A sample of the total lipid extract solution was placed in a screw-top test tube and dried in a heating block under nitrogen.  Fatty acid methyl esters (FAMES) were prepared by treating the dry lipid with anhydrous hydrochloric acid in methanol.  Fatty acids that occur in the sample as di- or triglycerides are detached from the glycerol molecule and converted to methyl esters.  After cooling to room temperature double-distilled water was added.  FAMES were recovered with petroleum ether and transferred to a vial.  The solvent was removed by heat under a gentle stream of nitrogen. The FAMES were dissolved in iso-octane, then transferred to a gas chromatography (GC) vial with a conical glass insert. Solvents and chemicals were checked for purity by running a sample blank.  The entire lipid extraction and methyl esterification process was performed and FAMES were dissolved iniso-octane.  Traces of contamination were subtracted from sample chromatograms.  The relative percentage composition was calculated by dividing the integrated peak area of each fatty acid by the total area of fatty acids present in the sample.

The step in the extraction procedure during which the chloroform, methanol, and lipid mixture is washed with water is a standard procedure for extracting lipids from modern samples.  This step was recently adopted to remove impurities so that clearer chromatograms could be obtained in the region where very-long-chain fatty acids occur.  It was anticipated that the detection and accurate assessment of these fatty acids could be instrumental in separating residues of animal origin from those of plant. To identify the residue, the relative percentage composition was determined first with respect to all fatty acids present in the sample (including very-long-chain fatty acids) and secondly with respect to the 10 fatty acids utilized in developing the identification criteria. The second step is necessary for the application of the identification criteria.