Emerging Mycotoxins– A Threat beyond Regulations? by romer-labs

Issue 5

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A magazine of Romer Labs®

Emerging Mycotoxins– A Threat beyond Regulations? How to Develop an LC-MS/MS-based Multi-Mycotoxin Method

Content

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Emerging Mycotoxins – A Threat beyond Regulations? The number of detectable mycotoxins has increased in recent years. Spot On investigates what is being done to measure and monitor them. By Lilian KUSTER, PhD, Product Manager at Romer Labs®

Ergot Alkaloids – an ancient story Currently there are no regulations for maximum ergot alkaloid levels but these are heavily under discussion and suitable methods are needed. By Lilian KUSTER, PhD, Product Manager at Romer Labs®

Alternaria toxins Alternaria toxins are amongst the group of emerging mycotoxins for which assessment and regulation are currently under discussion. By Lilian KUSTER, PhD, Product Manager at Romer Labs® Spot On is a quarterly publication of Romer Labs Division Holding GmbH, distributed free-of-charge. ISSN: 2414-2042

Editors: Cristian Ilea, Simone Schreiter Contributors: Georg Häubl, Lilian Kuster, Irene Hahn Graphic: Reinhold Gallbrunner Research: Kurt Brunner

Publisher: Romer Labs Division Holding GmbH Erber Campus 1 3131 Getzersdorf, Austria Tel: +43 2782 803 0 www.romerlabs.com

©Copyright 2023, Romer Labs® All rights reserved. No part of this publication may be reproduced in any material form for commercial purposes without the written permission of the copyright holder except in accordance with the provisions of the Copyright, Designs and Patents Act 1998. All photos herein are the property of Romer Labs or used with license.

How to Develop an LC-MS/MS-based Multi-Mycotoxin Method

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Romer Labs is developing a single method that can detect multiple mycotoxins simultaneously. And here is how we do it. By Irene HAHN, PhD, Quality Control Manager at Romer Labs®

Spot On Issue 5

Editorial The need to be on the front-line Experts in the field of food and feed safety have known for some time that the number of toxic metabolites derived from fungi can be tremendously high. In recent years, this list of harmful substances has been further extended with the addition of modified mycotoxins. Although modified mycotoxins have been discussed in the literature since the 1980s, they have only been brought into focus in food analysis since 2000 and are now also considered by regulatory bodies. But what are modified mycotoxins? We introduce them in the first article, alongside more detail on other emerging mycotoxins such as ergot alkaloids and Alternaria toxins. You will find a comprehensive discussion of these compounds before focus turns to the modern analytical methods which allow the determination and quantification of these otherwise neglected or overlooked contaminants. The application of mass spectrometers as detectors for liquid chromatography facilitated and allowed to gain more information on the occurrence of numerous analytes in a sample. The excellent sensitivity of modern mass spectrometer methods enables the detection of hundreds of toxins in a single analysis run. This cutting-edge technology has been used by Romer Labs for the development of a new multi-mycotoxin detection method. The second article describes how we harnessed the technology to develop such a method. Highly reliable analytical standard substances are a key requirement in achieving precise results. Romer Labs established the application of so called ‘stable isotope labeled’ standards which can further enhance the accuracy of a measurement. The production of analytical standards for modified mycotoxins can be particularly tricky as many of them cannot be obtained by the simple fermentation process of a mycotoxin-producing fungus. They can be obtained in other ways such as chemical synthesis or modifications usually carried out by nature. These can be performed in a test tube using the application of purified enzymes. Our work here is ongoing so watch out for more news in a future issue. We hope that you enjoy reading this issue of Spot On.

Georg Häubl R&D Scientist, Romer Labs®

A magazine of Romer Labs®

Emerging Mycotoxins – A Threat beyond Regulations? By Lilian KUSTER, Product Manager at Romer Labs®

Photo: Shutterstock_Nomad_Soul

The number of detectable mycotoxins has increased in recent years. Spot On investigates what is being done to measure and monitor them.

