The measurement of urinary digoxin and dihydrodigoxin by radioimmunoassay and by mass spectroscopy

Abstract

A radioimmunoassay for urinary digoxin is described which includes an initial solvent extraction to remove factors in urine which cause non-specific interference in the assay. The recoveries obtained using different solvents are compared and the non-specific factors influencing the assay investigated further. These effects were overcome by the use of a small urine volume (10 mul) in a direct, unextracted, urine assay and the results obtained correlated closely with those from the assay using prior extraction (r=0.99). No false positive results were obtained with unextracted urine samples from hospitalised patients not receiving digoxin. The specificity was also determined with regard to the natural steroids, spironolactone and the metabolites of digoxin including dihydrodigoxin. The metabolite dihydrodigoxin, with a saturated lactone ring, was not detected whereas the mono-, and bis-digitoxo-sides and digoxigenin metabolites did cross react in the assay. It was not possible to separate dihydrodigoxin and digoxin by thin-layer chromatography or solvent extraction due to their similar structures, however, mass spectroscopy was successful in this respect and was employed to obtain the ratio of dihydrodigoxin to digoxin in extracted urine samples. Levels of urinary digoxin excreted by patients maintained on different oral doses of the drug were measured. The percentage excreted in the urine as digoxin correlated closely with the oral dose (r = 0.96) but was found to be lower than that reported in most previous studies. Mass spectroscopy measurements showed that an average of 16.4% (range 12.2-19.7%) of the total oral dose was excreted as dihydrodigoxin in the urine of nine patients investigated.