LC-MS/MS Method for Mycotoxin Analysis

A special issue of Toxins (ISSN 2072-6651). This special issue belongs to the section "Mycotoxins".

Deadline for manuscript submissions: closed (15 October 2016) | Viewed by 82993

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Department of Chemistry, Università di Roma “La Sapienza”, Piazzale Aldo Moro 5, 00185 Rome, Italy
Interests: mycotoxins; liquid chromatography-tandem mass spectrometry; food contaminants; analytical chemistry

Special Issue Information

Dear Colleagues,

Various filamentous fungi can produce secondary metabolites that have no biochemical significance in fungal growth and development; however, some of these metabolites can cause deleterious effects on other organisms and are classified as mycotoxins. The main mycotoxin-producing fungi belong to species of Fusarium, Aspergillus, Penicillium, Claviceps and Alternaria genera. Mostly, human exposure to mycotoxins occurs through the intake of contaminated agricultural products or indirectly through the intake of residues or metabolite products present in foods of animal origin. Seldom, exposure may also occur through dermal contact and inhalation. About 400 mycotoxins belonging to heterogeneous chemical classes are known, and they can exert very diverse toxic effects (e.g., cancerogenic and immunosuppressive effects). The most known are aflatoxins, ochratoxin, trichothecenes A and B, fumonisins, and macrocyclic lactones.

Immunochemical methods (such as ELISA) can be used for rapid screening of mycotoxin presence, however, for confirmation purpose, analytical methods based on high performance liquid chromatography (LC) are preferred, especially when coupled with tandem mass spectrometry (MS/MS), which allows to determine multiclass mycotoxins in a single analysis.

This Special Issue aims to collect papers describing development or application of LC-MS/MS-based methods for the analysis of mycotoxins in various matrices, with attention also on sample preparation step. We welcome both research or review articles proposing novelties or overview, respectively, on this topic

Prof. Dr. Aldo Laganà
Guest Editor

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Keywords

  • mycotoxins
  • fungi
  • molds (moulds)
  • extraction
  • multiresidue determination
  • liquid chromatography
  • mass spectrometry
  • Fusarium
  • Aspergillus

Published Papers (12 papers)

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Editorial

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215 KiB  
Editorial
Introduction to the Toxins Special Issue on LC-MS/MS Methods for Mycotoxin Analysis
by Aldo Laganà
Toxins 2017, 9(10), 325; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins9100325 - 16 Oct 2017
Cited by 9 | Viewed by 4473
Abstract
Various filamentous fungi can produce secondary metabolites, whose biochemical significance in fungal growth and development has not always been fully clarified; however, some of these metabolites can cause deleterious effects on other organisms and are classified as mycotoxins [...]
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(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)

