Skip to main content
SpringerLink
Log in

Mycotoxin production by pure fungal isolates analysed by means of an uhplc-ms/ms multi-mycotoxin method with possible pitfalls and solutions for patulin-producing isolates

  • Original Paper
  • Published:
Mycotoxin Research Aims and scope Submit manuscript

Abstract

This study is the first report of applying an ultra high performance liquid chromatography/tandem mass spectrometric (UHPLC-MS/MS) multi-mycotoxin method to identify and quantify the mycotoxins produced by pure fungal isolates grown on Yeast Extract Sucrose (YES) agar. The method developed concerns a triple extraction procedure based on methanol, dichloromethane and ethyl acetate. The total extract was chromatographically separated on an UHPLC BEH C18 column and analyzed with a triple quadrupole mass spectrometer. Performance characteristics (specificity, linearity, possible matrix effects, recovery, repeatability, reproducibility and limit of detection) were evaluated by spiking experiments with blank agar plugs and the analytes. Verrucarol was used as internal standard. Recovery percentages varied between 56 and 125%, whereas the limit of detection ranged from 1 to 1,500 ng g−1 with the exception of NIV, PAT and ZEA. The method was successfully applied for examining the in vitro mycotoxin production by Aspergillus fumigatus, A. flavus and A. niger. The mobile phases used for chromatographic separation were slightly modified when studying patulin-producing molds due to signal interference between this mycotoxin and an unknown metabolite. This modified method was successfully applied for Penicillium roqueforti, P. paneum and P. carneum grown on YES agar medium. Application of the multi-mycotoxin UHPLC-MS/MS method developed may be of great importance for studying the mycotoxin capacity of fungal isolates under varying growth conditions, in order to obtain a better insight into the conditions which induce or suppress mycotoxin production by pure fungal isolates or from a chemotaxonomic point of view.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Beltran E, Ibanez M, Sancho JV, Hernandez F (2009) Determination of mycotoxins in different food commodities by ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry. Rapid Commun Mass Spectrom 23:1801–1809

    Article  CAS  PubMed  Google Scholar 

  • Bennet JW, Klich M (2003) Mycotoxins. Clin Microbiol Rev 16:497–516

    Article  Google Scholar 

  • Berger U, Oehme M, Kuhn F (1999) Quantitative determination and structure elucidation of type A- and B-trichothecenes by HPLC/ion trap multiple mass spectrometry. J Agric Food Chem 47:4240–4245

    Article  CAS  PubMed  Google Scholar 

  • Berthiller F, Schuhmacher R, Buttinger G, Krska R (2005) Rapid simultaneous determination of major type A- and B-trichothecenes as well as zearalenone in maize by high performance liquid chromatography-tandem mass spectrometry. J Chromatogr A 1062:209–216

    Article  CAS  PubMed  Google Scholar 

  • Betina V (1984) Biological effects of mycotoxins. In: Betina V (ed) Mycotoxins-production, isolation, separation and purification. Elsevier, Amsterdam, pp 25–36

    Google Scholar 

  • Bird BA, Campbell IM (1982) Disposition of mycophenolic acid, brevianamide A, asperphenamate and ergosterol in solid cultures of Penicillium brevicompactum. Appl Environ Microbiol 43:345–348

    CAS  PubMed  Google Scholar 

  • Biselli S, Hummert C (2005) Development of a multicomponent method for Fusarium toxins using LC-MS/MS and its application during a survey for the content of T-2 toxin and deoxynivalenol in various feed and food samples. Food Addit Contam 22:752–760

    Article  CAS  PubMed  Google Scholar 

  • Biselli S, Gartig L, Wegner H, Hummert C (2004) Analysis of Fusarium toxins using LC/MS-MS: application to various food and feed matrices. LC-GC Europe 17:25–30

    CAS  Google Scholar 

  • Boysen M, Skouboe P, Frisvad J, Rossen L (1996) Reclassification of the Penicillium roqueforti group into three species on the basis of molecular genetic and biochemical profiles. Microbiology 142:541–549

    Article  CAS  PubMed  Google Scholar 

  • Byrem TM, Pestka JJ, Chu FS, Strasburg GM (1999) Analysis and pharmacokinetics of cyclopiazonic acid in market weight pigs. J Anim Sci 77:173–179

    CAS  PubMed  Google Scholar 

  • Cavaliere C, Foglia P, Pastorini E, Samperi R, Lagana A (2005) Development of a multiresidue method for analysis of major Fusarium mycotoxins in corn meal using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 19:2085–2093

    Article  CAS  PubMed  Google Scholar 

  • Delmulle B, De Saeger S, Adams A, De Kimpe N, Van Peteghem C (2006) Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures. Rapid Commun Mass Spectrom 20:771–776

