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, ...