Simultaneous determination of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam in human serum using gas chromatography-mass spectrometry.

A method for the quantitation of midazolam and its metabolites 1-hydroxymidazolam and 4-hydroxymidazolam from human serum capable of monitoring concentrations achieved under therapeutic conditions is presented. The substances were extracted under basic conditions with toluene and the hydroxy metabolites transformed to their tert-butyldimethylsilyl derivatives with N-(tert-butyldimethylsilyl)-N-methyltrifluoroacetamide. The samples were measured by gas chromatography-mass spectrometry. The limits of detection are 0.2 ng ml(-1) for midazolam and 0.1 ng ml(-1) for 1-hydroxy- and 4-hydroxymidazolam. The coefficients of variation are 3.9% at 5 ng ml(-1) for midazolam, 6.7% at 2 ng ml(-1) for 1-hydroxymidazolam and 8.8% (22.2%) at 0.5 (0.2) ng ml(-1) for 4-hydroxymidazolam.