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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.
OBJECTIVE To describe a highly sensitive LC-ESI MS nmethod 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 (ie, a cocktail approach with caffeine 100 μg · kg -1, 100 μg · kg -1 of tolbutamide, 500 μg · kg -1 of omeprazole, 500 μg · kg -1 of dextromethorphan, 50 μg · kg -1 of midazolam and 100 μg · kg -1 of midazolam) acetate with diazepam and gliclazide as the IS. The assay was performed on an Agilent Eclipse Plus C18 column (2.1 × 50 mm, 3.5 μm). The mobile phase consisted of 0.01% formic acid (1 mmol·L -1 Ammonium formate) and acetonitrile The flow rate was 0.3 m L · min -1. The samples were analyzed by LC-20A & 5500 Qtrap ESI MSnin MRM mode. The MS / MS reaction selected 181.2 / 124.0 m / z for caffeine, 195.2 / 138.2 m / z for paraxanthine, 269.1 / 170.0 m / z for tolbutamide, 285.1 / 186.0 m / z for 4-hydroxytolbutamide, 346.1 / 198.1 m / z for o meprazole, 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 / 324.2 m / z for 1’-hydroxymidazolam.RESULTS The datashowed that the method was with good linearity in the range of 0.2-200 ng · m L-1 for caffeine, 0.1-25 ng · m L-1 for paraxanthine, 0.05- 100 ng · m L -1 for omeprazole, 0.01-25 ng · mL -1 for 5-hydroxyomeprazole, 0.1-200 ng · mL -1 for dextromethorphan, 0.05-12.5 ng · mL -1 for dextrophan, 0.2-200 ng · mL -1 for midazolam , and 0.2-25 ng · mL -1 for 1’-hydroxymidazolam, respectively. The% 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, 2E1 and 3A) research.