Doping control analysis of small peptides in human urine using LC-HRMS with parallel reaction monitoring mode: screening and confirmation

Abstract

This study described a reliable analytical method, which combines solid-phase extraction (SPE) with liquid chromatography-high resolution mass spectrometry (LC-HRMS) employing the parallel reaction monitoring (PRM) mode, for screening 41 small peptides and 3 non-peptide growth hormone secretagogues in human urine. Additionally 36 small peptides and 3 non-peptide growth hormone secretagogues were also confirmed in the same way. For the whole screening procedure, the PRM mode was applied to the HRMS detection of small peptides, which reduces the background noise from matrix compounds to a large extent and thus improves the selectivity and reliability of the peptide analytes. Meanwhile, competent chromatographic separation was achieved within a total runtime of 14 minutes, indicating an improvement in the detection efficiency. Moreover, the PRM mode could also be applied to the confirmation procedure due to its strong identification power with a low risk of generating false positives or negatives and good selectivity. Validation was performed according to the relevant World Anti-Doping Agency (WADA) criteria, including selectivity and reliability, limit of detection (LOD), limit of identification (LOI), recovery, extraction stability and carryover. The LODs of the peptide analytes ranged between 0.20 ng mL⁻¹ and 0.92 ng mL⁻¹ in urine, while their LOIs ranged between 0.20 ng mL⁻¹ and 2.00 ng mL⁻¹, which met the corresponding Minimum Required Performance Levels (MRPLs) as defined by WADA. The developed method furnished the rapid and sensitive detection of small peptides in urine for more than 5000 samples with no false-positive or false-negative, indicating that it is an eligible method for doping control analysis.