Research

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1527 KiB  
Article
Development of a LC-MS/MS Method for the Multi-Mycotoxin Determination in Composite Cereal-Based Samples
by Barbara De Santis, Francesca Debegnach, Emanuela Gregori, Simona Russo, Francesca Marchegiani, Gabriele Moracci and Carlo Brera
Toxins 2017, 9(5), 169; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins9050169 - 18 May 2017
Cited by 71 | Viewed by 7234
Abstract
The analytical scenario for determining contaminants in the food and feed sector is constantly prompted by the progress and improvement of knowledge and expertise of researchers and by the technical innovation of the instrumentation available. Mycotoxins are agricultural contaminants of fungal origin occurring [...] Read more.
The analytical scenario for determining contaminants in the food and feed sector is constantly prompted by the progress and improvement of knowledge and expertise of researchers and by the technical innovation of the instrumentation available. Mycotoxins are agricultural contaminants of fungal origin occurring at all latitudes worldwide and being characterized by acute and chronic effects on human health and animal wellness, depending on the species sensitivity. The major mycotoxins of food concern are aflatoxin B1 and ochratoxin A, the first for its toxicity, and the second for its recurrent occurrence. However, the European legislation sets maximum limits for mycotoxins, such as aflatoxin B1, ochratoxin A, deoxynivalenol, fumonisins, and zearalenone, and indicative limits for T-2 and HT-2 toxins. Due to the actual probability that co-occurring mycotoxins are present in a food or feed product, nowadays, the availability of reliable, sensitive, and versatile multi-mycotoxin methods is assuming a relevant importance. Due to the wide range of matrices susceptible to mycotoxin contamination and the possible co-occurrence, a multi-mycotoxin and multi-matrix method was validated in liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose to overcome specific matrix effects and analyze complex cereal-based samples within the Italian Total Diet Study project. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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1840 KiB  
Article
Simultaneous Determination of Multi-Mycotoxins in Cereal Grains Collected from South Korea by LC/MS/MS
by Dong-Ho Kim, Sung-Yong Hong, Jea Woo Kang, Sung Min Cho, Kyu Ri Lee, Tae Kyung An, Chan Lee and Soo Hyun Chung
Toxins 2017, 9(3), 106; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins9030106 - 16 Mar 2017
Cited by 68 | Viewed by 8594
Abstract
An improved analytical method compared with conventional ones was developed for simultaneous determination of 13 mycotoxins (deoxynivalenol, nivalenol, 3-acetylnivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, [...] Read more.
An improved analytical method compared with conventional ones was developed for simultaneous determination of 13 mycotoxins (deoxynivalenol, nivalenol, 3-acetylnivalenol, aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, fumonisin B1, fumonisin B2, T-2, HT-2, zearalenone, and ochratoxin A) in cereal grains by liquid chromatography-tandem mass spectrometry (LC/MS/MS) after a single immunoaffinity column clean-up. The method showed a good linearity, sensitivity, specificity, and accuracy in mycotoxin determination by LC/MS/MS. The levels of 13 mycotoxins in 5 types of commercial grains (brown rice, maize, millet, sorghum, and mixed cereal) from South Korea were determined in a total of 507 cereal grains. Mycotoxins produced from Fusarium sp. (fumonisins, deoxynivalenol, nivalenol, and zearalenone) were more frequently (more than 5%) and concurrently detected in all cereal grains along with higher mean levels (4.3–161.0 ng/g) in positive samples than other toxins such as aflatoxins and ochratoxin A (less than 9% and below 5.2 ng/g in positive samples) from other fungal species. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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2422 KiB  
Article
A Greener, Quick and Comprehensive Extraction Approach for LC-MS of Multiple Mycotoxins
by Andreas Breidbach
Toxins 2017, 9(3), 91; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins9030091 - 07 Mar 2017
Cited by 13 | Viewed by 7045
Abstract
In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, [...] Read more.
In food/feed control, mycotoxin analysis is often still performed “one analyte at a time”. Here a method is presented which aims at making mycotoxin analysis environmentally friendlier through replacing acetonitrile by ethyl acetate and reducing chemical waste production by analyzing four mycotoxins together, forgoing sample extract clean-up, and minimizing solvent consumption. For this, 2 g of test material were suspended in 8 mL water and 16 mL ethyl acetate were added. Extraction was accelerated through sonication for 30 min and subsequent addition of 8 g sodium sulfate. After centrifugation, 500 µL supernatant were spiked with isotopologues, dried down, reconstituted in mobile phase, and measured with LC-MS. The method was validated in-house and through a collaborative study and the performance was fit-for-purpose. Repeatability relative standard deviation (RSDs) between 16% at low and 4% at higher contaminations were obtained. The reproducibility RSDs were mostly between 12% and 32%. The trueness of results for T-2 toxin and Zearalenone were not different from 100%, for Deoxynivalenol and HT-2 toxin they were larger than 89%. The extraction was also adapted to a quick screening of Aflatoxin B1 in maize by flow-injection–mass spectrometry. Semi-quantitative results were obtained through standard addition and scan-based ion ratio calculations. The method proved to be a viable greener and quicker alternative to existing methods. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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1706 KiB  
Article
QuEChERS Purification Combined with Ultrahigh-Performance Liquid Chromatography Tandem Mass Spectrometry for Simultaneous Quantification of 25 Mycotoxins in Cereals
by Juan Sun, Weixi Li, Yan Zhang, Xuexu Hu, Li Wu and Bujun Wang
Toxins 2016, 8(12), 375; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8120375 - 15 Dec 2016
Cited by 33 | Viewed by 6104
Abstract
A method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) purification combined with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), was optimized for the simultaneous quantification of 25 mycotoxins in cereals. Samples were extracted with a solution containing 80% [...] Read more.
