Multi reax

The extraction step was carried out as described by Oruç et al. (2017 ) with slight modifications. Briefly, 1 g of propolis sample was extracted with 20 mL of 70% EtOH at ambient temperature for 1 h using a multi‐vortex (Multi Reax, Heidolph, Germany), and then the sample was ultrasonicated for 15 min (Wise Clean, DAIHAN Scientific, Korea). The propolis extract was filtered using Whatman filter paper (No: 1), and the filtrates were concentrated using a rotary evaporator at 40°C at 200 rpm. The dried extract was dissolved in 2 mL of absolute methanol and kept at 4°C until used. Appropriate dilutions were made, and the diluted extracts were used for the determination of phenolic compounds by LC–MS/MS and also for the determination of total phenolics (TP) content (Oruç et al., 2017 ).

The control lipid extraction method used in this experiment was based on the method developed by Folch et al.^{14 (link)}. Lyophilized biomass was divided into five replicates of 150 mg in 15 ml centrifuge tubes and added 20 volumes (3 ml) of either dichloromethane/methanol (2:1 v/v, DCM/MeOH), hexane/isopropanol (2:1 v/v, hexane/IPA) or hexane. Following the addition of solvent, the samples were subjected to the following treatments: No treatment (control), stirring using a shaker (Heidolph Multireax) at 1000 RPM for 60 min at room temperature (mixing), and sonication at 20 kHz for 10 min (sonication) on ice. The samples were then added 3 ml MiliQ water added 5% NaCl and centrifuged for 5 min at 3000 G, before the organic phase was transferred to a 4 ml vial and evaporated under nitrogen. The extraction procedure was repeated once without physical treatment for each sample, and the yield was determined gravimetrically for each extraction respectively as percent of AFDW. Finally, the samples were dissolved (10 mg/ml) in DCM/MeOH (2:1 v/v) and stored at -80 °C.

The baked and cooled snacks were ground in an analytical mill A 11 basic (IKA Poland Sp. z o.o., Warsaw, Poland). Approx. 0.3 g of ground material was weighed on an analytical balance to the nearest 0.0001 g into a 15 mL plastic falcon and mixed with 10 mL of extraction reagent (80% ethyl alcohol). Extraction was carried out on a shaker Multi Reax (Heidolph Instruments GmbH & Co., Schwabach, Germany) for 24 h at 6000 rpm at room temperature. Samples were centrifuged for 2 min at 5000 rpm using a laboratory centrifuge MegaStar 600 (VWR International Sp. z o.o., Gdańsk, Poland). The supernatant was transferred to resealable 0.2 mL tubes. Three extracts were made for each sample.

Mussel samples were transported to the laboratory in refrigerated conditions and processed within 6 h from the collection. Samples were externally cleaned with potable water. In aseptic working conditions, about 10 individuals were opened and the flesh meat and intervalvular fluids were pooled together, and 25 g of bivalve sample were homogenized in a blender and tenfold diluted with alkaline saline peptone water (ASPW) (10⁻¹ dilution). Sediment samples (10 g) were tenfold diluted with ASPW and vortex-mixed for 15 min at 2000× g with the Multi Reax (Heidolph, Germany) (10⁻¹ dilution). Water and phytoplankton-net samples were vortex-mixed for 15 min at 2000× g with the Multi Reax. From seawater and net-phytoplankton raw samples and the 10⁻¹ dilution of mussel and sediment samples further serial dilutions (10⁻¹ to 10⁻⁴) were prepared in ASPW (1 + 9 mL); then, 1 mL of each dilution was surface-spread plated on TCBS agar (Difco Laboratories, Detroit, MI, USA) and incubated at 37 ± 1 °C for 18 ± 3 h. After incubation, yellow colonies growing on TCBS agar plates were counted (only plates with 3 to 150 colonies were considered) for V. alginolyticus enumeration [51 (link),52 (link)]. The results were reported as UFC per gram of mussels/sediment or UFC per milliliter of water/phytoplankton-net sample.

