Shotgun lipidomic analysis of human meibomian gland secretions with electrospray ionization tandem mass spectrometry.

Lipids secreted from the meibomian glands (meibum) form the outmost layer of tear film covering the ocular surface. A primary function of this superficial lipid layer is to prevent evaporation of the aqueous component in the tear film.1 Changes in lipid profiles of meibum likely lead to ocular surface diseases such as dry eye disease, which is a prevalent condition affecting over 12 million Americans, or 6% to 27% of people worldwide.2 Therefore, it is important to know the identities of the components in meibum to better diagnose and manage ocular surface disease, with the additional goal of future therapeutic development. The conventional methods applied to the analysis of meibum samples include thin layer chromatography (TLC), nuclear magnetic resonance spectroscopy, gas chromatography (GC), and gas chromatography-mass spectrometry (GC-MS).3 With these methods, progress has been made in identifying the lipid components in meibum. Previously, the major components of meibum have been reported to be wax esters (WEs),4–8 cholesteryl esters (CEs),4–8 diesters (DEs),4,5,7 and triacylglycerols (TGs).4–8 The major fatty acid and fatty alcohol moieties of these lipids have also been determined.7,9 However, limitations still exist with these methods, including low sensitivity, low resolution, possible degradation due to prolonged exposure time, low sample recovery due to derivatization, isomerization, and/or decomposition due to sustained high temperature analysis and long analysis time.3 Furthermore, these methods are mainly based on hydrolysis and derivatization followed by GC-MS analysis of all the meibum lipids or a certain subclasses; thus information of the fatty acid or fatty alcohol moieties only has been obtained and the more important qualitative and quantitative information of individual intact lipids was missing. To circumvent these limitations, atmospheric pressure chemical ionization (APCI)-MS coupled with reversed-phase or normal phase high-performance liquid chromatography (HPLC) separation was recently applied for the direct analysis of the meibum lipids including WEs10,11 and CEs12 without derivatization. However, the high energy and the high temperature involved with APCI could cause the dissociation of some lipid species. For instance, saturated CE peaks are spontaneously fragmented with APCI-MS.12 This creates a challenge in providing accurate identification and quantitation. In addition, a large number of samples are required for APCI-MS analysis because only high flow rate (>0.2 mL/min) is compatible with this method. In contrast, electrospray ionization (ESI) is a “softer” ionization method13 and can be operated at low flow rate; thus it is more compatible for the analysis of unstable lipids as well as limited or small volume samples. The application of ESI-MS to meibum samples has been reported recently.11,14,15 In this work, direct ESI-MS and MS/MS analyses were performed for individual meibum samples collected by capillary tube after meibomian gland expression. After minimizing the interference peaks and increasing the signal-to-noise ratio, we confirmed the previously reported major lipid species, including WEs, CEs, DEs, TGs, and free fatty acids (FFAs) in meibum samples. More importantly, we also determined the major molecular components of these lipid species. To the best of our knowledge, this is the most detailed report of the major species in meibum on the molecular level. Furthermore, we also observed a set of peaks reported recently as those of (O-acyl)-ω-hydroxy fatty acids (OAHFAs).16 These OAHFAs may play important role in maintaining the stability of tear film.