Sofie Monbaliu

Occurrence of Mycotoxins in Feed as Analyzed by a Multi-Mycotoxin LC-MS/MS Method

Crops used for animal feed can be easily contaminated by fungi during growth, harvest, or storage, resulting in the occurrence of mycotoxins. Because animal feed plays an important role in the food safety chain, the European Commission has set maximum levels for aflatoxin B1 and recommended maximum levels for deoxynivalenol, zearalenone, ochratoxin A, and the sum of fumonisin B1 and B2. A multimycotoxin LC-MS/MS method was developed, validated according to Commission Decision 2002/657/EC and EN ISO 17025 accredited for the simultaneous detection of 23 mycotoxins (aflatoxin-B1, aflatoxin-B2, aflatoxin-G1, aflatoxin-G2, ochratoxin A, deoxynivalenol, zearalenone, fumonisin B1, fumonisin B2, fumonisin B3, T2-toxin, HT2-toxin, nivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, diacetoxyscirpenol, fusarenon-X, neosolaniol, altenuene, alternariol, alternariol methyl ether, roquefortine-C, and sterigmatocystin) in feed. The decision limits of the multimycotoxin method varied from 0.7 to 60.6 microg/kg. The apparent recovery and the results of the precision study fulfilled the performance criteria as set in Commission Decision 2002/657/EC. The analysis of three different feed matrices (sow feed, wheat, and maize) provided a good basis for the evaluation of the toxin exposure in animal production. In total, 67 samples out of 82 (82%) were contaminated; type B-trichothecenes and fumonisins occurred most often. The majority of the infected feed samples (75%) were contaminated with more than one type of mycotoxin.

LC-MS/MS multi-analyte method for mycotoxin determination in food supplements

A multi-analyte method for the liquid chromatography-tandem mass spectrometric determination of mycotoxins in food supplements is presented. The analytes included A and B trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin and T-2 toxin), aflatoxins (aflatoxin-B1, aflatoxin-B2, aflatoxin-G1 and aflatoxin-G2), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B1, fumonisin-B2 and fumonisin-B3), ochratoxin A, zearalenone, beauvericin and sterigmatocystin. Optimization of the simultaneous extraction of these toxins and the sample pretreatment procedure, as well as method validation were performed on maca (Lepidium meyenii) food supplements. The results indicated that the solvent mixture ethyl acetate/formic acid (95:5, v/v) was the best compromise for the extraction of the analytes from food supplements. Liquid–liquid partition with n-hexane was applied as partial clean-up step to remove excess of co-extracted non-polar components. Further clean-up was performed on Oasis HLB™ cartridges. Samples were analysed using an Acquity UPLC system coupled to a Micromass Quattro Micro triple quadrupole mass spectrometer equipped with an electrospray interface operated in the positive-ion mode. Limits of detection and quantification were in the range of 0.3–30 ng g−1 and 1–100 ng g−1, respectively. Recovery yields were above 60% for most of the analytes, except for nivalenol, sterigmatocystine and the fumonisins. The method showed good precision and trueness. Analysis of different food supplements such as soy (Glycine max) isoflavones, St Johns wort ( Hypericum perforatum), garlic (Allium sativum), Ginkgo biloba, and black radish (Raphanus niger) demonstrated the general applicability of the method. Due to different matrix effects observed in different food supplement samples, the standard addition approach was applied to perform correct quantitative analysis. In 56 out of 62 samples analysed, none of the 23 mycotoxins investigated was detected. Positive samples contained at least one of the toxins fumonisin-B1, fumonisin-B2, fumonisin-B3 and ochratoxin A.

Development of a multi‐mycotoxin liquid chromatography/tandem mass spectrometry method for sweet pepper analysis

A multi-mycotoxin method was developed for the simultaneous determination of trichothecenes (nivalenol, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, neosolaniol, fusarenon-X, diacetoxyscirpenol, HT-2 toxin, T-2 toxin), aflatoxins (aflatoxin-B(1), aflatoxin-B(2), aflatoxin-G(1) and aflatoxin-G(2)), Alternaria toxins (alternariol, alternariol methyl ether and altenuene), fumonisins (fumonisin-B(1), fumonisin-B(2) and fumonisin-B(3)), ochratoxin A, zearalenone, beauvericin and sterigmatocystin in sweet pepper. Sweet pepper was extracted with ethyl acetate/formic acid (99:1, v/v). After splitting up the extract, two-thirds of the extract was cleaned up using an aminopropyl column followed by an octadecyl column. The remaining part was cleaned up using a strong anion-exchange column. After recombination of both cleaned parts of the sample extract, the combined solvents were evaporated and the residue was dissolved in mobile phase; 20 microL was injected into the chromatographic system, so only one run was used to separate and detect the mycotoxins in positive electrospray ionization using selected reaction monitoring. The samples were analyzed with a Micromass Quattro Micro triple quadrupole mass spectrometer (Waters, Milford, MA, USA). The mobile phase consisted of variable mixtures of water and methanol, 1% acetic acid and 5 mM ammonium acetate. The limits of detection of the multi-mycotoxin method varied from 0.32 microg kg(-1) to 42.48 microg kg(-1). The multi-mycotoxin liquid chromatography/tandem mass spectrometry (LC/MS/MS) method fulfilled the method performance criteria required by the Commission Regulation (EC) No 401/2006. Sweet peppers inoculated by Fusarium species were analyzed using the developed method. Beauvericin (9-484 microg kg(-1)) and fumonisins (fumonisin-B(1) up to 4330 microg kg(-1), fumonisin-B(2) up to 4900 microg kg(-1), and fumonisin-B(3) up to 299 microg kg(-1)) were detected.

