Simultaneous detection of recombinant growth hormones in equine plasma by liquid chromatography/high-resolution tandem mass spectrometry for doping control

Growth hormone (GH), or somatotropin, is a protein that may enhance physical performance and facilitate growth and wound healing. For this reason, growth hormones and their recombinant analogues are prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing under Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horse Racing Authorities (IFHA). Identifying the illicit use of GHs in both human athletes and racehorses is challenging, especially when differentiating between endogenous and exogenous GHs, and between analogues of GH from different species. This paper describes a multiplexed mass spectrometric method for the simultaneous detection of three recombinant GHs, namely recombinant equine GH (reGH), recombinant human GH (rhGH) and recombinant porcine GH (rpGH), in equine plasma. Recombinant chicken GH (rcGH) was used as an internal standard. The recombinant GHs were extracted from equine plasma by automated C4 solid-phase extraction after ammonium sulfate precipitation, and then cleaned up by chloroform/methanol precipitation before trypsination. Proteotypic peptides were analyzed by liquid chromatography/high-resolution tandem mass spectrometry (LC-MS/HRMS). The limits of detection were estimated to be approximately 0.5 ng/mL for reGH, 2.5 ng/mL for rhGH and 1.25 ng/mL for rpGH. Confirmation at 1 ng/mL for reGH and 5 ng/mL each for rhGH and rpGH was successfully achieved by comparing the retention times and relative abundances of three major product-ions of the respective standards in accordance with industry criteria published by the Association of Official Racing Chemists. The developed method requires less plasma (2 mL) and has a shorter turnaround time as compared with other published mass spectrometric methods, and demonstrates good precision and reproducibility. To our knowledge, this is the first reported method for the simultaneous detection of different recombinant GHs (reGH, rhGH and rpGH) at low ng/mL level in horse plasma samples.