Development of solid-phase extraction and methylation procedures to analyse free fatty acids in lipid-rich seeds.

In order to develop a sensitive and reliable method for FFA quantification in lipid matrices of seeds, two SPE procedures employed in meat and dairy chemistry were compared using a 100/1 mixture of triolein/heptadecanoic acid. The overall efficiency of the SPE procedure retained was satisfactory since it allowed removal of 99.8% of triacylglycerols (TAG) and recovery of 99.2% of FFA as quantified by gas chromatography of fatty acid methyl esters (FAME). However, the low amount of TAG eluted in the FFA fraction represented a non-negligible percentage (17%) of FAME and the procedure thus required further improvement. TAG pollution was successively decreased to 12%, 8% and finally 1.5% by: i) modifying the volume of elution of TAG; ii) removing the saponification step initially performed according to the standard FAME procedure; and iii) reducing the duration of the BF(3)-catalyzed methylation reaction to 1 min. The new SPE/methylation procedure described here was then compared to the most widely used method for FFA measurement in plants which is based on thin-layer chromatography (TLC). Both procedures were applied to coffee seeds stored for 0-18 months at 15 degrees C under 62% relative humidity and provided consistent results. A very clear negative correlation was observed between the loss of seed viability and the accumulation of FFA in seeds during the course of storage independent of the method employed for FFA quantification. However, we demonstrated that the TLC/on-silica methylation procedure underestimates FFA contents in comparison with the new SPE/methylation procedure because of a selective loss of unsaturated FA.