Keywords

51.1 Introduction

Reverse-phase chromatography or RP-HPLC is a commonly used HPLC technique for the analysis of a wide range of compounds. In this technique, the stationary phase used in nonpolar while the mobile phase is aqueous or moderately polar in nature. Hydrocarbons are used as the stationary phase and water or acetonitrile is used as the mobile phase in this kind of high-performance liquid chromatography (RP-HPLC). Solutes are eluted in decreasing polarity order in RP-HPLC. For RP-HPLC, stationary phases for the silanol group are produced by treating the surface with an organo chloro silane. Non polar hydrocarbon chains in the stationary phase interact with sample molecules to provide a retention mechanism in RP-HPLC.

Gallic acid was determined by Carl Wilhelm Scheele in 1786. It is a naturally occurring low-molecular-weight tri phenolic compound. Its chemical name is 3,4,5-trihydroxy benzoic acid. It is also known as trihydroxy benzoic acid. It exists both independently and as a component of tannins (specifically, gallotannin). Bark, wood, leaves, fruits, roots, and seeds are only some of the plant parts that contain gallic acid or its derivatives. Mechanism of action: Gallic acid has been shown to be selectively hazardous for cancer cells while having no effect on normal cells, inducing the death of cancer cells via mitochondria-mediated mechanisms. Gallic acid has been credited with a number of health benefits, including antioxidant, anti-inflammatory, and anticancer actions. It has a specific beneficial role in the apoptosis of cancer cells and brain health.

A chemical structure of gallic acid.

Molecular structure of gallic acid

51.1.1 Experimental Work

Materials, Reagents, and Chemicals

The pure form of gallic acid, HPLC grade water, acetonitrile, triethyl amine, and formic acid (Rankem), and pipettes, beakers, burettes, and measuring cylinder which are manufactured by Borosil.

Equipment

HPLC (Alliance) was manufactured by Waters e 2695 – Empower software 2.0 versions, UV–vis spectrophotometer by UV-1700, and ultrasonicator (UCA 701) by Unichrome and PH meter by Eutech.

51.1.2 General Preparations

Preparation of Standard Stock Solution

Accurately weigh and transfer 100 mg of gallic acid working standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution). Further pipette 5 mL of the above stock solutions into a 50 ml volumetric flask and dilute up to the mark with diluent (100 ppm of gallic acid).

Preparation of Buffer (0.1% Formic Acid)

1 mL of the formic acid is dissolved in 1 L of HPLC water and filtered through 0.45 μ membrane filter paper.

Preparation of Mobile Phase

Mobile phase was prepared by mixing 0.1% formic acid and ACN taken in the ratio 70:30. It was filtered through a 0.45 μ membrane filter to remove the impurities, which may interfere in the final chromatogram.

Chromatographic Condition

Multiple trails were run to determine the optimal chromatographic settings for this approach use suitable high-performance liquid chromatographic equipped with PDA detector.

Column:

Waters X-Terra RP-18 (150 × 4.6 mm, 3.5 μ)

Movable phase:

Acetonitrile and 0.1% formic acid (30:70)

Wavelength:

275 nm

Flow rate:

1 mL/min

Injection volume:

10 μL

Run time:

5 min

Preparation of Diluent

Mobile phase was used as a diluent.

Preparation of Standard Solution

Accurately weigh and transfer 100 mg of gallic acid working standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution).

Further pipette 5 mL of the above stock solutions into a 50 mL volumetric flask and dilute up to the mark with diluent (100 ppm of gallic acid).

Procedure

Inject 10 μL of the standard, sample into the chromatographic system and measure the areas for gallic acid peak and calculate the %assay by using the formulae.

Determination of Working Wavelength (λmax)

The isosbestic wavelength was utilized to make an estimate of the drug’s potency. At the isosbestic point, the molar absorptivity of all interconvertible compounds is equal to one another. That is why we relied on this wavelength for our precise medication estimations.

