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.
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.
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