Development and validation of an analytical method based on QuEChERS followed by UHPLC-ToF-MS for the determination of tropane alkaloids in buckwheat (Fagopyrum esculentum L.) and buckwheat products.

A method was developed for the determination of tropane alkaloids (TAs), including atropine, scopolamine, anisodamine and homatropine in buckwheat and related products. This work presents an optimised methodology based on QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) extraction procedure followed by ultra-high performance liquid chromatography combined with time-of-flight mass spectrometry for the determination of TAs (atropine, scopolamine, anisodamine and homatropine) in buckwheat samples. The analytical methodology was successfully validated, demonstrating good linearity, low limit of quantification, repeatability (RSDr < 15%), inter-day precision (RSDR < 19%) and recovery (74-113%). Finally, 13 commercial samples of buckwheat were analysed and the results demonstrated that they were in compliance with the current European regulations regarding TAs.

The potential of citrus by-products in the development of functional food and active packaging.

Food by-product valorization has become an important research area for promoting the sustainability of the food chain. Citrus fruits are among the most widely cultivated fruit crops worldwide. Citrus by-products, including pomace, seeds, and peels (flavedo and albedo), are produced in large amounts each year. Those by-products have an important economic value due to the high content on bioactive compounds, namely phenolic compounds and carotenoids, and are considered a valuable bio-resource for potential applications in the food industry. However, green extraction techniques are required to ensure their sustainability. This chapter addresses the main components of citrus by-products and their recent applications in food products and active food packaging, towards a circular economy. In addition, the concern regarding citrus by-products contamination (e.g. with pesticides residues and mycotoxins) is also discussed.

Analysis of Antibiotics in Bivalves by Ultra-High Performance Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometry.

The presence of pharmaceuticals in aquatic ecosystems mostly originates from wastewater treatment plants (WWTPs) and such a situation can be responsible for significant negative impacts on natural ecosystems, such as estuarine and coastal areas. Bioaccumulation of pharmaceuticals, namely antibiotics, in exposed organisms is known to have remarkable effects on different trophic levels of non-target organisms such as algae, invertebrates and vertebrates, including the emergence of bacterial resistance. Bivalves are a highly appreciated seafood product, as they are fed by filtering water, and can bioconcentrate chemicals, being ideal for biomonitoring environmental health hazards in coastal and estuarine ecosystems. To use this sentinel species, an analytical strategy was developed to be used in accessing antibiotics, from human and veterinary medicine, and evaluate their occurrence as emerging pollutants in aquatic environments. The optimized analytical method was fully validated according to the European requirements defined by the Commission Implementing Regulation 2021/808. The validation comprised the following parameters: specificity, selectivity, precision, recovery, ruggedness, linearity, and the decision limit CCα, as well as the limit of detection (LoD) and limit of quantification (LoQ). The method was validated for 43 antibiotics to allow their quantification in both contexts, environmental biomonitoring and food safety.

The Bright and Dark Sides of Herbal Infusions: Assessment of Antioxidant Capacity and Determination of Tropane Alkaloids.

Herbal infusions are highly popular beverages consumed daily due to their health benefits and antioxidant properties. However, the presence of plant toxins, such as tropane alkaloids, constitutes a recent health concern for herbal infusions. This work presents an optimized and validated methodology based on the QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of tropane alkaloids (atropine, scopolamine, anisodamine, and homatropine) in herbal infusions, in accordance with criteria established by Commission Recommendation EU No. 2015/976. One of the seventeen samples was contaminated with atropine, exceeding the current European regulation regarding tropane alkaloids. In addition, this study evaluated the antioxidant capacity of common herbal infusions available on Portuguese markets, indicating the high antioxidant capacity of yerba mate (Ilex paraguariensis), lemon balm (Melissa officinalis), and peppermint (Mentha x piperita).

Mycotoxins in Rice Correlate with Other Contaminants? A Pilot Study of the Portuguese Scenario and Human Risk Assessment.