The wavelength of maximum absorption of the solution of the drug in a mixture of acetonitrile and 0.1% formic acid (30:70) was scanned using PDA detector within the wavelength region of 200–400 nm against acetonitrile and 0.1% formic acid (30:70) as blank. The absorption curve shows an isosbestic point at 275 nm. Thus 275 nm was selected as the detector wavelength for the HPLC chromatographic method.

A spectrum plots A U versus nanometer. It displays a fluctuating line. The peak values are 233, 246, 275, and 316 nanometers.

PDA – Spectrum of gallic acid

51.1.3 Syrup Formulation

Preparation of Simple Syrup

Weigh 66.35 g of sucrose. Add sucrose in hot purified water under continuous stirring until it dissolved. Kept aside for cooling.

A text reads, as per I P 666.7 g of sucrose in 1000 m l of water.

Preparation of Gallic Acid Syrup

Add gallic acid, preservative (sodium benzoate), diluents, and sweetener (glycerine) in the above simple syrup. Stir the solution for 20 min. After cooling, filter the final syrup through filter paper.

System Suitability

The tailing factor for the peak due to gallic acid in standard solution should not be more than 2.0.

Theoretical plates for the gallic acid peak in standard solution should not be less than 2000.

Formula for Assay

$$ \%\mathrm{Assay}={\frac{AT}{AS}}^{\ast }{\frac{WS}{DS}}^{\ast }{\frac{DT}{WT}}^{\ast }{\frac{\mathrm{Average}\kern0.5em \mathrm{weight}}{\mathrm{Label}\kern0.5em \mathrm{claim}}}^{\ast }{\frac{P}{100}}^{\ast }100 $$

where:

  • AT = average area counts of test (sample) preparation

  • AS = average area counts of standard preparation

  • WS = weight of working standard taken in mg

  • DS = dilution of working standard in mL

  • DT = dilution of test (sample) in mL

  • WT = weight of test (sample) taken in mg

  • P = percentage purity of working standard

  • LC = Label claim mg/mL

Procedure

Inject each level into the chromatographic system and measure the peak area.

Plot a graph of peak area versus concentration (on the X-axis concentration and on the Y-axis peak area) and calculate the correlation coefficient.

Range

The range of an analytical technique is the concentration range across which its precision, accuracy, and linearity have been shown.

Inclusion Criteria

Correlation coefficient should be not less than 0.999.

51.1.4 Preparation Accuracy Sample Solutions

For the Preparation of 50% Solution (with Respect to Target Assay Concentration)

Accurately weigh and transfer 50 mg of gallic acid standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution).

Further pipette 5 mL of the above stock solutions into a 50 mL volumetric flask and dilute up to the mark with diluent (50 ppm of gallic acid).

For the Preparation of 100% Solution (with Respect to Target Assay Concentration)

Accurately weigh and transfer 100 mg of gallic acid standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution).

Further pipette 5 mL of the above stock solutions into a 50 mL volumetric flask and dilute up to the mark with diluent.(100 ppm of gallic acid).

For the Preparation of 150% Solution (with Respect to Target Assay Concentration)

Accurately weigh and transfer 150 mg of gallic acid standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution).

Further pipette 5 mL of the above stock solutions into a 50 mL volumetric flask and dilute up to the mark with diluent (150 ppm of gallic acid).

Procedure

Inject the standard solution, accuracy – 50%, accuracy – 100%, and accuracy – 150% solutions.

Inclusion Criteria

The % recovery for each level should be between 98.0% and 102.0%.

Precision

Precision is the degree of repeatability of an analytical method under normal operating conditions. Precision is of three types:

  1. 1.

    System precision

  2. 2.

    Method precision

  3. 3.

    Intermediate precision (a. Intraday precision, b. Interday precision)

System precision is checked by using standard chemical substances to ensure that the analytical system is working properly.

In this peak area, % of drug of six determinations is measured and % RSD should be calculated.

In method precision, a homogenous sample of a single batch should be analyzed six times. This indicates whether a method is giving constant results for a single batch. In this, analyze the sample six times and calculate the % RSD.

