Anal. Chem. 1999, 71, 1981-1989
Acetonitrile Chemical Ionization Tandem Mass Spectrometry To Locate Double Bonds in Polyunsaturated Fatty Acid Methyl Esters Colleen K. Van Pelt and J. Thomas Brenna*
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853
A rapid method is presented for determining the location of double bonds in polyunsaturated fatty acid methyl esters (FAME) using an ion-trap mass spectrometer. The mass spectrum of the chemical ionization reagent acetonitrile in an ion trap includes a m/z 54 ion, identified previously as 1-methyleneimino-1-ethenylium ion. We show that it reacts with double bonds of polyunsaturated FAME to yield a series of covalent product ions all appearing at (M + 54)+. Collisional dissociation of these ions yields diagnostic fragments, permitting unambiguous localization of double bonds. For methylene-interrupted and conjugated FAME, one of these fragments results from loss of the hydrocarbon end of the chain, while the other involves loss of the methyl ester. Major diagnosticfragment ions for monoene and diene FAME occur as a result of cleavage adjacent to either allylic sites or double bonds in the original analyte and appear at one mass unit above the mass expected for homolytic cleavage. Fragmentation of polyene FAME yields major diagnostic ions resulting from cleavage between double bonds that appear one mass unit lower. The method is shown to produce highly characteristic spectra for FAME with 1 to 6 double bonds. Identification of double-bond position in highly unsaturated fatty acids is demonstrated in a mixture of unknown polyunsaturated FAME from an extract of cultured Y79 human retinoblastoma cells. The principal fatty acids found in mammalian tissue and fluids contain from zero to six double bonds, normally methyleneinterrupted and of cis stereochemistry. While the number of double bonds is easily determined by electron impact (EI) or chemical ionization (CI) mass spectrometry (MS) and by other methods, a convenient instrumental method for location of double bonds in FAME remains a challenge. The conventional method for fatty acid analysis is methylation to form fatty acid methyl esters (FAME) for analysis by gas chromatography (GC). It has long been known that EI ionization of FAME causes double-bond migration,1 resulting in ambiguous spectra, and CI with conventional proton-transfer reagents does not yield useful fragments. Schneider2 has summarized three general approaches for locating double-bond position in fatty acids using EI or CI-MS: chemical * Corresponding author: (Phone) (607) 255-9182; (fax) 607 255-1033; (E-mail)
[email protected]. (1) Budzikiewicz, H.; Busker, E. Tetrahedron 1980, 36, 255. 10.1021/ac981387f CCC: $18.00 Published on Web 04/09/1999
© 1999 American Chemical Society
