Polar pesticides in food of animal origin: interlaboratory validation to evaluate method fitness-for-purpose of official control.

The present work reports on the design, execution and evaluation of results of an interlaboratory validation study aimed at verifying the fitness-for-purpose of a LC-MS/MS method for the detection of polar pesticides in food of animal origin in official control and monitoring programmes. To this scope, five participant laboratories, with relevant expertise, were recruited. After passing a pre-trial test, the participants were asked to analyse test samples of bovine fat, chicken eggs and cow's milk, contaminated with 11 polar pesticides (group A: Aminomethyl phosphonic acid (AMPA), cyanuric acid, ethephon, glyphosate, fosetyl aluminium, 2-hydroxyethyphosphonic acid (HEPA), maleic hydrazide, N-acetyl-glyphosate, group B: N-acetyl glufosinate (NAG), 3-methylphosphinicopropionic acid (MPP) and glufosinate ammonium) at two different levels (0.05 and 0.25 mg/kg-1 and 0.01 and 0.05 mg/kg-1 for group A and B respectively. The method was based on acidified methanol/water extraction followed by dSPE clean up with C18 sorbent. For LC-MS/MS analysis isotopically labelled standards were used for all targeted analytes. With a couple of exceptions, average recoveries ranged from 85% to 110%, with repeatability (RSDr) ranging from 3% to 25%, and reproducibility (RSDR) from 4% to 26%. The assessment by different laboratories provided also insights on key factors impacting method performance characteristics and its implementation by new users.

Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities.

The present manuscript reports on monitoring data of 12 ergot alkaloids (EAs) in cereal and cereal-derived products, collected in Italy over the period 2017-2020, for official control purposes under the edge of the Commission Recommendation 2012/154/EU on the monitoring of the presence of EAs in feed and food. To these purposes, an LC-MS/MS method was set up and applied, after in-house verification of its analytical performance. Besides satisfactory recoveries and precision, the method's quantification limits proved suitable to assess the compliance of cereals and cereal-based foods with the recently issued EU maximum permitted levels (Commission Regulation 2021/1399/EU). The validity of the generated data was also evaluated through the adoption of four proficiency tests, from which acceptable z-score values (-2 ≤ z ≤ 2) were obtained. The method was then applied to analyse a total of 67 samples, collected in Italy over the period 2017-2020. The samples consisted of 18 cereal grains, 16 flours (14 of wheat and 2 of spelt) and 31 other types of cereals derivatives (including 9 for infants). Overall, the EAs analysis returned a high percentage of left-censored data (>86%). Among the positive samples, the highest contamination levels, up to 94.2 µg/kg, were found for ergocristine (12% incidence), followed by ergocristinine (7% incidence) with levels of up to 48.3 µg/kg.

Introduction to the Toxins Special Issue on Improved Analytical Technologies for the Detection of Natural Toxins and Their Metabolites in Food.

Food, by nature, is a biological substrate and is therefore capable of supporting the growth of microbials that are potential producers of toxic compounds [...].

Rapid and reliable detection of glyphosate in pome fruits, berries, pulses and cereals by flow injection - Mass spectrometry.

A flow injection - mass spectrometry method for rapid glyphosate detection in food commodities was developed and validated. The sample preparation protocol included a simple and rapid extract purification step through polymeric solid phase extraction cartridges followed by addition of isotopically labeled glyphosate to the final test sample. The optimized method was subjected to intra-laboratory validation (spiking range 0.5-100 mg/kg) in chickpeas, grapes and apples, as representatives of three different commodity groups as defined in SANTE/11813/2017 guidelines. Recoveries were in the range 60-111%, repeatability and within laboratory reproducibility were ≤17%.The trueness of the results generated with the developed method was evaluated by analysis of a set of incurred chickpea and wheat samples (glyphosate range 0.5-36 mg/kg) and comparison with the reference method (Quick Polar Pesticides Method), confirming the method fitness-for-purpose of rapid compliance testing.

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.

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.

Simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in maize by liquid chromatography/tandem mass spectrometry after multitoxin immunoaffinity cleanup.

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.

Analysis of T-2 and HT-2 toxins in cereal grains by immunoaffinity clean-up and liquid chromatography with fluorescence detection.

A sensitive, precise and accurate method has been developed for the simultaneous determination of T-2 and HT-2 toxins in cereal grains at ppb levels using high-performance liquid chromatography (HPLC) with fluorescence detection and 1-antroylnitrile (1-AN) as labeling reagent after immunoaffinity clean-up. Cereal samples were extracted with methanol/water (90:10, v/v), and the extracts were cleaned-up through commercially available immunoaffinity columns containing monoclonal anti-T-2 antibodies (T-2 test HPLC, Vicam). T-2 and HT-2 toxins were quantified by reversed-phase HPLC with fluorometric detection (excitation wavelength 381 nm, emission wavelength 470 nm) after derivatization with 1-AN. The monoclonal antibody showed 100% cross-reactivity with both T-2 and HT-2 toxin, and the immunoaffinity column clean-up was effective up to 1.4 microg of both toxins. The method was successfully applied to the analysis of T-2 and HT-2 toxins in wheat, maize and barley. Recoveries from spiked samples with toxin levels from 25 to 500 microg/kg ranged from 70% to 100%, with relative standard deviation generally lower than 8%. The limit of detection of the method was 5 microg/kg for T-2 toxin and 3 microg/kg for HT-2 toxin, based on a signal-to-noise ratio 3:1. HT-2 toxin was detected in ten naturally contaminated wheat samples out of 14 samples analyzed, with toxin levels ranging from 10 to 71 microg/kg; three of them contained also T-2 toxin up to 12 microg/kg.