Targeting misuse of 2‐amino‐N‐ethyl‐1‐phenylbutane in urine samples: in vitro–in vivo correlation of metabolic profiles and development of LC‐TOF‐MS method

Abstract

A phenyethylamine derivative, 2-amino-N-ethyl-1-phenylbutane (2-AEPB), has recently been detected in doping control and drugs-of-abuse samples, and identified as a non-labelled ingredient in a dietary supplement. To facilitate efficient control of this substance we have studied the in vitro metabolic behaviour of 2-AEPB with human liver preparation, compared these results with in vivo pathways in human, and finally propose an analytical strategy to target the potential misuse of 2-AEPB for toxicological, forensic and doping control purposes. The major in vitro formed metabolites originated from desethylation (M1) and monohydroxylation (M2). A minor metabolite with hydroxylation/N-oxidation was also observed (M3). In vitro-in vivo correlation was studied in an excretion study with a single, oral dose of 2-AEPB-containing supplement. An unmodified substance was the most abundant target compound and detected until the last point of sample collection (72 h), and the detection of M1 (40 h) and M2 (27 h) demonstrated good correlation to in vitro results. In the study with authentic cases (n = 6), 2-AEPB and M1 were mainly found in free urinary fraction, whereas higher inter-individual variability was observed for M2. It was predominantly conjugated and already within this limited number of cases, the ratio between glucuronide- and sulpho-conjugated fractions varied significantly. As a conclusion, hydrolysis is not mandatory in the routine sample preparation, and as the separation can be based on either gas chromatography or liquid chromatography, this study verifies that routine mass spectrometric detection methods targeted to amphetamine derivatives can be easily extended to control the misuse of 2-AEPB.

Annual banned‐substance review: analytical approaches in human sports drug testing

“…The routinely applied GC‐MS‐based test method was assessed concerning its LOD for NN‐DMPPA, which was determined at 30 ng/mL and, thus, fulfilling WADA MRPL criteria. Similarly, Kobayashi et al . and Wojtowicz et al .…”

“…[203] The routinely applied GC-MS-based test method was assessed concerning its LOD for NN-DMPPA, which was determined at 30 ng/mL and, thus, fulfilling WADA MRPL criteria. Similarly, Kobayashi et al [204] and Wojtowicz et al [205] investigated options to detect the newly identified phenethylamine derivative 2-amino-N-ethyl-1-phenylbutane (2-AEPB) in doping controls. Following in vitro and in vivo studies, the intact compound and its desethylated analogue were identified as appropriate target analytes, enabling the unequivocal detection of the prohibited substance down to approximately 3-5 ng/mL using both LC-HRMS and GC-MS.…”

“…--2 Diuretics - [182] --S6 Stimulants [63,191,203,205] [189,[192][193][194][196][197][198][199][200]202,204] [24, [186][187][188][189][190][191]195,198,201] S7 Narcotics ----S8 Cannabinoids [208][209][210] [ 95,[208][209][210][211][212][214][215][216][217] - [206][207][208][209][210]213,216] S9 Glucocorticosteroids - [221][222][223][224][225] - [24,...…”