LOW-LEVEL DETERMINATION OF DORZOLAMIDE AND ITS DE-ETHYLATED METABOLITE IN HUMAN PLASMA BY LIQUID-CHROMATOGRAPHY WITH ATMOSPHERIC-PRESSURE CHEMICAL-IONIZATION TANDEM MASS-SPECTROMETRY

A sensitive and specific method for the determination of dorzolamide ( I) and its de-ethylated metabolite (II) in human plasma has been devel oped utilizing high pressure liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. The analytes and internal standa rd (III) were isolated from the deproteinized pH 8.0 buffered plasma, using a liquid-liquid extraction with a mixture of ethyl acetate, tolu ene, and isopropanol. The analytes were then back extracted into 0.085 % phosphoric acid (200 mu l) and after washing the acidic extract with hexane, the organic layer was discarded and a fraction (50 mu l) of t he acid extract was injected into the LC/MS/MS system. The MS/MS detec tion was performed on a PE Sciex API III tandem mass spectrometer usin g a heated a nebulizer interface. Multiple reaction monitoring of the parent-->production combinations of mit 325-->199, 297-->199, and 397- ->306 were used to quantify I, II, and III, respectively. The assay wa s validated in the concentration ranges of 0.5-100 and 2.5-100 ng ml(- 1) of plasma for I and II, respectively. The precision of the assays, expressed as coefficients of variation (C.V.%), were less than 10% ove r the entire concentration range, with adequate assay specificity and accuracy. The LC/MS/MS method provided a 10-fold increase in the sensi tivity of I over the previously reported HPLC/UV method [1]. (C) 1997 Published by Elsevier Science B.V.