We characterized the performance of a micro-flow LC-ESI-MS² approach to analyze lipid mediators (LMs) and polyunsaturated fatty acids (PUFA) that was optimized for SPE free lipid extraction. Tandem mass spectrometry was exclusively performed in parallel reaction monitoring (PRM) mode using TOF and Orbitrap analyzers. This acquisition strategy allowed in addition to quantitation by specific quantifier ions to perform spectrum comparisons using full MS² spectra information of the analyte. Consequently, we developed a dedicated software SpeCS that allows to 1) process raw peak lists, 2) generate customized spectral libraries, 3) test specificity of quantifier ions and 4) perform spectrum comparisons. The dedicated scoring algorithm is based on signal matching and Spearman's rank correlation of intensities of matched signal. The algorithm was evaluated in respect of its specificity to distinguish structural related LMs on both instrument platforms. We show how high resolution mass spectrometry is beneficial to distinguish co-eluted LM isomers and provide a generalized quality control procedure for PRM. The applicability of the approach was evaluated analyzing the lipid mediator response during M. tuberculosis infection in the mouse lung.