Multiseparation of anthocyanins and anthocyanidins by high performance liquid chromatography combined with response surface methodology

View or download the full article: 
PDF icon health-risk-analysis-2019-3-14.pdf
UDC: 
613, 614, 616.9
DOI: 
10.21668/health.risk/2019.3.14.eng
Authors: 

Vu Thi Trang1,2, Le Hoang Duc2, Nguyen Hoai Thu2, Le Thi Hong Hao1,2, Nguyen Xuan Trung2

Organization: 

1National Institute for Food Control, 65 Pham ThanDuat, Hanoi, Vietnam
2VNU University of Science, Vietnam National University-Hanoi, 19 Le Thanh Tong, Hanoi, Vietnam

Abstract: 

A method has been developed for the separation of anthocyanins and anthocyanidins with high performance liquid chromatography (HPLC). Experimental designs were applied for multivariate optimization of the HPLC experimental conditions with using response surface methodology (RSM). Three independent factors, namely formic acid concentration, initial ratio of acetonitrile in gradient program and flow rate, were used to design mathematical models. The responses were resolutions of four peak couples which were the most difficult to separate including: pelargonidin-3-glucoside and delphinidin, delphinidin and peonidin-3-glucoside, peonidin-3-glucoside and malvidin-3-glucoside, peonidin and malvidin. Variance analysis proved a chosen model was highly fit and the RSM method yielded good results in improving separation of anthocyanins and anthocyanidins. The optimized HPLC parameters were as follows: C18 column (250 mm × 4.6 mm × 5 µm), mobile phase is FA 10 % and ACN used as a gradient; flow rate 0.8 mL.min-1. Using these optimum conditions, separation of compounds with good resolutions and a run time of less than 30 min were archived. The results for method validation satisfied the requirement of AOAC, linearity range from 0.2–10 ppm with R2 ≥ 0.9955, LOD from 0.05–0.1 mg/kg, RSD from 4.79–6.45 % and the recovery is from 85.4 – 109.6 %. The method was applied to determone anthocyanins and anthocyanidins in some fruits and vegetables samples with the content of anthocyanidins being from 5.74 – 218.27 mg/100g. Anthocyanins primarily concentrate in peel of fruits and vegetables, black bean peel contains most anthocyanins.

