Automated high-performance liquid chromatographic determination of amphetamine in biological fluids using column-switching and on-column derivatization

A rapid and simple liquid-chromatographic method has been developed for on- line quantification of amphetamine in biological fluids. Untreated samples (20 mu L) are injected directly into the chromatographic system and purifie d on a 20 mm x 2.1 mm i.d. pre-column packed with 30 mu m Hypersil Cis stat ionary phase. After clean-up the analyte is transferred to the analytical c olumn (125 mm x 4 mm i.d., 5 mu m LiChrospher 100 RP18) for derivatization and separation using a mixture of acetonitrile and the derivatization reage nt (o-phthaldialdehyde and N-acetyl-L-cysteine) as the mobile phase. The ex perimental conditions for on-line derivatization and resolution of the amph etamine have been optimized, and the results have been compared with those obtained by derivatizing the analyte in pre-column mode. The method describ ed has been applied to the determination of amphetamine in plasma and urine . Good linearity and reproducibility were obtained in the 0.1-10.0 mu g mL( -1) concentration range, and limits of detection were 25 ng mL(-1) and 10 n g mL(-1) with UV and fluorescence detection, respectively. The procedure de scribed is very simple anti rapid, because no off-line manipulation of the sample: is required; the total analysis time is approximately 8 min.