Rice is the second most important cereal crop and is vital for the diet of billions of people. However, its consumption can increase human exposure to chemical contaminants, namely mycotoxins and metalloids. Our goal was to evaluate the occurrence and human exposure of aflatoxin B1 (AFB1), ochratoxin A (OTA), zearalenone (ZEN), and inorganic arsenic (InAs) in 36 rice samples produced and commercialized in Portugal and evaluate their correlation. The analysis of mycotoxins involved ELISA, with limits of detection (LODs) of 0.8, 1 and 1.75 µg kg-1 for OTA, AFB1, and ZEN, respectively. InAs analysis was carried out by inductively coupled plasma mass spectrometry (ICP-MS; LOD = 3.3 µg kg-1). No sample showed contamination by OTA. AFB1 was present in 2 (4.8%) samples (1.96 and 2.20 µg kg-1), doubling the European maximum permitted level (MPL). Concerning ZEN, 88.89% of the rice samples presented levels above the LOD up to 14.25 µg kg-1 (average of 2.75 µg kg-1). Regarding InAs, every sample presented concentration values above the LOD up to 100.0 µg kg-1 (average of 35.3 µg kg-1), although none surpassed the MPL (200 µg kg-1). No correlation was observed between mycotoxins and InAs contamination. As for human exposure, only AFB1 surpassed the provisional maximum tolerable daily intake. Children were recognized as the most susceptible group.

Risk Assessment of Nine Coccidiostats in Commercial and Home-Raised Eggs.

The poultry industry, in order to prevent and control coccidiosis caused by Eimeria spp., widely uses coccidiostats as feed additives. The main objective of this study was to evaluate the presence of nine coccidiostats in 62 egg samples by UHPLC-MS/MS. Overall, detection frequency and average concentration were 90.3% (56/62) and 106.3 µg kg-1, respectively. Only diclazuril and nicarbazin were detected. Diclazuril, only found in home-raised eggs, showed an overall detection frequency of 8.1% (5/62), with average and maximum concentrations of 0.46 ± 1.90 µg kg-1 and 13.6 µg kg-1, respectively. Nicarbazin presented an overall higher frequency, 88.7% (55/62), with levels up to 744.8 µg kg-1. Additionally, four samples (6.5%) presented both nicarbazin and diclazuril. Home-raised egg samples (n = 28) showed a detection frequency of 89.3%, with nicarbazin found in more samples (85.7% vs. 17.9%) and at higher levels (266.3 ± 169.4 µg kg-1 vs. 0.91 ± 2.78 µg kg-1) when compared to diclazuril. In supermarket samples (n = 34), only nicarbazin was detected in 31 samples (91.1%), with an average of 167.6 ± 62.2 µg kg-1. Considering the average contamination scenario, consumers' health should not be adversely affected by egg consumption. In every scenario considered, children were the most vulnerable population group.

Occurrence of zearalenone in dairy farms - A study on the determinants of exposure and risk assessment.

The Azorean dairy industry is based on a semi-intensive model. Due to the few storage facilities and the large climatic fluctuations that characterize the Azores, the complete feed mixture (total mixed ration - TMR) is susceptible to the proliferation of a variety of fungi and mycotoxin contamination. Thus, chronic ingestion of these xenobiotics may lead to increased susceptibility to disease, loss of reproductive performance and, in the case of dairy cattle, decreased productivity and quality of milk produced. Since it is impossible to eliminate completely the presence of mycotoxins, it is essential to ensure the implementation of strategies to reduce their concentration in products intended for food and feed, as well as to monitor and control the levels present in food. This study aimed to evaluate the occurrence zearalenone (ZEA) in feed (TMR) in four dairy cattle farms on the Azorean Island of São Miguel and relate it to the occurrence of this mycotoxin in the milk produced, by associating several production and health indicators translated by the monthly milk contrast. To this end, the monthly milk contrast data, the determination of ZEA in the ingested feed and the (individual) milk samples from the four farms included in the study were used. Eighty-three (98.8%) of the milk samples under study had detectable ZEA levels (1.56 ± 1.36 µg/L), higher than reported in previous similar studies. Although ZEA concentration was not significantly associated with any production indicator analyzed (days in milk, age at calving, milk yield, protein content, butyrate content, somatic cell concentration and urea), it was found that the production regime and type of feed management is a major factor in the exposure of animals to high mycotoxin contents. Further studies are recommended to ensure continued monitoring and reduction of the risk associated with exposure of animals and humans to ZEA.

