INTRODUCTION:

Gallic acid (3, 4, and 5- trihydroxy benzoic acid) is a natural phenolic antioxidant compound widely distributed in herbal plants, fruits and vegetables. Structurally Gallic acid has phenolic groups that serve as a source of readily available hydrogen atoms such that the subsequent radicles produced can be delocalized over the phenolic structure.

The interest in Gallic acid is due to its pharmacological activities such as radical scavenging, Anti mutagenic, Anti carcinogenic and Antioxidant.^1,2

Fig. 1: Structure of Gallic acid

Gallic acid found united by ester linkages to a central glucose residue. Gallic acid is also known as pyrogallol tannins because on dry distillation Gallic acid converted into pyrogallol. Gallic acid is promising phytochemical and active ingredient of most of herbal formulations.^3-4Literature survey revealed that a variety of analytical methods such as TLC, HPLC, UV-visible^5-25 has been developed for their analytical application. The aim of present work is to develop and validate a method for the analysis by UV-visible spectrophotometer which is easily adaptable as the routine in quality testing laboratories. This has enabled us to reduce total time of analysis besides taking care of the error caused due to incomplete extraction and use of internal standard.

MATERIAL AND METHODS:

Reagents and Materials:

Triphala tablet was procured from Unja, Gujarat, as a gift sample, Analytical grade water used and purchased from chemical store of collage. Tablet formulation collected from local market. All the other chemicaland reagents used were of analytical grade.

Method development:

Instrumentation andApparatus:

Spectroscopic analysis was carried out using Double beam UV-visible spectrophotometer (Shimadzu, model 1800) havingtwo matched quartz cells with 1cm light path length matched quartz cells used for analytical purpose. Electronic analytical balance (Acculab ALC- 210.4) Ultra sonicator (EN 30 US, Genentech Fast Clear), all glasses wares were made of Borosil. All instruments and glass wares were calibrated.

Preparation of standard stock solution of Gallic acid:

Accurately weighted quantity of Gallic acid 50mg was transferred into 50mL volumetric flask dissolved and diluted up to the mark with distilled water. This was given a stock solution having strength of 1000μg/mL.

Preparation of working standard Solution of Gallic acid:

The Working standard solution 100μg/mL of Gallic acid was prepared by diluting 5mL of Standard Stock solution with distilled water in 50mL volumetric flask up to the mark.

Selection of wavelength for measurement:

The working solution containing 5µg/mL of Gallic acid was scanned between 200 - 400nm against distilled water. It is evident that Gallic acid show an absorbance at 258nm respectively (The spectra shown in fig: 6.1).

Preparations of Sample Solution:

Weigh accurately 20 tablets were powdered. Weigh 100 mg of tablet powder in a 100mL containing volumetric flask and dilute with distilled water and up to the mark with distil water. The solution was filtered using whatman filter paper no.42 and few drops of filtrate were discarded (1000µg/mL of Gallic acid). Transfer 1mL of this solution into a 10mL volumetric flask and diluted to mark with distilled water (100µg/mL of Gallic acid). From this solution pipette out 1mL of working standard solution of Gallic acid was diluted to 10mL volumetric flask and mark up to with distilled water (10µg/mL of Gallic acid.

Method optimization:

Selection and optimization of solvent:

It is well known that the solvent does exerts a profound influence on the quality and shape of the peak. The choices of solvent for UV method development are: Methanol, Water, 0.1 N NaOH, 0.1N HCl etc. Different of solvents were optimized. Out of which Distilled water satisfied all the condition relative to peak quality and non-interference at the specified wavelength.

Selection of Wavelength:

The working solution containing 5µg/mL of Gallic acid was scanned between 200-400nm against distilled water. It is evident that Gallic acid show an absorbance at 258nm respectively.

Method validation:

Validation of the method was carried out as per the international conference on harmonization (ICH) guidelines Q2(R1)²⁵and according the parameters evaluated was:

Sensitivity:

Sensitivity of the method was determined with respect to limit of detection (LOD) and limit of quantification (LOQ). Series of concentration of drug solutions (5-25µg/mL) were used and analysed to determine LOD and LOQ.

LOD and LOQ were experimentally verified by diluting known concentration of Gallic acid until the average response were approximately 3 or 10 times the standard deviation of the responses for six replicate determinations.

Specificity and selectivity:

Marketed triphala tablet of Gallic acid of concentration of 10µg/mL were prepared in distilled water and 10µg/mL of standard Gallic acid were analysed by the proposed method. The estimated amounts of marketed formulation were compared with that of pure Gallic acid of the same strength.

Linearity and Range:

Linearity is expressed in terms of correlation co-efficient of linear regression analysis. Calibration curve for Gallic acid consisted of different concentrations of standard solution ranging from 5-35µg/mL. The solutions were prepared by pipette out 0.5, 1, 1.5, 2, 2.5mL of working standard solution of Gallic acid 100µg/mL into series of 10mL volumetric flasks and the volume was adjusted to mark with distilled water. The linearity response was determined by analysing 7 independent levels of calibration curve in the range of 5-25µg/mL for Gallic acid was measured at 258nm against distilled water. Plot the calibration curve of absorbance v/s concentration and determine correlation co-efficient and regression line equation for Gallic acid.

