Expression of GM content in mass fraction from digital PCR data.

Nowadays the quantification of the content of genetically modified (GM) constituents in food or feed products is performed by using either quantitative real-time PCR (qPCR) or digital PCR (dPCR). The latter is increasingly used. Therefore, experimental protocols for the quantification of 52 GM events authorised in the EU have been converted into a digital format and minimum performance characteristics for dPCR methods are detailed. Because of the need to harmonise the transformation of PCR results between two different measurement scales, 50 conversion factors for Certified Reference Materials (CFCRM) have been experimentally determined by three and sometimes four independent expert laboratories. The uncertainty of each CFCRM has been estimated to express dPCR results in mass fraction with a consistent uncertainty contribution. In 38 out of 58 cases, the validated qPCR methods (for 52 event-specific and 6 taxon-specific measurements) could easily be transferred into dPCR methods by using the same oligo sequences, final oligo concentration or annealing temperatures for the dPCR procedure. Laboratories have nevertheless used different strategies to improve the resolution or to reduce the so-called rain in their dPCR outcome. Those modifications were needed for PCR procedures that could not be converted without changes into a digital format. Therefore, exclusion/quality criteria such as the maximum rate of partitions with intermediate fluorescence "rain", the minimum resolution and repeatability are suggested for dPCR methods. The CFCRM determined in this study were generally in agreement with the declared zygosity of the GM parental donor for hemizygous maize events. In a limited number of GM events the CFCRM values were significantly different when measured with different maize-specific (ZmAdh1 or hmgA) genes.

Single and multi-laboratory validation of a droplet digital PCR method.

The authorisation of genetically modified food and feed in the EU is subject to the provision of evidence of safety and of the availability of reliable analytical methods. These methods represent an essential tool for official laboratories to enforce a harmonised market control. Here the validation of droplet digital PCR (dPCR) methods has been performed for studying if the performance and acceptance parameters set by EU and other international guidelines for the analysis of genetically modified organisms (GMO) in food and feed are suitable and achievable also with such methods. The single-laboratory validation study showed that performance requirements set for GMO analysis by real time PCR can also be used to assess dPCR-based methods. Moreover, trueness and precision were assessed for both simplex and duplex formats in a multi-laboratory validation study organised according to international standards. Overall, the data on trueness, repeatability and reproducibility precision resulting from the collaborative study are satisfying the acceptance criteria for the respective parameters as stipulated in the EU and other international guidance such as the Codex Committee on Methods of Analysis and Sampling (CCMAS). For instance, the duplex droplet dPCR method for MON810 showed relative repeatability standard deviations from 1.8% to 15.7%, while the relative reproducibility standard deviation was found to be between 2.1% and 16.5% over the dynamic range studied. Moreover, the relative bias of the dPCR methods was well below 25% across the entire dynamic range. In addition, other aspects supporting the application of digital PCR for the control of GMOs on the market were experimentally assessed such as the conversion of the measurement results from copy number ratio to mass fraction, the influence of the DNA extraction step and of the ingredient content. It was found that the DNA extraction step added only a limited contribution to the variability of the measurement results under the studied conditions. The decreasing amount of the target ingredient content may decrease the level of precision of the method, although within the acceptance range of GMO performance parameters.

A journey towards whole water certified reference materials for organic substances: measuring polycyclic aromatic hydrocarbons as required by the European Union Water Framework Directive.

In 2000, the Water Framework Directive (WFD) came into force in the European Union with the aim of protecting and improving water quality. The priority substances established to be monitored are predominantly organic compounds, for which the WFD sets the requirement of 'whole water sample' analysis. This legislative requirement poses analytical challenges for the monitoring laboratories as well as technical challenges for reference materials producers. In the past, there were attempts to produce reference materials as quality assurance/quality control tools for measuring organic priority substances in whole water. A critical reflection on the approaches and solutions applied to prepare such kind of matrix reference materials is presented along with a discussion on the difficulties encountered by the analytical laboratories in analysing such complex matrices. The Certified Reference Material (CRM) ERM-CA100 can be considered as a pioneer for a 'whole water' CRM (containing humic acids) and has been designed for the analysis of polycyclic aromatic hydrocarbons (PAHs). Further developments seem to be necessary to upgrade the design towards a CRM which will also include suspended particulate matter, another basic constituent of natural surface water samples.

