Development and Validation of a Gradient RP-HPLC–DAD Method for the Simultaneous Determination of p-Hydroxybenzoic Acid, Benzoic Acid and Salicylic Acid in Gymnema Sylvestre Leaves and Extract

Vollständiger Abstract

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The use and monitoring of preservatives such as p-hydroxybenzoic acid (pHBA), benzoic acid (BA), and salicylic acid (SA) in food and pharmaceutical products require reliable and sensitive analytical procedures. HPLC-based methods have been widely reported for the determination of benzoic acid, salicylic acid, and related preservatives in food and pharmaceutical matrices. In herbal extracts, particularly those prepared using methanolic or alcoholic solvents, trace-level determination of these compounds can be analytically challenging because of the complexity of plant matrices and the presence of naturally occurring phenolic compounds. The present study aimed to develop and validate a simple, precise, and robust reverse-phase high-performance liquid chromatographic (RP-HPLC) method with diode-array detection (DAD) for the simultaneous determination of pHBA, BA, and SA in Gymnema sylvestre leaves and their corresponding extracts, and to evaluate their occurrence in the raw material and extracts. Chromatographic separation was achieved on a C18 column using a binary gradient system consisting of 0.1% orthophosphoric acid and a mixture of acetonitrile–methanol, with detection at 241 nm. The analytical method was validated according to ICH Q2(R1) guidelines for specificity, linearity, accuracy, precision, robustness, limit of detection (LOD), and limit of quantification (LOQ). The method demonstrated good linearity (r² > 0.99) over the tested concentration range, acceptable precision (RSD < 2%), and satisfactory recoveries. These validation characteristics demonstrate the suitability of the method for quantitative determination of the selected analytes in the investigated botanical matrix. Application of the validated method to G. sylvestre samples obtained from different geographical regions demonstrated that pHBA, BA, and SA were present at low concentrations in both the leaves and corresponding extracts. The detection of phenolic compounds in plant-derived materials is consistent with the established literature on the occurrence and analysis of plant phenolics. Furthermore, comparison of the raw materials with their corresponding extracts showed no consistent or substantial enrichment during extraction. These findings indicate and confirm that the detected preservatives are naturally present G. sylvestre Leaves.