Keywords: 
anthocyanin, anthocyanidin, response surface methodology (RSM), HPLC, antioxidant, chromatography.
Vu Thi Trang, Le Hoang Duc, Nguyen Hoai Thu, Le Thi Hong Hao, Nguyen Xuan Trung. Multiseparation of anthocyanins and anthocyanidins by high performance liquid chromatography combined with response surface methodology. Health Risk Analysis, 2019, no. 3, pp. 118–127. DOI: 10.21668/health.risk/2019.3.14.eng
References: 
  1. Bordonaba J.G., Crespo P., Terry L.A. A new acetonitrile-free mobile phase for HPLC-DAD determination of indi-vidual anthocyanins in blackcurrant and strawberry fruits: A comparison and validation study. Food Chemistry, 2011, no. 129, pp. 1265–1273. DOI: 10.1016/j.foodchem.2010.09.114
  2. Deineka V.I., Deineka L.A., Saenko I.I. Regularities of Anthocyanins Retention in RP HPLC for «Water–Acetonitrile–Phosphoric Acid Mobile» Phases. Journal of Analytical Methods in Chemistry, 2015, vol. 1, pp. 6. DOI: 10.1155/2015/732918
  3. Castañeda-Ovando A., Pacheco-Hernández L., Paez E., Rodríguez J.A., Galan-Vidal C.A. Chemical studies of anthocyanins: A review. Food Chemistry, 2009, vol. 113, pp. 859–871. DOI: 10.1016/j.foodchem.2008.09.001
  4. Casteele K.V. [et al.]. Separation of some anthocyanidins, anthocyanins, proanthocyanidins and related subtances by reversed phase high performance liquid chromatography. Journal of chromatography, 1983, vol. 259, pp. 291–300.
  5. Hertog M.G., Hollman P.C., Katan M.B., Kromhout D. Intake of potentially anticarcinogenic flavonoids and their determinants in adults in The Netherlands. Nutr. Cancer, 1993, no. 20, pp. 21–29. DOI: 10.1080/01635589309514267
  6. Catherine A.R., Nicholas J.M., George P. Structure-antioxidants activity relationships of flavonoids and phenolic acids. Free Radical Biology & Medicine, 1996, no. 20, pp. 933–956. DOI: 10.1016/0891-5849(95)02227-9
  7. Lee J., Wrolstad R.E., Durst R. AOAC Official Method 2005.02 Total Monomeric Anthocyanin Pigment Content of Fruit Juices, Beverages, Natural Colorants, and Wines pH Differential Method. Official Methods of Analysis of AOAC International, 2005, chapter 37, pp. 37–39.
  8. Cesa S., Carradori S., Bellagamba G., Locatelli M., Antonietta Casadei M., Masci A., Paolicelli P. Evaluation of pro-cessing effects on anthocyanin content and colour modifications of blueberry (Vaccinium spp.) extracts: Comparison between HPLC-DAD and CIELAB analyses. Food Chemistry, 2017, no. 232, pp. 114–123. DOI: 10.1016/j.foodchem.2017.03.153
  9. Fibigr J., Satínsky D., Solich P. A UHPLC method for the rapid separation and quantification of anthocyanins in acai berry and dry blueberry extracts. Journal of Pharmaceutical and Biomedical Analysis, 2017, no. 143, pp. 204–213. DOI: 10.1016/j.jpba.2017.05.045
  10. Canuto G.A.B., Oliveira D.R., Da Conceição L.S.M., Farah J.P.S., M.F.M. Tavare. Development and validation of a liquid chromatography method for anthocyanins in strawberry (Fragaria spp.) and complementary studies on stability, kinetics and antioxidant power. Food Chemistry, 2016, no. 192, pp. 566–574. DOI: 10.1016/j.foodchem.2015.06.095
  11. Li D., Li B., Ma Y., Sun X., Lin Y., Meng X. Polyphenols, anthocyanins, and flavonoids contents and the antioxidant capacity of various cultivars of highbush and half-high blueberries. Journal of Food Composition and Analysis, 2017, no. 62, pp. 84–93. DOI: 10.1016/j.jfca.2017.03.006
  12. Trikas E.D., Melidou M., Papi R.M., Zachariadis G.A., Kyriakidis D.A. Extraction, separation and identification of anthocyanins from red wine by-product and their biological activities. Journal of functional food , 2016, no. 25, pp. 548–558.
  13. Huang Zh., Wang B., Williams P., Pace R.D. Identification of anthocyanins in muscadine grapes with HPLC-ESI-MS. Food Science and Technology, 2009, no. 42, pp. 819–824. DOI: 10.1016/j.lwt.2008.11.005
  14. Ruiz A., Hermosín-Gutiérrez I., Vergara C., Von Baer D., Zapata M., Hitschfeld A., Obando L., Mardones C. Antho-cyanin profiles in south Patagonian wild berries by HPLC-DAD-ESI-MS/MS. Food Research International, 2013, no. 51, pp. 706–713. DOI: 10.1016/j.foodres.2013.01.043
  15. Bridle P., Garcia-Viguera C., Tomas-Barberan F.A. Analysis of Anthocyanins by Capillary Zone Electrophoresis. Journal of Liquid Chromatography & Related Technologies, 1996, vol. 19, no. 4, pp. 537–545. DOI: 10.1080/10826079608005518
  16. Caboni M.F., Comandini P., Giampaolo B., Cardinali A., Cerretani L. CZE separation of strawberry anthocyanins with axitic buffer and comparison with HPLC. Journal of separatrion of science, 2008, no. 31, pp. 3257–3264. DOI: 10.1002/jssc.200800199
  17. Shim Y., Kim S., Seo D., Park H., Ha J. Rapid method for determination of anthocyanin glucosides and free del-phinidin in grapes using u-HPLC. Journal of Chromatographic Science, 2014, no. 52, pp. 629–635. DOI: 10.1093/chromsci/bmt091
  18. Gao L., Mazza G. Rapid Method for Complete Chemical Characterization of Simple and Acylated Anthocyanins by High-Performance Liquid Chromatography and Capillary Gas-Liquid Chromatography. Journal of Agricultural and Food Chemistry, no. 42, pp. 118–125. DOI: 10.1021/jf00037a020
  19. Liu X., Mu T., Sun H., Zhang M., Chen J. Optimisation of aqueous two-phase extraction of anthocyanins from purple sweet potatoes by response surface methodology. Food Chemistry, 2013, no. 141, pp. 3034–3041. DOI: 10.1016/j.foodchem.2013.05.119
  20. Nyman N.A., Kumpulainen J.T. Determination of anthocyanidins in berries and red wine by high-performance liquid chromatography. Journal of Agricultural and Food Chemistry, 2001, no. 49, pp. 4183–4187. DOI: 10.1021/jf010572i
  21. Deividas B., Ivanauskas L., Dirse V. Study of diversity of anthocyanin composition in bilberry (Vaccinium myrtillus L.) fruits. Meditsina (Kaunas, Lithuania), 2007, vol. 43 no. 12, pp. 971–977.
  22. Welch C., Wu Q., Simon J. Recent Advances in Anthocyanin Analysis and Characterization. Current Analytical Chemistry, 2008, vol. 4, no. 2, pp. 75–101. DOI: 10.2174/157341108784587795
  23. Zhang Zh., Kou X., Fugal K. Comparison of HPLC Methods for Determination of Anthocyanins and Anthocyanidins in Bilberry Extracts. Journal of Agricultural and Food Chemistry, 2004, no. 52, pp. 688–691. DOI: 10.1021/jf034596w
Received: 
27.05.2019
Accepted: 
26.07.2019
Published: 
30.09.2019

You are here

Home