Carbon isotope ratios of phenethylamine and its urinary metabolite phenylacetylglutamine.

Abstract

Phenethylamine (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.