Research article Development of solid-phase extraction and methylation procedures to analyse free fatty acids in lipid-rich seeds Andre ´ina Laffargue, Alexandre de Kochko, Ste ´phane Dussert * IRD, UR 188, UMR DIA-PC, 911 Avenue d’Agropolis, BP 64501, F-34394 Montpellier Cedex, France Received 25 July 2006; accepted 22 January 2007 Available online 30 January 2007 Abstract 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 offatty 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 0e18 months at 15  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. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Ageing; Coffee seed; Free fatty acid; Plant; Purification; Solid-phase extraction; Thin-layer chromatography 1. Introduction De-esterification of fatty acids from glycerolipids, includ- ing phospholipids and triacylglycerols, has been identified as one of the major mechanisms of injury in various plant systems subjected to ageing, anoxia, drying, or freezing: e.g. potato cells under oxygen deprivation [16], dehydrated germi- nating soybean axes [24], rapidly dried carrot somatic embryos [27], aged Typha latifolia pollen [30], chilled non- orthodox neem seeds [22], and frozen Arabidopsis thaliana plants [33]. In particular, accumulation of free fatty acids (FFA) has been shown to play a crucial role in seed ageing [28,31]. Two hypotheses have been proposed for the origin of FFA: free radical attack [25,30] or lipase/phospholipase A 2 activity [33]. FFA are well-established membrane destabi- lizing agents leading to irreversible damage [3,12,36] but are also known to play a crucial role in plant biotic and abiotic stress signalling [32]. In plant physiology studies, FFA are classically analyzed using the following procedure [16,22,24,27,28,30]: FFA are separated from other lipid classes by thin-layer chromatogra- phy (TLC), scraped off TLC plates, derivatized (generally by Abbreviations: FA, fatty acid; FAME, fatty acid methyl ester; FFA, free fatty acid; GC, gas chromatography; RH, relative humidity; SPE, solid-phase extraction; TAG, triacylglycerol; TLC, thin-layer chromatography. * Corresponding author. Tel.: þ33 4 6741 6185; fax: þ33 4 6741 6330. E-mail address: dussert@mpl.ird.fr (S. Dussert). 0981-9428/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2007.01.012 Plant Physiology and Biochemistry 45 (2007) 250e257 www.elsevier.com/locate/plaphy