derivatization of double bonds yielding compounds that produce fragmentation a) near or b) remote from the modified site, and c) chemical ionization with specific reagents. There are many examples of the first general method. Double bonds may be derivatized by methyl thiolation,3-6 epoxidation,2,7-9 methoxy mercuration-demercuration with subsequent NaBH4 reduction,10 formation of Diels-Alder adducts with various reagents,11,12 ozonolysis,13 and osmium tetraoxide oxidation followed by conversion of the diol to a trimethylsilyl group14 or a methoxy group.15 All products yield fragment ions due to cleavage near the derivatization site. The second general method is based on derivatization to induce useful fragmentation from charged or radical sites remote from the double bonds. Examples where the derivatization is remote from the double bond include picolinyl esters,16-18 sulfate esters,19 pyrrolidides,20-22 and 2-alkenyl-4,4-dimethyloxazolines.23,24 The major disadvantage of all derivatization methods is that they (2) Schneider, B.; Budzikiewicz, H. Rapid Commun. Mass Spectrom. 1990, 4, 550. (3) Pepe, C.; Dizabo, P.; Dagaut, J.; Balcar, N.; Lautier, M. F. Eur. Mass Spectrom. 1997, 1, 209. (4) Pepe, C.; Balcar, N.; Dizabo, P.; Dagaut, J.; Saliot, A.; Couffignal, R.; Sayer, H. Rapid Commun. Mass Spectrom. 1995, 9, 1576. (5) Vincenti, M.; Guglielmetti, G.; Cassani, G.; Tonini, C. Anal. Chem. 1987, 59, 694. (6) Scribe, P.; Guezennec, J.; Dagaut, J.; Pepe, C.; Saliot, A. Anal. Chem. 1988, 60, 928. (7) Bieri-Leonhardt, B. A.; DeVilbiss, E. D.; Plimmer, J. R. J. Chromatogr. Sci. 1980, 18, 364. (8) Hogge, L. R.; Underhill, E. W.; Wong, J. W. J. Chromatogr. Sci. 1985, 23, 171. (9) Kuhn, G.; Weidner, S.; Decker, R.; Hollander, A. Rapid Commun. Mass Spectrom. 1997, 11, 914. (10) Blomquist, G. J.; Howard, R. W.; McDaniel, C. A.; Remaley, S.; Dwyer, L. A.; Nelson, D. R. J. Chem. Ecol. 1980, 6, 257. (11) Young, D. C.; Vouros, P.; Descosta, B.; Holick, M. F. Anal. Chem. 1987, 59, 1954. (12) Kidwell, D. A.; Biemann, K. Anal. Chem. 1982, 54, 2462. (13) Aveldano, M. I.; Sprecher, H. J. Biol. Chem. 1987, 262, 1180. (14) Murata, T.; Ariga, T.; Araki, E. J. Lipid Res. 1978, 19, 172. (15) Niehaus, W. G. J.; Ryhage, R. Anal. Chem. 1968, 40, 1840. (16) Lie Ken Jie, M. S. F.; Choi, Y. C. J. Am. Oil Chem. Soc. 1992, 69, 1245. (17) Harvey, D. J. Biomed. Mass Spectrom. 1984, 11, 340. (18) Harvey, D. J. Biomed. Mass Spectrom. 1982, 9, 33. (19) Nakamura, T.; Takazawa, T.; Maruyama-Ohki, Y.; Nagaki, H.; Kinoshita, T. Anal. Chem. 1993, 65, 837. (20) Andersson, B. A.; Christie, W. W.; Holman, R. T. Lipids 1975, 10, 215. (21) Andersson, B. A.; Heimermann, W. H.; Holman, R. T. Lipids 1974, 9, 443. (22) Vetter, W.; Walther, W.; Vecchi, M. Helv. Chim. Acta 1971, 54, 1599. (23) Mossoba, M. M.; Yurawecz, M. P.; Roach, J. A. G.; Lin, H. S.; McDonald, R. E.; Flickinger, B. D.; Perkins, E. G. Lipids 1994, 29, 893. (24) Yu, Q. T.; Liu, B. N.; Zhang, J. Y.; Huang, Z. H. Lipids 1989, 24, 79.
