Equine Urine Screening for SARMs Using Solid Phase Extraction Followed by Triple Quadrupole LC-MS

Historically, anabolic steroids have been abused in human and equine sports for those who want to gain an unfair advantage. The recent discovery of nonsteroidal selective androgen receptor modulators (SARMs) presents a beneficial substitute for athlete abuse with the ability to selectively stimulate anabolic tissues combined with lesser pharmacologic side effects compared to anabolic steroids. As a result, SARMs have a high potential for abuse in human and equine sports, and they are easily accessible via the black market. Although there has only been one reported case of a SARM candidate in an equine doping control sample, the number of cases is expected to be much higher with the belief that these compounds will go undetected by anti-doping laboratories. In this study, a sensitive and robust method utilizing solid phase extraction (SPE) followed by triple quadrupole LC-MS was successfully validated, allowing for the rapid screening and confirmation of SARMs Andarine, LGD-4033, and MK-2866 in equine urine. Drug-free equine urine aliquots were fortified with the compounds of interest. After overnight enzyme hydrolysis, the pH of the samples was adjusted to 6. The SPE columns were conditioned and the samples were added, followed by a wash with phosphate buffer (pH 6). After acidification, drugs were eluted, dried down, and reconstituted in 1:1 0.2% formic acid in water: methanol. Twenty five µL were injected onto the LC-MS QQQ for analysis. Results showed that the SPE column allows for successful extraction recovery of all compounds of interest. Limit of detection (LOD) was approximately 1 ng/µL for all compounds. Quantitative requirements were met for most compounds, with all precision values less than 20%. Bias requirements were met for two of the three compounds, with accuracy values lower than 25%, excluding LGD-4033. In conclusion, the use of SPE followed by LC-MS QQQ for instrumental screening of Andarine, LGD-4033, and MK-2866 in equine urine produces satisfactory results and is recommended. Adding these emerging SARM therapeutic candidates to routine urine screening will maintain integrity in equine sports and deter handlers who are tempted to administer these readily available drugs without sufficient research on the adverse health effects to the animal.