The precision of the instrument was checked by repeatedly injecting (n = 6) solutions of 100 ppm of gallic acid.

Acceptance Criteria

The % RSD for the absorbance of six replicate injection results should not be more than 2%.

Robustness

As part of the robustness, deliberate change in the flow rate, mobile phase composition, temperature variation was made to evaluate the impact on the method.

  1. A.

    The flow rate was varied from 0.9 mL/min to 1.1 m.

    • A standard solution of 100 ppm of gallic acid was prepared and analyzed using the varied flow rates along with the method flow rate.

    • On the evaluation of the above results, it can be concluded that the variation in flow rate affected the method significantly. Hence, it indicates that the method is robust even with a change in the flow rate ± 10%.

  2. B.

    The variation of the Organic Phase ratio.

    • A standard solution of 100 ppm of gallic acid was prepared and analyzed using the varied in mobile phase ratio.

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

The limit of detection (LOD) and limit of quantification (LOQ) of the drug carry were calculated using the following equation as per international conference harmonization (ICH) guidelines.

$$ \mathrm{LOD}=3.3 X\sigma /S $$
$$ \mathrm{LOQ}=10 X\sigma /S $$

LOD for gallic acid was found to be 0.3 μg/mL and LOQ for gallic acid was found to be 1 μg/mL.

51.1.5 Degradation Studies

Preparation of Stock

Accurately weigh and transfer 100 mg of Gallic acid working standard into a 100 mL clean dry volumetric flask, add diluent and sonicate to dissolve it completely, and make volume up to the mark with the same solvent (stock solution).

Acid Degradation

Pipette 5 mL of the aforementioned solution was added to a 50 mL vacuum flask, followed by 3 mL of 1 N HCl. The vacuum flask was then maintained at 60 °C for 6 h before being neutralized with 1 N NaOH and diluted to 50 mL with diluent. Filter the solution using 0.22-micron syringe filters and transfer it to bottles.

Alkali Degradation

Pipette 5 mL of the above solution into a 50 mL volumetric flask and add 3 mL of 1 N NaOH was added. Then, the volumetric flask was kept at 60 °C for 6 h and then neutralized with 1 N HCl and make up to 50 mL with diluent. Filter the solution with 0.22 microns syringe filters and place it in vials.

Thermal-Induced Degradation

Gallic acid sample was taken in Petri dish and kept in a hot air oven at 105 °C for 24 h. Then the sample was taken and diluted with diluents and injected into HPLC and analyzed.

Peroxide Degradation

Pipette 5 mL above stock solution was added to a 50 mL vacuum flask, 1 mL of 3% w/v hydrogen peroxide was added to the flask and the volume was built up to the mark using diluent. The vacuum flask was then maintained at 60 °C for 6 h. After that, the vacuum flask was left at room temperature for 15 min. Filter the solution using 0.45-micron syringe filters and transfer it to bottles.

Reduction Degradation

Pipette 5 mL of above-stock solution was added to a 50 mL vacuum flask, 1 mL of 10% sodium bisulfate was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60 °C for 6 h. After that, the vacuum flask was left at room temperature for 15 min. Filter the solution using 0.45-micron syringe filters and transfer it to bottles.

Photolytic Degradation

Gallic acid sample was placed in sunlight for 24 h. Then the sample was taken and diluted with diluents and injected into HPLC and analyzed.

Hydrolysis Degradation

Pipette 5 mL of above-stock solution was added to a 50 mL vacuum flask, 1 mL of HPLC grade water was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60 °C for 6 h. After that, the vacuum flask was left at room temperature for 15 min. Filter the solution using 0.45-micron syringe filters and transfer it to bottles.

51.2 Results and Discussion

Optimization of Chromatographic Conditions (Fig. 51.1 and Table 51.1)

Fig. 51.1
A chromatogram of trial 6 plots A U versus minutes. It illustrates a constant line with a peak at 3.580 minutes. The peak is labeled gallic acid.

