ABSTRACT: A simple method for regiospecific analysis of triglycerides was developed. It consists of partial deacylation of triglycerides by ethylmagnesium bromide followed by derivati- zation of monoglycerides with n-butyryl chloride, and direct analysis of dibutyrate derivatives of monoglycerides by gas chromatography. The chromatographic conditions were carried out with monoglycerides of C 12 to C 20 fatty acids and resulted in separation of dibutyrate derivatives between those bearing the medium- or long-chain fatty acid in the sn-1(3) and sn-2 po- sitions of glycerol. Beef tallow and grapeseed and cotton seed oils were analyzed using this new method, and their regiospe- cific distributions were compared with literature data. The method does not require separation of products by thin-layer chromatography or special analytical equipment other than a standard gas chromatograph, and it can thus be used for routine regiospecific analysis of triglycerides. Paper No. J8945 in JAOCS 76, 481–484 (April 1999). KEY WORDS: Acid chloride, beef tallow, cotton seed oil, ethylmagnesium bromide, fatty acid, gas chromatography, grapeseed oil, regiospecific analysis, triglyceride. Positional distribution of fatty acids on the glycerol moiety of triglycerides affects both the functional properties (1) and the metabolism of fats and oils (2,3). The methods used to carry out regio- or stereospecific analysis of fatty acids in triglyc- erides usually start with partial deacylation either by pancre- atic lipase (4–7), for specific removal of fatty acid, or by a Grignard reagent (4,5,8–10). Subsequently, two approaches can be used: one consists of analyzing 2-monoglycerides, usually isolated by thin-layer chromatography, for their fatty acid composition and estimating values in the sn-1(3) posi- tion from the fatty acid composition of 2-monoglycerides and triglycerides (11). The second approach consists of analyzing diglycerides for their fatty acid composition and determining the fatty acid composition in the sn-1(3) and sn-2 positions from the fatty acid composition of diglycerides and triglyc- erides (4,5). Though these are indirect methods, they allow accurate determination of regiospecific distribution of fatty acids in triglycerides. However, use of these methods on a routine basis is laborious, since they require isolation of ei- ther mono- or diglycerides, which are time-consuming opera- tions. Tandem mass spectrometry may also be used for re- giospecific analysis of triglycerides. Though very powerful, this method is not yet widely used because of the limited availability of the instruments (12). Hence, methods used to perform positional distribution analysis of fatty acid in triglycerides are either time consuming, limiting their use on a routine basis, or require analytical techniques which are pro- hibitive for general use. Thus, there is a need for a simple, fast, and reliable method for regiospecific analysis of triglyc- erides. In this study, we developed a method for regiospecific analysis of triglycerides based on their partial deacylation by a Grignard reagent followed by derivatization of the reaction products with n-butyryl chloride, in presence of a base such as triethylamine, and direct analysis of the so-formed dibu- tyrate derivatives of monoglycerides by gas chromatography. The method was applied to beef tallow, grapeseed oil, and cotton seed oil. MATERIALS AND METHODS Materials. Standard monoglycerides used were: 1-mono- lauroyl-rac-glycerol, 1-monomyristoyl-rac-glycerol, 1-mono- palmitoyl-rac-glycerol, 2-monopalmitoylglycerol, 1-mono- palmitoleoyl-rac-glycerol, 1-monostearoyl-rac-glycerol, 1- monooleoyl-rac-glycerol, 1-monolinoleoyl-rac-glycerol, 1- monolinolenoyl-rac-glycerol. Standard triglycerides were: trilaurin, trimyristin, tripalmitolein, trimargarin, tristearin, tri- olein, trilinolein, trilinolenin, and triarachidin. All were pur- chased from Sigma Chemicals (St. Louis, MO). Ethylmagne- sium bromide, n-butyryl chloride, and triethylamine were ob- tained from Aldrich (Milwaukee, WI). Deodorized beef tal- low and refined grapeseed and cotton seed oils were obtained from a local fat and oil processor. Deacylation of triglycerides and derivatization. Standard triglycerides, beef tallow, and grapeseed and cotton seed oils were partially deacylated with ethylmagnesium bromide, de- rivatized with n-butyryl chloride, and analyzed by gas chro- matography. A typical procedure was as follows: To a stirred solution of grapeseed oil (5 mg) in anhydrous diethyl ether (0.5 mL), contained in a flame-dried flask under inert atmo- sphere (N 2 ), a solution of ethylmagnesium bromide in the same solvent (3.0 M; 20 μL, 5 eq) was added. After stirring Copyright © 1999 by AOCS Press 481 JAOCS, Vol. 76, no. 4 (1999) *To whom correspondence should be addressed. E-mail: joseph.arul@aln.ulaval.ca A Simple Method for Regiospecific Analysis of Triacylglycerols by Gas Chromatography Paul Angers and Joseph Arul* Department of Food Science and Nutrition and Dairy Research Center (STELA), Université Laval, Québec G1K 7P4, Canada