Semi-automated determination of plasma stability of drug discovery compounds using liquid chromatography–tandem mass spectrometry

A simple procedure for the measurement of stability of drug candidates in plasma was developed to eliminate the traditional labor-intensive and time-consuming sample preparation procedures that are typically used for these studies. The procedure makes use of a thermostatic autosampler as an incubator combined with the direct plasma injection method based on high-performance liquid chromatography (HPLC) coupled to tandem mass spectrometry (MS–MS). Untreated human, monkey, mouse and rat plasma containing the test compound was directly injected into a mixed-function column for on-line protein removal and chromatography. The test compound and its biotransformation product were separated via HPLC and monitored using the tandem mass spectrometer. The need for adequate chromatographic separation of the test compound (M) from its carboxylic acid metabolite (M+1) is demonstrated. Plasma samples from four different species at specified incubation temperatures were sequentially assayed in one analytical procedure. The injection-to-injection time was about 6 min. The peak responses of the test compound in individual plasma samples were repeatedly determined every 24 min. The retention times and peak shape of all analytes were found to be consistent throughout the experiments. The stability of the test compound in plasma was found to be a function of animal species, incubation time and temperature. The test compound was rapidly degraded in rat plasma at 37 °C, but it could be stabilized by adding sodium thiosulfate.