SPECIFIC AND SENSITIVE DETERMINATION OF DIGOXIN AND METABOLITES IN HUMAN SERUM BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY WITH CYCLODEXTRIN SOLID-PHASE EXTRACTION AND PRECOLUMN FLUORESCENCE DERIVATIZATION

A precolumn fluorescence derivatization high performance liquid chroma tographic method has been developed for the simultaneous determination of digoxin and its metabolites digoxigenin bisdigitoxoside, digoxigen in monodigitoxoside digoxigenin, and dihydrodigoxin (20-R and 20-S epi mers) in human serum. Digoxin and its metabolites were extracted from serum samples (containing digitoxin as internal standard) with a cyclo dextrin solid-phase extraction (SPE) column. Fluorescent derivatives w ere formed by reaction of the analytes with 1-naphthoyl chloride in th e presence of 4-dimethylaminopyridine under a nitrogen atmosphere in a glove box with controlled relative humidity (26% r.h. or less). The d erivatives were isolated using cyclodextrin and Cl SPE columns sequent ially, and determined by HPLC using silica column separation and fluor escence detection. Calibration curves were linear over the concentrati on range from 0.25 to 4.0 ng ml(-1). Recoveries of digoxin and its met abolites from serum ranged from 62 to 86%, and coefficients of variati on from repetitive analyses ranged from 6.9 to 20.9% and from 5.8 to 1 2.2% at 0.5 ng ml(-1) and 2.0 ng ml(-1), respectively. This method has been shown capable of specifically determining digoxin and its major metabolites in serum, and has been successfully used in the determinat ion of digoxin and its metabolites in serum samples collected from pat ients undergoing digoxin therapy. This method thus permits the investi gation of digoxin metabolism and pharmacokinetics after the administra tion of commercial dosage forms.