Hplc plus grade

PGE2, PGE2-d9, PGD2, 15-keto-PGF2α, PGA2, PGB2, and PGJ2 analytical standards were purchased from Cayman Chemical (Ann Arbor, MI). All solvents used for LC-MS were HPLC Plus grade from Sigma Aldrich Corp. (St. Louis, MO).
Cell culture supernatants along with relevant standards were undergone extraction and samples were analyzed by LC-MS using a Shimadzu Nexera UHPLC (Shimadzu North America, Columbia, MD) interfaced to a Q-Exactive Plus ion trap mass spectrometer with a HESI source (ThermoFisher Scientific, San Jose, CA). Chromatographic separations were achieved by employing a 2.1 x 100 mm, 1.8 μm, Acquity HSS T3 column (Waters, Milford, MA) with gradient elution at 0.45 mL/min [20 (link)]. PGE2 was quantified against PGE2-d9 internal standard. The standard curve was generated using a constant concentration of PGE2-d9 (50 pg on column) and variable concentrations of PGE2 (2 pg to 10 ng on column). Peak areas for LC-MS quantification of PGE2 and PGE2-d9 were calculated using Xcalibur QuanBrowser software (ThermoFisher Scientific, San Jose, CA). The coefficient of determination for the standard curve (R2) was 0.9996.

Sapwood of spruce wood (Picea abies) from Switzerland was used. For wood functionalization, succinic anhydride and acetone were purchased from Sigma-Aldrich (St. Louis, MI, USA). Pyridine (anhydrous grade) was obtained from VWR (Radnor, PA, USA). Tip functionalization required 11-mercaptoundecanoic acid (HOOC(CH₂)₁₀SH, ≥ 95%, Sigma-Aldrich), 11-mercapto-1-undecanol (OH(CH₂)₁₁SH, ≥ 97%, Sigma-Aldrich) and ethanol (HPLC grade, ≥ 99.8%, Sigma-Aldrich). Buffer preparation in the pH range from pH 2 to pH 12 required water (HPLC plus grade, Sigma Aldrich), monobasic sodium phosphate (NaH₂PO₄ anhydrous, ≥ 99.0%, Sigma-Aldrich), phosphoric acid (H₃PO₄, 85wt%, Sigma-Aldrich), sodium phosphate dibasic dihydrate (Na₂HPO₄*2H2O, ≥ 99.5%, Riedel-de Haën, Seelze, Germany) and trisodium phosphate dodecahydrate (Na₃PO₄*12H₂O, ≥ 98%, Sigma-Aldrich).

From each group three fish were removed completely at random and 150 mg of dorsal muscle was removed from each with a scalpel and placed in a microcentrifuge tube. Subsequently, 300 µL of cold methanol (LC-MS-grade, Fisher Chemicals, Hampton, NH, USA) was added to each tube, and the sample was homogenized using a polypropylene plastic mortar. Next, 1 mL of cold methyl tert-butyl ether (MTBE) (HPLC Plus grade, Sigma Aldrich, Saint Louis MO, USA) was added to each sample. The tubes were then shaken for 1 h at room temperature. Afterward, 250 µL of water (LC-MS-grade, Fisher Chemicals, Hampton, NH, USA) was added and kept for 5 min at room temperature for phase separation. The samples were then centrifuged at 13,000× g for 7 min. The upper lipid phase was recovered in a sterile tube and placed in a speed vac for 40 min based on a procedure described by Matyash et al. [18 (link)]. Finally, the samples were transferred to an HPLC vial and placed in the UHPLC Autosampler at 4 °C.