Carbon isotope ratios of phenethylamine and its urinary metabolite phenylacetylglutamine

Abstract

AbstractPhenethylamine (PEA) is a naturally occurring trace amine that acts as a modulator in the central nervous system. It is widely sold as a dietary supplement and advertised for its mood enhancing effects and should support weight loss. It is prohibited in sports and itemized as a stimulant on the Prohibited List issued by the World Anti‐Doping Agency (WADA). After oral administration of PEA, its urinary concentration is found only slightly elevated while metabolites of PEA show a significant increase. Besides 2‐(2‐hydroxyphenyl)acetamide sulfate, especially phenylacetylglutamine (PAG) was found at significantly elevated urinary concentrations after the administration. Due to large inter‐ and intra‐individual variations in urinary concentrations of all metabolites, establishing a concentration or concentration ratio‐based threshold remained complicated to unambiguously identify post‐administration samples. In accordance with the approach employed in detecting testosterone misuse, the applicability of isotope ratio mass spectrometry to differentiate between endogenously elevated concentrations and PEA administrations was investigated. A method encompassing solid‐phase extraction combined with acetylation and high‐performance liquid chromatography (HPLC)‐based clean‐up was developed and validated for PEA. The more abundant metabolite PAG was purified by a direct injection approach on the HPLC and could be analyzed without the need for derivatization. Both methods were validated considering applicable WADA regulations. A reference population encompassing n = 57 samples was investigated to establish population‐based thresholds considering the carbon isotope ratios (CIRs) found at natural abundance for PAG. The derived threshold was tested for its applicability by re‐analysis of numerous post‐administration samples encompassing single‐ and multi‐dose trials.