Searching for new long-term urinary metabolites of metenolone and drostanolone using gas chromatography-mass spectrometry with a focus on non-hydrolysed sulfates.

Sulfated metabolites have been shown to have potential as long-term markers of anabolic-androgenic steroid (AAS) abuse. In 2019, the compatibility of gas chromatography-mass spectrometry (GC-MS) with non-hydrolysed sulfated steroids was demonstrated, and this approach allowed the incorporation of these compounds in a broad GC-MS initial testing procedure at a later stage. However, research is needed to identify which are beneficial. In this study, a search for new long-term metabolites of two popular AAS, metenolone and drostanolone, was undertaken through two excretion studies each. The excretion samples were analysed using GC-chemical ionization-triple quadrupole MS (GC-CI-MS/MS) after the application of three separate sample preparation methodologies (i.e. hydrolysis with Escherichia coli-derived ß-glucuronidase, Helix pomatia-derived ß-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1ß-methyl-5α-androstan-17-one-3ζ-sulfate, was documented for the first time to provide the longest detection time of up to 17 days. This metabolite increased the detection time by nearly a factor of 2 in comparison with the currently monitored markers for metenolone in a routine doping control initial testing procedure. In the second excretion study, it prolonged the detection window by 25%. In the case of drostanolone, the non-hydrolysed sulfated metabolite with the longest detection time was the sulfated analogue of the main drostanolone metabolite (3α-hydroxy-2α-methyl-5α-androstan-17-one) with a detection time of up to 24 days. However, the currently monitored main drostanolone metabolite in routine doping control, after hydrolysis of the glucuronide with E.coli, remained superior in detection time (i.e. up to 29 days).

New long term metabolite in human urine for metenolone misuse by liquid chromatography quadrupole time-of-flight mass spectrometry.

In this study, metenolone metabolic profiles were investigated. Metenolone was administered to one healthy male volunteer. Liquid-liquid extraction and direct-injection were applied to processing urine samples. Urinary extracts were analyzed by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOFMS) using full scan and product ion scan with accurate mass measurement for the first time. Due to the lack of useful fragment ion for structural elucidation, GC-MS instrumentation was employed to obtain structural details of the trimethylsilylated phase I metabolite released after hydrolysis, and the EI mass spectrum was always informative in steroidal structure studies owing to more useful fragment ions than the ESI mass spectrum. 16 metabolites including 6 glucuronide and 9 unreported sulfate conjugates were characterized and tentatively identified. All the metabolites were evaluated in terms of how long they could be detected. The sulfate conjugate S6 (1-methylen-5α-androst-3,17-dione-2ξ-sulfate) was considered to be a new long term metabolite for metenolone misuse that could be detected 40 days by liquid-liquid extraction and up to 30 days by direct-injection analysis after oral administration.

Comparison of sulfo-conjugated and gluco-conjugated urinary metabolites for detection of methenolone misuse in doping control by LC-HRMS, GC-MS and GC-HRMS.

Methenolone (17ß-hydroxy-1-methyl-5α-androst-1-en-3-one) misuse in doping control is commonly detected by monitoring the parent molecule and its metabolite (1-methylene-5α-androstan-3α-ol-17-one) excreted conjugated with glucuronic acid using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) for the parent molecule, after hydrolysis with ß-glucuronidase. The aim of the present study was the evaluation of the sulfate fraction of methenolone metabolism by LC-high resolution (HR)MS and the estimation of the long-term detectability of its sulfate metabolites analyzed by liquid chromatography tandem mass spectrometry (LC-HRMSMS) compared with the current practice for the detection of methenolone misuse used by the anti-doping laboratories. Methenolone was administered to two healthy male volunteers, and urine samples were collected up to 12 and 26 days, respectively. Ethyl acetate extraction at weak alkaline pH was performed and then the sulfate conjugates were analyzed by LC-HRMS using electrospray ionization in negative mode searching for [M-H](-) ions corresponding to potential sulfate structures (comprising structure alterations such as hydroxylations, oxidations, reductions and combinations of them). Eight sulfate metabolites were finally detected, but four of them were considered important as the most abundant and long term detectable. LC clean up followed by solvolysis and GC/MS analysis of trimethylsilylated (TMS) derivatives reveal that the sulfate analogs of methenolone as well as of 1-methylene-5α-androstan-3α-ol-17-one, 3z-hydroxy-1ß-methyl-5α-androstan-17-one and 16ß-hydroxy-1-methyl-5α-androst-1-ene-3,17-dione were the major metabolites in the sulfate fraction. The results of the present study also document for the first time the methenolone sulfate as well as the 3z-hydroxy-1ß-methyl-5α-androstan-17-one sulfate as metabolites of methenolone in human urine. The time window for the detectability of methenolone sulfate metabolites by LC-HRMS is comparable with that of their hydrolyzed glucuronide analogs analyzed by GC-MS. The results of the study demonstrate the importance of sulfation as a phase II metabolic pathway for methenolone metabolism, proposing four metabolites as significant components of the sulfate fraction.

