Simultaneous Determination of Testosterone and Testosterone Enanthate in Equine Plasma by UHPLC-MS-MS

Testosterone and testosterone enanthate are performance-enhancing substances that are banned in racehorses competing in the State of Pennsylvania (PA). A tolerance concentration of 2,000 pg mL^?1 plasma has been established for testosterone in intact colts and stallions at the time they are competing in PA. Testosterone enanthate is a precursor of testosterone and can be used to boost plasma testosterone concentration above natural, age and seasonally variable plasma concentration. To control abuse, a verifiable method for rapid determination of both substances in equine plasma was needed. For this reason, an ultra high performance liquid chromatography-tandem mass spectrometry method for high-throughput analysis of both analytes in equine plasma was developed. Analytes were recovered from plasma by liquid–liquid extraction using mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v), separated on a C₁₈ sub-2 μm column and detected on a triple quadrupole mass spectrometer using positive electrospray ionization mode with selected reaction monitoring scan. SRM ion transitions of m/z 289 → m/z 97, m/z 289 → m/z 109, m/z 289 → m/z 79 were used for testosterone identification while m/z 401 → m/z 253, m/z 401 → m/z 271, m/z 401 → m/z 97 were employed for testosterone enanthate. Retention time and product ion intensity ratio were used as confirmation criteria to ascertain the presence of both analytes in equine plasma. The limits of detection, quantification and confirmation were 50 pg 0.5 mL^?1, 100 pg 0.5 mL^?1 and 250 pg 0.5 mL^?1, respectively for both analytes. The method was validated for recovery efficiency, sensitivity, matrix effect, linearity, precision and accuracy. This method is routinely used in the PA program for androgenic anabolic steroids doping control in racehorses and in the on-going testosterone enanthate pharmacokinetics study. The method is defensible, fast, selective, specific and reproducibly reliable.