The Assay of Endogenous and Exogenous Anabolic Androgenic Steroids

According to the regulations of the World Anti-Doping Agency (WADA) [1], anabolic androgenic steroids are classified as prohibited substances in sports. They are covered in the section “S1. Anabolic Agents 1. Anabolic Androgenic Steroids (AAS)”. This section is further subdivided into “a. Exogenous AAS” and “b. Endogenous AAS”. AAS represent the class of substances most frequently detected in human sports doping control analyses for many years [2]. The analysis for AAS in doping control is generally performed using urine specimen as matrix. Most of the AAS are extensively metabolized in the human body and only small amounts of the parent substances are excreted in the urine. The most important metabolic phase-I reactions include oxidation or reduction in positions C-3 and C-17. Furthermore, D4 steroids in general are substrates for 5a reduction. Additional double bonds in ring A or B or additional substituents in position C-4 or C-6 push the reduction toward 5b orientation. The introduction of hydroxy functions and their subsequent oxidation especially in positions C-4, C-6, and C-16 is also observed and found most abundantly in steroids like metandienone, dehydrochloromethyltestosterone, stanozolol, and other steroids with additional condensed heterocycles on the A-ring [3–10]. Phase-II metabolism of AAS in humans includes mainly 3a-glucuronidation, while sulfatation mainly occurs at 3b-hydroxy functions. On 17-hydroxy functions, sulfatation as well as glucuronidation occurs equally. In case of 17-alkylated steroids, 17-epimerization occurs by hydrolysis of the phase-II metabolite, 17-sulfate. As a by-product of this reaction, 17,17-dimethyl18-nor-13-ene analogs are also detectable [11]. Endogenous AAS represent a special group of compounds. In a strict sense, the relevant steroids are not truly endogenous but are obtained by partial synthesis from plant sterols [12]. The number of compounds with potential of abuse is comparably restricted. Testosterone, biologically the most important anabolic androgenic hormone, is still