Automated trace enrichment for screening and/or determination of primary, secondary and tertiary amphetamines in biological samples by liquid chromatography

Abstract

A rapid and simple liquid chromatographic method for the automated determination of amphetamines in biological fluids was developed. The proposed procedure is based on the injection of 250 µL of sample into a 20 × 2.1 mm id precolumn (packed with a 30 µm Hypersil C₁₈ stationary phase) for enrichment and purification of the analytes. Next, the analytes are transferred to a 5 µm LiChrospher 100 RP₁₈, 125 × 4 mm id analytical column for their separation under reversed-phase conditions. Water was used to eliminate the matrix components from the precolumn and a 0.2 M phosphate buffer (pH 3) containing 2% triethylamine was the mobile phase for the resolution of the amphetamines. The UV detector was set at 210 nm. The method was applied to the determination of different primary, secondary and tertiary amphetamines in plasma and urine: β-phenylethylamine, norephedrine, ephedrine, N-methylpseudoephedrine, pseudoephedrine, N-methylephedrine, amphetamine, 3-phenylpropylamine and methamphetamine. The method provides satisfactory linearity and reproducibility within the tested concentration range (1.0–10.0 µg mL^–1) and limits of detection of 50–500 ng/mL^–1.