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Development of a HPLC method for the simultaneous analysis of riboflavin and other flavin compounds in liquid milk and milk products

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Abstract

A high-performance liquid chromatography method with fluorescence detection was developed for the quantification of riboflavin (RF), flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and their photodegradation products, lumichrome (LC) and lumiflavin (LF), in liquid milk and milk products. Both sample preparation and chromatographic separation were studied to avoid acidic conditions that proved to affect flavin stability and degrade FAD into FMN. The sample preparation includes centrifugal skimming and ultrafiltration steps and is suitable for routine application. Linear response was obtained for individual flavins in the respective concentration ranges of interest and relative standard deviation (RSD) was lower than 5%, except for FAD (RSD 11%). The recovery ranged between 80–100%. The proposed method proved to be suitable for assessing flavins in commercial liquid milk and fermented milk products, and for monitoring the degradation of FAD, FMN and RF and the formation of LF and LC in bottled milk exposed to light during shelf storage.

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References

  1. Ball GFM (2004) Flavins: riboflavin, FMN and FAD (vitamin B2). In: Ball GFM (ed) Vitamins: their role in the human body. Blackwell Publishing Ltd, Oxford

    Chapter  Google Scholar 

  2. Sheraz MA, Kazi SH, Ahmed S, Anwar Z, Ahmad I (2014) Photo, thermal and chemical degradation of riboflavin. Beilstein J Org Chem 10:1999–2012

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Gliszczynska-Swigło A, Koziołowa A (2000) Chromatographic determination of riboflavin and its derivatives in food. J Chromatogr A 881:285–297

    Article  PubMed  Google Scholar 

  4. Monteiro MC, Perrone D (2013) Chemistry and biochemistry of riboflavin and related compounds. In: Preedy VR (ed) B vitamins and folate chemistry, analysis, function and effects. RCS Publishing, Cambridge

    Google Scholar 

  5. Depeint F, Bruce WR, Shangari N, Mehta R, O’Brien PJ (2006) Mitochondrial function and toxicity: role of the B vitamin family on mitochondrial energy metabolism. Chem Biol Interact 163:94–112

    Article  CAS  PubMed  Google Scholar 

  6. Nollet LML, Toldrá F (2013) Water soluble vitamins. In: Nollet LML (ed) Food analysis by HPLC. Taylor & Francis Group, New York

    Google Scholar 

  7. Golbach JL, Ricke SC, O’Bryan CA, Crandall PG (2014) Riboflavin in nutrition, food processing and analysis—a review. J Food Res 3:23–35

    Article  Google Scholar 

  8. Bitsch R, Bitsch I (2011) HPLC determination of riboflavin in fortified foods. In: Rychlik M (ed) Fortified foods with vitamins: analytical concepts to assure better and safer products. Wiley, New York

    Google Scholar 

  9. Choe E, Huang R, Min DB (2005) Chemical reactions and stability of riboflavin in foods. J Food Sci 70:R28-R36

    Article  Google Scholar 

  10. Cardoso DR, Libardi SH, Skibsted LH (2012) Riboflavin as a photosensitizer. Effect on human health and food quality. Food Funct 3:487–502

    Article  CAS  PubMed  Google Scholar 

  11. Jung MY, Yooh SH, Lee HO, Min DB (1998) Single oxygen and ascorbic acid effects on dimethyl sulfide and off-flavour in skim milk exposed to light. J Food Sci 63:408–412

    Article  CAS  Google Scholar 

  12. Lee JH, Min DB (2009) Changes of headspace volatiles in milk with riboflavin photosensitization. J Food Sci 74:C563-C568

    Google Scholar 

  13. Shuping W, Zhiqin J, Heting L, Li Y, Daixun Z (2001) Sensitized photooxygenation of cholesterol and pseudocholesterol derivatives via singlet oxygen. Molecules 6:52–60

    Article  Google Scholar 

  14. Solah VA, Staines V, Honda S, Limley HA (2007) Measurement of milk color and composition: effect of dietary intervention on western Australian holstein-friesian cow’s milk quality. J Food Sci 72:S560-S566

    Article  CAS  Google Scholar 

  15. Albalá-Hurtado S, Veciana-Nogués MT, Izquierdo-Pulido M, Mariné-Font A (1997) Determination of water-soluble vitamins in infant milk by high-performance liquid chromatography. J Chromatogr A 778:247–253

