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Equine Plasma Screening for SARMs using Liquid-Liquid Extraction and Triple Quadrupole LC-MS
thesisposted on 01.12.2019, 00:00 by Lucy K Freitag
While androgenic steroids such as testosterone have been popular in the world of sports doping, they carry negative androgenic side effects that have led to the search for a replacement. Selective androgen receptor modulators (SARMs) have become an appealing doping agent for those looking to gain an unfair advantage. As SARMs cause stimulation in anabolic tissues without significant stimulation of androgenic tissues, they have a high potential for abuse in human and equine competition sports. With one reported case of a SARM in an equine doping control sample, laboratories must be prepared for the emergence SARMs in the horseracing of industry. This presents the need for a validated method to detect and confirm the presence of SARMs in equine plasma samples, so labs can report usage and authorities can take appropriate disciplinary action. In this study, the ease of a liquid-liquid extraction was combined with the sensitivity of triple quadrupole liquid chromatography-mass spectrometry (LC-MS QQQ) to produce a reliable and accurate method for the detection of SARMs Andarine, Ostarine, and LGD-4033 in equine plasma samples. Each sample was spiked with known concentrations of the drugs of interest. The sample then received internal standard solution and methyl tert-butyl ether before being mixed by rotorack. Samples were then centrifuged, and the top layer was transferred to a new tube. The top layers were dried down, reconstituted, and transferred to a well plate for analysis on LC-MS QQQ. Results showed that the extraction was successful in recovering the drugs of interest from the plasma samples. Samples were most stable when stored in frozen conditions, and extraction efficiencies were above 75% for all three SARMs. Limits of detection (LOD) were 0.0019 ng/mL for Andarine and Ostarine and 0.00039 ng/mL for LGD-4033. Quantitative requirements as set in the method validation procedure were met for all three drugs. Precision values for all three drugs at high and low concentrations stayed below 15%, and bias for all three drugs at high and low concentrations was less than 6%. Therefore, a liquid-liquid extraction procedure followed by analysis using LC-MS QQQ is a recommended screening protocol for Andarine, Ostarine, and LGD-4033 in equine plasma samples. Implementation of the protocol as outlined in this work would help to detect and prevent the use of SARMs in the horseracing industry.