LC-MS/MS Method for Simultaneous Analysis of Growth Hormone-Releasing Peptides and Secretagogues in Human Urine

Abstract

Growth hormone (GH)-releasing peptides (GHRPs) and GH secretagogues (GHSs) are listed in the World Anti-Doping Agency (WADA) Prohibited List. In the present study, we developed and validated a method for the simultaneous analysis of seven GHRPs (alexamorelin, GHRP-1, -2, -4, -5, -6, and hexarelin) and three GHSs (anamorelin, ibutamoren, and ipamorelin) in human urine. Method validation was performed at minimum required performance levels specified by WADA technical documents (2 ng/mL) for all substances, and the method was validated with regard to selectivity (no interference), linearity (R2 > 0.9986), matrix effects (50.0%-141.2%), recovery (10.4%-100.8%), and intra- (2.8%-16.5%) and inter-day (7.0%-22.6%) precisions. The limits of detection for screening and confirmation were 0.05-0.5 ng/mL and 0.05-1 ng/mL, respectively.