Spot On Issue 5

Modifying mycotoxins as plant defense Typically, mycotoxins are explicitly produced by fungi and their parent structure is often modified by the fungus itself which releases a cocktail of structurally related compounds. During infection, these substances are then often further modified by the host plant of the fungus. The living plant might change the chemical structure of toxins and produce so-called masked mycotoxins. The formation of these masked toxins is a major detoxification strategy of crops, as they are less toxic for the plant. Usually, a glucose molecule or a sulfate is involved in the conjugation and detoxification. Although these masked toxins do not further harm the plant, their toxicity to humans and animals might reemerge when the added masking molecule is cleaved in the gastrointestinal tract of mammals during digestion (Figure 1). In plant breeding, the increasing occurrence and production of some masked mycotoxins might be linked to novel resistant breeds. Deoxynivalenol-3-glucoside, for example, has been reportedly linked to resistance against Fusarium head blight. This means that Fusarium resistant plants have been proven to show higher deoxynivalenol-3-glucoside to deoxynivalenol ratios, but these are accompanied by lower levels of total deoxynivalenol and the modified form due to higher Fusarium resistance. A magazine of Romer Labs®

Figure 1. Cleavage reaction of deoxynivalenol-3-glucoside to native deoxynivalenol during digestion

Digestion

Box 1

Ergot Alkaloids – an ancient story Ergot alkaloids are secondary metabolites usually produced by fungi belonging to the genus Claviceps. The most commonly occurring species producing ergot alkaloids is Claviceps purpurea. “Ergot” is a french word meaning “spur”, and was chosen as the name since grains, when infected, present so-called sclerotia and often resemble the spurs on the legs of a rooster. Many different cereal plants and grasses, including rye, wheat and triticale among others, can become infected by these fungi during cool, wet weather conditions. These fungi then produce structures called sclerotia. These sclerotia contain different classes of ergot alkaloids, the most prominent being ergometrine, ergotamine, ergosine, ergocristine, ergocryptine and ergocornine together with their epimeric –inine forms. If grains containing sclerotia are processed by grinding into flour, high contamination levels of ergot alkaloids typically follow. Currently, available data on ergot alkaloids show that the intake of contaminated food or feed can severely affect animals and humans. Ergot poisioning is called ergotism, a severe pathological syndrome. Symptoms include hallucinations, itchy and burning skin, nausea, dizziness and even abortion. Ergotism is one of the oldest known diseases caused by mycotoxins and was first described in the Middle Ages as so-called St. Anthony’s fire. Furthermore, ergot alkaloids are not only known as mycotoxins. Ergotamine, for example, is one of the components of the psychoactive drug lysergic acid diethylamide (LSD). Ergot alkaloids are also used for medicinal purposes, including the treatment of migraines and the induction of birth process among others. Up to now, the amount of ergot alkaloids present in food and feed is not regulated, but regulations are under strong discussion in the European Union. Currently, the most widely used detection method for ergot alkaloids is based on HPLCFLD (High Performance Liquid Chromatography with fluorescence detection) and can be performed using calibrants and a one-step cleanup provided by Romer Labs. An official CEN LC-MS/MS (liquid chromatography – mass spectrometry) method is currently under development. Photo: shutterstock_Carmen Rieb

ycotoxins are naturally occurring, secondary metabolites produced by various molds. These compounds are toxic to humans and animals. Toxigenic molds contaminate a wide range of crops and produce mycotoxins as a result of the infection of plant tissues in the field. Unfortunately, the formation of these toxins can continue even after harvest and the level of mycotoxins in grains continues to increase during storage. Contaminated crops represent a major health risk to humans and animals. The most prominent mycotoxin-producing field-fungi are represented by fungi of the species Fusarium and Aspergillus. Beyond these, there are over 300 different fungi that are known to produce over 400 different mycotoxins. In recent years, more and more mycotoxins have been considered as relevant as they contribute to the risk posed to humans and animals. Risk assessment studies have been performed for various important mycotoxin groups including ergot alkaloids (see Box 1), Alternaria toxins (see Box 2) and modified or masked mycotoxins.