Coccidiostats and Poultry: A Comprehensive Review and Current Legislation.

Coccidiosis remains one of the major problems of the poultry industry. Caused by Eimeria species, Coccidiosis is a contagious parasitic disease affecting poultry with great economic significance. Currently, in order to prevent health problems caused by this disease, broiler farmers make extensive use of coccidiostats in poultry feed, maintaining animal health and, in some cases, enhancing feed conversion. The presence of unauthorized substances, residues of veterinary products and chemical contaminants in the food industry is of concern, since they may pose a risk to public health. As the use of coccidiostats has been increasing without any requirements for veterinary prescription, research and surveillance of coccidiostat residues in poultry meat is becoming imperative. This review presents an up-to-date comprehensive discussion of the state of the art regarding coccidiosis, the most used anticoccidials in poultry production, their mode of action, their prophylactic use, occurrence and the European Union (EU) applicable legislation.

Mycotoxins Exposure in Cabinda, Angola-A Pilot Biomonitoring Survey of Breastmilk.

Breast milk is considered the ideal form of nutrition for newborns and infants. However, it can carry over contaminants, namely mycotoxins, with biological effects to which this population is particularly vulnerable. Human biomonitoring and surveillance programs are particularly scarce in low-income countries, where food security is a more urgent priority in comparison with food safety. This pilot survey aims to assess exposure of breastfed infants to aflatoxin M1 (AFM1), zearalenone (ZEN), and ochratoxin A (OTA) in Angola, and to evaluate the main socio-demographical and food consumption determinants of lactating mothers. All 37 breast milk samples analyzed are found to be contaminated with ZEN and OTA, although none are found contaminated with AFM1. Contamination levels are lower than previously reported for ZEN but higher in the case of OTA. A significant association between ZEN levels in breast milk and the consumption of cookies by the lactating mothers is found. As for OTA, higher levels are observed in the milk from mothers with younger infants, for which high estimated daily intake (EDI) is determined. As far as the authors are aware, this is the first survey of the occurrence of mycotoxins in breast milk in Angola, so further human biomonitoring works should follow, given that mycotoxins are a global health issue that directly impact the health of populations.

Arsenic in Portuguese Rice: Is There Any Risk?

Arsenic is a metalloid with natural and anthropogenic sources and its inorganic form is toxic to humans. Rice is highly consumed worldwide and is prone to arsenic contamination; therefore, this study evaluated the inorganic arsenic content of 70 Portuguese rice samples. These were analysed through inductively coupled plasma mass spectrometry (ICP-MS) with a detection limit of 3.3 µg kg-1. The average contamination was of 29.3 µg kg-1, with brown and short rice presenting higher values than white and long rice. The highest concentration, 100 µg kg-1, equalled the maximum residue limit (MRL) for rice destined for infants' consumption. The estimated daily intake (EDI) surpassed the benchmark dose (lower confidence limit 10%) (BMDL10) of 0.3 µg kg-1 of bw/day considering children in the 95th percentile of rice consumption and the worst-case scenario concentration. However, other sources also contribute to the EDI and some population groups can exceed the BMDL10.

Glyphosate Use, Toxicity and Occurrence in Food.

Glyphosate is a systemic, broad-spectrum and post-emergent herbicide. The use of glyphosate has grown in the last decades, and it is currently the most used herbicide worldwide. The rise of glyphosate consumption over the years also brought an increased concern about its possible toxicity and consequences for human health. However, a scientific community consensus does not exist at the present time, and glyphosate's safety and health consequences are controversial. Since glyphosate is mainly applied in fields and can persist several months in the soil, concerns have been raised about the impact that its presence in food can cause in humans. Therefore, this work aims to review the glyphosate use, toxicity and occurrence in diverse food samples, which, in certain cases, occurs at violative levels. The incidence of glyphosate at levels above those legally allowed and the suspected toxic effects of this compound raise awareness regarding public health.

