Determination of a new reversible proton pump inhibitor,DBM-819, in human plasma and urine,and rat tissue homogenates by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method was developed for the determination of a new proton pump inhibitor, DBM-819, in human plasma and urine and rat tissue homogenates using KR-60461 as an internal standard. A 100-microl aliquot of acetonitrile (containing 0.5 microg/ml of the internal standard) and a 200-microl aliquot of 0.1 M Na(2)HPO(4) (adjusted pH 11 with 1 N NaOH) were added to a 100-microl aliquot of biological sample. After vortex-mixing, the mixture was extracted with 1 ml of ethylacetate. After centrifugation at 12000 x g for 3 min, the organic layer was collected and evaporated under nitrogen gas. The residue was then reconstituted with a 100-microl aliquot of mobile phase, and a 40-microl aliquot was injected onto the HPLC column. The mobile phase, 0.02 M phosphate buffer (pH 5): acetonitrile: methanol (46:44:10, v/v/v), was run at a flow rate of 0.5 ml/min and the column effluent was monitored by the fluorescence detector set at an excitation wavelength of 340 nm and an emission wavelength of 470 nm. The retention times for DBM-819 and the internal standard were approximately 10.5 and 12 min, respectively. The detection limits of DBM-819 in human plasma and urine, and rat tissue homogenates were 0.01, 0.02 and 0.02 (or 0.05) microg/ml. respectively. The coefficients of variation (CV) of the assay were below 11% for human plasma and urine, and rat tissue homogenates. No interferences from endogenous substances were found.