Development and Validation of an HPLC-DAD Method for the Simultaneous Analysis of Phenolic Compounds

Authors

  • Lloyd Earl L. Flandez Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines 4031 https://orcid.org/0000-0003-2674-9346
  • Katherine Ann T. Castillo-Israel Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines 4031
  • Joel P. Rivadeneira Institute of Food Science and Technology, College of Agriculture and Food Science, University of the Philippines Los Baños, College, Laguna, Philippines 4031
  • Arvin Paul P. Tuaño Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna, Philippines 4031
  • Amelia B Hizon-Fradejas Institute of Chemistry, College of Arts and Sciences, University of the Philippines Los Baños, College, Laguna, Philippines 4031

DOI:

https://doi.org/10.11113/mjfas.v19n5.3049

Abstract

Phenolic compounds are natural substances that exhibit different functional bioactivities and provide health-protective actions against chronic illnesses. The vast potential of these compounds in health and other sectors demands the establishment of analytical procedures for their immediate and simultaneous analysis. In this study, a high-performance liquid chromatography with diode-array detection (HPLC-DAD) method was developed and validated for the simultaneous analysis of gallic acid, catechin, epicatechin, rutin hydrate, caffeic acid, syringic acid, ellagic acid, p-coumaric acid, trans-ferulic acid, myricetin, resveratrol, and quercetin. The chromatographic separation of the selected polyphenols was carried out in a reversed-phase Inertsil ODS-3 column (250mm x 4.5mm x 5µm) at a flow rate of 0.8 mL/min, injection volume of 20 µL, and column temperature of 30°C. The detection and quantification of phenolic compounds were done at specific wavelengths (254, 275, 305, and 325 nm) using gradient elution for 40 minutes, with acidified water and acetonitrile solution as mobile phase. Validation of the established analytical procedure showed that the coefficient of determination (R2 > 0.99), limit of detection (0.01 to 0.35 µg/mL), limit of quantitation (0.03 to 1.07 µg/mL), recovery values (98.33 to 101.12%), and repeatability (RSD < 5%) respectively indicated a linear, sensitive, accurate, and precise analytical method for the simultaneous chromatographic analysis of the 12 phenolic compounds. Overall, the developed HPLC-DAD procedure can offer adequate confidence for the identification and quantification of specific polyphenols and can be modified or updated for future analysis of phenolic compounds in different plant extracts.

References

Joseph, S. V., Edirisinghe, I., Burton-Freeman, B. M. (2014). Berries: Anti-inflammatory effects in humans. J. Agric. Food Chem., 62(18), 3886-903. Doi: 10.1021/jf4044056.

Lizardo, R. C. M., Mabesa, L. B., Dizon, E. I., Aquino, N. A. (2015). Functional and antimicrobial properties of bignay [Antidesma bunius (L.) Spreng] extract and its potential as natural preservative in a baked product. Int. Food Res. J., 2(1), 88-95.

Luna-Guevara, M. L., Luna-Guevara, J. J., Hernández-Carranza, P., Ruíz-Espinosa, H., Ochoa-Velasco, C. E. (2018). Phenolic compounds: A good choice against chronic degenerative diseases. Stud. Nat. Prod. Chem., 59, 79-108. https://doi.org/10.1016/B978-0-444-64179-3.00003-7.

Mutha, R. E., Tatiya, A. U., Surana, S. J. (2021). Flavonoids as natural phenolic compounds and their role in therapeutics: An overview. Futur. J. Pharm. Sci., 7(1). https://doi.org/10.1186/s43094-020-00161-8.

Nour, V., Trandafir, I., Cosmulescu, S. (2013). HPLC determination of phenolic acids, flavonoids and Juglone in walnut leaves. J. Chromatogr. Sci., 51(9), 883-890. https://doi.org/10.1093/chromsci/bms180.

Baby, B., Antony, P., Vijayan, R. (2018). Antioxidant and anticancer properties of Berries. Crit. Rev. Food Sci. Nutr., 58(15), 2491-2507. https://doi.org/10.1080/10408398.2017.1329198.

Chen, T., Shi, N., Afzali, A. (2019). Chemopreventive effects of strawberry and black raspberry on colorectal cancer in inflammatory bowel disease. Nutrients, 11(6). https://doi.org/10.3390/nu11061261.

