Gas chromatography-mass spectrometry (GC–MS)

An aliquot of the dried hexane extract (2 gm) was refluxed with 15 ml alcoholic potassium hydroxide (10%) for 5 h. The reaction mixture is then cooled and diluted with 20 ml distilled water. The unsaponifiable matter is then extracted with petroleum ether (3 × 20 ml) through shaking in separating funnel. The petroleum ether extracts are combined and washed with distilled water till free from alkalinity. The extract is then dried over anhydrous calcium chloride and filtered. The unsaponifiable fraction is finally dried under vacuum using a rotary evaporator, to be ready for GC/MS analysis.

The remaining aqueous mother liquor left after removal of petroleum ether was acidified with 10% HCl to liberate free fatty acids. The mixture is extracted with petroleum ether (3 × 20 ml), extracts are combined and dehydrated over anhydrous calcium chloride. Petroleum ether is evaporated under pressure till having a constant weight of the saponifiable matter.

Methylation of the free fatty acids from the saponifiable fraction was performed by refluxing the dry saponifiable residue with 50 ml absolute methanol and 3 ml concentrated sulfuric acid. Reflux was done in a water bath for 2 h at 100 °C. The reaction mixture is cooled and then extracted with petroleum ether (3 × 20 ml). The extract is then dried to obtain a dry residue of fatty acids methyl ester.

The unsaponifiable fraction and the fatty acids methyl esters were subjected to GC/MS analysis under the following conditions; A capillary column packed with 70% Cyanopropyl Polysilphenylene Siloxane (30 m, 0.25 ID, 1.4 μm film thickness), Injector temperature: 300 °C, temperature transfer line: 350 °C, temperature programming: Initial temperature is 90 °C held for 2 min and then a gradual temperature increase at 3 °C/min till it reaches 350 °C with a hold time of 5 min., Carrier gas: He (1.5 ml/min), Sample injection volume: 1 μl, Ionization energy: 70 eV, Run time: 49 min. Identification of different constituents:

In the case of unsaponifiable constituents, identification was done by comparison of their retention times and relative retention time to 4-4(ethylcyclohexyl)-1-pentylcyclohexene and mass spectra to those indexed in the NIST library and the Wiley database. Only compounds of high matching probability were considered.

In the case of fatty acid methyl esters from the saponifiable fraction, identification was done by comparison of their retention times and relative to the retention time of palmitic acid also mass spectra to those of authentic fatty acid methyl esters co-injected with the sample.