Analysis for higenamine in urine by means of ultra‐high‐performance liquid chromatography–tandem mass spectrometry: Interpretation of results

Abstract

Higenamine (Norcoclaurine) is a very popular substance in Chinese medicine and is present in many plants. The substance may be also found in supplements or nutrients, consumption of which may result in violation of anti-doping rules. Higenamine is prohibited in sport at all times and included in Class S3 (β-2-agonists) of the World Anti-Doping Agency (WADA) 2017 Prohibited List. The presence of higenamine in urine samples at concentrations greater than or equal to 10 ng/mL constitutes an adverse analytical finding (AAF). This work presents a new metabolite of higenamine in urine sample which was identified by means of ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Samples were prepared according to 2 protocols - a Dilute and Shoot (DaS) approach and a method involving acid hydrolysis and double liquid-liquid extraction (LLE). To meet the requirements typical for a confirmatory analysis, the screening procedure was further developed. In samples prepared by the DaS method, 2 peaks were observed; the earlier one was specific for higenamine and the later one unknown. MS scan analysis showed mass about 80 Da higher than that of higenamine. In turn, in samples prepared in accordance with the protocol involving hydrolysis, an increase in the area under peak for higenamine was observed, while the second peak was absent. It seems that the described strategy of detection of higenamine in urine avoids false negative results.

The use of a valid and straightforward method for the identification of higenamine in dietary supplements in view of anti‐doping rule violation cases

“…Moreover, according to the International Standard for Laboratories, all WADA‐accredited laboratories shall analyze each dietary supplement specifically requested by an ADO as the part of a doping case investigation . Because of these requirements, the development of a simple and rapid method for the identification of higenamine in urine and dietary supplements was necessary. According to WADA technical document TD2018MRPL, the presence of higenamine in urine samples at concentrations greater or equal to 10 ng/mL constitutes an AAF.…”

Annual banned‐substance review: Analytical approaches in human sports drug testing

“…In addition, studies into metabolites as potential target analytes were conducted, and the inclusion of the sulfoconjugate of higenamine was suggested based on findings in authentic doping control samples. Whilst not yet being fully characterized, the presence of higenamine sulfate in an athlete's sample was confirmed by LC–MS/MS, offering a prolonged detection window for the prohibited substance that only constitutes an AAF if exceeding urinary concentrations of 10 ng/mL . The natural occurrence of higenamine in traditional medicinal plants such as Nandina domestica , Aconitum charmichaelii , etc, has necessitated further investigations in an anti‐doping context to clarify whether the administration of over‐the‐counter products such as throat lozenges or dietary supplements can result in AAFs and thus in (inadvertent) anti‐doping rule violations.…”

Detection of bemitil and its metabolite in urine by means of LC–MS/MS in view of doping control analysis

“…13,14 2.3 | Dilute-and-shoot approach Briefly, for DaS, 100 μL of urine was spiked with 5 μL of Amphetamine-D11 (internal standard) at final concentration of 500 ng/mL and diluted with 900 μL of water. 13,14 2.3 | Dilute-and-shoot approach Briefly, for DaS, 100 μL of urine was spiked with 5 μL of Amphetamine-D11 (internal standard) at final concentration of 500 ng/mL and diluted with 900 μL of water.…”