Evaluation and Quantitation of Intact Wax Esters of Human Meibum by Gas-Liquid Chromatography-Ion Trap Mass Spectrometry

PURPOSE. Wax esters (WE) of human meibum are one of the largest group of meibomian lipids. Their complete characterization on the level of individual intact lipid species has not been completed yet. We obtained detailed structural information on previously uncharacterized meibomian WE. METHODS. Intact WE were separated and analyzed by means of high-temperature capillary gas-liquid chromatography (GLC) in combination with low voltage (30 eV) electron ionization ion trap mass spectrometry (ITMS). 3D (mass-to-charge ratio [m/z] versus lipid sample weight versus signal intensity) calibration plots were used for quantitation of WE. RESULTS. We demonstrated that GLC-ITMS was suitable for analyzing unpooled/underivatized WE collected from 14 individual donors. More than 100 of saturated and unsaturated WE (SWE and UWE, respectively) were detected. On average, UWE represented about 82% of the total WE pool. About 90% of UWE were based on oleic acid, while less than 10% were based on palmitoleic acid. The amounts of poly-UWE were < 3% of their mono-UWA counterparts. SWE were based primarily on C-16-C-18 fatty acids (FA) in overall molar ratios of 22:65:13. A pool of C-16:0-FA was comprised of a 20:80 (mol/mol) mixture of straight chain and iso-branched isomers, while the corresponding ratio for C-18:0-FA was 43:57. Interestingly, C-17:0-FA was almost exclusively branched, with anteiso- and isoisomers found in a ratio of 93:7. CONCLUSIONS. GLC-ITMS can be used successfully to analyze more than 100 individual species of meibomian WE, which were shown to comprise 41 +/- 8% (wt/wt) of meibum, which made them the largest group of lipids in meibum. (Invest Ophthalmol Vis Sci. 2012;53:3766-3781) DOI:10.1167/iovs.11-9333