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999 1981
require a specialized wet chemical modification step prior to MS analysis and thus are much less convenient than a purely instrumental method applicable directly to FAME. CI single-stage MS methods have employed a variety of reagent gases including vinylamine,25,26 vinyl methyl ether either alone or as a mixture with N2, N2 and CS2, or CO2,27,28 methylamine,29 isobutane,30,31 OH- produced by EI in a 10:90 ratio of N2O to CH4,32 and NO.33-35 Budzikiewicz et al. report that the double bonds in methylene-interrupted triene and tetraene systems can be located by two radical-ion fragments using NO as the reagent gas.35 In general, the disadvantage to CI-based methods reported to date is that they are limited to special cases and have only been evaluated for localization of a maximum of four double bonds. An effective alternative based on fast atom bombardment (FAB) coupled to tandem MS (MS/MS) has been reported. Up to three double bonds have been localized by labeling with deuterium using N2D2 prior to analysis by negative ion FAB collision activated dissociation (CAD).36 Later work by Adams and Gross showed that alkali-metal-cationized molecules, formed in situ in a glycerol matrix and desorbed by FAB, undergo chargeremote fragmentation upon collisional activation.37,38 This method was shown to locate the positions of 6 double bonds in a fatty acid. The disadvantage of these methods is that they require a FAB source, which is becoming a specialized technique as liquid introduction by electrospray ionization and related techniques increasingly dominates. Acetonitrile (CH3CN, molecular weight (MW) ) 41) is an unconventional liquid CI reagent first reported by Yinon and Cohen.39 It has been reported recently as a proton-transfer CI reagent for several environmental40,41 and biological42-45 applications. With a proton affinity of 788 kJ/mol,46 acetonitrile is a slightly less energetic reagent than isobutane (proton affinity ) 683 kJ/mol).47 Its mass spectrum under ion-trap CI conditions includes ions at m/z 40 and 42, formed by loss or gain (25) Bouchoux, G.; Penaud-Berruyer, F. Org. Mass Spectrom. 1993, 28, 271. (26) Bouchoux, G.; Penaud-Berruyer, F. Org. Mass Spectrom. 1994, 29, 366. (27) Chai, R.; Harrison, A. G. Anal. Chem. 1981, 53, 34. (28) Ferrer-Correia, A. J. V.; Jennings, K. R.; Sen Sharma, D. K. Adv. Mass Spectrom. 1978, 7, 287. (29) Budzikiewicz, H.; Laufenberg, G.; Brauner, A. Org. Mass Spectrom. 1985, 20, 65. (30) Doolittle, R. E.; Tumlinson, J. H.; Proveaux, A. Anal. Chem. 1985, 57, 1625. (31) Vekey, K.; Baan, G.; Jennings, K. R. Biomed. Environ. Mass Spectrom. 1988, 16, 267. (32) Takeuchi, G.; Weiss, M.; Harrison, A. G. Anal. Chem. 1987, 59, 918. (33) Hunt, D. F.; Harvey, T. M. Anal. Chem. 1975, 47, 2136. (34) Mouloud, M.; Metro, F.; Goutefongea, R.; Dumont, J. P. Sci. Aliments 1992, 12, 371. (35) Brauner, A.; Budziliewicz, H.; Boland, W. Org. Mass Spectrom. 1982, 17, 161. (36) Jenson, N. J.; Tomer, K. B.; Gross, M. L. Anal. Chem. 1985, 57, 2018. (37) Adams, J.; Gross, M. L. Anal. Chem. 1987, 59, 1576. (38) Adams, J.; Gross, M. L. Org. Mass Spectrom. 1988, 23, 307. (39) Yinon, J.; Cohen, A. Org. Mass Spectrom. 1983, 18, 47. (40) Milde, D.; Plzak, Z.; Suchanek, M. Collect. Czech. Chem. Commun. 1997, 62, 1403. (41) Plzak, Z.; Polanska, M.; Suchanek, M. J. Chromatogr., A 1995, 699, 241. (42) Shetty, H. U.; Holloway, H. W.; Rapoport, S. I. Anal. Biochem. 1995, 224, 279. (43) Shetty, H. U.; Holloway, H. W. Biol. Mass Spectrom. 1994, 23, 440. (44) Shetty, H. U.; Daly, E. M.; Greig, N. H.; Rapoport, S. I.; Soncrant, T. T. J. Am. Soc. Mass Spectrom. 1991, 2, 168. (45) Lim, H. K.; Sakashita, C. O.; Foltz, R. L. Rapid Commun. Mass Spectrom. 1988, 2, 129. (46) Lias, S. G.; Bartmess, J. F.; Holmes, J. L.; Levin, R. D.; Mallard, W. G. J. Phys. Chem. Ref. Data 1988, 17, 1.
