Analysis of microsomal metabolic stability using high-flow-rate extraction coupled to capillary liquid chromatography–mass spectrometry

A method is described for on-line high-speed extraction of microsomal samples and analysis by capillary liquid chromatography–mass spectrometry (LC–MS) for the determination of metabolic stability in connection with the development of positron emission tomography (PET) tracers. The method allowed direct injections of large sample volumes at a fast extraction rate, providing a gain in both sensitivity and sample preparation time. The calibration curve of the test compound flumazenil (Ro 15–1788) was linear in the concentration range of 1–150 nM, with a correlation coefficient exceeding 0.999. The accuracy of the method ranged from 98 to 101%. A high precision was obtained, with mean intra-assay and inter-assay relative standard deviations of at most 1.4 and 1.5%, respectively, for quality control (QC) samples. The extraction efficiency was determined to be 99.4%, the total recovery 96% and the carryover to ≤0.23%. Extractions were performed in a concentration interval of 30–3000 nM without any sign of column overload. The method was successfully used for determining the microsomal metabolic stability of flumazenil. As a result, the described analysis system is currently used for metabolic screening of PET tracer candidates in our laboratory.