I routinely quantify the fatty acid composition from seed lipid extracts by transmethylation to fatty acid methyl esters with either methanolic HCl or sodium methoxide, followed by GC-FID. However, I am now looking for a method to analyse low concentrations (less than 1 nanogram per mL) of free fatty acids (C6 - C20) in seed extracts containing low microgram per mL quantities of lipids (mostly triacylglycerols).

At such low concentrations, analysis of underivitized free fatty acids by HPLC seems impractical. However, if I must derivitize (for HPLC or GC analysis)it becomes very important that I don't inadvertently measure fatty acids that may arise from lipid degradation during sample preparation or derivitization.

I've considered two possible routes:
1) Separate the free fatty acids from the lipids by SPE before derivitization and/or analysis
2) Use a derivitizing reagent that is specific for carboxylic acids and has no transesterifying activity with lipids

Does anyone have experience of either of these two procedures (or others) that will effectively discriminate between low concentrations of free fatty acids and high concentrations of lipid in the same sample? I'm aware that derivitization reagents such as diazomethane are supposed to be highly specific for free acids, but I've yet to find data on any "residual" transesterifying activity.

thanks
Tony

I'd try dissolving sample in DMF or pyridine and making trimethylsilyl derivatives by adding BSTFA, then capillary GC assay on methyl siloxane/DB-1/DB-5 type columns. We actually used heptanoic acid as internal standard when were especially interested in the C6 level.

The diazomethane derivatization should be the method of choice for your problem. I have looked at this extensively, some time ago:
J Chrom B, 228, 75 (82); Clinica Chimica Acta, 124, 343 (82); J Chrom B, 679, 208 (96); Lab Note in Aldrichimica Acta, 31, 2 (98).
The reports of unwanted reactions of diazomethane, including those of reas with the double bonds stem from confusions with reactions of the methylene carbene. The carbene is not formed unless you irradiate diazomethane with light or heat it.... Diazomethane reacts heterolytically only with highly activated double bonds (there are none in fatty acids). "Reactions" with the ester group are in actuality due to reactions with the carboxylate which has been formed by careless hydrolysis of esters.