RESUMO
Three different liquid chromatographic methods (two quantitative methods which employ fluorescence detection and one qualitative method which employs selected ion-monitoring detection) were developed and validated to provide complementary specificity for determination of CI-988, a cholecystokinin-B antagonist, in rat plasma. The first quantitative method involves isocratic separation of "non-ionized" CI-988 and internal standard on a C-18 column, whereas the alternative quantitative method involves isocratic separation of the "anionic" analytes. These two quantitative HPLC methods rely on the intrinsic fluorescence of CI-988 and internal standard for detection, and both methods are equally sensitive (linear range of 2.0-1000 ng ml-1), accurate (+/- 15% relative error), and precise (< or = 15% relative standard deviation). Plasma CI-988 concentrations for samples (N = 69) assayed with the "non-ionized" separation are linearly correlated with concentrations for the same samples assayed with the "anionic" separation (y = 1.08 chi - 0.57, R = 0.999). In addition, a third qualitative method, HPLC-thermospray mass spectrometry, was developed to provide complementary evaluation of assay specificity through the use of selected CI-988 fragment ion monitoring. When investigating an anomalous chromatographic result that calls into question the specificity of a method, the availability and use of alternative validated chromatographic separations and orthogonal detection schemes are beneficial.