1982 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
of H, and at m/z 54, identified as 1-methyleneimino-1-ethenylium, H2Cd+NdCdCH2.48-54 A 40-amu adduct has been observed55 upon reaction with C20-37 saturated hydrocarbons corresponding to addition of CH2CN+. For similar monounsaturated hydrocarbons, both a 40-amu and a 54-amu adduct were observed,56 suggesting that both the m/z 40 and 54 are useful as reagent ions, with the latter reactive toward double bonds. Recently it has been reported that the reagent ion with a m/z of 54 reacts selectively with the double bond in a C23 monounsaturated hydrocarbon, resulting in fragmentation about the double bond.54 We report here a rapid, general method for the identification of double-bond position in polyunsaturated FAME using acetonitrile CI-MS/MS implemented on a benchtop GC/ion-trap MS. Diagnostic fragment ions are identified and catalogued for all major long chain polyunsaturated FAME of mammalian origin, from monoenes to hexaenes. Finally, the method is demonstrated by analysis of a highly unsaturated FAME mixture from a culturedcell extract. EXPERIMENTAL SECTION Chemicals. The following fatty acid methyl esters were purchased from Sigma Chemical Co. (St. Louis, MO): cis-9octadecenoic acid methyl ester, trans-9-octadecenoic acid methyl ester, cis-11-octadecenoic acid methyl ester, trans-11-octadecenoic acid methyl ester, cis-12-octadecenoic acid methyl ester, cis-13octadecenoic acid methyl ester, cis-9,12-octadecadienoic acid methyl ester, all-trans-9,12-octadecadienoic acid methyl ester, 9,11octadecadienoic acid methyl ester (mixture of all geometrical isomers),10,12 all-cis-6,9,12-octadecatrienoic acid methyl ester, allcis-9,12,15-octadecatrienoic acid methyl ester, and all-trans-9,12,15-octadecatrienoic acid methyl ester. BF3 (14%) in methanol was also acquired from Sigma. FAME standard mixture “68A” was obtained from NuCheck Prep (Elysian, MN) and contained the following compounds relevant to this report: cis-11-eicosenoic acid methyl ester, all-cis-11,14-eicosadienoic acid methyl ester, all-cis11,14,17-eicosatrienoic acid methyl ester, all-cis-8,11,14-eicosatrienoic acid methyl ester, all-cis-5,8,11,14-eicosatetraenoic acid methyl ester, and all-cis-4,7,10,13,16,19-docosahexaenoic acid methyl ester. Acetonitrile was purchased from Aldrich Chemical Co. (Milwaukee, WI). The biological sample used in this study was an extract of Y79 human retinoblastoma cells acquired from the National Institutes of Health (NIH). Solvents used were acquired from Fischer Scientific (Pittsburgh, PA). For convenient reference we use the Miller notation for designating specific FAME. It is defined as n:dωz, where n is the (47) Lias, S. G.; Liebman, J. F.; Levin, R. D. J. Phys. Chem. Ref. Data 1984, 13, 695. (48) Gray, G. A. J. Am. Chem. Soc. 1968, 90, 6002. (49) Heerma, W.; Sarneel, M. M.; Dijkstra, G. Org. Mass Spectrom. 1986, 21, 681. (50) Wincel, H. Int. J. Mass Spectrom. Ion Processes 1998, 175, 283. (51) Wincel, H.; Fokkens, R. H.; Nibbering, N. M. M. Int. J. Mass Spectrom. Ion Processes 1990, 96, 321. (52) Wincel, H.; Wlodek, S.; Bohme, D. K. Int. J. Mass Spectrom. Ion Processes 1988, 84, 69. (53) Bortolini, O.; Pandolfo, L.; Tommaselli, C.; Traldi, P. Rapid Commun. Mass Spectrom. 1998, 12, 1425. (54) Moneti, G.; Pieraccini, G.; Favretto, D.; Traldi, P. J. Mass Spectrom. 1998, 33, 1148. (55) Moneti, G.; Pieraccini, G.; Dani, F. R.; Catinella, S.; Traldi, P. Rapid Commun. Mass Spectrom. 1996, 10, 167. (56) Moneti, G.; Pieraccini, G.; Dani, F.; Turillazzi, S.; Favretto, D.; Traldi, P. J. Mass Spectrom. 1997, 32, 1371.
