Veronica M.T. Lattanzio

Multiplex dipstick immunoassay for semi-quantitative determination of Fusarium mycotoxins in cereals.

A multiplex dipstick immunoassay based method for the simultaneous determination of major Fusarium toxins, namely zearalenone, T-2 and HT-2 toxins, deoxynivalenol and fumonisins in wheat, oats and maize has been developed. The dipstick format was based on an indirect competitive approach. Four test lines (mycotoxin-BSA conjugates) and one control line were located on the strip membrane. Labelled antibodies were freeze-dried within the microwell. Two matrix-related sample preparation protocols have been developed for wheat/oats (not containing fumonisins) and maize (containing fumonisins) respectively. The use of a methanol/water mixture for sample preparation allowed recoveries in the range 73-109% for all mycotoxins in all tested cereals, with relative standard deviation less than 10%. The optimized immunoassay was able to detect target mycotoxins at cut off levels equal to 80% of EU maximum permitted levels, i.e. 280, 400, 1400 and 3200 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins, deoxynivalenol and fumonisins in maize, and 80, 400 and 1400 μg kg(-1), respectively, for zearalenone, T-2/HT-2 toxins and deoxynivalenol in wheat and oats. Analysis of naturally contaminated samples resulted in a good agreement between multiplex dipstick and validated confirmatory LC-MS/MS. The percentage of false positive results was less than or equal to 13%, whereas no false negative results were obtained. Data on the presence/absence of 6 mycotoxins at levels close to EU regulatory levels were obtained within 30 min. The proposed immunoassay protocol is rapid, inexpensive, easy-to-use and fit for purpose of rapid screening of mycotoxins in cereals.

Determination of trichothecenes in cereals and cereal-based products by liquid chromatography-tandem mass spectrometry.

A sensitive, accurate and precise method for the simultaneous determination of nivalenol (NIV), deoxynivalenol (DON), T-2 toxin (T-2) and HT-2 toxin (HT-2) in different food matrices, including wheat, maize, barley, cereal-based infant foods, snacks, biscuits and wafers, has been developed. The method, using liquid chromatography coupled with atmospheric pressure chemical ionization triple quadrupole mass spectrometry (LC–APCI–MS/MS), allowed unambiguous identification of the selected trichothecenes at low µg per kg levels in such complex food matrices. A clean-up procedure, based on reversed phase SPE Oasis® HLB columns, was used, allowing good recoveries for all studied trichothecenes. In particular, NIV recoveries significantly improved compared to those obtained by using Mycosep® #227 columns for clean-up of the extracts. Limits of detection in the various investigated matrices ranged 2.5–4.0 µg kg−1 for NIV, 2.8–5.3 µg kg−1 for DON, 0.4–1.7 µg kg−1 for HT-2 and 0.4–1.0 µg kg−1 for T-2. Mean recovery values, obtained from cereals and cereal products spiked with NIV, DON, HT-2 and T-2 toxins at levels from 10 to 1000 µg kg−1, ranged from 72 to 110% with mean relative standard deviation lower than 10%. A systematic investigation of matrix effects in different cereals and cereal products was also carried out by statistically comparing the slopes of standard calibration curve with matrix-matched calibration curve for each of the four toxins and the eight matrices tested. For seven of the eight matrices tested, statistically significant matrix effects were observed, indicating that, for accurate quantitative analysis, matrix-matched calibration was necessary. The method was applied to the analysis of 57 samples of ground wheat originated from South Italy and nine cereal food samples collected from retail markets.

Identification and characterization of new Fusarium masked mycotoxins, T2 and HT2 glycosyl derivatives, in naturally contaminated wheat and oats by liquid chromatography–high-resolution mass spectrometry

The presence of glucoside derivatives of T-2 and HT-2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high-resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T-2 toxin and two monoglucoside derivatives of HT-2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C-3 position for T-2 toxin and at C-3 or C-4 position for HT-2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T-2 and HT-2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk.

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 µg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.

