Abstract
A reversed-phase high-performance liquid chromatography method with ultraviolet detection at 220 nm was developed to determine four carboxylic acid metabolites in plasma following therapeutic doses of the calcium antagonist felodipine. After the addition of an internal standard the analytes were isolated by liquid-liquid and solid-phase extraction. The metabolites were applied to a C2 cartridge in their free acid form, but they were transformed and retained as ion pairs with tetrabutylammonium during a wash with phosphate buffer (pH 7), prior to automated elution and injection by the Varian AASP system onto the analytical C18 column. Using a sample volume of 1 ml of plasma, the lower limit of determination for the metabolites was about 20 nmol/l. The influence of the pH of the mobile phase on the retention time of the metabolites and the structural requirements for the internal standard were studied. The method was applied to plasma samples from four dogs collected after an oral dose of felodipine. The plasma concentration-time profiles of the metabolites gave useful information about the mechanisms by which they were formed and eliminated.
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