Development and application of stir bar sorptive extraction with polyurethane foams for the determination of testosterone and methenolone in urine matrices.

This work describes the development, validation, and application of a novel methodology for the determination of testosterone and methenolone in urine matrices by stir bar sorptive extraction using polyurethane foams [SBSE(PU)] followed by liquid desorption and high-performance liquid chromatography with diode array detection. The methodology was optimized in terms of extraction time, agitation speed, pH, ionic strength and organic modifier, as well as back-extraction solvent and desorption time. Under optimized experimental conditions, convenient accuracy were achieved with average recoveries of 49.7 8.6% for testosterone and 54.2 ± 4.7% for methenolone. Additionally, the methodology showed good precision (<9%), excellent linear dynamic ranges (>0.9963) and convenient detection limits (0.2-0.3 µg/L). When comparing the efficiency obtained by SBSE(PU) and with the conventional polydimethylsiloxane phase [SBSE(PDMS)], yields up to four-fold higher are attained for the former, under the same experimental conditions. The application of the proposed methodology for the analysis of testosterone and methenolone in urine matrices showed negligible matrix effects and good analytical performance.

Metabolism of methenolone acetate in a veal calf.

The use of anabolic steroids has been banned in the European Union since 1981. In this study, the metabolism of the anabolic steroid methenolone acetate, was investigated in a male veal calf. After daily oral administration of methenolone acetate, three main metabolites were detected in both urine and faeces samples. Among these metabolites, alpha-methenolone was apparently the main one, but 1-methyl-5alpha-androstan-3,17-diol and 3alpha-hydroxy-1-methyl-5alpha-androstan-17-one were also observed. The parent compound was still detectable in faeces. As a consequence, abuse of methenolone acetate as growth promoter can be monitored by analysing urine and faeces samples. A few days after the last treatment, however, no metabolites were observed. Alpha-methenolone was detectable in urine until 5 days after the last treatment, but in faeces no metabolites were detectable after 3 days.

Effect on sports drug tests of ingesting meat from steroid (methenolone)-treated livestock.

Anabolic-androgenic steroids are widely misused in human sports and are also used as growth promoters in livestock. Athletes who consume meat containing such hormone residues may risk failing a sports drug test. Prompted by an athlete's defense case, we questioned whether the consumption of small livestock given doses of anabolic steroid, orally or intramuscularly, could generate positive results in samples tested by our analytical procedures. We analyzed urine from eight men who consumed chickens that had been either fed with methenolone acetate (1 mg/day) from day 0 to 21 or injected with methenolone heptanoate depot (1 mg/intramuscular injection) on days 0, 7, and 14 and slaughtered on day 22. No methenolone or characteristic major metabolite was detected in samples from subjects who ate meat from the orally dosed chickens. However, 50% of the samples collected 24 h after consumption of the intramuscularly dosed chickens were confirmed positive. Hence, eating meat containing small amounts of injected hormone may constitute a serious liability to the athlete.

Studies on anabolic steroids--11. 18-hydroxylated metabolites of mesterolone, methenolone and stenbolone: new steroids isolated from human urine.

