Carbon isotope ratios of phenethylamine and its urinary metabolites phenylacetylglutamine and phenylacetamide

Code: 21A03MT

Phenethylamine (PEA) is listed on WADA´s Prohibited List as a stimulant and therefore its administration is forbidden in competition. As the human body naturally produces PEA, its presence in urine samples alone is not an indication for its misuse. Already in 2015, a potential metabolite indicative for the administration of PEA ((2-(3-hydroxyphenyl)acetamide sulfate) has been reported, based on pilot study results conducted with two individuals. During a follow up study encompassing more volunteers and different administration protocols, other potential metabolites were identified. Both PEA and its metabolites show are large inter-individual variance, i.e. the urinary concentrations of these compounds can be substantially elevated even without any administration simply due to the individual´s metabolism. This phenomenon is well described for urinary steroids and their concentrations as demonstrated for testosterone. Here, the carbon isotope ratios (CIR) are used to confirm the exogenous origin of elevated concentrations if found in urine samples of athletes. Similarly, within this research project a method suitable to determine Page 2/8 the CIR of PEA and its metabolites will be developed and validated according to WADA guidelines. Besides the development of a sophisticated sample clean-up procedure possible derivatization techniques will be investigated to ensure valid results. Finally, a reference population will be investigated to figure out the expected range of isotopic ratios for PEA and its metabolites. This will enable a clear differentiation between endogenous (produced naturally inside the human body) and exogenous (administered) PEA found in urine specimens.

Main Findings

Phenethylamine (PEA) is itemized as a stimulant on the Prohibited List of the World Anti-Doping Agency since 2015. It represents a naturally occurring trace amine and has been investigated in the 1970s and 80s regarding its medical potential as a modulator in the central nervous system. Today it is sold as a dietary supplement, marketed for its mood enhancing effects and as a potential treatment for weight loss.

Its detection in sports drug testing remains complicated as PEA is an endogenous substance, i.e. it is present in urine, at least in trace amounts. Investigations into a reference population demonstrated that the found urinary concentrations show a large inter-individual variation. Interestingly, after the oral administration of PEA, its urinary concentrations were found only slightly elevated. Establishing a urinary concentration threshold for PEA was therefore not successful and further studies focused on potential metabolites of PEA identifying one sulphated minor metabolite and phenylacetylglutamine (PAG) as the most abundant metabolite of PEA in urine. Again, the biological variability of these urinary metabolites complicated establishing a threshold and only the combination of both parameters in a binary logistic regression enabled to identify post-administration samples.

Aim of this research was the development and validation of an isotope ratio mass spectrometry-based method for PEA and PAG to differentiate between the endogenous or exogenous source of these metabolites considering their carbon isotope ratio (CIR). Especially for PAG, the developed method met all expectations and reference population-based thresholds were established. Unfortunately, both endogenous PEA and PAG showed unexpectedly depleted CIR and the found difference between exogenous and endogenous PEA was only around 3 ‰. According to these relatively similar CIR, the opportunities for detecting an oral administration of PEA were very short, and after a single oral dose none of the volunteers was found with CIR outside the population-based thresholds.

In athletes with relatively enriched CIR the developed method will most probably not result in an adverse finding as here the impact of the exogenous PEA will not influence the determined CIR of urinary PAG beyond the established threshold.

Further investigations will be necessary to elucidate the potential of IRMS to detect the misuse of PEA, e.g. by means of stable isotopes other than carbon.