Chromatogram of Trial-6

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Table 51.1 Optimized chromatographic conditions
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Specificity (Figs. 51.2, 51.3 and 51.4)

Fig. 51.2
A chromatogram of blank plots A U versus minutes. It displays a constant line from (0, 0) to (5, 0). Values are approximated.

Chromatogram of blank

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Fig. 51.3
A chromatogram of placebo plots A U versus minutes. It exhibits a constant line from (0, 0) to (5, 0). Values are approximated.

Chromatogram of placebo

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Fig. 51.4
A chromatogram of standard plots A U versus minutes. It illustrates a constant line with a peak at 3.584 minutes. The peak is labeled gallic acid.

Chromatogram of standard

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51.3 Analytical Method Validation (HPLC)

The method’s linearity, accuracy, precision, and specificity were all confirmed to be satisfactory. The method was validated in accordance with ICH standards.

Linearity

A chromatogram of linearity plots A U versus minutes. It exhibits a stable line with a peak at 3.584 minutes. The peak is labeled gallic acid.

Chromatogram of linearity

Accuracy (Table 51.2, Figs. 51.6, 51.7 and 51.8)

Table 51.2 Accuracy results of gallic acid by RP-HPLC method
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Fig. 51.5
A line graph plots area counts versus concentration in p p m. It exhibits a linearly increasing line from (0, 0) to (160, 3500000). Values are estimated. The value of y equals 20135.78 x + 3105.61 and R square equals 0.9998.

Calibration curve for gallic acid at 275 nm

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Fig. 51.6
A chromatogram for accuracy 50% plots A U versus minutes. It illustrates a constant line with a peak at 3.589 minutes. The peak is labeled gallic acid.

Chromatogram for accuracy 50%

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Fig. 51.7
A chromatogram for accuracy 100% plots A U versus minutes. It displays a constant line with a peak at 3.559 minutes. The peak is labeled gallic acid.

Chromatogram for accuracy 100%

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Fig. 51.8
A chromatogram for accuracy 150% plots A U versus minutes. It exhibits a constant line with a peak at 3.575 minutes. The peak is labeled gallic acid.

Chromatogram for accuracy 150%

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Precision (Tables 51.3, 51.4 and Fig. 51.9)

Table 51.3 Standard results for gallic acid by RP-HPLC method
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Table 51.4 Method precision for gallic acid by RP-HPLC method
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Fig. 51.9
A chromatogram of method precision plots A U versus minutes. It illustrates a constant line with a peak at 3.577 minutes. The peak is labeled gallic acid.

Chromatogram of method precision

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Acceptance Criteria

The % RSD for the area of six standard injections results should not be more than 2% (Table 51.5 and Fig. 51.10).

Table 51.5 Intermediate precision for gallic acid by RP-HPLC method
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Fig. 51.10
A chromatogram of intermediate precision plots A U versus minutes. It displays a constant line with a peak at 3.581 minutes. The peak is labeled gallic acid.

Chromatogram of intermediate precision

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Acceptance Criteria

The % RSD for the area of six standard injection results should not be more than 2%.

Robustness (Table 51.6, Figs. 51.11, 51.12, 51.13 and 51.14)

Fig. 51.11
A chromatogram for less flow rate plots A U versus minutes. It exhibits a constant line with a peak at 3.955 minutes. The peak is labeled gallic acid.

Chromatogram for less flow rate (0.9 ml)

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Fig. 51.12
A chromatogram for more flow rate plots A U versus minutes. It illustrates a constant line with a peak at 3.277 minutes. The peak is labeled gallic acid.

Chromatogram for more flow rate (1.1 mL)

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Fig. 51.13
A chromatogram for less organic phase plots A U versus minutes. It displays a constant line with a peak at 4.814 minutes. The peak is labeled gallic acid.

Chromatogram for less organic phase (27:73)

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Fig. 51.14
A chromatogram for more organic phase plots A U versus minutes. It exhibits a constant line with a peak at 2.847 minutes. The peak is labeled gallic acid.