Precision:

Repeatability was done by analysing three different concentrations of Gallic acid (10µg/mL, 15µg/mL, 20µg/mL) in distilled water on a single day. Intermediate precision was done by analysing the same three concentrations on three different days.

Accuracy:

Accuracy was determined by calculating recovery of Gallic acid by the standard addition method. Amount of standard solutions (8, 10, 12µg/mL) were added to a prequantified test solution of Gallic acid (10µg/mL). Each solution was measured in triplicate, and the recovery was calculated by measuring absorbance.

Robustness:

Robustness of the proposed method was also determined by changing the λ_maxof the analysis (λ_{max 258 nm}) BY ±2.0 nm. % Mean recovery (± % confidence interval) as well as % relative error was reported.

RESULT AND DISCUSSION:

The proposed method was found to be simple, sensitive, precise, economical and rapid for the routine estimation of Gallic acid in pharmaceutical dosage form.

Analysis of the drug:

Melting point of Gallic acid found to be 177^°C. Drug was freely soluble in methanol, water, and sparingly soluble in 0.1N NAOH and 0.1N HCL and ACN. Spectral scan-λ_maxof Gallic acid was found to be at 258nm.

Analytical method validation parameter:

The method was validated as per ICH guidelines (Q2 (R1)

Specificity:

The presence of excipient in formulation does not interfere with the drug peak. Therefore, the proposed method was found specific and selective for the drug.

Linearity and Range

Linearity test solution of Gallic acid (5-25µg/mL) was prepared from stock solution at five different concentration levels. The calibration curve was constructed by plotting peak areas versus their corresponding concentration. Theslope, Y-intercept, and correlation coefficient of the calibration curve were calculated. The correlation coefficient was found to be 0.995 and the calibration curve of Gallic acid. The validation parameters are shown in the table 1.

Table 1: Result of validation parameters of obtained by the developed method

Validation parameter	Result obtained
λmax	258 nm
Linearity	5 – 25 µg/mL
Regression line equation	Y=0.0405x + 0.007
Slope	0.0405
Intercept	0.007
Correlation coefficient	0.996
LOD (µg/mL)	0.338
LOQ (µg/mL)	1.03

Precision:

Intraday precision refers to the use of analytical procedure within a laboratory over a short period of time using the same operator with the same equipment whereas interday precision involves estimation of variation in analysis when a method is used within a laboratory on different days, by different analysts. Repeatability (interday) was assessed by analysing these three different concentrations (10, 15, 20µg/mL), three times a day for at least three different days (table 2)

The %RSD for intraday precision and interday precision was found to be 0.414- 0.116 and 0.254 - 0.118 respectively.

Table 2: Intra and inter precision studies of developed method

Intraday Precision
Conc. (µg/mL)	Mean absorbance ±SD (n=3)	%RSD
10	0.508 ± 0.003	0.59
15	0.604 ± 0.002	0.41
20	0.857 ± 0.001	0.12
Interday Precision
Conc. (µg/mL)	Mean absorbance ±SD (n=3)	%RSD
10	0.492 ± 0.003	0.65
15	0.789 ± 0.002	0.25
20	0.844 ± 0.001	0.11

Accuracy:

The accuracy of an analytical procedure expresses the closeness of agreement between the value which is accepted either as a conventional true value or an accepted reference value and the value found. Thus, accuracy is a measure of the exactness of the analytical method. Accuracy can also be described as the extent to which test results generated by the method and the true value agree. The true value for accuracy assessment can be obtained in several ways.

The % Recovery for the standard analysis and reference analysis method for all three-concentration level ranged from 99.83 – 100.7% (table 3)

Robustness:

Robustness of the proposed method was also determined by changing the λ_max of the analysis (λ_max258nm) by ±2.0nm. The result of robustness was found that % RSD was less than 2%.

Analysis of Marketed formulation:

Applicability of the proposed method was tested by analysing the commercially available tablet formulation triphala tablet. The results are shown in the table 4.

CONCLUSION:

The analytical Method developed on UV Visible spectrophotometer was simple, reliable, Accurate and reproducible. The method eliminates extraction steps thus reduce analytical time, cost and minimize the extraction errors. Low cast, faster speed, satisfactory precision and good specificity, to assess unequivocally the analyte in the presence of components which may be expected to be present, are the main features of this method. The analytical Method was validated as per ICH Q2 Guidelines and can be successfully used for estimation of Gallic acid in triphala tablet.

ACKNOWLEDGEMENT:

I would like to express my special thanks tostudents who help me for completing my project. I also would also like to extend my gratitude to the principal sir Dr. C.N. Patel for providing me with all the facility that was required.

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Received on 04.07.2023 Modified on 11.11.2023

Asian J. Pharm. Ana. 2024; 14(3):122-126.

DOI: 10.52711/2231-5675.2024.00021

Level	Sample amount of Gallic acid (µg/mL)	Standard added amount of Gallic acid (µg/mL)	Observed concentration (µg/mL)	%Recovery
80%	10	8	8.04	100.5
100%	10	10	10.07	100.7
120%	10	12	11.98	99.83

Formulation	Amount of formulation tested	*Mean Absorbance at 258nm± SD**	% RSD	Amount of Gallic acid found	% Recovery of Gallic acid
Triphala tablet	5 µg/mL	0.152 ± 0.001	0.657%	3.538µg/mL	70.76%