Determination of amphetamine and related compounds in urine using on-line derivatization in octadecyl silica columns with 9-fluorenylmethyl chloroformate and liquid chromatography

A method for the determination of amphetamine and related compounds in urine based on on-line derivatization with 9-fluorenylmethyl chloroformate (FMOC) and high-performance liquid chromatography is described. Derivatization is performed in a 20 x 2.1 mm I.D. column packed with a Hypersil ODS C-18, 30 mu m stationary phase, which is also used for sample clean-up and enrichment of the analytes. Next, the derivatized analytes are transferred to a LiChrospher 100 RP-C-18 (5 mu m, 125 x 4 mm I.D.) analytical column for their separation and quantification, using reversed-phase conditions and fluorescence detection. The described assay was applied to the determination of norephedrine, ephedrine, pseudoephedrine, amphetamine, phenylpropylamine and methamphetamine at concentrations of 0.5-10.0 mu g/ml. Analyte conversions were about 55-96% of those obtained by the off-line derivatization mode under similar conditions, resulting in limits of detection in the 5-25 ng/ml range.