Equine Serum Screening for Thyroxine via Triple Quadrupole LC-MS

The use of levothyroxine supplements as a performance enhancer in horse racing has been reported anecdotally since the 1970s. Although no direct evidence of such purported enhancement abilities has yet been provided in the literature, high-profile cases of levothyroxine abuse have shown that the practice is still prevalent in the horse racing industry. Standard veterinary practice currently utilizes immunoassays for equine serum thyroxine analysis, while current forensic analyses largely rely on chromatographic and mass-spectral instrumentation. Here, a method for equine serum thyroxine analysis using liquid chromatography and mass-spectral instrumentation was developed and validated for use in forensic laboratories. Using a liquid-liquid extraction protocol, both qualitative and quantitative factors were examined for the developed analytical method. Overall, the method exhibited high specificity and sensitivity, and low carryover; however, significant serum matrix effects led to low extraction efficiency and process efficiency. Serum deproteination was briefly investigated and did exhibit significant improvements, but further optimization is required. The present method can be utilized for equine serum thyroxine analysis, although the practicality of such measurements for forensic analysts remains in question.