Objective: To establish a rapid,accurate and simultaneous quantitative analysis method for probe drugs of major cytochrome P450（CYP450） enzymes and their specific metabolites,in order to be used in clinical evaluation of drug-drug interaction（DDI）. Methods: A“ probe cocktail” approach was employed to evaluate the activities of CYP450s composed of caffeine（CYP1A2）,losartan（CYP2C19）,omeprazole（CYP2C9）, dextromethorphan（CYP2D6,CYP3A4） and midazolam（CYP3A4）. An online-SPE-LC-MS/MS method was developed to analyze the human plasma after“ probe cocktail” was administrated. Plasma samples were hydrolyzed by glucuronidase and subjected to online solid-phase extraction with Spark HySphere-C18HD（10 mm×2 mm, 7 μm） thereafter. Separation of 11 analytes was accomplished by a gradient elution on a Shiseido MGⅢ（2.0 mm×100 mm,5 μm） analytical column using methanol-acetonitrile（1∶1）-water with 0.01% formic acid as the mobile phase at a flow rate of 0.35 mL·min-1. The column temperature was maintained at 40 ℃ . Mass spectrometry was performed using a triple-quadrupole mass spectrometer operated in positive ion ESI mode, with MS/MS transitions of 5 probe drugs and the corresponding 6 metabolites monitored. Results: The established analysis method could complete the detection of 11 analytes in 5 minutes. The validation of methodology showed that the method had specificity for the analysis of plasma samples,exhibited good linearity in the quantitative ranges and little matrix effect. The RSD values of the intra-and inter-batch quality control samples of the analytes were 2.1%-14.2% at medium and high concentrations and 2.1%-15.4% at LLOQ level along with the accuracy in the range of 86.4% to 107.2%. The samples had good stability at room temperature,long-term freezing,repeated freeze-thaw and placement at autosampler. The method was applied to analyze plasma samples from subjects taken the“ probe cocktail”. The results showed that the concentrations of 5 probe drugs and 6 metabolites were all within the quantitative range. Among them,the concentration of dextromethorphan's metabolite 3-methoxymorphan was the lowest,all of which were lower than 2 ng·mL-1. Conclusion: The established online SPE-LC-MS/MS method provides a rapid and sensitive analytical tool to serve the investigation of multiple DDI and the CYP450 enzymes phenotyping in the clinic.
ZHANG Ying, LI Jun-mei, MIAO Lan, SUN Ming-qian, LIN Li, LIU Jian-xun, XU Li
. Simultaneous determination of 5 probe drugs for CYP450s and their 6 specific metabolites in human plasma by online SPE-LC-MS/MS*[J]. Chinese Journal of Pharmaceutical Analysis, 2021
, 41(4)
: 603
-612
.
DOI: 10.16155/j.0254-1793.2021.04.06
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