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Liquid chromatography coupled with tandem mass spectrometric detection has gained more importance for mycotoxin determination in recent years. In addition to instrumental improvements, the development of LC-MS-MS has also been a consequence of the availability of stable isotope internal standards, e.g., 13C-labelled mycotoxins. Thus, the LC-ESI-MS-MS method using a 13C15-deoxynivalenol internal standard as a state-of-the-art technique was selected as a reference procedure for an in-house method comparison study of the determination of deoxynivalenol (DON) in wheat materials. Alternative methods include LC-diode array detection, which is a frequently used routine procedure for DON-analysis, and gas chromatography after trimethylsilylation. For GC application, an electron capture detector and a negative chemical ionisation mass spectrometry detector were used, which have both been well described in the literature. The method comparison was conducted using t test statistics. Additionally, this study also calculates important method performance characteristics, including accuracy, linearity, limit of detection, limit of quantification, recovery, and variation coefficient. Furthermore, this is the first report of a GC-MS method for the determination of DON using a fully carbon-labelled 13C15-DON as an IS. The GC-MS using 13C15-DON as an IS has produced comparable results to the 13C-IS-LC-MS-MS reference method with a similar sensitivity. ECD detection was slightly less sensitive, but is also suited for DON analysis in wheat. Due to the high LOQ, the LC-DAD method seems highly applicable to the measurement of highly contaminated samples.
Mycotoxigenic fungal pathogens Fusarium and Alternaria are a leading cause of loss in cereal production. On wheat-ears, they are confronted by bacterial antagonists such as pseudomonads.
Studies on these groups’ interactions often neglect the infection process’s temporal aspects and the associated priority effects. In the present study, the focus was on how the first colonizer affects the subsequent ones. In a climate chamber experiment, wheat-ears were successively inoculated with two different strains (Alternaria enuissima At625, Fusarium graminearum Fg23, or Pseudomonas simiae Ps9).
Over three weeks, microbial abundances and mycotoxin concentrations were analyzed and visualized via Self Organizing Maps with Sammon Mapping (SOM-SM). All three strains revealed different characteristics and strategies to deal with co-inoculation: Fg23, as the first colonizer, suppressed the establishment of At625 and Ps9. Nevertheless, primary inoculation of At625 reduced all of the Fusarium toxins and stopped Ps9 from establishing. Ps9 showed priority effects in delaying and blocking the production of the fungal mycotoxins. The SOM-SM analysis visualized the competitive strengths: Fg23 ranked first, At625 second, Ps9 third. Our findings of species-specific priority effects in a natural environment and the role of the mycotoxins involved are relevant for developing biocontrol strategies.
Abstract Alternariol (AOH) and alternariol-9-monomethyl ether (AME) are two secondary metabolites of Alternaria fungi which can be found in various foodstuff like tomatoes, nuts, and grains. Due to their toxicity and potential mutagenic activity the need for the development of high-throughput methods for the supervision of AOH- and AME-levels is of increasing interest. As the availability of both native and labeled AOH and AME analytical standards is very limited we herein wish to present a novel concise approach towards their synthesis employing a ruthenium-catalyzed ortho-arylation as the key step. Finally, we demonstrate their suitability as internal standards in stable-isotope dilution assay (SIDA)-HPLC-MS/MS analysis commonly used for the quantification of the natural products in food and feed.
Phomopsin A (PHO-A), produced by the fungus Diaporthe toxica, is a mycotoxin known to be responsible for fatal liver disease of lupin-fed sheep. The füll spectrum of the toxic secondary metabolites produced by D. toxica is still unknown. PHO-A and the naturally occurring derivatives B—E have been subject to several studies to reveal their structures as well as chemical and toxicological properties. In this work, a methylated derivative of PHO-Aisolated firom lupin seeds inoculated %vith D. toxica is described. It was characterized by high-resolution mass and NMR data and shown to be the N-methylated derivative of PHO-A 1 is cytotoxic againstHepG2 cells.
Ergot alkaloids are a group of toxic compounds, formed by fungi on infested grasses. In 2022, the European Commission set into effect maximum levels for the sum of the twelve major ergot alkaloids in multiple foods. To facilitate the laborious and costly individual quantification of the twelve major ergot alkaloids by HPLC–MS/MS or -FLD, we recently reported a sum parameter method (SPM) for ergot alkaloid quantification. Here, derivatization to lysergic acid hydrazide—a derivative of the mutual ergoline backbone in all ergot alkaloids—allowed simplified determination of all ergot alkaloids in flour via HPLC-FLD. For the measurement of more complex matrices like processed foods, we now developed a MS/MS-based SPM. Two internal standards (IS), isotopically labelled at different positions of the molecule, were synthesized and employed in the MS/MS-measurements. Method performance using either the 13CD3-labelled or the 15N2-labelled IS was evaluated on naturally contaminated rye and wheat flour samples as well as on processed food matrices. Employing the 13CD3-labelled IS leads to lower variances and better consistency with the reference data (obtained by the FLD-based SPM) in flour samples compared to the 15N2-labelled IS. The novel method significantly improves the measurement of ergot alkaloids in complex food matrices, due to their increased selectivity and thus lower interferences. Furthermore, the application of isotope labelled IS obviates the need for time-consuming steps like the determination of recovery rate based, matrix specific correction factors as described in the MS/MS-based European standard method for ergot alkaloid quantification (EN 17425).