    Article  CAS  PubMed  Google Scholar 

  • Di Mavungu JD, Monbaliu S, Scippo ML et al (2009) LC-MS/MS multi-analyte method for mycotoxin determination in food supplements. Food Addit Contam A Chem Anal Control Expo Risk Assess 26:885–895

    Google Scholar 

  • D'Mello JPE, MacDonald AMC (1997) Mycotoxins. Anim Feed Sci Technol 69:155

    Article  Google Scholar 

  • Filtenborg O, Frisvad JC, Svendsen JA (1983) Simple screening method for molds producing intracellular mycotoxins in pure cultures. Appl Environ Microbiol 45:581–585

    CAS  PubMed  Google Scholar 

  • Fink-Gremmels J (1999) Mycotoxins: their implications for human and animal health. Vet Ext Q 21:115–120

    CAS  Google Scholar 

  • Fink-Gremmels J (2008) Mycotoxins in cattle feeds and carry-over to dairy milk: a review. Food Addit Contam 25:172–180

    Article  CAS  Google Scholar 

  • Frisvad JC, Filtenborg O (1983) Classification of Terverticillate Penicillia based on profiles of myco-toxins and other secondary metabolites. Appl Environ Microbiol 46:1301–1310

    CAS  PubMed  Google Scholar 

  • Gilbert J, Anklam E (2002) Validation of analytical methods for determining mycotoxins in foodstuffs. Trends Anal Chem 21:468–486

    Article  CAS  Google Scholar 

  • Hussein HS, Brasel JM (2001) Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology 167:101–134

    Article  CAS  PubMed  Google Scholar 

  • Jiang J, Yan L, Ma Z (2009) Molecular characterization of an atoxigenic Aspergillus flavus strain AF051. Appl Microbiol Biotechnol 83:501–505

    Article  CAS  PubMed  Google Scholar 

  • Klotzel M, Lauber U, Humpf HU (2006) A new solid phase extraction clean-up method for the determination of 12 type A and B trichothecenes in cereals and cereal-based food by LC-MS/MS. Mol Nutr Food Res 50:261–269

    Article  PubMed  Google Scholar 

  • Kokkonen M, Jestoi M, Rizzo A (2005) Determination of selected mycotoxins in mould cheeses with liquid chromatography coupled to tandem with mass spectrometry. Food Addit Contam 22:449–456

    Article  CAS  PubMed  Google Scholar 

  • Kupfahl C, Michalka A, Lass-Floerl C et al (2008) Gliotoxin production by clinical and environmental Aspergillus fumigatus strains. Int J Med Microbiol 298:319–327

    Article  CAS  PubMed  Google Scholar 

  • Monbaliu S, Van Poucke C, Van Peteghem C, Van Poucke K, Heungens K, De Saeger S (2009) Development of a multi-mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis. Rapid Commun Mass Spectrom 23:3–11

    Article  CAS  PubMed  Google Scholar 

  • Monbaliu S, Van Poucke C, Detavernier C et al (2010) Occurrence of mycotoxins in feed as analyzed by a multi-mycotoxin LC-MS/MS method. J Agric Food Chem 58:66–71

    Article  CAS  PubMed  Google Scholar 

  • Norred WP, Porter JK, Dorner JW, Cole RJ (1998) Occurrence of the mycotoxin cyclopiazonic acid in meat after oral administration to chickens. J Agric Food Chem 36:113–116

    Article  Google Scholar 

  • Peraica M, Radic B, Lucic A, Pavlovic M (1999) Toxic effects of mycotoxins in humans. Bull World Health Organ 77:754–766

    CAS  PubMed  Google Scholar 

  • Plattner RD, Maragos CM (2003) Determination of deoxynivalenol and nivalenol in corn and wheat by liquid chromatography with electrospray mass spectrometry. J AOAC Int 86:61–65

    CAS  PubMed  Google Scholar 

  • Rasmussen RR, Storm IMLD, Rasmussen PH, Smedsgaard J, Nielsen KF (2010) Multi-mycotoxin analysis of maize silage by LC-MS/MS. Anal Bioanal Chem 397:765–776

    Article  CAS  PubMed  Google Scholar 

  • Razzazi-Fazeli E, Bohm J, Luf W (1999) Determination of nivalenol and deoxynivalenol in wheat using liquid chromatography-mass spectrometry with negative ion atmospheric pressure chemical ionisation. J Chromatogr A 854:45–55

    Article  CAS  PubMed  Google Scholar 

  • Razzazi-Fazeli E, Rabus B, Cecon B, Bohm J (2002) Simultaneous quantification of A-trichothecene mycotoxins in grains using liquid chromatography atmospheric pressure chemical ionisation mass spectrometry. J Chromatogr A 968:129–142

