Determination of midazolam and two metabolites of midazolam in human plasma by gas chromatography--negative chemical-ionization mass spectrometry.

A method is described for measuring midazolam, a new anesthesia induction agent and hypnotic, and its hydroxymethyl and desmethyl metabolites in human plasma. Deuterated analogues of each compound are added to plasma as internal standards. The compounds are extracted from plasma with benzene containing 20% 1,2-dichloroethane and after removal of the extracting solvent are dissolved in a solution of bis-(trimethylsilyl)acetamide and acetonitrile. An aliquot of this solution is analyzed by gas chromatography--mass spectrometry with the mass spectrometer set to monitor in the gas chromatographic effluent the M.- ions of drug, metabolites and internal standards generated by methane electron-capture negative chemical ionization. For all three compounds, the limit of quantitation is 1 ng ml-1, and the precision (relative standard deviation) at a concentration of 5 ng ml-1 is less than 6%. Measurable amounts of the hydroxymethyl, but not the desmethyl, metabolite of midazolam could be found in the plasma of humans given either an intravenous or an oral dose of midazolam maleate.