Development, validation, and application of a multimatrix UHPLC-MS/MS method for quantification of Datura-type alkaloids in food.

A quantitative ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated for the determination of tropane alkaloids (TAs), atropine and scopolamine, in a variety of food products. The sample preparation of cereal-based food, oilseeds, honey, and pulses consisted of a solid-liquid extraction with an acidified mixture of methanol and water, while an additional step of solid-phase extraction on a cation-exchange sorbent was introduced in the treatment of teas and herbal infusions, aromatic herbs, spices and food supplements. The limits of quantification of the method varied from 0.5 to 2.5 µg kg-1. Apparent recovery was in the range of 70-120%, and repeatability and intermediate precision were below 20%. The method was successfully applied in a proficiency testing exercise as well as in the analysis of various commercial foods. Only 26% of the analysed food samples contained one or both TAs. The mean concentrations for atropine and scopolamine amounted to 21.9 and 6.5 µg kg-1, respectively, while the maximum concentrations were 523.3 and 131.4 µg kg-1, respectively. Overall, the highest levels of TA sum were found in an herbal infusion of fennel and a spice mix containing fennel and anise seeds.

The contribution of cacao consumption to the bioaccessible dietary cadmium exposure in the Belgian population.

Since 2019, EU limits apply to cadmium (Cd) concentrations in cacao-derived food products. The dietary risk assessment leading to that regulation used consumption surveys aggregated to a limited number of chocolate product categories and did not consider differences in Cd bioaccessibility. Here, the cacao-related dietary Cd exposure in the Belgian population was estimated with higher resolution and accounting for bioaccessibility. A food frequency questionnaire and a 24-h recall (N = 2055) were set up for the Belgian population, in combination with ICP-MS analysis of a large subset of cacao-containing products (N = 349). Both the average chocolate consumption (28 g day-1) and the relative contribution of chocolate to the total dietary Cd exposure (7-9%) were higher than previously estimated for the Belgian population, probably because of some selection bias towards chocolate consumers in the cohort. The Cd bioaccessibility in chocolate products was a factor 5 (cacao powder) and 2 (dark chocolate) lower compared to wheat flour, suggesting lower bioavailability in chocolate than in wheat, which is a main contributor to dietary Cd. This study suggests that Cd intake from cacao consumption has been underestimated because of hidden cacao in non-chocolate food categories but, in contrast, may have overestimated the true exposure because of lower bioavailability compared to the main foodstuffs contributing to Cd exposure.

LC-MS/MS Validation and Quantification of Cyanotoxins in Algal Food Supplements from the Belgium Market and Their Molecular Origins.

Food supplements are gaining popularity worldwide. However, harmful natural compounds can contaminate these products. In the case of algae-based products, the presence of toxin-producing cyanobacteria may cause health risks. However, data about the prevalence of algal food supplements on the Belgian market and possible contaminations with cyanotoxins are scarce. Therefore, we optimized and validated a method based on Ultra High Performance Liquid Chromatography-Tandem Mass Spectrometry to quantify eight microcystin congeners and nodularin in algal food supplements. Our analytical method was successfully validated and applied on 35 food supplement samples. Nine out of these samples contained microcystin congeners, of which three exceeded 1 µg g-1, a previously proposed guideline value. Additionally, the mcyE gene was amplified and sequenced in ten products to identify the taxon responsible for the toxin production. For seven out of these ten samples, the mcyE gene could be amplified and associated to Microcystis sp. EFSA and posology consumption data for algal-based food supplements were both combined with our toxin prevalence data to establish different toxin exposure scenarios to assess health risks and propose new guideline values.

Comparative profiling and exposure assessment of microplastics in differently sized Manila clams from South Korea by µFTIR and Nile Red staining.

