UC Davis

Anabolic Androgenic Steroids (AAS) are a group of compounds with high potential for abuse in sports including horse racing. Within the last three decades, both gas chromatography – mass spectrometry (GC-MS) and liquid chromatography – mass spectrometry (LC-MS) methods have been developed for routine qualitative and quantitative detection of endogenous or exogenous AAS in urine and serum to address their misuse in sports. However, most current screening methods for the detection of AAS require using enzymatic or chemical hydrolysis to detect free steroids rather than their phase II conjugates which constitute the majority of excreted drug for most anabolic agents. In recent decades, new long-term phase II metabolites of AAS that were previously not detected by GC-MS have been identified by LC-MS methods, including glucurono-conjugates and sulfo-conjugates that are difficult to cleave using enzymatic hydrolysis. This research project focuses on the development and validation of a method capable of the simultaneous detection of 32 steroid phase II metabolites using LC-MS/MS, with prior clean-up steps including protein precipitation and Solid Phase Extraction (SPE) through weak anion exchange cartridges. All compounds, including isomers and epimers, were chromatographically separated over a 20-minute run using a reversed phase C18 column using methanol and water as the organic and aqueous mobile phases. The mass spectrometer data acquisition was set to use selected reaction monitoring scans following introduction via electrospray ionization in both positive and negative modes. The method was validated, and the following parameters were determined: linearity, accuracy, precision, limit of detection (LOD), limit of quantitation (LOQ), recovery, matrix effect, and stability. Finally, a population study using gelding, mare, and stallion urine samples from horses actively competing was conducted to evaluate the endogenous production of the targeted compounds.