Tracking emerging mycotoxins in food: development of an LC-MS/MS method for free and modified Alternaria toxins

Hannes Puntscher,Benedikt Warth,Philipp Skrinjar,Hannes Mikula,Joachim Podlech,Doris Marko,Mary-Liis Kütt,Johannes Fröhlich

doi:10.1007/s00216-018-1105-8

Hannes Puntscher, Benedikt Warth + Show 6 more

Open Access

https://doi.org/10.1007/s00216-018-1105-8

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Abstract

Mycotoxins produced by Alternaria fungi are ubiquitous food contaminants, but analytical methods for generating comprehensive exposure data are rare. We describe the development of an LC-MS/MS method covering 17 toxins for investigating the natural occurrence of free and modified Alternaria toxins in tomato sauce, sunflower seed oil, and wheat flour. Target analytes included alternariol (AOH), AOH-3-glucoside, AOH-9-glucoside, AOH-3-sulfate, alternariol monomethyl ether (AME), AME-3-glucoside, AME-3-sulfate, altenuene, isoaltenuene, tenuazonic acid (TeA), tentoxin (TEN), altertoxin I and II, alterperylenol, stemphyltoxin III, altenusin, and altenuic acid III. Extensive optimization resulted in a time- and cost-effective sample preparation protocol and a chromatographic baseline separation of included isomers. Overall, adequate limits of detection (0.03–9 ng/g) and quantitation (0.6–18 ng/g), intermediate precision (9–44%), and relative recovery values (75–100%) were achieved. However, stemphyltoxin III, AOH-3-sulfate, AME-3-sulfate, altenusin, and altenuic acid III showed recoveries in wheat flour below 70%, while their performance was stable and reproducible. Our pilot study with samples from the Austrian retail market demonstrated that tomato sauces (n = 12) contained AOH, AME, TeA, and TEN in concentrations up to 20, 4, 322, and 0.6 ng/g, while sunflower seed oil (n = 7) and wheat flour samples (n = 9) were contaminated at comparatively lower levels. Interestingly and of relevance for risk assessment, AOH-9-glucoside, discovered for the first time in naturally contaminated food items, and AME-3-sulfate were found in concentrations similar to their parent toxins. In conclusion, the established multi-analyte method proved to be fit for purpose for generating comprehensive Alternaria toxin occurrence data in different food matrices.Graphical abstractᅟ

Highlights

Alternaria is an ubiquitously occurring fungal genus belonging to the division of Ascomycota
Alternaria strains are capable of producing mycotoxins, toxic secondary metabolites, which can be assigned to five substance classes (Fig. 1): dibenzo-α-pyrone derivatives, e.g., alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), isoaltenuene, altenusin (ALS); perylene quinone derivatives, e.g., altertoxin I, II, and III (ATX-I, ATX-II, ATX-III), alterperylenol (ALP), stemphyltoxin III (STTX-III); tetramic acid derivatives, e.g., tenuazonic acid (TeA), allo-tenuazonic acid, altersetin (AST); miscellaneous structures (tentoxin (TEN), altenuic acid III (AA-III); and aminopentol esters, e.g., A. alternata f. sp
ALT, isoALT, and AA-III were synthesized at the Institute of Organic Chemistry (Karlsruhe Institute of Technology, Germany) [41, 42], while AOH-3Glc, AOH-9-Glc, AOH-3-S, and AME-3-Glc were synthesized at the Institute of Applied Synthetic Chemistry (Vienna University of Technology (TU Wien), Vienna, Austria) [28]

Summary

Introduction

Alternaria is an ubiquitously occurring fungal genus belonging to the division of Ascomycota. (e.g., A. alternata, A. tenuissima, A. solani, and A. infectoria) can infest a wide variety of agricultural crops like cereals (wheat, barley, and sorghum), tomatoes, sunflower seeds, citrus fruits, apples, grapes, and olives [1,2,3,4,5]. Alternaria strains are capable of producing mycotoxins, toxic secondary metabolites, which can be assigned to five substance classes (Fig. 1): dibenzo-α-pyrone derivatives, e.g., alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), isoaltenuene (isoALT), altenusin (ALS); perylene quinone derivatives, e.g., altertoxin I, II, and III (ATX-I, ATX-II, ATX-III), alterperylenol (ALP), stemphyltoxin III (STTX-III); tetramic acid derivatives, e.g., tenuazonic acid (TeA), allo-tenuazonic acid (alloTeA), altersetin (AST); miscellaneous structures (tentoxin (TEN), altenuic acid III (AA-III); and aminopentol esters, e.g., A. alternata f.

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Conclusion