New RP-HPLC Method Development and Validation for Simultaneous Estimation and Forced Degradation Studies of Gallicacid and Curcumin in Solid Dosage Form

S.K. Godasu^1* and S.A. Sreenivas^2
*Correspondence: S.K. Godasu, Department of Pharmaceutics, Mewar University, Chittorgarh, Rajasthan, India, Email:

Keywords

Gallic acid and curcumin • RP‐HPLC • Simultaneous estimation

INTRODUCTION

Gallic acid: is a phenolic acid, also known as 3,4,5- trihydroxybenzoic acid, obtained from fruit of Embelicaofficinalis belonging to family Euphorbiaceae. Gallic acid is having different pharmacological activities like antiinflammatory, antimicrobial, antifungal, antidiabetic, anticancer, antioxidant, antiviral etc (Figure 1).

Curcumin: is a natural flavonoid obtained from the rhizome of Curcuma longa belonging to family Zingiberaceae. Curcumin is chemically 1,7-bis (4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5- dione) having various activities like antibacterial, antiprotozoal, antiviral, hypoglycemic, anticoagulant, antioxidant, antitumor, anticarcinogenic, coloring agent, flavoring agent [1].

Many methods have been described in the literature for the determination of Curcumin and Gallic acid individually andin combination with other drugs. However, there is no HPLC method reported for the simultaneous estimation of these drugs in combined dosage forms. There are different dosage forms like tablet, capsule, syrup and granules having these both drugs. The aim of this work was to develop an RP-HPLC method with ultraviolet detection for the simultaneous determination of Curcumin and Gallic acid in pharmaceutical dosage forms. The present RP-HPLC method was validated as per ICH guidelines [2].

Materials and Methods

Chemicals and reagents: Curcumin and Gallic acid were obtained as a gift sample from Pharma train lab, Hyderabad. KH₂PO₄ was analytical grade supplied by finer chemical LTD, Mumbai, Orthophosphoric acid (MERCK), Acetonitrile (Molychem, HPLC grade) and Water for HPLC (LICHROSOLV) (MERCK), Methanol for HPLC (LICHROSOLV (MERCK).

Equipment and chromatographic conditions: The chromatography was performed on a Waters 2695 HPLC system, equipped with an auto sampler, PDA detector and Empower 2 software. Analysis was carried out at 260 nm with column Inspire (4.6 × 150 mm, 5 m), dimensions at ambient temperature. The optimized mobile phase consists of 30% buffer 70% Acetonitrile. Buffer is 1 ml of orthophosphoric acid in 1000 ml water Flow rate was maintained at 1 ml/min and run time for 5 min (Figure 2).

Preparation of solutions: Pipette out 1 ml of Ortho Phosphoric Acid was taken in a 1000 ml volumetric flask, dissolved and diluted to 1000 ml with HPLC water and the volume was adjusted to pH 3.0 with NaOH.

Preparation of mobile phase: 300 ml (30%) of accurately measured buffer solution was mixed with 700 ml of Acetonitrile (70%) and degasified by sonicator. Finally, the solution was filtered through 0.45 μ filter.

The diluents: The Mobile phase was used as the diluent.

Preparation of standard stock solution: Accurately weigh 1000 mg of Gallic acid and 10 mg of Curcumin working standards and transfer into a 100 ml volumetric flask containing 60 mL of Diluent and sonicate to dissolve it completely. Make up the final volume to 100 mL with diluent (Stock solution). Pipette out 1.5 ml of the stock solution into a 10 ml volumetric flask and make up to the mark with diluent [3].

Preparation of Sample stock solution: Accurately weigh 1000 mg of Gallic acid and 10 mg Curcumin equivalent tablet powder and transfer into a 100 mL volumetric flask containing 60 mL of diluent and sonicate it for 30 mins to dissolve the drugs completely. Make up the final volume to 100 mL with diluent (Stock solution). After filtering the solution pipette out 1.5 ml of the stock solution into a 10 ml volumetric flask and make up to the mark with diluent [4].

