Figure 1. Workflow for untargeted lipid profiling with UHPLC-QTOF of tears using the two lipid extraction methods under comparison. Tear samples were collected individually from ten healthy subjects. A pool was made to evaluate in all cases the same sample and divided into five replicates of 10 µl to be extracted with the internal standard (IS) added before the extraction (prespiked) and another five replicates of 10 µl each with the IS added after the lipid extraction of the tear (post-spiked). The extracts were analyzed with ultrahigh-performance liquid chromatography-quadrupole time of flight (UHPLC-QTOF), and the lipid species obtained were identified and quantified.