Log transformation of proficiency testing data on the content of genetically modified organisms in food and feed samples: is it justified?

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.

Development and validation of a multi-analyte GC-MS method for the determination of 84 substances from plastic food contact materials.

Chemical substances shall not migrate from food contact materials (FCM) at levels that are potentially harmful for the consumers. Each of the current analytical methods applied to verify the migration of substances from FCM covers only one or few substances. There is a very limited number of publications on the development of analytical methods allowing the simultaneous determination of several classes of FCM substances, and almost none of them reported methods entirely dedicated to the ones in the positive list of Commission Regulation (EU) No. 10/2011 for plastic FCMs. Therefore, a simple, sensitive and reliable multi-analyte method was developed for the analysis of FCM substances in food simulants. It employs an optimised liquid-liquid extraction with dichloromethane as extraction solvent in the presence of 10% m/v NaCl, followed by quantitative analysis with gas chromatography coupled to mass spectrometry (GC-MS). A combination of total ion chromatograms (TICs) and extracted ion chromatograms (EICs) was used. The optimisation and validation of the method have been carried out according to current international guidelines. Adequate sensitivity was demonstrated in the selected concentration ranges for most of the analytes, with limits of quantification (LOQs) at least three times lower than the legislative limit, when existing. The results showed that the method is sufficiently accurate for the majority of substances, with recoveries between 70 and 115% and relative standard deviations (RSDs) smaller than 20% at three concentration levels. The method was applied to the analysis of some FCM multilayers. The method allows, for the first time, the simultaneous quantification of 84 FCM substances in two of the official food simulants (A and C) at levels of a few ng g-1. Graphical abstract.

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. Graphical abstract.

Ten years of proficiency testing reveals an improvement in the analytical performance of EU National Reference Laboratories for genetically modified food and feed.

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.

First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Towards metrologically traceable and comparable results in GM quantification.

The GM content in a food or feed product produced from or containing genetically modified organisms (GMO) has to be expressed in Europe in the form of a GM mass fraction. However, the most widely used quantification methods, based on PCR, are basically counting PCR-amplifiable DNA fragments in a sample extract. This paper outlines the requirements for obtaining comparable measurement results which are fit for regulatory decision-making. It introduces the concept of a reference measurement system which enables GMO analysis laboratories to relate their results to a universally accessible reference, thus establishing metrological traceability to a unique reference point. The conversion factors required for transforming data from one measurement unit into the other have to carry a minimum uncertainty and are anchored to specified certified reference materials. The establishment of such conversion factors and related calibration approaches to achieve comparable GM quantification results are sketched. Graphical abstract ᅟ.

An assessment of the impact of extraction and digestion protocols on multiplexed targeted protein quantification by mass spectrometry for egg and milk allergens.

The unintentional presence of even trace amounts of certain foods constitutes a major hazard for those who suffer from food allergies. For many food industries, product and raw ingredient surveillance forms part of their risk assessment procedures. This may require the detection of multiple allergens in a wide variety of matrices. Mass spectrometry offers a possible solution for the quantification of multiple allergens in a single analysis. The capability of MS to quantify many peptides from a complex protein digestion is well known. However, a lack of matrix certified reference materials has made the optimisation of extraction and digestion conditions for multiplexed allergen quantification difficult to assess. Here, we report a systematic study, using preliminary screening followed by a Design of Experiments approach, to find the optimal buffer and digestion conditions for detecting milk and egg protein markers in a model processed food matrix. Five of the most commonly used buffers, two chaotropic reagents and two reducing reagents were assessed for the optimal extraction of multiple protein markers. While the choice of background buffer had little impact, the use of chaotropic and reducing reagents showed significant benefits for the extraction of most proteins. A full factorial design experiment was applied to the parameters shown to have a significant impact on protein recovery. These studies suggest that a single optimal set of extraction conditions enabling the quantitative recovery of all proteins is not easily achieved. Therefore, although MS is capable of the simultaneous quantification of many peptides in a single run, greater consideration of protein extraction is required before these are applied for multiplex allergen quantification in food matrices. Graphical abstract.

