LC–MS–MS Method to Simultaneously Determine Six Probe Drugs for CYP450 Isozymes in Human Liver Microsomes

Here, we report a rapid and specific method based on high-performance liquid chromatography coupled with tandem mass spectrometry (LC–MS–MS) capable of quantifying six CYP450-specific probe substrates in human liver microsomal incubation mixtures simultaneously. These analytes were prepared by single-step extraction and detected in one run by switching polarity of electrospray ionization mode three times. Following optimization of the chromatographic conditions, the peaks were well separated, and retention times ranged between 2.0 and 8.4 min. The total run time for a single injection was within 9 min. This method was fully validated over linear range of 18.8–3,000.0 ng mL−1 for diclofenac, 0.8–3,000.0 ng mL−1 for dapson, 1.5–3,000.0 ng mL−1 for dextromethorphan, 2.0–4,000.0 ng mL−1 for omeprazole, 75.0–3,000.0 ng mL−1 for chlorzoxazone and 0.8–3,000.0 ng mL−1 for phenacetin using diazepam as internal standard. Samples were prepared by protein precipitation and analyzed on the LC–MS–MS equipped with ESI interface. For each analyte, inter- and intra-day precision (RSD%) were <15 % and accuracy was within 85–115 %. The specificity, precision, accuracy, stabilities and matrix effect were evaluated.