Simultaneous detection of different chemical classes of selective androgen receptor modulators in urine by liquid chromatography-mass spectrometry-based techniques

Analytical procedures to detect the misuse of selective Androgen Receptor modulators in human urine, targeting either the parent drugs and/or their main metabolites, were developed and validated. In detail, 19 target compounds belonging to 9 different chemical classes were considered: arylpropionamide (i.e., andarine (S4), ostarine (S22), S1, S6, S9 and S23), diarylhydantoin (i.e., GLPG0492), indole (i.e., LY2452473, GSK2881078), isoquinoline-carbonyle (i.e., PF-02620414), phenyl-oxadiazole (i.e., RAD140), pyrrolidinyl-benzonitrile (i.e., LGD4033), quinolinone (i.e., LGD2226, LGD3303), steroidal (i.e., Cl-4AS-1, MK0773 and TFM-4AS-1), and tropanol (i.e., AC-262536 and ACP105) derivatives. The metabolites of the target compounds considered were enzymatically synthesized by using human liver microsomes. Sample pre-treatment included enzymatic hydrolysis followed by liquid-liquid extraction at neutral pH. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. Validation was performed according to the ISO 17025 and the World Anti-Doping Agency guidelines. The analyses carried out on negative samples confirmed the method's selectivity, not showing any significant interferences at the retention times of the analytes of interest. Detection capability was determined in the range of 0.1-1.0 ng/mL for the screening procedure and 0.2-1.0 ng/mL for the confirmation procedure (except for GLPG0492 and GSK2881078). The recovery was greater than 80 % for all analytes, and the matrix effect was smaller than 35 %. The method also matched the criteria of the World Anti-Doping Agency in terms of repeatability of the relative retention times (CV% < 1.0) and of the relative abundances of the selected ion transitions (performed only in the case of triple quadrupole, CV% < 15), ensuring the correct identification of all the analytes considered. Urine samples containing andarine, ostarine, or LGD4033 were used to confirm the actual applicability of the selected analytical strategies. All target compounds (parent drugs and their main metabolites) were detected and correctly identified.