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 [used in cardiovascular pharmacology]. 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 1 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 2 well-separated, symmetrical peaks that represent the 2 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 4 days. The procedure appears to be suitable for metabolic investigations and as a prototype for future analytical developments.
