Abstract
BackgroundLow 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.
Highlights
The collective term “dioxin” covers a total of 75 polychlorinated dibenzo-p-dioxins (PCDDs) and 135 dibenzofurans (PCDFs), and generally refers to those specific congeners that produce a common spectrum of toxic and biological effects [1,2,3]
The resulting improved fit exhibiting smaller calibrator bias values in particular in the lower part of the curve considerably reduces the risk for over- or underestimation of Bioanalytical equivalent (BEQ) results in unknown food samples, especially in those with low Maximum level (ML) or action levels (ALs) set by European Union (EU) legislation
Under the EU’s General Food Law’s legislative framework [61], member states are required to monitor a wide range of food samples for dioxins and Decision limit (DL)-Polychlorinated biphenyl (PCB), some—such as pork—at low levels of contamination
Summary
The collective term “dioxin” covers a total of 75 polychlorinated dibenzo-p-dioxins (PCDDs) and 135 dibenzofurans (PCDFs), and generally refers to those specific congeners that produce a common spectrum of toxic and biological effects [1,2,3]. Dioxins are formed during combustion processes, when organic compounds oxidize in the presence of halogen compounds, especially chlorine or bromine This is the case with smelting, waste incineration, chlorine bleaching of paper pulp, and the production of some herbicides and pesticides, and with natural processes such as forest fires or thunderstorms. These toxic dioxin-like chemicals are frequently highly lipophilic and resistant to metabolic degradation and as such, once these compounds enter the body, they are difficult to eliminate, with a 2,3,7,8-TCDD half-life in adult humans ranging from 3 to 10 years [4]. 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
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