Development and Validation of HPTLC and RP-UHPLC Methods for Estimation of Silymarin in Herbal Formulation

Abstract

Background: Silymarin, a flavonolignan complex isolated from Silybum marianum L. (milk thistle), is widely incorporated into herbal formulations because of its hepatoprotective, antioxidant, and anti-inflammatory properties. Reliable and validated analytical methods are essential to ensure the quality, safety, and consistency of Silymarin-containing herbal products. Aim: The present study aimed to develop and validate simple, rapid, accurate, and reliable High-Performance Thin-Layer Chromatography (HPTLC) and Reverse Phase-Ultra High-Performance Liquid Chromatography (RP-UHPLC) methods for the quantitative estimation of Silymarin in herbal formulations. Methods: The identity of Silymarin was confirmed by FTIR spectroscopy. HPTLC analysis was performed using silica gel 60 F254 plates with hexane:ethyl acetate:ortho-phosphoric acid (6:3.5:0.5 v/v/v) as the mobile phase and detection at 287 nm. RP-UHPLC analysis was carried out on a C18 column using methanol:0.1% glacial acetic acid (85:15 v/v) at a flow rate of 1.0 mL/min with UV detection at 287 nm. Both methods were validated according to ICH Q2 (R2) guidelines for linearity, accuracy, precision, specificity, sensitivity, ruggedness, and robustness. Results: The HPTLC method produced a well-resolved peak with an Rf value of 0.52 and showed excellent linearity over 500–3000 ng/spot (r2 = 0.9992). The RP-UHPLC method exhibited a retention time of 2.6 min with linearity over 20–120 µg/mL (r2 = 0.9991). Both methods demonstrated recoveries ranging from 99–101%, precision with %RSD below 2%, satisfactory Limit of Detection (LOD) and Limit of Quantification (LOQ) values, and excellent specificity without interference from formulation excipients. Analysis of commercial herbal formulations showed Silymarin content within 98–103% of the labelled claim. Conclusion: The developed HPTLC and RP-UHPLC methods are simple, precise, accurate, sensitive, robust, and suitable for the routine quality control, standardisation, and quantitative estimation of Silymarin in herbal formulations. Major Findings: This study developed and validated simple HPTLC and RP-UHPLC methods for the estimation of Silymarin in herbal formulations. Both methods demonstrated excellent accuracy, precision, and specificity, making them suitable for routine quality control.