Identification of meibomian gland lipids by gas chromatography-mass spectrometry: application to the meibomian lipids of the mouse.

Methods are described for the structural determination of the constituent alcohols, steroids and fatty acids from meibomian gland esters. The compounds, obtained from hydrolysed extracts, were separated and quantified as trimethylsilyl and methyl ester-trimethylsilyl derivatives by fused-silica capillary column gas chromatography. Structural information relating to the position of chain branching and the position of double bonds in the aliphatic chains of the acids and alcohols was obtained from the mass spectra of a number of other derivatives. Thus pyrrolidide-trimethylsilyl and picolinyl ester-trimethylsilyl derivatives provided information on both branching and unsaturation. The double-bond position in unsaturated fatty acids was also examined by use of trimethylsilyl derivatives of the derived glycols. The positions of chain branching of the alcohols were determined by the spectra of their acetate and nicotinate derivatives and by their gas-liquid chromatographic retention times. In meibomian gland lipids from the mouse, cholesterol was the major constituent; alcohols from the n-, iso-, anteiso- and unsaturated series were present with iso-C26 and anteiso-C27 being the most abundant. Mono-unsaturated fatty acids belonged mainly to the omega 9 series and saturated acids belonged to the iso-, anteiso- and n-series. Several 1,2-diols were also identified. The most abundant of these had iso-C16 and iso-C20 chains. GC-MS studies on the intact wax esters showed them to be composed of the branched-chain alcohols and both branched-chain and unsaturated acids.