Isotopic, mycotoxin, and pesticide analysis for organic authentication along the production chain of wheat-derived products.

Wheat-based products are staples in diets worldwide. Organic food frauds continuously threaten consumer trust in the agri-food system. A multi-method approach was conducted for the organic authentication and safety assessment of pasta and bakery products along their production chain. Bulk and Compound-Specific (CS) Isotope Ratio Mass Spectrometry (IRMS) suggested the δ15Nbulk, δ15Nleucine and δ15Nproline as promising organic markers, with CS able to distinguish between pairs which bulk analysis could not. Processing significantly affected the values of δ15Nleucine, δ13Cproline and δ13Cleucine. Multi-mycotoxin analysis (HT-2, T-2, DON, ZEN, OTA, AFB1) revealed higher contamination in conventional than organic samples, while both milling and baking significantly reduced mycotoxin content. Lastly, from the evaluation of 400 residues, isopyrazam was present at the highest concentration (0.12 mg/kg) in conventional wheat, exhibiting a 0.12 Processing Factor (PF), while tebuconazole levels remained unchanged in pasta production (90 °C) and reduced below LOQ in biscuits and crackers (180-250 °C).

QuEChERS method combined to liquid chromatography high-resolution mass spectrometry for the accurate and sensitive simultaneous determination of pyrrolizidine and tropane alkaloids in cereals and spices.

Within the last decades, in the EU, there has been an increasing interest in toxic plant alkaloids as food contaminants, especially after the continuous and growing consumption of plant-based foods compared with food of animal origin. In this regard, the once neglected presence of these tropane alkaloids (TAs) and pyrrolizidine alkaloids (PAs) has recently been reconsidered by the European Food Safety Authority, highlighting the lack of data and the need to develop risk assessment strategies. For this reason, the emphasis has been placed on detecting their occurrence in food through the development of accurate and sensitive analytical methods to achieve the determination of these compounds. The present study aims to elaborate and validate an analytical method based on QuEChERS sample preparation approach, exploiting the UHPLC coupled to the HRMS to simultaneously identify and quantify 21 PAs and two TAs in cereals and spices. For TAs, the obtained limit of detection (LOD) is 0.1 µg·kg-1 and the limit of quantification (LOQ) is 0.4 µg·kg-1 , while for PAs, the LODs values ranging between 0.2 to 0.3 µg·kg-1 and the LOQ, between 0.4 and 0.8 µg·kg-1 , ensuring compliance with the recently established European Regulations. Several commercial samples were analysed to further verify the applicability of this comprehensive analytical approach.

Non-targeted high-resolution mass spectrometry study for evaluation of milk freshness.

Milk freshness is an important parameter for both consumers' health and quality of milk-based products. Up to now there have been neither analytical methods nor specific parameters to uniquely define milk freshness from a complete and univocal chemical perspective. In this study, 8 molecules were selected and identified as responsible for milk aging, using a liquid chromatography-high-resolution mass spectrometry approach followed by chemometric data elaboration. For model setup and marker selection, 30 high-quality pasteurized fresh milk samples were collected directly from the production site and analyzed immediately and after storage at 2 to 8°C for 7 d. The markers were then validated by challenging the model with a set of 10 milk samples, not previously analyzed. Our results demonstrated that the markers identified within this study can be successfully used for the correct classification of non-fresh milk samples, complementing and successfully enhancing parallel evaluations obtainable through sensory measures.

Practical approach to develop a multi-group screening method for detection of mycotoxins, pesticides and veterinary drugs in food.

A multi-group screening method to detect residues of veterinary drugs in meat and environmental contaminants in wheat flour has been developed using liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (LC-HRMS). The procedure was tested for over 300 representative compounds (173 veterinary drugs, 122 pesticides and 9 mycotoxins) analysing in parallel negative and positive (spiked) samples according to European validation rules. The Screening Target Concentrations (STCs) were chosen conservatively with respect to the method purposes. Interpretation of results was based on retention time, mass accuracy of precursor and MS2 spectral library. Evaluating the percentage of false negative results, 280 out of the 304 analytes were detectable at the STCs (false compliant rate ≤ 5%). In wheat flours, incurred levels of mycotoxins, deoxynivalenol and 3-acetyldeoxynivalenol, higher than STCs, were frequently found, whereas in meat, the most detected veterinary drugs were antibiotics generally at negligible concentrations (<10 µg kg-1 ). Finally, seven test materials from proficiency test schemes were successfully tested.

ESEM-EDS-based analytical approach to assess nanoparticles for food safety and environmental control.