The term “modified mycotoxin” includes both the modification of a parent toxin molecule by the fungus itself, and the masking of the toxin which only occurs in the plant tissue. Another type of modification takes place in mammals when aflatoxin B1 is consumed through contaminated feed and converted to aflatoxin M1. This

aflatoxin M1 migrates into the milk of lactating animals and is excreted with it. In addition, modifications of toxins can also occur during food processing, in particular heating and fermentation, increasing their prevalence. These modified mycotoxins might occur in relevant amounts in food and feed. The phenomenon of modifying mycotoxins is particularly related to Fusarium toxins (trichothecenes, zearalenone and fumonisins) but modified forms have also been reported for other mycotoxins like aflatoxins, ochratoxin A or patulin.

Photo: Shutterstock_ Jean Faucett

Altered and masked forms of deoxynivalenol – an example

Box 2

Alternaria toxins Alternaria toxins represent a possible health-endangering group of mycotoxins produced mainly by the Alternaria species. These are a widespread group of fungi contaminating mainly fruits and vegetables, but also other crop plants, during growth as well as storage. The most important mycotoxin-producing species is Alternaria alternata which occurs mainly on cereals and seeds but also on olives, various fruits and tomatoes. A vast number of Alternaria mycotoxins are known to occur naturally on infected crops, fruits and vegetables, including tenuazonic acid, alternariol, alternariol monomethyl ether, altenuene and altertoxin I. Structurally, these toxins are related to fumonisins. Even though Alternaria toxins are normally associated with fruits and vegetables that are visibly infected by Alternaria rot, they have also been found in cereals, such as wheat, rye, sorghum, rice and even tobacco. Alternaria toxins have been shown to exhibit both acute and chronic effects and therefore represent a threat to animal and human health. The most studied mycotoxin in the group of toxins produced by the species Alternaria is tenuazonic acid. Its main function is the inhibition of protein synthesis and results in antitumor, antiviral and antibacterial activity. Most of the other Alternaria toxins show cytotoxic activity in mammals, some of them are mutagenic like the altertoxins, while others are toxic to the unborn like alternariol and alternariol monomethyl ether. Currently no guidelines or legislative limits are set for Alternaria toxins. So far it has been of general belief that their occurrence in food is very low and therefore the risk of human exposure is very limited. Nevertheless, data for their risk assessment is currently collected and methods for the detection of Alternaria toxins based on liquid chromatography – mass spectrometry (LC-MS) are under development.

Deoxynivalenol is the mycotoxin with the most studies conducted on the different versions of frequently observed modifications. The modified forms of deoxynivalenol can be divided into two main groups: altered and masked forms. There are two main altered forms of deoxynivalenol secreted by the fungus itself: 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol, as found in Fusarium-contaminated cereals. Plants are able to mask the deoxynivalenol to deoxynivalenol-3-glucoside and as recent studies show, this may take on two sulfonated forms: deoxynivalenol-3-sulfate and deoxynivalenol-15-sulfate (Table 1).

How harmful are modified and emerging mycotoxins? Modified mycotoxins can be either more or less toxic than their parent compounds. For example, they may be more bioavailable due to modifications. Toxicological data on modified mycotoxins are scarce, and current results and knowledge on the real risks and effects of these compounds are insufficient. This lack of knowledge makes it difficult to conduct a proper risk assessment. Nevertheless, there have been studies describing their potential threat to food safety. Furthermore, it has to be highlighted that masked mycotoxins can be “unmasked” again in the digestive tract of animals and humans, releasing the parent compound with its toxicological effects again. A similar situation exists with emerging mycotoxins: toxicological data are scarce which makes it difficult to set up regulations and maximum tolerated limits to protect humans and animals from potential health risks.

Do regulations cover all mycotoxin risks? To ensure food and feed safety, many countries have established regulatory limits for mycotoxins in crops. Currently, in most developed countries, there are regulations on maximum levels or at least guidance levels Spot On Issue 5

Table 1. Modified forms of the trichothecene deoxynivalenol Altered deoxynivalenol