Identification of single target taxon-specific reference assays for the most commonly genetically transformed crops using digital droplet PCR.

Knowledge of the number of DNA sequences targeted by the taxon-specific reference assays is essential for correct GM quantification and is key to the harmonisation of measurement results. In the present study droplet digital PCR (ddPCR) was used to determine the number of DNA target copies of taxon-specific assays validated for real-time PCR for the four main genetically modified (GM) crops. The transferability of experimental conditions from real-time PCR to ddPCR was also explored, as well as the effect of DNA digestion. The results of this study indicate that for each crop at least one taxon-specific assay can be identified as having a single DNA target. A short list of taxon-specific reference assays is proposed as best candidates for the relative quantification of GM events for soybean, maize, cotton and oilseed rape. The investigated assays could be in most cases transferred to ddPCR without further optimisation. The use of DNA digestion did not improve ddPCR characteristics such as rain and resolution at the conditions tested.

Addressing Analytical Challenges of the Environmental Monitoring for the Water Framework Directive: ERM-CE100, a New Biota Certified Reference Material.

In the context of supporting the EU Member States in the implementation of the EU Water Framework Directive (WFD), a project for the production of a fish reference material (ERM-CE100) certified for its content of the two priority substances hexachlorobenzene (HCB) and hexachlorobutadiene (HCBD) was carried out at the Joint Research Centre (JRC) of the European Commission. The starting material was naturally contaminated Wels catfish (Silurus glanis), caught in the Ebro River (Spain). A novel approach for the processing of the fish was tested that resulted in a homogeneous and stable reference material in the form of a wet paste. The fresh-like texture of the matrix enhances the comparability of this material toward routinely analyzed environmental biota samples and facilitates its use as a quality assurance tool given that the WFD environmental quality standards (EQS) for biota are expressed as wet weight. Certified values for the mass fractions of HCB and HCBD were assigned with 120 ± 8 and 36 ± 4 µg/kg, respectively. The related interlaboratory comparison involved 13 expert laboratories applying a range of analytical methodologies. It is the first biota CRM ever available for HCBD. ERM-CE100 can be used to assess the performance of analytical methods employed in the mandatory monitoring of water bodies under the WFD, thus, providing a benchmark for establishing comparability among measurement results.

Development and validation of a method for mercury determination in seawater for the process control of a candidate certified reference material.

A simple, robust and reliable method for mercury determination in seawater matrices based on the combination of cold vapour generation and inductively coupled plasma mass spectrometry (CV-ICP-MS) and its complete in-house validation are described. The method validation covers parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), trueness, repeatability, intermediate precision and robustness. A calibration curve covering the whole working range was achieved with coefficients of determination typically higher than 0.9992. The repeatability of the method (RSDrep) was 0.5 %, and the intermediate precision was 2.3 % at the target mass fraction of 20 ng/kg. Moreover, the method was robust with respect to the salinity of the seawater. The limit of quantification was 2.7 ng/kg, which corresponds to 13.5 % of the target mass fraction in the future certified reference material (20 ng/kg). An uncertainty budget for the measurement of mercury in seawater has been established. The relative expanded (k = 2) combined uncertainty is 6 %. The performance of the validated method was demonstrated by generating results for process control and a homogeneity study for the production of a candidate certified reference material.

