• GYAMCHO TSHERING BHUTIA Laboratory of Nanomedicine, Division of Pharmaceutical Biotechnology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, West Bengal, India
  • ASIT KUMAR DE Department of Chemistry, Jadavpur University, Kolkata 700032, West Bengal, India
  • TANMOY BERA Laboratory of Nanomedicine, Division of Pharmaceutical Biotechnology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, West Bengal, India



RP-HPLC method development, Forced degradation, Stability studies, Curcumin, EGCG, Nanoformulation, Co-encapsulation


Objective: A new reverse-phase high-performance liquid chromatography (RP-HPLC) method was developed to simultaneously determine curcumin and epigallocatechin gallate (EGCG) in novel nanoformulation.

Methods: The high-performance liquid chromatography (HPLC) method was achieved by using a Thermo Scientific Hypersil Base Deactivated Silica (BDS) C18 column (25 cm X 4.6 mm, 5 µm) at 35 ºC column oven temperature. The chromatographic procedure was performed with a mobile phase of acetonitrile and 0.025 M (pH 4.0) potassium dihydrogen phosphate (KH2PO4) buffer by gradient mode of elution. The injection volume was 20 µl, and the flow rate was 1.5 ml/min, with ultraviolet (UV) detection using a diode array detector (DAD) at a 268 nm isosbestic wavelength.

Results: Drug entrapment efficiency studies were performed with co-encapsulated EGCG and curcumin nanoformulation, which were found to be 94.35 % and 95.12 %, respectively. This shows that the developed method is highly effective. EGCG and curcumin were eluted at 3.9 minutes and 10.7 minutes, respectively. The linearity range was 25-175 µg/ml for EGCG and 12.5-100 µg/ml for curcumin. The correlation coefficient was 0.991 for EGCG and 0.999 for curcumin from the linearity curve, which indicates that the method can produce good sensitivity. Forced degradation studies were conducted in acidic, basic, oxidative, thermal, photolytic, and UV stress conditions, where all the degradation peaks were monitored.

Conclusion: The developed method was linear, simple, rapid, robust, and precise. It could be used to quantify EGCG and curcumin simultaneously in various nanoformulations for in-vivo and in-vitro applications.


Download data is not yet available.


Guaadaoui A, Benaicha S, Elmajdoub N, Bellaoui M, Hamal A. What is a Bioactive Compound? A Combined Definition for a Preliminary Consensus. Int J Nutr Food Sci. 2014;3:174-9.

Hooper L, Cassidy A. A review of the health care potential of bioactive compounds. J Sci Food Agric. 2006;86:1805–13.

Hung HC, Joshipura KJ, Jiang R, Hu FB, Hunter D, Smith-Warner SA, Colditz GA, Rosner B, Spiegelman D, Willett WC. Fruit and vegetable intake and risk of major chronic disease. J Natl Cancer Inst. 2004;96:1577-84.

Kim MJ, Moon Y, Tou JC, Mou B, Waterland NL. Nutritional value, bioactive compounds, and health benefits of lettuce (Lactuca sativa L.). J Food Compos Anal. 2016;49:19-34.

Recharge N, Riaz M, Ko S, Park S. Novel technologies to enhance the solubility of food-derived bioactive compounds: A review. J Funct Foods. 2017;39:63-73.

Esfanjani AF, Assadpour E, Jafari SM. Improving the bioavailability of phenolic compounds by loading them within lipid-based nanocarriers. Trends Food Sci Technol. 2018;76:56-66.

Assadpour E, Jafari SM. A systematic review on nanoencapsulation of food bioactive ingredients and nutraceuticals by various nanocarriers. Crit Rev Food Sci Nutr. 2019;59:3129-51.

Rezaei A, Fathi M, Jafari SM. Nanoencapsulation of hydrophobic and low-soluble food bioactive compounds within different nanocarriers. Food Hydrocolloids. 2019;88:146-62.

