methenolone and dromostanolone

The World Anti-Doping Agency (WADA) publishes yearly their prohibited list, and sets a minimum required performance limit for each substance. To comply with these stringent requirements, the anti-doping laboratories have at least two complementary methods for their initial testing procedure (ITP), one using gas chromatography - mass spectrometry (GC-MS) and the other using liquid chromatography-MS (LC-MS). Anabolic androgenic steroids (AAS) have in previous years consistently been listed as the most frequently detected class of compounds. Over the last decade, evidence has emerged where a longer detection time is attained by focusing on sulfated metabolites of AAS instead of the conventional gluco-conjugated metabolites. Despite a decade of research on sulphated AAS using LC-MS, no LC-MS ITP has been developed that combines this class of compounds with the other mandatory targets. Such combination is essential for economical purposes. Recently, it was demonstrated that the direct injection of non-hydrolysed sulfates is compatible with GC-MS. Using this approach and by taking full use of the open screening capabilities of the quadrupole time of flight MS (QTOF-MS), this work describes for the first time a validated ITP that allows the detection of non-hydrolysed sulfated metabolites of AAS while, simultaneously, remaining capable of detecting a vast range of other classes of compounds, as well as the quantification of endogenous steroids, as required for an ITP compliant with the applicable WADA regulations. The method contains 263 compounds from 9 categories, including stimulants, narcotics, anabolic androgenic steroids and beta-blockers. Additionally, the advantages of the new method were illustrated by analysing excretion samples of drostanolone, mesterolone and metenolone. No negative effects were observed for the conventional markers and the detection time for mesterolone and metenolone increased by up to 150% and 144%, respectively compared to conventional markers.

Topics: Adult; Anabolic Agents; Androstanols; Doping in Sports; Gas Chromatography-Mass Spectrometry; Humans; Hydrolysis; Limit of Detection; Male; Mass Screening; Metabolome; Methenolone; Reproducibility of Results; Steroids; Substance Abuse Detection

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 β-glucuronidase, Helix pomatia-derived β-glucuronidase/arylsulfatase and non-hydrolysed sulfated steroids). For metenolone, a non-hydrolysed sulfated metabolite, 1β-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).

Topics: Adult; Anabolic Agents; Androstanols; Doping in Sports; Gas Chromatography-Mass Spectrometry; Humans; Male; Methenolone; Substance Abuse Detection; Sulfates; Tandem Mass Spectrometry

Many athletes use drugs, especially anabolic androgenic steroids (AAS), but there are few reports on the endocrinological and pathological changes in AAS abusers. In this study we reported the results of endocrinological examinations in rats administered AAS and also physical changes. We separated 37 male Wistar rats (7 weeks old) into 3 groups: Group A was medicated with nandrolone decanoate, metenolone acetate, and dromostanolone; Group B with nandrolone decanoate and saline; and Group C was given only saline. They were given subcutaneous injections of the medications or the control vehicle once a week for 6 weeks. Medications were stopped for 4 weeks, and then resumed for another 6 weeks. After that, rats were sacrificed. Serum testosterone level in Group A was significantly higher than that in Group C. Serum dihydrotestosterone in Group A was significantly higher than that in both Groups B and C. Serum estradiol-17beta levels in Groups A and B were significantly higher than that in Group C. In pathological evaluation, heart, testis, and adrenal gland were severely damaged. These findings indicate that there is a high degree of risk related to the use of AAS.

Topics: Adrenal Glands; Anabolic Agents; Androgens; Androstanols; Animals; Behavior, Animal; Dihydrotestosterone; Estradiol; Male; Methenolone; Myocardium; Nandrolone; Nandrolone Decanoate; Rats; Rats, Wistar; Testis; Testosterone

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.

Topics: Anabolic Agents; Androstanols; Biotransformation; Fluorocarbons; Gas Chromatography-Mass Spectrometry; Humans; Indicators and Reagents; Methenolone; Nandrolone; Reproducibility of Results; Sensitivity and Specificity; Substance Abuse Detection; Testosterone