Simultaneous determination of midazolam and flumazenil concentrations in human plasma by gas chromatography

A gas chromatographic method for the simultaneous determination of midazolam and flumazenil concentrations in human plasma has been developed, using clinazolam as the internal standard. A mixture of chloroform and ethyl acetate (80:20) was used to extract the two compounds. A 2-microliters volume of the reconstituted sample was injected using a 1:20 split injection mode. Intra-day coefficients of variation ranged from 2 to 6.9%. The assay was linear over the range 3-1000 ng/ml. This assay was subsequently used to analyze samples from a human pharmacokinetic study.