Validated HPLC/MS/MS assay for CI-1011 in rat plasma and a comparison with an HPLC/UV assay

Abstract

A liquid chromatographic/mass spectrometric (LC/MS/MS) method to quantitate CI-1011 in rat plasma has been validated and compared to an LC/UV assay. The analyte and internal standard were isolated from the plasma matrix by using liquid/liquid extraction with diethyl ether. The ether layer was evaporated to dryness and the residue reconstituted in acetonitrile–water (70:30, v/v). A 2.1×150 mm×5 μm Zorbax RX-C18 column with a mobile phase of acetonitrile–ammonium acetate (pH 8.0; 5 mM)–triethylamine (70:30:0.03, v/v/v) delivered at a flow rate of 0.2 ml min −1 was used for chromatography. Analyte and internal standard ion chromatograms were obtained by operating the mass spectrometer in the negative ion multiple reaction monitoring mode to detect the presence of a precursor–product ion pair for both the analyte and the internal standard. Samples were introduced into the mass spectrometer using electrospray ionization. Retention times of CI-1011 and of the internal standard (IS), [ 13C 6]CI-1011, were approximately 4.2 min. No peaks interfering with the quantitation of CI-1011 were observed throughout the validation process. Mean recoveries of CI-1011 from rat plasma ranged from 98.2 to 105%. The recovery of the IS was 100%. Assay precision for CI-1011, based on the percent relative standard deviation of replicate quality controls, was less than or equal to 5.60% with an accuracy of ±8.80%. The lower limit of quantitation for CI-1011 was 0.500 ng ml −1 for a 0.2-ml sample aliquot. CI-1011 is stable in rat plasma for 24 h at room temperature and for at least 34 days at −20°C. This assay has been proven suitable for routine quantitation of CI-1011 in rat plasma at concentrations from 0.500 (100 pg on-column) to 500 ng ml −1. The applicability of this method to determine CI-1011 concentrations in rat plasma is reported in this manuscript. CI-1011 concentrations, in plasma samples from cholesterol- and chow-fed rats administered single daily oral doses of CI-1011 in a CMC/Tween suspension, obtained using a validated LC/UV assay were compared to concentrations obtained using the reported LC/MS/MS assay over the concentration range 0.0806–12.3 μg ml −1. The concordance correlation coefficient determined for this comparison was 0.9977, suggesting that the CI-1011 concentrations obtained by the two assays are in excellent agreement.