SIMULTANEOUS DETERMINATION OF FLUMAZENIL, MIDAZOLAM AND METABOLITES IN HUMAN BIOLOGICAL-FLUIDS BY LIQUID-CHROMATOGRAPHY

A simple procedure for the simultaneous determination of flumazenil, m idazolam, 1-hydroxymethylmidazolam and 4-hydroxymidazolam in plasma or urine in surgical patients is described. The assay involves a prelimi nary extraction of the drugs, metabolites and internal standard (flura zepam) from biological fluid into an organic solvent mixture (dichloro methane-diethyl ether, 40:60 v/v). The extract was evaporated to dryne ss at 40-degrees-C under a gentle stream of nitrogen. The residue was re-dissolved in distilled methanol (80 mul) and a 30-mul aliquot was i njected via an automatic sampler into the liquid chromatograph and elu ted with the mobile phase (32% acetonitrile in 0.004 M sodium hydrogen phosphate buffer containing 1 ml of triethylamine and adjusted to pH 7 .2) at a flow-rate of 1.5 ml/min on a 30-mum C-8 precolumn linked to a 4-mum Nova-pak C18 cartridge column (100 mm x 8 mm I.D.) at ambient t emperature (25-degrees-C). The eluate was detected at 220 nm.