LC–MS/MS characterization of the urinary excretion profile of the mycotoxin deoxynivalenol in human and rat

The understanding of mycotoxins transfer to biological fluids is challenged by the difficulties in performing and replicating in vivo experiments as well as the lack of suitable methods of analysis to detect simultaneously a range of chemically different metabolites at trace levels. LC-MS/MS has been used herein to study the urinary excretion profile of the mycotoxin deoxynivalenol in human and Wistar rat. Deoxynivalenol and deoxynivalenol glucuronide were found in both human and rat urines, whereas de-epoxydeoxynivalenol and its glucuronide conjugate were only detected in rat urine. The presence of two deoxynivalenol glucuronide isomers in Wistar rat urine has been shown for the first time. Structure confirmation of the detected metabolites was provided by the analysis of fragmentation patterns. A solid phase extraction clean up procedure allowing recoveries in the range 72-102% for deoxynivalenol, de-epoxydeoxynivalenol, and their glucuronide conjugates was optimized. A multiple reaction monitoring method for the simultaneous determination of all investigated metabolites was elaborated allowing the direct detection of deoxynivalenol metabolites without the hydrolysis step. Deoxynivalenol urinary levels in the range 0.003-0.008 μg/ml were detected in healthy human subjects, whereas deoxynivalenol and de-epoxynivalenol levels between 1.9-4.9 μg/ml and 1.6-5.9 μg/ml, respectively were found in administered rat urine. These findings emphasize the relevance of the highly selective and sensitive LC-MS/MS technique for the direct detection and characterization of deoxynivalenol metabolites in complex biological matrices.

Improved method for the simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in cereals and derived products by liquid chromatography-tandem mass spectrometry after multi-toxin immunoaffinity clean up.

An improved method for the quantitative determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, fumonisins (B1, B2), zearalenone, deoxynivalenol, nivalenol, T-2 and HT-2 toxins in cereals and derived products, at levels comparable with EU maximum permitted levels, was developed. The effective co-extraction of the mycotoxins under investigation was achieved in 4min by a double extraction approach, using water followed by methanol. Clean up of the extract was performed by a new multi-toxin immunoaffinity column. Analytical performance characteristics were evaluated through single laboratory validation. Raw wheat and maize, corn flakes and maize snacks were chosen as representative matrices for method validation. The validation assay was carried out at 50, 100 and 150% of EU maximum permitted levels for each mycotoxin. Statistical analysis of the results (ANOVA) provided the within laboratory reproducibility and the error contributions from repeatability, between day effects, and influences from different matrix composition. Recoveries generally higher than 70% were obtained for all tested mycotoxins with relative standard deviation (within laboratory reproducibility) lesser than 37%. Limits of quantification (calculated as the lowest amount of each analyte which could be determined with a precision of 10%) ranged from 1μg/kg to 30μg/kg. The trueness of generated data was assessed by analysis of reference materials. The proposed method was proven to be suitable to assess, with a single analysis, compliance of the selected cereal based foods with the EU maximum permitted or recommended levels for all regulated mycotoxins.

Characterization of Fusarium verticillioides strains isolated from maize in Italy: Fumonisin production, pathogenicity and genetic variability

Fusarium verticillioides (teleomorph Gibberella moniliformis) is the main fungal agent of ear and kernel rot of maize (Zea mays L.) worldwide, including Italy. F.verticillioides is a highly toxigenic species since it is able to produce the carcinogenic mycotoxins fumonisins. In this study, 25 F. verticillioides strains, isolated from maize in different regions of Italy were analyzed for their ability to produce fumonisins, their pathogenicity and their genetic variability. A further referenced strain of G. moniliformis isolated from maize in USA was also used as outgroup. The fumonisins B₁, B₂, and B₃ were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pathogenicity tests were carried out by symptom observation and determination of growth parameters after inoculation of maize seeds, seedlings and wounded detached leaves. Total genomic DNA was used for Amplified Fragment Length Polymorphism (AFLP) analysis. About 20% of the analyzed strains were unable to produce fumonisins in in vitro experiments on inoculated maize flour, while, among fumonisin producers, a great variability was observed, with values ranging from 1 to 115 mg kg⁻¹. The different analyzed strains showed a wide range of pathogenicity in terms of effect on seed germination, seedling development and of symptoms produced on detached leaves, which were not correlated with the different in vitro fumonisin production. AFLP analysis indicated the presence of genetic diversity not only between the Italian strains and the American reference but also among the Italian isolates.

