Quantitative Estimation of Gallic Acid in Amla Extract by ... - IJPI

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031Quantitative Estimation of Gallic Acid in Amla Extract byGradient RP-HPLC methodAngshuman Biswas 1 , Ananya Chatterjee 2 , Sandip Kumar Bahdhopadhyay 21 Central Drugs Laboratory, 3, Kyd Street, Kolkata-162 Institution of Post Graduate Medical Education and Research, A.J.C. Bose Road, KolkataAbstractA Novel RP-HPLC assay method has been developed and validated for the estimation ofgallic acid. Chromatography was carried on C18 column (250 mm x 4.6 mm I.D., 5 μm) bygradient elution utilizing a mobile phase of acetonitrile and water containing 0.01 % v/v orthophosphoric acid (in the ratio of 80: 20% v/v) with UV detection at wavelength 272 nm at theflow rate 1ml/min. The proposed method was validated for sensitivity, specificity, linearity,accuracy, precision, ruggedness, robustness and solution stability. The response of the drugwas linear in the concentration range of 5-40 μg/ml. Limit of detection and limit ofquantification was found to be 0.22μg/ml and 6.77μg/ml respectively. The perentagerecovery ranged within 100.01-100.04%. Method, system, interday and intraday precisionwere also found to be within the limits of acceptance criteria. Method was found to be ruggedwhen analysis was carried out by different analyst. The proposed method is rapid, simple andalso it can be applied for the routine analysis of herbal formulations.Keywords: Gallic acid, Amla Ethanolic extract, RP-HPLC.IntroductionGallic acid (GA) is a phenolic compound.Structurally gallic acid has phenolic groupsthat serve as a source of readily availablehydrogen atoms such that the subsequentradicals produced can be delocalized overthe phenolic structure 1, 2 . The interest inthese compounds is due to itspharmacological activity as radicalscavengers 3.4 . It has been proved to havepotential preventive and therapeutic effectsin many diseases, where the oxidativestress has been implicated, includingcardiovascular diseases, cancer,neurodegenerative disorders and in aging.Volume 2, Issue 1, January− February 2012The phenolics are also of interest in food,cosmetic and pharmaceutical industries, assubstitutes for synthetic antioxidants 5 .Several chromatographic methods havebeen documented for determination ofgallic acid in plant extracts 6, 7 but due tothe complex nature and inherent variabilityof the chemical constituents of the plantbased drugs, it is difficult to establishquality control parameters and hencemodern analytical techniques are expectedto help in circumvention this problem. The*Corresponding AuthorAngshuman Biswas72 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031objective of the present investigation wasto establish and validate the fast andsensitive high performance liquidchromatography (HPLC) method fordetermination of gallic acid in ethanolicextract of amla.ExperimentalApparatusThe HPLC system consisted of a solventdelivery module Agilent 1100 SeriesGradient pump equipped with 20 µl loopand G1365B Multi Wavelength Detector.Integration was achieved by using thesoftware Chemstation. Separation wascarried out on a Zorbax, (250×4.6mm)5µm C-18 Column.Reagents and materialsEthanolic extract of amla, sodiumPhosphate Monobasic, Acetonitrile, OrthoPhosphoric Acid, MilliQ water. Allchemicals and solvents used were ofGR/HPLC grade of Rankem, India LimitedChromatographic ConditionThe mobile phase was prepared separatelywith 20mM monobasic Phosphate buffer(mobile phase A) and methanol (mobilephase B) and mobile phase is eluted as perfollowing gradient programming. The pHof the mobile phase (A) is maintained at4.5 with prior correction with 10%phosphoric acid. The prepared buffer wasfiltered through a Millipore 0.45 µmmembrane filter and ultrasonicallydegassed prior to use. Methanol and Waterin the ratio of 2:3 (v/v) was used as diluentthroughout the experiment. The detectionwavelength was set at 272 nm. The elutionwas done at a flow rate of 1.0 ml/min underambient condition.Volume 2, Issue 1, January− February 2012Standard Solution and CalibrationCurveA standard stock solution of gallic acid(1000 mcg/ml) was prepared in diluent.Subsequent dilutions were made in diluentto prepare the concentrations2.5,5,10,20,30 and 40 mcg/ml for gallicacid. The calibration curve was done byplotting peak area against sampleconcentration for each ingredient.AssayFinely powdered pure gallic acid wasweighed accurately on the electronicbalance (model Metler Toledo AG285) andvarying concentration of the analyte wastaken with proper