Singh S P, Moody D E
Department of Pharmacology and Toxicology, College of Pharmacy, University of Utah, Salt Lake City 84108, USA.
J Pharm Biomed Anal. 1995 Jul;13(8):1027-32. doi: 10.1016/0731-7085(95)01344-k.
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.
已设计出一种简单且灵敏的体外分析方法，用于测定右美沙芬O-去甲基化，这是人类（2D6）和大鼠（2D1）中P450 2D缺乏的一个标志物。使用市售的[N-甲基-³H] -右美沙芬开发了一种用于右美沙芬O-去甲基化的放射性薄层色谱分析方法。己烷 - 三乙胺能有效地从大鼠肝微粒体中提取右美沙芬及其代谢物，环己烷 - 甲苯 - 二乙胺（65:15:20，v/v/v）的溶剂系统能在两个放射性条带（右美沙芬和右啡烷）之间提供足够的分离距离（约2厘米），且不受未标记的N-去甲基化产物3-甲氧基吗啡喃和3-羟基吗啡喃的干扰。右啡烷从薄层板上的回收率随微粒体蛋白和孵育时间的增加而提高。相对于雄性Sprague-Dawley大鼠（分别为P450 2D代谢能力差和强的模型），雌性Dark Agouti大鼠的活性降低了八倍。该分析方法即使在放射性标记底物稀释约100倍的情况下，检测限仍约为100 pmol。批内和批间不精密度分别为12.4%和7.2%。用于右美沙芬O-去甲基化的放射性薄层色谱分析方法灵敏且简便，使用的是易于获得的设备。