To improve the accuracy of the measurements, for the analytes for which U-[¹³C]-labelled homologues were available, standard isotope dilution assay (SIDA) was performed. In the case of the sulfates derivates, matrix-matched calibration was carried out. For that, several blank oat samples were extracted by weighting 5 g of oat flour and mixing it with 10 mL acetonitrile and 10 mL water, shaking for 30 min (Multi Reax, Heidolph Instruments GmbH and Co.KG, Schwabach), centrifuging at 3800 g for 30 min (Megafuge 16, Thermo Fisher Scientific, Braunschweig, Germany), collecting 1 mL of the supernatant and mixing it with 250 mg of anhydrous magnesium sulfate. Then, the samples were centrifuged at 17,000 g for 5 min (Microfuge R, Beckmann, Krefeld, Germany) and all the resulting extract collected in a vial for further use.
Finally, three eight-point series of calibration solutions were prepared: one containing ZEN (8.008–500.5 ng/mL), α-ZEL (1.1047–69.074 ng/mL), β-ZEL (2.776–173.5 ng/L) and their respective U-[¹³C]-labelled homologues, dissolved in acetonitrile:water (25:75, v/v); one containing a series of calibration solutions of ZEN-14-S (0.858–546.25 ng/mL) and another one containing α-ZEL-14-S (0.853–947.1 ng/mL) and β-ZEL-14-S (0.6092–676.35 ng/mL), both dissolved in blank matrix extract (25%).

Bones, rind, subcutaneous fat, and innards were removed from the meat samples. Following mixing, the samples were freeze-dried and ground in a cryomill (SamplePrep6870 Freezer Mill, C3 Process and Analysis Technology GmbH, Haar, Germany). The ground, dry samples were stored in a freezer (−20°C) until use. The meat powder (500 mg) was extracted with 6 mL of water. After the samples were mixed on a test tube shaker (Multi Reax, Heidolph, Schwabach, Germany) for 10 min, samples were centrifuged at 3,000 rpm [relative centrifugal force (RCF), 1,690 x g] for 15 min. The aqueous supernatant was passed through a syringe filter (Chromafil Xtra PET −45/25, Macherey-Nagel, Düren, Germany) into a centrifuge tube. The 3 kDa ultrafiltration filters (Vivaspin^®, Sartorius, Goettingen, Germany) were rinsed three times with 2 mL of water each to remove glycerol [10 min at 3,000 rpm, relative centrifugal force (RCF), 1,690 × g]. After the cleaning step, 800 μL of the meat extract was transferred to the 3 kDa ultrafiltration unit, and samples were centrifuged at 3,000 rpm [relative centrifugal force (RCF), 1690 × g] for 1.5 h. An aliquot of the obtained filtrate (500 μL) was mixed with 250 μL of 3 M sodium dihydrogen phosphate buffer (pH 6) and 75 μL of TSP (dissolved in D₂O). A 600 μL-aliquot of this mixture was transferred to a 5-mm Boro 300-5-8 (Deutero, Bad Kreuznach, Germany) NMR tube.

The official EU-RL (European Union Reference Laboratory) LC-MS/MS method [81 ,82 ] was adopted. Shellfish homogenate (2.00 ± 0.05 g) was extracted twice with 9 mL of methanol. After the first methanol addition, the sample was vortex-mixed for 3 min at 2000 rpm with a Multi Reax (Heidolph, Germany), the extract centrifuged for 10 min at 2000 × g and the supernatant transferred to a 20 mL volumetric flask. During the second methanol addition, the mixture was homogenized with an Ultra Turrax T25 mixer (IKA Works, Wilmington, NC, USA) for 1 min at 10,000 rpm, the extract centrifuged (10 min at 2000 × g), the supernatant was combined with the first extract and the whole extraction volume brought to final volume of 20 mL with methanol. An aliquot of the methanolic extract was filtered through a 0.2-µm syringe filter (Minisart, Sartorius, Germany) and submitted to AZAs determination.