Lcad u hplc system

The extraction protocol and analysis of bioactive lipid mediators were performed as described by Le Faouder et al.,⁽¹⁵⁾ adapted by the Ambiotis SAS (Toulouse, France) standard operating procedure. Briefly, plasma (1 mL) was mixed with 0.4 mL of ice‐cold methanol and held at −80°C for protein precipitation. Samples were then centrifuged and supernatants collected. After removal of the organic solvent under a stream of nitrogen, samples were suspended in methanol and rapidly acidified to pH 3.5 with HCl. Acidified samples were then loaded into C‐18 SPE cartridges (Waters, Milford, MA), rapidly neutralized, and eluted with methyl formate. Eluate solvents were evaporated under a stream of nitrogen and residues suspended in mobile phase for LC/MS‐MS analyses using an Exion LCAD U‐HPLC system coupled with a Sciex QTRAP 6500+ MS‐MS system (AB Sciex, Framingham, MA), equipped with an ESI source in negative ion mode. Quantification was performed using calibration curves with synthetic standards for each of the SPMs included in the analysis.