Per- and polyfluoroalkyl substances (PFAS) are persistent environmental contaminants with known toxic effects. Among emerging PFAS, certain compounds, such as ClPFPECA, GenX, NaDONA, 9Cl-PF3ONS, and 11Cl-PF3OUdS, have attracted attention due to their involvement in major environmental contamination events. In this study, an LC-HRMS/MS method was developed and validated for their simultaneous determination in food. The method showed satisfactory precision and trueness, with LOQs between 10 and 100 ng/kg. It was then applied to 35 food samples, including eggs, vegetables and meat, collected from local markets and home production. All analytes were below the LOQ, except for the sporadic presence of three ClPFPECA in home-produced eggs. The four PFAS currently regulated in the EU were also analyzed to assess co-occurrence. Five non-commercial eggs out of the nine tested exceeded the maximum limits, confirming the results of previous studies that home-produced eggs may have higher PFAS levels than commercially farmed eggs.

The availability of routinely applicable analytical methods is a prerequisite for performing quantitative risk assessment of allergens due to accidental food contamination. Over the past 15 years, liquid-chromatography coupled to tandem mass-spectrometry (LC-MS/MS) has become one of the techniques of choice, mainly due to its superior selectivity and ability to simultaneously detect multiple allergenic proteins in complex matrices. This paper reviews the LC-MS/MS procedures published to date for the quantitative determination of six major allergens (milk, eggs, soy, almonds, hazelnuts, and peanuts) in baked goods in order to evaluate and compare their trueness, precision, and limits of detection and quantification. Quantifying proteins through bottom-up approaches is a complex challenge because the measurand (protein) does not coincide with the entity that is actually measured (signature peptide). This inherent complexity is often not accompanied by the application of rigorous analytical methodology and terminology, which has prevented an exhaustive assessment of whether the procedures examined are fit for purpose. Some papers do not provide a comprehensive and clear description of validation design and performed experiments; different types of measurement standards are used, as well as different calibration strategies that are often not fully detailed. Overall, although the sensitivity of methods is generally adequate, the quantification of allergens in processed foods using LC-MS/MS does not currently appear to have achieved a sufficient degree of reliability. To address all these issues, harmonized and internationally recognized guidelines on calibration strategies and validation of quantitative allergen methods would be required.

Pain management in horses plays a pivotal role in the therapeutic approach to several diseases. Horses have cannabinoid receptors at the level of dorsal root ganglia, blood vessels, and synoviocytes that can be up or down- regulated by inflammatory conditions, justifying the possible efficacy of exogenous cannabinoids (i.e., phytocannabinoids) in managing several painful pathologies in this animal species. However, the current use of supplements containing cannabidiol (CBD) in equines is based on anecdotal evidence, without the support of sufficient pharmacokinetic studies. In humans, the concentration peak of CBD and the area under the concentration-time curve (AUC) are both strongly influenced by food administration. Also, in equids, the oral bioavailability of some drugs can be influenced by the meal but no information is available about CBD. This study investigated the pharmacokinetics of CBD following single oral administration of two different formulations of pure CBD (oil and paste), dosed at 1 mg/kg, at two different times about food administration. CBD oil and CBD paste were administered orally at 1 mg/kg to eight healthy horses according to a cross over design, and blood samples were taken at pre-fixed time-points for the pharmacokinetic analyses. The obtained pharmacokinetic data did not allow for statistically significant differences between formulations (paste or oil) and feeding time (fed and fasted status). However, following treatment with the paste, the Cmax was achieved in a shorter time range compared to the oily formulation, indicating that it could be a better formulation to consider in future equine studies.

Food products are prone to microbial contamination able to affect their safety and quality attributes and their nutritional value. The interest in the potential use of bioactive compounds deriving from natural matrices, especially agro-industrial wastes, as alternatives to classical food preservatives has rapidly increased. In the present study a food grade olive mill wastewater polyphenolic extract and a commercial mix were characterised and their antioxidant and antimicrobial capacity were assessed. The antimicrobial activity was preliminary assessed in vitro by agar well diffusion, subsequently by microdilution method to define the minimum inhibitory and bactericidal concentration. The olive mill wastewater polyphenolic extract registered a higher antioxidant capacity [(13.3 +/- 1.0) 102 mu gTE/(100 g)] and antimicrobial efficacy (max MBC value 0.2500 g/mL) compared to commercial mix with wide potential application in food industry.

