Integrated HPTLC, HPLC, and LC–MS Approach for the Identification and UV-Based Antioxidant Evaluation of Gallic Acid

Gallic acid is a naturally occurring phenolic compound abundantly present in medicinal plants and herbal formulations, where it plays significantly role in contributing to a wide range of pharmacological activities. Therefore, the development of robust and reliable analytical techniques for its identification and quantification is essential. The objective of the present study was to develop and validate analytical methods for the identification and quantification of gallic acid using HPTLC, HPLC and LC–MS. For HPTLC analysis, gallic acid was separated on silica gel 60 F254 precoated plates using a mobile phase of toluene: ethyl acetate: formic acid (4.5:5:0.5, v/v/v), which produced well-defined and compact bands. The plates were visualized under ultraviolet light at 254 nm and 366 nm, followed by densitometric scanning. HPLC conditions were optimized to ensure accurate, precise, and reproducible quantification of gallic acid in the sample matrix. LC–MS analysis was employed to confirm the molecular identity of gallic acid based on its characteristic mass spectral profile. All analytical methods were validated as per standard guidelines, including parameters such as linearity, precision, accuracy, and specificity and demonstrated satisfactory reproducibility and consistency. The present study introduces a novel integrated analytical approach combining HPTLC, HPLC, and LC–MS for the precise identification and validation of gallic acid, coupled with UV-based antioxidant evaluation. Unlike existing methods that rely on single or dual techniques, this work establishes a unified, cross-validated platform that enhances analytical accuracy, structural confirmation, and bioactivity correlation, thereby addressing key limitations in current phytochemical analysis and advancing the field of analytical chemistry. In conclusion, the developed methods are reliable, validated, and suitable for the routine estimation of gallic acid in plant-based formulations, with potential applications in quality control and standardization of herbal products.