Analytical and Bioanalytical Chemistry

, Volume 408, Issue 12, pp 3319–3329 | Cite as

A fully automated and fast method using direct sample injection combined with fused-core column on-line SPE–HPLC for determination of ochratoxin A and citrinin in lager beers

  • Ivona Lhotská
  • Dalibor Šatínský
  • Lucie Havlíková
  • Petr Solich
Research Paper


A new fast and sensitive method based on on-line solid-phase extraction on a fused-core precolumn coupled to liquid chromatography with fluorescence detection has been developed for ochratoxin A (OTA) and citrinin (CIT) determination in lager beer samples. Direct injection of 100 μL filtered beer samples into an on-line SPE–HPLC system enabled fast and effective sample extraction including separation in less than 6 min. Preconcentration of OTA and CIT from beer samples was performed on an Ascentis Express RP C18 guard column (5 × 4.6 mm), particle size 2.7 μm, with a mobile phase of methanol/0.5 % aqueous acetic acid pH 2.8 (30:70, v/v) at a flow rate of 2.0 mL min−1. The flow switch from extraction column to analytical column in back-flush mode was set at 2.0 min and the separation was performed on the fused-core column Ascentis Express Phenyl-Hexyl (100 × 4.6 mm), particle size 2.7 μm, with a mobile phase acetonitrile/0.5 % aqueous acetic acid pH 2.8 in a gradient elution at a flow rate of 1.0 mL min−1 and temperature of 50 °C. Fluorescence excitation/emission detection wavelengths were set at 335/497 nm. The accuracy of the method, defined as the mean recoveries of OTA and CIT from light and dark beer samples, was in the range 98.3–102.1 %. The method showed high sensitivity owing to on-line preconcentration; LOQ values were found to be 10 and 20 ng L−1 for OTA and CIT, respectively. The found values of OTA and CIT in all tested light, dark and wheat beer samples were significantly below the maximum tolerable limits (3.0 μg kg−1 for OTA and 2000 μg kg−1 for CIT) set by the European Union.

Graphical Abstract

The beer contamination - potential sources of mycotoxins


On-line SPE–HPLC Ochratoxin A Citrinin Mycotoxins Beer Fused-core column 



The authors are grateful to the Charles University Grant Agency for grant GAUK no. 1316213. I. Lhotská would like to acknowledge financial support of the project of specific research, no. SVV 260 292. The work was co-financed by the project GAČR no. 15-10781S.

Compliance with ethical standards

Conflict of interest

The authors report no conflicts of interest in this work.