Risk Assessment of Nine Coccidiostats in Commercial and Home Raised Poultry.

For the first time, this paper aimed to evaluate nine ionophore and synthetic coccidiostat residues in poultry muscle samples, obtained from different production types, by solid-liquid extraction followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The fully validated methodology was successfully applied to a total of 101 chicken and turkey samples obtained from canteens, supermarkets, and home productions in Portugal. Halofuginone, diclazuril, decoquinate, narasin, lasalocid, and salinomycin were detected in 20.8% of the samples. Home raised samples showed a greater frequency, 47.1%. The synthetic coccidiostats halofuginone, diclazuril, and decoquinate were found in averages of 0.7 µg kg-1,2.9 µg kg-1, and 3.7 µg kg-1, respectively, while averages of 1.2 µg kg-1, 1.6 µg kg-1, and 1.3 µg kg-1 were found regarding the ionophores narasin, lasalocid, and salinomycin. As for the risk assessment, values lower than 8.06% of the acceptable daily intake (ADI) were observed, indicating that exposure to coccidiostats through consumption of poultry meat does not represent risk to consumers.

Mycotoxins in Pistachios (Pistacia vera L.): Methods for Determination, Occurrence, Decontamination.

The consumption of pistachios (Pistacia vera L.) has been increasing, given their important benefit to human health. In addition to being an excellent nutritional source, they have been associated with chemical hazards, such as mycotoxins, resulting in fungal contamination and its secondary metabolism. Aflatoxins (AFs) are the most common mycotoxins in pistachio and the most toxic to humans, with hepatotoxic effects. More mycotoxins such as ochratoxin A (OTA), fumonisins (FBs), zearalenone (ZEA) and trichothecenes (T2, HT2 and DON) and emerging mycotoxins have been involved in nuts. Because of the low levels of concentration and the complexity of the matrix, the determination techniques must be very sensitive. The present paper carries out an extensive review of the state of the art of the determination of mycotoxins in pistachios, concerning the trends in analytical methodologies for their determination and the levels detected as a result of its contamination. Screening methods based on immunoassays are useful due to their simplicity and rapid response. Liquid chromatography (LC) is the gold standard with new improvements to enhance accuracy, precision and sensitivity and a lower detection limit. The reduction of Aspergillus' and aflatoxins' contamination is important to minimize the public health risks. While prevention, mostly in pre-harvest, is the most effective and preferable measure to avoid mycotoxin contamination, there is an increased number of decontamination processes which will also be addressed in this review.

Development and Validation of QuEChERS Followed by UHPLC-ToF-MS Method for Determination of Multi-Mycotoxins in Pistachio Nuts.

Pistachios are one of the types of tree nut fruits with the highest mycotoxin contamination, especially of aflatoxins, worldwide. This study developed a Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method that was followed by Ultra-High Performance Liquid Chromatography combined with Time-of-Flight Mass Spectrometry (UHPLC-ToF-MS) for the determination of mycotoxins in pistachios. Different approaches to dispersive solid phase extraction as a clean-up method for high lipid matrices were evaluated. For this, classic sorbents such as C18 (octadecyl-modified silica) and PSA (primary secondary amine), and new classes of sorbents, namely EMR-Lipid (enhanced matrix removal-lipid) and Z-Sep (modified silica gel with zirconium oxide), were used. The QuEChERS method, followed by Z-Sep d-SPE clean-up, provided the best analytical performance for aflatoxins (AFB1, AFB2, AFG1 and AFG2), ochratoxin A (OTA), zearalenone (ZEA), toxin T2 (T2) and toxin HT-2 (HT2) in pistachios. The method was validated in terms of linearity, sensitivity, repeatability, interday precision and recovery; it achieved good results according to criteria imposed by Commission Regulation (EC) no. 401/2006. The method was applied to real samples and the results show that pistachios that are available in Portuguese markets are safe from mycotoxins that are of concern to human health.

