Can the ingestion of fruits from the Annona family lead to the detection of the Beta 2-Agonist Higenamine in doping control urine samples?

Code: T20M01MT

Higenamine, also known as norcolaurine, is a non-selective β2-agonist naturally occuring in different plants such as Nandida domestica, Tinospora crispa, and Annona quamosa [1-5]. Due to its bronchodilatative and stimulating effects, the misuse of higenamine in sports is prohibited at all times [6]. According to the relevant WADA technical document, a reporting level of 10 ng/mL (50% of the MInimum Required Performance Level (MRPL)) applies for the detection fo β2-agonists in doping control urine samples [7]. From 2016 till 2019, 201 samples werer reported as an adverse analytical finding (AAF) for higenamine [8-11].

Although higenamine has never been approved as a drug by the US Food and Drug Administration (FDA) [11], it plays an important role in traditional Chinese herbal medicine [1, 4]. Moreover, it was found tto be an (un)labeled ingredient of different weight loss and sports supplements [2-4, 12], which have caused several cases of unintentional doping during the last years [13-18].

As also some tropical fruit plants of the Annonaceae family were founhd to contain higenamine [2, 5], the aim of this study was to investigate whether the ingestion of such fruits can lead to AAFs in sports. The results of this reserach project can be of great value for anti-doping routine work, as they will help to ensure fair result management and decision-making processes in case of higenamine findings in sports drug testing programs.

Therefore, elimination studies were conducted in order to characterize the time-dependent urinary excretion of higenamine. Previous unpublished reports for L. C. Cameron's laboratory showed that the consumption of A. muricata (n=3) or A. squamosa (n=4) produced detectable higenamine in 100% of the subjects' urine (n=7). Otherwise, higenamine was not detectable in the C. papaya (control) group (n=3). In total, two administration studies with single dose of a fruit puree from Anonna muricata (330 g) and Anonna cherimola (330 g) were conducted, and the collected urine samples wre analyzed by means of liquid chromatography - tandem mass spectrometry (LC-MS/MS). HIgenamine detection times in general and, specifically, urinary concentrations of higenamine were desirable in support of an improved interpretation of AAFs, especially when scenarios of proven supplement contamination are debated and supplement administration protocols exist.

Main findings

The β2-agonist higenamine is prohibited in sports at all times and available, amongst others, as ingreditnets in various dietary supplements.
Further, a variety of plants including tropical fruits of the Annonaceae family have been reported to contain higenamine, and the aim of this study is to investigate whether the ingestion of such fruits can lead to AAFs in sports. For that purpose, single-dose elimination studies were conducted with A. muricata and a second higenamine-containing fruit (A. cherimola). Post-administration urine samples were analyzed concerning their higenamine content and assessed with regards to the currently enforced reporting level of 10 ng/mL.
All study volunteers produced urine samples containing higenamine after ingestion of the fruit preparations; however, under the chosen conditions, all observed urinary concentrations ranged exclusively below 5 ng/mL and, thus, the established MRL.

The herein obtained data can be considered in result management and decision-making processes in case of higenamine findings in sports drug testing programs. The outcome supports the position that single-dose administrations of these fruit species are rather unlikely to lead to AAFs in sports. Yet, substantial variability of the natural higenamine content in fruits exists, potentially influenced by seasonal/spatiotemporal factors, and also fruit processing and storage might affect the overall dietary higenamine availability. Hence, whilst less likely, it cannot be excluded that under specific circumstances the current MRL is exceeded by nutrition-derived higenamine uptake.