Thin-layer chromatographic and column liquid chromatographic analyses of morphine in urine via dabsylation

A semiquantitative screening method for morphine in urine and a quantitative assay method for the drug were developed. In the semiquantitative method, morphine in urine was directly reacted with 4-dimethylaminoazobenzene-4'-sulphonyl chloride (dabsyl chloride) in a slightly alkaline medium. The orange-coloured dabsyl morphine was separated by silica gel thin-layer chromatography and the spot intensity was visually compared with that of the standards. The limit of detection is 0.075 microgram/ml. In the quantitative method, morphine was extracted from urine before dabsylation. The dabsylation reaction is very fast and is complete within 5-10 min at room temperature. Dabsylation yield is maximum at a dabsyl chloride concentration of 6.2 mM. Total recovery of morphine using the extraction and dabsylation procedures described is 66%. Dabsyl morphine, thus formed, was analysed using high-performance liquid chromatography by monitoring its absorbance at 436 nm on a normal-phase mu Porasil column. The limit of quantitation using high-performance liquid chromatography is 0.26 microM (0.075 microgram/ml), which corresponds to 10.5 pmol of injected dabsyl morphine. Quantitative assay was also carried out by thin-layer chromatography on silica gel followed by densitometry. The limit of quantitation is 1.3 microM (0.375 microgram/ml).