number of C in the acid, d is the number of double bonds, and z is the location of the first double bond from the terminal methyl group. Most fatty acids belong to one of three “families” designated ω9, ω6, or ω3. In mammals, desaturation and elongation enzymes operate on the carboxyl side of the hydrocarbon chain exclusively, so that fatty acids of a particular family are derived from one another, while interfamily conversion is not observed. Numbering of double bonds in this notation is conventional in the biomedical literature and is more convenient than systematic numbering because it emphasizes the relationship between fatty acids physiologically derived from one another. This nomenclature system is used only for FAME containing methylene-interrupted double bonds. Therefore, the two conjugated FAME will be referred to by their systematic names. Instrumentation. A tabletop Varian Saturn 2000 ion trap coupled to a Varian 3400 gas chromatograph (GC) equipped with a Varian 8200 autoinjector (Varian, Walnut Creek, CA) was used for all analyses. The GC employed a septum-equipped temperature-programmable injector (SPI) which was initially held at 60 °C for 0.2 min before being ramped to 260 °C at a rate of 300 °C/min. It was held at 260 °C for 13 min for single-component FAME analysis or for 35 min for analysis of a complex FAME mixture. The SPI injector was cooled between runs with CO2. The column was a 60-m SGE Chromatography Supplies (Austin, TX) BPX-70 (0.32-mm id, 0.25-µm film thickness). The GC oven temperature profile was adjusted depending on the sample. For a single-component FAME, the GC oven was initially held at 60 °C for 3 min and then ramped at 50 °C/min to 220 °C and held for 7.2 min. For the FAME standard mixture 68A and the Y79 cell extract, the initial GC oven temperature was 80 °C and was held for 6 min. The temperature was then ramped to 170 °C at a rate of 50 °C/min and held for 5 min. The oven temperature was then increased to 200 °C at 4 °C/min. After 5 min at 200 °C, the oven was ramped to 220 °C at a rate of 50 °C/min and held for 2 min. Finally, the temperature was increased to 240 °C at 5 °C/min and held for 3 min. The injection volume was 1 µL, and the column head pressure was constant at 20 psi. Typical concentrations that gave abundant signal were 5 µg/mL for single-stage MS and 50 µg/mL for MS/MS experiments. Automatic reaction control (ARC) was enabled during these experiments. ARC adjusts the ionization time to maintain the number of ions in the trap within the trap’s narrow space charge limited capacity, thereby widening the dynamic range per scan. For MS experiments, the ARC target value was set to 20 000, while for MS/MS experiments the target was set to 5000. The parention masses of the product ions isolated in the MS/MS experiments along with the resonant excitation amplitudes used to collisionally dissociate the ions are listed in Table 1. Other MS/MS parameters used were: mass isolation window, 3 m/z units; excitation time, 30 ms; isolation time, 8 ms; bandwidth, 2 kHz; excitation storage level, 85 m/z units; parent prescan ionization type. Other trap parameters are as follows: electron multiplier, 105 setting; axial modulation amplitude, 4.0 V; trap temperature, 160 °C; manifold temperature, 40 °C; transfer-line temperature, 200 °C; and scan rate, 1000 ms. Liquid acetonitrile CI reagent was placed in a reservoir plumbed directly into the CI inlet leading to the ion trap. The acetonitrile pressure was adjusted to produce a ratio of m/z 40 to
