Establishment of a method for the analysis of five CYP450 enzyme substrates in biological samples
Objective To establish a method to determine CYP450 enzyme substrates in biological samples using HPLC.Methods Liver microsomes were prepared by calcium precipitation method,the probe substrates were incubated with rat liver microsomes in vitro using the probe substrate approach,Dextromethamine hydrobromide,omeprazole,phenacetin,nifedipine,and tolbutamide were used as probe drugs for CYP2D6,CYP2C19,CYP1A2,CYP3A4,and CYP2C9,respectively,using rat liver microsomes as the matrix.and the detection method for the five probe drugs was established using HPLC.Results Methodological investigations showed that the method had good specificity and was not affected by blank liver microsomes or internal standards.The linear regression equations were as follows:Dextromethorphan(Y=0.001 6X+0.003 7,r=0.994 8),Omeprazole(Y=0.005 3X+0.016 2,r=0.996 3),Phenacetin(Y=0.008 1X+0.019 9,r=0.995 0),Nifedipine(Y=0.009 3X+0.011 8,r=0.991 7),and Tolbutamide(Y=0.008 1X+0.022 0,r=0.995 1).Intra-day and inter-day precision were good with RSD ≤8.75％,and sample stability at 4,12,and 24 hours was good with RSD ≤ 14.31％.The extraction recovery rate was greater than 71.36％,with RSD ≤6.98％.All results met the requirements for sample content determination.Conclusion The probe substrate method established in this experiment can be used for rapid in vitro evaluation of the effects of drugs on CYP450 isozyme activities,providing a reliable detection method for clinical drug-drug interaction studies.
High-performance liquid chromatographyCytochrome P450Methodological examinationDrug drug interactions
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