Moghadamtousi SZ, Kadir HA, Hassandarvish P, Tajik H, Abubakar S, Zandi K. A Review on Antibacterial, Antiviral, and Antifungal Activity of Curcumin. BioMed Res Int. 2014;2014:1-12.

Salehi B, Stojanović-Radić Z, Matejić J, Sharifi-Rad M, Anil Kumar NV, Martins N, Sharifi-Rad J. The therapeutic potential of curcumin: A review of clinical trials. Eur J Med Chem. 2019;163:527-45.

Maheshwari RK, Singh AK, Gaddipati J, Srimal RC. Multiple biological activities of curcumin: a short review. Life Sci. 2006;78:2081-7.

Johnson R, Bryant S, Huntley AL. Green tea and green tea catechin extracts: an overview of the clinical evidence. Maturitas. 2012;3:280-7.

Shafabakhsh R, Milajerdi A, Reiner Ž, Kolahdooz F, Amirani E, Mirzaei H, Barekat M, Asemi Z. The effects of catechin on endothelial function: A systematic review and meta-analysis of randomized controlled trials. Crit Rev Food Sci Nutr. 2019;60:2369-78.

Tamura M, Ochiai K. Exploring the possible applications of catechin (gel) for oral care of the elderly and disabled individuals. Japanese Dental Science Review. 2012;48:126-34.

Cai ZY, Li XM, Liang JP, Xiang LP, Wang KR, Shi YL, Yang R, Shi M, Ye JH, Lu JL, Zheng XQ, Liang YR. Bioavailability of Tea Catechins and Its Improvement. Molecules. 2018;23:2346.

Manikandan R, Beulaja M, Arulvasu C, Sellamuthu S, Dinesh D, Prabhu D, Babu G, Vaseeharan B, Prabhu NM. Synergistic anticancer activity of curcumin and catechin: an in vitro study using human cancer cell lines. Microsc Res Tech. 2012;75:112-6.

Lam WH, Kazi A, Kuhn DJ, Chow LM, Chan AS, Dou QP, Chan TH. A potential prodrug for a green tea polyphenol proteasome inhibitor: Evaluation of the peracetate ester of (−)-epigallocatechin gallate [(−)-EGCG]. Bio organ Med Chem. 2004;12:5587-93.

Chakraborty S, Kar N, Kumari L, De A, Bera T. Inhibitory effect of a new orally active cedrol-loaded nanostructured lipid carrier on compound 48/80-induced mast cell degranulation and anaphylactic shock in mice. Int J Nanomed. 2017;12:4849-68.

Jayaprakasha GK, Rao LJ, Sakariah KK. Improved HPLC method for the determination of curcumin, demethoxycurcumin, and bisdemethoxycurcumin. J Agric Food Chem. 2002;50:3668-72.

Ansari MJ, Ahmad S, Kohli K, Ali J, Khar RK. Stability-indicating HPTLC determination of curcumin in bulk drug and pharmaceutical formulations. J Pharm Biomed Anal. 2005;39:132-8.

Yu W, Wen D, Cai D, Zheng J, Gan H, Jiang F, Liu X, Lao B, Yu W, Guan Y, Zhong G. Simultaneous determination of curcumin, tetrahydrocurcumin, quercetin, and paeoniflorin by UHPLC-MS/MS in rat plasma and its application to a pharmacokinetic study. J Pharm Biomed Anal. 2019;172:58-66.

Ramalingam P, Ko YT. A validated LC-MS/MS method for quantitative analysis of curcumin in mouse plasma and brain tissue and its application in pharmacokinetic and brain distribution studies. J Chromatogr B Analyt Technol Biomed Life Sci. 2014;969:101-8.

Wang H, Provan GJ, Helliwell K. HPLC determination of catechins in tea leaves and tea extracts using relative response factors. Food Chemistry. 2003;81:307-12.

Spark B, Krauze-Baranowska M, Pobłocka-Olech L. HPTLC Determination of Catechins in In-Vitro Cultures of Two Species of the Genus Phyllanthus. J Planar Chromatogr. 2008;21:103–6.