Particulate contamination in the form of nanoparticles (NPs) can occur not only in the environment but also in the food production chain, making human exposure almost inevitable. In the present study the potentialities of Environmental Scanning Electron Microscopy coupled with Energy-Dispersive X-ray Spectroscopy (ESEM-EDS) were exploited for the detection and visualization of inorganic NPs in air, raw materials and food products along the pasta production chain (wheat ear, wheat, semolina and pasta). Investigation of the elemental composition of NPs in the same samples was also carried out by using two independent techniques: ESEM-EDS and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). As for airborne particles, size-fractionated sampling was performed by an eight-stage cascade impactor located near the production plant with different exposition times for both ESEM-EDS and gravimetric analysis, respectively. In the case of raw materials and food products, the particles were collected on polycarbonate filters after immersion in milli-Q water in order to recover the NPs deriving from external environmental contamination. Only particle sizes lower than 0.8 µm were taken into consideration when ESEM-EDS analysis was carried out. NPs containing mainly Fe and Ti having dimensions <0.15 µm were identified in the filters used for wheat ear, wheat, semolina and pasta samples. As for air monitoring, gravimetric analysis showed an increase in the concentration levels of total particles during winter, particularly in the case of fine particulate matter (PM2.5). ESEM-EDS analysis, performed on filters of the VII and final stages, evidenced the same trend observed by gravimetric analysis, the majority of Fe-containing NPs being detected.

Egg product freshness evaluation: A metabolomic approach.

Egg products' freshness is a crucial issue for the production of safe and high-quality commodities. Up to now, this parameter is assessed with the quantification of few compounds, but the possibility to evaluate more molecules simultaneously could help to provide robust results. In this study, 31 compounds responsible of freshness and not freshness of egg products were selected with a metabolomic approach. After an ultrahigh-pressure liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) analysis, different chemometric models were created to select gradually the most significant features that were finally extracted and identified through HRMS data. Sample lots were collected directly from their arrival at the production plant sites, extracted immediately after, then left at room temperature, and extracted again after 24 and 48 hours (first day and second day, respectively). A total amount of 79 samples was used for the model creation. Furthermore, the same compounds were detected in seven new egg products sample lots not used for the model creation and treated with the same experimental design (total amount of samples, 21). The results obtained clearly demonstrate that these 31 molecules can be considered real freshness or not freshness chemical markers. Furthermore, this UHPLC-HRMS metabolomic approach allows for the detection of a larger set of metabolites clearly related to possible microbial growth over time, which is a relevant point for also ensuring food safety.

In-house validation and small-scale collaborative study to evaluate analytical performances of multimycotoxin screening methods based on liquid chromatography-high-resolution mass spectrometry: Case study on Fusarium toxins in wheat.

A strong trend toward using highly selective mass spectrometry technologies for screening of multiple mycotoxins has been observed in recent years. In the present study, the process of validation of a multimycotoxin screening method based on liquid chromatography-high-resolution mass spectrometry method is presented. The method was intended for the simultaneous screening of the major Fusarium toxins (deoxynivalenol, 3- and 15-acetyl deoxynivalenol, T-2 and HT-2 toxins, zearalenone, enniatins A, A1, B, and B1, and beauvericin) in wheat. The sample preparation protocol was based on a double extraction (methanol followed by acetonitrile/water mixture) and purification through solid-phase extraction C18 column. To provide insights for full exploitation of the potential of the double-stage high-resolution mass spectrometry detection, a full-scan acquisition event followed by a sequence of 5 fragmentation events (variable data-independent acquisition) was set for mycotoxin detection, the latter to be exploited for confirmatory purposes. Method analytical performances were evaluated through in-house validation and small-scale interlaboratory study, designed according to Commission Regulation 519/2014/EU, setting performance requirements for screening methods for mycotoxins. Screening target concentrations were close to European Union maximum permitted or indicative levels. The in-house validation provided the precision of the response under repeatability conditions and the intermediate precision (both resulting lower than 30%), the cutoff value, and the rate of false suspect results for negative (free of the mycotoxin of interest) samples, which resulted lower than 0.1% in all cases. The collaborative study provided reproducibility and laboratory independent cutoff values. Analysis of reference materials proved method trueness and suitability for screening of the major Fusarium mycotoxins in wheat. Finally, the applicability of the full-scan/variable data-independent acquisition detection approach was successfully tested on a set of naturally contaminated wheat samples, where 2 characteristic product ions could be detected for all identified mycotoxins even at levels in the low µg/kg range.

LC-HRMS for Characterizing Durum Wheat Pasta Production Variability and Consumer Overall Liking.

Semolina pasta represents one of the most important dishes in Italian cuisine worldwide. Italy is the leader in its production and, recently, the worldwide diffusion of its production has begun to grow tremendously. The perceived quality of a food product, such as pasta, is a key feature that allows a company to increase and maintain the competitive advantage of a specific brand. The overall flavor perception of the consumer, therefore, has become as important as other key quality factors such as texture and color; thus, the food industry needs to meet consumer expectations and needs the tools to objectively "measure" the quality of food products. Untargeted fingerprinting by means of coupling LC with high-resolution MS (HRMS) has been well received within the analytical community, and different studies exploiting this approach for the characterization of high-value food products have recently been reported in the literature. In the present work, a tentative application of the sensomics approach to cluster analysis of semolina pasta obtained using different production conditions was developed to objectively define target molecules that correlate with consumer overall liking of an industrial standard product. Principal component analysis of chemical and physical testing, GC-MS, LC-HRMS, and sensory data were performed with the aim of identifying the main parameters to discern similarities and differences among samples and clustering them according to these features. The correlation between analytical data and compounds related to sensory data was further investigated, and lastly, a partial least-squares regression model for the prediction of consumer overall liking was reported.

