Alternative long-term markers for the detection of methyltestosterone misuse.

Methyltestosterone (MT) is one of the most frequently detected anabolic androgenic steroids in doping control analysis. MT misuse is commonly detected by the identification of its two main metabolites excreted as glucuronide conjugates, 17α-methyl-5α-androstan-3α,17ß-diol and 17α-methyl-5ß-androstan-3α,17ß-diol. The detection of these metabolites is normally performed by gas chromatography-mass spectrometry, after previous hydrolysis with ß-glucuronidase enzymes, extraction and derivatization steps. The aim of the present work was to study the sulphate fraction of MT and to evaluate their potential to improve the detection of the misuse of the drug in sports. MT was administered to healthy volunteers and urine samples were collected up to 30days after administration. After an extraction with ethyl acetate, urine extracts were analysed by liquid chromatography tandem mass spectrometry using electrospray ionisation in negative mode by monitoring the transition m/z 385 to m/z 97. Three diol sulphate metabolites (S1, S2 and S3) were detected. Potential structures for these metabolites were proposed after solvolysis and mass spectrometric experiments: S1, 17α-methyl-5ß-androstan-3α,17ß-diol 3α-sulphate; S2, 17ß-methyl-5α-androstan-3α,17α-diol 3α-sulphate; and S3, 17ß-methyl-5ß-androstan-3α,17α-diol 3α-sulphate. Synthesis of reference compounds will be required in order to confirm the structures. The retrospectivity of these sulphate metabolites in the detection of MT misuse was compared with the obtained with previously described metabolites. Metabolite S2 was detected up to 21days after MT administration, improving between 2 and 3 times the retrospectivity of the detection compared to the last long-term metabolite of MT previously described, 17α-hydroxy-17ß-methylandrostan-4,6-dien-3-one.

Long-term detection of methyltestosterone (ab-) use by a yeast transactivation system.

The routinely used analytical method for detecting the abuse of anabolic steroids only allows the detection of molecules with known analytical properties. In our supplementary approach to structure-independent detection, substances are identified by their biological activity. In the present study, urines excreted after oral methyltestosterone (MT) administration were analyzed by a yeast androgen screen (YAS). The aim was to trace the excretion of MT or its metabolites in human urine samples and to compare the results with those from the established analytical method. MT and its two major metabolites were tested as pure compounds in the YAS. In a second step, the ability of the YAS to detect MT and its metabolites in urine samples was analyzed. For this purpose, a human volunteer ingested of a single dose of 5 mg methyltestosterone. Urine samples were collected after different time intervals (0-307 h) and were analyzed in the YAS and in parallel by GC/MS. Whereas the YAS was able to trace MT in urine samples at least for 14 days, the detection limits of the GC/MS method allowed follow-up until day six. In conclusion, our results demonstrate that the yeast reporter gene system could detect the activity of anabolic steroids like methyltestosterone with high sensitivity even in urine. Furthermore, the YAS was able to detect MT abuse for a longer period of time than classical GC/MS. Obviously, the system responds to long-lasting metabolites yet unidentified. Therefore, the YAS can be a powerful (pre-) screening tool with the potential that to be used to identify persistent or late screening metabolites of anabolic steroids, which could be used for an enhancement of the sensitivity of GC/MS detection techniques.

Determination of the hormonal growth promoter 17alpha-methyltestosterone in food-producing animals: bovine hair analysis by HPLC-MS/MS.

This paper describes the development, validation and application of a confirmatory method to detect 17alpha-methyltestosterone (MT) in bovine hair, to aid in controlling the administration of this growth promoter in meat-producing animals. After cryogenic grinding, MT was removed from the hair matrix using a single step extraction procedure with acetonitrile. Hydroxylamine derivatisation was used to enhance analyte determination with an electrospray ionisation (ESI) source. Determination was carried out using a triple quadrupole liquid chromatography tandem mass spectrometer (LC-MS/MS) in multiple reaction monitoring mode (MRM). The method was validated in accordance with the criteria defined in Commission Decision 2002/657/EC and using deuterated testosterone (T-d(3)) as the internal standard. The decision limit (CCalpha) was 0.07 ng g(-1) and the detection capability (CCbeta) was 0.12 ng g(-1). Repeatability was CV% (7%), within-laboratory reproducibility was CV% (11.0%), and trueness was (87%). Applicability of the method was demonstrated in an animal study. Samples obtained from animal experiments were analyzed and the presence of MT was confirmed.