dilution. The powderequivalent to 64.1 mg of ethanolic extractwas weighed accurately and dissolved in1.5 ml diluent. The solution was filteredthrough 0.45 µm Millex-HV syringe drivenmembrane filter unit. Twenty µl of thissolution was injected in triplicate under thespecified conditions. The peak areasobtained were related to slopes andintercepts from the calibration data tocalculate concentration of the gallic acid inethanolic extract.Results and DiscussionValidation of AssayTo validate the developed methodaccuracy, reproducibility and recoveryexperiments were carried out. The recoveryof the known amount of added standardwas studied at three levels. To an aliquot ofthe analyzed formulation a knownconcentration of standard solution wasadded. The content of gallic acid wasdetermined (Table 3).In the present study, a simple, precise,accurate and rapid reverse phase HPLCmethod has been developed and validated73 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031for the determination of gallic acid inherbal formulation. The developedanalytical method was validated as per ICHmethod validation guidelines 8 .The validation parameters addressed wereLOD, LOQ, linearity, accuracy, precision,robustness, ruggedness. System suitabilitytests were carried out on freshly preparedstandard stock solutions of pure gallic acid(Table 4). The calibration curve was linearin the range of 5-40 µg/ml for gallic acid.The limit of detection (LOD) and limit ofquantification (LOQ) for gallic acid wasfound 0.22 µg and 6.77 µg/ml respectively.ConclusionIn the present study, a simple andreproducible method for the estimation ofgallic acid in herbal formulation by reversephase HPLC method is developed. Thegallic acid content in ethanolic extracttwas quantified. The advantage of themethod lies in the simplicity of the samplepreparation and less run time. Thevalidated parameters indicate that thedeveloped method is quick, highselectivity and economic. Hence thedeveloped method is more suitable for theestimation of gallic acid in multicomponentherbal formulation.Acknowledgment: Director, CentralDrugs LaboratoryReferences:1. K. Robards , PD Prenzler , G Tucker , PSwatsitang and W Glover. Phenoliccompounds and their role in oxidativeprocesses in fruits, Food Chem(66)1999401-436.2. KM Nikolic.Theoretical study ofphenolic antioxidants properties inreaction with oxygen-centered radicals,J Mol Struc: THEOCHEM. (774)200695-105.3. M. Karamaæ .,A. Kosiñska andR.B.Pegg , Comparison of radical–scavenging activities of selectedphenolic acids.Pol, J. Food Nutr. Sci.(14)2005 165–170.4. S. Kaur , H. Michael , S Arora , P.LHarkonen and S Kumar,The in vitrocytotoxic and apoptotic activity ofTriphala –an Indian herbal drug, J.Ethnopharm. (97)2005 15–20.5. YY. Soong and PJ.Barlow , Antioxidantactivity and phenolic content of selectedfruit seeds, Food Chem.(88) 2004 411-417.6. Y. Amakura , M. Okada , S.Tsuji andY. Tonogai, Determination of phenolicacids in fruit juices by isocratic columnliquid chromatography, J ChromatogrA. (891) 2000 183-188.7. M. Rizzo , D. Ventrice , MA.Varone ,R. Sidari and A Carini , HPLCdetermination of phenolics adsorbed onyeasts, JPharm Biomed Anal.(42) 200646-55.8. ICH Harmonized Tripartite Guidelines,Validation of Analytical Procedures:Text and Methodology, 1994, Q2R (1).Volume 2, Issue 1, January− February 201274 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031Table-1 Gradient programming of HPLC SystemTime (minutes) Mobile phase (A) Mobile phase(B)0 95 58 90 1012 95 5Table-2 Assay of Gallic acid in the ethanolic extract of AmlaWeight of Ethanolicextract of Amla (mg)Amount of gallic acid found in AmlaExtract (%)64.1 0.04465 0.044765.6 0.045Mean 0.0445RSD 1.14Table- 3 Recovery study of Gallic acid by HPLC methodGallic acidadded (mg)AmountAmountfound (mg)PercentageRecovery10 20 3010.0045 20.0042 30.0047100.04 100.02 100.01Mean 100.023Volume 2, Issue 1, January− February 201275 | P a g ehttp://www.ijpi.org

INTERNATIONAL JOURNAL OFRESEARCH ARTICLEPHARMACEUTICAL INNOVATIONS ISSN 2249-1031Table-4Parametersresults of validation parameters of Gallic acidLinearityLinear equationRangeInterceptCorrelation coefficientResults foundY=19189X+6.205mcg/ml to 40mcg/ml6.200.9901Precision(%RSD)Ruggedness(%RSD)RobustnessLimit of Detection (LOD)Limit of Quantification (LOQ)System Suitability ParameterCalibration range (mcg/ml)Theoretical PlateTailing Factor0.330.45Robust0.22mcg/ml6.77mcg/ml5mcg/ml to 40mcg/ml40001.2Figure-1 Chromatogram of Ethanolic extract of Gallic acidVolume 2, Issue 1, January− February 201276 | P a g ehttp://www.ijpi.org