High-performance liquid chromatographic assay for CI-1004, a dual-inhibitor antiinflammatory agent, in rat, rabbit, dog, monkey and human plasma

Abstract

CI-1004 and PD 138389 (internal standard, I.S.), were isolated from rat, rabbit, dog, monkey and human plasma by solid-phase extraction with Bond-Elut C18 cartridges. Liquid chromatographic separation was achieved isocratically on a Zorbax Rx-C8 analytical column (250 mm×4.6 mm I.D.). The mobile phase consisted of acetonitrile-20 m M ammonium acetate (65:35, v/v), (pH 4.0). Column temperature was either 40°C (human assay) or 45°C and column effluent was monitored spectrophotometrically at 360 nm. Specificity, chromatographic performance parameters, system repeatability, recovery from matrix, linearity, precision, accuracy and stability were evaluated. Mean retention times (0S.D.) of CI-1004 and I.S. were 7.8±0.1 and 10.9 ±0.2 at 40°C and 7.7±0.2 min and 10.7±0.2 min at 45°C. No interfering peaks were observed at the retention time of CI-1004 throughout the validation process. Peak height ratios were proportional to CI-1004 over the concentration range of 7.5–5000 ng/ml in rat, rabbit and monkey plasma and 2.5–5000 ng/ml in dog and human plasma. Recovery of low, medium and high standards of CI-1004 ranged from 82.8–107% from all animal species and recovery of I.S. from rat, rabbit, dog and monkey plasma ranged from 77.5–82.0% and from human plasma was 111%. Assay precision for CI-1004 based on quality control samples was less than or equal to 8.5% C.V. with an accuracy (percentage relative error) of ±4.7% for all species. Minimum quantitation limit of CI-1004 was 7.5 ng/ml for 0.2 ml rat, rabbit and monkey plasma samples and 2.5 ng/ml for 0.5 ml dog and human plasma samples. The method is suitable for studying the preclinical and clinical pharmacokinetics of CI-1004;