Huang, H., Chen, G., Liao, D., Zhu, Y., Xue, X. (2016). Effects of Berries consumption on cardiovascular risk factors: A meta-analysis with trial sequential analysis of randomized controlled trials. Sci. Rep., 6(March), 1-11. https://doi.org/10.1038/srep23625.

Mattioli, V., Zanolin, M. E., Cazzoletti, L., Bono, R., Cerveri, I., Ferrari, M., Pirina, P., Garcia-Larsen, V. (2020). Dietary flavonoids and respiratory diseases: A population-based multi-case-control study in Italian adults. Public Health Nutr., 23(14), 2548-2556. https://doi.org/10.1017/S1368980019003562.

Mehta, A. J., Cassidy, A., Litonjua, A. A., Sparrow, D., Vokonas, P., Schwartz, J. (2016). Dietary anthocyanin intake and age-related decline in lung function: longitudinal findings from the VA normative aging study. Am. J. Clin. Nutr., 103(2), 542-550. https://doi.org/10.3945/ajcn.115.121467.

Xiao, D., Zhu, L., Edirisinghe, I., Fareed, J., Brailovsky, Y., Burton-Freeman, B. (2019). Attenuation of postmeal metabolic indices with red raspberries in individuals at risk for diabetes: A randomized controlled trial. Obesity, 27(4), 542-550. https://doi.org/10.1002/oby.22406.

De la Rosa, L. A., Moreno-Escamilla, J. O., Rodrigo-García, J., Alvarez-Parrilla, E. (2018). Phenolic compounds. Postharvest Physiol. Biochem. Fruits Veg., 253-271. https://doi.org/10.1016/B978-0-12-813278-4.00012-9.

Pandey, K. B., Rizvi, S. I. (2009). Plant polyphenols as dietary antioxidants in human. Oxid. Med. Cell. Longev., 2(5), 270-278.

Skrovankova, S., Sumczynski, D., Mlcek, J., Jurikova, T., Sochor, J. (2015). Bioactive compounds and antioxidant activity in different types of Berries. Int. J. Mol. Sci., 16(10), 24673-24706. https://doi.org/10.3390/ijms161024673.

Zuiter, A. S. (2014). Proanthocyanidin: Chemistry and Biology: From phenolic compounds to proanthocyanidins. Chemistry, Molecular Sciences and Chemical Engineering. https://doi.org/10.1016/b978-0-12-409547-2.11046-7.

Vasantha Rupasinghe, H. P., Nair, S. V. G., Robinson, R. A. (2014). Chemopreventive properties of fruit phenolic compounds and their possible mode of actions. Studies in Natural Products Chemistry. 42, 229-266. https://doi.org/10.1016/b978-0-444-63281-4.00008-2.

Aires, A. (2017). Phenolics in foods: Extraction, analysis and measurements. In Phenolic Compounds - Natural sources, importance and applications. InTech. https://doi.org/10.5772/66889.

Petrova, O. E., Sauer, K. (2017). High-performance liquid chromatography (HPLC)-based detection and quantitation of cellular c-Di-GMP. Methods Mol. Biol., 1657, 33-43. https://doi.org/10.1007/978-1-4939-7240-1_4.

Naczk, M., Shahidi, F. (2006). Phenolics in cereals, fruits and vegetables: Occurrence, extraction and analysis. J. Pharm. Biomed. Anal., 41(5), 1523-1542. https://doi.org/10.1016/j.jpba.2006.04.002.

Coskun, O. (2016). Separation tecniques: Chromatography. North. Clin. Istanbul, 3(2), 156-160. https://doi.org/10.14744/nci.2016.32757.

Ignat, I., Volf, I., Popa, V. I. (2011). A critical review of methods for characterisation of polyphenolic compounds in fruits and vegetables. Food Chem., 126(4), 1821-1835. https://doi.org/10.1016/j.foodchem. 2010.12.026.

Singh, D. P., Govindarajan, R., Khare, A., Rawat, A. K. S. (2007). Optimization of a high-performance liquid chromatography method for the separation and identification of six different classes of phenolics. J. Chromatogr. Sci., 45(10), 701-705. https://doi.org/10.1093/chromsci/45.10.701.

