Rapid extraction of total lipids and lipophilic POPs from all EU-regulated foods of animal origin: Smedes’ method revisited and enhanced

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.

Bioanalytical screening of low levels of dioxins and dioxin-like PCBs in pig meat (pork) for checking compliance with EU maximum and action levels using highly sensitive “third generation” recombinant H4L7.5c2 rat hepatoma cells

“…Within-laboratory reproducibility calculated as relative residual standard deviation of BEQ results at the respective GC/HRMS decision limits (DLs) is 19.8% for the PCDD/F-DL ML , 25.7% for the PCDD/F-DL AL , 26.5% for the DL-PCB-DL AL , and 16.9% for the PCDD/F-PCB-DL ML (Table 6 ), indicating that method precision is fully compliant with legal requirements (RSD R < 30%) [ 18 , 22 ]. Measurement uncertainties (MU) were not calculated for reasons explained elsewhere [ 35 ].…”

“…Following a fast and efficient modification of Smedes’ method [ 34 ], “total” lipids were extracted from each sample within just 6 min. Details of the procedure, which has been demonstrated to be applicable to each EU-regulated sample of animal origin, were published elsewhere [ 35 ].…”

“…In comparison to keeping the sample material under reflux with a suitable solvent (mixture) for a period of 4 h or longer on a Twisselmann extractor, a method widely used in traditional dioxin analysis, our rapid extraction yielded 99–105% of extractable lipids, more than 99.9% of which were re-dissolvable, e.g., in n-hexane [ 35 ]. Moreover, the short contact times between solvents and sample surface may largely prevent co-extraction of compounds structurally similar to the target analytes or activating the AhR signaling pathway for any other reason.…”

“…Moreover, the short contact times between solvents and sample surface may largely prevent co-extraction of compounds structurally similar to the target analytes or activating the AhR signaling pathway for any other reason. Together with various other features, this method significantly improves the selectivity of the assay [ 35 ].…”

“…The volumes of PCDD/F and DL-PCB eluates were reduced to 0.5 mL, transferred to a conical 1.2 mL vial, and carefully reduced further to 2–3 µL, followed by exchange of the solvent for 7 µL of DMSO in the case of the PCB-fraction, and for 14 µL of DMSO in the case of the dioxin/furan-fraction, following a well-established procedure, which keeps vial-to-vial variability, expressed as relative standard deviation (RSD), below 5%. The smaller volume for the final extract containing DL-PCBs was chosen due to a reduced relative potency (REP) or response of the cell bioassay to PCB 126, the most abundant DL-PCB, which is approximately 40% compared to the response of the assay to the most potent compound, 2,3,7,8-TCDD [ 21 , 35 – 38 ]. The small final extract volumes provide concentration factors of, roughly, 700 for DL-PCBs and 350 for PCDD/Fs, the magnitude of which may pose a challenge to the control of unwanted and potentially interfering co-extractives or co-eluents altering the cell response.…”