Separation of mephentermine from other structural analogues of phenethylamine and amphetamine in biological matrices using Liquid Chromatography-Tandem Mass Spectrometry.

Stimulants exert euphorigenic and relaxing effects which warrant intervention of regulatory authorities in non-medical usage of these doping agents. Thus, a semi-quantitative method utilizing Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry (UHPLC-MS/MS) was developed to monitor potential misuse of 10 structural analogues of amphetamine and phenylethylamine derivatives. The extraction protocol for urine was dilute and shoot (DS) with 1 mL aliquot size, while that for plasma/serum was solid-phase extraction (SPE) with a 100 µL aliquot size. Chromatographic separation was achieved over an 18-minute gradient on a Phenomenex Luna Omega UHPLC C18 column. The mobile phase consisted of water and acetonitrile, both containing formic acid as a modifier, specifically at 1 % (v/v) for urine and 0.1 % (v/v) for plasma/serum analyses. Positive electrospray ionization (ESI+) was employed for mass spectrometric detection with acquisition in multiple reaction monitoring (MRM) mode. Method validation was performed in accordance with World Anti-Doping Agency (WADA) requirements for analyzing prohibited substances with minimum reporting limits (MRLs). Limits of identification (LOI) determined for urine and plasma/serum matrices were as follows: 0.5 ng/mL and 1 ng/mL for Oxethazaine; 1 ng/mL and 2.5 ng/mL for BHPT and BHMPT; 12.5 ng/mL and 25 ng/mL for PT and 5 ng/mL and 12.5 ng/mL for MPT, NEAMP, DMAMP, 4MMAMP, NEBMPEA and NNBTPEA, respectively. An analytical method characterized by rapidity, simplicity and cost-effectiveness was implemented for the chromatographic separation of stimulant structural analogues, facilitating the determination of potential stimulant abuse within human performance sports.