Validated HPLC method for determination of sennosides A and B in senna tablets

Abstract

This study developed an efficient and reliable ion-pair liquid chromatographic method for quantitation of sennosides A and B in commercial senna tablets. Separation was conducted on a Hypersil C 18 column (250×4.6 mm, 5 μm) at a temperature of 40 °C, using a mixture of 0.1 M acetate buffer (pH 6.0) and acetonitrile (70:30, v/v) containing 5 mM tetrahexylammonium bromide as mobile phase. Sennosides A and B were completely separated from other constituents within 14 min. The developed method was validated. Both run-to-run repeatability ( n=10) and day-to-day reproducibility ( n=3) of peak area were below 0.4% RSD. Linearity of peak area was tested in the range 30–70 μg/ml ( r>0.9997). Accuracy was assessed with recovery and the recoveries for sennosides A and B were 101.73±1.30% and 101.81±2.18% ( n=3×6), respectively. Robustness of the analytical method was tested using a three-leveled Plackett–Burman design in which 11 factors were assessed with 23 experiments. Eight factors (column, concentration of ion pair reagent, % of organic modifier (acetonitrile), buffer pH, column temperature, flow rate, time constant and detection wavelength) were investigated in a specified range above and below the nominal method conditions. It was found that: (1) column and % acetonitrile affected significantly resolution and retention time, (2) column, % acetonitrile, column temperature, flow rate and time constant affected significantly the plate number of sennoside A, and (3) column and time constant affected significantly the tailing factor.