The accurate quantification of food allergens is crucial for ensuring consumer safety and compliance with regulatory requirements. A proficiency test (PT) was organised to evaluate the performance of laboratories in quantifying total egg and total milk protein in cookies. The PT involved 20 laboratories, which reported results using mainly commercial ELISA kits and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The findings indicate a satisfactory performance for milk protein determination among the majority of participant laboratories. However, the quantification of egg proteins in heated products remains a challenge, with most laboratories reporting results significantly below the reference value. Several potential factors contributing to this challenge are discussed, including the denaturation of egg proteins during heat treatment, differences in extraction strategies and the antibodies used in ELISA kits, and the lack of standardised methods and conversion factors for LC-MS/MS analysis. These findings underscore the importance of regular PT exercises to evaluate laboratory performance and ensure compliance with WHO/FAO recommendations. The results of this study aim to guide the development of improved analytical methods and strategies for ensuring the accurate quantification of food allergens.

A large variety of statistical methods can be used for proficiency testing (PT) programs in various areas of laboratory testing. Statistical methods described in ISO 13528 and other international standards address PT in a wide variety of applications. The most significant difference in statistical techniques is whether performance evaluations are determined from the participant results using consensus statistics from the current round, or whether the performance criteria are determined independently. For schemes evaluated by consensus, the next most significant factor is the experience of both the scheme and its participants. This is evidenced in the proportion of results reported by participants who lack competence, are newly enrolled, or do not understand the instructions provided. For example, statistical techniques that are necessary for novel schemes (e.g. run for the first time) may not be appropriate for a similar scheme after several rounds with the same participants. Similarly, different techniques may apply for closed schemes that have regular technical review of a limited group of experienced laboratories. Techniques that make allowances for high levels of “contamination” from incompetent or inexperienced participants, such as the z’ score with consensus assigned values, should not be used in experienced schemes using consensus statistics. Other techniques that are more sensitive to small systematic errors should be employed for closer monitoring of experienced laboratories, including statistical techniques that consider the measurement uncertainty of the results. Mature PT schemes and closed schemes for special purposes should evaluate the measurement uncertainty of participant results in any PT scheme used by laboratories that make decisions on conformity assessment, or where improvement of participant agreement is an objective for the scheme. Oversight bodies that require compliance with ISO/IEC 17043 should consider these recommendations, to better ensure global compatibility of measurements.

Inconsistent quantification results obtained from various analytical methods for food allergen testing hamper an accurate quantitative risk assessment and its regulatory implementation. In order to overcome such problems, a concept aiming at ensuring the comparability of quantitative food allergen measurement results is presented here. It is based on an approach called reference measurement system for food allergens, which uses a commonly agreed reference, namely the 'mass fraction of total protein of the allergenic ingredient in food'. The necessary system components are outlined, consisting of a primary reference measurement method, a certified reference material and a reference laboratory. This metrology-based concept can be applied to quantify various food allergens determined with different analytical procedures. The example of 'milk in cookies' is used to demonstrate the approach.

GMO control laboratories in the EU routinely monitor the presence and content of genetically modified organisms (GMOs) in food and feed products collected from the EU market. As the vast majority of GMOs comprize genetically modified plants, most control samples have a plant-based origin. For the first time, a pilot proficiency test was organised requiring the analysis of GMOs in a meat matrix. Meat pâté, a product in which soybean is occasionally identified, was spiked with GM soybean event MON89788, homogenised by mixing, aliquoted in sachets and frozen. The assigned value was determined by two independent expert laboratories. Several DNA extraction methods were tested and proved to be insufficient for the removal of PCR inhibitors present in the DNA extracts, resulting in a GM content underestimated by at least 30%. This problem was solved either by using hot-start qPCR chemistry or by applying the same method in a digital PCR format. A total of 52 laboratories participated in the study. They were requested to verify the presence of any GM soybean in the test item and to quantify the GM event(s) identified by their method of choice. All but one laboratory identified the MON89788 soybean event present in the pâté matrix. The majority of the quantitative results reported were below the assigned value, but did not deviate more than 50% from it. This study demonstrated the proficiency of most GMO control laboratories for the analysis of GMOs in a meat-based product. It also shows that method optimisation for GMO analysis in meat products is nevertheless advisable.