Carmines (E120) in coloured yoghurts: a case-study contribution for human risk assessment.

Carmines (E120) are used worldwide as natural food colouring agents of animal origin, with a widespread application, including yoghurts. Despite being considered safe for human health, carmines are known to cause allergic reactions. Our goal was to evaluate the presence of carmines in different yoghurts with a label declaration of E120, purchased in Portugal, and, for the first time, to assess the human risk. The analytical methodology, recommended by JECFA, was based on acid hydrolysis of the samples followed by spectrophotometric UV-Vis analysis at 494 nm. This methodology allowed for a limit of quantification (LOQ) of 39.0 mg/kg and recovery rates higher than 97.7%. All the samples had carmines at levels above the LOQ, ranging between 43.8 and 193.8 mg/kg, with an average of 125.2 ± 34.5 mg/kg. In total, 8 (26.7%) samples exceeded the European Union (EU) maximum permitted level (MPL) established for carmines in this foodstuff, 150 mg/kg. Solid yoghurts presented higher average levels, 137.2 mg/kg, when compared to liquid samples, 107.2 mg/kg, with a significant statistical difference (p= 0.0236) being observed. No significant statistical difference was observed between white and private labels, whose average levels were very similar, 125.4 vs 125 mg/kg, respectively. Although some samples were above the allowable values, the estimated daily intake (EDI), designed for the different scenarios of different yoghurt types, did not exceed the established ADI, 5 mg/kg bw/day. According to the obtained results, carmine ingestion through the consumption of yoghurt poses low risk to the Portuguese consumers. However, children were the most vulnerable population group with a calculated risk value of up to 10% considering the mean content scenario. These first findings point out the need to reinforce surveillance programmes and monitoring studies, contributing to an increased awareness regarding carmine exposure, however it must be emphasised that yoghurt has evident nutritional benefits depending on a healthful consumer choice.

Urine biomonitoring of glyphosate in children: Exposure and risk assessment.

The use of glyphosate has been increasing over the years, making it one of the most consumed herbicides in the world. Although children are considered a vulnerable population, only four previous published studies determined glyphosate in the urine of non-occupationally exposed children. The paucity of epidemiological data and biomonitoring surveys are considered major gaps, that hinder the implementation of science driven policies in the protection of public health. The aim of the present study was to determine glyphosate in the urine of 41 Portuguese children (2-13 years old) and identify potential determinants of exposure. Glyphosate was detected in 95.1% of the samples (1.77 ± 0.86 µg/L), up to a maximum value of 4.35 µg/L. Glyphosate concentrations were higher in the urine of children aged 7-9 years, living near agricultural areas (<1 km), with a higher percentage of consumption of home-produced foods, and whose parents applied herbicides in the backyard. Risk assessment revealed an exposure representing 1-5.58% of the established Acceptable Daily Intake (ADI) of glyphosate (0.5 mg/kg bw/day). The results should be further analyzed considering the age of the participants, for which no adjusted ADI exists. This was the first published report of glyphosate exposure in the urine of Portuguese children.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part II-Lessons Learned on Mycotoxins.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by means of determining the parent compounds, their metabolites, or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are dispersed and limited. Thus, to overcome this knowledge gap, this work reviews the published Portuguese HBM information concerning mycotoxins detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative approach to the available HBM data allows us to analyze the main determinants and patterns of exposure of the Portuguese population to the selected hazardous compounds, as well as to assess the potential health risks. We also aimed to identify the main difficulties and challenges of HBM through the analysis of the enrolled studies. Ultimately, this study aims to support national and European policies in promoting human health by summarizing the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Human Biomonitoring of Selected Hazardous Compounds in Portugal: Part I-Lessons Learned on Polycyclic Aromatic Hydrocarbons, Metals, Metalloids, and Pesticides.

