OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After ad...OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.展开更多
基金The project supported by National Natural Science Foundation of China(81473579,81273654,81102879)the National Science and Technology Major Projects for"Major New Drugs Innovation and Development"(2013ZX09103002-022)
文摘OBJECTIVE To describe a highly sensitive LC-ESI MSnmethod that was developed to simultaneously detect six CYP isoform-specific probes and their metabolites in rat plasma for microdosing cocktail study.METHODS After administration of a mixture of six probes(i.e.,a cocktail approach with caffeine 100μg·kg-1,tolbutamide100μg·kg-1,omeprazole 500μg·kg-1,dextromethorphan 500μg·kg-1,chlorzoxazone 50μg·kg-1and midazolam 100μg·kg-1)to SD rats.The plasma samples were extracted using ethyl acetate with diazepam and gliclazide as the IS.The assay was performed on an Agilent Eclipse Plus C18 column(2.1×50 mm,3.5μm).The mobile phase consisted of 0.01%formic acid(1 mmol·L-1ammonium formate)and acetonitrile.The flow rate was0.3 m L·min-1.The samples were analyzed by LC-20A&5500Qtrap ESI MSnin MRM mode.The MS/MS reaction selected 181.2/124.0 m/z ions for caffeine,195.2/138.2m/z for paraxanthine,269.1/170.0 m/z for tolbutamide,285.1/186.0 m/z for 4-hydroxytolbutamide,346.1/198.1m/z for omeprazole,362.2/214.2 m/z for 5-hydroxyomeprazole,272.3/147.1 m/z for dextromethorphan,258.2/157.0 m/z for dextrorphan,168.1/132.1 m/z for chlorzoxazone,326.1/291.2 m/z for midazolam,and 342.1/324.2m/z for 1′-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng·m L-1for caffeine,0.1-25 ng·m L-1for paraxanthine,0.05-100 ng·m L-1for omeprazole,0.01-25 ng·mL-1for 5-hydroxyomeprazole,0.1-200 ng·mL-1for dextromethorphan,0.05-12.5 ng·mL-1for dextrophan,0.2-200 ng·mL-1for midazolam,and 0.2-25 ng·mL-1for 1′-hydroxymidazolam,respectively.The stability%RSD for al probes was less than 15%and matrix effects in plasma on the ionization were negligible.CONCLUSION This highly sensitive and quantitative method allowed a pharmacokinetic study in subjects receiving doses 10-100 times lower than typical therapeutic doses.The established LCMS/MS method was suitable for pharmacokinetic study of this mixture cocktail probe group and could be applied deeply to CYP isoforms(1A2,2C9,2C19,2D6,2E1and 3A)research.