Masked deoxynivalenol

3-acetyl-deoxynivalenol

deoxynivalenol-3-glucoside

15-acetyl-deoxynivalenol

for mycotoxins in food and feed. These regulations only cover some of the known mycotoxins such as aflatoxins B1, B2, G1, G2 and M1; fumonisins B1, B2 and B3; ochratoxin A, deoxynivalenol, zearalenone, HT-2 toxin and T-2 toxin. As modified mycotoxins behave differently in their chemical reactions to parent mycotoxins, they can be easily missed in routine analysis. Current detection methods for regulated mycotoxins in food and feed do not include routine screening for these modified mycotoxins as they are not covered by legislation. Such standard methods may show up contamination levels below legislative limits, while contaminations from modified mycotoxins go undetected. This represents a correct result, but from a toxicological point of view the integration of modified toxins (e.g. as a sum parameter) would provide more sound data for risk assessment. Together, all these facts point to the possible hazards posed by modified mycotoxins to human health. Regulations on the maximum levels of modified mycotoxins as well as other emerging mycotoxins are currently under discussion in the European Union.

Analytical methods for mycotoxin quantification Mycotoxins are commonly analyzed by chromatographic methods like liquid chromatography–mass spectrometry (LC-MS) and immunochemical methods like enzyme-linked immunosorbent assay (ELISA). Immunochemical methods can, depending on the cross-reactivity of the antibody, respond to more than one compound (e.g. native mycotoxins and their modified forms) leading to a single result. In contrast LCbased separation methods might underestimate the total toxin levels as those methods resolve each compound as a single parameter and are usually only developed for the parent mycotoxins.

Limits of analytical methods There are two ways to detect and quantify modified mycotoxins: A “direct” approach that measures the whole modified compound, and an “indirect” approach that measures the parent compound after chemical A magazine of Romer Labs®

deoxynivalenol-3-sulfate

deoxynivalenol-15-sulfate

or enzymatic treatments that lead to the cleavage of modified mycotoxins, mainly by hydrolysis. Among the advantages of the indirect method are that reference materials for modified mycotoxins are not needed for correct quantification, and that all modified forms are included in the final result. The main disadvantages are that the efficiency of the hydrolysis process cannot be verified easily, and that there is no access to the quantities of the different forms of a toxin. Therefore, it is important to develop direct methods to obtain further insight into the occurrence of modified mycotoxins. All chromatographic technologies for parent mycotoxins are also potentially suitable for their modified forms as long as they are soluble and directly available for analysis. One major constraint of the direct determination and quantification of modified mycotoxins is the limited availability of reference materials (pure substances or calibrants in addition to isotope-labeled internal standards). Another drawback is that most methods require an adequate cleanup prior to the analysis procedure. Commercially available purification devices are currently designed for native mycotoxins and might not be necessarily suitable for modified forms. Work is currently underway to develop new reference standards as well as innovative cleanup devices to determine modified mycotoxins directly.

In recent years emerging mycotoxins have become more and more important in regards to their possible threat to humans and animals.

Emerging mycotoxins A threat beyond regulations? With the current gaps in routine mycotoxin analysis driven by missing regulations for emerging mycotoxins, many of these compounds can go undetected and pose a threat to both human and animal health. The extent of this threat, thought to be considerable, is however hard to estimate since toxicological data is still scarce, despite increasing research efforts in this direction.

References are available on request from the author.

Spot On Issue 5

How to Develop an LC-MS/MS-based Multi-Mycotoxin Method By Irene HAHN, Quality Control Manager at Romer Labs®

Frequently, cereal crops and raw materials are contaminated with more than one mycotoxin. Romer Labs is developing a single method that can detect multiple mycotoxins simultaneously. Irene Hahn describes how.

here are approximately 400 compounds of low molecular weight that are recognized as mycotoxins, each with different toxic effects for humans and animals. The high number of possible contaminants, as well as repeated reports of mycotoxin co-occurrence makes it necessary to develop suitable detection methods, like liquid chromatography – mass spectrometry (LC-MS/MS)-based multi-mycotoxin methods, to simultaneously analyze several mycotoxins. Developing such methods is challenging because A magazine of Romer Labs®

including multiple toxins with different chemical properties in one analysis means that compromises must be made when choosing optimal method parameters.

Analysis of mycotoxins based on LC-MS/MS Analytical methods based on reversed-phase LC coupled to MS (LC-MS/MS) have become a powerful and state-of-the-art technique in the qualitative and quantitative analysis of mycotoxins over the last decade.