DNA copy number concentration measured by digital and droplet digital quantitative PCR using certified reference materials.

The value assignment for properties of six certified reference materials (ERM-AD623a-f), each containing a plasmid DNA solution ranging from 1 million to 10 copies per µL, by using digital PCR (dPCR) with the BioMark™ HD System (Fluidigm) has been verified by applying droplet digital PCR (ddPCR) using the QX100 system (Bio-Rad). One of the critical factors in the measurement of copy number concentrations by digital PCR is the partition volume. Therefore, we determined the average droplet volume by optical microscopy, revealing an average droplet volume that is 8 % smaller than the droplet volume used as the defined parameter in the QuantaSoft software version 1.3.2.0 (Bio-Rad) to calculate the copy number concentration. This observation explains why copy number concentrations estimated with ddPCR and using an average droplet volume predefined in the QuantaSoft software were systematically lower than those measured by dPCR, creating a significant bias between the values obtained by these two techniques. The difference was not significant anymore when the measured droplet volume of 0.834 nL was used to estimate copy number concentrations. A new version of QuantaSoft software (version 1.6.6.0320), which has since been released with Bio-Rad's new QX200 systems and QX100 upgrades, uses a droplet volume of 0.85 nL as a defined parameter to calculate copy number concentration.

The importance of commutability of reference materials used as calibrators: the example of ceruloplasmin.

BACKGROUND: Different methods for ceruloplasmin tend to give different results in external quality assessment schemes. During the production of the certified reference material ERM-DA470k/IFCC discrepant measurement results were also found for ceruloplasmin measured with different methods, and consequently the protein could not be certified in the material. METHODS: We performed a commutability study with 30 serum samples and the reference materials ERM-DA470, ERM-DA470k/IFCC, and ERM-DA472/IFCC, using 6 different methods. Data were analyzed according to the CLSI Guideline C53-A to assess whether the reference materials had the same behavior as the serum samples with respect to measurement results obtained with combinations of the methods used. RESULTS: Measurement results from different methods showed a good linear correlation for the serum samples. ERM-DA470 showed marked noncommutability for certain combinations of methods. ERM-DA470k/IFCC and ERM-DA472/IFCC were commutable for more combinations of methods. The lack of commutability of ERM-DA470 for certain combinations of methods correlates with results from the UK National External Quality Assessment Service showing discrepancies between results from these methods. For serum stored in the presence of sodium azide the results from different methods are essentially equivalent. CONCLUSIONS: Ceruloplasmin in ERM-DA470 is a fully documented example of a situation in which, due to lack of commutability, the use of a common material for calibration did not lead to harmonization .

Food allergen analysis: Considerations for establishing a reference measurement system to implement EU legislation.

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.

Determination of Alternaria Toxins in Tomato, Wheat, and Sunflower Seeds by SPE and LC-MS/MS-A Method Validation Through a Collaborative Trial.

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.

Analysis of PBT and PET cyclic oligomers in extracts of coffee capsules and food simulants by a HPLC-UV/FLD method.

A HPLC-UV/FLD method was validated for the quantification of six polyethylene terephthalate (PET) and four polybutylene terephthalate (PBT) oligomers. PBT oligomers are EU regulated, while the PET ones are considered non-intentionally added substances (NIAS). LOQs were higher than 0.4 and 3.5 µg kg-1 for the simulants and in the polymer extracts, respectively. Recoveries ranged from 95 to 114 % with RSDs below 12%. Migration testing of PBT and polypropylene coffee capsules were performed with H2O and simulant C, and extracts were obtained with accelerated solvent extraction (ASE). For the latter legislative limits weren't surpassed. As no migration limits are existing for the analytes, both EFSA's toxicological threshold of concern (TTC) and sum of oligomers approaches were applied. The majority of oligomers were below the TTC (90 µg/person/day), but the limit value of 50 µg/kg food was surpassed for some capsules, which indicates a significant intake in both single and multiple consumption.