The impact of soil fertilization with animal manure on the spread and persistence of antibiotic resistance in the environment is far from being fully understood. To add knowledge about persistence and correlations between antibiotic residues and antibiotic resistance genes (ARGs) in fertilized soil, a longitudinal soil mesocosm study was conducted. Soil samples were collected from the mesocosms immediately before spreading and then afterward at fifteen time points during a 320-day observation period. Eight ARGs (ermB, sul1, tetA, tetG, tetM, cfr, fexA, and optrA) and the class 1 integron-integrase gene, intI1, were determined in both pig slurry and soil, as well as residues of 36 antibiotics. Soil chemical and biochemical parameters were also measured. Twelve antibiotics were detected in the slurry in the range of 3 µg kg−1–3605 µg kg−1, with doxycycline, lincomycin, and tiamulin being the most abundant, whereas ermB, sul1, and tetM were the predominant ARGs. Before spreading, neither antibiotic residues nor ARGs were detectable in the soil; afterwards, their concentrations mirrored those in the slurry, with a gradual decline over the duration of the experiment. After about three months, the effect of the amendment was almost over, and no further evolution was observed.

• A pool of 28 wild boar livers analysed for emerging PFAS, by LC-HRMS. • Chloropolyfluoro polyethers carboxilates substances (ClPFPECAs) were identified. • Six ClPFPECA congeners of the product CAS # 329238-24-6 determined at 4.1 ng g −1 . • ClPFPECA dechlorinated forms and C6O4 substance estimated at 7.9 and 1.8 ng g −1 . • Results suggest a bioaccumulative behaviour of ClPFPECAs in wild boar liver.

Tyrosol (T) and hydroxytyrosol (HT) are phenyl alcohol polyphenols with well-recognized health-promoting properties. They are widely diffused in several vegetables, especially in olive products (leaves, fruits and oil). Therefore, they could be present in food produced from herbivorous animals such as in milk and cheese. In this study, an analytical method to determine T, HT and some of their phase II metabolites (sulphates and glucuronides) in cheese was developed and validated. Samples were extracted with an acidic mixture of MeOH/water 80/20 (v/v) and, after a low temperature clean-up, the extracts were evaporated and injected in a liquid-chromatography coupled with high resolution mass spectrometry (LC-Q-Orbitrap). A validation study demonstrated satisfactory method performance characteristics (selectivity, linearity, precision, recovery factors, detection and quantification limits). The developed protocol was then applied to analyze 36 Italian cheeses made from ewe, goat and cow milk. The sum of detected compounds (T, tyrosol sulfate, hydroxytyrosol-3-O-sulfate and hydroxytyrosol-4-O-sulfate) reached as high as 2300 µg kg−1 on a dry weight basis, although in about 45% of cow cheeses it did not exceed 50 µg kg−1. Ewe cheeses were significantly richer of polyphenols (sum) as well as HT sulfate metabolites than cow cheeses. In conclusion, results shows that cheese cannot be considered an important dietary source of these valuable compounds.

A "two-procedure approach " was developed to detect and confirm residues of 64 antibiotic substances in honey using liquid-chromatography coupled to quadrupole time -of-flight mass spectrometry. Eight antimicrobial classes were determined (amphenicols, lincosamides, macrolides, nitroimidazoles, pleuromutilins, quinolones, sulfonamides and tetracyclines) together with single drugs. The procedures were validated in the range 0.2-50 mu g kg(-1), obtaining recovery factors from 67 to 100% and coefficients of variation in within-lab reproducibility conditions lower than 20%. Limits of detection and quantification were in the ranges 0.1-3.3 mu g kg(-1) and 0.2-11 mu g kg(-1), respectively, satisfying the current European levels for analytical control of antibiotic residues in honey. An extensive matrix effect study was performed considering acacia, chestnut and multiflower honey types. Method performances were confirmed through participation in proficiency tests obtaining z-scores between-0.7 and +1.08. The proposed approach was applied to honey samples collected in local markets and three out of fifty-five samples tested positive. The found molecules were sulfonamides (sulfamethazine and sulfathiazole) and tetracyclines (oxytetracycline and tetracycline) at concentrations lower than 2 mu g kg(-1). Two out of three contaminated samples contained honey from extra-EU countries.

