A comprehensive and high-throughput screening method for multiple prohibited substances by UPLC-QE Plus-HRMS and HPLC-QQQ-MS in human urine for doping control

Abstract

Since the World Anti-Doping Agency's (WADA) Prohibited List is updated on an annual basis, screening methods must be continually adapted to align with these changes. In accordance with Technical Document-MRPL 2022, a newly combined, comprehensive, rapid and high-throughput doping control screening method has been developed for the analysis of 350 substances with different polarities in human urine using ultra-high performance liquid chromatography coupled with Q Exactive Plus Hybrid Quadrupole-Orbitrap mass spectrometer (UPLC-QE Plus-HRMS) and ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-QQQ-MS). The limits of detection were in the range of 0.12–50 ng mL⁻¹ for beta-2 agonists, hormone and metabolic modulators, narcotics, cannabinoids and glucocorticoids, 0.1–14 ng mL⁻¹ for the manipulation of blood and blood components, beta blockers, anabolic agents and hypoxia-inducible factor (HIF) activating agents, and 2.5–100 000 ng mL⁻¹ for substances of Appendix A, diuretics & masking agents and stimulants. The sample preparation consisted of two parts: one is the dilute & shoot part analyzed in UPLC-QQQ-MS, another is a mixture of the dilute & shoot part and a liquid–liquid extraction part of hydrolyzed human urine analyzed in UPLC-QE Plus-HRMS in full scan mode with polarity switching and parallel reaction monitoring (PRM) mode. The method has been fully validated for doping control purposes. All the substances were compliant with WADA's required 1/2 minimum requirement performance level (MRPL) or minimum reporting level (MRL), and this method was successfully used in the 2022 Beijing Winter Olympic Games and Winter Paralympic Games for anti-doping purpose.