Determination Of Urinary Higenamine Excretion After Higenamine-containing Chinese Herbal Medicine Administration

Higenamine, a compound found in many Chinese herbs, is classified as a banned substance by the World Anti-Doping Agency (WADA). Chinese herbal medicine is commonly used in traditional Chinese medicine for treating various diseases. Therefore, it is important to evaluate the potential risk of athletes testing positive for higenamine after using Chinese herbal medicine containing this substance. This evaluation is necessary to prevent the occurrence of adverse analytical findings (AAF) in doping tests. PURPOSE: To determine the levels of urinary higenamine by ultra-performanceliquid chromatography–tandem mass spectrometry (UPLC–MS/MS) after the administration of higenamine-containing Chinese herbal medicine. METHODS: Recruited healthy participants received orally higenamine-containing Chinese herbal medicine three times a day for three consecutive days, and the multiple doses were equivalent to 966 μg of higenamine per day. Urine samples were collected before, during, and up to one day after discontinuing the administration. The sample preparation protocols involved enzymatic hydrolysis and purification via the use of β-glucuronidase and solid phase extraction. RESULTS: The UPLC-MS/MS analysis detected higenamine and sulfonated higenamine after the administration of higenamine-containing Chinese herbal medicine. Average levels of detected higenamine in urine samples were 0.57 ± 0.27 ng/mL. The highest level of higenamine detected was 1.56 ng/mL, and it was found on the evening of the third day of administration. On the other hand, our results showed that sulfonated higenamine produced a 2-5 times stronger signal response than higenamine in most samples. Furthermore, it was not detected in urine samples collected prior to drug administration, suggesting its potential as the important metabolite of higenamine ingestion. CONCLUSION: According to the current WADA Technical Document for higenamine, when the concentration of higenamine in urine samples is above a minimum reporting level of 10 ng/mL, the dosage and administration method used in this study do not constitute an AAF. Furthermore, the study suggests that sulfonated higenamine, detected in urine samples can be a valuable target analyte for identifying potential misuse or abuse of higenamine. This research was funded by the Ministry of Science and Technology, Taiwan (MOST 109-2410-H-037-016-MY3, MOST 109-2811-H-037-500-MY2, and MOST 111-2811-H-037-003).