Chromatogram for more organic phase (33:67)

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Results of Linearity for Gallic Acid (Fig. 51.5)

Table 51.6 Robustness results of gallic acid by RP-HPLC
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S. no

Gallic acid

Conc. (μg/mL)

Peak area

1

25.00

512,492

2

50.00

1,033,316

3

75.00

1,474,077

4

100.00

2,016,525

5

125.00

2,533,019

6

150.00

3,023,594

Regression equation

y = 20135.78x + 3105.61

Slope

20135.78

Intercept

3105.61

R 2

0.9998

LOD and LOQ (Table 51.7, Figs. 51.15 and 51.16)

Fig. 51.15
A chromatogram of L O D plots A U versus minutes. It displays a constant line with a peak at 3.555 minutes. The peak is labeled gallic acid.

Chromatogram of LOD

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Fig. 51.16
A chromatogram of L O Q plots A U versus minutes. It illustrates a constant line with a peak at 3.541 minutes. The peak is labeled gallic acid.

Chromatogram of LOQ

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Degradation Studies (Figs. 51.17, 51.18, 51.19, 51.20, 51.21, 51.22, 51.23, 51.24 and Table 51.8)

Fig. 51.17
A chromatogram of control degradation plots A U versus minutes. It exhibits a constant line with a peak at 3.572 minutes. The peak is labeled gallic acid.

Chromatogram of control degradation

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Fig. 51.18
A chromatogram of acid degradation plots A U versus minutes. It illustrates a constant line with 2 peaks. The shorter peak is at 0.864 minutes, and the higher peak at 3.580 minutes is labeled gallic acid.

Chromatogram of acid degradation

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Fig. 51.19
A chromatogram of alkali degradation plots A U versus minutes. It displays a constant line with 2 peaks. The shorter peak is at 1.219 minutes, and the higher peak at 3.581 minutes is labeled gallic acid.

Chromatogram of alkali degradation

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Fig. 51.20
A chromatogram of peroxide degradation plots A U versus minutes. It illustrates a constant line with 3 peaks. The shorter peak is at 1.911 minutes, a higher peak is at approximately 0.50 minutes, and the peak at 3.580 minutes is labeled gallic acid.

Chromatogram of peroxide degradation

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Fig. 51.21
A chromatogram of reduction degradation plots A U versus minutes. It exhibits a constant line with 2 peaks. The shorter peak is at 4.087 minutes, and the higher peak at 3.587 minutes is labeled gallic acid.

Chromatogram of reduction degradation

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Fig. 51.22
A chromatogram of thermal degradation plots A U versus minutes. It displays a constant line with 2 peaks. The shorter peak is at 2.819 minutes, and the higher peak at 3.581 minutes is labeled gallic acid.

Chromatogram of thermal degradation

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Fig. 51.23
A chromatogram of hydrolysis degradation plots A U versus minutes. It displays a constant line with a peak at 3.579 minutes. The peak is labeled gallic acid.

Chromatogram of hydrolysis degradation

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Fig. 51.24
A chromatogram of photolytic degradation plots A U versus minutes. It displays a constant line with a peak at 3.570 minutes. The peak is labeled gallic acid.

Chromatogram of photolytic degradation

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51.4 Conclusion

Table 51.7 Sensitivity parameters (LOD and LOQ) by RP-HPLC
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The devised HPLC technique for estimating the target drug is easy to use, quick to implement, highly reliable, and cheap. Both the mobile phase and the solvents are easily accessible, inexpensive, dependable, sensitive, and quick to prepare. The sample recoveries revealed noninterference of formulation excipients in the estimate, and they may be utilized in labs for the regular analysis of chosen medications, all of which were in excellent agreement with their individual label claims. Since the HPLC method’s system validation parameters have demonstrated good, accurate, and repeatable findings (without any interference of excipients), it can be inferred that the quick and easy procedures presented will be most beneficial for analysis. This study found that the stability indicating test technique by RP-HPLC was straightforward, reproducible, sensitive, and specific, with no cross-contamination from placebo or degradation products. So, they are suitable for regular gallic acid testing.

Table 51.8 Forced degradation results for gallic acid
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