A comprehensive approach to detecting multitudinous bioactive peptides in equine plasma and urine using hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry

Bioactive peptides possess pharmacological effects and can be illicitly used in sports. To deter such misuse, an untargeted method using high resolution mass spectrometry (HRMS) has been developed for comprehensive detection of multitudinous exogenous peptides in equine plasma and urine. Forty‐four peptides were extracted using mixed‐mode solid‐phase extraction (SPE) from plasma and urine, separated with a hydrophilic interaction liquid chromatography (HILIC) column, and detected on an HRMS instrument. Ammonium formate as a mobile phase additive had effects on HILIC retention and charge state distribution of the peptides. The acetonitrile percentage in the reconstitution solution affected the solubility of peptide neat standards and peptides in plasma and urine extracts differently. The stability of the peptides in plasma at ambient temperature was assessed. The limit of detection (LOD) was 10–50 pg/mL for most of the peptides in plasma, and ≤ 500 pg/mL for the remaining. LOD was 100–400 pg/mL for the majority of the analytes in urine, and ≤ 4000 pg/mL for the others. The method was used successfully to analyze incurred plasma and urine samples from research horses administered dermorphin. Even in the absence of reference standards, dermorphin metabolites (aFGYPS‐NH₂, YaFG, and YaF) were identified. These results demonstrate that data generated with this method can be retrospectively reviewed for peptides that are unknown at the time of sample analysis without requiring re‐analysis of the sample. This method provides a powerful novel tool for detection of numerous bioactive peptides and their metabolites in equine plasma and urine for doping control.