Anal. Chem. 2003, 75, 4925-4930
Technical Notes
Identification and Characterization of Conjugated Fatty Acid Methyl Esters of Mixed Double Bond Geometry by Acetonitrile Chemical Ionization Tandem Mass Spectrometry Anthony L. Michaud,†,| Martin P. Yurawecz,‡ Pierluigi Delmonte,‡ Benjamin A. Corl,§ Dale E. Bauman,§ and J. Thomas Brenna*,†
Division of Nutritional Sciences, Cornell University, Savage Hall, Ithaca, New York 14853, U.S. FDA, Center for Food Safety & Applied Nutrition, College Park, Maryland 20740, and Department of Animal Science, Cornell University, Ithaca, New York, 14853
Fatty acids with conjugated double bonds have attracted great interest because of their reported potent bioactivities. However, there are currently no rapid methods for their structural characterization. We report here a convenient mass spectrometry-based strategy to establish double bond geometry by analysis of collisional dissociation products of cis/trans and trans/cis conjugated linoleic acids (CLAs), as methyl esters, and to distinguish CLAs from homoallylic (methylene-interrupted) fatty acids in a single-stage mass spectrum. A series of CLA standards with double bond positions 6,8; 7,9; 8,10; 9,11; 10,12; 11,13; 12,14; and 13,15, with all four possible geometries (cis/trans; trans/cis; cis/cis; trans/trans) were analyzed. The m/z 54 (1-methyleneimino)-1-ethenylium ion, generated by self-reaction of acetonitrile under chemical ionization conditions, reacts with unsaturated fatty acids to yield an [M + 54]+ ion, which decomposes in the single-stage mass spectrum by loss of neutral methanol to form [M + 54 - 32]+. The ratio of [M + 54]+/[M + 54 - 32]+ in the single-stage mass spectra of CLA isomers is 1 order of magnitude less than for homoallylic diene FAME. Collisional dissociation of the [M + 54]+ ion yields two diagnostic ions that contain the r- and ω-carbon atoms and is characteristic of double bond position in the analyte. The fragment vinylic to the trans double bond is significantly more abundant than that for the cis double bond, revealing double bond geometry. The ratio of r to ω diagnostic ion abundances is >4.8 for cis/trans isomers, 4.8 for cis/trans isomers, 4.8, as with other cis/trans isomers, the value of 0.9 obtained for the trans/cis isomer is not less than 0.5 as was observed for other trans/cis CLA isomers. A standard containing only the trans/cis isomer is necessary to demonstrate whether the currently observed high value is due to some contribution of the overlapping cis/trans isomer to the MS/MS spectrum. These results demonstrate that the double bond geometry of CLAs can be assigned to one of three categories, cis/trans, trans/ cis, and cis/cis or trans/trans, from acetonitrile CIMS/MS data. Due to interferences observed with the m/z 320 ion, it is not (18) Van Pelt, C. K.; Carpenter, B. K.; Brenna, J. T. J. Am. Soc. Mass Spectrom. 1999, 10, 1253-1262.
Figure 5. Possible mechanism for R diagnostic ion formation in 9c,11t-18:2. Adduct formation occurs by a two-step cycloaddition. The resulting six-membered ring favors H transfer from a site that is two carbons away from a trans double bond over transfer from a cis double bond.
possible to obtain a reliable R/ω ratio for the 13,15 CLA isomers; however, double bond positions can still be assigned based on observation of the m/z 136 diagnostic ion. The structure and fragmentation mechanism of the [M + 54]+ parent ion must be consistent with the observed differences in diagnostic ion abundances for the CLAs with mixed double bond geometry. The mechanism for diagnostic ion formation in homoallylic dienes proposed previously18 is not applicable in this case as that mechanism requires a single C-C bond where a double bond is present in the conjugated diene in order to produce the observed diagnostic ions. Studies of the addition of vinylamine radical cation with conjugated dienes and methylene-interrupted dienes19 allow us to speculate on a possible mechanism for CLA diagnostic ion formation. In those studies, it is suggested that the formation of a six-membered-ring adduct between the vinylamine radical cation and conjugated dienes is more favorable than the formation of a four-membered ring that was observed with nonconjugated dienes. Other work with vinyl methyl ether CI of hydroxy conjugated dienes containing both a cis and a trans double bond also suggests a [4 + 2] cycloaddition for adduct formation between analyte and CI reagent ion.20 On the basis of these observations, we suggest a similar adduct between MIE and the CLA conjugated diene unit. Figure 5 shows a possible mechanism for R-diagnostic ion formation from the reaction of 9c,11t-18:2 with MIE. The three-dimensional structure about the cis double bond makes it more accessible for reaction to form the [M + 54]+ than the trans double bond. The electropositive terminal carbon of MIE undergoes nucleophilic (19) Bouchoux, G.; Penaud-Berruyer, F. Org. Mass Spectrom. 1994, 29, 366371. (20) Einhorn, J.; Duffault, J. M.; Virelizier, H.; Tabet, J. C. Rapid Commun. Mass Spectrom. 1988, 2, 112-115.
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attack by the cis double bond of the CLA. The side chain containing the ω end of the FAME rotates, and the trans double bond is available for attack by the nitrogen lone pair to form a six-membered ring, restricted by three sp2 hybridized atoms to a boatlike conformation. When the ω side chain is in an equatorial position, an H located two carbons away from the position of the original trans double bond is held close to the imine and transfer can occur, resulting in formation of the R diagnostic ion upon CAD. The ω diagnostic ion can form when MIE reacts with the CLA in a position that is antiparallel to that shown in the top reaction. This mechanism clearly favors the formation of diagnostic ions produced by C-C bond cleavage adjacent to the trans double bond in CLAs with cis/trans or trans/cis double bond geometry. For cis/cis and trans/trans CLAs, an equal abundance of both diagnostic ions is expected. Further experimental work with labeled compounds and molecular modeling studies are in progress to test this hypothesis. In conclusion, acetonitrile CIMS/MS, in conjunction with relative GC retention times, can be used for the rapid and nearly complete positive structural identification of all CLAs. Acetonitrile CIMS/MS provides information on double bond positions for all
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CLAs with the possible exception of 13c,15t-18:2. Acetonitrile CIMS/MS alone can separate CLAs into three categories of double bond geometries: cis/trans, trans/cis and then cis/cis or trans/ trans. Double bond geometry in cis/cis and trans/trans isomers can be deduced from relative retention times. Ambiguities remain for cis/cis and trans/trans 13,15-18:2 and 12,14-18:2 due to chromatographic peak overlap. Mechanistic studies to establish the structure and dissociation of the [M + 54]+ and diagnostic ions are in progress. ACKNOWLEDGMENT A special thanks to Gavin L. Sacks for help with the processing of raw data files to generate figures. This work was supported by NIH Grant GM49209. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review March 5, 2003. Accepted July 10, 2003. AC034221+
