Published: Journal of Chromatographic Science, Volume 35, Number 4, April 1997, pp. 169–175.

Liquid Chromatographic Analysis of Amphetamine and Related Compounds in Urine Using Solid-Phase Extraction and 3,5-Dinitrobenzoyl Chloride for Derivatization
R. Herráez-Hernández, P. Campíns-Falcó, and A. Sevillano-Cabeza

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, b-phenylethylamine, amphetamine, methamphetamine, and 3-phenylpropylamine. The derivatives formed are separated in a LiChrospher 100 RP₁₈ (125 x 4-mm i.d., 5-µm 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 p-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-µm 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-µg/mL concentration range; the limit of detection is 50 ng/mL.

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