Thin-layer chromatographic method for the determination of digitoxin in human serum

Abstract

A specific assay for therapy control purposes and the toxicology of digitoxin is described. Digitoxin is isolated from serum (2 ml) by means of a single extraction with chloroform. Part of the organic phase is evaporated to dryness at 40° in a stream of dry air and the residue is dissolved in a small volume of chloroform. Conventional thin-layer chromatography (TLC) is used for the separation of digitoxin and its metabolites and digoxin. Fluorescence of the spots can be generated by treatment with hydrogen chloride vapour under the influence of a quartz-halogen lamp. Interpolation between two reference standards gives the concentration in the sample. Linearity is observed between 2 and 20 ng. Extensive recovery studies in the therapeutic range of 10–50 ng/nl have been performed. The results showed an overall recovery of 99.1% with a standard deviation of 11.2%. The sensitivity is 1–2 ng of digitoxin when standards are applied on a conventional TLC plate with a small diameter. The time needed for one analysis is about 412 h, the real time of analysis being 212 h; in serial studies, 20–24 determinations could be made daily. Whereas in the descriptions of most methods it is not mentioned whether digitoxin metabolites are co-determined, the present assay separates the digitoxin completely from the other compounds in serum, and thus enables the total fate of digitoxin in relation to the clinical effect to be studied more specifically than by radioimmunoassay.