Detection and confirmation of hypoxia-inducible factor activating agents in equine plasma and urine by liquid chromatography coupled to mass spectrometry.

Hypoxia-inducible factor (HIF) activating agents can promote the transcription of the erythropoietin gene, enhancing an athlete's performance, and are therefore prohibited in sports. A liquid chromatography coupled to high-resolution mass spectrometry (LCHRMS) method was developed for the detection and confirmation of 11 HIF-activating substances in equine plasma and urine. Ammonium formate added to the mobile phase system suppressed the responses of all the analytes in either positive or negative ion mode and thus was not used. The starting organic percentage in the mobile phase gradient and sample reconstitution solution were optimized to minimize the carryover of the analytes. The analytes were extracted with solid-phase extraction. No interference from plasma or urine matrix was observed in the detection of the analytes by negative ion HRMS. Ten calibrators containing all 11 analytes at 10 to 10,000 pg/mL in plasma or urine were used to evaluate calibration ranges and limits of detection (LODs) by HRMS, and limits of confirmation (LOCs) by parallel reaction monitoring. LOD was 10 to 50 pg/mL for all analytes in plasma and 10 to 200 pg/mL in urine. LOC was 50 to 200 pg/mL for all analytes in plasma, except daprodustat and IOX 3 at 500 pg/mL, and 20 to 500 pg/mL in urine, except daprodustat and IOX 3 at 1000 pg/mL. The developed LC-MS method was successfully applied to the analysis of plasma and urine samples from a research horse following oral administration of JNJ 42041935.