Identification and differentiation of somapacitan, a long-acting growth hormone derivative, and recombinant human growth hormone in equine plasma by LC-HRMS for doping control purpose.

Somapacitan, a long-acting growth hormone derivative, and recombinant human growth hormone (rhGH) are protein-based drugs generally used to treat growth disorders and GH deficiency in humans. Due to their potential to enhance the horse performance, the use of these drugs is prohibited at-all-times by the International Federation of Horseracing Authorities for horseracing and the Fédération Equestre Internationale for equestrian sports. In this study, we developed a test method for the identification and differentiation of somapacitan and rhGH in equine plasma by using liquid chromatography high-resolution mass spectrometry (LC-HRMS). The method involved C4 solid-phase extraction after ammonium sulfate precipitation, followed by chloroform/methanol precipitation, trypsin digestion, and analysis by LC-HRMS. The discriminative identification of somapacitan and rhGH was successfully achieved through the detection of their respective unique T10 peptide fragments. Noteworthy, the T10 peptide fragment of somapacitan was detected with its side chain structure remaining intact. The limit of identification (confirmation) (LOI) was determined to be 5 ng/mL for somapacitan and 2 ng/mL for rhGH in equine plasma, while the limit of detection (LOD) was 5 ng/mL for somapacitan and 1 ng/mL for rhGH. Furthermore, using the peptide fragments T1, T8, and T9 (which are shared between somapacitan and rhGH), the presence of somapacitan in equine plasma could be confirmed with higher sensitivity, achieving down to LOIs of 2 ng/mL and LODs of 1 ng/mL. The method was validated with respect to specificity, identification capability, robustness, precision, and reproducibility, paving the way for the analysis of post-administration samples from our already planned administration trials and future potential positive cases.