Assessment of the genotoxic and mutagenic effects induced by T-2 mycotoxin in HepG2 cells.

The T-2 toxin is a mycotoxin produced by molds belonging to Fusarium. Among the Fusarium mycotoxins, trichothecenes are frequently reported in food and feed, being the T-2 toxin (T-2) the mycotoxin which possesses the highest toxicity. According to EFSA, T-2 is found in various cereal grains used in food and feed products, mainly in oats, and it has a high environmental impact due to its mechanisms of toxicity. However, recent information on its genotoxic and mutagenic effects is lacking. This work aimed to evaluate the genotoxic and mutagenic potential of T-2 in vitro. For this purpose, HepG2 cells were exposed to 15, 30, and 60 nM T-2 for 24 h, then the DNA damage was evaluated by the micronucleus and the comet assays. In addition, point mutation analysis was performed by the bacterial reverse mutation test using 0.15-60 nM of T-2 concentrations. The results showed chromosomal damage at 60 nM T-2 since significantly more MN appeared at this concentration than in the control samples. Regarding the comet assay, DNA double helix breaks appeared at all concentrations tested and, in a concentration-dependent manner. However, no mutagenic effects were observed at any of the concentrations tested for the Salmonella typhimurium (S. Typhimurium) strains TA98, TA100, TA1535, TA1537, or the Escherichia coli (E. Coli) WP2 strain in the absence or presence of a metabolic activation system. Therefore, these results showed that T-2 mycotoxin produced genotoxic effects by MN and comet assay, while no mutagenicity was observed. However, further research simulating different metabolic activation pathways and the combined exposure of this mycotoxin with other mutagenic chemicals that could be present in the diet is necessary to discard the mutagenic potential of T-2 fully. These results highlight the carcinogenic potential and danger associated with T-2 exposure and should be considered to prevent associated food risks for the human population.

Evaluation of the Bioaccessible Fraction of T-2 Toxin from Cereals and Its Effect on the Viability of Caco-2 Cells Exposed to Tyrosol.

The bioaccessibility of mycotoxins is an important factor that has to be considered when assessing the risk they pose to human health. Bioactive compounds like phenolics could play a protective role against the toxic effects of contaminants. In this work, the bioaccessible fraction of the T-2 toxin (T-2) contained in breakfast cereals and its effect on the viability of Caco-2 cells were investigated. Furthermore, the effect of tyrosol (a polyphenol abundant in EVOO) on T-2-induced cytotoxicity was evaluated in the same cell line. After standardized in vitro gastrointestinal digestion, the T-2 toxin was released from T-2-spiked breakfast cereals and further quantified by UHPLC-MS/MS. The bioaccessible fraction of T-2 was 51 ± 4%. The cell viability study was performed by pre-treating the cells for 24 h with tyrosol (25, 50 and 100 µM) and subsequently adding T-2 at 15 nM or by treating the cells with a combination of tyrosol and T-2. In the simultaneous treatment, 25 µM tyrosol prevented the toxic effects produced by the exposure to T-2 at 15 nM; however, cytotoxic effects were observed for the other combinations tested. The pre-treatment of Caco-2 cells with tyrosol did not attenuate the cytotoxic effects caused by exposure to T-2. These results suggest that tyrosol at low concentrations (25 µM) could exert a cytoprotective effect on Caco-2 cells against 15 nM T-2 when administered simultaneously with T-2. However, more studies are required to corroborate this hypothesis.

Women's Involvement in Steady Exercise (WISE): Study Protocol for a Randomized Controlled Trial.

BACKGROUND: Physical inactivity is a serious public health problem for people of all ages and is currently the fourth highest global risk factor for mortality. The transition period from adolescence to adulthood coincides with a marked reduction in participation in physical activity, with more than 50% (and up to 80%) of young adults stopping physical activity. This decrease in physical activity is more evident in women than in men. Despite efforts, existing programs face challenges in effectively initiating and maintaining physical activity among individuals, particularly women, for extended durations. To address these limitations, the Women's Involvement in Steady Exercise (WISE) randomized controlled trial (RCT) seeks to assess the efficacy of a digital high-intensity training intervention complemented by nutritional plans and other health-related advice. METHODS: The study will be a three-center, randomized (1:1), controlled, parallel-group trial with a six-month intervention period. A total of 300 participants will be recruited at three study sites in Spain, Serbia and Italy. The participants will be randomized to one of the two groups and will follow a six-month program. The primary outcome of the study is the daily step count. Self-reported physical activity, the adherence to the exercise program, body composition, physical activity enjoyment, quality of sleep and physical capacities will also be evaluated.