The present study evaluated the effects of feed supplementation with olive oil by-products on the microbial quality of rabbit meat. Thirty-three New Zealand White rabbits were randomly assigned to three experimental grower–finisher diets. Each dietary group consisted of three experimental treatments: (1) a basal control diet (C), (2) a C diet supplemented with a low dosage of polyphenol (150 mg/kg, L), and (3) a C diet supplemented with a high dose of polyphenols (280 mg/kg, H). Polyphenol analyses in feed and meat were performed using the liquid-chromatography coupled to tandem mass spectrometry technique (LC-MS/MS). Higher amounts of sulphate metabolites were detected in the H group. Microbiological quality was evaluated on Longissimus lumborum muscles stored under aerobic conditions at 4 °C. The H diet exerted an inhibitory effect on microbial growth (p < 0.001), notably for Pseudomonas spp., when compared to C and L diets; differences among the groups were observed starting from 6 days of storage. In the H group, the Pseudomonas spp. population showed an increase in the latency phase and a decrease in the maximum growth rate of the fitted curves in comparison with the C and L groups. The use of dietary polyphenols could be a strategy to reduce spoilage during meat storage.

A multiclass method for the determination of antimicrobial substances in eggs has been developed and validated, covering sixty-three antibiotics belonging to ten families. After extraction with acidified acetonitrile and EDTA, the analytes were injected into a liquid-chromatography high-resolution mass-spectrometry system operating in positive ionization mode. The procedure was successfully validated as confirmatory method evaluating selectivity, linearity, precision, recovery, decision limit, detection capability, limits of detection and quantitation. The here developed is the first method including all the antibiotics with Maximum Residue Limit (MRL) in eggs, except very polar drugs (aminoglycosides and colistins), which require specific protocols. Finally, a wide survey was carried out analyzing 100 commercial eggs samples from local markets. Only 3% contained residues of authorized substances at concentrations lower than the relevant MRLs.

Sample manipulation for storage and storage itself, interfere with the stability of labile lipids in human plasma, including vitamin E (alpha-tocopherol), polyunsaturated fatty acids (PUFAs), and their enzymatic and free radicalderived oxidation metabolites. This remains a main limit of lipidomics studies that often lack of sufficient standardization and validation at the pre-analytical level. In order to characterize the stability of these lipids in human plasma and to develop a standardized preanalytical protocol for lipidomics methods, the oxidation metabolites of alpha-tocopherol, the free form of omega 3 and omega 6 PUFAs, and some arachidonic acid (AA)-derived eicosanoids were investigated in human plasma during storage at different freezing temperatures. The effect of a protection/defense cocktail of antioxidants and lipoxygenase inhibitors (PD solution) on these lipid parameters was also evaluated. The temperature of storage markedly affected the formation of alpha-tocopheryl quinone (alpha-TQ), the main lipoperoxyl radical-derived oxidation metabolite of vitamin E, with the lowest production rate observed in samples stored at -80 degrees C or in liquid nitrogen. A similar effect of the storage temperature was observed for the free form of the omega-3 species eicosapentaenoic and docosahexaenoic acid, and for the omega-6 AA. Freezing samples at -20 degrees C resulted in a time-dependent formation of the pro-inflammatory eicosanoid LTB4. The PD solution prevents nonspecific alterations of these lipid parameters in samples that are processed for direct analysis and protects from the temperature-dependent modifications of free PUFAs. Combining PD solution and preservation at -80 degrees C or in liquid nitrogen, resulted in levels of alpha-TQ and PUFAs that remained stable over 1 month and up to 8 months of storage, respectively. This method paper provides indications for the optimal processing and storage of human plasma utilized in lipidomics studies.

The metabolism of α-tocopherol (α-TOH, vitamin E) shows marked interindividual variability, which may influence the response to nutritional and therapeutic interventions with this vitamin. Recently, new metabolomics protocols have fostered the possibility to explore such variability for the different metabolites of α-TOH so far identified in human blood, i.e., the “vitamin E metabolome”, some of which have been reported to promote important biological functions. Such advances prompt the definition of reference values and degree of interindividual variability for these metabolites at different levels of α-TOH intake. To this end, a one-week oral administration protocol with 800 U RRR-α-TOH/day was performed in 17 healthy volunteers, and α-TOH metabolites were measured in plasma before and at the end of the intervention utilizing a recently validated LC-MS/MS procedure; the expression of two target genes of α-TOH with possible a role in the metabolism and function of this vitamin, namely pregnane X receptor (PXR) and the isoform 4F2 of cytochrome P450 (CYP4F2) was assessed by immunoblot in peripheral blood leukocytes. The levels of enzymatic metabolites showed marked interindividual variability that characteristically increased upon supplementation. With the exception of α-CEHC (carboxy-ethyl-hydroxychroman) and the long-chain metabolites M1 and α-13′OH, such variability was found to interfere with the possibility to utilize them as sensitive indicators of α-TOH intake. On the contrary, the free radical-derived metabolite α-tocopheryl quinone significantly correlated with the post-supplementation levels of α-TOH. The supplementation stimulated PXR, but not CYP4F2, expression of leucocytes, and significant correlations were observed between the baseline levels of α-TOH and both the baseline and post-supplementation levels of PXR. These findings provide original analytical and molecular information regarding the human metabolism of α-TOH and its intrinsic variability, which is worth considering in future nutrigenomics and interventions studies.

