Quantitative Estimation of Gallic Acid in Amla Extract by Gradient RP-HPLC method

Transcription

1 Quantitative Estimation of Gallic Acid in Amla Extract by Gradient RP-HPLC method Angshuman Biswas 1, Ananya Chatterjee 2, Sandip Kumar Bahdhopadhyay 2 1 Central Drugs Laboratory, 3, Kyd Street, Kolkata-16 2 Institution of Post Graduate Medical Education and Research, A.J.C. Bose Road, Kolkata Abstract A Novel RP-HPLC assay method has been developed and validated for the estimation of gallic acid. Chromatography was carried on C18 column (250 mm x 4.6 mm I.D., 5 μm) by gradient elution utilizing a mobile phase of acetonitrile and water containing 0.01 % v/v ortho phosphoric acid (in the ratio of 80: 20% v/v) with UV detection at wavelength 272 nm at the flow rate 1ml/min. The proposed method was validated for sensitivity, specificity, linearity, accuracy, precision, ruggedness, robustness and solution stability. The response of the drug was linear in the concentration range of 5-40 μg/ml. Limit of detection and limit of quantification was found to be 0.22μg/ml and 6.77μg/ml respectively. The perentage recovery ranged within %. Method, system, interday and intraday precision were also found to be within the limits of acceptance criteria. Method was found to be rugged when analysis was carried out by different analyst. The proposed method is rapid, simple and also it can be applied for the routine analysis of herbal formulations. Keywords: Gallic acid, Amla Ethanolic extract, RP-HPLC. Introduction Gallic acid (GA) is a phenolic compound. Structurally gallic acid has phenolic groups that serve as a source of readily available hydrogen atoms such that the subsequent radicals produced can be delocalized over the phenolic structure 1, 2. The interest in these compounds is due to its pharmacological activity as radical scavengers 3.4. It has been proved to have potential preventive and therapeutic effects in many diseases, where the oxidative stress has been implicated, including cardiovascular diseases, cancer, neurodegenerative disorders and in aging. The phenolics are also of interest in food, cosmetic and pharmaceutical industries, as substitutes for synthetic antioxidants 5. Several chromatographic methods have been documented for determination of gallic acid in plant extracts 6, 7 but due to the complex nature and inherent variability of the chemical constituents of the plant based drugs, it is difficult to establish quality control parameters and hence modern analytical techniques are expected to help in circumvention this problem. The *Corresponding Author Angshuman Biswas 72 P a g e

2 objective of the present investigation was to establish and validate the fast and sensitive high performance liquid chromatography (HPLC) method for determination of gallic acid in ethanolic extract of amla. Experimental Apparatus The HPLC system consisted of a solvent delivery module Agilent 1100 Series Gradient pump equipped with 20 µl loop and G1365B Multi Wavelength Detector. Integration was achieved by using the software Chemstation. Separation was carried out on a Zorbax, ( mm) 5µm C-18 Column. Reagents and materials Ethanolic extract of amla, sodium Phosphate Monobasic, Acetonitrile, Ortho Phosphoric Acid, MilliQ water. All chemicals and solvents used were of GR/HPLC grade of Rankem, India Limited Chromatographic Condition The mobile phase was prepared separately with 20mM monobasic Phosphate buffer (mobile phase A) and methanol (mobile phase B) and mobile phase is eluted as per following gradient programming. The ph of the mobile phase (A) is maintained at 4.5 with prior correction with 10% phosphoric acid. The prepared buffer was filtered through a Millipore 0.45 µm membrane filter and ultrasonically degassed prior to use. Methanol and Water in the ratio of 2:3 (v/v) was used as diluent throughout the experiment. The detection wavelength was set at 272 nm. The elution was done at a flow rate of 1.0 ml/min under ambient condition. Standard Solution and Calibration Curve A standard stock solution of gallic acid (1000 mcg/ml) was prepared in diluent. Subsequent dilutions were made in diluent to prepare the concentrations 2.5,5,10,20,30 and 40 mcg/ml for gallic acid. The calibration curve was done by plotting peak area against sample concentration for each ingredient. Assay Finely powdered pure gallic acid was weighed accurately on the electronic balance (model Metler Toledo AG285) and varying concentration of the analyte was taken with proper dilution. The powder equivalent to 64.1 mg of ethanolic extract was weighed accurately and dissolved in 1.5 ml diluent. The solution was filtered through 0.45 µm Millex-HV syringe driven membrane filter unit. Twenty µl of this solution was injected in triplicate under the specified conditions. The peak areas obtained were related to slopes and intercepts from the calibration data to calculate concentration of the gallic acid in ethanolic extract. Results and Discussion Validation of Assay To validate the developed method accuracy, reproducibility and recovery experiments were carried out. The recovery of the known amount of added standard was studied at three levels. To an aliquot of the analyzed formulation a known concentration of standard solution was added. The content of gallic acid was determined (Table 3). In the present study, a simple, precise, accurate and rapid reverse phase HPLC method has been developed and validated 73 P a g e

