Lockspray unit

A Waters Synapt G2-Si high resolution quadrupole Time-of-Flight (qToF) mass spectrometer equipped with IonSabre-II atmospheric pressure chemical ionization (APCI) ion source and a LockSpray unit were used (all from Waters Corp., Milford, MA, USA). Chromatographic separation of the analytes was achieved on a Waters M-Class UPLC system using an Acquity UPLC C₁₈ BEH column (1 mm×100 mm, 1.7 μm; also from Waters). Raw UPLC/MS and MS^E files were analyzed in Waters MassLynx (v.4.1) and MS^E DataViewer (v.1.4) software packages and the elemental CHNO compositions of lipids were computed using the MassLynx EleComp (v.4.0) as described earlier (19 (link)–21 (link)).
A reversed phase (RP) UPLC/MS protocol that had been used in a preceding publication (16 (link)) was precisely followed. Pooling of samples from different mice was unnecessary as the sensitivity of the instrumentation was adequate to work with individual TP. WE, CE, and TAG standards were analyzed alongside meibomian lipid samples for comparison purposes in targeted supervised experiments. The meibum lipid samples were also compared using untargeted, unsupervised approach which was based on the Principal Component Analysis (PCA) of the raw UPLC/MS data using Progenesis QI software package (from Waters).