The accumulation of microplastics in marine organisms is an emerging concern. Due to trophic transfer, the safety of seafood is under investigation in view of the potential negative effects of microplastics on human health. In this study, market samples of Manila clams (Ruditapes philippinarum) from South Korea were segregated into two groups of considerably different size (p < 0.05), namely small clams with shell length of 40.69 ± 3.97 mm, and large clams of shell length 51.19 ± 2.86 mm. Comparative profiling of the number, size, shape, and polymer type of microplastics were performed using µFTIR imaging and Nile red staining. Overall, µFTIR detected only 1559 microplastics while 1996 microplastics were counted based on staining from 61 Manila clams (30 small and 31 large), leading to an overestimation of 18 to 75 %. Comparable microplastics concentration, based on µFTIR, were observed at 2.70 ± 1.66 MP/g or 15.64 ± 9.25 MP/individual for the small samples, and 3.65 ± 1.59 MP/g or 41.63 ± 16.90 MP/individual for the large ones (p > 0.05). Particle diameters of 20-100 µm was the most dominant, accounting for 44.6 % and 46.5 % of all microplastics from the small and large groups, respectively. Particles, with a circularity (resemblance to a circle) value between 0.6 and 1.0, were the most prevalent, followed by fragments and fibers. At least 50 % of microplastics from the small and large samples were polystyrene, making it the most abundant polymer type. Despite the substantial difference in the size of the animals, only a weak to moderate correlation was observed between microplastics content and the physical attributes of the clams such as shell length and weight, (soft) tissue weight, and total weight (Spearman's coefficient < 0.5). The estimated intake of microplastics by the Korean population was 1232 MP/person/year via small clams, 1663 MP/person/year via large clams, and 1489 MP/person/year via clams independent of size.

A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations.

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L-1) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L-1 to 2798.81 µg L-1 total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.

Cyanotoxins and Food Contamination in Developing Countries: Review of Their Types, Toxicity, Analysis, Occurrence and Mitigation Strategies.

Cyanotoxins have gained global public interest due to their potential to bioaccumulate in food, which threatens human health. Bloom formation is usually enhanced under Mediterranean, subtropical and tropical climates which are the dominant climate types in developing countries. In this context, we present an up-to-date overview of cyanotoxins (types, toxic effects, analysis, occurrence, and mitigation) with a special focus on their contamination in (sea)food from all the developing countries in Africa, Asia, and Latin America as this has received less attention. A total of 65 publications have been found (from 2000 until October 2021) reporting the contamination by one or more cyanotoxins in seafood and edible plants (five papers). Only Brazil and China conducted more research on cyanotoxin contamination in food in comparison to other countries. The majority of research focused on the detection of microcystins using different analytical methods. The detected levels mostly surpassed the provisional tolerable daily intake limit set by the World Health Organization, indicating a real risk to the exposed population. Assessment of cyanotoxin contamination in foods from developing countries still requires further investigations by conducting more survey studies, especially the simultaneous detection of multiple categories of cyanotoxins in food.

Concentrations and distribution of chlorinated paraffins in Belgian foods.

This study reports on concentrations of short- and medium-chain chlorinated paraffins (SCCPs and MCCPs, respectively) in a wide range of food samples (n = 211) purchased in Belgium during 2020. Samples were analysed by gas chromatography-mass spectrometry (GC-MS) and quantified using chlorine content calibration. ∑SCCPs were present above LOQ in 25% of samples with an overall range of

Dietary exposure assessment and risk characterization of citrinin and ochratoxin A in Belgium.

