Abstract
Human meibomian gland secretions (also known as meibum) were analyzed for the presence of cholesteryl esters (Chl-E) using HPLC in combination with atmospheric pressure chemical ionization mass spectrometry. A special procedure based on detection of the in-source generated ion m/z 369 was developed to monitor all Chl-E simultaneously. The structures of the detected compounds were studied using in-source and postsource fragmentation of the precursor (M+H)(+) ions. In concordance with previous studies, Chl-E were found in all of the tested samples and comprised approximately 31% of the entire lipid pool (w/w, dry weight). There were at least 20 different saturated and unsaturated Chl-E species observed, whose fatty acid residues ranged from C18 to C34. Monounsaturated fatty acids were the most visible components of the Chl-E pool. The eleven most prominent compounds were: C20:0-, C22:1-, C22:0-, C24:1-, C24:0, C25:0-, C26:1-, C26:0-, C28:1-, C28:0-, and C30:1-Chl-E. Other Chl-containing compounds were detected but not identified at the time. Therefore, Chl-E are a depot for very long chain saturated and monounsaturated fatty acids in human meibum.
Highlights
Human meibomian gland secretions were analyzed for the presence of cholesteryl esters (Chl-E) using HPLC in combination with atmospheric pressure chemical ionization mass spectrometry
As 1) wax ester (WE) and Chl-E were estimated to comprise more than two-thirds of the entire lipid pool of human meibum [7], and 2) WE detected in our experiments [25, 26] were mostly C18-based, there was a distinctive possibility that it is the Chl-E that have very long chain fatty acid (VLCFA) in their structures
Further HPLC-MS2 experiments revealed that upon collision-induced dissociation (CID), ion m/z 369 produced a family of product ions presented in Fig. 1D, which matched the fragmentation pattern of free cholesterol reported earlier [26]
Summary
Human meibomian gland secretions ( known as meibum) were analyzed for the presence of cholesteryl esters (Chl-E) using HPLC in combination with atmospheric pressure chemical ionization mass spectrometry. Our experiments with several authentic Chl-E showed that the signal m/z 369 originated from a Chl-E after it underwent a neutral loss of its fatty acid component, and/or from free Chl upon its dehydration, both happening spontaneously in the ion source of a mass spectrometer This dual origin of m/z 369 signal could be advantageous when analyzing meibum samples as it can be used for easy and simultaneous monitoring of the elution profiles of various Chl-E and free Chl. Earlier, human meibum was repeatedly reported to have large quantities of long chain and very long chain fatty acids (LCFA and VLCFA) of the C16 and above family esterified into various lipid species. As 1) WE and Chl-E were estimated to comprise more than two-thirds of the entire lipid pool of human meibum [7], and 2) WE detected in our experiments [25, 26] were mostly C18-based, there was a distinctive possibility that it is the Chl-E that have VLCFA in their structures
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