Amitraz and Its Metabolites: Oxidative Stress-Mediated Cytotoxicity in HepG2 Cells and Study of Their Stability and Characterization in Honey.

The population decrease of bees that has been observed in recent years due to the Varroa destructor parasite may endanger the production of bee-products whose demand is on the rise. To minimize the negative effects caused by this parasite, the pesticide amitraz is commonly used by beekeepers. Based on these, the objectives of this work are to determine the toxic effects caused by amitraz and its metabolites in HepG2 cells, as well as its determination in honey samples and the study of its stability with different heat treatments commonly used in the honey industry and its relationship with the amount of 5-hydroxymethylfurfural (HMF) produced. Amitraz significantly decreased cell viability by MTT assay and total protein content (PC) assay, being more cytotoxic than its metabolites. Amitraz and its metabolites caused oxidative stress by Lipid Peroxidation (LPO) production and Reactive Oxygen Species (ROS) generation. Residues of amitraz and/or its metabolites were found in analyzed honey samples, with 2,4-Dimethylaniline (2,4-DMA) being the main metabolite confirmed by high-performance liquid chromatography-high resolution mass spectrometry (HPLC-QTOF HRMS). Amitraz and its metabolites resulted as unstable even at moderate heat treatments. Additionally, a positive correlation in terms of HMF concentration in samples and the severity of heat treatment was also observed. However, quantified amitraz and HMF were within the levels set in the regulation.

Enhancement of the Antioxidant Effect of Natural Products on the Proliferation of Caco-2 Cells Produced by Fish Protein Hydrolysates and Collagen.

A large amount of fish side streams are produced each year, promoting huge economic and environmental problems. In order to address this issue, a potential alternative is to isolate the high-added-value compounds with beneficial properties on human health. The objectives of this study were to determine the effect of hydrolyzed fish protein and collagen samples on cell proliferation, as well as to determine the specific influence of minerals and metals on this effect and whether dietary antioxidants can enhance cell proliferation. The results of hydrolyzed fish protein and collagen samples showed negative effects on Caco-2 cell proliferation at the highest concentrations tested. Moreover, the pre-treatment of these hydrolyzates with vitamin C and E, quercetin and resveratrol increased the proliferation of bioaccessible fractions of hydrolyzated fish protein and collagen samples compared to the bioaccessible fractions without pre-treatment. The highest mineral concentrations were found for P, Ca and Mg. The metals found in the pure hydrolyzates were As, Cd, Hg and Pb; however, they appeared at almost undetectable levels in bioavailable fractions. It can be concluded that the consumption of hydrolyzates of fish by-products is an interesting strategy for complying with EFSA recommendations regarding fish consumption while at the same time reducing fish waste.

Multi-Mycotoxin Method Development Using Ultra-High Liquid Chromatography with Orbitrap High-Resolution Mass Spectrometry Detection in Breakfast Cereals from the Campania Region, Italy.

Breakfast cereals have been reported as one of the most susceptible cereal-based products to mycotoxin contamination. These products pose an even more concerning risk to human health since they are marketed as a ready-to-eat product and one of its main population targets is children. Therefore, the main goal of the present study was to conduct a monitoring study of multiple mycotoxins contained in breakfast cereals samples marketed in Italy through ultra-high performance liquid chromatography coupled to high-resolution Q-Orbitrap tandem mass spectrometry. An acetonitrile-based methodology was validated for quantifying 24 mycotoxins in breakfast cereals. The results showed that 93% of the samples contained at least one mycotoxin. Beauvericin was the most prevalent toxin (86% of samples; mean concentration: 30.66 µg/kg), although the main enniatins, zearalenone-derived forms and fumonisins B1 and B2 were also detected. Co-occurrence was observed in 73% of the positive samples with up to five mycotoxins simultaneously occurring, mainly due to the combination of beauvericin and enniatins. These results provided more evidence about the high impact of non-regulated mycotoxins, such as the emerging Fusarium toxins, in breakfast cereals, and encourages the development of analytical methodologies including these and zearalenone-derived forms that could be going unnoticed with current methodologies.

In Vitro and Predictive Computational Toxicology Methods for the Neurotoxic Pesticide Amitraz and Its Metabolites.

