Dispersive SPE, an alternative to traditional SPE for extraction of 43 doping peptides from equine urine prior to LC–MS screening

Dermorphin, growth hormone releasing peptide (GHRP), TB-500 and their analogues have been used illegally in the horse racing industry to improve the performance of the horses. This study aims to present dispersive solid phase extraction (dSPE) as an alternative to solid phase extraction (SPE) for the clean-up of equine urine samples prior to liquid chromatography combined with tandem mass spectrometry (LC–MS/MS) screening of 43 illegal performance enhancing peptides. Sorbent types and mass, washing and eluting solvents were tested to obtain the optimal clean-up conditions for these peptides in horse urine matrices. The resulting dSPE clean-up method gave optimal recovery and reproducibility of 43 target peptides; for the first time dSPE is proven as a viable alternative to SPE and achieves limits of detection (LOD) that are sufficient for the screening of these peptides. The LODs for all dermorphin, TB-500 and GHRP peptides were 1 ng/mL. Recoveries of the 43 target analytes extracted from 3 spiked urine samples ranged from 8.9 to 58.8%. The intra-day and inter-day precision for all target analytes ranged from 0.6 to 24.1% and 1.4 to 27.8% respectively. Using dSPE as a clean-up method, 43 peptide analytes of interest were successfully screened by LC–MS/MS.