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

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 x 4.6 mm I.D). The mobile phase consisted of acetonitrile-20 mM ammonium acetate (65:35, v/v), (pH 4.0). Column temperature was either 40 degrees C (human assay) or 45 degrees 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 (+/-S.D.) of CI-1004 and I.S. were 7.8+/-0.1 and 10.9+/-0.2 at 40 degrees C and 7.7+/-0.2 min and 10.7+/-0.2 min at 45 degrees 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.