INTERNATIONAL JOURNAL OF

PHARMACEUTICAL INNOVATIONS

RESEARCH ARTICLE
ISSN 2249-1031

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 m) 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 g/ml. Limit of detection and limit of
quantification was found to be 0.22g/ml and 6.77g/ml respectively. The perentage
recovery ranged within 100.01-100.04%. 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.
Volume 2, Issue 1, January February 2012

The phenolics are also of interest in food,

cosmetic and pharmaceutical industries, as
substitutes for synthetic antioxidants5.
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
http://www.ijpi.org

INTERNATIONAL JOURNAL OF
PHARMACEUTICAL INNOVATIONS
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 l loop
and G1365B Multi Wavelength Detector.
Integration was achieved by using the
software Chemstation. Separation was
carried out on a Zorbax, (2504.6mm)
5m 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 m
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.

Volume 2, Issue 1, January February 2012

RESEARCH ARTICLE
ISSN 2249-1031
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 m Millex-HV syringe driven
membrane filter unit. Twenty l 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
http://www.ijpi.org

INTERNATIONAL JOURNAL OF
PHARMACEUTICAL INNOVATIONS
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 g/ml for gallic acid.
The limit of detection (LOD) and limit of
quantification (LOQ) for gallic acid was
found 0.22 g and 6.77 g/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

RESEARCH ARTICLE
ISSN 2249-1031

2.

3.

4.

5.

6.

7.

8.
References:

compounds and their role in oxidative

processes in fruits, Food Chem(
66)1999401-436.
KM Nikolic.Theoretical study of
phenolic antioxidants properties in
reaction with oxygen-centered radicals,
J Mol Struc: THEOCHEM. (774)2006
95-105.
M. Karama .,A. Kosiska and
R.B.Pegg , Comparison of radical
scavenging activities of selected
phenolic acids.Pol, J. Food Nutr. Sci.
(14)2005 165170.
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)2005 1520.
YY. Soong and PJ.Barlow , Antioxidant
activity and phenolic content of selected
fruit seeds, Food Chem.(88) 2004 411417.
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) 2000 183-188.
M. Rizzo , D. Ventrice , MA.Varone ,
R. Sidari and A Carini , HPLC
determination of phenolics adsorbed on
yeasts, JPharm Biomed Anal.(42) 2006
46-55.
ICH Harmonized Tripartite Guidelines,
Validation of Analytical Procedures:
Text and Methodology, 1994, Q2R (1).

1. K. Robards , PD Prenzler , G Tucker , P

Swatsitang and W Glover. Phenolic

Volume 2, Issue 1, January February 2012

74 | P a g e
http://www.ijpi.org

INTERNATIONAL JOURNAL OF
PHARMACEUTICAL INNOVATIONS

RESEARCH ARTICLE
ISSN 2249-1031

Table-1 Gradient programming of HPLC System

Time (minutes)

Mobile phase (A)

Mobile phase(B)

95

90

10

12

95

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 (%)

64.1

0.044

65

0.0447

65.6

0.045

Mean

0.0445

RSD

1.14

Table- 3 Recovery study of Gallic acid by HPLC method

Gallic acid
Amount

10

20

30

Amount
found (mg)

10.0045

20.0042

30.0047

Percentage
Recovery

100.04

100.02

100.01

added (mg)

Mean

Volume 2, Issue 1, January February 2012

100.023

75 | P a g e
http://www.ijpi.org

INTERNATIONAL JOURNAL OF
PHARMACEUTICAL INNOVATIONS
Table-4

RESEARCH ARTICLE
ISSN 2249-1031

results of validation parameters of Gallic acid

Parameters
Linearity
Linear equation
Range
Intercept
Correlation coefficient

Results found

Precision(%RSD)
Ruggedness(%RSD)
Robustness
Limit of Detection (LOD)
Limit of Quantification (LOQ)
System Suitability Parameter
Calibration range (mcg/ml)
Theoretical Plate
Tailing Factor

0.33
0.45
Robust
0.22mcg/ml
6.77mcg/ml

Y=19189X+6.20
5mcg/ml to 40mcg/ml
6.20
0.9901

5mcg/ml to 40mcg/ml
4000
1.2

Figure-1 Chromatogram of Ethanolic extract of Gallic acid

Volume 2, Issue 1, January February 2012

76 | P a g e
http://www.ijpi.org