A radiometric TLC assay of liver microsomal dextromethorphan O-demethylation

A simple and sensitive assay for in vitro analysis of dextromethorphan O-demethylation, a marker for P450 2D deficiency in both humans (2D6) and rats (2D1), has been devised. Commercially available [N-methyl-3H]-dextromethorphan was used to develop a radiometric TLC assay for dextromethorphan O-demethylation. Hexane-triethylamine efficiently extracted dextromethorphan and metabolites from rat liver microsomes, and a solvent system of cyclohexane-toluene-diethylamine (65:15:20, v/v/v) provided sufficient separation (approximately 2 cm) between the two radioactive bands, dextromethorphan and dextrorphan, and no interference from the unlabeled N-demethylation products, 3-methoxymorphinan and 3-hydroxymorphinan. The recovery of dextrorphan from TLC plates increases with microsomal protein and incubation time. An eight-fold decrease in activity was noted in female Dark Agouti relative to the male Sprague-Dawley rats, respective models for poor and extensive P450 2D metabolizers. The assay, even with an approximately 100-fold dilution of radiolabeled substrate, had an approximate limit of detection of 100 pmol. Within- and between-run imprecision was 12.4% and 7.2%, respectively. The radiometric TLC assay for dextromethorphan O-demethylation was sensitive and easy, and used readily available equipment.