletes has been controlled by analysis of urine extracts by gas chromatography—mass spectrometry (GC—MS). The excreted steroids or their metabolites, or both, are isolated from urine by XAD-2 adsorption, enzymatic hydrolysis of conjugated excreted metabolites with β-glucuronidase from Escherichia coli, liquid-liquid extraction with diethyl ether, and converted into trimethylsilyl (TMS) derivatives. The confirmation of an anabolic steroid misuse is based on comparison of the electron impact ionization (EI) mass spectrum and GC retention time of the isolated steroid and/or its metabolite with the EI mass spectrum and GC retention time of authentic reference substances. For this purpose excretion studies with the most common anabolic steroids were performed and the main excreted metabolites were synthesized for bolasterone, boldenone, 4-chlorodehydromethyltestosterone, clostebol, drostanolone, fluoxymesterone, formebolone, mestanolone, mesterolone, metandienone, methandriol, metenolone, methyltestosterone, nandrolone, norethandrolone, oxandrolone and stanozolol. The metabolism of anabolic steroids, the synthesis of their main metabolites, their GC retention and El mass spectra as TMS derivatives are discussed.

A simple and convenient method has been developed to prepare sulfates of anabolic 17 beta-hydroxy-17 alpha-methyl steroids. The sulfates of methandienone, 17 alpha-methyltestosterone, mestanolone, oxandrolone, and stanozolol were prepared. Different A-ring functions were not affected under the sulfation condition. The buffered hydrolyses of these sulfates provided the 17-epimers of the original steroids and 17,17-dimethyl-18-nor-13(14)-ene steroids, presumably via the 17-carbocations.

The epimerization and dehydration reactions of the 17 beta-hydroxy group of anabolic 17 beta-hydroxy-17 alpha-methyl steroids have been investigated using the pyridinium salts of 17 beta-sulfate derivatives of methandienone 1, methyltestosterone 4, oxandrolone 7, mestanolone 10 and stanozolol 11 as model compounds. Rearrangement of the sulfate conjugates in buffered urine (pH 5.2) afforded the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes in a ratio of 0.8:1. These data indicated that both epimerization and dehydration of the 17 beta-sulfate derivatives were not dependent upon the respective chemical features of the steroids studied, but were instead inherent to the chemistry of the tertiary 17 beta-hydroxy group of these steroids. Interestingly, in vivo studies carried out with human male volunteers showed that only methandienone 1, methyltestosterone 4 and oxandrolone 7 yielded the corresponding 17-epimers 2, 5 and 8 and the 18-nor-17,17-dimethyl-13(14)-enes 3, 6 and 9 in ratios of 0.5:1, 2:1 and 2.7:1, respectively. No trace of the corresponding 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of mestanolone 10 and stanozolol 11 was detected in urine samples collected after administration of these steroids. These data suggested that the in vivo formation of the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes derivatives of 17 beta-hydroxy-17 alpha-methyl steroids is also dependent upon phase I and phase II metabolic reactions other than sulfation of the tertiary 17 beta-hydroxyl group, which are probably modulated by the respective chemical features of the steroidal substrates. The data reported in this study demonstrate that the 17-epimers and 18-nor-17,17-dimethyl-13(14)-enes are not artifacts resulting from the acidic or microbial degradation of the parent steroids in the gut as previously suggested by other authors, but arise from the rearrangement of their 17 beta-sulfate derivatives. Unchanged oxandrolone 7 was solely detected in the unconjugated steroid fraction whereas unchanged steroids 1, 4 and 11 were recovered from the glucuronide fraction. These data are indirect evidences suggesting that the glucuronide conjugates of compounds 1 and 4 are probably enol glucuronides and that of compound 11 is excreted in urine as a N-glucuronide involving its pyrazole moiety. The urinary excretion profiles of the epimeric and 18-nor-17,17-dimethyl-13(14)-ene steroids are presented and discussed on the basis of their structural features.

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.

The biotransformation of methandienone (17β-hydroxy-17α-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 Δ4- and 3-oxo groups and of the Δ1-function were characterized. The major diols were 17α-methyl-5α-androstane-3α,17β-diol and 17α-methyl-5β-androstane-3α,17β-diol, which were both excreted in the conjugate fraction in a 1:3.8 ratio. The immediate metabolic precursors of the 5β-diol, namely 17β-hydroxy-17α-methyl-5β-androsta-1-en-3-one and 17α-methyl-5β-androsta-1-en-3α,17β-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. Δ4-, 3-oxo- and Δ1-. The A-ring reduction products of the structurally related steroids mestanolone, 17α-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 17β-sulfate conjugates of methandienone and 17α-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.

