Suchergebnisse

AbstractBackgroundLow maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times.ResultsBased on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive "3rd generation" recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%).ConclusionsWe present a fast and efficient bioanalytical routine method validated according to the European Union's legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and turn-around times of 52 h. This was facilitated in particular by a quick and efficient extraction step followed by selective clean-up, use of a highly sensitive "3rd generation" H4L7.5c2 recombinant rat hepatoma cell CALUX bioassay, and optimized assay performance with improved calibrator precision and reduced lack-of-fit errors. New restrictions are proposed for the calibrator bias and the unspecific background contribution to reportable results. The procedure can utilize comparably small sample amounts and allows an annual throughput of 840–1000 samples per lab technician. The described bioanalytical method contributes to the European Commission's objective of generating accurate and reproducible analytical results according to Commission Regulation (EU) 2017/644 across the European Union.

BACKGROUND: Low maximum and action levels set by the European Union for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) in pig meat (pork) have led to a demand for reliable and cost-effective bioanalytical screening methods implemented upstream of gas chromatography/high-resolution mass spectrometry confirmatory technology, that can detect low levels of contamination in EU-regulated foods with quick turn-around times. RESULTS: Based on the Chemically Activated LUciferase gene eXpression (CALUX) bioassay, extraction and clean-up steps were optimized for recovery and reproducibility within working ranges significantly lower than in current bioassays. A highly sensitive "3rd generation" recombinant rat hepatoma cell line (H4L7.5c2) containing 20 dioxin responsive elements was exposed to pork sample extracts, and their PCDD/Fs and DL-PCBs levels were evaluated by measuring luciferase activity. The method was validated according to the provisions of Commission Regulation (EU) 2017/644 of 5 April 2017 with spiking experiments performed selectively for PCDD/Fs and DL-PCBs and individual calibration for PCDD/Fs, DL-PCBs and the calculated sum of PCDD/Fs and DL-PCBs. The resulting performance parameters met all legal specifications as confirmed by re-calibration using authentic samples. Cut-off concentrations for assessing compliance with low maximum levels and action levels set for PCDD/Fs and DL-PCBs within a range of 0.50–1.25 pg WHO-TEQ/g fat were derived, ensuring low rates of false-compliant results (ß-error < 1%) and keeping the rate of false-noncompliant results well under control (α-error < 12%). CONCLUSIONS: We present a fast and efficient bioanalytical routine method validated according to the European Union's legal requirements on the basis of authentic samples, allowing the analyst to reliably identify pork samples and any other EU-regulated foods of animal origin suspected to be noncompliant with a high level of performance and ...

Abstract
Background
Persistent organic pollutants (POPs) such as dioxins, dioxin-like chemicals and non-dioxin-like PCBs causing adverse effects to human health bio-accumulate through the food web due to their affinity for adipose tissues. Foods of animal origin are therefore the main contributors to human dietary exposure. The European Union's (EU) food safety policy requires checking of a wide range of samples for compliance with legal limits on a regular basis. Several methods of varying efficiency are applied by official control laboratories for extraction of the different classes of lipids and associated POPs, bound to animal tissue and animal products in varying degrees, sometimes leading to discrepancies especially in fresh weight based analytical results.

Results
Starting from Smedes' lipid extraction from marine tissue, we optimized the extraction efficiency for both lipids and lipophilic pollutants, abandoning the time-consuming centrifugation step. The resulting modified Smedes extraction (MSE) method was validated based on multiple analyses of a large number of real-world samples, matrix calibration and performance assessment in proficiency testing utilizing both instrumental and bioanalytical methodologies. Intermediate precision in 12 different foods was below 3% in chicken eggs, egg powder, animal fat, fish, fish oil, poultry, whole milk, milk fat and milk powder, and below 5% in bovine meat, liver, and infant food. In comparison to Twisselmann hot extraction, results presented here show an increased efficiency of MSE by + 25% for bovine liver, + 14% for chicken eggs, + 13% for poultry meat, + 12% for fish, 8% for bovine meat, and 6% for infant food.

Conclusions
For the first time, a fast and reliable routine method is available that enables the analyst to reproducibly extract "total" lipids from any EU-regulated food sample of animal origin within 6 to 8 min. Increased efficiency translates into a considerable increase in both lipid and wet weight-based analytical results measured for associated POPs, reducing the risk of false non-compliant results. Compared to a 4 h Twisselmann extraction, the extraction of 1000 samples using MSE would result in annual savings of about 250 h or 32 working days. Our MSE procedure contributes to the European Commission's objective of harmonizing analytical results across the EU generated according to Commission Regulation (EU) 2017/644.

