Development and validation of a method using supported liquid extraction for the simultaneous determination of midazolam and 1′-hydroxy-midazolam in human plasma by liquid chromatography with tandem mass spectrometry detection

摘要: The metabolic conversion of midazolam (MDZ) to its main metabolite 1'-hydroxy-midazolam (1-OH-MDZ) can be used as a probe drug for cytochrome P450 3A (CYP3A) activity. A sensitive method the simultaneous determination MDZ and 1-OH-MDZ in human plasma using supported liquid extraction (SLE) combination with chromatography-tandem mass spectrometry (LC-MS/MS) detection was developed validated. Plasma samples (100 μL) were diluted 0.5M NH(3) (aq) containing deuterated internal standards. extracted ethyl acetate on 96-well SLE-plate. Separation performed Symmetry Shield RP18 column an acidic gradient running from 2% 95% methanol 3 min. Detection triple quadrupole spectrometer positive electrospray selected reaction monitoring (SRM) mode. validated dynamic range 0.2-100 nmol/L both analytes. In concentration 0.6-75 recoveries ranges 91.2-98.6% 94.5-98.3% 1-OH-MDZ, respectively. Matrix effects more pronounced than but response still 75.4% or higher compared reference. overall repeatability within 2.2-7.6% analytes, reproducibility 3.1-10.2% analytes accuracy bias -1.1 7.5% successfully applied determine concentrations 14 healthy volunteers up 24h after administration single oral dose 2mg MDZ. SLE technology found convenient suitable sample preparation, rapid, selective reproducible plasma.