Proficiency testing providers, accreditation bodies and testing laboratories should be aware that a laboratory participating in a proficiency testing round might have reported a biased result despite a satisfactory performance indicated by an assessment using uniquely the z score. A complementary performance evaluation, based on the ζ score and the assessment of the measurement uncertainty, is therefore highly recommended. This work presents an intuitive graphical tool (the Naji2 plot) that combines z and ζ scores together with the reported measurement uncertainties. This tool allows a comprehensive assessment of the laboratory performance and enables to identify the need for corrective actions. The concerned laboratory should then perform a root cause analysis and investigate their bias and/or their measurement uncertainty evaluation.

The importance of laboratory performance evaluation in qualitative and interpretative proficiency testing (PT) and external quality assessments (EQA) has increased significantly over recent years. Unfortunately, the relevant ISO standards provide limited guidance on how these assessments could be harmonised. In 2014, the Eurachem PT Working Group performed an initial survey on current practices applied by PT/EQA providers in these areas. This paper presents the results of this survey along with the findings of an extensive literature review on the subject extended to 2020. Both the survey results and the literature search show that, even though a majority of scoring systems are based on simple yes/no or absence/presence responses, multiple types of performance evaluation criteria are in use for qualitative and interpretative PT/EQA schemes. This represents a clear disadvantage compared to the more aligned approach in quantitative PT/EQA schemes since laboratory proficiency between schemes is not as easily comparable. The paper presents current practices and scoring systems grouped by categories with defined criteria and may serve as a basis for further discussion and investigation towards the harmonisation of performance evaluation in qualitative and interpretative PT/EQA schemes.

Abstract Background Alternaria toxins are ubiquitous contaminants in highly consumed food products. Therefore, they are candidates to be regulated by EU legislation. In this context, the availability of reliable analytical methods is a keystone both for protecting the health of citizens and smooth functioning of the European market. Objective This paper describes an advanced LC-MS/MS method based on isotope dilution quantification suitable for the determination of altenuene, alternariol, alternariol monomethyl ether, tenuazonic acid, and tentoxin in tomato puree, wheat, and sunflower seeds. Methods The method has been validated in an interlaboratory study that included the analysis of both spiked and naturally contaminated food commodities. Twenty-three participants contributed with analytical data. Results The average recoveries and relative standard deviations for repeatability and reproducibility obtained across the tested matrixes were: 97, 8.0, and 23%, for altenuene, respectively; 95, 9.2, and 17% for alternariol, respectively; 98, 6.4, and 13% for alternariol monomethyl ether, respectively; 97, 4.2, and 9.3% for tenuazonic acid, respectively; and 102, 5.6, and 15% for tentoxin, respectively. The method enabled the determination of all tested Alternaria toxins close to or below 1 µg/kg. Conclusion Overall, the method showed a satisfactory trueness and precision, complying with the requirements for the monitoring of mycotoxins in food in the EU. It is currently under evaluation by the European Committee for Standardization for adoption as a standard method. Highlights Isotope dilution mass spectrometry method for the determination of Alternaria toxins in food.

A feasibility interlaboratory comparison (ILC) was organised by the European Commission’s (EC) Joint Research Centre (JRC) on the determination of the mass fraction of total cow’s milk protein in baked cookies. The ILC was organised to support Commission Regulation (EU) 1169/2011 on the provision of food information to consumers, including the mandatory allergen food labelling and the harmonisation of measurement procedures used for food allergen analysis. An incurred baked cookie was prepared at the JRC and samples were sent to participants for analysis. The laboratories were asked to report their results as “mass fraction of total cow’s milk protein in baked cookies”. This common well-defined measurand was used for the first time in such an ILC to assess the equivalence of measurement results. Homogeneity and stability of the test material were demonstrated to be adequate. An indicative assigned value was established using the JRC single-laboratory validated method based on liquid chromatography coupled to mass spectrometry (LC–MS), independently from the results reported by the participants. Twenty-three laboratories from the European Network of Food Allergen Detection Laboratories (ENFADL), representing 20 European Union (EU) Member States, reported results. Their laboratory performance was assessed using the percent difference D i score. The participating laboratories applied either enzyme-linked immunosorbent assays (ELISA) or LC–MS. Even though the majority of the laboratories used commercially available ELISA test kits, a significant scatter of all the reported results was observed. This indicates that further harmonisation is required for measurement procedures aiming to determine potentially allergenic constituents in food.