There is a strong trend towards the application of multimycotoxin methods achieved by LC-MS/MS.

Advantages of this method are the high sensitivity and selectivity, the application to multi-mycotoxin analysis as well as the delivery of additional information about mass-to-charge ratios (m/z) and fragment ions of the investigated analytes. Currently, there is a strong trend towards the application of multi-mycotoxin methods achieved by LC-MS/MS. The simultaneous determination of a wide range of mycotoxins belonging to different chemical families within one single measurement can be achieved using this technique. However, issues like the chemical diversity of the compounds themselves, the wide range of agricultural commodities being tested, varying concentration ranges and different occurrence distributions all challenge method development and optimization. Therefore, compromises must be made in the choice of extraction solvent and mobile phase, and conditions may be far from optimal for certain analytes, which include acidic (fumonisins), basic (ergot alkaloids), as well as polar (moniliformin, nivalenol) and apolar compounds (zearalenone, beauvericin). In addition, the lack of suitable commercially available analytical standards for certain analytes results in only qualitative screening statements rather than quantitative results.

General challenges for the determination of mycotoxins Usually, mycotoxin contaminations are heterogeneously distributed in agricultural crops and may be concentrated in ‘hot-spots’. Therefore, sampling represents an important and crucial step as a representative sample is essential for the precise and accurate determination of mycotoxin levels. With the majority of analytical techniques, a direct detection of mycotoxins in milled cereal samples cannot be achieved and therefore sample preparation procedures are required. Another important step is the sample extraction. During a conventional solid-liquid extraction, mycotoxins are extracted from ground cereal samples by mechanical shaking with different mixtures of solvents (aqueous and organic), sometimes also with acidic or alkaline modifiers. The resulting extracts can then be further used for analysis. Most of the developed analytical methods for regulatory and scientific purposes are based on chromatographic separation, mainly liquid chromatography (LC), in combination with a variety of detectors. LC detectors for the continuous monitoring and detection of analytes eluting from the chromatographic column are based on measurements of UV/Vis-absorbance, fluorescence and mass spectrometry (MS). Due to the different chemical and physicochemical properties of mycotoxins, the majority of such analytical methods have been optimized for one target compound, or at best, a group of closely related mycotoxins. In addition, these targeted methods often include extraction and cleanup steps to reduce or eliminate unwanted matrix components. Hence, the generated occurrence estimation always depends on the analyzed samples as well as the mycotoxins covered with the used analytical methods.

Figure 1. Phases and considered parameters during the set-up of an LC-MS/MS multi-mycotoxin method

Method development Method optimization Method validation

Method implementation

• LC and MS parameters • Sample preparation procedure • Stability testing • Selectivity • Working range

• LODs, LOQs • Linearity, accuracy • Precision, robustness • Matrix effects, recoveries • Real samples • Quality control samples • LIMS implementation

Development of a multi-mycotoxin LC-MS/MS method In general, methods for the quantification of mycotoxins in cereals and cereal-based products are comprised of a representative sampling, the optimized sample preparation procedure and a clean-up step as well as the analytical technique including separation and detection. The set-up of a multi-mycotoxin method based on LC-MS/MS follows usually a four-phase process. These and the considered parameters are summarized in Figure 1. During method development and optimization, the parameters significantly influence the quality and reliability of the results and should be evaluated carefully. For this purpose, analytical standards of each compound with stated purity must be used. However, for certain analytes, analytical standards are not commercially available. In those cases, it might be possible to access standards that are still under research, or work with available material that is less well characterized.