The use of anabolic steroids in racehorses is strictly regulated. We have developed a method for the simultaneous analysis of 11 anabolic steroids: fluoxymesterone, 17α-methyltestosterone, mestanolone, methandienone, methandriol, oxymetholone, boldenone, furazabol, methenolone, nandrolone, and stanozolol, for possible application to a doping test in racehorses. We selected 15 kinds of target substances for a doping test from the main metabolites of these anabolic steroids, and established a method for simultaneous analysis. Urine was hydrolyzed and subjected to solid-phase extraction. Then, the residue from the extracts was derivatized by trimethylsilylation. The derivatized samples were subjected to ion-trap gas chromatography-tandem mass spectrometry, and their mass chromatograms and product ion spectra were obtained. The limit of detection of the target substances was 5 - 50 ng/mL, and the mean recovery and coefficient of variation were 71.3 - 104.8% and 1.1 - 9.5%, respectively.

Anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, which were developed as oral formulations for therapeutic purposes, have been abused in the field of human sports. These anabolic steroids are also used to enhance racing performance in racehorses. In humans, structurally related 17alpha-methyltestosterone (MTS) and mestanolone (MSL), which are anabolic steroids with the 17alpha-methyl,17beta-hydroxyl group, have metabolites in common. The purpose of this study was to determine metabolites common to these two steroids in horses, which may serve as readily available screening targets for the doping test of these steroids in racehorses. Urine sample collected after administering MTS and MSL to horses was treated to obtain unconjugated steroid, glucuronide, and sulfate fractions. The fractions were subjected to gas chromatography/mass spectrometry (GC/MS), and 17alpha-methyl-5alpha-androstan-3beta,17beta-diol, 17alpha-hydroxymethyl-5alpha-androstan-3beta,17beta-diol, 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol, and 17alpha-methyl-5alpha-androstan-3beta,16alpha,17beta-triol were detected as the common metabolites by comparison with synthesized reference standards. The urinary concentrations of these metabolites after dosing were determined by GC/MS. 17Alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol was mainly detected in the sulfate fractions of urine samples after administration. This compound was consistently detected for the longest time in the urine samples after dosing with both steroids. The results suggest that 17alpha-methyl-5alpha-androstan-3beta,16beta,17beta-triol is a very useful screening target for the doping test of MTS and MSL in racehorses.

An easily available hypervalent iodine(V) reagent, 2-iodoxybenzoic acid (IBX) immobilized in the ionic liquid [bmim][Br] was found to be an efficient and eco-friendly protocol for the oxidation of 17α-methylandrostan-3β,17β-diol (1). At ambient temperature oxidation of 1 with IBX gave mestanolone (2) in good yield and with an increased stoichiometric amount of IBX, oxidation adjacent to the carbonyl functionality (α,β-unsaturation) occurred to give dehydrogenated 17β-hydroxy-17α-methyl-Δ1-androsten-3-one (3) as the major product in a one-pot reaction. The product is easily obtained by extraction with diethyl ether and evaporation of the solvent.

Due to the potential for misuse of a wide range of anabolic steroids in horse racing, a screening test to detect multiple compounds, via a common class of metabolites, would be a valuable forensic tool. An enzyme-linked immunosorbent assay (ELISA) has been developed to detect 17alpha-alkyl anabolic steroid metabolites in equine urine. 16beta-Hydroxymestanolone (16beta,17beta-dihydroxy-17alpha-methyl-5alpha-androstan-3-one) was synthesised in six steps from commercially available epiandrosterone (3beta-hydroxy-5alpha-androstan-17-one). Polyclonal antibodies were raised in sheep, employing mestanolone (17beta-hydroxy-17alpha-methyl-5alpha-androstan-3-one) or 16beta-hydroxymestanolone conjugated to human serum albumin, via a 3-carboxymethyloxime linker, as antigens. Antibody cross-reactivities were determined by assessing the ability of a library of 54 representative steroids to competitively bind the antibodies. Antibodies raised against 16beta-hydroxymestanolone showed excellent cross-reactivities for all of the 16beta,17beta-dihydroxy-17alpha-methyl steroids analysed and an ELISA has been developed to detect these steroid metabolites. Using this 16beta-hydroxymestanolone assay, urine samples from horses administered with stanozolol (17alpha-methyl-pyrazolo[4',3':2,3]-5alpha-androstan-17beta-ol), were analysed raw, following beta-glucuronidase hydrolysis, and following solid-phase extraction (SPE) procedures. The suppressed absorbances observed were consistent with detection of the metabolite 16beta-hydroxystanozolol. Positive screening results were confirmed by comparison with standard LCMS analyses. Antibodies raised against mestanolone were also used to develop an ELISA and this was used to detect metabolites retaining the parent D-ring structure following methandriol (17alpha-methylandrost-5-ene-3beta,17beta-diol) administration. The ELISA methods developed have application as primary screening tools for detection of new and known anabolic steroid metabolites.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.

