High-performance thin-layer chromatographic determination of digoxin and related compounds, digoxigenin...

A high-performance thin-layer chromatographic (HPTLC) method for the determination of digoxin and its related compounds digoxigenin bisdigitoxoside (DBD) and gitoxin in digoxin drug substance and tablets was developed. Separation of the three compounds was accomplished on a C18 wettable reversed-phase plate using water-methanol-ethyl acetate (50:48:2, v/v/v) as the mobile phase. The analytes were determined by densitometry using absorbance for digoxin and fluorescence for the two related compounds. All peaks were quantified by peak-height analysis. Linear regression analysis of the data was performed for all three compounds. The calibration range for digoxin was set at 320-480 ng per 5-mm band, equivalent to 80-120% (w/w) of a 400-ng band load, that for DBD was set at 4-12 ng per 5-mm band, equivalent to 1-3% (w/w) of the digoxin load, and that for gitoxin was set at 0.4-1.6 ng per 5-mm band, equivalent to 0.1-0.4% (w/w) of the digoxin load. The limit of quantification (LOQ) for digoxin was 64 ng per 5-mm band with a limit of detection (LOD) of 8 ng per 5-mm band. The LOQs for both DBD and gitoxin were 0.12 ng per 5-mm band with LODs of 0.4 ng per 5-mm band. The linearity range for the digoxin peak height in the absorbance mode was 0-5000 ng per 5-mm band. The linearity range for DBD and gitoxin peak heights in the fluorescence mode was 0-2000 ng per 5-mm band.