2.4. Sample collection and mass spectrometry

Following a rest period, tear and meibum samples were collected from the right eye of each subject using polytetrafluoroethylene and glass microcapillary tubes, respectively [59]. Details of the procedures, including sample collection, storage, extraction, and mass spectrometry analyses, have been described extensively in our previous reports [24,59–64]. In brief, samples were infused into the TripleTOF mass spectrometer (SCIEX 5600, Framingham, MA, USA) and a semi-targeted direct infusion electrospray MS and MS/MS^ALL was performed in the positive ion mode to determine the abundance of CEs (Fig. 2) and WEs (Fig. 3) in the tear and meibum samples [47]. Lipid intensities were identified with Analyst1.7 TF and LipidView1.3 (SCIEX, Framingham, MA, USA).

Illustration of masses that contain the cholesterol + NH4 ion of 369.352 m/z from MS/MS spectra of cholesteryl ester derived from a meibum (top) and tear (bottom) sample.

Illustration of a MS/MS of 652.69 m/z spectrum of wax ester 43:0 demonstrating the composition of varying acyl lengths.