Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the network of National Reference Laboratories: Determination of aflatoxin B1 in defatted peanut powder

The Joint Research Centre (JRC), a Directorate-General of the European Commission, operates the European Union Reference Laboratory (EURL) for Mycotoxins. One of its core tasks is to organise proficiency tests (PTs) among appointed National Reference Laboratories (NRLs). This report presents the results of the PT on the determination of aflatoxins in defatted peanut powder. The test items for this PT were two contaminated defatted peanut powder samples. The materials were produced by the JRC and dispatched to the participants mid October 2016. Each participant received one bottle per test material containing approximately 55 g each. Fifty-six participants from thirty countries (among them 40 NRLs and 16 official food control laboratories) registered for the exercise and 54 sets (Sample A and B) of results were reported. The assigned values, established by an exact-matching double isotope dilution mass spectrometric technique, were 2.80 µg/kg (± 0.19 µg/kg) aflatoxin B1 in sample A and 3.20 µg/kg (± 0.20 µg/kg) in sample B. Participants' results were rated with z-scores and zeta-scores for aflatoxin B1 in accordance with ISO 13528:2015. The z-score compares the participant's deviation from the reference value with the target standard deviation accepted for the PT, whereas the zeta-score provides an indication of whether the participant's estimate of uncertainty is consistent with the observed deviation from the assigned value. Only z-scores were used for the evaluation whether an individual laboratory underperformed. In total, 96 % of the attributed z scores were below an absolute value of two for sample A and and 92 % for sample B. This indicates that most of the participants performed satisfactorily. The few participants that had z-scores above an absolute value of 2 will have to investigate the reasons for the deviation (root-cause analysis) and report the planned corrective actions to the EURL.JRC.F.5-Food and Feed Complianc

Assignment of a Reference Value of Total Cow’s Milk Protein Content in Baked Cookies Used in an Interlaboratory Comparison

Interlaboratory comparisons (ILC) in the food allergens field mainly rely on the use of consensus values per applied methodology or even per type of an ELISA test kit. Results suggest good reproducibility; however, possible biases may not be recognized since metrological traceability to an independent reference is lacking. The work presented here utilizes isotope dilution mass spectrometry (IDMS) to assign a reference value of the total cow’s milk protein (TCMP) content in a baked cookie and its associated uncertainty. TCMP consists of several individual proteins, of which five (representing 92%) served us as markers for TCMP. Per marker, one to four proteotypic peptides were selected for the quantification. These were synthesized, and the mass fractions of respective reference solutions were determined with peptide-impurity-corrected amino acid analysis to establish traceability to SI units. Stable isotope labelled (“heavy”) analogues of the proteotypic peptides were also synthesized and blended with extracts of the test material or the reference solutions for IDMS. Through careful measurement design minimizing biases, well-defined model equations were developed, allowing appropriate estimation of the associated uncertainty. The determined reference value of 11.8 ± 1.1 mg TCMP/kg cookie was used for scoring of a novel ILC