In vivo biotransformation of 17 alpha-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry.

The in vivo phase I biotransformation of 17 alpha-methyltestosterone in the horse leads to the formation of a complex mixture of regio- and stereoisomeric C(20)O(2), C(20)O(3) and C(20)O(4) metabolites, excreted in urine as glucuronide and sulphate phase II conjugates. The major pathways of in vivo metabolism are the reduction of the A-ring (di- and tetrahydro), epimerisation at C-17 and oxidations mainly at C-6 and C-16. Some phase I metabolites have been identified previously by positive ion electron ionisation capillary gas chromatography/mass spectrometry (GC/EI + MS) mainly from the characteristic fragmentation patterns of their methyloxime-trimethylsilyl ether (MO-TMS), enol-TMS or TMS ether derivatives. Following oral administration of 17 alpha-methyltestosterone to two castrated thoroughbred male horses, the glucuronic acid conjugates excreted in post-administration urine samples were selectively hydrolysed by E. coli beta-glucuronidase enzymes. Unconjugated metabolites and the steroid aglycones obtained after enzymatic deconjugation were isolated from urine by solid-phase extraction, derivatised as MO-TMS ethers and analysed by GC/EI + MS. In addition to some of the known metabolites previously identified from the characteristic mass spectral fragmentation patterns of 17 alpha-methyl steroids, some isobaric compounds exhibiting a diagnostic loss of 103 mass units from the molecular ions with subsequent losses of trimethylsilanol or methoxy groups and an absence of the classical D-ring fragment ion were detected. From an interpretation of their mass spectra, these compounds were identified as 17-hydroxymethyl metabolites, formed in vivo in the horse by oxidation of the 17-methyl moiety of 17 alpha-methyltestosterone. This study reports on the GC/EI + MS identification of these novel 17-hydroxymethyl C(20)O(3) and C(20)O(4) metabolites of 17 alpha-methyltestosterone excreted in thoroughbred horse urine.

Methyltestosterone pharmacokinetics and oral bioavailability in rainbow trout (Oncorhynchus mykiss).

14C-methyltestosterone pharmacokinetics after intraarterial (2 and 20 mg/kg) and oral (30 mg/kg) administration were investigated in rainbow trout at 15 degrees C. Plasma concentrations of methyltestosterone were determined by reverse phase high performance liquid chromatography (HPLC) in combination with reverse isotope dilution for up to 6 and 12 days after oral and intraarterial administration, respectively. Methyltestosterone pharmacokinetic parameter values after intraarterial administration were determined using a two compartment model (WinNonlin). For the 2 and 20 mg/kg doses, respectively, the parameter values were, area under the plasma concentration-time curve (11.2 and 82.3 micromol h/l), total body clearance (0.640 and 0.903 l/h per kg), distribution half-life (4.13 and 8.23 h), elimination half-life (54.9 and 58.6 h), volume of the central compartment (3.83 and 13.9 l/kg), volume of distribution at steady state (6.06 and 26.8 l/kg), and the mean residence time (9.57 and 22.7 h). After oral administration, the following parameter values were assessed using a model-independent method, peak concentration (3.03 micromol/l), time of concentration peak (8.80 h), mean absorption time (13.8 h), and area under curve (AUC)(0-->infinity) (90.2 micromol h/l). A two compartment model analysis of the average plasma concentration-time profile after oral administration showed that absorption followed first-order kinetics with a half-life of 4.7 h. The oral bioavailability of methyltestosterone from food was about 70%.

Stable isotope dilution analysis of human urinary metabolites of 17alpha-methyltestosterone.

A method based on gas chromatography-mass spectrometry-selected-ion monitoring was developed to measure the main metabolites of 17alpha-methyltestosterone, 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, in human urine. 17alpha-Methyl-[(2)H3]-5alpha-androstan-3alpha,1 7beta-diol and 17alpha-methyl-[(2)H3]-5beta-androstan-3alpha,17 beta-diol were used as internal standards. The methods involved purification using a Sep-Pak C(18) cartridge, hydrolysis by beta-glucuronidase from Ampullaria and derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide/dithioerythriol/ammon ium iodide. Quantitation was achieved by selected-ion monitoring of the characteristic fragment ions ([(M+H)-2xTMSOH]+) of the di-TMS derivatives on the chemical ionization mode. The method provides a specific, sensitive and reliable technique to determine the urine levels of 17alpha-methyl-5alpha-androstan-3alpha,17beta-di ol and 17alpha-methyl-5beta-androstan-3alpha,17beta-dio l, and can be applied to pharmacokinetic studies of 17alpha-methyltestosterone.