Zhang, Y., Cai, P., Cheng, G., Zhang, Y. (2022). A brief review of phenolic compounds identified from plants: Their extraction, analysis, and biological activity. Nat. Prod. Commun., 17(1). https://doi.org/10.1177/ 1934578X211069721.

Magnusson, B. and Örnemark, U. (2014). Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics. 2nd ed.

Jorjong, S., Butkhup, L., Samappito, S. (2015). Phytochemicals and antioxidant of Mao-Luang (Antidesma bunius L.) cultivars from Northeastern Thailand. Food Chem., 181, 248-255. https://doi.org/10.1016/j.foodchem.2015.02.093.

Seal, T., Pillai, B., Chaudhuri, K. (2016). Identification and quantification of phenolic acids by HPLC, in three wild edible plants viz. Viburnum foetidum, Houttuynia cordata and Perilla ocimoides collected from North-Eastern Region in India. Int. J. Curr. Pharm. Rev. Res., 7(5), 267-274.

Boligon, A. A., Athayde, M. L. (2014). Importance of HPLC in analysis of plants extracts. Austin Chromatogr., 1(3), 2-3.

Chen, H. J., Inbaraj, B. S., Chen, B. H. (2012). Determination of phenolic acids and flavonoids in Taraxacum formosanum Kitam by liquid chromatography-tandem mass spectrometry coupled with a post-column derivatization technique. Int. J. Mol. Sci., 13(1), 260-285. https://doi.org/10.3390/ijms13010260.

Sanches, V. L., Cunha, T. A., Viganó, J., de Souza Mesquita, L. M., Faccioli, L. H., Breitkreitz, M. C., Rostagno, M. A. (2022). Comprehensive analysis of phenolics compounds in citrus fruits peels by UPLC-PDA and UPLC-Q/TOF MS using a fused-core column. Food Chem. X, 14. https://doi.org/10.1016/j.fochx.2022. 100262.

Svedström, U., Vuorela, H., Kostiainen, R., Laakso, I., Hiltunen, R. (2006). Fractionation of polyphenols in hawthorn into polymeric procyanidins, phenolic acids and flavonoids prior to high-performance liquid chromatographic analysis. J. Chromatogr. A, 1112(1-2), 103-111. https://doi.org/10.1016/j.chroma. 2005.12.080.

Agrahari, V., Bajpai, M., Nanda, S. (2013). Essential concepts of mobile phase selection for reversed phase HPLC. Res. J. Pharm. Technol., 6(5), 459-464.

Francisco, M. L. d. L., Resurreccion, A. V. A. (2009). Development of a reversed-phase high performance liquid chromatography (RP-HPLC) procedure for the simultaneous determination of phenolic compounds in peanut skin extracts. Food Chem., 117(2), 356-363. https://doi.org/10.1016/j.foodchem.2009.03.110.

Tasioula-Margari, M., Tsabolatidou, E. (2015). Extraction, separation, and identification of phenolic compounds in virgin olive oil by HPLC-DAD and HPLC-MS. Antioxidants, 4(3), 548-562. https://doi.org/10.3390/antiox4030548.

Boonen, J., D’Hondt, M., Veryser, L., Peremans, K., Burvenich, C., De Spiegeleer, B. (2013). A critical quality parameter in quantitative fused-core chromatography: The injection volume. J. Pharm. Anal., 3(5), 330-334. https://doi.org/10.1016/j.jpha.2013.02.002.

Dembek, M., Bocian, S. (2022). Stationary phases for green liquid chromatography. Materials (Basel)., 15(2). https://doi.org/10.3390/ma15020419.

Butkhup, L., Samappito, S. (2008). Analysis of anthocyanin, flavonoids, and phenolic acids in tropical bignay berries. Int. J. Fruit Sci., 8(1-2), 15-34. https://doi.org/10.1080/15538360802365913.

Lata, S., Mittal, S. K. (2017). In vitro and in vivo hepatoprotective activity of flavonoids rich extracts on Cucumis dipsaceus Ehrenb. (Fruit). Int. J. Pharmacol., 13(6), 563-572. https://doi.org/10.3923/ijp.2017.563.572.

