Digoxin and metabolites in urine: a derivatization--high-performance liquid chromatographic method capable of quantitating individual epimers of dihydrodigoxin.

A high-performance liquid chromatographic method is described for the determination of digoxigenin, digoxigenin monodigitoxoside, digoxigenin bis-digitoxoside, digoxin, and dihydrodigoxin as the 3,5-dinitrobenzoyl esters. The method is applied to a 10 ml urine sample by adding digitoxigenin as internal standard, extracting with methylene chloride, derivatizing with 3,5-dinitrobenzoyl chloride in pyridine, chromatographing with a normal-phase system and detecting at 254 nm. Derivatized digoxigenin, digoxigenin mono- and bis-digitoxoside, and digoxin each yielded one symmetrical peak with the limit of sensitivity of the method being approximately 100 ng/ml. Analysis of a commercially obtained sample of dihydrodigoxin resulted in two well-separated, symmetrical peaks that represent the two epimers of derivatized dihydrodigoxin. Data indicate rapid and complete esterification of all primary and secondary alcohol moieties in the various molecules and the derivatives are shown to be stable in chloroform for at least four days. The procedure appears to be suitable for metabolic investigations and as a prototype for future analytical developments.