FATTY ACID ASSAY OF TWO SAMPLES BY GC-MS

University Of Georgia, Athens GA

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Lipid Extraction and Transesterification The analysis was initially performed on two standard mixtures containing 1 ug and 0.1 ug individual fatty acids, respectively, to estimate the limit of detection of this method. Then the two samples and a water blank were analyzed together. The samples (1.6 mL, 150 mg/mL) and blank (1.6 mL) were extracted according to the method of Bligh and Dyer (E.G. Bligh and W.J. Dyer 1959 Can. J. Biochem. Physiol. 37:911) with 1.85 mL chloroform-methanol (1:2) by vortexing for 15 min. Then, 0.62 mL chloroform was added, followed by 0.62 mL water. The mixtures were vortexed for 5 min after each addition. The organic layers of each sample were separated and dried down under a stream of nitrogen. The samples were treated with 2 mL 1.5 M HCl in 4:1 methanol-toluene at 100 [unreadable]C for 1 h (G. Lepage and C. C. Roy 1986 Journal of Lipid Research 27:114). After cooling, 5 mL 6 % aqueous K2CO3 was added and the organic layer was separated for GC-MS. GC-MS GC/MS analysis for fatty acid methyl esters was performed on an Agilent 6890N gas chromatograph interfaced to a 5975B mass selective detector, using an Alltech EC-1 column (30m x 0.25 mm ID). Injection volume was 5 uL.