Method development During the development of an LC-MS/MS based method, the MS and LC parameters, as well as the sample preparation procedure must be worked out. For the optimization of the MS parameters, each compound should be injected as a pure analytical standard directly into the mass spectrometer. Subsequently, the ideal ionization mode (positive or negative), the most abundant precursor and product ions as well as the ideal declustering potentials, collision energies and collision cell exit potentials must be evaluated. During the optimization of the LC parameters, the ideal mobile phases and gradient as well as the optimal chromatographic Spot On Issue 5

column have to be evaluated. The method of choice for sample preparation procedure when analyzing multiple mycotoxins is a dilute-and-shoot approach without any sample cleanup so as not to adulterate the mycotoxin pattern during sample preparation. For instance, a cleanup (e.g. solid phase extraction) which does not suppress any of the required analytes is rarely available. Nevertheless, if a cleanup is not used, co-eluting and interfering matrix components may affect the ionization efficiency of target analytes resulting in poorer repeatability and lower accuracy. Therefore, the determination and compensation of such matrix effects is essential. This can be achieved by determination of the apparent recovery followed by a correction of the results with this value, matrix-matched calibration or the use of isotope-labeled internal standards. The latter will lead to results with the highest accuracy and reliability, yet with minimal time and cost investment.

Method optimization The optimization of the analytical method includes stability testing of the analytes in standard solution and samples, as well as proofing selectivity and determination of the working range.

Method validation Method validation is a prerequisite for the production of reliable results in terms of comparability and traceability. Method validation must be performed separately for each target analyte in all required matrices. Typical performance characteristics that should be evaluated during validation of a quantitative method are limits of detection (LOD), limits of quantification (LOQ), linearity, precision, selectivity, robustness, accuracy, matrix effects and recoveries. The validation of the method can be performed by spiking blank samples with each required analyte at a range of concentrations in replicate. When available, the trueness of the method should be confirmed using certified reference materials. Furthermore, matrix matched materials and the participation in proficiency testing, enable additional quality assurance. Among others, the limited number of reference materials is responsible for the semi-quantitative character of such screening methods. Although multi-toxin methods are already implemented in routine analysis, the high investment and maintenance costs must be considered.

Method implementation During the implementation, real samples as well as quality control samples should be measured with the validated method. In addition, the validated method A magazine of Romer Labs®

Currently, a multi-mycotoxin LC-MS/MS method is under development at Romer Labs. Table 1 summarizes the analytes included in the method, illustrating what is possible. Table 1. Analytes included in the multi-mycotoxin LC-MS/MS method at Romer Labs

Mycotoxin group

Included analytes

Aflatoxins

Enniatins

Enniatin A, enniatin A1, enniatin B, enniatin B1

AFB1, 13C-AFB1, AFB2, 13C-AFB2, AFG1, 13C-AFG1, AFG2, C-AFG2, AFM1, 13C-AFM1,

Alternaria toxins

AOH, tenuazonic acid

Ergometrine, ergosine, ergotamine, ergocornine, ergocristine, ergocryptine, ergine and –inine forms, agroclavine, dihydrolysergol

Ergot alkaloids Fumonisins

FB1, 13C-FB1, FB2, 13C-FB2, FB3, 13C-FB3

Masked mycotoxins

D3G, 13C-D3G

Others

STG, 13C-STG, MON, fusaric acid, gliotoxin, beauvericin

Ochratoxins

PAT, 13C-PAT, mycophenolic acid, 13C-mycophenolic acid, roquefortine C, 13C-roquefortine C, penicillic acid

Penicillium toxins Trichothecenes

OTA, 13C-OTA, OTB

Zearalenone and derivatives

DON, 13C-DON, NIV, 13C-NIV, 3-AcDON, 13C-3-AcDON, 15-AcDON, T-2, 13C-T-2, HT-2, 13C-HT-2, T-2 triol, T-2 tetraol, DAS, 13C-DAS, FusX, NEO, 15-MAS

ZON, 13C-ZON, α-ZOL, β-ZOL, ZAN

should be implemented and used in routine laboratories, which may be difficult in terms of availability of laboratory staff and instrumentation.

Conclusion In conclusion, the development of a multi-mycotoxin method based on LC-MS/MS is challenging when aiming to achieve reliable and comparable quantitative data. A vast number of different parameters that significantly influence the quality and reliability of the results must be considered carefully for each analyte in each matrix separately. In addition, the chemical diversity of the mycotoxins means that compromises have to be made during method development, which may be far from optimal for certain analytes. Moreover, the wide range of agricultural commodities as well as the varying concentration ranges and different occurrence distributions further challenge method development and optimization. Nevertheless, the development of multi-mycotoxin methods is certainly required and advances in this technology will further extend its application.

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