Development and Validation of a UV Spectrophotometric Method for Simultaneous Estimation of Gallic Acid and Tannic Acid in Herbal Extract

5.1.2 Instruments and Apparatus

2.2 METHODS

2.2.1 Procurement and Authentication of Herbal Material

The herbal material selected for the study will be procured from a local herbal market or authenticated supplier. The plant material will be identified and authenticated by a qualified botanist. The collected material will be washed properly to remove dust and foreign particles and then shade dried at room temperature for several days. After complete drying, the plant material will be powdered using a mechanical grinder and stored in airtight containers for further study.

2.2.2 Identification and Characterization of Standard Drugs

A. Organoleptic Evaluation

Gallic acid and tannic acid standards will be evaluated for organoleptic characteristics such as color, appearance, texture, and odor.

B. Melting Point Determination

The melting point of gallic acid and tannic acid will be determined using capillary fusion method. Small quantity of powdered sample will be filled in a capillary tube sealed at one end. The capillary tube will be placed in melting point apparatus. Temperature at which sample starts melting and completely melts will be recorded. Obtained melting point will be compared with standard reported values.

2.2.3 Solubility Analysis

Solubility study of gallic acid and tannic acid will be performed in various solvents such as distilled water, methanol, ethanol, and phosphate buffer.

Procedure:

Excess amount of drug will be added into 5 mL solvent. The mixtures will be shaken continuously for 24 hours at room temperature. Solutions will be visually observed for solubility behavior. Solubility will be classified as soluble, slightly soluble, or insoluble. The solvent showing good solubility and minimum spectral interference will be selected for analysis.

2.2.4 Selection of Solvent System

Different solvent systems will be evaluated based on:

• • • • Drug solubility
      • Stability of solution
      • UV transparency
      • Spectral interference
      • Methanol and distilled water mixture will be selected as optimized solvent system due to:
      • Good solubility of both drugs
      • Stable absorbance
      • Clear spectral characteristics in UV region

2.2.5 Preparation of Standard Stock Solutions

A. Preparation of Gallic Acid Stock Solution

Accurately weighed 10 mg of gallic acid will be transferred into 100 mL volumetric flask. About 50 mL methanol will be added and sonicated for complete dissolution. Final volume will be adjusted to 100 mL with methanol. Final concentration obtained 100 µg/mL

B. Preparation of Tannic Acid Stock Solution

Accurately weighed 10 mg of tannic acid will be transferred into 100 mL volumetric flask. Drug will be dissolved in methanol with sonication. Volume will be adjusted up to mark with methanol. Final concentration obtained 100 µg/mL

2.2.6 Determination of Absorption Maxima (λmax)

A. Determination of λmax of Gallic Acid

Procedure:

From stock solution, 1 mL will be pipetted into 10 mL volumetric flask. Volume will be adjusted using methanol to obtain 10 µg/mL solution. Solution will be scanned between 200–400 nm against methanol blank using UV spectrophotometer. Wavelength showing maximum absorbance will be recorded as λ-max. Expected λ-max Around 270–280 nm.

B. Determination of λmax of Tannic Acid

Appropriate dilution of stock solution will be prepared. Solution will be scanned between 200–400 nm. Maximum absorbance wavelength will be recorded. Expected λ-max: Around 260–300 nm.

2.2.7 Overlay Spectral Study

The UV spectra of gallic acid and tannic acid will be overlain to identify: Isoabsorptive point Analytical wavelengths Spectral overlap The selected wavelengths will be used for simultaneous equation and absorbance ratio methods.

2.2.8 Method Development

A. Simultaneous Equation Method

The simultaneous equation method will be developed using absorbance values measured at two selected wavelengths.

The concentration of gallic acid and tannic acid will be calculated using simultaneous equations:

Cx = 𝐀𝟐𝐚𝐲𝟏−𝐀𝟏𝐚𝐲𝟐

              𝐚𝐱𝟐𝐚𝐲𝟏−𝐚𝐱𝟏𝐚𝐲𝟐

Cy = 𝐀𝟏𝐚𝐱𝟐−𝐀𝟐𝐚𝐱𝟏

              𝐚𝐱𝟐𝐚𝐲𝟏−𝐚𝐱𝟏𝐚𝐲𝟐

Where, Cx = Concentration of SX

Cy =Concentration of FP

A1 = Absorbance of mixture at 216.5 nm

A2 =Absorbance of mixture at 236 nm

ax1 & ax2 Absorptivity of SX at 216.5 nm and 236 nm

ay1 & ay2 = Absorptivity of FP at 216.5nm and 236 nm

The absorbance of mixed standards will be measured and concentration will be calculated using the above equations.

0. 1. 1. Preparation of Calibration Curve
1. Calibration Curve for Gallic Acid

Aliquots of stock solution equivalent to 2–12 µg/mL will be prepared. Absorbance will be measured at selected wavelength. Calibration graph will be plotted between concentration and absorbance. Beer-Lambert’s law will be obeyed within selected concentration range.