Table 1. Molecular Weights and CID Amplitudes Used To Analyze Various FAME FAME
MW
MW of adduct
CID amplitude (V)
18:1 18:2 conj. 18:2 18:3 20:1 20:2 20:3 20:4 22:5 22:6 24:5 24:6
296 294 294 292 324 322 320 318 344 342 372 370
350 348 348 346 378 376 374 372 398 396 426 424
1.65 1.65 1.45 1.62 1.70 1.68 1.68 1.63 1.62 1.50 1.65 1.65
42 of approximately 1:6. The selective ejection chemical ionization (SECI) scan-mode parameters were as follows: CI ionization storage level, m/z 22; reagent ion eject amplitude, 8 V; CI reaction storage level, m/z 25; and CI background mass, m/z 65. RESULTS Single-Stage CI-MS. In Figure 1a-d a series of single-stage mass spectra for 20:1ω6-20:4ω6 FAME analyzed with acetonitrile CI is presented. The major ions are MH+, (MH - 32)+, (MH 32 - 18)+, and (M + 54)+. The MH+ ion is the base peak for 20:1, 20:3, and 20:4; however, (MH - 32)+ is the base peak for the diene. As the degree of unsaturation increases from 1 to 4 double bonds, the (M + 54)+ peak increases in intensity relative to the base peak. For 20:4, the (M + 54)+, (MH - 32)+, and (MH - 32 - 18)+ peaks are increased in intensity compared with those for 20:3. The CI-MS spectra of 22:5 and 22:6 presented in Figure 2a and b, respectively, demonstrate that the (M + 54)+ ion, at m/z 398 and m/z 396, respectively, becomes the base peak for the most unsaturated FAME. The general features of these data are typical of FAME mass spectra using acetonitrile CI. In the spectra of Figures 1 and 2, there are no ions that are diagnostic of double-bond position. In addition, single-stage spectra are indistinguishable for FAME of the same length and containing the same number of double bonds, similar to electron impact spectra (spectra not shown). CI-MS/MS of Monoenes. Figure 3 shows the CI-MS/MS spectra of four isomers of cis-18:1, generated by collisional dissociation of the isolated (M + 54)+ peak. At the CID voltages used for monounsaturates, decomposition of the (M + 54)+ ion is complete, and the (M + 54 - 32)+ peak at m/z 318 is the base peak. In addition, each isomer yields two fragments characteristic of double-bond position. These ions correspond to cleavage beyond the methylene group on either side of the double bond and appear at a mass one unit higher than would be expected from homolytic cleavage at the bonds, as indicated on the structures in Figure 3. Each of the four positional isomers has a unique CI-MS/MS spectrum that permits unambiguous identification of the double-bond location. These spectra are highly reproducible. The proposed site of bond breakage for diagnostically important ions and the ion mass expected for homolytic bond breakage, with and without the m/z 54 adduct, is displayed on the structure of the FAME in each spectrum. We define “R” ions as those that Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
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Figure 1. CI-MS spectra of (A) 20:1ω6, (B) 20:2ω6, (C) 20:3ω6, and (D) 20:4ω6. The major ions observed in these spectra are MH+, (MH - 32)+, and (M + 54)+. For 20:1, 20:3, and 20:4 the base peak is MH+ and for 20:2 the base peak is (MH - 32)+. With the exception of 20:2, as the degree of unsaturation increases, (MH - 32)+ and (M + 54)+ grow in intensity relative to MH+. The relative intensities of these ions appear to be very sensitive to the degree of unsaturation of FAME.
