‘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 + 6beta-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 microg ml(-1), in various sets was linear (r(2)>0.99) and absolute recoveries of all analytes were >90%. The lower limit of quantification was 0.1 microg 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.

Applicability of bioanalysis of multiple analytes in drug discovery and development: review of select case studies including assay development considerations

“…Rao et al (2003) have reported an HPLC assay for the simultaneous determination of probe substrates as well the metabolites formed due to the activity of some important CYP isozymes (namely CYP1A2, CYP2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4). The developed method offers several advantages, including the use of conventional instruments, simple and straightforward handling procedures, simplified data analysis and increased sample throughput by provision of a platform for continuous analysis of different combination sets in a single method.…”

Analysis of five HMG‐CoA reductase inhibitors— atorvastatin, lovastatin, pravastatin, rosuvastatin and simvastatin: pharmacological, pharmacokinetic and analytical overview and development of a new method for use in pharmaceutical formulations analysis and in vitro metabolism studies

“…A C 18 Inertsil ® ODS 3V column (4.6 × 250 mm, 5 µm, GL Sciences Inc., Tokyo, Japan) was used for the analysis. The ternary mobile phase system, consisting of reservoir A (0.01 M ammonium acetate buffer, pH 5.0-acetonitrile; 90:10), reservoir B (0.01 M ammonium acetate buffer, pH 5.0-acetonitrile; 5:95) and reservoir C (0.01 M ammonium acetate buffer, pH 5.0-methanol; 10:90), was run as per the gradient program (Rao et al, 2003) with a total flow rate of 1 mL/min through the column to elute the analytes. The eluate was monitored by the PDA detector (scan range 200-400 nm) and data integration was carried out by Millennium 32 software (version 4).…”

Altered intravenous pharmacokinetics of topotecan in rats with acute renal failure (ARF) induced by uranyl nitrate: Do adenosine A1antagonists (selective/non-selective) normalize the altered topotecan kinetics in ARF?

“…Plasma was harvested by centrifuging the blood using a Biofuge (Hereaus, Germany) at 7500g for 5 min and concentrations of total TPT (lactone plus hydroxy acid) were determined using a modified bioanalytical method earlier reported by us (Rao et al 2003).…”