Development and validation of a liquid chromatography–mass spectrometry procedure after solid-phase extraction for detection of 19 doping peptides in human urine

Abstract

A liquid chromatography–tandem mass spectrometry method was developed and used to simultaneously detect 19 small peptide hormones prohibited in sport and their main metabolites in urine after solid-phase extraction. Detection was achieved using a triple-quadrupole mass spectrometric detector coupled with an electrospray ionization interface after chromatographic separation with an octadecyl column based on fused-core particle technology. Sample pretreatment was performed by solid-phase extraction. The extraction procedure was optimized by comparison of different sorbents and washing/elution protocols. The best results were obtained using a mixed-mode weak cation exchange sorbent, two washing steps (ultrapurified water and methanol), and elution using 300 mM ammonium formate in 25 % ammonia/methanol (10/90) or 25 % ammonia/10 % formic acid/methanol (8/12/80). The procedure was validated in terms of sensitivity (lower limits of detection: 0.05–2.0 ng/ml depending on the target analyte), specificity, recovery [>60 %, coefficient of variation (CV) <15 % except for TB500 17–23 fragment, AOD9604, and ARA290 for which recovery was <50 %), ion suppression/enhancement (<35 %), robustness, carryover, stability of the target analytes [stable for at least for 2 days (25 °C, 2 weeks (4 °C), 2 months (−20 °C)], and repeatability of retention times (CV <0.1 %) and relative abundances of the selected ion transitions (CV <15 %). The suitability of the method was confirmed by analyzing spiked and excreted urines, the latter collected after intravenous injection of 0.1 mg of GHRP-2.