Here we present the application of a cross-platform approach, combining rapid direct infusion high-resolution/accurate mass ESI-FTICRMS with in-depth data-dependent LC-MS2 and LC-MS3 analysis for lipid profiling. The analytical approach as well as the subsequent data handling is described. The method was applied to human synovial fluid samples from osteo- and rheumatoid arthritis patients. Multivariate statistical analysis revealed esterified oxylipids as molecular features in a subset of the patient samples. Employing LC-MS2 and LC-MS3 analysis of these species, we were able to clarify the hypothesized lipid structures initially based on the accurate mass measurements performed on the ESI-FTICRMS platform. LC-MS3 analysis of intact esterified oxy-lipids and LC-MS2 analysis of the hydrolysis products allowed for the detection of positional isomers. The approach led to the structural elucidation of hydroxylated docosapentaenoic acid-containing diacyl-phosphatidylcholine type phospholipids in human synovial fluid.