Expanding analytical possibilities concerning the detection of stanozolol misuse by means of high resolution/high accuracy mass spectrometric detection of stanozolol glucuronides in human sports drug testing

Abstract

Anabolic-androgenic steroids (AAS) represent one of the most frequently detected classes of prohibited substances in doping controls. Due to their long-lasting beneficial effects on athletic performance, utmost retrospectivity via urine analysis is desirable and accomplished by targeting long-term metabolites of the respective drugs. In case of stanozolol, a substantial variety of metabolites has enabled the identification of numerous adverse analytical findings in the past, and recent studies concerning complementary phase-I and phase-II metabolites has further expanded the windows of opportunity for detecting the abuse of stanozolol. In this study, the utility of liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry (LC-MS/MS) for the detection of 3'-OH-stanozolol glucuronide in sports drug testing is presented and the identification of two additional and so far unreported metabolites is shown. The structures of the complementary glucuronic acid conjugates were attributed to stanozolol-N-glucuronide and 17-epistanozolol-N-glucuronide. By means of chemical synthesis, stanozolol-N-glucuronide was prepared and used to corroborate the suggested structures. The 3'-OH-stanozolol glucuronide and the newly identified target compounds were implemented into routine sports drug test assays consisting of direct injection LC-MS/MS or solid-phase extraction (SPE) followed by LC-MS/MS. A considerably expanded detection window for stanozolol abuse was demonstrated compared to the use of conventional phase-I metabolites and methodologies based on, for example, low resolution LC-MS/MS or gas chromatography-tandem mass spectrometry (GC-MS/MS). The commercial availability of 3'-OH-stanozolol glucuronide has been of great value for confirmatory purposes, and 17-epistanozolol-N-glucuronide was found to be a favourable long-term metabolite for doping controls as it was observed up to 28 days post-administration of the drug. Applying the established methodology over a period of six months to 659 routine sports drug testing samples, a total of 85 adverse analytical findings was uncovered, 72 of which would have remained undetected using earlier employed GC-MS/MS approaches.

Screening for anabolic steroids in sports: Analytical strategy based on the detection of phase I and phase II intact urinary metabolites by liquid chromatography tandem mass spectrometry

“…Boldenone, boldione, methyltestosterone and metandienone metabolites conjugated with sulfate have been recently identified [5][6][7][8]12]. Glucuronoconjugated metabolites poorly or not hydrolyzable using ␤-glucuronidases have been described for testosterone and stanozolol [13,14]. These results demonstrate the need to re-evaluate AAS metabolism to study new phase II metabolites not systematically studied up to now, to look for new long-term metabolites, and to include these new metabolites in the routine screening procedures.…”

“…New long-term phase II metabolites have been recently described for some AAS (e.g., boldenone, metandienone, methyltestosterone and stanozolol), for which standards are not commercially available [6,7,12,14]. For this reason, urines obtained after administration of these AAS were used to optimize the analysis conditions of the metabolites and to incorporate them into the screening procedure.…”

“…However, for many AAS most of the metabolic profile remains unknown [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. Studies on phase II steroid metabolism have been traditionally performed using gas chromatography-mass spectrometry (GC-MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS), after hydrolysis to release the phase I metabolites [2][3][4][9][10][11].…”

Urine Fingerprints of Stanozolol Treated Horses by Liquid Chromatography High Resolution Mass Spectrometry

“…The response to this request is straightforward in the case of negative samples easily attainable in routine laboratory, but more demanding in the case of positive samples since in routine screening Nevertheless, the discrimination of control and stanozolol-treated population is efficient and thus almost 4 months after anabolic steroid administration which is significantly longer than the detection window of stanozolol obtained by different target analysis. Although, the detection window is greatly improved with the screening of stanozolol long-term metabolites [28] it is mandatory to carry on further.…”

Fast, sensitive, and reliable

“…According to the prevailing International Standard for Laboratories (ISL), the doping control samples can be re-analyzed within a time period of up to eight years following the reporting of the original result. [6] This is particularly interesting when new instrument technologies are introduced in the analysis routine for the purpose of monitoring new drugderivatives or new chemical entities emerging onto market, [7] and also when new long-term excreted metabolites are reported for already known prohibited substances. [8] Although the flexibility of the ISL increases the risk of an adverse analytical finding, it also requires adequate sample storage condition and knowledge on the long-term effects on various analytes.…”