Development of a high-performance liquid chromatographic assay for digoxin using post-column fluorogenic derivatization.

An efficient high-performance liquid chromatographic (HPLC) separation for digoxin and its metabolites has been developed. Quantitation of digoxin at plasma levels was possible after the column effluent was passed through a fluorogenic post-column reactor. A study of the optimum post-column conditions was undertaken using a combination of ascorbic acid, hydrogen peroxide and hydrochloric acid, which was known to induce fluorescence in the digoxin molecule. Digoxin and its metabolites were separated on a 15 cm X 4.6 mm I.D., 3-microns reversed-phase (C18) HPLC column using methanol--ethanol--isopropanol--water (52:3:1:45) as the mobile phase at a flow-rate of 0.3 ml/min. A solution of 1.1 X 10(-3) M hydrogen peroxide in a 0.1% ascorbic acid solution and concentrated hydrochloric acid were added into the post-column reactor through a peristaltic pump at a combined flow with a flow-rate of 0.23 ml/min. The mixture was passed into the 20-m reaction coil maintained at 79 +/- 1 degrees C. The resulting digoxin fluorophore was monitored with a fluorescence detector. Detector responses were linear from 1.5 to 10 ng injected on-column. The overall performance demonstrated that this system has the sensitivity, linearity and stability desired in a digoxin plasma level determination. The total chromatographic time including the postcolumn derivatization step was about 40 min.