HPLC method validation for Digitalis and its analogue by pulsed amperometric detection

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.