Doping control analysis of trenbolone and related compounds using liquid chromatography–tandem mass spectrometry

Abstract

Trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one) and its derivatives such as 17α-methyltrenbolone represent a class of highly potent anabolic–androgenic steroids, which are prohibited in sports according to the regulation of the World Anti-Doping Agency (WADA). Due to marginal gas chromatographic properties of these compounds but excellent proton affinities resulting from a large and conjugated π-electron system, liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been the method of choice for the detection of these analytes in sports drug testing. Recent findings of trenbolone and methyltrenbolone in doping control urine samples of elite athletes demonstrated the importance of a sensitive and robust analytical method, which was based on an enzymatic hydrolysis of target compounds, liquid–liquid extraction and subsequent LC–MS/MS measurement. Diagnostic product ions obtained after collision-induced dissociation of protonated molecules were found at m/z 227, 211, 199 and 198, which enabled targeted screening using multiple reaction monitoring. Using 7 model compounds (trenbolone, epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone and altrenogest), the established method was validated for specificity, lower limits of detection (0.3–3 ng/mL), recovery (72–105%), intraday and interday precision (≤20%).

Introduction

Since more than 20 years, anabolic agents have been the most frequently detected prohibited compounds in doping control samples. Among these agents, synthetic anabolic steroids represent a major subgroup, and the detection of selected analytes, particularly those bearing a large and conjugated π-electron system such as trenbolone (17β-hydroxy-estra-4,9,11-trien-3-one, Fig. 1 (1)) and its derivatives, was demonstrably a complex task using conventional gas chromatographic–mass spectrometric (GC–MS) systems [1], [2], [3]. Various approaches using different derivatizations were evaluated to make the target analytes amenable to GC–MS and GC–MS/MS [4], [5], [6], [7], [8], e.g., employing trimethylsilyl-, methoxime-, trifluoroacetyl-, heptafluorobutyl-derivatives as well as mixed modifications. The major breakthrough in terms of sensitivity and traceability of the misuse of these agents was accomplished with the availability of robust instruments based on liquid chromatography–tandem mass spectrometry (LC–MS/MS) as demonstrated in human and veterinary drug testing [9], [10], [11], [12], [13], [14].

The enormous anabolic potential of trenbolone and its analogs, especially 17α-methyltrenbolone (methyltrienolone, metribolone, Fig. 1 (3)), was reported in the late 1960s [15], and 3 has been used in androgen binding assays ever since its high affinity to androgen receptors was observed [16], [17]. However, in contrast to trenbolone, methyltrenbolone has never been marketed as an anabolic agent due to its extreme liver toxicity causing intrahepatic cholestasis at orally administered amounts of 1 mg per day [18]. Nevertheless, a total of 11 adverse analytical findings for methyltrenbolone were reported in 2008, which once more demonstrated the importance of comprehensive and sensitive screening assays for anabolic agents in professional and amateur sport. A method based on conventional enzymatic hydrolysis of urine samples, liquid–liquid extraction (LLE) and LC–MS/MS using electrospray ionization was established and validated for doping control purposes. The method was applied to determine trenbolone and 6 structurally related compounds including epitrenbolone, methyltrenbolone, ethyltrenbolone, propyltrenbolone, 17-ketotrenbolone, and altrenogest (Fig. 1 (2–7)) in human urine, and allowed for the unambiguous determination of methyltrenbolone in cases of suspicious doping control test results.