Determination of MDMA and its three metabolites in the rat perfused liver.

3,4-Methylenedioxymethamphetamine (MDMA) is one of the most commonly abused illicit drugs in the world. We developed a rapid and simple high-performance liquid chromatography with a fluorescence (FL) detector method to determine MDMA and its metabolites, such as 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxyamphetamine (HMA) and its main unstable metabolite 3,4-dihydroxymethamphetamine (HHMA) besides the internal standards, in a perfusion medium. The separation of analytes was performed at 25°C on a Chromolith® C18 (100 × 4.6 mm) column from Merck (Darmstadt, Germany) without any derivatization. The FL detector wavelength was fixed at 285 nm for excitation and at 320 nm for emission. Acetonitrile:phosphate buffer (0.02 M) at pH = 3 (5:95 v/v) was used as the mobile phase. The elution order was HHMA, HMA, MDA and MDMA with a retention time of 1.7, 2.6, 6.1 and 7.4 min, respectively. The method was validated according to the FDA bioanalytical method validation guideline. The limits of quantifications (LOQs) obtained for MDMA, MDA, HMA and HHMA were 1, 1, 1.5 and 5 ng/mL, respectively. The repeatability of relative standard deviation was  <11% (except for LOQs). This method was applied successfully to determine MDMA and its metabolites in rat liver perfusion samples. To our knowledge, this is the first method introduced for the determination of HHMA as a free form with an FL detector.