Three complementary liquid chromatographic methods for determination of the peptoid cholecystokinindashB antagonist, CI-988, in rat plasma

Abstract

Three different liquid chromatographic methods (two quantitative methods which employ fluorescence detection and one qualitative method which employs selected iondashmonitoring detection) were developed and validated to provide complementary specificity for determination of CI-988, a cholecystokinindashB antagonist, in rat plasma. The first quantitative method involves isocratic separation of “nondashionized” 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 (⩽15% relative standard deviation). Plasma CI-988 concentrations for samples ( N = 69) assayed with the “nondashionized” separation are linearly correlated with concentrations for the same samples assayed with the “anionic” separation ( y = 1.08 x − 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.