‘Open access’ generic method for continuous determination of major human CYP450 probe substrates/metabolites and its application in drug metabolism studies

Abstract

1. An ‘open access’ generic high-performance liquid chromatography method was developed for different combination sets each containing specific cytochrome P450 probe substrate and the corresponding metabolite. Method development, optimization and validation were carried out with the following combinations: phenacetin + paracetamol + internal standard (IS, celecoxib), bufuralol + hydroxybufuralol + IS, testosterone + 6β-hydroxytestosterone + IS, chlorzoxazone + 6-hydroxychlorzoxazone + IS, coumarin + 7-hydroxycoumarin + IS, tolbutamide + hydroxytolbutamide + IS, and diazepam + desmethyldiazepam + IS.

2. The assay procedure involved a simple one-step liquid/liquid extraction followed by reverse phase chromatography (Inertsil ODS 3V column) employing a ternary gradient system and the eluate was monitored by a photodiode array/fluorescence detector. The standard curve for each compound, in the concentration range 0.1–10 μg ml⁻¹, in various sets was linear (r²>0.99) and absolute recoveries of all analytes were >90%. The lower limit of quantification was 0.1 μg ml⁻¹. The intraday precision and accuracy in the measurements of quality control were <15% relative standard deviation and <15% deviation from nominal values, respectively.

3. Each combination set was tested with individual chemical inhibitors (furafylline, quinidine, ketoconazole, disulfiram, diethyldithiocarbamate, sulphaphenazole and tranylcypromine) and all analytes were well resolved. Overall, the assay is simple, uses conventional instrumentation and provides a scope to analyse all cytochrome P450 combination sets continuously. The application of the method in the cytochrome P450 liability screen of novel compounds is also presented.