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K. B. Green-Church, J. Chen, K. Nichols; Identification of Individual Lipid Components in Human Meibomian Gland Secretions With High Resolution Mass Spectrometry. Invest. Ophthalmol. Vis. Sci. 2009;50(13):533.
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We report the identification of the molecular components of lipids observed in meibum by high resolution mass spectrometry (MS) and collision-induced dissociation (CID). The effect of electrospray conditions and mass analyzer settings were examined.
Human meibum samples from five non-dry eye patients were collected in 0.5 µL x 32 mm Drummond Scientific Microcaps and stored at -20oC. Meibum stock solutions were prepared by adding 1 mL of 2:1 chloroform:methanol in glass vials using glass syringes. Samples were diluted tenfold and analyzed by electrospray ionization (ESI), nanospray on a quadrupole time-of-flight (QTOF), and by microspray on an orbitrap MS. CID was performed for structure analysis/verification. The ESI cone voltage was set at 5 or 15 V, and QTOF collision voltage ranged from 5 - 40 V.
The ionization conditions were found to have a noteworthy effect on the mass spectra and can perhaps explain the differences observed between labs. Lipids were observed primarily as protonated or sodiated species depending on the MS conditions employed. By lowering the cone voltage, the ionization is softer and a greater number of high molecular weight lipids are observed. It is believed that a higher cone voltage causes more in-source fragmentation. Using accurate mass data, individual lipids from fatty acyls, glycerolipids and sterol categories are all identified in a single mass spectrum including the specific information of the corresponding chain lengths and number of double bonds. This is significant because we are able to report the actual structure of the lipids in one mass spectrum from one subject. A series of wax esters, fatty acids, diesters (fatty acyls category); triacylglycerols (glycerolipids category); and cholesteryl esters (sterol lipids category) are identified. Interestingly, the fatty acids were only observed in small amounts, only in certain ESI conditions and are believed to be a result from in source fragmentation from more complex lipids. Wax ester and cholesteryl ester peaks were further supported by MS/MS analysis.
Differences in instrument settings in addition to patient variation confounds comparisons between research findings between research teams. Optimization of mass spectrometry protocol will aid in the future development of the field of meibum lipidomics. With more sensitive and higher resolution mass spectrometry methods, minimum sample preparation is needed and the intact lipid molecules can be analyzed directly without derivation.
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