  1. 1.
    Xu B, Jia X, Gu L, Sung C. Review on the qualitative and quantitative analysis of the mycotoxin citrinin. Food Control. 2006;17:271–85.CrossRefGoogle Scholar
  2. 2.
    Turner NW, Subrahmanyam S, Pilatesky SA. Analytical methods for determination of mycotoxins: a review. Anal Chim Acta. 2009;632:168–80.CrossRefGoogle Scholar
  3. 3.
    Mikulíková R, Běláková S, Benešová K, Svoboda Z. Study of ochratoxin A content in South Moravian and foreign wines by the UPLC method with fluorescence detection. Food Chem. 2012;133:55–9.CrossRefGoogle Scholar
  4. 4.
    Mateo R, Medina Á, Mateo EM, Mateo F, Jimenéz M. An overview of ochratoxin A in beer and wine. Int J Food Microbiol. 2007;119:79–83.CrossRefGoogle Scholar
  5. 5.
    Ringot D, Chango A, Schneider YJ, Larondelle Y. Toxicokinetics and toxicodynamics of ochratoxin A, an update. Chem Biol Interact. 2006;159:18–46.CrossRefGoogle Scholar
  6. 6.
    Běláková S, Benešová K, Mikulíková R, Svoboda Z. Determination of ochratoxin A in brewing materials and beer by ultra performance liquid chromatography with fluorescence detection. Food Chem. 2011;126:321–5.CrossRefGoogle Scholar
  7. 7.
    Klarić MF, Rašić D, Peraica M. Deleterious effects of mycotoxin combinations involving ochratoxin A. Toxins. 2013;5:1965–87.CrossRefGoogle Scholar
  8. 8.
    Peraica M, Domijan AM, Miletić-Medved M, Fuchs R. The involvement of mycotoxins in the development of endemic nephropathy. Wien Klin Wochenschr. 2008;120:402–7.CrossRefGoogle Scholar
  9. 9.
    Ostrý V, Malíř F, Ruprich J. Producers and important dietary sources of ochratoxin A and citrinin. Toxins. 2013;5:1574–86.CrossRefGoogle Scholar
  10. 10.
    Flajs D, Peraica M. Toxicological properties of citrinin. Arch Ind Hyg Toxicol. 2009;60:457–67.Google Scholar
  11. 11.
    EU. Commission Regulation (EC) No 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Union L. 2006;364:16.Google Scholar
  12. 12.
    EU. Commission Regulation (EU) No 212/2014 of 6 March 2014 amending Regulation (EC) No 1881/2006 as regards maximum levels of the contaminant citrinin in food supplements based on rice fermented with red yeast Monascus purpureus. Off J Eur Union L. 2014;67:4.Google Scholar
  13. 13.
    Odhav B, Naicker V. Mycotoxins in South African traditionally brewed beers. Food Addit Contam. 2002;19:55–61.CrossRefGoogle Scholar
  14. 14.
    Trivedi AB, Hirota M, Doi E, Kitabatake N. Formation of a new toxic compound, citrinin H1, from citrinin on mild heating in water. J Chem Soc Perkin Trans. 1993;1:2167–71.CrossRefGoogle Scholar
  15. 15.
    Tessini C, Mardones C, Baer D, Vega M, Herlitz E, Saelzer R, et al. Alternatives for sample pre-treatment and HPLC determination of ochratoxin A in red wine using fluorescence detection. Anal Chim Acta. 2010;660:119–26.CrossRefGoogle Scholar
  16. 16.
    Mao J, Lei S, Yang X, Xiao D. Quantification of ochratoxin A in red wines by conventional HPLC–FLD using a column packed with core–shell particles. Food Control. 2013;32:505–11.CrossRefGoogle Scholar
  17. 17.
    Cao J, Zhou S, Kong W, Yang M, Wan L, Yang S. Molecularly imprinted polymer-based solid phase clean-up for analysis of ochratoxin A in ginger and LC–MS/MS confirmation. Food Control. 2013;33:337–43.CrossRefGoogle Scholar
  18. 18.
    Duarte SC, Lino CM, Pena A. Novel IAC-LC–ESI-MS2 analytical set-up for ochratoxin A determination in pork. Food Chem. 2013;138:1055–61.CrossRefGoogle Scholar
  19. 19.
    Wang L, Wang Z, Gao W, Chen J, Yang M, Kuang Y, et al. Simultaneous determination of aflatoxin B1 and ochratoxin A in licorice roots and fritillary bulbs by solid-phase extraction coupled with high-performance liquid chromatography–tandem mass spectrometry. Food Chem. 2013;138:1048–54.CrossRefGoogle Scholar
  20. 20.
    Huang LC, Zheng N, Zheng BQ, Wen F, Cheng JB, Han RW, et al. Simultaneous determination of aflatoxin M1, ochratoxin A, zearalenone and α-zearalenol in milk by UHPLC–MS/MS. Food Chem. 2014;146:242–9.CrossRefGoogle Scholar
  21. 21.
    Arroyo-Manzanares N, Huertas-Pérez JF, Gámiz-Gracia L, García-Campaña AM. A new approach in sample treatment combined with UHPLC–MS/MS for the determination of multiclass mycotoxins in edible nuts and seeds. Talanta. 2013;115:61–7.CrossRefGoogle Scholar
  22. 22.
    Llorent-Martínez EJ, Ortega-Barrales P, Fernández-de Córdova ML, Ruiz-Medina A. Quantitation of ochratoxin a in cereals and feedstuff using sequential injection analysis with luminescence detection. Food Control. 2013;30:379–85.CrossRefGoogle Scholar
  23. 23.
    González-Peñas E, Leache C, López de Cerain A, Lizarraga E. Comparison between capillary electrophoresis and HPLC-FL for ochratoxin A quantification in wine. Food Chem. 2006;97:349–54.CrossRefGoogle Scholar
  24. 24.
    Arroyo-Manzanares N, Gámiz-Gracia L, García-Campaña AM. Determination of ochratoxin A in wines by capillary liquid chromatography with laser induced fluorescence detection using dispersive liquid–liquid microextraction. Food Chem. 2012;135:368–72.CrossRefGoogle Scholar
