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

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 x 4.6 mm, 5 mum) at a temperature of 40 degreesC, 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 mug/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 x 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 sermoside A, and (3) column and time constant affected significantly the tailing factor. (C) 2002 Elsevier Science B.V. All rights reserved.