Detection time comparison of non-hydrolysed sulphated metabolites of metenolone, mesterolone and 17α-methyltestosterone analysed by four different mass spectrometric techniques.

The frequent detection of anabolic androgenic steroids (AAS) indicates their popularity among rule-breaking athletes. The so called long-term metabolites play a crucial role in their detection, and non-hydrolysed sulphated metabolites have gained renewed interest, as research has demonstrated their extended detection time compared to the more conventional markers (e.g., for metenolone and mesterolone). Their potential has been investigated using liquid and gas chromatography-mass spectrometry (LC- and GC-MS). However, due to their complementary nature, chances are that the most promising metabolite on one technique does not necessarily exhibit the same behaviour on the other and vice versa. Therefore, a comparison was carried out where as a trial model, metenolone, mesterolone and 17α-methyltestosterone were selected and the most likely long-term sulphated metabolites identified on four mass spectrometric instruments. Additionally, using a modified sample preparation procedure, comparison between conventional and non-hydrolysed sulphated metabolites between different GC-MS instruments was also included. When focusing on each individual marker, no cases were observed where a single metabolite provided a superior detection time on all instruments. Furthermore, for each AAS, there were incidences where a metabolite provided the best detection time on one instrument but could only be detected for a shorter period or not at all on other instruments. This demonstrates that metabolite detection windows and hence their added-value as target substance are unique and dependent on the analytical technique and not only on their pharmacokinetic behaviour. Consequently, in each case, a metabolite versus instrument evaluation is needed to maximise the probabilities of detecting doping offences.

Endocrine characterization of the designer steroid methyl-1-testosterone: investigations on tissue-specific anabolic-androgenic potency, side effects, and metabolism.

Various products containing rarely characterized anabolic steroids are nowadays marketed as dietary supplements. Herein, the designer steroid methyl-1-testosterone (M1T) (17ß-hydroxy-17α-methyl-5α-androst-1-en-3-one) was identified, and its biological activity, potential adverse effects, and metabolism were investigated. The affinity of M1T toward the androgen receptor (AR) was tested in vitro using a yeast AR transactivation assay. Its tissue-specific androgenic and anabolic potency and potential adverse effects were studied in a Hershberger assay (sc or oral), and tissue weights and selected molecular markers were investigated. Determination of M1T and its metabolites was performed by gas chromatography mass spectrometry. In the yeast AR transactivation assay, M1T was characterized as potent androgen. In rats, M1T dose-dependently stimulated prostate and levator ani muscle weight after sc administration. Oral administration had no effect but stimulated proliferation in the prostate and modulated IGF-I and AR expression in the gastrocnemius muscle in a dose-dependent manner. Analysis of tyrosine aminotransferase expression provided evidence for a strong activity of M1T in the liver (much higher after oral administration). In rat urine, 17α-methyl-5α-androstane-3α,17ß-diol, M1T, and a hydroxylated metabolite were identified. In humans, M1T was confirmed in urine in addition to its main metabolites 17α-methyl-5α-androst-1-ene-3α,17ß-diol and 17α-methyl-5α-androstane-3α,17ß-diol. Additionally, the corresponding 17-epimers as well as 17ß-hydroxymethyl-17α-methyl-18-nor-5α-androsta-1,13-dien-3-one and its 17-epimer were detected, and their elimination kinetics was monitored. It was demonstrated that M1T is a potent androgenic and anabolic steroid after oral and sc administration. Obviously, this substance shows no selective AR modulator characteristics and might exhibit liver toxicity, especially after oral administration.

Detection of Δ6-methyltestosterone in a "dietary supplement" and GC-MS/MS investigations on its urinary metabolism.

Since a few years more and more products have appeared on the market for dietary supplements containing steroids that had never been marketed as approved drugs, mostly without proper labeling of the contents. Syntheses and few data on pharmacological effects are available dated back mainly to the 1950s or 1960s. Only little knowledge exists about effects and side effects of these steroids in humans. The present study reports the identification of Δ6-methyltestosterone in a product named "Jungle Warfare", which was obtained from a web-based supplement store. The main urinary metabolites, 17α-hydroxy-17ß-methylandrosta-4,6-dien-3-one (Δ6-epimethyl-testosterone), 17α-methyl-5ß-androstane-3α,17ß-diol (3α,5ß-THMT), and 17ß-methyl-5ß-androstane-3α,17α-diol, as well as the parent compound excreted after a single oral administration were monitored by GC-MS/MS. Δ6-Epimethyltestosterone and 3α,5ß-THMT served for long-term detection (still present in the 181-189 h urine). 17α-Methyltestosterone and its 17-epimer were not detected in the urines (LOD 0.3ng/mL). The highest concentrations were found in the 14-20.5h urine for Δ6-epimethyltestosterone (600 ng/mL), and 3α,5ß-THMT (240 ng/mL) and in the 36-44.5h urine for 17ß-methyl-5ß-androstane-3α,17α-diol (7 ng/mL). For reference methyltestosterone and epimethyltestosterone were dehydrogenated with chloranil. The characterization of the products was performed by GC-MS(/MS) and NMR.

Using the protein chip to screen agonists and antagonists of the androgen receptor.

Based on its important physical and pathological function, the androgen receptor (AR) is regarded as a significant drug target. In this report, the authors describe a novel strategy of protein chip technology to screen agonists and antagonists of AR. First, the AR ligand binding domain (AR-LBD) was expressed in Escherichia coli, purified, and then immobilized on a silane-polysaccharide surface of a protein chip. Second, the affinities of methyltestosterone (MT) and fluorescent-labeled testosterone for the AR-LBD protein chip were determined. Third, a converse strategy of the protein chip was tested to evaluate its reliability as a drug screening method. Fourth, a 10,067-compound library was screened to find new ligands of AR. From the results, the K(d) of testosterone and the IC(50) of MT are consistent with the literature (0.61 vs. 0.49 nM 2.88 vs. 3.90 nM, respectively). The Z' factor of the high-throughput screening (HTS) method was 0.76, which meets the requirement of drug screening (>0.4). Finally, 3 active ligands of AR were identified with their IC( 50) values of 3.63, 2.19, and 1.71 microM, respectively. In summary, the novel strategy of the AR-LBD protein chip was suitable for HTS at the molecular level.