Analytical and Bioanalytical Chemistry

, Volume 405, Issue 7, pp 2371–2377 | Cite as

Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system

  • Michela Quaranta
  • Endry Nugroho PrasetyoEmail author
  • Klaus Koren
  • Gibson S. Nyanhongo
  • Michael Murkovic
  • Ingo Klimant
  • Georg M. Guebitz
Original Paper


It is estimated that up to 50 % of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O2. The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t 90 = 1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.


Online measurements Antioxidant Tetramethoxy azobismethylene quinone/syringaldazine Optical oxygen sensor 



Financial support from the European Commission (grant agreement number 264772 – ITN CHEBANA) is gratefully acknowledged.


  1. 1.
    Jomova K, Valko M (2011) Advances in metal-induced oxidative stress and human disease. Toxicology 283:65–87CrossRefGoogle Scholar
  2. 2.
    Valko M, Rhodes CJ, Moncol J, Izakovic M, Mazur M (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. Chem Biol Interact 160:1–40CrossRefGoogle Scholar
  3. 3.
    Barreira JCM, Ferreira ICFR, Oliveira MBPP, Pereira JA (2008) Antioxidant activity and bioactive compounds of ten Portuguese regional and commercial almond cultivars. Food Chem Toxicol 46:2230–2235CrossRefGoogle Scholar
  4. 4.
    Gorinstein S, Poovardom S, Leontowicz H, Leontowicz M, Namiesnik J, Vearasilp S, Haruenkit R, Ruamsuke P, Katrich E, Tashma Z (2011) Antioxidant properties and bioactive constituents of some rare exotic Thai fruits and comparison with conventional fruits: in vitro and in vivo studies. Food Res Int 44:2222–2232CrossRefGoogle Scholar
  5. 5.
    Ou B, Huang D, Hampsch-Woodill M, Flanagan JA, Deemer EK (2002) Analysis of antioxidant activity of common vegetables employing oxygen radical absorbance (ORAC) and ferric reducing antioxidant power (FRAP) assay: a comparative study. J Agric Food Chem 50(11):3122–3128CrossRefGoogle Scholar
  6. 6.
    Tabart J, Kevers C, Pincemail J, Defraigne JO, Dommes J (2009) Comparative antioxidant capacities of phenolic compounds measured by various tests. Food Chem 113:1226–1233CrossRefGoogle Scholar
  7. 7.
    Moreira FTC, Guerreiro JRL, Barros R, Sales MGF (2012) The effect of method, standard and sample components on the total antioxidant capacity of commercial waters assessed by optical conventional assays. Food Chem 134:564–571CrossRefGoogle Scholar
  8. 8.
    Prior RL, Wu X, Schaich K (2005) Standardized methods for the detemination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem 53:4290–4302CrossRefGoogle Scholar
  9. 9.
    Nugroho Prasetyo E, Kudanga T, Steiner W, Murkovic M, Nyanhongo GS, Guebitz GM (2009) Antioxidant activity assay based on laccase-generated radicals. Anal Bioanal Chem 393(2):679–687CrossRefGoogle Scholar
  10. 10.
    Nugroho Prasetyo E, Kudanga T, Steiner W, Murkovic M, Wonisch W, Nyanhongo GS, Guebitz GM (2010) Cellular and plasma antioxidant activity assay using tetramethoxy azobismethylene quinone. Free Radic Biol Med 49(7):1205–1211CrossRefGoogle Scholar
  11. 11.
    Nugroho Prasetyo E, Kudanga T, Steiner W, Murkovic M, Nyanhongo GS, Guebitz GM (2010) Laccase-generated tetramethoxy azobismethylene quinone (TMAMQ) as a tool for antioxidant activity measurement. Food Chem 118(2):437–444CrossRefGoogle Scholar
  12. 12.
    Nugroho Prasetyo E, Willibald W, Nyanhongo GS, Guebitz GM (2012) A unique two-way approach for the validation of total antioxidant capacity of serum samples. Eur J Clin Investig 42(4):432–438CrossRefGoogle Scholar
  13. 13.
    Alamansa E, Kandelbauer A, Pereira L, Cavaco P, Guebitz GM (2004) Influence of structure on dye degradation with laccase mediator system. Biocatal Biotransform 22:315–324CrossRefGoogle Scholar
  14. 14.
    Borisov SM, Nuss G, Haas W, Saf R, Schmuck M, Klimant I (2009) New NIR-emitting complexes of platinum(II) and palladium(II) with fluorinated benzoporphyrins. J Photochem Photobiol A Chem 201(2–3):128–135CrossRefGoogle Scholar
  15. 15.
    Carraway ER, Demas JN, DeGraff BA (1991) Luminescence quenching mechanism for microheterogeneous systems. Anal Chem 63(4):332–336CrossRefGoogle Scholar
  16. 16.
    Battino R, Rettich TR, Tominaga T (1983) The solubility of oxygen and ozone in liquids. J Phys Chem Ref Data 12(2):163–178CrossRefGoogle Scholar
  17. 17.
    Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C (1999) Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26(9–10):1231–1237CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Michela Quaranta
    • 1
  • Endry Nugroho Prasetyo
    • 2
    Email author
  • Klaus Koren
    • 1
  • Gibson S. Nyanhongo
    • 2
  • Michael Murkovic
    • 3
  • Ingo Klimant
    • 1
  • Georg M. Guebitz
    • 2
    • 4
  1. 1.Institute of Analytical Chemistry and Food ChemistryGrazAustria
  2. 2.Institute of Environmental BiotechnologyGrazAustria
  3. 3.Institute of BiochemistryGrazAustria
  4. 4.Institute for Environmental BiotechnologyUniversity of Natural Resources and Life Sciences, ViennaTullnAustria

Personalised recommendations