digoxin and acetonitrile

We developed a highly sensitive and selective reversed-phase HPLC-pulsed amperometric detection (RP-HPLC-PAD) method for cardiac glycoside detection. Eight cardiac glycosides were completely separated within 45 min on a reversed-phase column using a water-acetonitrile gradient, and were detected using a PAD under NaOH alkaline conditions. The detection (S/N=3) and quantification (S/N=10) limits for the cardiac glycosides were 0.1-0.3 and 0.3-0.8 ng, respectively. The linear regression coefficient was 0.9962-0.9998 for concentrations of 1-25 μg/mL. Cardiac glycosides in the Digitalis purpurea leaf displayed intra- and inter-day precisions (RSDs) of <9.30% and average recoveries of 98.63-99.94%. The contents of gitoxin, digitonin, and digitoxin in the D. purpurea were 0.197, 0.11, and 0.379 mg/g for leaf dried at 60 °C, 0.058, 0.11, and 0.090 mg/g for leaf dried at ambient temperature, and N.D. (not detected), and 18.379 mg/g, N.D. for seed, respectively. We conclude that our method shows good precision and accuracy.

Topics: Acetonitriles; Cardiac Glycosides; Chemistry Techniques, Analytical; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Digitalis; Digitonin; Digitoxin; Digoxin; Electrochemistry; Models, Chemical; Plant Leaves; Reproducibility of Results; Solvents; Temperature

We describe the optimization and validation of a clinically feasible radioreceptor assay to detect endogenous digitalis-like factor(s) (EDLF) in human plasma and urine. The assay is based on the competitive replacement of 125I-labeled digoxin on human placenta membranes by ligands present in sample extracts. Digoxin and ouabain were used as calibrators. We also describe simple and effective methods for extraction and enrichment of EDLF from human plasma and urine. Assay sensitivity and precision were enhanced by using a sequential saturation technique with appropriate concentrations of tracer and receptors. Filtration was used to separate bound from free ligand. A two-step solid-state extraction with acetonitrile allowed the separation of two EDLFs with different polarity (EDLF-1 and EDLF-2) from the same plasma sample. A one-step solid-state extraction with methanol was suitable for urine. EDLF-1 and EDLF-2 in healthy adults were respectively 204 +/- 155 and 207 +/- 423 pmol/L ouabain equivalents, or 312 +/- 241 and 302 +/- 581 pmol/L digoxin equivalents. Plasma concentrations of EDLFs in newborns and pregnant women were higher than in healthy adults, and the concentrations in urine were higher than in plasma. Several cross-reactivity experiments showed that physiological concentrations of endogenous steroids and lipids did not inhibit binding, and supported the hypothesis that EDLFs are endogenous compounds other than the steroids and lipids also investigated.

Topics: Acetonitriles; Adult; Binding, Competitive; Blood Proteins; Cardenolides; Cell Membrane; Digoxin; Female; Humans; Infant, Newborn; Iodine Radioisotopes; Male; Methanol; Middle Aged; Ouabain; Placenta; Pregnancy; Radioligand Assay; Reference Values; Saponins; Sensitivity and Specificity

Urinary samples were collected from individuals not taking cardiac glycosides. Aliquots of 30 ml were passed through preparative octadecylsilane-bonded phase columns and eluted in fractions by stepwise increasing concentrations of acetonitrile. Eluted fractions were analysed for their contents of endogenous digoxinlike substance (EDLS) by radioimmunoassay of digoxin and by a bioassay of cardiac glycosides, which measures the uptake of rubidium (86Rb) by erythrocytes as an index of Na+, K+-ATPase activity. In both assays, digoxinlike activity was found in several fractions, but the highest values were consistently measured in the fractions eluted with 40% acetonitrile. Greater amounts of EDLS were recovered from the urine of pregnant women than from the urine of men and nonpregnant women.

Topics: Acetonitriles; Adult; Blood Proteins; Cardenolides; Digoxin; Female; Humans; Indicator Dilution Techniques; Male; Pregnancy; Radioimmunoassay; Rubidium Radioisotopes; Saponins