Studies on digitalis. IV. A method for thin-layer chromatographic separation and determination of digitoxin and cardioactive metabolites in human blood and urine.
A thin-layer chromatographic method for the separation of digitoxin and its cardioactive metabolites in one system is described. Pre-coated silica gel plates impregnated with 15% formamide solution in acetone were developed twice in the same direction (running distance 18cm) with ethyl methyl ketone-xylene (50:50) as solvent. The system showed no border-zone effects, and the reproducibility was good. Samples (5 ml) of serum  or urine were extracted with dichloromethane, the extracts were evaporated, the residues were dissolved in 70% ethanol, the ethanol solutions were washed twice with light petroleum and then evaporated, and the residues were dissolved in chloroform-methanol for application to the thin-layer plates. After development, the metabolites were scraped from the plates and analyzed by means of a modified rubidium-86 method. The recovery for the whole procedure was 59%, and the sensitivity of the method permitted the determination of down to 0.5 ng per spot. The method will facilitate the study of digitoxin metabolism in patients undergoing treatment with the drug.