Determination of MDMA and MDA in rat urine by semi-micro column HPLC-fluorescence detection with DBD-F and their monitoring after MDMA administration to rat

A simultaneous semi-micro column HPLC method with fluorescence detection of abused drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), amphetamine (AP) and methamphetamine (MP) in rat urine was examined by using 4-(N,N-dimethylaminosulphonyl)-7-fluoro-1,2,3-benzoxadiazole (DBD-F) as a labelling reagent and α-phenylethylamine as an internal standard (IS). A sample (50 µL) of rat urine was added to 5 µL IS and 100 µL 100 mmol[sol ]L borate buffer (pH 12) and extracted with 1.5 mL n-hexane. After evaporation, 50 µL 75 mmol[sol ]L borate buffer (pH 8.5) and 50 µL 20 mmol[sol ]L DBD-F in CH3CN were added to the residue and mixed well. The resultant solution was heated for 20 min at 80°C and then cooled in an ice bath. A good separation of DBD-derivatives could be achieved within 45 min using a semi-micro ODS column with an eluent of CH3CN[sol ]CH3OH[sol ]10 mmol[sol ]L imidazole–HNO3 buffer (pH 7.0) (= 45:5:50, v[sol ]v[sol ]v %). The DBD derivatives were monitored at 565 nm with an excitation at 470 nm. The calibration curves showed good linearity (r = 0.997) with 0.5–15 ng[sol ]mL detection limits at a S[sol ]N ratio of 3. MDMA and MDA in rat urine could be monitored for 15 h after a single administration of MDMA to rat (2.0 mg[sol ]kg, i.p.). The concentrations for MDMA and MDA (n = 3) were 0.13–160.1 and 0.17–10.9 µg[sol ]mL, respectively. Copyright © 2005 John Wiley & Sons, Ltd.