An improved strategy for analysis of lipid molecules utilising a reversed phase C UHPLC column and scheduled MS/MS acquisition.

Measuring physiochemically diverse molecules (including lipids) which vary significantly in their concentrations poses a great analytical challenge. In untargeted lipidomics studies, reversed phase chromatography coupled with data-dependent MS/MS acquisition (DDA) is frequently applied. The optimal assay should deliver a high number of detected compounds with associated fragmentation data. In this work, we introduce novel 30 and 50 min UHPLC assays utilising lipid separation on a C stationary phase with a modified DDA strategy using smaller precursor m/z ranges scheduled for different lipid classes across the retention time range (defined as scheduled MS/MS). To evaluate the efficiency of the novel assays, mammalian tissue extracts (lamb liver, kidney and heart) were analysed and data were compared to a 15 min reversed phase C assay with multiple traditional DDA injections. The 30 min C assay detected double the number of detected compounds compared to the 15 min C assay. Applying the scheduled MS/MS DDA strategy with a single injection, a similar number of annotated lipids were reported compared to the traditional DDA strategy applied with five replicate injections on a C column. A longer 50 min C chromatographic assay did not result in an expected improvement in the chromatographic separation of overlapping isomer peaks compared to the 30 min method but did result in loss of accuracy of peak picking algorithms. We recommend the 30 min C assay with scheduled MS/MS acquisition as an efficient tool to analyse complex biological matrices and to annotate lipid species based on MS/MS data.