HPTLC Method Development and Validation for the Simultaneous Estimation of Embelin, Gallic Acid and Piperine in an <i>Ayurvedic</i> Formulation: A DOE Approach

Background: India has a longstanding legacy in Ayurveda, a holistic system of medicine that relies profoundly on herbal formulations. As worldwide interest in plant-based therapeutics grows, the need for scientific validation and quality control of Ayurvedic formulation has become increasingly important. Standardization through analytical methods ensures therapeutic consistency, safety, and efficacy. Embelin (from Embelia ribes), Gallic acid (from Emblica officinalis and other sources), and Piperine (from Piper nigrum) are well-documented phytoconstituents known for their anti-inflammatory, anti-oxidant, and bio-enhancing activities. These phytoconstituents are often present together in polyherbal formulations used in treating metabolic and inflammatory disorders. However, simultaneous estimation of these markers is rarely reported using a validated, robust analytical method. Aim: This study aimed to develop and validate a novel High-Performance Thin-Layer Chromatography (HPTLC) method for the simultaneous estimation of embelin, gallic acid, and piperine in an Ayurvedic formulation, using a Design of Experiments (DoE) approach for method optimization. Method: Chromatographic separation was achieved on silica gel 60 F254 plates using a mobile phase of toluene: ethyl acetate: petroleum ether: formic acid (7:1:1:1.5 v/v/v/v). Detection wavelengths were optimized to 284 nm (embelin), 278 nm (gallic acid), and 333 nm (piperine). Method validation was carried out per ICH Q2 (R2) guidelines. Robustness was assessed using a 2⁴⁻¹ fractional factorial design. Results: The method showed linearity (1000–5000 ng/band) with r² values of 0.9982, 0.9984, and 0.9986 for embelin, gallic acid, and piperine, respectively. LOD/LOQ values were within acceptable limits. Accuracy ranged from 99.90–101.02%, and precision showed %RSD <2%. The robustness study identified petroleum ether as a critical factor influencing gallic acid and piperine estimation. Conclusion: The validated HPTLC method is accurate, precise, robust, and suitable for routine quality control of Ayurvedic formulations containing these phytoconstituents. Major Findings: A validated HPTLC method was developed for the simultaneous estimation of embelin, gallic acid, and piperine with excellent linearity, accuracy, and precision as per ICH guidelines. DoE-based robustness testing identified petroleum ether as a critical factor affecting the resolution of gallic acid and piperine.