Liquid chromatographic analysis of amphetamine and related compounds in urine using solid-phase extraction and 3,5-dinitrobenzoyl chloride for derivatization.

A chromatographic method for the analysis of amphetamine and related compounds in urine using 3,5-dinitrobenzoyl chloride (3,5-DNB) as a labeling reagent is presented. This assay is based on the employment of solid-phase extraction (SPE) cartridges for sample cleanup and derivatization. Experimental conditions are optimized for the simultaneous derivatization of ephedrine, norephedrine, pseudoephedrine, beta-phenylethylamine, amphetamine, methamphetamine, and 3-phenylpropylamine. The derivatives formed are separated in a LiChrospher 1000 RP18 (125 x 4-mm i.d., 5-microns film thickness) analytical column using a water-acetonitrile gradient elution and detected at 254 nm. Derivatization in C18 SPE disks is found to be the best option for analysis of urine samples; this method provides analyte conversions that are about 85-102% of those obtained by the analogous solution derivatization. Because the 3,5-DNB reagent is a strong pi-acid, the described method can be used in combination with a Pirkle-type donor column for chiral analysis. The practicality of the described approach is illustrated by determining amphetamine enantiomers using a Supelcosil LC-(S)-naphtylurea (250 x 4.6-mm i.d., 5-microns film thickness) column and a mobile phase of n-hexane-acetonitrile-ethyl acetate. Under these conditions, good linearity and reproducibility are observed over the 0.5-10 micrograms/ml concentration range; the limit of detection is 50 ng/mL.