New metabolites of mesterolone, methenolone and stenbolone bearing a C18 hydroxyl group were isolated from the steroid glucuronide fraction of urine specimens collected after administration of single 50 mg doses of these steroids to human subjects. Mesterolone gave rise to four metabolites which were identified by gas chromatography/mass spectrometry as 18-hydroxy-1 alpha-methyl-5 alpha-androstan-3,17-dione 1, 3 alpha,18-dihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 2, 3 beta,18-dihydroxy-1-alpha-methyl-5 alpha-androstan-17-one 3 and 3 alpha,6 xi,18-trihydroxy-1 alpha-methyl-5 alpha-androstan-17-one 4. These data suggest that mesterolone itself was not hydroxylated at C18, but rather 1 alpha-methyl-5 alpha-androstan-3,17-dione, an intermediate metabolite which results from oxidation of mesterolone 17-hydroxyl group. In addition to hydroxylation at C18, reduction of the 3-keto group and further hydroxylation at C6 were other reactions that led to the formation of these metabolites. It is of interest to note that in the case of both methenolone and stenbolone, only one 18-hydroxylated urinary metabolite namely 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 5 and 18-hydroxy-1-methyl-5 alpha-androst-1-ene-3,17-dione 6 were both detected in post-administration urine specimens. These data indicate that the presence of a methyl group at the C1 or C2 positions in the steroids studied is a structural feature that seems to favor interaction of hepatic 18-hydroxylases with these steroids. These data provide further evidence that 18-hydroxylation of endogenous steroids can also occur in extra-adrenal sites in man.

Studies on anabolic steroids--4. Identification of new urinary metabolites of methenolone acetate (Primobolan) in human by gas chromatography/mass spectrometry.

The metabolism of methenolone acetate (17 beta-acetoxy-1-methyl-5 alpha-androst-1-en-3-one), a synthetic anabolic steroid, has been investigated in man. After oral administration of a 50 mg dose of the steroid to two male volunteers, twelve metabolites were detected in urine either in the glucuronide, sulfate or free steroid fractions. Methenolone, the parent steroid was detected in urine until 90 h after administration. Its cumulative urinary excretion accounted for 1.63% of the ingested dose. With the exception of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major biotransformation product of methonolone acetate, metabolites were excreted in urine at lower levels, through minor metabolic routes. Most of methenolone acetate metabolites were isolated from the glucuronic acid fraction, namely methenolone, 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, 3 alpha-hydroxy-1 alpha-methyl-5 alpha-androstan-17-one, 17-epimethenolone, 3 alpha,6 beta-dihydroxy-1-methylen-5 alpha-androstan-17-one, 2 xi-hydroxy-1-methylen-5 alpha-androstan-3,17-dione, 6 beta-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione, 16 alpha-hydroxy-1-methyl-5 alpha-androst-1-en-3,17-dione and 3 alpha,16 alpha-dihydroxy-1-methyl-5 alpha-androst-1-en-17-one. Interestingly, the metabolites detected in the sulfate fraction were isomeric steroids bearing a 16 alpha- or a 16 beta-hydroxyl group, whereas 1-methyl-5 alpha-androst-1-en-3,17-dione was the sole metabolite isolated from the free steroid fraction. Steroids identity was assigned on the basis of the mass spectral features of their TMS ether, TMS enol-TMS ether, MO-TMS, and d9-TMS ether derivatives and by comparison with reference and structurally related steroids. The data indicated that methenolone acetate was metabolized into several compounds resulting from oxidation of the 17-hydroxyl group and reduction of A-ring substituents, with or without concomitant hydroxylation at the C6 and C16 positions.

Detection and quantitation of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major urinary metabolite of methenolone acetate (Primobolan) by isotope dilution--mass spectrometry.

A highly accurate method has been developed for detection and quantitation of 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one, the major urinary metabolite of methenolone acetate (Primobolan) in man. Unlabelled as well as 2H-labelled 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one were synthesized from 1-methylen-5 alpha-androstane-3,17-dione. A fixed amount of the internal standard was added to a fixed amount of urine and the mixture was treated with Helix pomatia for 24 h. After extraction and purification by t.l.c., the mixture was converted into methoxime--trimethylsilyl derivative and analyzed by combined GC--MS. Unlabelled 3 alpha-hydroxy-1-methylen-5 alpha-androstan-17-one could be quantitated from the ratio between the tracings of the ions at m/z 372 and m/z 375 (corresponding to the M-31 ions). In alternative procedures, the ions at m/z 403 and m/z 406 (molecular ions) as well as m/z 282 and m/z 285 (M-90-31 ions) could be used. Under the conditions employed, the metabolite could be identified and quantitated in concentrations exceeding 10 ng/ml. Significant amounts of the metabolite could be detected in urine during 5 days after a single oral ingestion of 10 mg of Primobolan. The method has been successfully used for analyses of urine samples obtained from athletes involved in competition.