A method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) purification combined with ultrahigh performance liquid chromatography tandem mass spectrometry (UPLC–MS/MS), was optimized for the simultaneous quantification of 25 mycotoxins in cereals. Samples were extracted with a solution containing 80% acetonitrile and 0.1% formic acid, and purified with QuEChERS before being separated by a C18 column. The mass spectrometry was conducted by using positive electrospray ionization (ESI+) and multiple reaction monitoring (MRM) models. The method gave good linear relations with regression coefficients ranging from 0.9950 to 0.9999. The detection limits ranged from 0.03 to 15.0 µg·kg−1, and the average recovery at three different concentrations ranged from 60.2% to 115.8%, with relative standard deviations (RSD%) varying from 0.7% to 19.6% for the 25 mycotoxins. The method is simple, rapid, accurate, and an improvement compared with the existing methods published so far. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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267 KiB  
Article
Harmonized Collaborative Validation of Aflatoxins and Sterigmatocystin in White Rice and Sorghum by Liquid Chromatography Coupled to Tandem Mass Spectrometry
by Hyun Ee Ok, Fei Tian, Eun Young Hong, Ockjin Paek, Sheen-Hee Kim, Dongsul Kim and Hyang Sook Chun
Toxins 2016, 8(12), 371; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8120371 - 13 Dec 2016
Cited by 19 | Viewed by 3918
Abstract
An interlaboratory study was performed in eight laboratories to validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white [...] Read more.
An interlaboratory study was performed in eight laboratories to validate a liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of aflatoxins and sterigmatocystin (STC) in white rice and sorghum (Sorghum bicolor). Fortified samples (at three different levels) of white rice and sorghum were extracted, purified through a solid-phase extraction (SPE) column, and then analyzed by LC/MS/MS. The apparent recoveries (ARs) ranged from 78.8% to 95.0% for aflatoxins and from 85.3% to 96.7% for STC. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of aflatoxins were in the ranges 7.9%–33.8% and 24.4%–81.0%, respectively. For STC, the RSDr ranged from 7.1% to 40.2% and the RSDR ranged from 28.1% to 99.2%. The Horwitz ratio values for the aflatoxins and STC ranged from 0.4 to 1.2 in white rice and from 0.3 to 1.0 in sorghum, respectively. These results validated this method for the simultaneous determination of aflatoxins and STC by LC/MS/MS after SPE column cleanup. The percentages of satisfactory Z-score values (|Z| ≤ 2) were the following: for white rice, 100% for aflatoxins and STC; for sorghum, 100%, except in data from two laboratories for STC (0.3 μg/kg). This validated that the LC/MS/MS method was successfully applied for the determination of aflatoxins and STC in 20 white rice and 20 sorghum samples sourced from Korean markets. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
6287 KiB  
Article
Co-Occurrence of Regulated, Masked and Emerging Mycotoxins and Secondary Metabolites in Finished Feed and Maize—An Extensive Survey
by Paula Kovalsky, Gregor Kos, Karin Nährer, Christina Schwab, Timothy Jenkins, Gerd Schatzmayr, Michael Sulyok and Rudolf Krska
Toxins 2016, 8(12), 363; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8120363 - 06 Dec 2016
Cited by 164 | Viewed by 10214
Abstract
Global trade of agricultural commodities (e.g., animal feed) requires monitoring for fungal toxins. Also, little is known about masked and emerging toxins and metabolites. 1926 samples from 52 countries were analysed for toxins and metabolites. Of 162 compounds detected, up to 68 metabolites [...] Read more.
Global trade of agricultural commodities (e.g., animal feed) requires monitoring for fungal toxins. Also, little is known about masked and emerging toxins and metabolites. 1926 samples from 52 countries were analysed for toxins and metabolites. Of 162 compounds detected, up to 68 metabolites were found in a single sample. A subset of 1113 finished feed, maize and maize silage samples containing 57 compounds from 2012 to 2015 from 44 countries was investigated using liquid chromatography and mass spectrometry. Deoxynivalenol (DON), zearalenone (ZEN) and fumonisins showed large increases of annual medians in Europe. Within a region, distinct trends were observed, suggesting importance of local meteorology and cultivars. In 2015, median DON concentrations increased to 1400 μ g·kg 1 in Austria, but were stable in Germany at 350 μ g·kg 1 . In 2014, enniatins occurred at median concentrations of 250 μ g·kg 1 in Europe, at levels similar to DON and ZEN. The latter were frequently correlated with DON-3-glucoside and ZEN-14-sulfate. Co-occurrence of regulated toxins was frequent with e.g., enniatins, and moniliformin. Correlation was observed between DON and DON-3-glucoside and with beauvericin. Results indicate that considerably more than 25% of agricultural commodities could be contaminated with mycotoxins as suggested by FAO, although this is at least partly due to the lower limits of detection in the current survey. Observed contamination percentages ranged from 7.1 to 79% for B trichothecenes and 88% for ZEN. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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1341 KiB  
Article
Metabolism of HT-2 Toxin and T-2 Toxin in Oats
by Jacqueline Meng-Reiterer, Christoph Bueschl, Justyna Rechthaler, Franz Berthiller, Marc Lemmens and Rainer Schuhmacher
Toxins 2016, 8(12), 364; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8120364 - 05 Dec 2016
Cited by 31 | Viewed by 6701
Abstract
The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive [...] Read more.
The Fusarium mycotoxins HT-2 toxin (HT2) and T-2 toxin (T2) are frequent contaminants in oats. These toxins, but also their plant metabolites, may contribute to toxicological effects. This work describes the use of 13C-assisted liquid chromatography–high-resolution mass spectrometry for the first comprehensive study on the biotransformation of HT2 and T2 in oats. Using this approach, 16 HT2 and 17 T2 metabolites were annotated including novel glycosylated and hydroxylated forms of the toxins, hydrolysis products, and conjugates with acetic acid, putative malic acid, malonic acid, and ferulic acid. Further targeted quantitative analysis was performed to study toxin metabolism over time, as well as toxin and conjugate mobility within non-treated plant tissues. As a result, HT2-3-O-β-d-glucoside was identified as the major detoxification product of both parent toxins, which was rapidly formed (to an extent of 74% in HT2-treated and 48% in T2-treated oats within one day after treatment) and further metabolised. Mobility of the parent toxins appeared to be negligible, while HT2-3-O-β-d-glucoside was partly transported (up to approximately 4%) through panicle side branches and stem. Our findings demonstrate that the presented combination of untargeted and targeted analysis is well suited for the comprehensive elucidation of mycotoxin metabolism in plants. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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2999 KiB  