    Article  PubMed  Google Scholar 

  16. Hall NK, Chapman TM, Jung Kim H, Min DB (2010) Antioxidant mechanisms of Trolox and ascorbic acid on the oxidation of riboflavin in milk under light. Food Chem 118:534–539

    Article  CAS  Google Scholar 

  17. Gatti R, Gioia MG (2005) Liquid chromatographic determination with fluorescence detection of B6 vitamers and riboflavin in milk and pharmaceuticals. Anal Chim Acta 538:135–141

    Article  CAS  Google Scholar 

  18. Severo Silva L Jr, Trevisan MG, Rath S, Poppi RJ, Feyes FGR (2005) Chromatographic determination of riboflavin in the presence of tetracyclines in skimmed and full cream milk using fluorescence detection. J Braz Chem Soc 16:1174–1178

    Google Scholar 

  19. Viñas P, Balsalobre N, López-Erroz C, Hernández-Córdoba M (2004) Liquid chromatographic analysis of riboflavin vitamers in foods using fluorescence detection. J Agric Food Chem 52:1789–1794

    Article  CAS  PubMed  Google Scholar 

  20. Russell LF, Vanderslice JT (1992) Comments on the standard fluorometric determination of riboflavin in foods and biological tissues. Food Chem 43:79–82

    Article  CAS  Google Scholar 

  21. Gentili A, Caretti F, D’Ascenzo G, Marchese S, Perret D, Di Corcia D, Mainero Rocca L (2008) Simultaneous determination of water-soluble vitamins in selected food matrices by liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 22:2029–2043

    Article  CAS  PubMed  Google Scholar 

  22. Phillips MM (2015) Liquid chromatography with isotope-dilution mass spectrometry for determination of water-soluble vitamins in foods. Anal Bioanal Chem 407:2965–2974

    Article  CAS  PubMed  Google Scholar 

  23. Cellar AN, McClure SC, Salvati ML, Reddy TM (2016) A new sample preparation and separation combination for precise, accurate, rapid, and simultaneous determination of vitamins B1, B2, B3, B5, B6, B7, and B9 in infant formula and related nutritionals by LC–MS/MS. Anal Chim Acta 934:180–185

    Article  CAS  PubMed  Google Scholar 

  24. Hampel D, Allen LH (2016) Analyzing B-vitamins in human milk: methodological approaches. Crit Rev Food Sci Nutr 56:494–511

    Article  CAS  PubMed  Google Scholar 

  25. Redeuil K, Bénet S, Affolter M, Thakkar SK, Campos-Giménez E (2017) A novel methodology for the quantification of B-vitamers in breast milk. J Anal Bioanal Tech 8:1000352

    Article  CAS  Google Scholar 

  26. Cataldi TRI, Nardiello D, De Benedetto GE, Bufo SA (2002) Optimizing separation conditions for riboflavin, flavin mononucleotide and flavin adenine dinucleotide on capillary zone electrophoresis with laser-induced fluorescence detection. J Chromatogr A 968:229–239

    Article  CAS  Google Scholar 

  27. Webster JB, Duncan SE, Marcy JE, O’Keefe SF (2009) Controlling light oxidation flavor in milk by blocking riboflavin excitation wavelengths by interference. J Food Sci 74:S390-S398

    Article  CAS  Google Scholar 

  28. Osório MV, Marques SS, Oliveira HM, Barreiros L, Segundo MA (2016) Fluorometric method based on molecular recognition solid-phase extraction for determination of riboflavin in milk and infant formula. J Food Compos Anal 45:141–146

    Article  CAS  Google Scholar 

  29. Wold JP, Skaret J, Dalsgaard TK (2015) Assessment of the action spectrum for photoxidation in full fat bovine milk. Food Chem 179:68–75

    Article  CAS  PubMed  Google Scholar 

  30. Zandomeneghi M, Carbonaro L, Zandomeneghi G (2007) Biochemical fluorimetric method for the determination of riboflavin in milk. J Agric Food Chem 55:5990–5994

    Article  CAS  PubMed  Google Scholar 

  31. Drössler P, Holzer W, Penzkofer A, Hegemann P (2002) pH dependence of the absorption and emission behavior of riboflavin in aqueous solution. Chem Phys 282:429–439

    Article  Google Scholar 

  32. Magnusson B, Örnemark U (2014) Eurachem guide: the fitness for purpose of analytical methods—a laboratory guide to method validation and related topics. ISBN 978-91-87461-59-0http://www.eurachem.org. Accessed 21 Jan 2018