  25. 25.
    Dohnal V, Dvořák V, Malíř F, Ostrý V, Roubal T. A comparison of ELISA and HPLC methods for determination of ochratoxin A in human blood serum in the Czech Republic. Food Chem Toxicol. 2013;62:427–31.CrossRefGoogle Scholar
  26. 26.
    Flajs D, Domijan AM, Ivić D, Cvjetković B, Peraica M. ELISA and HPLC analysis of ochratoxin A in red wines of Croatia. Food Control. 2009;20:590–2.CrossRefGoogle Scholar
  27. 27.
    O'Mahony J, Clarke L, Whelan M, Kennedy R, Lehotay SJ, Danaher M. The use of ultra-high pressure liquid chromatography with tandem mass spectrometric detection in the analysis of agrochemical residues and mycotoxins in food - challenges and applications. J Chromatogr A. 2013;1292:83–95.CrossRefGoogle Scholar
  28. 28.
    Köppen R, Koch M, Siegel D, Merkel S, Maul R, Nehls I. Determination of mycotoxins in foods: current state of analytical methods and limitations. Appl Microbiol Biotechnol. 2010;86:1595–612.CrossRefGoogle Scholar
  29. 29.
    Songsermsakul P, Rizzazi-Fazeli E. A review of recent trends in applications of liquid chromatography-mass spectrometry for determination of mycotoxins. J Liq Chromatogr Relat Technol. 2008;31:1641–86.CrossRefGoogle Scholar
  30. 30.
    Cigić IK, Prosen H. An overview of conventional and emerging analytical methods for the determination of mycotoxins. Int J Mol Sci. 2009;10:62–115.CrossRefGoogle Scholar
  31. 31.
    Reiter E, Zentek J, Razzazi E. Review on sample preparation strategies and methods used for the analysis of aflatoxins in food and feed. Mol Nutr Food Res. 2009;53:508–24.CrossRefGoogle Scholar
  32. 32.
    Vazquez BI, Fente C, Franco C, Cepeda A, Prognon P, Mahuzier G. Simultaneous high-performance liquid chromatographic determination of ochratoxin A and citrinin in cheese by time-resolved luminescence using terbium. J Chromatogr, A. 1996;727:185–93.CrossRefGoogle Scholar
  33. 33.
    Franco CM, Fente CA, Vazquez B, Cepeda A, Lallaoui L, Prognon P, et al. Simple and sensitive high-performance liquid chromatography-fluorescence method for the determination of citrinin application to the analysis of fungal cultures and cheese extracts. J Chromatogr, A. 1996;723:69–75.CrossRefGoogle Scholar
  34. 34.
    Arévalo FJ, Granero AM, Raba HFJ, Zón MA. Citrinin determination in rice samples using micro fluidic electrochemical immunosensor. Talanta. 2011;83:966–73.CrossRefGoogle Scholar
  35. 35.
    Li Y, Wu H, Guo L, Zheng Y, Guo Y. Microsphere-based flow cytometric immunoassay for the determination of citrinin in red yeast rice. Food Chem. 2012;134:2540–5.CrossRefGoogle Scholar
  36. 36.
    Nigović B, Sertić M, Mornar A. Simultaneous determination of lovastatin and citrinin in red yeast rice supplements by micellar electrokinetic capillary chromatography. Food Chem. 2013;138:531–8.CrossRefGoogle Scholar
  37. 37.
    Rogatsky E, Braaten K, Cruikshank G, Jayatillake H, Zheng BN, Stein DT. Flow inconsistency: the evil twin of column switching-hardware aspects. J Chromatogr, A. 2009;1216:7721–7.CrossRefGoogle Scholar
  38. 38.
    Fernández-Ramos C, Šatínský D, Šmídová B, Solich P. Analysis of trace organic compounds in environmental, food and biological matrices using large-volume sample injection in column-switching liquid chromatography. Trend Anal Chem. 2014;62:69–85.CrossRefGoogle Scholar
  39. 39.
    Anli E, Alkis İM. Ochratoxin A and brewing technology: a review. J Inst Brew. 2010;116:23–32.CrossRefGoogle Scholar
  40. 40.
    Tölgyesi Á, Stroka J, Tamosiunas V, Zwickel T. Simultaneous analysis of Alternaria toxins and citrinin in tomato: an optimised method using liquid chromatography-tandem mass spectrometry. Food Add Contam Part A. 2015;32:1512–22.CrossRefGoogle Scholar
  41. 41.
    Skrbić B, Koprivica S, Godula M. Validation of a method for determination of mycotoxins subjected to the EU regulations in spices: the UHPLC-HESI-MS/MS analysis of the crude extracts. Food Control. 2013;31:461–6.CrossRefGoogle Scholar
  42. 42.
    Prelle A, Spadaro D, Garibaldi A, Gullino ML. Co-occurrence of aflatoxins and ochratoxin A in spices commercialized in Italy. Food Control. 2014;39:192–7.CrossRefGoogle Scholar
  43. 43.
    EU. Commission Regulation (EU) No. 105/2010 of 5 February 2010 amending Regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. Off J Eur Union L. 2010;35:8.Google Scholar
  44. 44.
    Škarková J, Ostrý V, Malíř F, Roubal T. Determination of ochratoxin A in food by high performance liquid chromatography. Anal Lett. 2013;46:1495–504.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Ivona Lhotská
    • 1
  • Dalibor Šatínský
    • 1
  • Lucie Havlíková
    • 1
  • Petr Solich
    • 1
  1. 1.Department of Analytical Chemistry, Faculty of PharmacyCharles UniversityHradec KrálovéCzech Republic

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