Novel, Rapid, Isocratic RP-HPLC Method for Simultaneous Estimation of Piperine and Embelin in Herbal Formulation
The objective of this paper was to develop and validate a novel, simple, rapid, precise and accurate, reverse-phase high-performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of piperine and embelin in the herbal formulation as per the International Conference on Harmonization guidelines (ICH). Chromatographic separation was achieved using a Cosmosil C-18 (250*4.6mm) SH 5.0 µm column with a mobile phase consisting of methanol and 0.02 M phosphate buffer in ratio 98:2 v/v, (pH adjusted to 2.3 with ortho-phosphoric acid) at a flow rate of 1 mL/min and column temperature maintained at 28°C and ultraviolet (UV) detection at 288 nm. The retention time of piperine and embelin was found to be 4.15 and 5.69 min respectively. The linearity of piperine and embelin was tested in the range of 5-40 ?g/mL. The correlation coefficient for piperine and embelin was found to be 0.997 and 0.995, respectively. The recovery values (98-102%) indicate a satisfactory accuracy. The method was found to be precise as the percentage relative standard deviation was found to be <2 %. The proposed novel isocratic RP-HPLC method is rapid (short run time below 10 min), precise, accurate and sensitive. The method was successfully applied for the simultaneous analysis of piperine and embelin in herbal formulation.