Quantitative targeted and retrospective data analysis of relevant pesticides, antibiotics and mycotoxins in bakery products by liquid chromatography-single-stage Orbitrap mass spectrometry.

In addition to 'traditional' multi-residue and multi-contaminant multiple reaction monitoring (MRM) mass spectrometric techniques devoted to quantifying a list of targeted compounds, the global food industry requires non-targeted methods capable of detecting other possible potentially hazardous compounds. Ultra-high-performance liquid chromatography combined with a single-stage Orbitrap high-resolution mass spectrometer (UHPLC-HRMS Exactive™-Orbitrap Technology) was successfully exploited for the complete selective and quantitative determination of 33 target compounds within three major cross categories (pesticides, antibiotics and mycotoxins) in bakery matrices (specifically milk, wheat flour and mini-cakes). Resolution was set at 50 000 full width at half maximum (FWHM) to achieve the right compromise between an adequate scan speed and selectivity, allowing for the limitations related to the necessary generic sample preparation approach. An exact mass with tolerance of 5 ppm and minimum peak threshold of 10 000 units were fixed as the main identification conditions, including retention time and isotopic pattern as additional criteria devoted to greatly reducing the risk of false-positive findings. The full validation for all the target analytes was performed: linearity, intermediate repeatability and recovery (28 analytes within 70-120%) were positively assessed; furthermore, limits of quantification between 5 and 100 µg kg(-1) (with most of the analytes having a limit of detection below 6 µg kg(-1)) indicate good performance, which is compatible with almost all the regulatory needs. Naturally contaminated and fortified mini-cakes, prepared through combined use of industrial and pilot plant production lines, were analysed at two different concentration levels, obtaining good overall quantitative results and providing preliminary indications of the potential of full-scan HRMS cluster analysis. The effectiveness of this analytical approach was also tested in terms of the formulation of hypotheses for the identification of other analytes not initially targeted which can have toxicological implications (e.g. 3-acetyl-deoxynivalenol and deoxynivalenol-3-glucoside), opening a window on retrospective investigation perspectives in food safety laboratories.

Reliable liquid chromatography-mass spectrometry method for investigation of primary aromatic amines migration from food packaging and during industrial curing of multilayer plastic laminates.

Primary aromatic amines (PAAs) can migrate from packaging into food from different sources such as polyurethanic adhesives used for the manufacture of multilayer films, which may contain residual aromatic isocyanates, or recycled paperboard, because of the presence of azo dyes in the printed paper massively used in the recycling process. In the present work, a reliable analytical method, exploiting a conventional high-performance liquid chromatography-(selected ion monitoring)-mass spectrometry system, for PAAs compliance assessment in food contact materials was developed as an effective alternative to the current standard spectrophotometric one, moving in this way from the screening to the accurate and selective quantitation perspective for the analysis of PAAs both in aqueous and acidic food simulants. The main validation parameters were verified achieving very satisfactory results in terms of linearity range, limit of detection (ranging from 0.1 to 1.0 µg kg(-1)) and quantitation (ranging from 0.1 to 3.6 µg kg(-1)), repeatability and accuracy. Suitability of the method was demonstrated for a wide range of commercial samples, chosen among different producers of the most common used food packaging plastic and paperboard categories and then analyzed to assess the risk related to PAAs migration. Finally, the method was also successfully exploited to monitor the evolution of potential PAAs migration during the industrial curing process of multilayer plastic laminates, prior to their release for delivery to the food industry end user.

Liquid chromatography-electrospray ionization-tandem mass spectrometry method for the determination of epoxidized soybean oil in food products.

Epoxidized soybean oil (ESBO) is widely used as a plasticizer and stabilizer in such polymers [poly(vinyl chloride) in particular] commonly adopted for manufacturing of gaskets of the lids for glass jars and plastic films for food packaging. Human exposure to ESBO and its derivatives is likely to occur over a lifetime with a significant variation according to life stage. A reversed phase liquid chromatography interfaced with electrospray ion trap tandem mass spectrometry method for the determination of ESBO in foods was developed. A simple sample treatment procedure entailing the use of an extraction step with dichloromethane without any further cleanup was proved. Chromatographic separation was performed using two C18 columns with an aqueous acetic acid-acetone-acetonitrile mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (4 mg kg(-1)), quantitation limits, linearity (established over 2 orders of magnitude), recovery (good mean recoveries, higher than 90% for all of the signals detected), precision (RSD% < 8), and trueness. The applicability of the method to the determination of ESBO in different food matrices (in particular those rich in edible oil) was demonstrated, and the performances were compared to those reachable by the commonly well-known gas chromatography-mass spectrometry procedure.