In general, fatty acid profiling is performed using GC-FID or GC-MS. The lipidic fraction in blood or plasma samples is hydrolyzed and methylated into its fatty acid methyl esters (FAMEs). A 1 µL sample of the iso-octane extract containing the FAMEs is separated using a polar column, providing separation according to carbon number and number of double bonds. Separation of cis-trans isomers is also obtained using

a cyanopropyl silicone column with high cyano content (e.g. HP-88).

Separate steps are used for hydrolysis and methylation. Fatty acids have broad metabolic functions and exist in free forms or integrated into more complex lipids. They are present in all organisms and constitute essential structural elements of biological membranes, regulate the activity of enzymes and fulfill important roles as signaling molecules. Because of their highly reduced chemical structure, fatty acids yield more than twice as much energy upon oxidation compared with polysaccharides making fat the most efficient form for living organisms to store excess energy. They may be either methylated yielding fatty acid methyl esters (FAME) or reacted surfaces. A derivatization reagent is applied for the determination of free fatty acids in mixture os acids (C1 to C5) in biological specimens need a special treatment taking into. A reliable and sensitive method for comprehensive analysis of free fatty acids and fatty acid composition of complex lipids in biological material. The separation and quantitation of fatty acids is achieved by capillary gas chromatography. The analytical method uses pentafluorobenzyl bromide derivatization and negative chemical ionization gas chromatography-mass spectrometry. The chromatographic procedure provides base line separation between saturated and unsaturated fatty acids of different chain lengths as well as between most positional isomers. Fatty acids are extracted in the presence of isotope-labeled internal standards for high quantitation accuracy. Mass spectrometer conditions are optimized for broad detection capacity and sensitivity capable of measuring trace amounts of fatty acids in complex biological samples.

 Thank you Prof. Upadhyay for your suggestions.

Hi,

For the simple derivatization of fatty acids into methyl esters, I would recommend to use TMS-diazomethane and MeOH. This is easily available by Sigma and the reaction itself is quite straightforward. The reaction products are quite clean and fully compliant with GC-MS. I don't know however how it behaves with your matrix.

'hope this helps.

Kind regards,

Cedric.

Thanks Guignard Cedric for your valuable information.

Best regards,

Jitendra

Thanks Andre.

Best regards,

Jitendra

Extract plasma lipids into mixture of organic solvents (1:2) Methanol and Dichloromethane as per modified method of Folch. Evaporate solvents under nitrogen atmosphere. The residue is  treated with methanolic sodium hydroxide (0.2M)  warmed for few minutes  followed by BF3/Methanol treatment. Add water and extract with n-hexane.Wash hexane layer  with water and dry over sodium sulphate. The n-haxane layer containing FAME  can be injected to GC-MS analysis. This method  is simple and feasible.

Thanks Abdur Mirajkar for your suggestation.

Regards,

Jitendra

se:

Journal of Separation Science (Impact Factor: 2.59). 11/2009; 32(21):3738-45. DOI: 10.1002/jssc.200900455

and:

International Journal of Food Science & Technology 11/2014; 50(2). DOI:10.1111/ijfs.12699

Dissolve your lipids in 1 mL of toluene, then add 3mL of 2% sulfuric acid in methanol.  Cap under nitrogen, then put parafilm around the cap to prevent any evaporation of the short chained FAMEs.  Cook in a scientific oven or water bath at 55C for 16 hours.  Neutralize with 3mLs of a bicarb/carbonate solution.  Add 5mLs hexane and centrifuge at 3000xg at 10C for 15 min.  Take off organic layer and dry under nitrogen.  Resuspend in either 5ul/mg or 10ul/mg (hexane ul, mg amount of sample) .  I've done this with 50mgs of food oil and gotten awesome results, and with the isolated phospholipids from 200mg human plasma.  It has a good working range.  Best of luck.

Ausitn

esterification with methanol in the presence of BF3, then extract with non-polar solvent and evaporate it.