The aim of the study was to define the chemical characteristics, antioxidant capacity, oxidative status, sensory properties, and the presence of polyphenols in ovine cheese obtained after dietary administration of spray-dried olive mill wastewater (SDP). SDP is a waste from olive oil production rich in bioactive molecules obtained by further processing the olive mill wastewater through a spray-drying system. Thirty-six sheep were randomly assigned to two experimental groups that received a standard diet based on hay and concentrate. The concentrate fed to the SDP group was supplemented with SDP at a rate 25 g/kg (as fed). The trial lasted 9 weeks. Milk from the two treatment groups was separately collected and used for manufacturing cheese. Cheese quality parameters and proximate composition were not affected by the dietary treatment, whereas the antioxidant status and oxidative stability of cheese were positively affected. Polyphenol analyses in cheese were performed through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The concentration of hydroxytyrosol and tyrosol, and their sulphate metabolites, were higher in cheese from supplemented sheep. These findings suggest that polyphenol metabolites can play a major role in the beneficial effects observed in food produced from sheep fed SDP.

Antimicrobial resistance (AMR) arises mostly because of the use and abuse of antibiotics in both the human and veterinary sectors. Furthermore, antibiotic residues can be discharged into the environment, promoting the spread of AMR as well as becoming a potential ecotoxicological risk. Aquaculture is one such source of environmental pollution. In the present work, we evaluated the correlation between antibiotic consumption in 11 selected aquafarms located along the Nera River, and the detection of their residues in surface water and sediment samples gathered monthly from the same stream. Antibiotic consumption was calculated using the ESVAC defined daily doses for animals (DDDvet) method, and a chemical analysis was conducted using a multiclass method. Therefore, the ecological risk of antibiotics being detected in surface waters was established based on the risk quotient (RQ). A strong positive correlation was identified between the concentrations detected in surface waters and the consumption of flumequine (r = 72%,p-value = 0.0085) and florfenicol (r = 83%,p-value = 0.0008). The RQ, however, was low for all the detected antibiotics in surface waters. Therefore, we proved that the antibiotics used in aquaculture can reach the near aquatic environment, but, in this work, they did not represent a toxicological risk to the surrounding ecosystem.

An analytical method for the quantification of thirty-three perfluoroalkyl and polyfluoroalkyl substances (PFASs) in animal liver was developed applying the isotopic dilution methodology with twenty-one labelled isotopologues of native compounds. The proposed protocol involved the determination of short and long aliphatic chain PFASs (C4C18) extracting liver with acetonitrile followed by two clean-up steps. The instrumental analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. The acquisition method combined full MS/dd-MS2, t-SIM/dd-MS2 and SIM experiments with variable resolution in order to maximize in one chromatographic run accuracy, sensitivity and selectivity. An eight-level validation study was performed evaluating linearity, trueness, precision, quantification and detection limits. Trueness was from 94 to 126% with intra-laboratory reproducibility lower than 20%. Limits of quantification were in the range 2–100 pg g−1, except for 2,3,3,3-tetrafluoro-2-(1,1,2,2,3,3,3-heptafluoropropoxy)-propanoic acid, HFPO-DA (500 pg g−1). The analysis of a certified reference material (IRMM-427) and participation in a proficiency test scheme (FAPAS – 0687) confirmed these satisfactory performances. Finally, the application of the developed procedure to detect PFASs in sixteen liver samples of farm animals revealed that chicken was the less contaminated species.

