DEVELOPMENT AND VALIDATION OF NOVEL RP-HPLC METHOD FOR THE SIMULTANEOUS ESTIMATION OF ELLAGIC ACID AND QUERCETIN IN AN AYURVEDIC FORMULATION
Objective: To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) method for simultaneous qualitative and quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation and validate as per international conference on harmonization (ICH) guidelines.
Methods: In the present work, good chromatographic separation was achieved isocratically using a shim-pack HPLC C18 column (4.6 x 250 mm, 5Î¼m) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3.5 with orthophosphoric acid) and acetonitrile in the ratio 60:40, at flow rate of 1.2 ml/min and column temperature maintained at 35 Â°C. The effluents obtained were monitored at 255 nm with UV-visible detector.
Results: The retention time of ellagic acid and quercetin were found to be 1.65 min and 2.94 min respectively. Linearity of ellagic acid and quercetin were tested in the range of 6-14 ppm and 3-11 ppm respectively. The correlation coefficient for ellagic acid and quercetin were 0.997 and 0.993 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveals that the method is precise.
Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of ellagic acid and quercetin in an ayurvedic formulation. Hence the developed method can be used for quantitative analysis and quality control of extracts and commercial samples of other plant species and formulation containing these two markers.
2. Shobhen R, Patel NM, Patel PM. A review on modification of analytical techniques in herbal research. Int J Res Ayurveda Pharm 2011;2:1483-5.
3. Nowak R. Determination of ellagic acid in pseudofruits of some species of roses. Acta Pol Pharm 2006;63:289-92.
4. Tiwari P, Patel RK. Quantification of gallic acid and ellagic acid in arjunarishta by validated HPTLC densitometry. Int J Pharm Sci Res 2012;3:2215-23.
5. Walid E, Hassan M, Mona M. HPLC analysis of quercetin and antimicrobial activity of comparative methanol extracts of shinus molle L. Int J Curr Microbiol Appl Sci 2015;4:550-8.
6. Jain V, Saikh S. Simultaneous RP-HPLC analysis of quercetin and kaempferol in different plant parts of Cissus Quadrangularis. Int J Pharm Pharm Sci 2016;8:138-42.
7. Lee FA, Mun FY, Yvonne T, Peh K, Yusrida D. HPLC method for simultaneous quantitative detection of quercetin and curcuminoids in traditional chinese medicines. J Pharmacopuncture 2014;17:36-49.
8. Thakur AK, Jain V, Hingorani L, Laddha KS. Improved high-performance liquid chromatographyâ€“DAD method for the simultaneous analysis of quercetin and kaempferol in the stems of Cissus quadrangularis Linn. Acta Chromatogr 2009;21:95-103.
9. Olena IG. Simultaneous determination of flavonoids and phenolic acids in different parts of prunella vulgaris L. by HPLC-DAD. J Pharmacogn Phytochem 2014;29:1248-54.
10. Vemula VR, Sharma PK. RP-HPLC method development and validation for simultaneous estimation of diclofenac and tolperisone in tablet dosage form. Asian J Pharm Clin Res 2013;6 Suppl 3:186-9.
11. International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human use. Validation of Analytical Procedures: Text and Methodology ICH Q2 (R1); 2005.
12. Sarat M, Murali PK, Rambabu C. Development and validation of RPHPLC method for simultaneous estimation of amlodipine besylate and benazepril Hcl in tablet dosage form. Int J Curr Pharm Res 2012;4:80-4.
13. FDA, Guidance R. Validation of chromatographic methods. center for drug evaluation and research (CDER), Food Drug Administration; 1994. p. 2.
14. FDA, ORA validation and verification guidance for human drug analytical methods. Food and Drug Administration; 2003. p. 1.