Automated determination of amphetamine enantiomers using a two-dimensional column-switching chromatographic system for derivatization and separation
A method for the on-line quantification of amphetamine enantiomers was developed. The assay uses a 20 mm × 2.1 mm id pre-column packed with a Hypersil C18 (30 µm) material for purification and derivatization of the analytes and a mixture of o-phthaldialdehyde and N-acetyl-L-cysteine as derivatizing reagent. The resulting derivatives were subsequently separated in a conventional C18 achiral column using a mobile phase of acetonitrile–methanol–acetate buffer (6 + 48 + 46 v/v), and detected by fluorescence. Under optimized conditions, the system provided average responses of 88 ± 3 and 87 ± 3% compared with those of the analogous solution derivatization for the L- and D-amphetamine enantiomers, respectively. The method shows good linearity and reproducibility in the 0.25–10.0 µg ml–1 concentration range. The limits of detection were 50 ng ml–1 for both isomers. The method was applied to the quantification of amphetamine enantiomers in pharmaceuticals and urine samples.