Enantioselective high-performance liquid chromatography with fluorescence detection of methamphetamine and its metabolites in human urine

A simple, rapid and highly sensitive high-performance liquid chromatographic method with fluorescence detection for determining the enantiomers of methamphetamine and its major metabolites, amphetamine and p-hydroxymethamphetamine, in urine samples was developed. Using a newly developed reagent for amines, namely, 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride, six enantiomers were derivatized under mild conditions (i.e., 10 min at room temperature, pH 9.0) and separated isocratically on a cellulose tris(3,5-dimethylphenylcarbamate) coated silica gel column following a pre-separation on an ODS column within 42 min, and the effluent was monitored at 440 nm (lambda(ex) 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05-100 mu mol dm(-3) with correlation coefficients greater than or equal to 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8-8.8 fmol per 5 mu l injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were less than or equal to 7.4%. The method was successfully applied to discriminate between (S)-(+)-methamphetamine and its corresponding metabolites found in abusers' urine and their antipodes in a sample taken from a Parkinsonian patient on selegiline (Deprenyl) therapy.