Direct high-performance liquid chromatographic and high-performance liquid chromatographic-thermospray-mass spectrometric determination of enantiomers of methamphetamine and its main metabolites amphetamine and p-hydroxymethamphetamine in human urine

For the identification of drug abuse, a simple and rapid method which allows us to distinguish enantiomers of methamphetamine (MA) and its metabolites amphetamine (AP) and p-hydroxymethamphetamine (p-OHMA) in human urine was explored by coupling direct HPLC and HPLC-thermospray-mass spectrometry (HPLC-TSP-MS) both of which employ a β-cyclodextrin phenylcarbamate-bonded silica column. HPLC analysis was performed after the solid-phase extraction from the urine sample with Bond Elut SCX, and d- and l-enantiomers of MA, AP and p-OHMA could be separated well. The proposed conditions are as follows: eluent, acetonitrile-methanol-50 mM potassium phosphate buffer (pH 6.0) (10:30:60, v/v) flow-rate, 1.0 ml/min temperature, 25°C. The linear calibration curves were obtained for d- and l- MA and AP in the concentration range from 0.2 to 20 μg/ml; the relative standard deviation for d- and l-AP and d- and, l-MA ranged from 1.67 to 2.35% at 2 μg/ml and the detection limits were 50 ng/ml for d- and l-AP and d-MA and 100 ng/ml for l-MA. For the verification of the direct HPLC identification, HPLC-TSP-MS was also carried out under the same conditions except that acetonitrile-methanol-100 mM ammonium acetate (pH 6.0) (10:30:60, v/v) was used as an eluent. Upon applying the scan mode, 10 ng/ml for d- and l-AP and d-MA and 20 ng/ml for l-MA were the detection limits. Using the selected ion monitoring mode, 0.5 ng/ml, 0.8 ng/ml and 1 ng/ml could be detected for d- and l-AP, d-MA and l-MA, respectively.