The Varroa destructor parasite is responsible for varroasis in honeybees worldwide, the most destructive disease among parasitic diseases. Thus, different insecticides/acaricides have been widely used within beehives to control these parasitic diseases. Namely, amitraz is the most used acaricide due to its high efficacy shown against Varroa destructor. However, pesticides used for beehive treatments could be incorporated into the honey and accumulate in other hive products. Hence, honeybee health and the impairment of the quality of honey caused by pesticides have gained more attention. Amitraz and its main metabolites, N-(2,4-dimethylphenyl) formamide (2,4-DMF) and 2,4-dimethylaniline (2,4-DMA), are known to be potent neurotoxicants. In this research, the cytotoxicity of amitraz and its metabolites has been assessed by MTT and PC assays in HepG2 cells. In addition, possible target receptors by in silico strategies have been surveyed. Results showed that amitraz was more cytotoxic than its metabolites. According to the in silico ADMEt assays, amitraz and its metabolites were predicted to be compounds that are able to pass the blood-brain barrier (BBB) and induce toxicity in the central and peripheral nervous systems. The main target class predicted for amitraz was the family of A G protein-coupled receptors that comprises responses to hormones and neurotransmitters. This affects, among other things, reproduction, development, locomotion, and feeding. Furthermore, amitraz and its metabolites were predicted as active compounds interacting with diverse receptors of the Tox21-nuclear receptor signaling and stress response pathways.

Evaluation of cytotoxicity, analysis of metals and cumulative risk assessment in microalgae.

Microalgae are one promising source for the production of bioactive compounds. However, microalgae can accumulate harmful substances. So, our objectives were (i) to evaluate cell viability after Phaeodactylum triconutum (0% and 65% cell disruption, DR) and Tetraselmis chuii (0% and 67% DR) freeze-dried exposure in HepG2 cells by MTT assay; (ii) to evaluate cell viability after P. triconutum and T. chuii extract exposure; (iii) to assess the effect in cell viability when they were simultaneously exposed to T-2 toxin and, (iv) to evaluate if inflammatory response is related to the mechanism of toxicity of these microalgae by qPCR assays. Results demonstrated that cell viability did not increase after freeze-dried microalgae exposure in HepG2 cells. And, no IC50 values were observed. However, an increase in HepG2 cell viability after exposure of T. chuii 0% DR extract at 5, 25 and 100 µg/mL was observed. Additionally, 1:64 diluted T. chuii 0% DR with IC50/4 T-2 and with IC50/2 T-2 and 1:32 diluted T. chuii 0% DR with IC50/4 T-2 showed an increase in cell viability. Both microalgae increased the relative TNF-α, IL-1ß and IL-6 mRNA expression. Concluding, no cytotoxic effect was evidenced but, it was noted up-regulation of inflammatory genes after T. chuii exposure in HepG2 cell. Thus, more studies related the mechanistic toxicity of microalgae are needed to evaluate the potential toxicological risk of inflammation of these novel foods. .

Target analysis and retrospective screening of contaminants in ready-to-eat cooked ham samples through UHPLC-Q-Orbitrap HRMS.

The use of veterinary drugs (VDs) is widely administered to animals for both therapeutic and prophylactic purposes. However, their improper use may involve their occurrence in the final products intended for human consumption. In this scientific work, a method for the investigation of target (n = 30) VDs residues and retrospective suspect screening followed by confirmation using analytical standards of others 38 contaminants in ready-to-eat cooked ham by ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was developed. The extraction was performed based on the QuEChERS approach and validated in accordance with the European Regulation 2021/808. The application of the in-house validated method to ready-to-eat cooked ham showed the occurrence of fourteen VDs residues. Despite the important incidence, the concentration levels found were below the maximum residue limits set for VDs in porcine muscle, except for colchicine. Constant monitoring of animals derived food is strongly recommended to ensure the food safety of consumers.

Stressful Effects of T-2 Metabolites and Defense Capability of HepG2 Cells.

The T-2 toxin (T-2), a mycotoxin produced by several species of Fusarium which belongs to group A of trichothecenes, is rapidly metabolized, and its main metabolites are HT-2, Neosolaniol (Neo), T2-triol and T2-tetraol. In this work, the antioxidant defense system of HepG2 cells against oxidative stress induced by T-2 and its metabolites was evaluated. The results obtained demonstrated that there is an overall decrease in glutathione (GSH) levels after all mycotoxins exposure. Moreover, the GSH levels and the enzymatic activities related to GSH (GPx and GST) increased with NAC pre-treatment (glutathione precursor) and decreased with BSO pre-treatment (glutathione inhibitor). The GPx activity is increased by T2-tetraol. The GST activity increased after T-2 and T2-triol exposure; however, T2-tetraol decreased its activity. Furthermore, CAT activity increased after T-2 and T2-triol; nevertheless, Neo decreased its activity. Finally, SOD activity is increased by all mycotoxins, except after T-2 exposure. So, the damage associated with oxidative stress by T-2 and its metabolites is relieved by the antioxidant enzymes system on HepG2 cells.

