Sultana Shaikh, Vandana Jain


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 quercetin, ellagic acid and rutin 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, 5μm) and a mobile phase consisting of 0.02 M potassium dihydrogen orthophosphate buffer (pH adjusted to 3 with orthophosphoric acid) and methanol in the ratio 55:45, at flow rate of 1 ml/min and column temperature maintained at 35 °C. The effluents obtained were monitored at 254 nm with a UV-visible detector.


Results: The retention time of quercetin, ellagic acid and rutin were found to be 7.52 min, 9.10 min and 12.47 min respectively. Linearity of quercetin, ellagic acid and rutin were found in the range of 8-12 ppm, 9-17 ppm and 7-11 ppm respectively. The correlation coefficient for quercetin, ellagic acid and rutin were 0.997, 0.999 and 0.999 respectively. The high recovery values (98 %-102 %) indicate a satisfactory accuracy. The low percent relative standard deviation (% RSD) values in the precision study reveal that the method is precise.


Conclusion: The developed method is novel, simple, precise, rapid, accurate and reproducible for simultaneous quantitative estimation of quercetin, ellagic acid and rutin 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 three markers.



Quercetin, Ellagic acid, Rutin, Ayurvedic formulation, RP-HPLC, Validation, ICH.


Lokhande S, Chougule A, Patil S, Patil V. Need of herbal drug standardization. International Ayurvedic Medical Journal 2015; 03: 874-7.

Shobhen R., Patel NM, Patel PM. A review on modification of analytical techniques in herbal research. Int J Res Ayurveda Pharm 2011; 02:1483-5.

Pawar NP, Salunkhe VR. Development and validation of UV spectrophotometric method for simultaneous estimation of rutin and gallic acid in hydroalcoholic extract of triphala churna. Int J PharmTech Res 2013; 05:724-9.

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 App Sci, 2015; 04(11):550-8.

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(4):36-49.

Nowak R. Determination of ellagic acid in pseudofruits of some species of roses. Acta Pol Pharm, 2006; 63:289-92.

Tiwari P, Patel RK. Quantification of gallic acid and ellagic acid in arjunarishta by validtaed HPTLC densitometry. Int J Pharm Sci Res, 2012; 03:2215-23.

Kuntic V, Pejic N, Ivkovic B, Vujic Z, Ilic K, Micic S, Vukojevic V. Isocratic RP-HPLC method for rutin determination in solid oral dosage forms. J Pharm Biomed Anal 2007; 43:718-21.

Weerasak S, Vorarat S. Simultaneous determination of gallic acid, catechin, rutin, ellagic acid and quercetin in flower extracts of Michelia alba, Caesalpinia pulcherrima and Nelumbo nucifera by HPLC. Thai Pharm Health Sci J 2007; 02:131-7.

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; 06 Suppl 3: 186-9.

Shah R, Shah R. Development and validation of RP-HPLC method for phenytoin sodium and phenobarbitone in bulk and pharmaceutical dosage form. Int J Pharm Pharm Sci 2017; 09:224-9.

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.

Sarat M, Murali PK and 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; 04: 80-4.

Madhukar A, Kannappan N, Kumar CB. Analytical method development and validation for the determination of hydrochlorothiazide, amlodipine besylate and telmisartan hydrochloride in multicomponent tablet dosage form and in biorelevant media (fassif) by RP-HPLC techniques. Int J Pharm Pharm Sci 2015; 07:218-25.

FDA, Guidance R. Validation of chromatographic methods. center for drug evaluation and research (CDER), Food and Drug Administration; 1994. p. 2.

FDA, ORA validation and verification guidance for human drug analytical methods. Food and Drug Administration; 2003. p. 1.

Fatal error: Call to a member function getGalleyLabel() on null in /home/innowar1/public_html/journals/cache/t_compile/%%38^38D^38D7420B%%article.tpl.php on line 189