Exposure to mycotoxins is a worldwide problem. To ensure public health, it is imperative to characterize the risks related to these toxins. The present study aims to conduct a dietary exposure assessment of citrinin (CIT) and ochratoxin A (OTA) in the Belgian population using consumption data of a variety of foodstuffs. A total of 367 food samples from different food categories were collected in Belgian supermarkets and analysed for CIT and OTA using a validated liquid chromatography-tandem mass spectrometry method. Daily CIT and OTA exposure to the Belgian population was calculated based on the analytical results and food consumption data in three age categories (3-9, 10-17 and 18-64 years), obtained from a national food consumption survey. Furthermore, a risk characterization was performed for CIT, in which no intake values exceeded the tolerable daily intake (TDI) of 200 ng kg-1 bw day-1, indicating no health risk. However, a CIT intake level of 187 ng kg-1 bw day-1 was detected for children in the age category of 3-9 years in the worst case scenario for rice, indicating that rice consumption could contain a potential health hazard for young children. For OTA, a potential health risk was detected in several food categories (biscuits, croissants, rice, flour, meat imitates, herbs and spices) in the higher percentiles (P99) or at maximum found concentrations when calculating the margin of exposure (MoE) for neoplastic effects. An attempt to perform a cumulative health risk assessment for both toxins was done. Although a high number of uncertainties is involved, combined margin of exposure (MoET) values indicated a potential health risk related to the combined exposure to CIT and OTA. For the first time, our study demonstrated the potential health risks of CIT and OTA after individual and combined exposure, in particular related to rice consumption. Moreover, further research is recommended concerning multiple mycotoxin exposure in young children.

Detection of toxins involved in foodborne diseases caused by Gram-positive bacteria.

Bacterial toxins are food safety hazards causing about 10% of all reported foodborne outbreaks in Europe. Pertinent to Gram-positive pathogens, the most relevant toxins are emetic toxin and diarrheal enterotoxins of Bacillus cereus, neurotoxins of Clostridium botulinum, enterotoxin of Clostridium perfringens, and a family of enterotoxins produced by Staphylococcus aureus and some other staphylococci. These toxins are the most important virulence factors of respective foodborne pathogens and a primary cause of the related foodborne diseases. They are proteins or peptides that differ from each other in their size, structure, toxicity, toxicological end points, solubility, and stability, types of food matrix to which they are mostly related to. These differences influence the characteristics of required detection methods. Therefore, detection of these toxins in food samples, or detection of toxin production capacity in the bacterial isolate, remains one of the cornerstones of microbial food analysis and an essential tool in understanding the relevant properties of these toxins. Advanced research has led into new insights of the incidence of toxins, mechanisms of their production, their physicochemical properties, and their toxicological mode of action and dose-response profile. This review focuses on biological, immunological, mass spectrometry, and molecular assays as the most commonly used detection and quantification methods for toxins of B. cereus, C. botulinum, C. perfringens, and S. aureus. Gathered and analyzed information provides a comprehensive blueprint of the existing knowledge on the principles of these assays, their application in food safety, limits of detection and quantification, matrices in which they are applicable, and type of information they provide to the user.

Gantenerumab reduces amyloid-ß plaques in patients with prodromal to moderate Alzheimer's disease: a PET substudy interim analysis.

BACKGROUND: We previously investigated low doses (105 or 225 mg) of gantenerumab, a fully human monoclonal antibody that binds and removes aggregated amyloid-ß by Fc receptor-mediated phagocytosis, in the SCarlet RoAD (SR) and Marguerite RoAD (MR) phase 3 trials. Several lines of evidence suggested that higher doses may be necessary to achieve clinical efficacy. We therefore designed a positron emission tomography (PET) substudy to evaluate the effect of gantenerumab uptitrated to 1200 mg every 4 weeks on amyloid-ß plaques as measured using florbetapir PET in patients with prodromal to moderate Alzheimer's disease (AD). METHODS: A subset of patients enrolled in the SR and MR studies who subsequently entered the open-label extensions (OLEs) were included in this substudy. Patients were aged 50 to 90 years with a clinical diagnosis of probable prodromal to moderate AD and were included based on a visual read of the original screening scan in the double-blind phase. Patients were assigned to 1 of 5 titration schedules (ranging from 2 to 10 months) with a target gantenerumab dose of 1200 mg every 4 weeks. The main endpoint of this substudy was change in amyloid-ß plaque burden from OLE baseline to week 52 and week 104, assessed using florbetapir PET. Florbetapir global cortical signal was calculated using a prespecified standard uptake value ratio method converted to the Centiloid scale. RESULTS: Sixty-seven of the 89 patients initially enrolled had ≥ 1 follow-up scan by August 15, 2018. Mean amyloid levels were reduced by 39 Centiloids by the first year and 59 Centiloids by year 2, a 3.5-times greater reduction than was seen after 2 years at 225 mg in SR. At years 1 and 2, 37% and 51% of patients, respectively, had amyloid-ß plaque levels below the amyloid-ß positivity threshold. CONCLUSION: Results from this exploratory interim analysis of the PET substudy suggest that gantenerumab doses up to 1200 mg resulted in robust amyloid-ß plaque removal at 2 years. PET amyloid levels were consistent with sparse-to-no neuritic amyloid-ß plaques in 51% of patients after 2 years of therapy. Amyloid reductions were similar to those observed in other placebo-controlled studies that have suggested potential clinical benefit. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01224106 (SCarlet RoAD) and NCT02051608 (Marguerite RoAD).

