Evaluation of cassette analysis in pharmacokinetic studies

Abstract

In Drug Discovery the first pharmacokinetic in vivo study of new compounds is of great importance to provide an initial assessment of the drug pharmacokinetik (PK) parameters. Data from these studies is used to optimize the PK properties of the chemical series and to calculate the initial doses in further in vivo studies. Because of the everexisting demand to increase throughput in Drug Discovery, there is a constant need for improved methods that provide faster bioanalysis or a reduced amount of samples. The aim of this study was to develop a method for sample pooling of three standardized pharmacokinetic in vivo studies. Strategies for sample reduction, fast chromatography, sensitivity and avoiding ionization suppression was investigated.

In this study six reference compounds were selected to examine how sample pooling influences accuracy, precision, LOQ and PK-parameters. Sample preparation was performed by protein precipitation followed by HPLC-MS/MS and UPLC-MS/MS analysis. Diazepam, propranolol and imipramine were analysed in positive ESI mode whilst naproxen, diclophenac and rofecoxib were analysed in negative ESI mode. As warfarin can be detected both modes it was used as internal standard for all compounds. Spiked plasma (simulated to PK profiles) and samples from in vivo studies was used for the assessment. Equal volumes of three plasma samples corresponding to each time point of three individually dosed rats were pooled. The matrix effect of different formulations, e.g. cyclodextrine in terms of ionization suppression was examined.