Abstract
Purpose: :
To investigate the use of electrospray ion trap (ESI–IT/MS) and quadrupole time of flight mass spectrometry (Q–TOF/MS) for the identification of lipid species in normal human meibum and tear and gas chromatography electron impact mass spectrometry (GC/EI–MS) for the quantitation of cis–9–octadecenamide (oleamide), a primary fatty acid amide.
Methods: :
For lipid identification, mass spectrometry studies were performed on chloroform/methanol extracted meibum and tear samples using lithium [M+Li]+ and acid [M+H]+ adducts in the positive mode and deprotonated species [M–H]– in the presence of ammonium acetate in the negative mode. For quantitation of oleamide in meibum and tear, an in–house internal standard methodology was developed using stereoscopic microscopy and linear–volumetric measurement with calibrated microcapillaries.
Results: :
A number of the major lipids observed by ESI/MS were identified including free fatty acids (including myristic, palmitic, stearic, and oleic acid) and the primary fatty acid amide cis–9–octadecenamide (oleamide). The fragmentation pathways for oleamide were characterized by ESI–IT/MS which included both charge driven and charge remote pathways. Other fatty acid amides (myristamide, stearamide, palmatide) have not been observed in the meibum or tear film. The free fatty acid oleic acid was observed to undergo a reduction in the electrospray process which can be attributed to a novel reduction of the olefin bond via saturation thus producing stearic acid. Quantitative analyses suggest a relatively abundant amount of oleamide in meibum at approximately 19% of the total volume analyzed.
Conclusions: :
ESI/MS is a powerful tool for the investigation of the major lipid components in meibum and tear, and we have identified a number of the major lipid species using this technology. The observance of the expression of oleamide in such a continuously high manner suggests that oleamide may play a major role in relation to the normal structure, function, and stability of the tear film. The previously reported functions of oleamide in other central nervous systems as a messenger or signaling molecule, an endogenous sleep–inducing lipid, or an inhibitor of gap junction communication are unlikely relative to its role in the tear film or ocular surface activity, but warrant further investigation.
Keywords: lipids • conjunctiva