A Broad-Spectrum Peptide Screening Method Using an Optimized Solid-Phase Extraction and Liquid Chromatography–High-Field Asymmetric Ion Mobility Spectrometry–Mass Spectrometry for Doping Control in Equine Urine

The abuse of prohibited peptide-based drugs with a broad spectrum of chemical characteristics poses a significant concern for the horseracing industry. Recently, there has been a notable increase in positive cases of small-peptide drugs reported in equine and canine sports. In addition to small peptides, large peptides (over 2 kDa) with structural diversity have also entered the market in increasing numbers as drugs for humans and livestock. However, the simultaneous analysis of both small- and large-peptide-based drugs is still challenging. In this study, a screening method was developed to cover 74 analytes, including peptides, their catabolites, and/or their mimetics, with molecular weights ranging from 0.3 kDa to greater than 5 kDa. The simultaneous extraction of both small and large peptides was achieved using a weak cation-exchange solid-phase extraction cartridge with a mixture of different pore sizes (suitable for large peptides), followed by analysis using liquid chromatography electrospray ionization high-field asymmetric ion mobility spectrometry tandem mass spectrometry (LC–FAIMS–MS/MS). For method validation, the limits of detection (LoDs), reproducibility, recovery, matrix effect, selectivity, and carryover were evaluated. Remarkably, the LoDs of ~80% of the analytes were less than or equal to 50 pg/mL, with the lowest LoD (1 pg/mL) being observed for selected peptides in horse urine. These results indicate a substantial advancement in achieving comprehensive coverage for both small and large peptides with high sensitivity for the purpose of doping control in horseracing and equestrian sports.