The ubiquitous Fusarium genus is responsible for the spoilage of vast amounts of cereals and fruits.
Besides the economic damage, the danger to human and animal health by the concomitant exposure to mycotoxins represents a serious problem. A large number of Fusarium species produce a variety of different mycotoxins of which the class of trichothecenes are of particular importance due to their toxicity.
Being identified as the common volatile precursor during the biosynthesis of trichothecenes, (−)-trichodiene (TD) is considered to be a biomarker for the respective mycotoxin content in food samples. We postulated that the development of a non-invasive, on-site GC-IMS method for the quantification of (−)-trichodiene supplemented with a stationary SIDA headspace GC-MS reference method would allow circumventing the laborious and expensive analyses of individual trichothecenes in large cereal samples.
In this work we present the syntheses of the required native calibration standard and an isotope labeled (TD-D3) internal standard.
Distribution of Trichothecenes, Zearalenone, and Ergosterol in a Fractionated Wheat Harvest Lot
(2008)
To investigate possible co-occurrences of type B trichothecenes and zearalenone within a Fusarium culmorum-infected wheat harvest lot, kernels were fractionated into six groups by visual criteria. The Fusarium-damaged kernels were subdivided into white, shrunken, and red kernel groups, and the remaining kernels were sorted into healthy, black spotted, and nonspecific groups. The distribution patterns of nivalenol, deoxynivalenol, zearalenone, and ergosterol were determined for possible correlations. Significant correlations between the distribution patterns were found for the mycotoxins and ergosterol for the grouped kernels (r = 0.997-0.999, p < 0.0001). Additionally, remarkably outstanding levels of nivalenol (24-fold more than the mean at 1.16 mg/kg), deoxynivalenol (27-fold more than the mean at 0.16 mg/kg), zearalenone (25-fold more than the mean at 77 µg/kg), and ergosterol (17-fold more than the mean at 13.4 mg/kg) were found in the red kernel group. Further, detailed mycotoxin and ergosterol analyses were carried out on various segments (kernel surface, conidia, bran, and flour) of the red kernels. However, the mycotoxin and ergosterol distribution profiles revealed nonsignificant correlations for these kernel segments, with the exception of deoxynivalenol and nivalenol, which were moderately correlated (r = 0.948, p = 0.035).
Mycotoxins occur widely in foodstuffs and cause a variety of mold-related health risks to humans and animals. Elucidation of the metabolic fate of mycotoxins and the growing number of newly discovered mycotoxins have enhanced the demand for fast and reliable simulation methods. The viability of electrochemistry coupled with mass spectrometry (EC/ESI-MS), Fenton-like oxidation, and UV irradiation for the simulation of oxidative phase I metabolism of the mycotoxins citrinin (CIT) and dihydroergocristine (DHEC) was investigated. The specific reaction products are compared with metabolites produced by human and rat liver microsomes in vitro. Depending on the applied potential between 0 and 2000 mV vs. Pd/H-2 by using a flow-through cell, CIT and DHEC are oxidized to various products. Besides dehydrogenation and dealkylation reactions, several hydroxylated DHEC and CIT species are produced by EC and Fenton-like reaction, separated and analyzed by LC-MS/MS and ESI-HRMS. Compared to reaction products from performed microsomal incubations, several mono- and dihydroxylated DHEC species were found to be similar to the reaction products of EC, Fenton-like reaction, and UV-induced oxidation. Consequentially, nonmicrosomal efficient and economic simulation techniques can be useful in early-stage metabolic studies, even if one-to-one simulation is not always feasible.
Ergot alkaloids (EAs) formed by Claviceps fungi are one of the most common food contaminants worldwide, affecting cereals such as rye, wheat, and barley. To accurately determine the level of contamination and to monitor EAs maximum levels set by the European Union, the six most common EAs (so-called priority EAs) and their corresponding epimers are quantified using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The quantification of EAs in complex food matrices without appropriate internal tandards is challenging but currently carried out in the standard method EN 7425:2021 due to their commercial unavailability. To address the need for isotopically labeled EAs, we focus on two semi-synthetic approaches for the synthesis of these reference standards. Therefore, we investigate the feasibility of the N6-demethylation of native ergotamine to yield norergotamine, which can subsequently be remethylated with an isotopically labeled methylating reagent, such as iodomethane (13CD3-I), to yield isotopically labeled ergotamine and its C8-epimer ergotaminine. Testing the isotopically labeled ergotamine/-inine against native ergotamine/-inine with HPLC coupled to high-resolution HR-MS/MS proved the structure of ergotamine-13CD3 and ergotaminine-13CD3. Thus, for the first time, we can describe their synthesis from unlabeled, native ergotamine. Furthermore, this approach is promising as a universal way to synthesize other isotopically labeled EAs.