Effect of Different Coffee Brews on Tryptophan Metabolite-Induced Cytotoxicity in HT-29 Human Colon Cancer Cells.

Coffee consumption positively influences colon health. Conversely, high levels of tryptophan metabolites such as skatole released from intestinal putrefactive fermentation in the presence of excessive dietary animal protein intake, and gut microbiota alterations, may have several adverse effects, including the development of colorectal cancer. Therefore, this study aimed to elucidate the potential protective effects of coffee in the presence of different skatole levels. The results showed that skatole exposure induced reduced cell viability and oxidative stress in the HT-29 human colon cancer cell line. However, co-treatment of cells with skatole and coffee samples was able to reduce ROS production (up to 45% for espresso) compared to cells not treated with coffee. Real-time PCR analysis highlighted that treating HT-29 cells with skatole increased the levels of inflammatory cytokines and chemokines TNF-α, IL-1ß, IL-8, and IL12, whereas exposure to coffee extracts in cells that were pretreated with skatole showed anti-inflammatory effects with decreased levels of these cytokines. These findings demonstrate that coffee may counteract the adverse effects of putrefactive compounds by modulating oxidative stress and exerting anti-inflammatory activity in colonocytes, thus suggesting that coffee intake could improve health conditions in the presence of altered intestinal microbiota metabolism.

Foodomics: Current and Future Perspectives in Food Analysis.

Climate change, an increase in population, and the recent pandemic crisis triggered by SARS-CoV-2 have all contributed to a period of global problems [...].

Effect of Phenolic Extract from Red Beans (Phaseolus vulgaris L.) on T-2 Toxin-Induced Cytotoxicity in HepG2 Cells.

Red beans contain human bioactive compounds such as polyphenols. Several in vitro studies have proposed the natural compounds as an innovative strategy to modify the toxic effects produced by mycotoxins. Hence, in this work, a complete investigation of the polyphenolic fraction of red beans was performed using a Q-Orbitrap high-resolution mass spectrometry analysis. Notably, epicatechin and delphinidin were the most detected polyphenols found in red bean extracts (3.297 and 3.108 mg/Kg, respectively). Moreover, the red bean extract was evaluated against the T-2 toxin (T-2) induced cytotoxicity in hepatocarcinoma cells (HepG2) by direct treatment, simultaneous treatment, and pre-treatment assays. These data showed that T-2 affected the cell viability in a dose-dependent manner, as well as observing a cytotoxic effect and a significant increase in ROS production at 30 nM. The simultaneous treatment and the pre-treatment of HepG2 cells with red bean extract was not able to modify the cytotoxic T-2 effect. However, the simultaneous treatment of T-2 at 7.5 nM with the red bean extract showed a significant decrease in ROS production, with respect to the control. These results suggest that the red bean extract could modulate oxidative stress on HepG2 cells.

Multiclass and multi-residue screening of mycotoxins, pharmacologically active substances, and pesticides in infant milk formulas through ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry analysis.

Infant milk formulas are designed to substitute human milk when breastfeeding is unavailable. In addition to human milk and milk-derived products, these formulas can be a vehicle of contaminants. In this work, a multiclass method based on the QuEChERS (quick, easy, cheap, effective, rugged, and safe) approach was developed for the simultaneous determination of contaminants (n = 45), including mycotoxins and veterinary drug residues, occurring in infant milk formulas. By using an ultra-high-performance liquid chromatography quadrupole-Orbitrap coupled with high-resolution mass spectrometry analysis (UHPLC-Q-Orbitrap HRMS; Thermo Fisher Scientific), further retrospective analysis of 337 contaminants, including pesticides, was achieved. The method was validated in accordance with European regulations and applied for the analysis of 54 infant milk samples. Risk assessment was also performed. Dexamethasone was detected in 16.6% of samples (range: 0.905-1.131 ng/mL), and procaine benzyl penicillin in 1 sample at a concentration of 0.295 ng/mL. Zearalenone was found in 55.5% of samples (range: 0.133-0.638 ng/mL) and α-zearalenol in 16.6% of samples (range: 1.534-10.408 ng/mL). Up to 49 pesticides, 11 veterinary drug residues, and 5 mycotoxins were tentatively identified via retrospective analysis based on the mass spectral library. These findings highlight the necessity of careful evaluation of contaminants in infant formulas, considering that they are intended for a vulnerable part of the population.

