Automated determination of amphetamine enantiomers using a two-dimensional column-switching chromatographic system for derivatization and separation

Abstract

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.