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Simultaneous separation and determination of 16 testosterone and nandrolone esters in equine plasma using ultra high performance liquid chromatography–tandem mass spectrometry for doping control

You, Youwen, Uboh, Cornelius E., Soma, Lawrence R., Guan, Fuyu, Li, Xiaoqing, Liu, Ying, Rudy, Jeffrey A., Chen, Jinwen, Tsang, Deborah
Journal of chromatography 2011 v.1218 no.26 pp. 3982-3993
athletic performance, detection limit, ethers, ethyl acetate, ionization, ions, issues and policy, liquid chromatography, liquid-liquid extraction, methanol, monitoring, nandrolone, racehorses, screening, steroids, tandem mass spectrometry, testosterone
The potential for using testosterone and nandrolone esters in racehorses to boost the biological concentrations of these steroids and enhance athletic performance is very compelling and should be seriously considered in formulating regulatory policies for doping control. In order to regulate the use of these esters in racehorses, a sensitive and validated method is needed. In this paper, we report such a method for simultaneous separation, screening, quantification and confirmation of 16 testosterone and nandrolone esters in equine plasma by ultra high performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS). Analytes were extracted from equine plasma by liquid–liquid extraction using a mixture of methyl tert-butyl ether and ethyl acetate (50:50, v/v) and separated on a sub-2 micron C₁₈ column. Detection of analytes was achieved on a triple-quadrupole mass spectrometer by positive electrospray ionization mode with selected reaction monitoring (SRM). Mobile phase comprised 2mM ammonium formate and methanol. Deuterium-labeled testosterone enanthate and testosterone undecanoate were used as dual-internal standards for quantification. Limits of detection (LOD) and quantification (LOQ) were 25–100pg/mL and 100–200pg/mL, respectively. The linear dynamic range of quantification was 100–10,000pg/mL. For confirmation of the presence of these analytes in equine plasma, matching of the retention time with mass spectrometric ion ratios from MS/MS product ions was used. The limit of confirmation (LOC) was 100–500pg/mL. The method is sensitive, robust, selective and reliably reproducible.