Human biomonitoring (HBM) data provide information on total exposure regardless of the route and sources of exposure. HBM studies have been applied to quantify human exposure to contaminants and environmental/occupational pollutants by determining the parent compounds, their metabolites or even their reaction products in biological matrices. HBM studies performed among the Portuguese population are disperse and limited. To overcome this knowledge gap, this review gathers, for the first time, the published Portuguese HBM information concerning polycyclic aromatic hydrocarbons (PAHs), metals, metalloids, and pesticides concentrations detected in the urine, serum, milk, hair, and nails of different groups of the Portuguese population. This integrative insight of available HBM data allows the analysis of the main determinants and patterns of exposure of the Portuguese population to these selected hazardous compounds, as well as assessment of the potential health risks. Identification of the main difficulties and challenges of HBM through analysis of the enrolled studies was also an aim. Ultimately, this study aimed to support national and European policies promoting human health and summarizes the most important outcomes and lessons learned through the HBM studies carried out in Portugal.

Citrinin in Foods and Supplements: A Review of Occurrence and Analytical Methodologies.

Citrinin (CIT) deserves attention due to its known toxic effects in mammalian species and its widespread occurrence in food commodities, often along with ochratoxin A, another nephrotoxic mycotoxin. Human exposure, a key element in assessing risk related to food contaminants, depends upon mycotoxin contamination levels in food and on food consumption. Commercial supplements, commonly designated as red rice, usually used in daily diets in Asiatic countries due to their medicinal properties, may pose a health problem as a result of high CIT levels. In addition to the worldwide occurrence of CIT in foods and supplements, a wide range of several analytical and detection techniques with high sensitivity, used for evaluation of CIT, are reviewed and discussed in this manuscript. This review addresses the scientific literature regarding the presence of CIT in foods of either vegetable or animal origin, as well as in supplements. On what concerns analytical methodologies, sample extraction methods, such as shaking extraction and ultrasonic assisted extraction (UAE), clean-up methods, such as liquid-liquid extraction (LLE), solid phase extraction (SPE) and Quick, Easy, Cheap, Effective, Rugged and Safe (QuECHERS), and detection and quantification methods, such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC), capillary electrophoresis (CE), biosensors, and ELISA, are also reviewed.

Artificial sweeteners in non-alcoholic beverages: Occurrence and exposure estimation of the Portuguese population.

Artificial sweeteners such as acesulfame-potassium (ACE-K). saccharin (SAC), and aspartame (ASP), play a vital role in the food industry. Despite apparent benefits, several studies report adverse reactions on consumers. EFSA is currently re-evaluating several individual sweeteners, including ASP, Ace-K and SAC that shall be completed by 31 December 2020. Our goal was to evaluate, by liquid chromatography with ultraviolet detection (LC-UV), their levels in non-alcoholic beverages from the Portuguese market (soft drinks, sport drinks, energy drinks, and nectars), and estimate the exposure of the Portuguese adolescent/adult population. ACE-K was found in 85.7% of the samples, in mean levels of 97.4 mg/L. ASP and SAC, detected in 37.5% and 33.9% of the samples, were found in averages of 161.5 and 55.0 mg/L, respectively. The maximum limits set for ASP were exceeded in one energy drink. SAC exceeded the maximum permitted level (MPL) level in 3 soft drinks based on tea extracts. In 4 samples SAC was found but not mentioned in the label, and in one of these samples exceeded the MPL. The estimated daily intake (EDI), calculated in mg per kilogram of body weight per day, of ACE-K, SAC and ASP was found to be below the acceptable daily intake (ADI), both for Portuguese adolescents and adult population. Traditional soft drinks accounted for the highest EDIs representing 1.8%, 2.3% and 0.6% for ACE-K, SAC and ASP, respectively, of the ADI. Considering the previous report in Portugal it is observed that the population is currently exposed to a greater amount of artificial sweeteners, which may indicate a trend for more artificial sweeteners' usage in order to reduce sugar intake.