Analysis of digoxin at therapeutic concentrations using high-performance liquid chromatography with post-column...

A high-performance liquid chromatographic (HPLC) procedure has been developed for the analysis of digoxin in plasma at therapeutic concentrations. The assay method provides resolution of digoxin from its metabolites using a 15 cm X 4.6 mm HPLC column containing 3-micron octadecylsilane-bonded stationary phase. The effluent of the column is passed through a post-column reactor in which a fluorescent derivative is formed by the co-addition of hydrochloric acid and dehydroascorbic acid. Detection of the derivative is accomplished in a fluorometer with excitation at 336 nm and emission at 425 nm. The extraction efficiency for recovery of digoxin from plasma samples was 70% using chloroform-isopropanol (9:1) following a pre-wash with isooctane to remove endogenous substances. The calibration curve was linear (r = 0.9999) over the range 0.5-4 ng/ml digoxin in plasma using digitoxigenin as internal standard. The minimum detectable quantity of digoxin in plasma was 0.5 ng/ml at a signal-to-noise ratio of 4:1. Split-samples of digoxin control sera were assayed by the HPLC procedure and by the prescribed radioimmunoassay procedure. Excellent correlation was observed between the two methods (r = 0.999). No interference was noted when a selection of commonly co-prescribed drugs were evaluated for chromatographic co-elution or interference in detection with that of digoxin or the internal standard.