Novel quadrupole-time of flight-based methodology for determination of multiple mycotoxins in human hair.

The potential of hair as matrix for assessing long-term exposure to mycotoxins remains scarcely explored. Therefore, this study aimed to develop and validate an analytical methodology for the simultaneous determination of aflatoxins, enniatins, beauvericin and T-2 toxin in human hair, based on a pretreatment stage prior to salt-assisted liquid-liquid extraction and followed by high performance liquid chromatography coupled to high resolution Q-TOF mass spectrometry for the first time. Washing with a non-ionic detergent was successfully applied, whereas enzymatic digestion with Pronase E was mandatory for releasing mycotoxins from the hair matrix. The methodology was validated according to Commission Decision 2002/657/EC, with limits of quantification ranging from 0.6 to 8.7 ng/g. The analysis of 10 samples showed at least one mycotoxin occurring in 67% of samples, including the carcinogenic aflatoxins. This is the first validated methodology for the quantification of multiple mycotoxins in human hair.

High-Throughput Determination of Major Mycotoxins with Human Health Concerns in Urine by LC-Q TOF MS and Its Application to an Exposure Study.

Human biomonitoring constitutes a suitable tool to assess exposure to toxins overcoming the disadvantages of traditional methods. Urine constitutes an accessible biological matrix in biomonitoring studies. Mycotoxins are secondary metabolites produced naturally by filamentous fungi that produce a wide range of adverse health effects. Thus, the determination of urinary mycotoxin levels is a useful tool for assessing the individual exposure to these food contaminants. In this study, a suitable methodology has been developed to evaluate the presence of aflatoxin B2 (AFB2), aflatoxin (AFG2), ochratoxin A (OTA), ochratoxin B (OTB), zearalenone (ZEA), and α-zearalenol (α-ZOL) in urine samples as exposure biomarkers. For this purpose, different extraction procedures, namely, the Solid Phase Extraction (SPE); Dispersive Liquid-Liquid Microextraction (DLLME); and Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) methods were assessed, followed by Liquid Chromatography coupled to Quadrupole Time of Flight Mass Spectrometry with Electrospray Ionization (LC-ESI-QTOF-MS) determination. Then, the proposed methodology was applied to determine mycotoxin concentrations in 56 human urine samples from volunteers and to estimate the potential risk of exposure. The results obtained revealed that 55% of human urine samples analyzed resulted positive for at least one mycotoxin. Among all studied mycotoxins, only AFB2, AFG2, and OTB were detected with incidences of 32, 41, and 9%, respectively, and levels in the range from

Human Biomonitoring of T-2 Toxin, T-2 Toxin-3-Glucoside and Their Metabolites in Urine through High-Resolution Mass Spectrometry.

The metabolic profile of T-2 toxin (T-2) and its modified form T-2-3-glucoside (T-2-3-Glc) remain unexplored in human samples. Therefore, the present study aimed to investigate the presence of T-2, T-2-3-Glc and their respective major metabolites in human urine samples (n = 300) collected in South Italy through an ultra-high performance liquid chromatography (UHPLC) coupled to Q-Orbitrap-HRMS methodology. T-2 was quantified in 21% of samples at a mean concentration of 1.34 ng/mg Crea (range: 0.22-6.54 ng/mg Crea). Almost all the major T-2 metabolites previously characterized in vitro were tentatively found, remarking the occurrence of 3'-OH-T-2 (99.7%), T-2 triol (56%) and HT-2 (30%). Regarding T-2-3-Glc, a low prevalence of the parent mycotoxin (1%) and its metabolites were observed, with HT-2-3-Glc (17%) being the most prevalent compound, although hydroxylated products were also detected. Attending to the large number of testing positive for T-2 or its metabolites, this study found a frequent exposure in Italian population.

Cytoprotective Effects of Fish Protein Hydrolysates against H2O2-Induced Oxidative Stress and Mycotoxins in Caco-2/TC7 Cells.

