Sample preparation and gas chromatography of primary fatty acid amides

A method for the isolation of bio-active primary fatty acid amides (PFAMʹs) from total lipid extract by solid-phase extraction (SPE) was developed and validated. The lowest mass of amide to be loaded and recovered by this method was detected as 0.5 μg using 500 mg of normal phase adsorbent. The isolated PFAMʹs were separated and quantified by GC/MS and percent recoveries were calculated. An HP-5MS column was able to provide base line separation between the saturated and unsaturated PFAMʹs whereas clear resolution between geometric and positional isomers having the same number of carbons was obtained using a BPX70 column. The separated amides were all 18 carbon analogs of cis-9-octadecenoamide (oleamide). Detection limits in the single ion monitoring mode were found to be on the order of 10 pg in a 1 μl injection. Solid phase extraction of amides from total lipid extract before GC/MS analysis provides clean detection and interference free analysis.