The outcome of proficiency tests (PTs) is influenced, among others, by the evaluation procedure chosen by the PT provider. In particular for PTs on GMO testing a log-data transformation is often applied to fit skewed data distributions into a normal distribution. The study presented here has challenged this commonly applied approach. The 56 data populations from proficiency testing rounds organised since 2010 by the European Union Reference Laboratory for Genetically Modified Food and Feed (EURL GMFF) were used to investigate the assumption of a normal distribution of reported results within a PT. Statistical evaluation of the data distributions, composed of 3178 reported results, revealed that 41 of the 56 datasets showed indeed a normal distribution. For 10 datasets, the deviation from normality was not statistically significant at the raw or log scale, indicating that the normality assumption cannot be rejected. The normality of the five remaining datasets was statistically significant after log-data transformation. These datasets, however, appeared to be multimodal as a result of technical/experimental issues with the applied methods. On the basis of the real datasets analysed herein, it is concluded that the log transformation of reported data in proficiency testing rounds is often not necessary and should be cautiously applied. It is further shown that the log-data transformation, when applied to PT results, favours the positive performance scoring for overestimated results and strongly penalises underestimated results. The evaluation of the participants' performance without prior transformation of their results may highlight rather than hide relevant underlying analytical problems and is recommended as an outcome of this study. Graphical abstract.

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.

National Reference Laboratories (NRLs) in the Member States of the European Union (EU) monitor the implementation of the EU legislation on the presence of genetically modified organisms (GMOs) in food and feed. The EU Reference Laboratory for GM Food and Feed (EURL GMFF) supports the harmonisation of measurement procedures and the improvement of the analytical performance of these laboratories, among others through the organisation of a proficiency testing (PT) scheme. The PT results reported over 10 years have been analysed using common criteria applied to the reported data. The outcome revealed a gradual decrease of the relative standard deviation within the sets of the reported data with time. The extent of the deviation of the results from the assigned value also diminished between 2010 and 2019. The average deviation from the assigned value was independent of the GM content in the later PT rounds but it was affected by the complexity of the test item matrix. Performance scores were calculated for all results reported by the 62 NRLs. The number of unsatisfactory performance scores obtained decreased with time. The trends observed indicate an improvement in the analytical performance and an increased harmonisation of GMO testing within the EU enforcement laboratories.

The outcome of a proficiency test (PT) organised by the European Union Reference Laboratory for Food Contact Materials (EURL-FCM) is presented. The PT was set up to assess the analytical performance of National Reference Laboratories (NRLs) and Official Control Laboratories (OCLs) in the determination of mass fractions of polyethylene terephthalate (PET) and polybutylene terephthalate (PBT) cyclic dimers and trimers in the official food simulant D1 containing ethanol and water (50:50 v/v). The EURL-FCM had developed and validated an analytical method based on HPLC-UV to monitor the homogeneity and stability of the target oligomers in the PT test items and to determine the respective assigned values, as prescribed in ISO 17043, 2010ISO 17043, 2010. The standard operating procedure of the method was provided to the participants and could be used instead of their own routine methods. Laboratory results were rated using z, z' and zeta scores in accordance with ISO 13528, 2015. The standard deviation for proficiency assessment, sigma(pt), was set to 20 % of the respective assigned value, for all the four studied oligomers, based on the perception of experts. A total of 36 participants from 26 countries have registered to the exercise. They received two test items. Solution 1 consisted of food simulant D1 fortified with a known mass fraction of the four oligomers, while Solution 2 was obtained by a migration experiment with PET bottles and food simulant D1 and further fortification of the resulting solution with the four oligomers. The majority of the participating laboratories presented satisfactory results for the four PET and PBT oligomers. For the analysis of Solution 1, 79-88 % of the participants obtained vertical bar z (or z')-scores vertical bar below 2, while the satisfactory performances ranged from 71 to 85 % for Solution 2. This PT has been organised for the first time at EU level for the analysis of polyester oligomers and confirms that most of the NRLs are able to monitor properly these oligomers in the frame of Regulation (EU) No 10/2011.