Many studies report the potent antioxidant capacity for fish protein hydrolysates, including radical scavenging activity and inhibition ability on lipid peroxidation (LPO). In this study, the in vitro cytotoxicity of protein hydrolysates from different salmon, mackerel, and herring side streams fractions was evaluated in the concentration range from 1 to 1:32 dilution, using cloned human colon adenocarcinoma cells TC7 (Caco-2/TC7) by MTT and PT assays. The protein hydrolysates' antioxidant capacity and oxidative stress effects were evaluated by LPO and reactive oxygen species (ROS) generation, respectively. The antioxidant capacity for pure and bioavailable hydrolysate fraction was also evaluated and compared. Additionally, mycotoxin levels were determined in the fish protein hydrolysates, and their cytoprotective effect against T-2 toxin was evaluated. Both hydrolysates and their bioavailable fraction induced similar cell viability rates. The highest cytoprotective effect was obtained for the salmon viscera protein hydrolysate (HSV), which increased the cell viability by 51.2%. ROS accumulation induced by H2O2 and LPO was suppressed by all pure hydrolysates. The cytoprotective effect of hydrolysates was observed against T-2. Moreover, the different fish fraction protein hydrolysates contain variable nutrients and unique bioactive peptide composition showing variable bioactivity, which could be a useful tool in developing dietary supplements with different target functional properties.

Citrinin Dietary Exposure Assessment Approach through Human Biomonitoring High-Resolution Mass Spectrometry-Based Data.

Citrinin (CIT) is a scarcely studied mycotoxin within foodstuffs, so the biomonitoring of this toxin and its metabolite dihydrocitrinone (DH-CIT) in biological samples represents the main alternative to estimate the exposure. Hence, this study aimed to evaluate the presence of CIT and DH-CIT in 300 urine samples from Italian individuals in order to assess the exposure. Quantification was performed through an ultrahigh-performance liquid chromatography high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS)-based methodology. CIT was quantified in 47% of samples (n = 300) up to 4.0 ng/mg Crea (mean = 0.29 ng/mg Crea), whereas DH-CIT was quantified in 21% of samples up to 2.5 ng/mg Crea (mean = 0.39 ng/mg Crea). Considering different age groups, average exposure ranged from 8% to 40% of the provisional tolerable daily intake, whereas four individuals surpassed the limits suggested by the European Food Safety Authority. These results revealed non-negligible exposure levels to CIT, encouraging further investigation in foodstuffs monitoring studies.

Mycotoxin Occurrence and Risk Assessment in Gluten-Free Pasta through UHPLC-Q-Exactive Orbitrap MS.

Celiac disease (CD) is a genetic-based autoimmune disorder which is characterized by inflammation in the small intestinal mucosa due to the intolerance to gluten. Celiac people should consume products without gluten, which are elaborated mainly with maize or other cereals. Contamination of cereals with mycotoxins, such as fumonisins (FBs) and aflatoxins (AFs) is frequently reported worldwide. Therefore, food ingestion is the main source of mycotoxin exposure. A new analytical method was developed and validated for simultaneous analysis of 21 mycotoxins in gluten-free pasta, commonly consumed by celiac population as an alternative to conventional pasta. Ultrahigh-performance liquid chromatography coupled to quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Exactive Orbitrap MS) was used for analyte separation and detection. The mycotoxins included in this work were those widely reported to occur in cereal samples, namely, ochratoxin-A (OTA), aflatoxins (AFB1, AFB2, AFG1 and AFG2), zearalenone (ZON), deoxynivalenol (DON), 3-acetyl-deoxynivalenol and 15-acetyl-deoxynivalenol (3-AcDON and 15-AcDON, respectively), nivalenol (NIV), neosolaniol (NEO), fusarenone-X, (FUS-X), T-2 toxin (T-2) and HT-2 toxin (HT-2), fumonisin B1 and B2 (FB1 and FB2, respectively), enniatins (ENN A, ENN A1, ENN B and ENN B1) and beauvericin (BEA). The validated method was successfully applied to 84 gluten-free pasta samples collected from several local markets of Campania region (Italy) during September to November 2020 to monitor the occurrence of mycotoxins and to assess the exposure to these food contaminants. A significant number of samples (95%) showed mycotoxin contamination, being Fusarium mycotoxins (FB1, ZON and DON) the most commonly detected ones. Regarding the risk assessment, the higher exposures were obtained for NIV, DON and FB1 for children and teenagers age group which can be explained due to their lower body weight.