2. Calibration Curve for Tannic Acid

Different concentrations ranging from 2–12 µg/mL will be prepared. Absorbance will be recorded. Calibration graph will be constructed. Regression equation and correlation coefficient (R²) will be calculated.

0. 1. 2. Preparation of Herbal Extract
1. Drying and Powdering

The collected plant material will be shade dried and pulverized into coarse powder.

2. Extraction Procedure

Soxhlet Extraction Method About 50 g powdered drug will be packed in Soxhlet apparatus. Methanol will be used as extraction solvent. Extraction will be carried out for 6–8 hours. Extract will be filtered and concentrated using water bath. Concentrated extract will be dried and stored in desiccator.

1. 1. 3. Preparation of Sample Solution

Accurately weighed quantity of herbal extract equivalent to required concentration will be dissolved in methanol. Solution will be sonicated for complete dissolution. Filtered through Whatman filter paper. Appropriate dilution will be prepared for UV analysis.

1. 1. 4. Application of Developed Method to Herbal Extract

The developed UV spectrophotometric method will be applied for simultaneous estimation of gallic acid and tannic acid present in herbal extract. The absorbance of sample solution will be measured at selected wavelengths and concentrations will be calculated using developed equations.

1. 1. 5. Validation of Developed Method (ICH Q2(R1))

The developed analytical method will be validated according to ICH guidelines.

1. Linearity

Linearity will be evaluated by analyzing different concentrations of gallic acid and tannic acid.

2. Accuracy

Accuracy will be determined using recovery study by standard addition method.

Recovery studies will be performed at: 80%, 100%, 120% Percentage recovery will be calculated using equation:

3. Precision
   1. Intraday Precision Analysis will be performed three times within same day.
   2. Interday Precision Analysis will be repeated on three different days.
4. Specificity

Specificity will be evaluated by observing interference from: Solvent, Excipients, Herbal matrix.

The developed method should selectively estimate both compounds without interference.

5. Limit of Detection (LOD)

LOD indicates minimum detectable concentration.

6. Limit of Quantification (LOQ)

LOQ indicates minimum quantifiable concentration.

7. Robustness

Robustness will be evaluated by introducing small deliberate variations.

3. RESULTS AND DISCUSIONS

3.1 Identification of Standard Drugs

Table Organoleptic Properties of Gallic Acid and Tannic Acid

Figure Structure of Gallic Acid

Figure Structure of Tannic Acid

The observed organoleptic properties of gallic acid and tannic acid were found to comply with reported literature values. The appearance, colour and nature of both standards confirmed their identity and suitability for analytical studies. No visible impurities were observed, indicating acceptable purity of the reference standards used for method development.

3.2 Melting Point Determination

Table Melting Point of Standard Drugs Gallic acid and tannic acid

The melting point values obtained were in close agreement with reported standards. This confirms the purity and authenticity of both marker compounds. The results suggest that the standards were suitable for further analytical investigations.

1. 1. Solubility Studies

Table Solubility Profile

Methanol exhibited excellent solubilizing capacity for both compounds and produced clear solutions without precipitation. Therefore methanol was selected as the analytical solvent for further studies.

1. 2. Determination of λmax

Figure UV Spectrum of Gallic Acid

Gallic acid exhibited a prominent absorption peak at 272 nm due to electronic transitions associated with its aromatic phenolic structure. The wavelength was selected because it provided maximum sensitivity and reproducibility.

Figure UV Spectrum of Tannic Acid

Tannic acid exhibited maximum absorbance at 276 nm. The broad absorption peak is attributed to multiple phenolic hydroxyl groups present in the structure.

3.5 Overlay Spectral Study

Figure Overlay Spectrum of Gallic Acid and Tannic Acid

result

• λ max of Gallic Acid = 272 nm
• λ max of Tannic Acid = 276 nm
• Isoabsorptive Point = 274 nm

The overlay spectrum revealed sufficient spectral overlap between gallic acid and tannic acid. However, significant differences in absorptivity were observed at selected wavelengths, enabling successful application of the simultaneous equation method.

1. 6. Calibration Curve
      1. Gallic Acid

Figure calibration Curve of Gallic Acid

Regression Equation: Y=0.0603X+0.001

R² = 0.9992

The calibration curve demonstrated excellent linearity within the concentration range of 2–12 µg/ml. The high correlation coefficient confirms adherence to Beer-Lambert's law.

3.6.2 Tannic Acid

Figure 7.7 Calibration Curve of Tannic Acid

Regression Equation: 𝑌 = 0.0565𝑋 + 0.002

R² = 0.9988

The calibration plot showed excellent linearity and reproducibility. The high R² value indicates reliability of the developed method.

1. 7. Linearity

The linearity of the developed UV spectrophotometric method was evaluated by analyzing standard solutions of Gallic Acid and Tannic Acid in the concentration range of 2–12 µg/mL. The absorbance values obtained at the selected analytical wavelengths showed a direct proportional relationship with concentration for both analytes. The calibration curves exhibited excellent linearity with correlation coefficient (R²) values of 0.9992 for Gallic Acid and 0.9988 for Tannic Acid.

