An analytical method was developed for hypoxia-inducible factor (HIF) stabilizers based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) sample preparation and liquid chromatography–high resolution mass spectrometry analysis. HIF stabilizers potentially enhance the performance of athletes, and hence, they have been prohibited. However, the analysis of urinary HIF stabilizers is not easy owing to their unique structure and characteristics. Hence, we developed the QuEChERS preparation technique for a complementary method and optimized the pH, volume of extraction solvent, and number of extractions. We found that double extraction with 1% of formic acid in acetonitrile provided the highest recovery of HIF stabilizers. Moreover, the composi-tion of the mobile phase was also optimized for better separation of molidustat and IOX4. The developed method was validated in terms of its precision, detection limit, matrix effect, and recovery for ISO accreditation. To the best of our knowledge, this is the first demonstration of the application of the QuEChERS method, which is suitable as a complementary analytical method, in antidoping.
Glycerol was identified and isolated from endogenous interferences during analysis of human urine using high-resolution mass spectrometry (HRMS) for doping control. Urinary sample preparation was simple; the samples were diluted with an organic solvent and then analyzed using a liquid chromatography-mass spectrometry (“dilute and shoot” method). Although the interfering ion peaks were observed at the similar retention time of glycerol, the inference could be identified by isolation with HRMS and further investigation. Thus, creatinine was identified as the endogenous interference for glycerol analysis and it also caused ion suppression resulting in the decrease of glycerol signal. This study reports the first identification and efficient isolation of endogenous interferences in human urine for “dilute and shoot” method. The information about ion suppression could be novel to prevent overestimation or a false result for antidoping analysis.