Identification and characterization of higenamine metabolites in human urine by quadrupole-orbitrap LC-MS/MS for doping control.

Higenamine is an alkaloid found in aconite, Annona squamosa, nanzhu (sacred bamboo), and other plants. It can be used to treat coughing, asthma, heart failure, and erectile dysfunction as well as aid in weight loss. It is also a banned substance in and out of competition as defined by the World Anti-Doping Agency (WADA). In this work, higenamine metabolic profiles were investigated in detail. Two healthy volunteers (one male and one female) took a higenamine tablet (5 mg), and urine samples were collected for two weeks. Solid-phase extraction (SPE) without enzymatic hydrolysis was used to clean the urine samples, and the urine extracts were then analyzed by liquid chromatography-quadrupole-orbitrap mass spectrometry (quadrupole-orbitrap LC-MS/MS) with accurate mass measurements. Higenamine and 32 metabolites were detected: 6 methylated, 10 sulfated and 16 glucuronidated metabolites. The chemical structures were elucidated by their fragmentation patterns, and accurate molecular formula determination was obtained for these newly reported metabolites. Three metabolic pathways containing methylation, glucuronidation, sulfation, and combinations of these were provided with methylation as the main metabolic pathway. The post-dose detection windows within urine of all 32 metabolites were compared with that of the parent drug, and a new potential biomarker (M7) was suggested for higenamine misuse. All urine samples were processed by two sample preparation methods: the dilute-and-shoot (DS) procedure and acid hydrolysis followed by SPE, and the time periods for a higenamine positive trails of two methods were compared. Although the DS method used to process the urine samples of athletes in the most of WADA-accredited laboratories to detect only free higenamine, acid hydrolysis followed by SPE is preferable and offers routine analysis to avoid false-negative results.