Add time:07/17/2019         Source:sciencedirect.com

Simple and precise high-performance liquid chromatographic (HPLC) assays were developed and validated for the determination of a renin inhibitor (RI), 4-cyclohexyl-2-hydroxy-3-(3-methylsulfanyl-2-{2-[(morphine-4-carbonyl)amino]- 3-phenylpropionylamino}propionylamino)butyric acid isopropyl ester (CP-80, 794, I), and its hydrolytic cleavage metabolite, 2-[(morpholine-4-carbonyl)amino]-3-phenylpropionic acid (CP-84, 364, II) in dog and human plasma. The internal standard for I, CP-83,092 (III, a carbon-substituted derivative of I) and analyte were extracted by liquid-liquid extraction using n-butyl chloride, cleaved to form a fragment containing a primary amine, and subsequently fluorescently derivatized for measurement by HPLC. Samples were analyzed by reversed-phase HPLC using a Waters C18 column with fluorescence detection at 390 nm excitation/440 nm emission. The quantitation limit of I was 10 ng/ml and the calibration curve was linear over the range of 0.01–1.0 μg/ml (r2>0.99). In dog and human plasma, intra-and inter-assay precision ranged from 2.3 to 16% and 3.0 to 18%, respectively. The average recoveries were similar (≥63%) for both I and III and the upper limit of quantification of I can be as high as 4 μg/ml. The internal standard for II, CP-96,452 (IV, a methoxy derivative of II) and analyte were extracted by anion-exchange solid-phase extraction and subsequent liquid-liquid extraction using methyl tert.-butyl ether. Samples were analyzed by reversed-phase HPLC using a Waters C18 column with ultraviolet detection at 214 nm. The quantification limit of II was 20 ng/ml and the calibration curve was linear over the range of 0.02–2.0 μg/ml (r2>0.99). In dog and human plasma, intra- and inter-assay precision ranged from 2.6 to 13.0% and 1.8 to 2.0%, respectively. The average recoveries were similar (≥75%) for both II and IV and the upper limit of quantification of II can be as high as 20 μg/ml. The methods described have been successfully applied to the quantification of II can dog and human plasma samples over a 2 year period.

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