Oxylipin Quantification in Synovial Fluid

Concentrations of 34 oxylipins (see S1 Table) were quantified by liquid chromatography–mass spectrometry (LS-MS/MS) using a validated quantitative method based on that described by Wong et al.[21 (link)]. Lipids not included in [21 (link)] were measured using the following LC-MS/MS settings (precursor / product ions / collision energy): 17R resolvin D1 (375.3 / 141.0 / 22), 6-ketoPGF1a (369.1 / 162.8 / 36), resolvin D2 (375.3 / 175.0 / 30), 17-HDoHE (343.1 / 343.1 / 9). The HPLC system used was a Shimadzu series 10AD VP LC system (Shimadzu, Columbia, MD, USA) and the MS system used was an Applied Biosystem MDS SCIEX 4000 Q-Trap hybrid triple-quadrupole–linear ion trap mass spectrometer (Applied Biosystem, Foster City, CA, USA) equipped with an electrospray ionisation (ESI) interface. Quantification of the eicosanoids was calculated using fully extracted calibration standards for each of the analytes. Before quantification of lipid-derived inflammatory mediators of rat synovial fluid, blank filter papers were stained with equivalent concentrations of calibration standards (100pM, 500pM, 1nM, 2.5 nM, 5nM, 10nM) and were compared with actual standard calibration curves. Quantification was performed using Analyst 1.4.1. Identification of each compound in plasma samples was confirmed by LC retention times of each standard and precursor and product ion m/z ratios.