Enabling the inclusion of non-hydrolysed sulfated long term anabolic steroid metabolites in a screening for doping substances by means of gas chromatography quadrupole time-of-flight mass spectrometry.

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.

Quantitative analysis of total methenolone in animal source food by liquid chromatography-tandem mass spectrometry.

Methenolone, an anabolic androgenic steroid, has been applied to improve the quality and protein content of meat in animal husbandry. However, the usage of methenolone in sports is banned for its doping effects. Several methods have been reported to monitor the content of methenolone in serum and urine samples, but a highly sensitive detection system has not been developed for the determination of methenolone in animal source food due to its constituent complexity. In this study, a novel detection system was developed to quantify the contents of both free and conjugated methenolone in animal source food including pork, beef, mutton, milk, and eggs by using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) coupled with delicate pretreatment procedures. The conjugated methenolone in the above food samples was released by dual enzyme digestion, and the total methenolone was extracted by 1% formic acid in acetonitrile, followed by the purification using a PRiME HLB column or QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) salt. The compound d3 -methyltestosterone was used as an internal standard to minimize matrix interference. Finally, a wide linear range (0.5-20 µg/kg), low limit of detection (LOD) (0.3 µg/kg), good precision (<7% relative standard deviation), and high recovery (>90%) were obtained in the study of method validation. In summary, this analytical method provides a practicable monitoring tool for the quantification of methenolone in animal source food.

In vitro metabolic studies using homogenized horse liver in place of horse liver microsomes.

The study of the metabolism of drugs, in particular steroids, by both in vitro and in vivo methods has been carried out in the authors' laboratory for many years. For in vitro metabolic studies, the microsomal fraction isolated from horse liver is often used. However, the process of isolating liver microsomes is cumbersome and tedious. In addition, centrifugation at high speeds (over 100 000 g) may lead to loss of enzymes involved in phase I metabolism, which may account for the difference often observed between in vivo and in vitro results. We have therefore investigated the feasibility of using homogenized horse liver instead of liver microsomes with the aim of saving preparation time and improving the correlation between in vitro and in vivo results. Indeed, the preparation of the homogenized horse liver was very simple, needing only to homogenize the required amount of liver. Even though no further purification steps were performed before the homogenized liver was used, the cleanliness of the extracts obtained, based on gas chromatography-mass spectrometry (GC-MS) analysis, was similar to that for liver microsomes. Herein, the results of the in vitro experiments carried out using homogenized horse liver for five anabolic steroids-turinabol, methenolone acetate, androst-4-ene-3,6,17-trione, testosterone, and epitestosterone-are discussed. In addition to the previously reported in vitro metabolites, some additional known in vivo metabolites in the equine could also be detected. As far as we know, this is the first report of the successful use of homogenized liver in the horse for carrying out in vitro metabolism experiments. Copyright © 2011 John Wiley & Sons, Ltd.

Doping control for methenolone using hair analysis by gas chromatography-tandem mass spectrometry.

A sensitive, specific and reproducible method for the quantitative determination of methenolone in human hair has been developed. The sample preparation involved a decontamination step of the hair with methylene chloride. The hair sample (about 100 mg) was solubilized in 1 ml 1 M NaOH, 15 min at 95 degrees C, in presence of 1 ng testosterone-d3 used as internal standard. The homogenate was neutralized and extracted using consecutively a solid-phase (Isolute C18 eluted with methanol) and a liquid-liquid (pentane) extraction. The residue was derivatized by adding 50 microl MSTFA-NH4I-2-mercaptoethanol (1000:2:5, v/v/v), then incubated for 20 ml at 60 degrees C. A 1.5-microl aliquot of the derivatized extract was injected into the column (HP5-MS capillary column, 5% phenyl-95% methylsiloxane, 30 m x 0.25 mm I.D., 0.25 microm film thickness) of a Hewlett-Packard (Palo Alto, CA, USA) gas chromatograph (6890 Series). Methenolone was detected by its parent ion at m/z 446 and daughter ions at m/z 208 and 195 through a Finnigan TSQ 700 MS-MS system. The assay was capable of detecting 1 pg/mg of methenolone when approximately 100 mg hair material was processed. Linearity was observed for methenolone concentrations ranging from 2 to 100 pg/mg with a correlation coefficients of 0.965-0.981. Intra-day and between-day precisions at 2, 10 and 25 pg/mg were 10.9-14.1% and 13.7-16.8%, respectively, with an extraction recovery of 97.6%. The analysis of a strand of hair obtained from two bodybuilders, revealed the presence of methenolone at the concentrations of 7.3 and 8.8 pg/mg.