  33. Andrés-Lacueva C, Mattivi F, Tonon D (1998) Determination of riboflavin, flavin mononucleotide and flavin adenine dinucleotide in wine and other beverages by high-performance liquid chromatography with fluorescence detection. J Chromatogr A 823:355–363

    Article  PubMed  Google Scholar 

  34. Islam MS, Honma M, Nakabayashi T, Kinjo M, Ohta N (2013) pH dependence of the fluorescence lifetime of FAD in solution and in cells. Int J Mol Sci 14:1952–1963

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Capo-chichi CD, Guéant JL, Feillet F, Namour F, Vidailhet M (2000) Analysis of riboflavin and riboflavin cofactor levels in plasma by high-performance liquid chromatography. J Chromatogr B 739:219–224

    Article  CAS  Google Scholar 

  36. Koop J, Monschein S, Macheroux EP, Knaus T, Macheroux P (2014) Determination of free and bound riboflavin in cow’s milk using a novel flavin-binding protein. Food Chem 146:94–97

    Article  CAS  PubMed  Google Scholar 

  37. Kanno C, Kanehara N, Shirafuji K, Tanji R, Imai T (1991) Binding form of vitamin B2 in bovine milk: its concentration, distribution and binding linkage. J Nutr Sci Vitaminol 37:15–27

    Article  CAS  PubMed  Google Scholar 

  38. Gliszczynska-Swigło A, Rybicka I (2015) Simultaneous determination of caffeine and water-soluble vitamins in energy drinks by HPLC with photodiode array and fluorescence detection. Food Anal Method 8:139–146

    Article  Google Scholar 

  39. Cattaneo S, Masotti F, Pellegrino L (2009) Liquid infant formulas: technological tools for limiting heat damage. J Agric Food Chem 57:10689–10694

    Article  CAS  PubMed  Google Scholar 

  40. Schmidt A, Schreiner MG, Mayer HK (2017) Rapid determination of the various native forms of vitamin B6 and B2 in cow’s milk using ultra-high performance liquid chromatography. J Chromatogr A 1500:89–95

    Article  CAS  PubMed  Google Scholar 

  41. Sunaric S, Denic M, Kocic G (2012) Evaluation of riboflavin content in dairy products and non-dairy substitutes. Ital J Food Sci 24:352–357

    CAS  Google Scholar 

  42. Huang R, Kim HJ, Min DB (2006) Photosensitizing effect of riboflavin, lumiflavin, and lumichrome on the generation of volatiles in soy milk. J Agric Food Chem 54:2359–2364

    Article  CAS  PubMed  Google Scholar 

  43. LeBlanc JG, Laiño JE, del Valle MJ, Vannini V, van Sinderen D, Taranto MP, de Valdez GF, de Giori GS, Sesma F (2011) B-Group vitamin production by lactic acid bacteria—current knowledge and potential applications. J Appl Microbiol 111:1297–1309

    Article  CAS  PubMed  Google Scholar 

  44. Fracassetti D, Gabrielli M, Encinas J, Manara M, Pellegrino L, Tirelli A (2017) Approaches to prevent light-struck taste in white wine. Aust J Grape Wine Res 23:329–333

    Article  CAS  Google Scholar 

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Acknowledgements

We are grateful to Dr. Claudio Gardana from our Department for performing the UPLC MS/MS analysis.

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Authors and Affiliations

  1. Department of Food, Environmental and Nutritional Sciences, Università degli Studi di Milano, Via G. Celoria 2, 20133, Milan, Italy

    Daniela Fracassetti, Sara Limbo, Paolo D’Incecco, Antonio Tirelli & Luisa Pellegrino

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  1. Daniela Fracassetti
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  2. Sara Limbo
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  3. Paolo D’Incecco
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  4. Antonio Tirelli
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  5. Luisa Pellegrino
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Correspondence to Daniela Fracassetti.

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Fracassetti, D., Limbo, S., D’Incecco, P. et al. Development of a HPLC method for the simultaneous analysis of riboflavin and other flavin compounds in liquid milk and milk products. Eur Food Res Technol 244, 1545–1554 (2018). https://doi.org/10.1007/s00217-018-3068-6

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  • DOI: https://doi.org/10.1007/s00217-018-3068-6

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