Determination of 3,4-methylenedioxymethamphetamine and its five main metabolites in rat urine by solid-phase extraction and high performance liquid chromatography with on line mass spectrometry.
The consumption of psychostimulant amphetamine-like drugs has increased significantly in recent years. Some MDMA metabolites are probably involved in the neurotoxicity and neurodegeneration caused by prolonged use rather than MDMA itself. We recently developed a method to analyze MDMA and its five main metabolites in rat plasma 7. We have now fully validated this method to the quantification of these drugs in rat urine. We extracted MDMA and its metabolites with Oasis WCX cartridges, separated them on a Nucleodur C18 analytical column and quantified them by ion-trap mass spectrometry. Linearity was excellent 12.5-1250ngmL urine for HMA, HMMA, MDA and MDMA, 25-2500ngmL for HHMA, and 150-7500ngmL for HHA (r(2)>0.993 for all analytes). The lower limits of quantification were 12.5ngmL urine for MDMA, MDA, HMA and HMMA, 25ngmL for HHMA and 150ngmL for HHA. Reproducibility was good (intra-assay precision1.7-6.1% inter-assay precision0.6-5.7%), as was accuracy (intra-assay deviation0.1-4.8% inter-assay deviation0.7-7.9%). Average recoveries were around 85.0%, except for HHMA (66.2%) and HHA (53.0%) (CV<8.3%). We also checked the stability of stock solutions and the internal standards after freeze-thawing and in the autosampler. Lastly, we measured the MDMA, MDA, HHMA, HHA, HMMA and HMA in urine samples taken over 24h from rats given subcutaneous MDMA.