2 6 di tert butyl 4 methylphenol bht

The solvents n-hexane, 2-propanol (iPrOH), formic acid (FA), acetonitrile (ACN), and methanol (MeOH) of ultrahigh-performance LC/MS grade were purchased from Biosolve (Valkenswaard, Netherlands). Water was produced in-house with a Barnstead GenPure System from Thermo Scientific (Waltham, MA, USA). Methyl tert-butyl ether (MTBE) of pro analysis quality was obtained from Carl Roth (Karlsruhe, Germany). 2-Butanol of extra-pure quality was purchased from Thermo Fisher Scientific (Waltham, MA, USA). 2,6-Di-tert-butyl-4-methylphenol (BHT) of gas chromatography quality and EDTA of ACS reagent grade were purchased from Sigma-Aldrich (St Louis, MO, USA). Ethyl acetate and chloroform (CHCl₃) of LC grade, zinc sulfate heptahydrate of pro analysis quality, and phosphate-buffered saline (PBS) of liquid, sterile-filtered, suitable-for-cell-culture grade were obtained from Merck Chemicals (Darmstadt, Germany). Tris (hydroxymethyl)aminomethane–HCl buffer was obtained from Bioanalytic (Umkirch, Germany). Unlabeled and deuterium-labeled PUFA and eicosanoid standards were purchased from Cayman Chemicals (Ann Arbor, MI, USA); the detailed information can be found in Table S1.

Absolute ethanol and hydrochloric acid were purchased from RCI Labscan Ltd. (Bangkok, Thailand). Trolox (6-hydroxy-2,5,7,8- tetramethylchroman-2-carboxylic acid), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) or ABTS, Folin-Ciocalteu reagent and 2,6-di-tert-butyl-4-methylphenol (BHT) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Gallic acid, sodium carbonate, potassium persulfate and potassium chloride were procured from Merck (Darmstadt, Germany). Linoleic acid was purchased from TCI: Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Iron (II) chloride tetrahydrate was supplied from Qrec (Auckland, New Zealand). Ammonium thiocyanate was purchased from Ajax Finechem Pty Ltd. (Taren Point, NSW, Australia).

The PUFA-plasmalogen (vinyl ether) derivative 1-(1Z-octadecenyl)-2-docosahexaenoyl-sn-glycero-3-phosphoethanolamine (PL-DHA-PE) and the fluorescent lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt) (18:1 Liss Rhod PE) were purchased from Avanti Polar Lipids, Inc. (Alabama). The composition of the scallop-derived plasmalogen extract was characterized by the provider as a mixture of ethanolamine vinyl ether phospholipid (49.4%), choline vinyl ether phospholipid (24.9%), cholesterol (16.0%), and ceramide aminoethyl phosphonate (9.7%). This natural plasmalogen combination with 70% vinyl ether phospholipid content is referred as scPL70. Monoolein (MO, C18:1c9, powder, ≥99%), retinoic acid (RA), vitamin E (VitE), 2,6-di-tert-butyl-4-methylphenol (BHT), and D-α-tocopherol polyethylene glycol-1000 succinate (TPGS-PEG₁₀₀₀) were purchased from Sigma-Aldrich. Water of MilliQ quality (Millipore Corp., Molsheim, France) was used for preparation of a phosphate buffer solution (NaH₂PO₄/Na₂HPO₄, 1 × 10⁻² M, pH 7, p.a. grade, Merck).

700 μl of sample (4 μl of plasma diluted in water, or 700 μl of homogenized cells) was mixed with 800 μl 1 N HCl:CH3OH 1:8 (v/v), 900 μl CHCl3 and 200 μg/ml of the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT; Sigma Aldrich). 3 μl of SPLASH LIPIDOMIX Mass Spec Standard (#330707, Avanti Polar Lipids) was spiked into the extract mix. The organic fraction was evaporated using a Savant Speedvac spd111v (Thermo Fisher Scientific) at room temperature and the remaining lipid pellet was stored at - 20°C under argon.

Chemicals used in this study were: Yeast Extract (Biokar Diagnostics, France); Peptone (Biokar Diagnostics, France); Glucose (Sigma, Germany); Chlormaphenicol (Biomatik, USA); Ethyl Methane Sulfonate (Sigma, Germany); Sodium Thiosulfate (Sigma, Germany); Sodium Phosphate (Sigma, Germany); Sodium Chloride (Sigma, Germany); 2,6-Di-tert-butyl-4-methylphenol (BHT) (Sigma, Germany); Methanol (Carlo-Herba, France); Hydrochloric acid 37% (Carlo Herba, France); n-Hexane (VWR chemical, France); Dichloromethane (VWR chemical, France); 2,2-dimethoxypropane (Sigma, Germany); Hydrochloric acid solution (Sigma, Germany); Sodium Methoxide solution (Sigma, Germany) and Nonadecanoic acid (Sigma, Germany).