A NOVEL RP-HPLC METHOD FOR SIMULTANEOUS ESTIMATION OF BERBERINE, QUERCETIN, AND PIPERINE IN AN AYURVEDIC FORMULATION
Keywords:Berberine, Quercetin, Piperine, Ayurvedic formulation, Validation, RP-HPLC, ICH
Objective: The objective of this study was to develop and validate a novel, simple, rapid, precise and accurate reversed-phase high performance liquid chromatographic (RP-HPLC) method for simultaneous quantitative estimation of berberine, quercetin, and piperine in Ayurvedic formulation.
Methods: The chromatographic separation was achieved using a stationary phase C18 shim-pack (150 mm x 4.6 mm, 5Âµ) column and mobile phase consisted of acetonitrile: 0.04 M potassium dihydrogen phosphate buffer (pH 3.0 adjusted using orthophosphoric acid) in a ratio of 65:35 v/v, with a flow rate of 1 ml/min and UV detection at 255 nm.
Results: The retention time of berberine, quercetin, and piperine were found to be 2.7, 3.0 and 6.3 min respectively. Linearity for berberine, quercetin, and piperine were found in the range of 12-28 Âµg/ml. All calibration curve showed good linear correlation coefficients (r2Ëƒ 0.999) within the tested ranges. Mean percent recoveries for berberine, quercetin, and piperine were found to be within the acceptance limits (98-120%). The percent relative standard deviation (% RSD) for precision was found to be less than 2% which indicates method is precise.Conclusion: The developed method is novel, simple, precise, accurate and can be used for quantitative analysis and quality control of the raw material as well as other commercial formulations containing these three markers.
Ong ES. Extraction methods and chemical standardization of botanicals and herbal preparations. J Chromatogr B: Anal Technol Biomed Life Sci 2004;812:23-33.
Lokhande S, Chougule A, Patil S, Patil V. Need of herbal drug standardization. Int Ayurvedic Med J 2015;3:874-7.
Asokar LV, Kakkar KK, Chakra OG. Glossary of Indian medicinal plants with active principles, Publication and Information Directorate, New Delhi; 1992. p. 122.
Quality Standards of Indian Medicinal Plants. Vol. I. Indian Council of Medical Research, New Delhi; 2003. p. 10-50.
Sheu SJ, Lin KL. Liquid chromatographic determinations of the constituents in shao-yao-tang and related Chinese herbal preparations. J High Resol Chromatogr 1998;21:569-73.
Jain V, Shaikh 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.
Jain V, Shaikh S. Simultaneous RP-HPLC analysis of quercetin and kaempferol in different plant part of Cissus quadrangularis. Int J Pharm Pharm Sci 2015;4:550-8.
Jain V, Shaikh S. Development and validation of RP-HPLC method for the simultaneous estimation of quercetin, ellagic acid and rutin in the hydroalcoholic extract of triphala churna. Int J Appl Pharm 2018;5:123-4.
Singh NK, Kumar P, Gupta DK, Singh VK. UV-spectrophotometric method development for estimation of piperine in chitrakadi vati. Der Pharm Lett 2011;3:178-82.
Sheikh ZA, Sadia S, Khan SS, Haider FZ. A novel HPTLC method for quantitative estimation of biomarkers in the polyherbal formulation. Asian Pac J Trop Biomed 2015;5:955-9.
ICH, Stability testing of new drug substances and products (Q1AR2), International Conference on Harmonization, IFPMA, Geneva; 2003.
Vemula VR, Sharma PK. RP-HPLC method development and validation for simultaneous estimation of diclofenac and tolperisone in the tablet dosage form. Asian J Pharm Clin Res 2013;6 Suppl 3:186-9.
Shah R, Shah R. Development and validation of an RP-HPLC method for phenytoin sodium and phenobarbitone in bulk and pharmaceutical dosage form. Int J Pharm Pharm Sci 2017;9:224-9.
FDA, Guidance R. Validation of chromatographic methods. Center for drug evaluation and research (CDER), Food Drug Administration; 1994. p. 2.
FDA, ORA validation and verification guidance for human drug analytical methods. Food and Drug Administration; 2003. p. 1.