Determinants of persistent organic pollutant (POP) concentrations in human breast milk of a cross-sectional sample of primiparous mothers in Belgium.

BACKGROUND: Bio-accumulation of persistent organic pollutants (POPs) in the environment and in the food chain can lead to high pollutant concentrations in human fat-containing tissues and breast milk. OBJECTIVES: We aimed to identify the maternal characteristics that determined POP concentrations in breast milk of primiparous mothers in Belgium. METHODS: Breast milk samples were obtained from a cross-sectional sample of 206 primiparous mothers in 2014. POP concentrations in breast milk samples were determined by GC-ECNI-MS and GC-EI-MS/MS depending on the analytes' sensitivity. Associations between POP concentrations in breast milk and potential determinants were investigated using two-way contingency tables and multivariable generalized linear models. RESULTS: Fifteen of the 23 screened POPs were detected in the breast milk samples. Four organochlorine compounds (p,p'-DDT, p,p'-DDE, HCB and ß-HCH) and two brominated flame retardant congeners (BDE-47, BDE-153) were detected at concentrations above the limit of quantification in >50% of the breast milk samples. Maternal age and BMI were usually associated with higher POP concentrations. Rural residency and consumption of home-produced eggs, fatty fish and fish oil supplements were associated with higher concentrations of DDT and DDE. Consumption of fatty fish and being breastfed during childhood were associated with higher concentrations of HCB and ß-HCH. Fish oil supplements and home-produced eggs were associated with higher concentrations of BDEs, but for BDE congeners exposure routes other than diet require further investigation. CONCLUSIONS: Dietary and non-dietary determinants predict individual POP concentrations in breast milk.

Correction to: A phase III randomized trial of gantenerumab in prodromal Alzheimer's disease.

Following publication of the original article [1], the author reported errors in the formatting of the table. The details of the errors are as follows.

Development and validation of a quantitative UHPLC-MS/MS method for selected brominated flame retardants in food.

An ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS) method was developed and validated (in-house) for the quantification of selected brominated flame retardants (BFRs), including tetrabromobisphenol A (TBBPA), hexabromocyclododecanes (HBCDs), tetrabromobisphenol S (TBBPS) and bromophenols (BPs), in various food matrices. The sample preparation consisted of extraction of TBBPS with acidified acetonitrile followed by a fast dispersive solid-phase extraction (dSPE) clean-up and extraction of the other BFRs with a mixture of hexane and dichloromethane (1:1, v/v) with subsequent clean-up using acidified silica (44%, w/w). The limits of quantification of the method varied widely for the types of food matrices and the different classes of BFRs from 4 pg g-1 wet weight (ww) to 8 ng g-1 ww. For most of the analytes the apparent recovery was in the range 70-120%, and the method precision (under repeatability conditions) was below 20%. The method was successfully applied in proficiency testing exercises as well as for analysis of various food items. Only 25% of the collected food samples contained BFRs, with 4-bromophenol and α-HBCD as the only detected compounds. The contaminated foodstuffs were fish and eggs with concentrations in the range from 48 to 305 pg g-1 ww.

