In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize
A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple “salting-out liquid-liquid extraction” (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n = 13) and maize (n = 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.
KeywordsMycotoxins Co-occurrence Cereals Ultra-high performance liquid chromatography Mass spectrometry QuEChERS SO-LLE
The authors thank the Spanish Ministry of Economy and Competitiveness (Project Ref: AGL2015-70708-R). Natalia Arroyo-Manzanares received a post-doctoral grant from the University of Granada.
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
- 1.RASFF annual report. 2015. https://ec.europa.eu/food/sites/food/files/safety/docs/rasff_annual_report_2015.pdf. Accessed 10 Nov 2016.
- 2.Miller JD. Significance of grain mycotoxins for health and nutrition. In: Champ BR, Highley E, Hocking AD, Pitt JI, editors. Fungi and mycotoxins in stored products. Canberra: ACIAR Proceedings; 1991. p. 126–35.Google Scholar
- 8.Regulation (EC) No. 1881/2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Communities 2006;L364:5–24.Google Scholar
- 13.Commission Recommendation of 15 March 2012 on the monitoring of the presence of ergot alkaloids in feed and food (2012/154/EU). Off J Eur Communities 2012;L77:20–21.Google Scholar
- 15.Malachová A, Sulyok M, Beltrán E, Berthiller F, Krska R. Optimization and validation of a quantitative liquid chromatography–tandem mass spectrometric method covering 295 bacterial and fungal metabolites including all regulated mycotoxins in four model food matrices. J Chromatogr A. 2014;1362:145–56.CrossRefPubMedGoogle Scholar
- 17.Diana Di Mavungu J, Malysheva SV, Sanders M, Larionova D, Robbens J, Dubruel P, et al. Development and validation of a new LC–MS/MS method for the simultaneous determination of six major EAs and their corresponding epimers. Application to some food and feed commodities. Food Chem. 2012;135:292–303.CrossRefGoogle Scholar
- 18.Commission Decision of 12 August 2002 implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results (2002/657/EC). Off J Eur Communities 2002;L221:8–36.Google Scholar
- 20.De Boevre M, Diana Di Mavungu J, Maene P, Audenaert K, Deforce D, Haesaert G, et al. Development and validation of an LC-MS/MS method for the simultaneous determination of deoxynivalenol, zearalenone, T-2-toxin and some masked metabolites in different cereals and cereal-derived food. Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2012;29:819–35.CrossRefPubMedGoogle Scholar
- 21.Wenzl T, Haedrich J, Schaechtele A, Robouch P, Stroka J, Guidance document on the estimation of LOD and LOQ for measurements in the field of contaminants in feed and food; EUR 28099, Publications Office of the European Union, Luxembourg, ISBN 978-92-79-61768-3; 2016 https://doi.org/10.2787/8931.
- 28.Commission Recommendation of 27 March 2013 on the presence of T-2 and HT-2 toxin in cereals and cereal products (2013/165/EU). Off J Eur Communities 2013;L91:12–15.Google Scholar
- 30.Commission Regulation (EC) No. 401/2006 of laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Off J Eur Communities 2006;L70:12–34.Google Scholar