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.
Kwon
Ha-Jeong
HJ
Department of Preventive and Social Dentistry, Graduate School, Kyung Hee University, Hoegi-dong, Dongdaemoon-gu, Seoul 130-701, South Korea.
Sim
Hee-Jung
HJ
Lee
Sa-im
SI
Lee
Yong-Moon
YM
Park
Yong-Duk
YD
Hong
Seon-Pyo
SP
D000097:Acetonitriles / Q000737:chemistry; D002301:Cardiac Glycosides / Q000737:chemistry; D002623:Chemistry Techniques, Analytical; D002626:Chemistry, Pharmaceutical / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004070:Digitalis / Q000378:metabolism*; D004072:Digitonin / Q000032:analysis; D004074:Digitoxin / Q000032:analysis; D004077:Digoxin / Q000031:analogs & derivatives / Q000032:analysis; D004563:Electrochemistry / Q000379:methods*; D008956:Models, Chemical; D018515:Plant Leaves / Q000378:metabolism; D015203:Reproducibility of Results; D012997:Solvents; D013696:Temperature
D000097:Acetonitriles; D002301:Cardiac Glycosides; D012997:Solvents; D004077:Digoxin; D004074:Digitoxin; D004072:Digitonin; C005003:gitoxin; C032159:acetonitrile