Monitoring 2‐phenylethanamine and 2‐(3‐hydroxyphenyl)acetamide sulfate in doping controls

Abstract

2-Phenylethanamine (phenethylamine, PEA) represents the core structure of numerous drugs with stimulant-like properties and is explicitly featured as so-called specified substance on the World Anti-Doping Agency (WADA) Prohibited List. Due to its natural occurrence in humans as well as its presence in dietary products, studies concerning the ability of test methods to differentiate between an illicit intake and the renal elimination of endogenously produced PEA were indicated. Following the addition of PEA to the Prohibited List in January 2015, retrospective evaluation of routine doping control data of 10 190 urine samples generated by combined gas chromatography-mass spectrometry and nitrogen phosphorus-specific detection (GC-MS/NPD) was performed. Signals for PEA at approximate concentrations > 500 ng/mL were observed in 31 cases (0.3%), which were subjected to a validated isotope-dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) test method for accurate quantification of the target analyte. Further, using elimination study urine samples collected after a single oral administration of 250 mg of PEA hydrochloride to two healthy male volunteers, two tentatively identified metabolites of PEA were observed and evaluated concerning their utility as discriminative markers for PEA intake. The ID-LC-MS/MS approach was extended to allow for the simultaneous detection of PEA and 2-(3-hydroxyphenyl)acetamide sulfate (M1), and concentration ratios of M1 and PEA were calculated for elimination study urine samples and a total of 205 doping control urine samples that returned findings for PEA at estimated concentrations of 50-2500 ng/mL. Urine samples of the elimination study with PEA yielded concentration ratios of M1/PEA up to values of 9.4. Notably, the urinary concentration of PEA did increase with the intake of PEA only to a modest extent, suggesting a comprehensive metabolism of the orally administered substance. Conversely, doping control urine samples with elevated (>50 ng/mL) amounts of PEA returned quantifiable concentrations of M1 only in 3 cases, which yielded maximum ratios of M1/PEA of 0.9, indicating an origin of PEA other than an orally ingested drug formulation. Consequently, the consideration of analyte abundance ratios (e.g. M1/PEA) is suggested as a means to identify the use of PEA by athletes, but further studies to support potential decisive criteria are warranted.

Dietary Supplement and Food Contaminations and Their Implications for Doping Controls

“…The natural monoamine alkaloid phenylethylamine (PEA) and its synthetic derivatives function as neuromodulators in the CNS, resulting in stimulating effects similar to amphetamine [80][81][82]. Since 2015, these agents are found among the specified stimulants on the WADA Prohibited List [41,81]. PEA and related compounds are widely distributed as dietary supplements promising positive effects on energy and exercise duration [79,80].…”

“…As the detected concentrations of PEA in some of these products (0.7-171.6 mg/g) were significantly higher than the natural levels of this compound in Acacia rigidula extracts (up to 1.5 µg/g), it can be assumed that also here admixtures of synthetic PEA to these products occurred. But as PEA is also produced by the human body, the differentiation of an illicit administration of the drug from endogenous levels is a complicated analytical task and requires the consideration of PEA metabolite profiles indicative for oral ingestion [81].…”

Overview of regulation of dietary supplements in the USA and issues of adulteration with phenethylamines (PEAs)

“…[49] The addition of PEA itself to the WADA Prohibited List in January 2015 [50] necessitates the development of a test method for urine that could discriminate between illicit intakes and metabolic elimination of endogenously produced PEA. Although consideration of this topic is beyond the scope of this review, the interested reader is referred to the recent work of Sigmund et al [51] in evaluating the use of analyte abundance ratios as a means of potentially identifying the use of PEA by athletes.…”

The 33rd Manfred Donike Workshop on Doping Analysis

“…[1] Forsdahl et al illustrated the advantage of analyzing testosterone conjugates from serum as unequivocal evidence regarding the use of synthetic steroids. [2] This has increasing importance considering the growing number of testosterone preparations consisting of 'natural' carbon isotope signatures.…”

“…The importance of testing for phenylethylamine administrations by means of its metabolite(s), rather than by phenylethylamine itself (due to the natural endogenous occurrence of the substance) was discussed and the tentatively identified 2‐(3‐hydroxyphenyl)acetamide sulfate suggested as a potential target analyte . Forsdahl et al .…”