Table Linearity Data

Precision

Table Intraday Precision

Gallic Acid

Tannic Acid

Table Intraday Precision

Gallic Acid

Tannic Acid

Figure Precision Graph Data

3.8 Precision

The precision of the developed UV spectrophotometric method was evaluated through intraday and interday studies at three concentration levels (4, 8, and 12 µg/mL) for both Gallic Acid and Tannic Acid. The intraday %RSD values ranged from 0.53–0.73% for Gallic Acid and 0.55–0.69% for Tannic Acid, while the interday %RSD values ranged from 0.65–0.87% and 0.70–0.88%, respectively. All %RSD values were well below the acceptable limit of 2.0% recommended by ICH Q2(R2) guidelines, indicating excellent repeatability and intermediate precision. These findings demonstrate that the developed method is precise, reproducible, and reliable for the routine quantitative estimation of Gallic Acid and Tannic Acid in herbal extracts.

3.9 Specificity

Specificity of the developed UV spectrophotometric method was evaluated by comparing the spectra of blank solvent, standard solutions of Gallic Acid and Tannic Acid, and herbal extract samples. No significant absorbance was observed from the solvent or matrix components at the selected analytical wavelengths. The spectra of Gallic Acid and Tannic Acid were well resolved and free from interference.

Figure 7.10: Overlay spectra of blank, Gallic Acid standard, Tannic Acid standard, and herbal sample demonstrating absence of interference from solvent and matrix components, confirming specificity of the developed method.

The specificity of the developed UV spectrophotometric method was evaluated using blank solvent, standard solutions, and herbal extract samples in accordance with ICH Q2(R2) guidelines. No interfering absorbance was observed from the solvent or herbal matrix at the selected analytical wavelengths, indicating that the analyte response was free from matrix interference. The overlay spectra showed well-defined absorption peaks for both Gallic Acid and Tannic Acid, confirming their selective identification and quantification. These findings demonstrate that the developed method is highly specific and suitable for the routine simultaneous estimation of Gallic Acid and Tannic Acid in herbal extracts.

3.10 Limit of Detection (LOD) and Limit of Quantification (LOQ)

The sensitivity of the developed UV spectrophotometric method was evaluated by determining the Limit of Detection (LOD) and Limit of Quantification (LOQ) for both Gallic Acid and Tannic Acid. The calculated LOD values were 0.18 µg/mL for Gallic Acid and 0.21 µg/mL for Tannic Acid, whereas the LOQ values were 0.55 µg/mL and 0.64 µg/mL, respectively.

Table LOD and LOQ

Figure Comparison of LOD and LOQ values for Gallic Acid and Tannic Acid showing high sensitivity of the developed UV spectrophotometric method.

The sensitivity of the developed UV spectrophotometric method was assessed by determining the Limit of Detection (LOD) and Limit of Quantification (LOQ) in accordance with ICH Q2(R2) guidelines. The LOD values were 0.18 µg/mL for Gallic Acid and 0.21 µg/mL for Tannic Acid, while the corresponding LOQ values were 0.55 µg/mL and 0.64 µg/mL, respectively. These low values indicate that the method is sufficiently sensitive to detect and accurately quantify both analytes at trace concentration levels. Since the LOD and LOQ values were considerably lower than the lower limit of the calibration range (2 µg/mL), the developed method demonstrates excellent analytical sensitivity and is suitable for the routine quantitative estimation of Gallic Acid and Tannic Acid in herbal extracts.

3.10.1 Robustness

The robustness of the developed UV spectrophotometric method was evaluated by introducing small deliberate variations in analytical conditions such as wavelength (±2 nm), solvent composition, and analysis time. The absorbance values obtained under modified conditions were compared with those obtained under optimized conditions.

Table Robustness Study

Figure Effect of deliberate dvariations in analytical conditions on absorbance values of Gallic Acid and Tannic Acid.

Robustness

The robustness of the developed UV spectrophotometric method was evaluated by introducing small deliberate variations in analytical conditions, including wavelength (±2 nm), solvent composition, and analysis time, as recommended by ICH Q2(R2) guidelines. Under these modified conditions, the absorbance values for Gallic Acid ranged from 0.569 to 0.577, while Tannic Acid ranged from 0.520 to 0.527. The observed variations were minimal, indicating that minor changes in experimental parameters did not significantly affect the analytical performance. These results confirm that the developed method is robust, reliable, and suitable for the routine quantitative estimation of Gallic Acid and Tannic Acid in herbal extracts.

The validated UV spectrophotometric method was successfully applied for simultaneous quantification of Gallic Acid and Tannic Acid in the selected herbal extract. The concentrations of both marker compounds were determined using the corresponding calibration curves and regression equations developed during the linearity study.

Figure Quantitative estimation of Gallic Acid and Tannic Acid in herbal extract using the developed UV spectrophotometric method

Table Quantitative Estimation of Gallic Acid in Herbal Extract

Table  Quantitative Estimation of Tannic Acid in Herbal Extract

Table Quantification of Marker Compounds in Herbal Extract

Figure Quantitative estimation of Gallic Acid and Tannic Acid in herbal extract using the developed UV spectrophotometric method.