Results and Discussion

Procedure: 10 L of standard and sample solutions were injected into the LC-system and measure the peak areas for Gallic acid and Curcumin.

The developed chromatographic method was validated for system suitability, linearity accuracy, precision, ruggedness and robustness as per ICH guidelines.

System suitability parameters: To evaluate system suitability parameters such as retention time, tailing factor and USP theoretical plate count, the mobile phase was allowed to flow through the column at a flow rate of 1.0 ml/min for 5 minutes to equilibrate the column at ambient temperature. Chromatographic separation was achieved by injecting a volume of 10 μL of standard into Inspire (4.6 × 150 mm, 5 m), the mobile phase of composition 0.1% OPA buffer and acetonitrile in the (30:70) was allowed to flow through the column at a flow rate of 1.0 ml per minute. Retention time, tailing factor and USP theoretical plate count of the developed method are shown in (Table 1).

Assay of pharmaceutical formulation: The proposed validated method was successfully applied to determine Gallic acid and Curcumin in their tablet dosage form. The result obtained for Gallic acid and Curcumin was comparable with the corresponding labeled amounts and they were shown in Table 2.

Validation of Analytical method: Linearity and Range: Stock solution was prepared by dissolving the appropriate amount of Gallic acid and Curcumin in 10 ml of diluent and further diluted to the required concentrations with diluent. The solution was prepared at five concentration levels ranging from 500 μg/ml to 2500 μg/ml of Gallic acid and 5 μg/ml to 25 μg/ml of Curcumin. Inject each level into the chromatographic system and measure the peak area. Plot a graph of peak area versus concentration (on X-axis concentration and on Y-axis Peak area) and calculate the correlation coefficient [5]. The results are shown in (Table 3).

Accuracy studies: The accuracy was determined by help of recovery study. The recovery method carried out at three level 50%, 100%, 150%. Inject the standard solutions into chromatographic system. Calculate the Amount found and Amount added for Gallic acid and Curcumin and calculate the individual recovery and mean recovery values. The results are shown in (Table 4).

Precision Studies: precision was calculated from Coefficient of variance for six replicate injections of the standard. The standard solution was injected for six times and measured the area for all six Injections in HPLC. The %RSD for the area of six replicate injections was found. The results are shown in Table 5.

Ruggedness: To evaluate the intermediate precision of the method, Precision was performed on different day. The standard solution was injected for six times and measured the area for all six injections in HPLC. The %RSD for the area of six replicate injections was found. The results are shown in (Table 5-10).

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. The flow rate was varied at 0.8 ml/min to 1.2 ml/min. The Organic composition in the Mobile phase was varied from 10% to 10%. LOD and LOQ: The sensitivity of RP-HPLC was determined from LOD and LOQ. Which were calculated from the calibration curve using the following equations as per ICH guidelines [6].?

• LOD=3.3 σ/S and

• LOQ=10 σ/S, where

• σ= Standard deviation of y intercept of regression line,

• S= Slope of the calibration curve

Forced degradation studies and Acid degradation condition:

Accurately 3.0 ml of stock solution into a 1 0 ml volumetric flask and 3 ml of 0.1 N HCl was added. Then, the volumetric flask was kept at 60°C for 6 h and then neutralized with 0.1 N NaOH and makeup to 10 ml with diluent. The solution was filtered through 0.45 μ filter, and then filtrate was injected into system and percentage of degradation was calculated [7].

Alkali degradation condition: Accurately 3.0 ml of stock sample into a 10 ml volumetric flask and add 3 ml of 0.1 N NaOH was added. Then, the volumetric flask was kept at 60°C for 6 h and then neutr alized with 0.1 N HCl and makeup to 10 ml with diluent. The solution was filtered through 0.45 filters and then the filtrate was injected into the system and percentage of degradation was calculated (Figure 3-6).

Thermal-induced degradation condition: 3 ml of stock sample was taken in petri dish and kept in hot air oven at 110°C for 24 h. The samples were then placed in a desiccator till reaching the room temperature. The content in the flasks was dissolved using methanol and diluted up to the mark. Then the sample was taken and diluted with diluents and injected and percentage of degradation was calculated [8].