Anomericity of T-2 Toxin-glucoside: Masked Mycotoxin in Cereal Crops

T-2 toxin is a trichothecene mycotoxin produced when Fusarium fungi infect grains, especially oats and wheat. Ingestion of T-2 toxin contaminated grain can cause diarrhea, hemorrhaging, and feed refusal in livestock. Cereal crops infected with mycotoxin-producing fungi form toxin glycosides, sometimes called masked mycotoxins, which are a potential food safety concern because they are not detectable by standard approaches and may be converted back to the parent toxin during digestion or food processing. The work reported here addresses four aspects of T-2 toxin-glucosides: phytotoxicity, stability after ingestion, antibody detection, and the anomericity of the naturally occurring T-2 toxin-glucoside found in cereal plants. T-2 toxin-β-glucoside was chemically synthesized and compared to T-2 toxin-α-glucoside prepared with Blastobotrys muscicola cultures and the T-2 toxin-glucoside found in naturally contaminated oats and wheat. The anomeric forms were separated chromatographically and differ in both NMR and mass spectrometry. Both anomers were significantly degraded to T-2 toxin and HT-2 toxin under conditions that mimic human digestion, but with different kinetics and metabolic end products. The naturally occurring T-2 toxin-glucoside from plants was found to be identical to T-2 toxin-α-glucoside prepared with B. muscicola. An antibody test for the detection of T-2 toxin was not effective for the detection of T-2 toxin-α-glucoside. This anomer was produced in sufficient quantity to assess its animal toxicity.

Distribution of T-2 and HT-2 Toxins in Milling Fractions of Durum Wheat

The effect of processing on mycotoxin content in milling fractions has been investigated in 10 samples of durum wheat contaminated with T-2 and HT-2 toxins at levels ranging from 97 to 5,954 μg/kg (sum of T-2 and HT-2 toxins). Either naturally contaminated samples or samples artificially inoculated with Fusarium sporotrichioides under field conditions were used. A method based on liquid chromatography-tandem mass spectrometry coupled with immunoaffinity column cleanup was validated in-house for the simultaneous analysis of both toxins in a variety of matrices, including uncleaned wheat, cleaned wheat, screenings, bran, red dog, fine middlings, and semolina. Mean recoveries from samples spiked with T-2 and HT-2 toxins at levels of 100 μg/kg ranged from 85 to 107%, with relative standard deviations (RSDs) lower than 14%. The milling process led to an increase of T-2 and HT-2 toxin contents up to 13- and 5-fold in screenings and bran, respectively, compared with occurrence in the uncleaned wheat; however, an overall reduction of T-2 and HT-2 toxins by 54% (RSD, 20%) and 89% (RSD, 3%) was observed in cleaned wheat and in semolina, respectively.

Quantitative analysis of mycotoxins in cereal foods by collision cell fragmentation-high-resolution mass spectrometry: performance and comparison with triple-stage quadrupole detection.

A liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method for the simultaneous determination of aflatoxins (B1, B2, G1, G2), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in wheat flour, barley flour and crisp bread was developed. Mycotoxin fragmentation patterns obtained by high-energy collision dissociation (HCD) were investigated to obtain quantitative and confirmatory information (two characteristic masses per mycotoxin) using Orbitrap™-based high-resolution mass spectrometry. LC-HRMS (full-scan) detection carried out by HCD allows the monitoring of the pseudo-molecular ion and an additional characteristic fragment (for each mycotoxin) with mass accuracy in the range 0.1–3.9 ppm, meeting current European regulatory requirements for LC-MS confirmatory analysis. A sample preparation procedure based on polymeric solid-phase extraction cartridges was applied, allowing recoveries higher than 74% for nine mycotoxins, with a relative standard deviation lower than 13%. Detection limits in the range 0.5–3.4 µg kg−1 were obtained for three cereal matrices. A critical comparison between the proposed method and a validated method based on triple quadrupole mass spectrometry showed similar performance in terms of detection limits, recoveries and repeatability, and matrix effects. Based on an efficient sample extraction and clean-up, the LC-HCD-HRMS method reported here represents a reliable and robust alternative tool for mycotoxin analysis in food matrices as compared with well-established triple quadrupole-based approaches.