Naldi M, Fiori J, Gotti R, Périat A, Veuthey JL, Guillarme D, Andrisano V. UHPLC determination of catechins for the quality control of green tea. J Pharm Biomed Anal. 2014;88:307-14.

Chang CL, Wu RT. Quantification of (+)-catechin and ()-epicatechin in coconut water by LC-MS. Food Chemistry. 2011;126:710-7.

Aswathy SR, Muhas C, Anjali Sruthy S, Devi Swapna PV, Gopinath U. Validation and application of RP-HPLC method for quantification of enrofloxacin in pure and veterinary dosage forms. Int J Pharm Pharm Sci. 2022;14:42-7.

De AK, Bera T. Analytical method development, validation and stability studies by RP-HPLC method for simultaneous estimation of andrographolide and curcumin in co-encapsulated nanostructured lipid carrier drug delivery system. Int J Appl Pharm. 2021;13:73-86.

Sesharao M, Madhavarao V. A new validated simultaneous reversed-phase high-performance liquid chromatography assay method for estimation of two flavones (baicalein and chrysin) in API drugs. Asian J Pharm Clin Res. 2018;11:351-6.

Rao N, Gawde KD. Method development and forced degradation studies for simultaneous estimation of salbutamol sulfate, etofylline, and bromhexine hydrochloride in pharmaceutical dosage form using reversed-phase high-performance liquid chromatography method. Asian J Pharm Clin Res. 2018;11:378-82.

Hassan DH, Shohdy JN, El-Nabarawi M, El-Setouhy DA, Abdellatif MM. Nanostructured lipid carriers for transdermal drug delivery. Int J Appl Pharm. 2022;14:88–93.

Sai PN, Venkateswarlu BS, Kumudhavalli MV, Muruganantham V. Bio-analytical method development and validation for the simultaneous estimation of decitabine and cedazuridine in human plasma using LC-MS/MS. Int J Appl Pharm. 2021;13:257–62.

Ramesh D, Habibuddin M. Development and validation of RP-HPLC method for the determination of alvimopan in rat plasma. Int J Pharm Pharm Sci. 2018;10:124-9.

Madriwala B, Jays J. Analytical method development and validation for estimation of residual solvents in gliclazide using gas chromatography. Int J Curr Pharm Res. 2022;14:68-73.

Alumuri T, Amarababu NL, Kurnool A, Kanuparthy PR, Merugu K. Development and validation of a stability indicating related substances of atenolol and nitrendipine by RP-HPLC. 2022;14:265-73.

Pal VK, Pal Y. Analytical method development, and method validation for determination assay and content uniformity of levonorgestrel by Reversed-Phase High-Performance Liquid Chromatography. Asian J Pharm Clin Res. 2020;13:101-7.

Mishra S, Sarker K, Ghosh A, Saha A, Sen S. A validated stability indicating RP-HPLC method for estimation of avapritinib in bulk and tablet dosage form. Int J Appl Pharm. 2022;14:95-101.

Rayudu S, Manoranjani M, Reddy DR. Analytical method development and validation of dexmethylphenidate and serdexmethylphenidate by using RP-HPLC in the bulk and pharmaceutical dosage form. Int J App Pharm. 2022;14:110-5.

Sura RS, Subrahmanyam CVS, Rachamalla SS. Bioanalytical RP-HPLC method development and validation of clopidogrel bisulfate in Wistar rat plasma and its application to pharmacokinetic study. Int J App Pharm. 2022;14:106-11.

Devi PR, Rambabu K. Rapid determination of carboplatin and docetaxel using RP-HPLC with PDA detector. Int J App Pharm. 2022;14:186-92.

Reddy CS, Rao BT. Development and validation of a stability-indicating related substances of trandolapril by RP-HPLC and its degradation. Int J App Pharm. 2021;13: 115-21.



How to Cite




Original Article(s)