We developed a gas chromatography/mass spectrometry method for detection and quantitation of anabolic steroids in head hair. Following alkaline digestion and solid-phase extraction, the MO-TMS derivatives gave a specific fragmentation pattern with EI ionization. For stanozolol, the TMS-HFBA derivative showed several diagnostic ions. For androstanolone, mestanolone (methylandrostanolone), and oxymetholone two chromatographic peaks for cis and trans isomers of derivatives were seen. Recoveries were 35 to 45% for androstanolone, oxymetholone, chlorotestosterone-acetate, dehydromethyltestosterone, dehydrotestosterone, fluoxymesterone, mestanolone, methyltestosterone, and nandrolone; 52% for mesterolone, trenbolone; 65% for bolasterone; 24% for methenolone and 17% for stanozolol. Limits of detection were 0.002 to 0.05 ng/mg and of quantitation were 0.02 to 0.1 ng/mg. Seven white male steroid abusers provided head hair samples (10 to 63 mg) and urine. In the hair samples, methyltestosterone was detected in two (confirmed in urine); nandrolone in two (also confirmed in urine); dehydromethyltestosterone in four (but not found in urine); and clenbuterol in one (but not in urine). Oxymethalone was found in urine in one, but not in the hair. One abuser had high levels of testosterone: 0.15 ng/mg hair, and 1190 ng/mL urine. We conclude that head hair analysis has considerable potential for the detection and monitoring of steroid abuse.

X-Ray diffraction and IR spectrophotometry showed that testosterone crystallized from octanol had a different form from hat crystallized from the other C5 to C10 alkanols. Differential scanning calorimetry thermograms and melting points were similar for all crystallizates, but the heat of fusion of the octanol product was greater than the others. Purity determinations by DSC revealed that the samples were at least 0.99 mole fraction pure, indicating they were polymorphs, rather than solvates. This was confirmed by thermogravimetric and elemental analysis. Methyltestosterone behaved in a similar manner, but the properties of methandienone, mestanolone and nandrolone were not affected by the nature of the crystallizing solvent.

The inhibitory effect of an anabolic steroid, stanozolol, on testicular microsomal cytochrome P-450 (17 alpha-hydroxylase/C17,20-lyase) (P-450(17 alpha/lyase] and the nature of the interaction were compared with those of other anabolic steroids, furazabol and mestanolone. Stanozolol markedly inhibited delta 16-C19-steroid synthesizing activity, 17 alpha-hydroxylase and C17,20-lyase activities, which were mediated by oxygenase activities of testicular microsomal cytochrome P-450(17 alpha/lyase). In addition, stanozolol was a competitive inhibitor of 17 alpha-hydroxylase (Ki = 6.31 microM) and C17,20-lyase (Ki = 1.30 microM) activities in the reconstituted enzyme system. The interaction of cytochrome P-450&17 alpha/lyase) with stanozolol induced a type I difference spectrum (peak at 387 nm and trough at 418 nm) with a dissociation constant (Ks) of 1.47 microM.

The solubilities of mestanolone, methandienone, nandrolone and testosterone have been determined in the n-alkanes between C7 and C16 and in n-alkanols between C5 and C12. The mole fraction solubilities in the alkanes increased rectilinearly with the carbon number of the solvent, indicating a progressively decreasing increment in free energy of mixing for each additional methylene group. The solubilities in the alkanols of all the steroids except nandrolone, followed a parabolic relationship with carbon number and with solubility parameter of the solvent, and approximately fitted regular solution theory predictions. Divergences between observed and predicted solubilities were explained in terms of an interaction between solute and solvent which invalidated the geometric mean assumption. The suggestion was supported by thermodynamic measurements. Nandrolone showed decreasing solubility with increasing solvent carbon number. Its behaviour fitted the theory suggested for the other steroids and was confirmed by the thermodynamic data.

A controlled metabolic study of the steroid mestanolone was conducted on 20 females with chronic pulmonary tuberculosis, of whom 5 served as controls and 15 received 100 mg. of mestanolone daily. A statistically significant increase in weight occurred in the treated patients and was associated with a significant retention of nitrogen, potassium and phosphorus in the ratio in which they are normally found in protoplasm. The mean gain in body weight resulting from two courses of mestanolone (eighteen days each with an interval of eighteen days between) was 2.10 Kg., and the mean nitrogen retention was 1.89 Gm. daily.

SUMMARY The ratios of the relative anabolic and androgenic activities, based on assays measuring nitrogen retention and on the increase in weight of the ventral prostate gland of rats, are presented for the following steroids: 7α:17α-dimethyltestosterone (4·2:1·3 = 3·2); oxymesterone (4-hydroxy-17α-methyltestosterone) (1·8:0·36 = 5·0); mestanolone (17α-methylandrostanolone) (0·8:1·0 = 0·8); and fluoxymesterone (9α-fluoro-11β-hydroxy-17α-methyltestosterone) (3·8:1·4 = 2·7). The degree of dissociation of the two activities appears to be considerably less than that reported earlier for certain 19-nor steroids and some heterocyclic steroids of current clinical interest.