Hexane

All reagents were of analytical grade. Water was obtained with a Milli-Q system from Millipore (Bedford, MA, USA). Acetone, hexane, hydrochloric acid (HCl), acetic acid (AA), acetonitrile (ACN), and methanol (MeOH) were from Scharlau (Barcelona, Spain). Tris(hydroxymethyl)aminomethane (Tris), sodium dodecyl sulfate (SDS), DL-dithiothreitol (DTT), sodium chloride (NaCl), urea, sodium hydroxide (NaOH), trifluoroacetic acid (TFA), β-mercaptoethanol (β-M), bovine serum albumin (BSA), lysozyme from chicken eggs (LYS), myoglobin from equine heart (MYO), and concanavalin (CONC) were from Merck (Darmstadt, Germany). Laemmli buffer, Mini-Protean precast gels, Tris/glycine/SDS running buffer, Bio-Safe Coomassie stain, and Bradford reagent (Coomassie Blue G-250) were acquired at Bio-Rad (Hercules, CA, USA).
Raw olives of Picual variety were kindly donated by the World Olive Germplasm Bank of IFAPA (Córdoba, Spain) and cheese whey was kindly donated by a cheese factory.

HPLC grade methanol, acetone, and methyl tert-butyl ether were acquired from Fisher Scientific Scharlau Chemie (Loughborough, UK). Sodium chloride was purchased from POCH S.A. (Sowińskiego, Poland). Ultrapure water was obtained with a Milli-Q system (Millipore Ibérica, Madrid, Spain). Ammonium carbonate, acetonitrile, hexane, ethanol (HPLC grade), magnesium chloride hexahydrate, acetic acid, and ammonium acetate were acquired from Scharlab (Sentmenat, Spain). Butyl hydroxytoluene (BHT) was purchased from Scharlau Chemie S.A. (Barcelona, Spain). Calcium chloride dihydrate was obtained from Merck (Darmstadt, Germany). Sodium hydrogen carbonate and potassium dihydrogen phosphate were acquired from VWR (Llinars del Vallès, Spain). Potassium chloride was obtained from Panreac (Castellar del Vallès, Spain). Digestive enzymes (porcine pepsin, porcine bile extract, porcine pancreatin, and porcine lipase) were acquired from Sigma-Aldrich (Darmstadt, Germany).
Caffeic acid, ferulic acid, and p-coumaric acid commercial patterns were obtained from Sigma-Aldrich (St. Louis, MO, USA). B-carotene standard was acquired from Carote-Nature (Ostermundigen, Switzerland), and α-carotene was acquired from Supelco-Merck (Darmstadt, Germany).

In order to extract the oil, the shell was removed from the samples manually until obtaining 25 g of walnut. This quantity was mixed with 250 mL of hexane (1:10 w/v) (Scharlau, Sentmenat, Spain) and homogenized in Ultraturrax T-18 basic for 3 min. The lipid fraction was taken to a rotary evaporator, distilling under vacuum until the removal of the hexane. The hexane-free oil was dried over sodium sulfate (Merck Darmstadt, Germany) and filtered (Whatman # 4 filter paper). The samples were stored in the dark at −80 °C until further analysis (up to 24 h).

The monomer 2-(4-methylthiazol-5-yl)ethyl methacrylate (MTA) was synthesized as previously reported [20 (link)]. The monomer 2-hydroxyethyl methacrylate (HEMA, 99%; Aldrich, Steinheim, Germany) was distilled prior to use. 2,2′-Azobisisobutyronitrile (AIBN, 98%; Acros, Buch, Switzerland) was recrystallized twice from methanol (MeOH, 99.9%; Aldrich) prior to use. Anhydrous dimethyl sulfoxide (DMSO, 99.8%) and N,N-dimethylformamide (DMF, 99.8%) were purchased from Alfa-Aesar (Karlsruhe, Germany) and were used as received. 1-Iodobutane (BuI, 99%, Aldrich), iodomethane (MeI, 99%; Aldrich), and hexane (96%; Scharlau, Sentmenat, Spain) were used as received.
For the microbiological assays, sodium chloride (NaCl, 0.9%, BioXtra, Steinheim, Germany, suitable for cell cultures) and phosphate buffered saline (PBS, pH 7.4) were obtained from Aldrich. BBL^TM Mueller Hinton broth used as microbial growth media was purchased from Becton, Dickinson and Company (Madrid, Spain). Sheep blood (5%) and Columbia Agar plates were acquired from BioMérieux (Madrid, Spain). American Type Culture Collection (ATCC) Gram-positive Staphylococcus aureus (S. aureus, ATCC 29213) and Gram-negative Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) bacteria were obtained from Oxoid^TM (Wesel, Germany).

Stock solution preparation, 100 mg/mL reference standard solutions of examined pesticides (Augsburg, Germany) were prepared in 100 mL hexane (Scharlau, Barcelona, Spain) in a volumetric flask. Stock solutions were kept in a refrigerator at 4°C. Preparation of 10 mg/mL individual standards for each pesticide was achieved by diluting 5.0 mL of stock solution in 50 μL hexane. The intermediate solution was maintained in the refrigerator at 4°C. Calibration standards of OCs mixture were also achieved by dissolving appropriate amounts of intermediate solutions in hexane and matrix extract.