Background: Phytase-based preparations are important feed additives currently authorised in the European Union (EU). The European Standard (EN) and International Organization for Standardization (ISO) standard 30024 describes a harmonized method for the determination of phytase activity and is fit-for-purpose for official control of a group of phytase products. However, it is not suitable for the determination of the phytase activity of a new feed additive encoded as 4a16 in the EU Register of Feed Additives, to which a slightly different phytase activity definition has been attributed. Objective: To establish a robust conversion factor to support official control laboratories that apply the EN ISO method when monitoring feed products containing 4a16. Methods: The phytase activity of test materials was determined by the participants using the EN ISO and/or the “applicant” methods. Results: Robust relative SDs for repeatability and for reproducibility of the methods applied for the determination of the phytase activity in the materials containing the 4a16 feed additive ranged from 2.6 to 22% (EN ISO method) and from 2.4 to 39% (applicant method). Conclusions: The data obtained confirmed the performance characteristics published for other phytase-based feeds in the related standard methods. These results allowed us to estimate a factor of 2.68 to convert phytase activities measured with the EN ISO method into the enzyme activity measured with the applicant method. Highlights: The obtained conversion factor will allow EU official laboratories to screen feed samples supplemented with the 4a16 phytase by applying EN ISO Standard 30024.

A summary of the working group discussions on proficiency testing (PT) and external quality assessment (EQA) held at the Eurachem Workshop, Berlin, Germany, 6–9 October 2014 is provided. The working groups covered a range of issues concerned with current practice and future directions: review of ISO/IEC 17043; user perspective of PT/EQA; sampling PT/EQA; the revised ISO 13528; PT/EQA in developing countries; and harmonisation of performance assessment in qualitative PT/EQA. Delegates from 64 countries attended the workshop, and this diversity of different backgrounds was represented on each of the working groups in order to capture a range of views and experience from a number of different measurement sectors. Working group representatives included PT/EQA providers, participants in PT/EQA schemes, end-users of PT/EQA results such as accreditation bodies and regulatory authorities, national measurement institutes, laboratory associations, universities and independent consultants from countries around the world.

A proficiency testing (PT) round dedicated to assess the competence of European Union (EU) National Reference Laboratories (NRLs), EU Official Control Laboratories (OCLs) and OCLs from associated countries for the determination of the insecticide fipronil in eggs is described. The content of the target analytes (sum of fipronil plus its metabolite fipronil sulfone, expressed as fipronil in the PT test material) was set around the regulated Maximum Residue Level (MRL) set by EU legislation. The PT was organised by the European Commission's (EC) Directorate General (DG) Joint Research Centre (JRC) - in agreement with the EC DG for Health and Food Safety (SANTE) - following a request by the Belgian Authorities. Eighty-six NRLs and OCLs from 22 EU Member States, Norway, Serbia and Albania participated. The test items used were two materials made of frozen liquid eggs, processed and characterised at the JRC facilities in Geel (Belgium). The majority of the participants (94%) were shown to have a satisfactory performance, expressed as z scores, thus demonstrating the analytical capability of most of the participating NRLs and OCLs to enforce the relevant EU Regulations ((EC) 396/2005 and 1127/2014). Furthermore, 93% of the participants who provided a compliance statement classified, correctly, one of the test items as non-compliant.