carry the ester end of the molecule and “ω” ions as those that carry the terminal-carbon end. Both R and ω ions from the C18 monoenes appear at one amu above the expected mass, thus implying hydrogen transfer from neutral to ion. The mass spectra of two trans 18:1 isomers are presented in Figure 4. The stereoisomeric pairs, cis-9-18:1 and trans-9-18:1 and cis-11-18:1 and trans-11-18:1, produce very similar spectra. The only significant minor difference is the presence of reproducible small peaks in the cis spectra that are not observed in the trans spectra. For instance, cis-9-18:1 yields a reproducible peak at m/z 180 and cis-11-18:1 gives an ion present at m/z 220, neither of which is observed for the corresponding trans isomers. It may be possible to distinguish the cis and trans isomers by these subtle differences. Dienes: Conjugated and Trans. In Figure 5 we present the CI-MS/MS mass spectra of all-cis 18:2ω6 and 20:2ω6 observed upon dissociation of (M + 54)+. For 18:2, major peaks are the (M + 54)+ at m/z 348 and the (M + 54 - 32)+ at m/z 316 as well as the two diagnostic ions at m/z 192 and 278. These ions correspond to cleavage immediately before the first double bond and directly after the second, as indicated in Figure 5. As with the monounsaturated FAME, these ions appear one mass unit higher than expected from homolytic cleavage. The 20:2 spectrum shows analogous peaks. Figure 6 is the spectrum of all-trans 18:2ω6 showing that the major ions are observed at the same 1984 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
Figure 2. CI-MS spectra of (A) 22:5ω3 and (B) 22:6ω3. The base peak in these highly unsaturated FAME is no longer the MH+ as seen in Figure 1, but rather the (M + 54)+ ion. In the case of 22:5 (A), MH+, m/z 345, is abundant; however, for 22:6 (B), MH+ at m/z 343 is of low intensity.
masses as for all-cis-18:2. We note that m/z 150 is more abundant in all-trans-18:2 compared with all-cis-18:2. The (M + 54)+ ion of the 18:1 isomers, shown in Figures 3 and 4, was collisionally dissociated using the same MS/MS parameters as the (M + 54)+ ion of the 18:2 isomers of Figure 5. Since the (M + 54)+ ion of the 18:1 FAME was quantitatively dissociated while the (M + 54)+ present in the 18:2 FAME spectra was not, it appears that the methylene-interrupted dienes are more stable than the monoenes. In ion traps it is common for some parent ion to remain after collisional dissociation. Figure 7 shows the CI-MS/MS spectra of methyl esters of two conjugated 18:2 isomers, 9,11-octadecadienoic acid and 10,12octadecadienoic acid. Each positional isomer was a mixture of all four possible geometrical isomers. Both of the diagnostic ions expected from analysis of the methylene-interrupted dienoates were present; however, one of the ions was of much lower abundance. Figure 7a displays the CI-MS/MS spectrum of 9,11octadecadienoic acid methyl ester and reveals that the diagnostic ion m/z 264 is in high abundance, while m/z 192 is present but in low abundance. The spectrum of 10,12-octadecadienoic methyl ester in Figure 7b shows a major m/z 178 ion and a minor m/z 278 ion. As with the methylene-interrupted dienoate species, these ions appear at a mass one unit greater than that expected from homolytic bond cleavage. Trienes and Higher Polyenes. Figures 8 and 9 present the CI-MS/MS spectra from a series of trienes with 18 and 20 carbons, respectively. In Figure 8a and b, 18:3ω3 (all-cis-6,9,12-18:3) yields m/z 190 and 234 diagnostic ions and can readily be distinguished from 18:3ω6 (all-cis-9,12,15-18:3), where m/z 148 and 276 are the diagnostic ions. As shown in Figure 8, these ions originate from cleavages on either side of the center double bond. In contrast to
Figure 3. CI-MS/MS spectra of (A) 18:1ω9, (B) 18:1ω7, (C) 18:1ω6, and (D) 18:1ω5. Specific cleavages at the allylic carbons shown in the structures at the top of each spectrum give rise to a pair of diagnostic ions that unambiguously identify the position of the double bond. Each of these pairs of diagnostic ions is unique to a particular isomer. The designated cleavage sites on the FAME structures include the mass of the fragment ion for homolytic cleavage and the mass of this ion + 54 amu expected in the CI-MS/MS spectrum. The diagnostic ions actually observed for monoenes are 1 amu higher than expected.
