Dietary higenamine from Annonaceae family fruits as a possible source of unintentional doping

Members of the genus Aconitum have been used for millennia, both as poisons and medicines, in Eastern culture. Higenamine has non-selective beta-agonist effects, activating both β₁ and β₂ adrenoreceptors, and is present in a variety of plants. The World Anti-Doping Agency has banned Higenamine both in competition and out of competition. Due to the common uses of higenamine in Brazilian culture, both as medicine and food, we studied the urinary concentrations of higenamine after the consumption of fruits of the Annona genus. We evaluated whether the ingestion of these fruits has the potential to cause anti-doping code violations. We measured higenamine concentrations for a 72 h period in the urine of ten healthy, physically active males (age 20-30; weight 70-80 kg; not consuming supplements or medications) after eating a unique meal containing fruits. Fruit consumption ranges were: Carica papaya (control) 348 ± 98 g; A. muricata 450 ± 282 g; and A. squamosa 314 ± 60 g. (all mean± SD). Higenamine was measured using ultra-performance liquid chromatography coupled with electrospray-tandem mass spectrometry. The appearance of urinary higenamine occurred within the first 12 h after eating A. muricata (n = 3), and the maximum concentration found was 1.9 ng/mL. The ingestion of A. squamosa has also been shown to cause higenamine urinary excretion. The elimination kinetics of the subjects who ingested A. squamosa (n = 4) were different from each other. After ingestion of the control fruit, C. papaya, we detected no higenamine in the urine of any participants (n = 3). Although the kinetics varied by individuals and fruits, A. muricata ingestion produced higher higenamine excretion; however, the A. squamosa portion weighed ∼66 % of the A. muricata portion. We conclude that eating Annonaceae family fruits cause detectable higenamine excretion. Conversely, single ingestion did not reach the WADA's threshold to cause adverse analytical findings.