Silicone Wristband Passive Samplers Yield Highly Individualized Pesticide Residue Exposure Profiles.

Monitoring human exposure to pesticides and pesticide residues (PRs) remains crucial for informing public health policies, despite strict regulation of plant protection product and biocide use. We used 72 low-cost silicone wristbands as noninvasive passive samplers to assess cumulative 5-day exposure of 30 individuals to polar PRs. Ethyl acetate extraction and LC-MS/MS analysis were used for the identification of PRs. Thirty-one PRs were detected of which 15 PRs (48%) were detected only in worn wristbands, not in environmental controls. The PRs included 16 fungicides (52%), 8 insecticides (26%), 2 herbicides (6%), 3 pesticide derivatives (10%), 1 insect repellent (3%), and 1 pesticide synergist (3%). Five detected pesticides were not approved for plant protection use in the EU. Smoking and dietary habits that favor vegetable consumption were associated with higher numbers and higher cumulative concentrations of PRs in wristbands. Wristbands featured unique PR combinations. Our results suggest both environment and diet contributed to PR exposure in our study group. Silicone wristbands could serve as sensitive passive samplers to screen population-wide cumulative dietary and environmental exposure to authorized, unauthorized and banned pesticides.

A phase III randomized trial of gantenerumab in prodromal Alzheimer's disease.

BACKGROUND: Gantenerumab is a fully human monoclonal antibody that binds aggregated amyloid-ß (Aß) and removes Aß plaques by Fc receptor-mediated phagocytosis. In the SCarlet RoAD trial, we assessed the efficacy and safety of gantenerumab in prodromal Alzheimer's disease (AD). METHODS: In this randomized, double-blind, placebo-controlled phase III study, we investigated gantenerumab over 2 years. Patients were randomized to gantenerumab 105 mg or 225 mg or placebo every 4 weeks by subcutaneous injection. The primary endpoint was the change from baseline to week 104 in Clinical Dementia Rating Sum of Boxes (CDR-SB) score. We evaluated treatment effects on cerebrospinal fluid biomarkers (all patients) and amyloid positron emission tomography (substudy). A futility analysis was performed once 50% of patients completed 2 years of treatment. Safety was assessed in patients who received at least one dose. RESULTS: Of the 3089 patients screened, 797 were randomized. The study was halted early for futility; dosing was discontinued; and the study was unblinded. No differences between groups in the primary (least squares mean [95% CI] CDR-SB change from baseline 1.60 [1.28, 1.91], 1.69 [1.37, 2.01], and 1.73 [1.42, 2.04] for placebo, gantenerumab 105 mg, and gantenerumab 225 mg, respectively) or secondary clinical endpoints were observed. The incidence of generally asymptomatic amyloid-related imaging abnormalities increased in a dose- and APOE ε4 genotype-dependent manner. Exploratory analyses suggested a dose-dependent drug effect on clinical and biomarker endpoints. CONCLUSIONS: The study was stopped early for futility, but dose-dependent effects observed in exploratory analyses on select clinical and biomarker endpoints suggest that higher dosing with gantenerumab may be necessary to achieve clinical efficacy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01224106 . Registered on October 14, 2010.

Application of LC-MS/MS MRM to Determine Staphylococcal Enterotoxins (SEB and SEA) in Milk.

Staphylococcus aureus is one of the important aetiological agents of food intoxications in Europe and can cause gastro-enteritis through the production of various staphylococcal enterotoxins (SEs) in foods. Due to their stability and ease of production and dissemination, some SEs have also been studied as potential agents for bioterrorism. Therefore, specific and accurate analytical tools are required to detect and quantify SEs. Online solid-phase extraction liquid chromatography electrospray ionization tandem mass spectrometry (online SPE-LC-ESI-MS/MS) based on multiple reaction monitoring (MRM) was used to detect and quantify two types of SE (A and B) spiked in milk and buffer solution. SE extraction and concentration was performed according to the European Screening Method developed by the European Reference Laboratory for Coagulase Positive Staphylococci. Trypsin digests were screened for the presence of SEs using selected proteotypic heavy-labeled peptides as internal standards. SEA and SEB were successfully detected in milk samples using LC-MS/MS in MRM mode. The selected SE peptides were proteotypic for each toxin, allowing the discrimination of SEA and SEB in a single run. The detection limit of SEA and SEB was approximately 8 and 4 ng/g, respectively.