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in tea and herbal infusions

Tropane alkaloids (TAs) are toxins found in a wide variety of plant species growing in mild climates. The most well-known are Datura, Atropa and Hyoscyamus sp., belonging to the Solanaceae family. The TAs family comprises more than 200 compounds, of which atropine and scopolamine are the most active producing anticholinergic symptoms (e.g. blurred vision, dry mouth, muscle spasms, tachycardia and death) if ingested in toxic quantities. The presence of botanical impurities (e.g. seeds, leaves and roots) has been reported in a variety of tea and herbal blends, stressing the need to control the quality of these products in the EU market. The European Union Reference Laboratory (EURL) for Mycotoxins organised a proficiency test (PT) on the determination of TAs (atropine and scopolamine) in tea and herbal infusions upon request from DG SANTE. The measurand levels were targeted to provide insight on the measurement capabilities of EU Member States' laboratories at concentrations close to the recommended limit of quantification (LOQ) established by the Commission Recommendation 2015/976 (preferably below 5 μg/kg and not higher 10 μg/kg). Additionally, the ratio of atropine to scopolamine was kept as native in the plant materials in some samples. Three matrices appropriately processed were provided to the participants: black tea, peppermint leaves and fennel seeds. The concentrations of atropine varied from 8.3 to 42.2 μg/kg while those of scopolamine ranged from 1.5 to 20.8 μg/kg. The participants were asked to determine atropine and scopolamine in 6 contaminated samples (2 per matrix) and 3 blank materials spiked by them with a TAs solution of unknown concentration. This setup was also aimed to allow a preliminary assessment of the robustness of the EURL-developed method. Thirty-three laboratories from 11 Member States joined the PT, with a very significant participation from Germany. The performance of the laboratories was assessed using z-scores with regard to the assigned values obtained by exact matching double isotope dilution mass spectrometry (EMD-IDMS), in line with the ISO 13528:2015. In all cases, the consensus values derived from the participants' data were within the range of the assigned values, considering the respective confidence intervals. On average, eighty-seven percent of the z-scores for atropine and 84 % for scopolamine fell in the acceptable range (|z| ≤ 2). The success rate varied from 83 to 94 % for atropine and from 67 to 94 % for scopolamine, across the distributed matrices and concentration levels. The robust standard deviations of the reported results for both TAs were in good agreement with the target standard deviation (22 %). The results of this PT indicate that EU Member States’ laboratories can determine atropine and scopolamine reliably in tea and herbal infusions at levels relevant to the current legislation (Commission Recommendation 2015/976).JRC.F.5-Food and Feed Complianc

Report on the 2016 Proficiency Test of the European Union Reference Laboratory for Mycotoxins for the Network of National Reference Laboratories: Determination of tropane alkaloids in cereal products for infants and young children

Tropane alkaloids (TAs) are plant toxins that occur mainly in Datura, Atropa and Hyoscyamus sp, belonging to the Solanaceae family, besides a variety of other families such as Erythroxylaceae, Brassicaceae, Proteaceae, Euphorbiaceae, Rhizophoraceae, Convolvulaceae and Cruciferae. The TAs occur in all parts of the plants and botanical impurities have been found in a range of crops due to accidental contamination during harvesting. The intoxication via the food leads to anticholinergic effects (e.g. blurred vision, pupil dilation, dry mouth, vomiting, muscle spasms, tachycardia, etc.), culminating in severe intoxications and death. The EFSA CONTAM Panel established a group Acute Reference Dose (ARfD) of 0.016 μg/kg body weight (b.w.) expressed as the sum of (-)-hyoscyamine and ( ) scopolamine, assuming equivalent potency. Infants and young children are the most exposed age classes as they consume a higher amount of cereal-based products per body weight. EFSA estimated that the dietary exposure of toddlers could be up to seven times the group ARfD. Recently, two European legislation acts were published in this field: Commission Recommendation (EU) 2015/976, recommending the monitoring of tropane alkaloids in certain food categories, and Commission Regulation (EU) 2016/239, enforcing maximum levels of tropane alkaloids in certain cereal-based foods for infants and young children. The EURL-Mycotoxins organised a proficiency test (PT) concerning the determination of atropine and scopolamine in cereal-based baby food, aiming to underpin and assess the measurement capability of Member States' (MS) laboratories. Particular focus was given to levels relevant for enforcement of legislation. Two samples were distributed to the participants: one sample labelled “C” – biscuits for infants containing approx. 1.2 µg/kg of atropine and 0.2 µg/kg of scopolamine, and one sample labelled “E” – cereals for porridge containing approx. 7.4 µg/kg of atropine and 1.0 µg/kg of scopolamine. Forty-eight datasets from 18 EU MS laboratories were received. Overall, 81 % of the z-scores were in the range of -2 to 2 and 90 % were in the range of -3 to 3. For the lowest TA level (sample C) still 75 % of z-scores fell into an acceptable range (|z| ≤ 2), while the mass fraction of scopolamine was far below the target level of 1 µg/kg. In line with this observation, the vast majority of reported LOQs were below 1.0 µg/kg. The methodologies used by the participants can be clustered into three groups: the method supplied by the EURL; the RIKILT SOP A1070 and methods based on QuEChERS. The instrumental determination was by LC-MS/MS, with one exception (GC-MS). The recoveries reported by the participants were close to 95 %. No statistically significant dependence of the z scores on the analytical methodology was observed. These results support the assumption that atropine and scopolamine can be reliably determined at the maximum levels proposed by the EU to ensure the protection of infants and young children's health using state-of-the-art analytical instrumentation.  JRC.F.5-Food and Feed Complianc

