Determination Of The CYP3A4and CYP2E1Enzyme Activity In Rat Liver Microsomes By High Performance Liquid Chromatography And Its Application

Objective To establish a sensitive, stable high-performance liquid chromatography with midazolam and Chlorzoxazone as probe drugs, rat liver microsomal incubation system Midazolam and its metabolites1-hydroxymidazolam, Chlorzoxazone and its metabolites6-hydroxy Chlorzoxazone was measured to calculate the CYP3A4and CYP2E1enzyme kinetic parameters in rat liver microsomes, to evaluate the impact of the traditional Chinese medicine Shenkang and Danhong injection in different volume concentration to CYP3A4and CYP2E1enzyme activity in rats. Method To1-hydroxymidazolam,it employed a Hypersil BDS C18column (150mm×4.60mm,5.00μm) at30℃. The mobile phase consisted of (A)10.0mmol/L dipotassium hydrogen phosphate buffer (pH8.4)-(B) Acetonitrile (60:40, v/v). The flow ratewas set at1.00mL/min and the ultravioletdetectorwas operated at230nm. Put the midazolam into the rat liver microsomal incubation system and determine the concentration of1-hydroxy midazolam, to calculate Km and Vmax values using the Lineweaver-Burk plot. Using the different volumetric concentration of Shuxuetong injection to incubate with rat liver microsomes, to examine its effect on the activity of rat CYP3A4. To6-hydroxy Chlorzoxazone,it employed a Hypersil BDS C18column (150mm×4.60mm,5.00μm) at30℃. The mobile phase consisted of (A)50.0mmol/L dipotassium hydrogen phosphate buffer (pH4.5)-(B) Acetonitrile. The flow ratewas set at1.00mL/min and the ultravioletdetectorwas operated at290nm. Put the Chlorzoxazone into the rat liver microsomal incubation system and determine the concentration of6-hydroxy Chlorzoxazone, to calculate Km and Vmax values using the Lineweaver-Burk plot. Using the different volumetric concentration of Shenkang and Danhong injection to incubate with rat liver microsomes, to examine its effect on the activity of rat CYP3A4and CYP2E1. Results Midazolam,1-hydroxy midazolam and its internal standard carbamazepine separating well and no interference from endogenous. The linear range of1-hydroxy midazolam is from0.300to20.0μmol/L. The day and the another day precision were less than10%and the recovery rate was more than85%. The kinetic parameters Km is56.5μmol/L, and the Vmax is1.01nmol/min/mg. The injection of Shenkang and Danhong have inbibitional effect on the activity of rat microsomal CYP3A4. Chlorzoxazone,6-hydroxy Chlorzoxazone and its internal standard phenacetinum separating well and no interference from endogenous. The linear range of6-hydroxy Chlorzoxazone is from0.500to30.0μmol/L. The day and the another day precision were less than10%and the recovery rate was more than85%. The kinetic parameters Km is138.8μmol/L, and the Vmax is1.68nmol/min/mg. The injection of Danhong have inbibitional effect on the activity of rat microsomal CYP2E1.But the injection of Shenkang have no effect on the activity of rat microsomal CYP2E1.Conclusion The method is rapid, stable and efficient. It’s suitable for determination of Midazolam and its metabolites1-hydroxymidazolam, Chlorzoxazone and its metabolites6-hydroxy Chlorzoxazone, and it can be used in enzyme kinetics and drug interaction studies.