Article
Spotlight on the Underdogs—An Analysis of Underrepresented Alternaria Mycotoxins Formed Depending on Varying Substrate, Time and Temperature Conditions
by Theresa Zwickel, Sandra M. Kahl, Horst Klaffke, Michael Rychlik and Marina E. H. Müller
Toxins 2016, 8(11), 344; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8110344 - 19 Nov 2016
Cited by 26 | Viewed by 6072
Abstract
Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not [...] Read more.
Alternaria (A.) is a genus of widespread fungi capable of producing numerous, possibly health-endangering Alternaria toxins (ATs), which are usually not the focus of attention. The formation of ATs depends on the species and complex interactions of various environmental factors and is not fully understood. In this study the influence of temperature (7 °C, 25 °C), substrate (rice, wheat kernels) and incubation time (4, 7, and 14 days) on the production of thirteen ATs and three sulfoconjugated ATs by three different Alternaria isolates from the species groups A. tenuissima and A. infectoria was determined. High-performance liquid chromatography coupled with tandem mass spectrometry was used for quantification. Under nearly all conditions, tenuazonic acid was the most extensively produced toxin. At 25 °C and with increasing incubation time all toxins were formed in high amounts by the two A. tenuissima strains on both substrates with comparable mycotoxin profiles. However, for some of the toxins, stagnation or a decrease in production was observed from day 7 to 14. As opposed to the A. tenuissima strains, the A. infectoria strain only produced low amounts of ATs, but high concentrations of stemphyltoxin III. The results provide an essential insight into the quantitative in vitro AT formation under different environmental conditions, potentially transferable to different field and storage conditions. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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2139 KiB  
Article
Development and Validation of a LC-ESI-MS/MS Method for the Determination of Alternaria Toxins Alternariol, Alternariol Methyl-Ether and Tentoxin in Tomato and Tomato-Based Products
by Yelko Rodríguez-Carrasco, Jordi Mañes, Houda Berrada and Cristina Juan
Toxins 2016, 8(11), 328; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8110328 - 11 Nov 2016
Cited by 54 | Viewed by 7642
Abstract
Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), [...] Read more.
Alternaria species are capable of producing several secondary toxic metabolites in infected plants and in agricultural commodities, which play important roles in food safety. Alternaria alternata turn out to be the most frequent fungal species invading tomatoes. Alternariol (AOH), alternariol monomethyl ether (AME), and tentoxin (TEN) are some of the main Alternaria mycotoxins that can be found as contaminants in food. In this work, an analytical method based on liquid chromatography (LC) tandem mass spectrometry (MS/MS) detection for the simultaneous quantification of AOH, AME, and TEN in tomato and tomato-based products was developed. Mycotoxin analysis was performed by dispersive liquid-liquid microextraction (DLLME) combined with LC-ESI-MS/MS. Careful optimization of the MS/MS parameters was performed with an LC/MS system with the ESI interface in the positive ion mode. Mycotoxins were efficiently extracted from sample extract into a droplet of chloroform (100 µL) by DLLME technique using acetonitrile as a disperser solvent. Method validation following the Commission Decision No. 2002/657/EC was carried out by using tomato juice as a blank matrix. Limits of detection and quantitation were, respectively, in the range 0.7 and 3.5 ng/g. Recovery rates were above 80%. Relative standard deviations of repeatability (RSDr) and intermediate reproducibility (RSDR) were ≤ 9% and ≤ 15%, respectively, at levels of 25 and 50 ng/g. Five out of 30 analyzed samples resulted positive to at least one Alternaria toxin investigated. AOH was the most common Alternaria toxin found, but at levels close to LOQ (average content: 3.75 ng/g). Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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1260 KiB  
Article
Natural Occurrence of Alternaria Toxins in the 2015 Wheat from Anhui Province, China
by Wenjing Xu, Xiaomin Han, Fengqin Li and Lishi Zhang
Toxins 2016, 8(11), 308; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8110308 - 26 Oct 2016
Cited by 56 | Viewed by 4695
Abstract
The exposure to Alternaria toxins from grain and grain-based products has been reported to be related to human esophageal cancer in China. In this study, a total of 370 freshly harvested wheat kernel samples collected from Anhui province of China in 2015 were [...] Read more.
The exposure to Alternaria toxins from grain and grain-based products has been reported to be related to human esophageal cancer in China. In this study, a total of 370 freshly harvested wheat kernel samples collected from Anhui province of China in 2015 were analyzed for the four Alternaria toxins tenuazonic acid (TeA), tentoxin (TEN), alternariol (AOH) and alternariol monomethyl ether (AME) by high performance liquid chromatography-tandem mass spectrometry method (HPLC-MS/MS). TeA was the predominant toxin detected followed by TEN, AOH and AME. The concentrations of the four Alternaria toxins varied geographically. The samples from Fuyang district showed higher TEN concentration levels than the other regions studied (p < 0.05). Furthermore, 95% (352/370) of the wheat samples were positive for more than one type of Alternaria toxins. Positive correlation was observed between concentration levels of TeA and TEN, AOH and AME, TeA and AOH, and the total dibenzopyrone derivatives (AOH + AME) and TeA. Results indicate that there is a need to set the tolerance limit for Alternaria toxins in China, and more data on the contamination of these toxins in agro-products is required. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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Review

