Mass spectrometry based metabolomics approaches for food safety, quality and nutrition

In the last years, the research in food science and nutrition has grown parallel to the consumers’ concern about the quality and safety of the food they eat. As a result, ensuring food safety, food quality and investigating on nutrition has never been more necessary than today. More powerful analytical procedures are now required to detect and identify undesired and toxic compounds. Moreover, consumers demand is now moving toward the valorization of food with potential health benefits. The identification of bioactive compounds is crucial to provide customers a healthy diet. Finally, one of the main challenges is to improve our limited understanding of the roles of nutritional compounds at molecular level. Food scientists have to face a large number of challenges to adequately answer the new emerging questions of food science. In this context, mass spectrometry showed to be a powerful tool for the investigation on food quality, safety and nutrition. The technical developments of chromatography coupled to mass spectrometry allowed several progresses for the detection of both endogenous compounds and contaminants in food. However, the main progress was due to high resolution mass spectrometry, a promising technique that has opened new horizons in screening and identification of a wide range of unknowns compounds. Mass spectrometry-based metabolomics is a key tool that has been involved, nowadays, in the study of the food and nutrition domains. As per definition, metabolomics includes the exhaustive study of the whole small metabolite composition of a particular system, the food metabolome is not an exception, being rich of endogenous and exogenous compounds with different properties and abundances. Nowadays, three main approaches for the screening of substances can be pointed out: targeted screening (analysis of few known compounds), suspected screening (analysis of a class of expected compounds) and untargeted screening (analysis of a wide range of unknown and unexpected compounds). In this thesis, the three approaches have been studied in details to understand their difficulties, advantages and disadvantages for the analysis of food metabolome. Several steps have been proved to be necessary for the development of a reliable analysis such as sample preparation, chromatographic and mass spectrometric method, data analysis and identification. The thesis is then divided in three main sections: targeted approach for food safety, suspected approach for food quality, untargeted approach for nutrition. In each section, one of the three main approaches used in food metabolomics has been studied for its application on one of the three different food analysis fields. Moreover, each topic has been treated focusing the attention on a specific step of the method development. The topic of metabolomics for food safety has been studied in a targeted approach by developing a method for the analysis of secondary metabolites of fungi, namely mycotoxins, in food (Paper I and II). A particular material for the clean-up of mycotoxins for their quantification in milk and cereals was presented. The topic of metabolomics for food quality has been treated in a suspected approach by developing a method for the analysis of several classes of secondary metabolites of plants such as phenolic acids, flavonoids, and glucosinolates in food (Paper III, IV and V). The development of a chromatographic and mass spectrometric method for the metabolic profiling of strawberry and cauliflower was presented. The topic of metabolomics for nutrition has been treated in an untargeted approach by developing a method for the analysis of the human urinary metabolome after the consumption of meat and dairy products. Several data analysis approaches have been shown for the investigation of the whole urinary metabolome. Ultimately, it has been shown how several aspects should be taken into account when analyzing complex matrices like food through different approaches. Sample preparation, chromatographic and mass spectrometric methods and data analysis have to be treated in different ways based on the used approach. In each case, the advantages of each technique can be exploited based on the purpose of the study. However, aside from the challenges that have to be faced, mass spectrometry-based metabolomics can definitely represent a powerful tool for food safety, quality and nutrition

Liquid chromatographic strategies for separation of bioactive compounds in food matrices

Nowadays, there is an increasing attention for nutraceuticals and, in general, bioactive compounds naturally present in food. Indeed, the possibility of preserving human health and preventing disease (e.g., cardiovascular diseases, cancer etc.) by the intake of healthy food is attractive for both consumers and food industries. In turn, research in this field was also prompted significantly, with the aim of characterizing these bioactive compounds and ascribe to them a specific activity. The bioactive compounds can belong to several chemical classes. However, their chemical diversity and presence in complex matrices, such as food, make it challenging both their isolation and characterization. To tackle this issue, efficient separation systems are needed, which are mainly based on chromatography. In this context, this mini-review aims to provide the reader with an overview of the most relevant and recent approaches for the separation of the most common bioactive compounds in food, in particular polyphenols, phenols, carotenoids, and peptides, by liquid chromatography approaches. © 2018 by the authors

A rapid magnetic solid phase extraction method followed by liquid chromatography-tandem mass spectrometry analysis for the determination of mycotoxins in cereals

