RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF SILYMARIN AND PIPERINE IN A POLYHERBAL FORMULATION

Abstract

The current research paper explains the designed & validated a simple, specific & powerful Reverse-Phase High-Performance Liquid Chromatographic (RP-HPLC) technique to estimate the amount of Silymarin (Silybin) & Piperine in Polyherbal formulation. Chromatographic technique for the separation is developed using an Enable C18G column (250 mm × 4.6 mm×5 µm) with a mobile phase consisting phosphate buffer (pH 3.52) & acetonitrile (30:70 v/v) under isocratic elution & the  flow rate is 1.0 mL/min. Detection was performed on 288nm using a UV detector. Silymarin and Piperine had a retention time of about 8.1 min and 9.5 min respectively, which is a good showing of both the peak resolution and selectivity.

The method that was developed was tested in accordance with the ICH Q2 (R1) guideline evaluating such parameters of system suitability, as accuracy, precision, linearity, Limit of Detection (LOD), Limit Of Quantification (LOQ), robustness and ruggedness. The Linearity was observed in the concentration range of 5-25 µg per mL for both analyte with correlation coefficients (r²) of 0.9992 for Silymarin and 0.9994 for Piperine. The recovery percentages fell within 98102 and the values of the RSD were equal to minor than 2 which validated the accuracy and precision of the method. The LOD_s & LOQs values were found to be 0.78 µg per mL and 2.35 µg per mL for Silymarin, and 0.65 µg per mL and 2.00 µg per mL for Piperine demonstrating high sensitivity.

The validated technique has been successfully used on five commercially prepared polyherbal preparations with assay results of 95-105% of the stated claim. All chromatograms showed several small peaks which were the herbal constituents but only Silymarin and Piperine were determined as the marker compounds concerned since they had pharmacological importance. The suggested RP-HPLC technique is reproducible, economical, and efficient, and thus can be applied to normal quality control and standardization of Silymarin and Piperine herbal preparations.