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2211 KiB  
Review
Recent Advances and Future Challenges in Modified Mycotoxin Analysis: Why HRMS Has Become a Key Instrument in Food Contaminant Research
by Laura Righetti, Giuseppe Paglia, Gianni Galaverna and Chiara Dall’Asta
Toxins 2016, 8(12), 361; https://0-doi-org.brum.beds.ac.uk/10.3390/toxins8120361 - 02 Dec 2016
Cited by 53 | Viewed by 7584
Abstract
Mycotoxins are secondary metabolites produced by pathogenic fungi in crops worldwide. These compounds can undergo modification in plants, leading to the formation of a large number of possible modified forms, whose toxicological relevance and occurrence in food and feed is still largely unexplored. [...] Read more.
Mycotoxins are secondary metabolites produced by pathogenic fungi in crops worldwide. These compounds can undergo modification in plants, leading to the formation of a large number of possible modified forms, whose toxicological relevance and occurrence in food and feed is still largely unexplored. The analysis of modified mycotoxins by liquid chromatography–mass spectrometry remains a challenge because of their chemical diversity, the large number of isomeric forms, and the lack of analytical standards. Here, the potential benefits of high-resolution and ion mobility mass spectrometry as a tool for separation and structure confirmation of modified mycotoxins have been investigated/reviewed. Full article
(This article belongs to the Special Issue LC-MS/MS Method for Mycotoxin Analysis)
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