Photolytic degradation condition: Accurately 3.0 ml of stock sample was exposed to sunlight for about 6 h and then the sample diluted with 5 ml of mobile phase and percentage of degradation [9,10].

Oxidative degradation condition: Accurately 3.0 ml of stock sample into a 10 ml volumetric flask, 1 ml of 3.0 ml of 3% H2O2 was added and the volume was made up to the mark with diluents. The volumetric flask was then kept at room temperature for 15 min. The solution was filtered through 0.45 μ filter and then filtrate was injected into the chromatography system and the percentage of degradation was calculated. Force degradation results are shown in (Tables 11-13).

Conclusion

The proposed HPLC method was found to be simple, precise, accurate and sensitive for the simultaneous estimation of Gallic acid and curcumin in pharmaceutical dosage forms. Hence, this method can easily and conveniently adopt for routine quality control analysis of Gallic acid and curcumin in pure and its pharmaceutical dosage forms.

References

1. Sobhanardakani, S, A Farmany, and S Abbasi. "A new modified multiwalled carbon nanotube paste electrode for quantification of tin in fruit juice and bottled water samples." J Ind Eng Chem 20 (2014): 3214-3216.

Google Scholar, Crossref, Indexed at 

2. Lutomski, J, Kedzia B, and Debska W. "Effect of an alcohol extract and of active ingredients from Curcuma longa on bacteria and fungi (author's transl)." Planta Med 26 (1974): 9-19.

Google Scholar, Crossref, Indexed at 

3. Bhavanishankar, T N, and V Sreenivasamurthy. "Effect of turmeric (Curcuma longa) fractions on the growth of some intestinal and pathogenic bacteria in vitro." Indian J Exp Biol 17 (1979): 1363-1366.

Google Scholar, Crossref, Indexed at 

4. Son wane, S D, S A Nirmal, A N Patil, and S R Pattan et al. "Development and validation of HPTLC method to detect curcumin and gallic acid in polyherbal formulation." J Liq Chromatogr Relat Technol 34 (2011): 2664-2673.

Google Scholar, Crossref, Indexed at 

5. Pundarikakshudu, Kilambi, and N Dave Hiral. "Simultaneous determination of curcumin and berberine in their pure form and from the combined extracts of Curcuma longa and Berberis aristata." Int J Appl Sci Eng 8 (2010): 19-26.

Google Scholar, Crossref, Indexed at 

6. Himesh, Soni, Sita Sharan Patel, Mishra K, and Govind Nayak, et al. "Qualitative and quantitative profile of curcumin from ethanolic extract of Curcuma longa." Int Res J Pharm 2 (2011): 180-184.

Google Scholar, Indexed at 

7. Jasim, Fadhil, Ali Fatima. "A novel method for the spectrophotometric determination of curcumin and its application to curcumin spices." J Microchem 38 (1988): 106-110.

Google Scholar, Crossref, Indexed at

8. Ashraf, Kamran, Mujeeb Mohd, Ahmad Altaf, and Amir Mohd, et al. "Validated HPTLC analysis method for quantification of variability in content of curcumin in Curcuma longa L (turmeric) collected from different geographical region of India." Asian Pac J Trop Biomed 2 (2012): 584-588.

Google Scholar, Crossref, Indexed at

9. Kim, Dong Wuk, Mehmood Yousaf Abid, Xun Li Dong, and Jong Oh Kim, et al. "Development of RP-HPLC method for simultaneous determination of docetaxel and curcumin in rat plasma: Validation and stability." Asian J Pharma Sci 12 (2017): 105-113.

Google Scholar, Crossref, Indexed at

10. Tejaswi JK, and Rajan RG. RP-HPLC method development and validation for simultaneous estimation and forced degradation studies of lamivudine and raltegravir in solid dosage form. Int J App Pharm 10 (2018): 242-248.

Google Scholar, Crossref, Indexed at