Biotransformation and branchial excretion of 17 alpha-methyltestosterone in trout.

This study was designed to characterize the branchial excretion of 17 alpha-methyltestosterone (17MT) metabolites in rainbow trout (Oncorhynchus mykiss). Spinally transected trout were force-fed 0.4 mg/kg [1,2-3H]17MT and the branchial, fecal, and urinary excreta were collected during 24 hr. Branchial elimination was the primary route of excretion, because it contributed to 68% of the excreted radioactivity within 24 hr after ingestion. The radio-HPLC metabolic profile obtained from branchial excreta showed that 17MT was partly eliminated across the gills as parent compound. In addition to unchanged 17MT, several unconjugated metabolites have been isolated and identified by GC/MS. Two major pathways were involved in the biotransformation of 17MT: hydroxylation and/or reduction of the androstene structure. Reduction of the 4-ene functionality leading to the formation of methyldihydrotestosterone and its further reduction leading to the formation of methylandrostane-diol metabolites was observed. Other metabolites resulted from hydroxylation of 17MT at C-6 and C-7 positions and eventual further reduction of the 3-oxo-delta 4 group. They were tentatively assigned the structures of 17 alpha-methyl-4-androsten-6 beta,17 beta-ol-3-one, 17 alpha-methyl-4-androsten-7 xi,17 beta-ol-3-one, and 17 alpha-methyl-5-xi-androstan-3 xi,7 xi-triol. In addition, 17 alpha-methyl-4-androsten-17 beta-ol-3,11-dione and 17 alpha-methyl-17 beta-hydroxy-4,6-androstadiene-3-one were also identified and resulted probably from the oxidation of 11-hydroxy-17MT and dehydration of 6-hydroxy-17MT, respectively.

Effect of hydrotropic substances on the complexation of sparingly soluble drugs with cyclodextrin derivatives and the influence of cyclodextrin complexation on the pharmacokinetics of the drugs.

The influence of hydrotropic compounds on complex formation by 2-hydroxypropyl-beta-cyclodextrin (2-HP-beta-CD) was investigated with methyltestosterone (MeT). Various representatives of the lyotropic series were used for this purpose. Additive hydrotropic effects were observed for nicotinamide and urea, which disrupt the water structure, while structure formers such as sorbitol exerted negative effects. The effects of hydrotropic substances on the phase solubility relationship of MeT showed that inclusion complex formation with 2-HP-beta-CD depends on the degree of ordering of the solvent and is apparently subject to entropy effects. Combined systems comprising 2-HP-beta-CD and auxiliary substances with various underlying solubilizing principles were also investigated. Combination of 2-HP-beta-CD with conventional solubilizers, such as 1,2-propylene glycol or sodium deoxycholate, reduced the solubilization capacity of 2-HP-beta-CD. Competitive displacement of the inclusion molecule from its 2-HP-beta-CD complex by sodium deoxycholate suggested that cholesterol participates in the release mechanism of the inclusion molecule under in vivo conditions. The spontaneous release of complexed drug molecules could indirectly be shown on the basis of the spontaneous action of a complexed dihydropyridine derivative after iv administration in rats. The bioavailability of an investigational drug in cynomolgus monkeys could be enhanced sevenfold by inclusion complexation with 2-HP-beta-CD.

Stable isotope methodology in the pharmacokinetic studies of androgenic steroids in humans.

The use of stable isotopically labeled steroids combined with gas chromatography/mass spectrometry (GC/MS) has found a broad application in pharmacologic studies. Initially, stable isotopically labeled steroids served as the ideal analytic internal standard for GC/MS analysis; however, their in vivo use has expanded and has proven to be a powerful pharmacokinetic tool. We have successfully used stable isotope methodology to study the pharmacokinetic/bioavailability of androgens. The primary advantage of the technique is that endogenous and exogenous steroids with the same basic structure can be differentiated by using stable isotopically labeled analogs. The method was used to examine the pharmacokinetics of testosterone and testosterone propionate, and to clarify the influence of endogenous testosterone. Another advantage of the isotope methods is that steroidal drugs can be administered concomitantly in two formulations (e.g., solution and solid dosage). A single set of blood samples serves to describe the time course of the formulations being compared. This stable isotope coadministration technique was used to estimate the relative bioavailability of 17 alpha-methyltestosterone.