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 method using high-performance liquid chromatography with fluorescence detection was developed to measure the abused drugs MDMA, MDA, amphetamine, and methamphetamine in rat urine. The drugs were labeled with a fluorescent reagent and separated within 45 minutes. Detection limits ranged from 0.5 to 15 ng/mL. After a single injection of MDMA to rats, both MDMA and its metabolite MDA could be detected in urine for up to 15 hours, with concentrations ranging from 0.13 to 160.1 µg/mL for MDMA and 0.17 to 10.9 µg/mL for MDA.

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Abstract

Abstract 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 CH 3 CN 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 CH 3 CN[sol ]CH 3 OH[sol ]10 mmol[sol ]L imidazole–HNO 3 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.