Development of multiresidue analysis for prohibited substances in antidoping analysis by liquid chromatography and mass spectrometry: for worldwide sports events

Abstract

World Anti-Doping Agency (WADA) requires to the doping laboratory to analyze prohibited substances and their metabolites in various classes and a screening method which can detect multisubstances is an important challenge in the field of doping control. Particularly for global sports events like the Olympics, where numerous samples must be analyzed in a short period, it is very important to integrate various existing analytical methods into a single method to save time, manpower, and resources. In this study, we developed an analytical method that integrates three existing methods: dilute and inject, peptide analysis, and LLE-based analysis. Many drugs excreted in urine exist as metabolites of glucuronide form, and we used the enzyme glucuronidase, which can hydrolyze them to their parent form. We also used a mixed-mode SPE of a weak cation exchange mechanism, to extract peptides and polar substances simultaneously. Furthermore, we introduced a method of diluting the dried samples with the urine to recover specific drug of glucuronide form or polar drugs that are not extracted. We conducted various optimizations and validation for SPE extraction and enzyme reaction efficiency depending on buffer composition, temperature and pH. In particular, we were able to remove matrix interference using a high-resolution mass spectrometer, the orbitrap, and performed high-efficiency analysis of highly polar drugs like meldonium. The developed analytical method can simultaneously analyze over 350 prohibited drugs and their metabolites, and using this method, we successfully performed sample analysis for large-scale sports events like the Pyeongchang Winter Olympics.