A procedure based on liquid chromatography–mass spectrometry (LC–MS) is described for determination of 3,4-methylenedioxymethamphetamine (MDMA), 2,5-dimethoxy-4-methyl-phenethylamine (2C-D), 4-bromo-2,5-dimethoxy-β-phenethylamine (2C-B), 1-(8-bromo-2,3,6,7-tetrahydrobenzo[1,2-b:4,5-b’] difuran-4-yl)-2-aminoethane (2C-B-Fly), 4-ethylthio-2,5-dimethoxy-β-phenethylamine (2C-T-2), 4-iodo-2,5-dimethoxy-β-phenethylamine (2C-I), and 4-ethyl-2,5-dimethoxy-β-phenethylamine (2C-E), 1-(m-chlorophenyl)piperazine (m-CPP), 4-hydroxy-N,N-diisopropyltryptamine (4-OH-DIPT) and 4-acetoxy-N,N-diisopropyltryptamine (4-acetoxy-DIPT) in urine of consumers using 3,4 methylendioxypropylamphetamine (MDPA) as internal standard.
Sample preparation involved a solid-phase extraction procedure at pH 6 of both non-hydrolyzed and enzymatically hydrolyzed urine samples. Chromatography was performed on a C18 reversed-phase column using a linear gradient of 10 mM ammonium bicarbonate, pH 7.3 and acetonitrile as a mobile phase. Separated analytes were determined in LC–MS single ion monitoring mode using an atmospheric pressure ionization–electrospray ionization (ESI) interface. The assay was tested on urine samples from consumers of compounds under investigation (n = 32).
Limits of quantification varied between 20 and 60 ng/mL for the different analytes under investigation. Calibration curves were linear to 2000 ng/mL for all the substances under investigation, with a minimum r2 > 0.99. At three concentrations spanning the linear dynamic range of the assay, mean recoveries ranged between 55.4 and 95.6% for the different analytes. Higher analytes concentrations in hydrolyzed samples showed the presence of conjugated compounds in urine.