Validated UHPLC-MS-MS method for rapid analysis of capsaicin and dihydrocapsaicin in equine plasma for doping control.

A method involving ultra high-performance liquid chromatography-tandem mass spectrometry was developed and validated for the analysis of capsaicin and dihydrocapsaicin in equine plasma. The analytes were recovered from plasma by liquid-liquid extraction using methyl tert-butyl ether and separated on a sub-2 micron column. The mobile phase was composed of 2 mM ammonium formate and methanol. A triple quadrupole mass spectrometer was used to detect the analytes in positive electrospray ionization mode with selected reaction monitoring. The limits of detection, quantification and confirmation for both analytes were 0.5, 1.0 and 2.5 pg/mL, respectively. The linear dynamic range of quantification was 1.0-1,000 pg/mL. During storage, both analytes in equine plasma were unstable at room temperature but stable at -20 and -70°C. The retention time and product ion ratios were employed as the criteria for confirmation of the presence of the analytes in plasma. The total analysis time was 2 min. The method is fast, selectively sensitive, reproducible, reliable and fully validated.