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Fluorometric determination of primary and secondary amines in blood and urine after thin-layer chromatography.

7-Chloro-4-nitrobenzo-2-oxa -1,3-diazole reacts with primary and secondary amines to produce highly fluorescent compounds. The resulting emission spectra for primary and secondary amines are similar; however, when the excitation spectra of these compounds are determined when the emission wavelength is kept at 520 nm, distinctly different results are obtained. Thin-layer chromatography of suitably concentrated aliquots of these solutions yield characteristic spots, the Rf values of which can be used to help identify the substances involved. This reaction was applied to the quantitative determination of amphetamine in biological extracts. If amphetamine is the only drug present it can be quantitated by fluorometry and confirmed by the thin-layer chromatographic procedure. If more than one drug is present, the fluorometric procedure is unsatisfactory for quantitative analysis. Thin-layer chromatography can be useful to identify qualitatively the drugs involved. Gas chromatography is the procedure of choice in these situations.