Esi triple quadruple g6460 mass spectrometer

To 100 μL of plasma 300 μL of cold methanol and 5 μL of internal standard (Deuterium labeled compounds) were added. After centrifugation at 900 g for 15 min at 4°C, supernatants were transferred into 2 mL 96-well deep plates and diluted in H₂O to 2 mL. Samples were then submitted to solid phase extraction (SPE) using OASIS HLB 96-well plate (30 mg/well, Waters) pretreated with MeOH (1mL) and equilibrated with 10% MeOH (1 mL). After sample application, extraction plate was washed with 10% MeOH (1 mL). After drying under aspiration, lipids mediators were eluted with 1 mL of MeOH. Prior to LC-MS/MS analysis, samples were evaporated under nitrogen gas and reconstituted in 10 μL on MeOH.
LC-MS/MS analyses of eicosanoids were performed as described [20 (link)]. Briefly, lipid mediators were separated on a ZorBAX SB-C18 column (2.1 mm, 50 mm, 1.8 μm) (Agilent Technologies) using Agilent 1290 Infinity HPLC system (Technologies) coupled to an ESI-triple quadruple G6460 mass spectrometer (Agilent Technologies). Data were acquired in Multiple Reaction Monitoring (MRM) mode with optimized conditions (ion optics and collision energy). Peak detection, integration and quantitative analysis were done using Mass Hunter Quantitative analysis software (Agilent Technologies) based on calibration lines built with commercially available eicosanoids standards (Cayman Chemicals).

Prostaglandin E2 (PGE2) was quantified from BMDM supernatant using liquid chromatography–tandem mass spectrometry (LC-MS/MS). Briefly, 1 ml of BMDM cell culture supernatant was added to 300 µl of cold methanol and 5 µl of internal standards (deuterium labeled compounds). Samples were centrifuged at 900 g for 15 min at 4°C, supernatants were diluted in H₂O to 2 ml and submitted to solid phase extraction using a HRX 96-well plate (50 mg/well, Macherey Nagel, Germany). LC-MS/MS analyses were performed on a Agilent 1290 Infinity HPLC system coupled to an ESI-triple quadruple G6460 mass spectrometer (Agilent Technologies) as previously described (43 (link)).