Bioactive peptides are recognised for being highly specific and selective to the target receptors while being safe and potent. The availability of bioactive peptides in the global market has increased rapidly in recent years. As such, they pose significant doping threats to both human and horse sports, and the development of a sensitive detection method for bioactive peptides is imminent in order to control their misuse. Owing to the wide range of basicity of the peptides with varying amino acid sequences and side chain modification, the extraction of these peptide drugs is often challenging. In view of this, we have developed a sensitive detection method in horse urine based on two consecutive solid-phase extractions (SPE) and analysed on ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC/HRMS).
This novel extraction method involved consecutive SPEs from horse urine using two cartridges to recover peptide drugs with diverse chemical properties. Urine samples were first extracted with a mixed-mode weak cation exchange cartridge (WCX). In addition to the eluate, the methanol wash was collected and subjected to another extraction by a mixed-mode strong cation exchange cartridge (MCX) to provide complementary coverage of the peptide drugs. The extracts from the WCX and MCX extractions are then separately analysed on UHPLC/HRMS after disulfide bond reduction by tris(2-carboxyethyl)phosphine (TCEP). Simultaneous screening of over 90 bioactive peptide drugs and/or their metabolites at low to sub parts-per-billion (ppb) levels in horse urine could be achieved. Key peptide drugs including [Dmt1]DALDA, EMP-1, GHRP-1 to GHRP-6, TB-500 and/or their metabolites are all covered in this method with good sensitivity. The use of two extraction cartridges also greatly increases the potential to expand the target scope coverage for peptides with different chemical properties.