3 for the determination of gallic acid in herbal formulation. The developed analytical method was validated as per ICH method validation guidelines 8. The validation parameters addressed were LOD, LOQ, linearity, accuracy, precision, robustness, ruggedness. System suitability tests were carried out on freshly prepared standard stock solutions of pure gallic acid (Table 4). The calibration curve was linear in the range of 5-40 µg/ml for gallic acid. The limit of detection (LOD) and limit of quantification (LOQ) for gallic acid was found 0.22 µg and 6.77 µg/ml respectively. Conclusion In the present study, a simple and reproducible method for the estimation of gallic acid in herbal formulation by reverse phase HPLC method is developed. The gallic acid content in ethanolic extractt was quantified. The advantage of the method lies in the simplicity of the sample preparation and less run time. The validated parameters indicate that the developed method is quick, high selectivity and economic. Hence the developed method is more suitable for the estimation of gallic acid in multicomponent herbal formulation. Acknowledgment: Director, Central Drugs Laboratory References: 1. K. Robards, PD Prenzler, G Tucker, P Swatsitang and W Glover. Phenolic compounds and their role in oxidative processes in fruits, Food Chem( 66) KM Nikolic.Theoretical study of phenolic antioxidants properties in reaction with oxygen-centered radicals, J Mol Struc: THEOCHEM. (774) M. Karamaæ.,A. Kosiñska and R.B.Pegg, Comparison of radical scavenging activities of selected phenolic acids.pol, J. Food Nutr. Sci. (14) S. Kaur, H. Michael, S Arora, P.L Harkonen and S Kumar,The in vitro cytotoxic and apoptotic activity of Triphala an Indian herbal drug, J. Ethnopharm. (97) YY. Soong and PJ.Barlow, Antioxidant activity and phenolic content of selected fruit seeds, Food Chem.(88) Y. Amakura, M. Okada, S.Tsuji and Y. Tonogai, Determination of phenolic acids in fruit juices by isocratic column liquid chromatography, J Chromatogr A. (891) M. Rizzo, D. Ventrice, MA.Varone, R. Sidari and A Carini, HPLC determination of phenolics adsorbed on yeasts, JPharm Biomed Anal.(42) ICH Harmonized Tripartite Guidelines, Validation of Analytical Procedures: Text and Methodology, 1994, Q2R (1). 74 P a g e

4 Table-1 Gradient programming of HPLC System Time (minutes) Mobile phase (A) Mobile phase(b) Table-2 Assay of Gallic acid in the ethanolic extract of Amla Weight of Ethanolic extract of Amla (mg) Amount of gallic acid found in Amla Extract (%) Mean RSD 1.14 Table- 3 Recovery study of Gallic acid by HPLC method Gallic acid Amount Amount found (mg) Percentage Recovery Mean P a g e

5 Table-4 Parameters results of validation parameters of Gallic acid Linearity Linear equation Range Intercept Correlation coefficient Results found Y=19189X mcg/ml to 40mcg/ml Precision(%RSD) Ruggedness(%RSD) Robustness Limit of Detection (LOD) Limit of Quantification (LOQ) System Suitability Parameter Calibration range (mcg/ml) Theoretical Plate Tailing Factor Robust 0.22mcg/ml 6.77mcg/ml 5mcg/ml to 40mcg/ml Figure-1 Chromatogram of Ethanolic extract of Gallic acid 76 P a g e