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

Abstract

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 (λ_ex 330 nm). Calibration curves for these derivatives using spiked human urine were linear in the range 0.05–100 µmol dm^–3 with correlation coefficients 0.999. The detection limits at a signal-to-noise ratio of 3 were 2.8–8.8 fmol per 5 µl injection. The relative standard deviations of within- (n = 6) and between-day (n = 5) variations were ⩽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.