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 developed utilizing high pressure liquid chromatography (HPLC) with tandem mass spectrometric (MS/MS) detection. The analytes and internal standard (III) were isolated from the deproteinized pH 8.0 buffered plasma, using a liquid-liquid extraction with a mixture of ethyl acetate, toluene, and isopropanol. The analytes were then back extracted into 0.085% phosphoric acid (200 microliters) and after washing the acidic extract with hexane, the organic layer was discarded and a fraction (50 microliters) of the acid extract was injected into the LC/MS/MS system. The MS/MS detection was performed on a PE Sciex API III tandem mass spectrometer using a heated nebulizer interface. Multiple reaction monitoring of the parent-->product ion combinations of m/z 325-->199, 297-->199, and 397-->306 were used to quantify I, II, and III, respectively. The assay was 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% over the entire concentration range, with adequate assay specificity and accuracy. The LC/MS/MS method provided a 10-fold increase in the sensitivity of I over the previously reported HPLC/UV method [1].