Magiera, S., Zaręba, M. (2015). Chromatographic determination of phenolic acids and flavonoids in Lycium barbarum L. and evaluation of antioxidant activity. Food Anal. Methods, 8(10), 2665-2674. https://doi.org/10.1007/s12161-015-0166-y.

Natividade, M. M. P., Corrêa, L. C., Souza, S. V. C. de, Pereira, G. E., Lima, L. C. de O. (2013). Simultaneous analysis of 25 phenolic compounds in grape juice for HPLC: Method validation and characterization of São Francisco Valley samples. Microchem. J., 110, 665-674. https://doi.org/10.1016/j.microc.2013.08.010.

Seal, T. (2016). HPLC determination of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of Zanthoxylum acanthopodium, a wild edible plant of Meghalaya State of India. Int. J. Pharm. Pharm. Sci., 8(3), 103-109.

Lakka, N. S., Kuppan, C. 2020. Principles of chromatography method development. Biochem. Anal. Tools - Methods Bio-Molecules Stud., https://doi.org/10.5772/intechopen.89501.

Urbstaite, R., Raudone, L., Liaudanskas, M., Janulis, V. (2022). Development, validation, and application of the UPLC-DAD methodology for the evaluation of the qualitative and quantitative composition of phenolic compounds in the fruit of American Cranberry (Vaccinium macrocarpon Aiton). Molecules, 27(2). https://doi.org/10.3390/molecules27020467.

Gonçalves, J., Ramos, R., Rosado, T., Gallardo, E., Duarte, A. P. (2019). Development and validation of a HPLC–DAD method for quantification of phenolic compounds in different sweet cherry cultivars. SN Appl. Sci., 1(9), 1-11. https://doi.org/10.1007/s42452-019-0680-4.

Sellappan, S., Akoh, C. C., Krewer, G. (2002). Phenolic compounds and antioxidant capacity of Georgia-Grown blueberries and blackberries. J. Agric. Food Chem., 50(8), 2432-2438. https://doi.org/10.1021/ jf011097r.

Latasa, M. (2014). A simple method to increase sensitivity for RP-HPLC phytoplankton pigment analysis. Limnol. Oceanogr. Methods, 12(1 JAN), 46-53. https://doi.org/10.4319/lom.2014.12.46.

Park, J. S., Kim, I. S., Rehman, S. U., Na, C. S., Yoo, H. H. (2016). HPLC determination of bioactive flavonoids in Hovenia dulcis Fruit Extracts. J. Chromatogr. Sci., 54(2), 130-135. https://doi.org/10.1093/chromsci/ bmv114.

Chanda Gupta, P. (2015). Method validation of analytical procedures. PharmaTutor, 3(1), 32-39.

Ciric, A., Jelikic-Stankov, M., Cvijovic, M., Djurdjevic, P. (2018). Statistical optimization of an RP-HPLC method for the determination of selected flavonoids in berry juices and evaluation of their antioxidant activities. Biomed. Chromatogr., 32(4). https://doi.org/10.1002/bmc.4150.

Tang, W., Li, W., Yang, Y., Lin, X., Wang, L., Li, C., Yang, R. (2021). Phenolic compounds profile and antioxidant capacity of Pitahaya fruit peel from two red-skinned species (Hylocereus polyrhizus and Hylocereus undatus). Foods, 10(6). https://doi.org/10.3390/foods10061183.

Waters Corporation. (1997). Performance PerSPECtives. No. 34 Maple Street, Milford, MA 01757, 1-2.

Al-Rimawi, F., Odeh, I. (2015). Development and validation of an HPLC-UV method for determination of eight phenolic compounds in date palms. J. AOAC Int., 98(5), 1335-1339. https://doi.org/10.5740/jaoacint. 15-010.

Luaces, P., Pascual, M., Pérez, A. G., Sanz, C. (2021). An easy-to-use procedure for the measurement of total phenolic compounds in olive fruit. Antioxidants, 10(11). https://doi.org/10.3390/antiox10111656.

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Published

19-10-2023

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Vol. 19 No. 5 (2023): September-October

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Copyright (c) 2023 Lloyd Earl L. Flandez, Katherine Ann T. Castillo-Israel, Joel P. Rivadeneira, Arvin Paul P. Tuaño, Amelia B Hizon-Fradejas

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