Vitamin E metabolism is a complex and highly regulated process intimately associated with lipoprotein metabolism. Hepatic steps of this process markedly differ between alpha-tocopherol (α-TOH) and other forms of this vitamin, including the cytochrome P450-dependent transformation to a series of metabolites that are now utilized as both nutritional and functional indicators of the vitamin status and metabolism. In the last three decades a number of protein interactions and gene modulation effects that characterize these differences have identified. These aspects are described in this chapter along with the recently identified role of enzymatic and free radical-derived metabolites of this vitamin as bioactive lipids. Compelling in vitro and in vivo data demonstrate that long-chain metabolites of α-TOH are more potent than their precursor in modulating peroxidation reactions and lipid metabolism, inflammatory genes, cell cycle regulation, and antiatherogenic pathways. The majority of these studies took advantage of the recently developed lipidomics technology allowing unbiased identifications and quantification of the entire spectrum of physiological metabolite in biological fluids, cell culture samples, and tissues. Other achievements also included the recognition of new metabolites, the identity of which are currently under investigation. These studies on the functional metabolome of vitamin E have led to speculation that, similarly to other fat-soluble vitamins, vitamin E may provide some of its biological roles by behaving as a provitamin. If confirmed this would represent a real breakthrough in human nutrition and a major step forward in defining vitamin E essentiality and adequacy, and their assessment strategy as well.

Two recent randomized controlled trials demonstrated improved radiographic, histological and hepatometabolic cues of non-alcoholic steatohepatitis (NASH) in pediatric patients treated with the ω-3 fatty acid docosahexaenoic acid (DHA) in combination with vitamin D (VD) or with choline (CHO) and vitamin E (VE), the DHA-VD and DHA-CHO-VE trials, respectively). In the present study we verified the nutritional compliance to these DHA-based multivitamin treatments; lipidomics biomarkers of the reported outcome on NASH indicators were also investigated. Samples were obtained from 30 biopsy-proven pediatric NASH patients of the DHA-CHO-VE trial randomized in multivitamin treatment group and placebo group (n = 15 each), and from 12 patients of the treatment group of the DHA-VD trial. All patients underwent 6-month therapy plus 6 months of follow-up. Plasma samples and clinical data were obtained at baseline and at the end of the study (12 months). Selected biomarkers included the free form of DHA and other ω-3 fatty acid arachidonic acid (AA), indices of the vitamin E status, and some hepatic metabolites of these lipids. Radiographic and histological improvements of treated patients were associated with increased concentrations of DHA, α-linolenic acid and α-tocopherol (i.e. VE), and with decreased AA that was also investigated in complex lipids by untargetd lipidomics. As a result a significantly lowered AA/DHA ratio was observed to represent the main indicator of the response to the DHA-based therapy. Furthermore, baseline levels of AA/DHA showed strong association with NAS and US improvement. A stable correction of DHA AA metabolism interaction is associated with the curative effect of this therapy and may represent a key nutritional endpoint in the clinical management of pediatric NASH.

alpha-Linolenic acid (ALA) and its most important positional isomer gamma-linolenic acid (GLA), are essential fatty acids (vitamin F). Therefore, ALA- and GLA-rich edible oils hold great potential in human and animal nutrition, as well as in nutraceutics and cosmetics. Quality control and nutritional validation of oil products is thus of increasing importance. In the present study, the cellulose tris(3,5-dichlorophenylcarbamate)-based chiral stationary phase was successfully used for separation of ALA and GLA, a major challenge in the liquid chromatography of these isomers. The chromatographic conditions were firstly optimized on a HPLC system with UV detection, and the use of a reversed-phase eluent system made up of aqueous 10 mM ammonium acetate/acetonitrile (40/60, v/v; (s)(w)pH 6.0) with a 25 degrees C column temperature resulted optimal for the simultaneous discrimination of the two isomers at a 0.5 mL/min flow rate (alpha = 1.10; R-S = 1.21). The method was then optimized for LC MS/MS implementation. The proposed innovative separation method holds a great potential for the quantification of ALA and GLA in food and biological matrices, thus opening the way to further investigations involving the two positional isomers. (C) 2019 Elsevier B.V. All rights reserved.

Enzymatic and non-enzymatic (free-radical-derived) metabolites of vitamin E are objects of increasing interest as both are indicators of the metabolism and biological functions of this fat-soluble vitamin. These include bioactive long-chain metabolites of alpha-tocopherol formed by the activity of cytochrome P450, which have recently been measured for the first time in human plasma using a new unbiased LC-MS/MS procedure. This chapter summarises the available knowledge on analysis strategies and techniques currently in use to measure the different types of metabolites of this vitamin so far identified and studied in biological samples.