    Article  CAS  PubMed  Google Scholar 

  • Ren YP, Zhang Y, Shao SL, Cai ZX, Feng L, Pan HF, Wang ZG (2007) Simultaneous determination of multi-component mycotoxin contaminants in foods and feeds by ultra-performance liquid chromatography tandem mass spectrometry. J Chromatogr A 1143:48–64

    Article  CAS  PubMed  Google Scholar 

  • Richard E, Heutte N, Sage L, Pottier D, Bouchart V, Lebailly P, Garon D (2007) Toxigenic fungi and mycotoxins in mature corn silage. Food Chem Toxicol 45:2420–2425

    Article  CAS  PubMed  Google Scholar 

  • Rosinska D, Vierikova M, Lehotay J (2009) Determination of patulin in apple products using HPLC with photodiode array detector and ultra performance liquid chromatography with electrospray-tandem mass spectrometry. J Liq Chromatogr Relat Technol 32:500–511

    Article  CAS  Google Scholar 

  • Royer D, Humpf HU, Guy PA (2004) Quantitative analysis of Fusarium mycotoxins in maize using accelerated solvent extraction before liquid chromatography atmospheric pressure chemical ionization tandem mass spectrometry. Food Addit Contam 21:678–692

    Article  CAS  PubMed  Google Scholar 

  • Samson RA, Hoekstra ES, Frisvad JC (2004) Mycological media. In: Samson RA, Hoekstra ES, Frisvad JC (eds) Introduction of food- and airborne fungi. Ponsen & Looyen, Wageningen, pp 378–381

    Google Scholar 

  • Smedsgaard J (1997) Micro-scale extraction procedure for standardized screening of fungal metabolite production in cultures. J Chromatogr A 760:264–270

    Article  CAS  PubMed  Google Scholar 

  • Sorensen LK, Elbaek TH (2005) Determination of mycotoxins in bovine milk by liquid chromatography tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 820:183–196

    Article  CAS  PubMed  Google Scholar 

  • Spanjer MC, Rensen PM, Scholten JM (2008) LC-MS/MS multi-method for mycotoxins after single extraction, with validation data for peanut, pistachio, wheat, maize, cornflakes, raisins and figs. Food Addit Contam A Chem Anal Control Expo Risk Assess 25:472–489

    CAS  Google Scholar 

  • Sulyok M, Berthiller F, Krska R, Schuhmacher R (2006) Development and validation of a liquid chromatography/tandem mass spectrometric method for the determination of 39 mycotoxins in wheat and maize. Rapid Commun Mass Spectrom 20:2649–2659

    Article  CAS  PubMed  Google Scholar 

  • Sulyok M, Krska R, Schuhmacher R (2007) A liquid chromatography/tandem mass spectrometric multi-mycotoxin method for the quantification of 87 analytes and its application to semi-quantitative screening of moldy food samples. Anal Bioanal Chem 389:1505–1523

    Article  CAS  PubMed  Google Scholar 

  • Tuomi T, Saarinen L, Reijula K (1998) Detection of polar and macrocyclic trichothecene mycotoxins from indoor environments. Analyst 123:1835–1841

    Article  CAS  PubMed  Google Scholar 

  • Vasanthi S, Bhat RV (1998) Mycotoxins in foods - occurrence, health & economic significance & food control measures. Indian J Med Res 108:212–224

    CAS  PubMed  Google Scholar 

  • Veldman A, Meijs JAC, Borggreve GJ, Heeresvandertol JJ (1992) Carry-over of aflatoxin from cows food to milk. Anim Prod 55:163–168

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful to Mieke Van Tomme, Petra De Neve, Patricia Van Herreweghe, Luc Batjoens, and Els Verween for their excellent technical assistance. The research was funded by a PhD grant (IWT-SB/63435) of the Agence for Innovation by Science and Technology in Flanders (IWT).

Author information

Authors and Affiliations

  1. Technology and Food Science Unit, Flemish Government Institute for Agricultural and Fisheries Research (ILVO), Brusselsesteenweg 370, 9090, Melle, Belgium

    Els Van Pamel, Geertrui Vlaemynck, Marc Heyndrickx, Lieve Herman & Els Daeseleire

  2. Department of Biology, Research Group Mycology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium

    Annemieke Verbeken

Authors
  1. Els Van Pamel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Geertrui Vlaemynck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marc Heyndrickx
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Lieve Herman
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Annemieke Verbeken
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Els Daeseleire
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Els Van Pamel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Van Pamel, E., Vlaemynck, G., Heyndrickx, M. et al. Mycotoxin production by pure fungal isolates analysed by means of an uhplc-ms/ms multi-mycotoxin method with possible pitfalls and solutions for patulin-producing isolates. Mycotox Res 27, 37–47 (2011). https://doi.org/10.1007/s12550-010-0073-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12550-010-0073-4

Keywords

Search

Navigation