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

Abstract

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.

Identification of exvivo catabolites of peptides with doping potential in equine plasma by HILIC‐HRMS

“…After another centrifugation as mentioned above, 90 μL of the supernatant was taken for HILIC‐HRMS analysis. The second reconstitution step aimed at better dissolving hydrophilic peptides and catabolites from the plasma extracts 21 …”

“…A stock solution of ammonium formate (HCOONH 4 , 1.0 M, pH 3.70), a working HCOONH 4 solution (20 mM, pH 3.70), and a working aqueous ammonium hydroxide solution (0.59 M) were made, as described elsewhere 21 . Mobile phase solutions of ACN/HCOONH 4 (20 mM)/FA (20/80/0.1, and 95/5/0.1, v/v/v) were prepared.…”

“…Immediately after the collection of blood, plasma was harvested by centrifuging at 3000 × g for 15 min and stored at −20°C until use (blank plasma). The stability of the peptides in equine plasma was assessed by a procedure described elsewhere 13,21 . Briefly, plasma from four research horses (twelve 1.00 mL aliquots from each horse) was spiked with the 27 peptides (5.0 ng in 50 μL ACN/H 2 O/FA (50/50/0.1, v/v/v), final concentration 5.0 ng/mL each), and the spiked plasma samples were incubated at 4°C, ambient temperature (22°C), and 37°C for different periods of time (2, 24, 48, and 72 hr).…”

“…Control plasma samples (no incubation period) were prepared using a procedure described previously 21 . Briefly, a 1.00 mL aliquot of blank plasma was transferred into a glass culture tube, and 2.0 mL of 0.59 M ammonium hydroxide was added.…”

“…Thus, assessment of the stability of peptides in plasma at different temperatures for various periods of time is warranted for studying their detectability in biological matrices. For this purpose, in a previous study we evaluated the stability of 44 bioactive peptides in equine plasma at ambient temperature 21 . As indicated by the results from that study, peptides comprising all l ‐amino acids without any protection at either terminus are rapidly degraded in equine plasma.…”