A reference method for determining the total allergenic protein content in a processed food: the case of milk in cookies as proof of concept

The establishment of a reference method for the determination of the allergen protein content in a processed food material has been explored. An analytical approach was developed to enable the comparability of food allergen measurement results expressed in a decision-relevant manner. A proof of concept is here presented, resulting in quantity values for the common measurand, namely ‘mass of total allergen protein per mass of food’. The quantities are determined with SI traceability to enable the comparability of reported results. A method for the quantification of total milk protein content in an incurred baked food at a concentration level clinically relevant is presented. The strategy on how to obtain the final analytical result is outlined. Challenges associated with this method are discussed, in particular the optimal extraction of the marker proteins, the complete digestion and release of the peptides in an equimolar fashion, the use of conversion factors to translate the amount of measured proteins into total milk protein and the estimation of the uncertainty contributions as well as of the combined uncertainty of the final result. The implementation of such a reference method for the determination of the total allergen content in a processed food is an important step, which will provide comparable measurement data of relevance to risk assessors

Intersex related gene expression profiles in clams Scrobicularia plana : molecular markers and environmental application

Intersex, the appearance of female characteristics in male gonads, has been identified in several aquatic species. It is a widespread phenomenon in populations of the bivalve, Scrobicularia plana, from the southwest coast of the U.K. Genes previously identified as differentially expressed (ferritin, testicular haploid expressed gene, THEG, proliferating cell nuclear antigen, PCNA; receptor activated protein kinase C, RACK; cytochrome B, CYB; and cytochrome c oxidase 1, COX1) in intersex clams relative to normal male clams, were selected for characterisation and an environmental survey of the Channel region. Transcripts were significantly differentially expressed at sites with varying intersex incidence and contaminant burdens. Significant correlations between specific gene expressions, key contaminants and sampling locations have been identified, though no single gene was associated with intersex incidence. The results highlight the difficulty in understanding the intersex phenomenon in molluscs where there is still a lack of knowledge on the control of normal reproduction

Identification of Reproduction-Specific Genes Associated with Maturation and Estrogen Exposure in a Marine Bivalve Mytilus edulis

Background: While it is established that vertebrate-like steroids, particularly estrogens (estradiol, estrone) and androgens (testosterone), are present in various tissues of molluscs, it is still unclear what role these play in reproductive endocrinology in such organisms. This is despite the significant commercial shellfishery interest in several bivalve species and their decline. Methodology/Principal Findings: Using suppression subtraction hybridisation of mussel gonad samples at two stages (early and mature) of gametogenesis and (in parallel) following controlled laboratory estrogen exposure, we isolate several differentially regulated genes including testis-specific kinases, vitelline lysin and envelope sequences. Conclusions: The differentially expressed mRNAs isolated provide evidence that mussels may be impacted by exogenous estrogen exposure

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation

Integrated Metabolomic and gene expression study of estrogenic response mechanisms in the marine bivalve mytilus edulis

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Review on metabolomics for food authentication

European and global food policies require that food put on the market is authentic, which means that the label declarationmatches the composition and provenance of the food item. During the last decades conventional approaches such as the determination of certain major and minor components by wet chemistry of chromatographic methods have been used for food authentication. Although many of the traditional methods are still used because they are part of product standards, new approaches that could complement existing methodologies, such as metabolomics are emerging in food authentication applications. Metabolomics aims to study multiple metabolites in a cell, a tissue or an organism. The main advantage of metabolomic applications in food authentication makes use of its untargeted nature, which can enable the detection of emerging frauds. The purpose of this review is to summarise and describe the recent metabolomic applications in the area of food authentication and to discuss its current limitations and future potential.JRC.D.5-Standards for Food Bioscienc