Department of Pharmacology, Faculty of Medicine, Masaryk University, Komenskeho nam. 2, 662 43 Brno, Czech Republic.
J Pharm Biomed Anal. 2010 Aug 1;52(4):557-64. doi: 10.1016/j.jpba.2010.01.028. Epub 2010 Jan 21.
A new, simple, rapid, sensitive, and repeatable reversed-phase HPLC method was developed and validated for the simultaneous determination of tolbutamide, phenacetin and their metabolites in rat liver perfusate. Chlorpropamide was used as an internal standard to ensure the precision and accuracy of this method. Analytes were extracted into diethyl ether using a two-step liquid-liquid extraction. A C18 analytical column and a mobile phase composed of acetonitrile and potassium phosphate buffer were used for the chromatographic separation with UV detection. Limits of detection varied between 20 and 46ng/mL for phenacetin, tolbutamide and their metabolites. The overall extraction recovery for the analytes varied from 65.4% in paracetamol to 88.0% in tolbutamide for concentrations within the expected range of concentrations from previous experimental samples. In terms of precision, the intra- and inter-day variation at three different concentrations in all analytes never exceeded 7.6 and 11.4%, respectively. This method is applicable for the modeling and description of possible pharmacological interactions on rat cytochromes P450 1A2 and 2C6/11 or can be used for in vitro evaluation of both cytochromes 1A2 and 2C.
建立并验证了一种新的、简单的、快速的、灵敏的和可重复的反相高效液相色谱法，用于同时测定大鼠肝灌流液中甲苯磺丁脲、非那西丁及其代谢物。氯丙磺脲被用作内标，以确保该方法的精密度和准确度。分析物用二乙醚通过两步液-液萃取提取。采用 C18 分析柱和由乙腈和磷酸钾缓冲液组成的流动相进行色谱分离，并用紫外检测。对于非那西丁、甲苯磺丁脲及其代谢物，检测限在 20 到 46ng/mL 之间变化。在预期浓度范围内，分析物的总提取回收率在对乙酰氨基酚中为 65.4%，在甲苯磺丁脲中为 88.0%。就精密度而言，所有分析物在三个不同浓度下的日内和日间变化均不超过 7.6%和 11.4%。该方法适用于大鼠细胞色素 P450 1A2 和 2C6/11 可能发生的药理学相互作用的建模和描述，也可用于细胞色素 1A2 和 2C 的体外评估。
