Detection of β-methylphenethylamine, a novel doping substance, by means of UPLC/MS/MS

Abstract

Novel substances of expected doping activity are constantly introduced to the market. β-Methylphenethylamine (BMPEA) is classified as a doping agent by the World Anti-Doping Agency as it is a positional isomer of amphetamine. In this work, the development and application of a simple and rapid analytical procedure that enables discrimination between both isomers is described. The analytes of interest were extracted from urine by a two-step liquid-liquid extraction and then analyzed by UPLC/MS/MS under isocratic conditions. The entire analytical procedure was validated by evaluating its selectivity, discrimination capabilities, carry-over, sensitivity, and influence of matrix effects on its performance. Application of the method resulted in detection of BMPEA in eight anti-doping samples, including the first report of adverse analytical finding regarding its use. Further analysis showed that BMPEA may be eliminated unchanged along with its phase II conjugates, the hydrolysis of which may considerably improve detection capabilities of the method. Omission of the hydrolysis step may therefore, produce false-negative results. Testing laboratories should also carefully examine their LC/MS/MS-based amphetamine and BMPEA findings as both isomers fragment yielding comparable collision-induced dissociation spectra and their insufficient chromatographic separation may result in misidentification. This is of great importance in case of forensic analyses as BMPEA is not controlled by the public law, and its manufacturing, distribution, and use are legal.

Fig. 1

ToF MS/MS spectra for the m/z 136.11 precursor ions of amphetamine (a) and BMPEA (b)

Fig. 2

Discrimination capabilities of the method at different concentration levels (a) and chromatograms of a real case sample prepared with hydrolysis (H) or shortened hydrolysis (sH) of metabolic conjugates (b). Amphetamine (AMPH) was used as an internal standard to quantify BMPEA concentration in excretion urines. Extracted ion chromatograms were recorded for the precursor ion of m/z 136.11

Fig. 3

Analytical performance of the dilute-and-shoot method (a) and a chromatogram of excretion sample (b). Amphetamine (AMPH) was used as an internal standard to quantify BMPEA concentration and to correct for matrix-dependent shifts of BMPEA retention time