Quantitative Profiling of Major Neutral Lipid Classes in Human Meibum by Direct Infusion Electrospray Ionization Mass Spectrometry

Abstract

PURPOSE. The purpose of this investigation was to better understand lipid composition in human meibum.METHODS. Intact lipids in meibum samples were detected by direct infusion electrospray ionization mass spectrometry (ESI-MS) analysis in positive detection mode using sodium iodide (NaI) as an additive. The peak intensities of all major types of lipid species, that is, wax esters (WEs), cholesteryl esters (CEs), and diesters (DEs) were corrected for peak overlapping and isotopic distribution; an additional ionization efficiency correction was performed for WEs and CEs, which was simplified by the observation that the corresponding ionization efficiency was primarily dependent on the specific lipid class and saturation degree of the lipids while independent of the carbon chain length. A set of WE and CE standards was spiked in meibum samples for ionization efficiency determination and absolute quantitation.RESULTS. The absolute amount (lmol/mg) for each of 51 WEs and 31 CEs in meibum samples was determined. The summed masses for 51 WEs and 31 CEs accounted for 48 6 4% and 40 6 2%, respectively, of the total meibum lipids. The mass percentages of saturated and unsaturated species were determined to be 75 6 2% and 25 6 1% for CEs and 14 6 1% and 86 6 1% for WEs. The profiles for two types of DEs were also obtained, which include 42 a,x Type II DEs, and 21 x Type I-St DEs.CONCLUSIONS. Major neutral lipid classes in meibum samples were quantitatively profiled by ESI-MS analysis with NaI additive.

A Comparison of Patient Matched Meibum and Tear Lipidomes

“…Similar to WE FA chains, the CE species detected at the highest abundance contained esterified FA chains of 24:0, 25:0, and 26:0. This is in agreement with the recent study by Chen, 16 as well as earlier studies of Chen, 14 Lam, 13 and Nicolaides. 17 Butovich et al 18 characterized CE speciation in meibum by HPLC atmospheric pressure chemical ionization MS, and their study reported CE 24:0, CE 25:0, and CE 26:0 as highly abundant, but also reported CE 26:1 and 28:1 as highly abundant.…”

“…15 This is also in agreement with the recent ESI-MS study by Chen et al, 16 who report the highest abundance WE species of WE 42:1, WE 43:1, and WE 44:1. Similar to WE FA chains, the CE species detected at the highest abundance contained esterified FA chains of 24:0, 25:0, and 26:0.…”

Characterization of Wax Esters by Electrospray Ionization Tandem Mass Spectrometry: Double Bond Effect and Unusual Product Ions

“…[6, 29] However, for each peak of a certain m / z there could be multiple isomers. Dissociation of each peak generated mixed fragments from different isomers.…”

“…The double bond effect and neutral molecule adduct formation observed for dissociation of wax esters could be helpful for identification and quantification of wax esters in biological samples. Current work is ongoing to utilize these fragmentation patterns in conjunction with our earlier work of profiling wax esters in meibum [29] to identify and quantify each isobaric species composition in meibum samples from the corresponding MS/MS spectra. [6]…”

“…Meibum sample collection and solution preparation has been described previously. [6, 29] Briefly, meibum was collected in 0.5 μL (32 mm length, 0.0220″ OD, 0.00560″ ID) glass microcapillary tubes (Drummond, Broomall, PA) with the length of about 1 mm, and was then dissolved in chloroform-methanol solvent mixture (2:1, vol/vol) with a concentration of approximately 6.7 µg/mL of total lipids and 1 mM ammonium acetate additive.…”