Development of Targeted and Non-targeted Analysis for Illegal Doping Drugs by Liquid Chromatography-Mass Spectrometry

Abstract

In 2016, growth hormone-releasing peptides, growth hormone secretagogues, and insulin-like growth factor-1 are listed by the World Anti-Doping Agency as illegal doping drugs based on increasing of its usage in athletes to promote the secretion of growth hormone. This study developed and validated the targeted and non-targeted methods for the simultaneous analysis of illegal doping drugs. First section of this study was focused on qualification analysis (PART 1), targeted method using HPLC-MS/MS for doping control was developed for growth hormone-releasing peptides and growth hormone secretagogues, and validated 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 with the limit of detection (0.05 to 0.5 ng/mL). Furthermore, new non-targeted qualitative method that has a traceability for unknown compounds was developed with growth hormone-related drugs include small chemicals. The feature of non-targeted method is direct application for unknown drugs without additional optimization, and is expected to contribute to next generation of doping test. The method was validated in terms of selectivity (no interferences), recovery (29-131%), matrix effect (35-237%) and intra- and inter-day precision (%CV lower than 25%) with limit of detection (0.0002 to 100 ng/mL). In second part (PART 2), endogenous insulin-like growth factor-1 that is an endogenous biomarker and increases when an athlete injects growth hormone, was quantitatively analyzed using a calibration curve in human serum. This method was validated with selectivity (no interferences), matrix effect (90-94%), carry-over (lower than 5%) and intra- and inter-day precision (%CV lower than 15%) to target substances (T1 and T2) with limit of detection (10 ng/mL). Newly developed targeted and non-targeted methods were accredited by KOLAS and are playing important role of anti-doping tests in Korea.