An emetic Bacillus cereus outbreak in a kindergarten: detection and quantification of critical levels of cereulide toxin.

A Bacillus cereus-related emetic outbreak was reported in a Belgian kindergarten. High levels of emetic B. cereus (>1.5E+07 colony-forming units/g) were detected in the food leftovers, and the presence of an emetic strain was confirmed in feces. Emetic toxin levels ranging up to 4.2 µg/g were also quantified in the leftovers by liquid chromatography coupled to tandem mass spectrometry (LC-MS(2)) analysis. Those levels, although moderate in comparison with earlier published intoxications, provoked profuse-vomiting episodes in 20 toddlers aged between 10 and 18 months. Few studies have focused on the levels of emetic toxin implicated in food intoxications. This publication emphasizes the importance of defining toxic doses of emetic toxin among high-risk population groups.

Foodborne cereulide causes beta-cell dysfunction and apoptosis.

AIMS/HYPOTHESIS: To study the effects of cereulide, a food toxin often found at low concentrations in take-away meals, on beta-cell survival and function. METHODS: Cell death was quantified by Hoechst/Propidium Iodide in mouse (MIN6) and rat (INS-1E) beta-cell lines, whole mouse islets and control cell lines (HepG2 and COS-1). Beta-cell function was studied by glucose-stimulated insulin secretion (GSIS). Mechanisms of toxicity were evaluated in MIN6 cells by mRNA profiling, electron microscopy and mitochondrial function tests. RESULTS: 24 h exposure to 5 ng/ml cereulide rendered almost all MIN6, INS-1E and pancreatic islets apoptotic, whereas cell death did not increase in the control cell lines. In MIN6 cells and murine islets, GSIS capacity was lost following 24 h exposure to 0.5 ng/ml cereulide (P<0.05). Cereulide exposure induced markers of mitochondrial stress including Puma (p53 up-regulated modulator of apoptosis, P<0.05) and general pro-apoptotic signals as Chop (CCAAT/-enhancer-binding protein homologous protein). Mitochondria appeared swollen upon transmission electron microscopy, basal respiration rate was reduced by 52% (P<0.05) and reactive oxygen species increased by more than twofold (P<0.05) following 24 h exposure to 0.25 and 0.50 ng/ml cereulide, respectively. CONCLUSIONS/INTERPRETATION: Cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects. Thus, exposure to cereulide even at concentrations too low to cause systemic effects appears deleterious to the beta-cell.

6-Alkoxy-5-aryl-3-pyridinecarboxamides, a new series of bioavailable cannabinoid receptor type 1 (CB1) antagonists including peripherally selective compounds.

We identified 6-alkoxy-5-aryl-3-pyridinecarboxamides as potent CB1 receptor antagonists with high selectivity over CB2 receptors. The series was optimized to reduce lipophilicity compared to rimonabant to achieve peripherally active molecules with minimal central effects. Several compounds that showed high plasma exposures in rats were evaluated in vivo to probe the contribution of central vs peripheral CB1 agonism to metabolic improvement. Both rimonabant and 14g, a potent brain penetrant CB1 receptor antagonist, significantly reduced the rate of body weight gain. However, 14h, a molecule with markedly reduced brain exposure, had no significant effect on body weight. PK studies confirmed similarly high exposure of both 14h and 14g in the periphery but 10-fold lower exposure in the brain for 14h. On the basis of these data, which are consistent with reported effects in tissue-specific CB1 receptor KO mice, we conclude that the metabolic benefits of CB1 receptor antagonists are primarily centrally mediated as originally believed.