A validated multianalyte LC-MS/MS method for quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples.

This study was designed to develop a sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous detection and quantification of 25 mycotoxins in cassava flour, peanut cake and maize samples with particular focus on the optimization of the sample preparation protocol and method validation. All 25 mycotoxins were extracted in a single step with a mixture of methanol/ethyl acetate/water (70:20:10, v/v/v). The method limits of quantification (LOQ) varied from 0.3 μg/kg to 106 μg/kg. Good precision and linearity were observed for most of the mycotoxins. The method was applied for the analysis of naturally contaminated peanut cake, cassava flour and maize samples from the Republic of Benin. All samples analyzed (fifteen peanut cakes, four maize flour and four cassava flour samples) tested positive for one or more mycotoxins. Aflatoxins (total aflatoxins; 10-346 μg/kg) and ochratoxin A (<LOQ-2 μg/kg) were detected in peanut cake samples while fumonisin B(1) (4-21 μg/kg), aflatoxin B(2) (<LOQ-8 μg/kg), aflatoxin B(1) (<LOQ-9 μg/kg), diacetoxyscirpenol (<LOQ-6 μg/kg) and zearalenone (<LOQ-12 μg/kg) were detected and quantified in cassava flour samples. Fumonisin B(1) (13-836 μg/kg), fumonisin B(2) (5-221 μg/kg), fumonisin B(3) (<LOQ-375 μg/kg) and beauvericin (<LOQ-25 μg/kg) were detected in the maize samples.

Multimycotoxin UPLC−MS/MS for Tea, Herbal Infusions and the Derived Drinkable Products

In recent years the consumption of tea and herbal infusions has increased. These hot drinks are consumed as daily drinks as well as for medicinal purposes. All tea varieties (white, yellow, green, oolong, black and puerh) originate from the leaves of the tea plant, Camellia sinensis. All extracts made of plant or herbal materials which do not contain Camellia sinensis are referred as herbal infusions or tisanes. During processing and manufacturing fungal contamination of the plant materials is possible, enabling contamination of these products with mycotoxins. In this study a multimycotoxin UPLC-MS/MS method was developed and validated for the analysis of the raw tea and herbal infusion materials as well as for their drinkable products. The samples were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), with a mobile phase consisting of variable mixtures of water and methanol with 0.3% formic acid. The limits of detection for the different mycotoxins varied between 2.1 μg/kg and 121 μg/kg for raw materials and between 0.4 μg/L and 46 μg/L for drinkable products. Afterward 91 different tea and herbal infusion samples were analyzed. Only in one sample, Ceylon melange, 76 μg/kg fumonisin B(1) was detected. No mycotoxins were detected in the drinkable products.

Synthesis and application of a T-2 toxin imprinted polymer.

The synthesis of a T-2 toxin imprinted polymer and its application in food analysis are reported for the first time. A molecularly imprinted polymer (MIP) for the selective recognition of T-2 toxin (T-2) was synthesized by bulk polymerization. Methacrylamide and ethyleneglycol dimethacrylate were applied as functional monomer and cross-linker, respectively. Molecularly imprinted solid-phase extraction (MISPE) procedures were optimized for further application in the analysis of T-2. Scatchard plot analysis revealed that two classes of imprinted binding sites were formed in the imprinted polymer. The dissociation constant (KD) of the higher affinity binding sites was 7.0 micromol/l, while the KD of the lower affinity binding sites was 54.7 micromol/l. The performance of the MIP throughout the clean-up of spiked maize, barley and oat sample extracts was compared with the results obtained when using non-imprinted polymer, OASIS HLB and immunoaffinity columns (IAC). Depending on the food matrix and the spiked concentration, recoveries after MISPE and non-imprinted solid-phase extraction varied respectively from 60% to 73% and from 21% to 57%. Recoveries obtained after clean-up using OASIS HLB and IAC were in the range of 74-104% and 60-85%, respectively. Although highest recoveries were obtained with OASIS HLB sorbents, the designed MIP and the IAC were superior regarding selectivity, cross-reactivity, matrix effect, limits of detection (LOD) and limits of quantification (LOQ). Depending on the matrix, LOD after MISPE ranged from 0.4 microg/kg to 0.6 microg/kg and LOQ from 1.4 microg/kg to 1.9 microg/kg. LOD and LOQ after OASIS HLB clean-up varied from 0.9 microg/kg to 3.5 microg/kg and from 3.1 microg/kg to 11.7 microg/kg, respectively. The LOD and LOQ values obtained with IAC were in the range of 0.3-2.3 microg/kg and 1.0-7.7 microg/kg, respectively. Analysis of 39 naturally contaminated samples (maize, barley and oat) by liquid chromatography tandem mass spectrometry revealed that the MIP could be an excellent alternative for clean-up of contaminated food samples.

Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates from sweet pepper.

An internal fruit rot disease of sweet peppers was first detected in Belgium in 2003. Research conducted mostly in Canada indicates that this disease is primarily caused by Fusarium lactis Pirotta. Ninety-eight Fusarium isolates obtained from diseased sweet peppers from Belgium, as well as from other countries (Canada, the Netherlands and the United Kingdom) were identified by sequencing the translation elongation factor 1α (EF). Of these 98 isolates, 13 were identified as F. oxysporum Schltdl., nine as F. proliferatum (Matsush.) Nirenberg and two belonged to clade 3 of the F. solani species complex. Of the 74 remaining isolates, the EF sequence showed 97% to 98% similarity to F. lactis. Of these isolates, the β-tubulin (TUB), calmodulin (CAM) and the second largest subunit of RNA polymerase II (RPB2) genes were also sequenced. Analysis of the combined sequences revealed that the 74 isolates share nine combined sequences that correspond to nine multilocus sequence types (STs), while the F. lactis neotype strain and one other strain, both isolated from figs, form a separate ST. Together, these 10 STs represent a monophyletic F. lactis species complex (FLASC). An unusually high level of genetic diversity was observed between (groups of) these STs. Two of them (ST5 and ST6) fulfilled the criteria for species recognition based on genealogical exclusivity and together represent a new monophyletic species lineage (FLASC-1). The seven other STs, together with the F. lactis neotype ST, form a paraphyletic species lineage in the African clade of the Gibberella fujikuroi species complex (GFSC). From each of the 10 STs, the mycotoxin production was assessed using a multi-mycotoxin liquid chromatography mass spectrometry method. Out of the 27 analyzed mycotoxins, beauvericin and fumonisins were detected in sweet pepper tissue and in maize kernels. The 10 STs clearly differed in the amount of mycotoxin produced, but there was only limited congruence between the production profile and the phylogenetic analysis. Furthermore, the morphological characterization (based on mycelial growth rate and the length of macroconidia) showed distinct differences between the 10 STs, but again there was limited congruence with the phylogenetic results. In conclusion, the data presented in this study demonstrate that 75% of the isolates obtained from sweet pepper with internal fruit rot belong to a F. lactis species complex (FLASC), including a new FLASC-1 monophyletic species, and that the members of this complex display great genetic and phenotypic diversity.

Production and migration of mycotoxins in sweet pepper analyzed by Multimycotoxin LC-MS/MS

In this work the presence and migration behavior of mycotoxins formed in sweet pepper, inoculated by Fusarium species involved in internal fruit rot, were investigated. Two different commercial sweet pepper cultivars were inoculated with two different Fusarium proliferatum isolates that were sampled from diseased peppers. After 10 days of incubation at 20 °C in a closed container, the lesion caused by the fungal infection was dissected. Around the lesion, up to three concentric rings of pepper fruit tissue with a width of 5 mm were cut out and analyzed using a multimycotoxin LC-MS/MS method. The analyses resulted in the detection of beauvericin and fumonisins B(1), B(2), and B(3). Beauvericin was detected only in the lesions (95%), and the levels varied between 67 and 73800 μg/kg. Fumonisins B(1), B(2), and B(3) were detected in the lesions and in the surrounding tissue, indicating migration of these toxins into healthy parts of the sweet pepper. In the lesion the fumonisin B(1) level varied between 690 and 104000 μg/kg. Even in the outer ring fumonisin B(1) was still present. Mostly it was present at a lower level than in the lesion, with a maximum level of 556 μg/kg. A similar migration behavior was obtained for fumonisins B(2) and B(3), but lower levels were detected in the lesions, up to 10900 and 1287 μg/kg, respectively. The analysis of 20 pepper samples resulted in the detection of beauvericin or alternariol. Seven samples were contaminated, and the level of beauvericin was 124 μg/kg (N = 1), whereas the level of alternariol varied from below the LOQ (6.6 μg/kg) to 101 μg/kg (N = 6).

Detection and determination of natural toxins (mycotoxins and plant toxins) in feed

Abstract: Animal feed is often contaminated with different types of mycotoxins and plant toxins. These toxic substances can cause adverse effects on animal health, and carry-over of some toxins and/or their metabolites into edible tissues, milk and eggs may contribute to human exposure. Therefore risk assessments and official controls of feed samples have to be performed. In order to carry out these assignments, different tools such as rapid screening tests and chromatographic confirmatory tests have been developed to analyse and detect the toxic substances in different commodities. An overview of the detection and determination of mycotoxins and plant toxins in feed is presented.