BACKGROUND: Persistent organic pollutants (POPs) such as dioxins, dioxin-like chemicals and non-dioxin-like PCBs causing adverse effects to human health bio-accumulate through the food web due to their affinity for adipose tissues. Foods of animal origin are therefore the main contributors to human dietary exposure. The European Union's (EU) food safety policy requires checking of a wide range of samples for compliance with legal limits on a regular basis. Several methods of varying efficiency are applied by official control laboratories for extraction of the different classes of lipids and associated POPs, bound to animal tissue and animal products in varying degrees, sometimes leading to discrepancies especially in fresh weight based analytical results. RESULTS: Starting from Smedes' lipid extraction from marine tissue, we optimized the extraction efficiency for both lipids and lipophilic pollutants, abandoning the time-consuming centrifugation step. The resulting modified Smedes extraction (MSE) method was validated based on multiple analyses of a large number of real world samples, matrix calibration and performance assessment in proficiency testing utilizing both instrumental and bioanalytical methodologies. Intermediate precision in 12 different foods was below 3% in chicken eggs, egg powder, animal fat, fish, fish oil, poultry, whole milk, milk fat and milk powder, and below 5% in bovine meat, liver, and infant food. In comparison to Twisselmann hot extraction, results presented here show an increased efficiency of MSE by +25% for bovine liver, +14% for chicken eggs, +13% for poultry meat, +12% for fish, 8% for bovine meat, and 6% for infant food. CONCLUSIONS: For the first time, a fast and reliable routine method is available that enables the analyst to reproducibly extract "total" lipids from any EU-regulated food sample of animal origin within 6 to 8 minutes. Increased efficiency translates into a considerable increase in both lipid and wet weight-based analytical results measured for associated POPs, ...

This document on measurement uncertainty was developed within the network of the European Union Reference Laboratory (EURL) for Dioxins and PCBs in Feed and Food and the respective National Reference Laboratories (NRLs) of member states. Detailed guidance is given on the evaluation of measurement uncertainty in the quantitative analysis of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) and polychlorinated biphenyls (PCBs), to assist laboratories performing official feed and food control within the European Union, especially National Reference Laboratories (NRLs) and Official Laboratories (OFLs). It provides useful key elements contributing to further harmonization of compliance assessment and outlines practical aspects related to measurement uncertainty estimation. A new concept placing special emphasis on the inclusion of current method performance data is presented. The concept covers the full analytical process from sample receipt at the laboratory through sample storage, preparation and analysis, to data processing and reporting. In particular, it focuses on the role of analytical variability generally known as "measurement uncertainty" (MU) in the interpretation of analytical results for assessment of their compliance with a specification. Effects from sampling [EURACHEM/CITAC 2007, /18/] and transport also contributing to MU are acknowledged but not treated within the scope of this document. Two selected approaches for measurement uncertainty estimation are proposed for the determination of PCDD/Fs and PCBs in food and feed by gas chromatography-mass spectrometry (GC-MS) using internal standard stable isotope labelled analogues. An empirical, or "top-down", approach combines contributions from intermediate (intra-laboratory) precision and trueness (expressed as bias) to estimate measurement uncertainty, both for individual congeners and for sum parameters. The working group recommends the use of the empirical approach as described in this document as the main option for MU estimation, because it is designed and developed to cover the whole analytical process and also includes the opportunity to reassess or update MU on a regular basis. However, an alternative methodology based on a semi-empirical approach following the EURACHEM/CITAC guide [EURACHEM/CITAC 2012, /12/] is also presented. It has been designed for laboratories new to this type of analysis that have generated data from initial validation studies. In this case the semi-empirical approach may be a good starting point, however the authors recommend implementing the empirical or top-down approach once enough data have been gathered.

The European Union Reference Laboratory (EURL) for Polycyclic Aromatic Hydrocarbons (EURL PAH), the EURL for Heavy Metals in Feed and Food (EURL HM), the EURL for Mycotoxins (EURL Mycotoxins), and EURL for Dioxins and PCBs in Feed and Food aim to provide with this document guidance to official food control in the EU on the estimation of the limit of quantification of analytical methods for the determination of individual substances in the field of contaminants in feed and food. The document focusses on estimation of the limit of detection (LOD) and/or limit of quantification (LOQ) of polycyclic aromatic hydrocarbons (PAHs), heavy metals (HM), mycotoxins, and polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs) and polychlorinated biphenyls (PCBs). The concept presented here consists of two major routes: The first route responds to the requirements for determination of PAHs, HMs and Mycotoxins. The second route corresponds to PCDD/F and PCB analysis, where results are calculated as sumparameters and expressed in toxic equivalents (TEQs), converting thereby congener concentrations together with estimated LOQs using toxic equivalency factor (TEFs) into TEQs. Due to the nature of this more complex procedure, emphasis is put in the area of PCDD/F and PCB analysis on the LOQs, while LODs are of minor consequence. Terminology in this guidance document was adapted to the chemical analysis of contaminants in feed and food; hence, some of the general terms defined and used in international standards were replaced by more specific terms applicable to analytical chemistry. The presented statistical-mathematical approach is based on elements taken mainly from DIN 32645:2008-11 (DIN 2008) and ISO 11843-2:2000 (ISO 2000). This document covers only quantitative methods of analysis. The authors believe that LOD and LOQ values derived from the application of the presented experimental methodologies converge to a certain degree. Estimation of LOD/LOQ values based on blank measurements, and from calibration data is described. Signal-to-noise ratios are applied in the area of PCDD/Fs and PCBs. Mathematical terms and statistical background are presented as well. This guide document shall be applied systematically if measurement results are used for monitoring purposes and exposure modelling. However, authors are aware that precise knowledge of LOD/LOQ may not be required when assessing compliance with maximum levels exceeding LOD/LOQ. This guidance document shall be applied if maximum levels and analysis results are close to the expected LOQ. The authors acknowledge that the presented approach has its limitations and may not be generally applicable to all cases.