Mycotoxins can contaminate various food commodities, including cereals. Moreover, mycotoxins of different classes can co-contaminate food, increasing human health risk. Several analytical methods have been published in the literature dealing with mycotoxins determination in cereals. Nevertheless, in the present work, the aim was to propose an easy and effective system for the extraction of six of the main mycotoxins from corn meal and durum wheat flour, i.e., the main four aflatoxins, ochratoxin A, and the mycoestrogen zearalenone. The developed method exploited magnetic solid phase extraction (SPE), a technique that is attracting an increasing interest as an alternative to classical SPE. Therefore, the use of magnetic graphitized carbon black as a suitable extracting material was tested. The same magnetic material proved to be effective in the extraction of mycoestrogens from milk, but has never been applied to complex matrices as cereals. Ultra high-performance liquid chromatography tandem mass spectrometry was used for detection. Recoveries were > 60% in both cereals, even if the matrix effects were not negligible. The limits of quantification of the method results were comparable to those obtained by other two magnetic SPE-based methods applied to cereals, which were limited to one or two mycotoxins, whereas in this work the investigated mycotoxins belonged to three different chemical classes

Polydopamine-coated magnetic nanoparticles for isolation and enrichment of estrogenic compounds from surface water samples followed by liquid chromatography-tandem mass spectrometry determination

Estrogens, phytoestrogens, and mycoestrogens may enter into the surface waters from different sources, such as effluents of municipal wastewater treatment plants, industrial plants, and animal farms and runoff from agricultural areas. In this work, a multiresidue analytical method for the determination of 17 natural estrogenic compounds, including four steroid estrogens, six mycoestrogens, and seven phytoestrogens, in river water samples has been developed. (Fe3O4)-based magnetic nanoparticles coated by polydopamine (Fe3O4@pDA) were used for dispersive solid-phase extraction, and the final extract was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry. The Fe3O4 magnetic nanoparticles were prepared by a co-precipitation procedure, coated by pDA, and characterized by scanning electron microscopy, infrared spectroscopy, and elemental analysis. The sample preparation method was optimized in terms of extraction recovery, matrix effect, selectivity, trueness, precision, method limits of detection, and method limits of quantification (MLOQs). For all the 17 analytes, recoveries were >70 % and matrix effects were below 30 % when 25 mL of river water sample was treated with 90 mg of Fe3O4@pDA nanoparticles. Selectivity was tested by spiking river water samples with 50 other compounds (mycotoxins, antibacterials, conjugated hormones, UV filters, alkylphenols, etc.), and only aflatoxins and some benzophenones showed recoveries >60 %. This method proved to be simple and robust and allowed the determination of natural estrogenic compounds belonging to different classes in surface waters with MLOQs ranging between 0.003 and 0.1 μg L(-1). Graphical Abstract Determination of natural estrogenic compounds in water by magnetic solid phase extraction followed by liquid chromatography-tandem mass spectrometry analysis

microbeMASST: A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

microbeMASST, a taxonomically informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbe-derived metabolites and relative producers without a priori knowledge will vastly enhance the understanding of microorganisms’ role in ecology and human health

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms’ role in ecology and human health

UNTARGETED METABOLIC PROFILING BY MEANS OF UHPLC-QTOF/MS FOR THE IDENTIFICATION OF MEAT AND DAIRY PRODUCTS INTAKE BIOMARKERS

Meat intake has been associated with positive as well as negative health outcomes. Meat is essential from a nutritional point of view but, on the other hand, has been associated with increased risk of several diseases. However, a causal relation between meat consumption and negative health outcomes is still controversial. In order to shed light on this issue, a reliable method for assessment of meat intake is needed. Meat intake has often been assessed using very imprecise methods. Dietary biomarkers measured in biological fluids by means of untargeted metabolomics have been proposed as a possible way to find objective biomarkers for measurement of the actual intake of specific foods.A meat intake marker should be able to differentiate meat consumption from that of other protein-rich foods, such as dairy products. For this purpose, a randomized, controlled, cross-over single meal study was conducted on 17 subjects to collect urine samples at several time points after the ingestion of red meat or dairy product based meals.An ultra-high-performance-liquid chromatograph coupled via an electrospray source to a Q-TOF mass spectrometer was used for the separation and metabolic profiling of the urine samples. Data were analyzed and group differences compared by both univariate and multivariate data analysis and results combined. As a result, 37 compounds were found to be highly important for discriminating the two diets; in particular 20 markers were higher after the dairy-based meal, while 17 markers were higher after the meat-based meal. These markers were fragmented and tentatively identified. Some of them were synthesized to allow confirmation of their identity. Combinations of these markers should be further validated as meat or dairy product intake biomarkers