the monoenes and dienes, these ions appear at masses 1 unit lower than those expected from homolytic cleavage. As will be shown, the major diagnostic ions for all FAME with g3 double bonds behave in this way, indicating that a second, more favorable decomposition channel is available for trienes. The all-trans-9,12,15-18:3 CI-MS/MS spectrum is shown in Figure 8c. As compared with the all-cis-9,12,15-18:3 spectrum in Figure 8b, the diagnostic ions are strong; they again confirm the double-bond position. The peak at m/z 250 observed in the cis spectrum is missing in the spectrum of the trans isomer; however, the peak at m/z 134 in the trans spectrum is of greater abundance relative to the diagnostic ion, m/z 148. Figure 9 shows the CI-MS/MS spectra for the all-cis isomers 20:3ω3 (all-cis-11,14,17-20:3) and 20:3ω6 (all-cis-8,11,14-20:3). For 20:3ω3 the diagnostic ions m/z 148 and 304 are observed in Figure 9a; while in Figure 9b, the diagnostic ions are m/z 190 and 262 for 20:3ω3. Consistent with the C18 trienes, these strong peaks are found one mass unit below the expected masses, as shown in Figure 9. The CID energies used to collisionally dissociate the (M + 54)+ ion of 18:3 and 20:3 FAME were 1.62 and 1.68 V, respectively, as shown in Table 1, and were the only parameters varied between analysis of the two FAME. Since a more abundant (M + 54)+ remains after CID for 20:3, as seen in Figure 9, as
Figure 4. CI-MS/MS spectra of (A) trans-18:1ω9 and (B) trans-18:1ω7. The diagnostic ions observed in the trans isomers are the same as those seen in the cis isomers, as shown in Figure 3A,B. However, cis-18:1ω9 has a reproducible ion at m/z 180, which is not present in trans-18:1ω9, and similarly, cis-18:1ω7 has an ion at m/z 220 which is absent in the spectrum of trans-18:1ω7. These differences may aid in the identification of stereoisomers.
compared with that for 18:3 (Figure 8), it appears that the (M + 54)+ ions of 20:3 FAME are more stable than those of the 18:3 FAME. For FAME containing six double bonds, the CI-MS/MS spectra become only slightly more complex. Figure 10 is the mass spectrum of 22:6ω3 (all-cis-4,7,10,13,16,19-22:6). The (M + 54)+ and (M + 54 - 32)+ ions at m/z 396 and 364, respectively, are highly abundant as are the two diagnostic ions m/z 268 and 326. Figure 10 also shows the sites where this highly unsaturated FAME fragments to produce these two major diagnostic ions. Analysis of a Cultured-Cell Extract. Figure 11 is a polyunsaturate-rich region of a total ion chromatogram (TIC) of a FAME mixture from a Y79 human retinoblastoma cell extract. The section shown was divided into six different segments, each with its own ion-preparation file that permits a particular (M + 54)+ to be isolated for collisional dissociation. In this way FAME of various masses expected to emerge within these time windows can be analyzed by CI-MS/MS in a single run. Seven FAME with MS/ MS TIC abundance above 25% were identified. Figure 12 displays the CI-MS/MS spectra of the first three major peaks of the total ion chromatogram. The spectrum of the first peak, shown in Figure 12a, produced the diagnostic ions m/z 220 and 232 and was determined to be 20:3ω9 (all-cis-5,8,11-20:3). This compound was the most difficult of the seven to identify because of the large peak at m/z 300, which was identified as an unusually stable ion resulting from loss of CH2CO2CH3 from the (M + 54)+. The spectrum of Figure 12b matches that of the standard 20:3ω6 shown in Figure 9b. Figure 12c produces diagnostic ions at m/z 230 and 260 that both match the spectrum from a standard of 20:4ω6 (not shown). Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
1985
Figure 5. CI-MS/MS spectra of (A) cis-18:2ω6 and (B) cis-20:2ω6. Cleavage directly before the first double bond and immediately after the second, as shown in the structures, gives rise to the diagnostic ions m/z 192 and 278 and m/z 192 and 306, respectively. The base peaks, m/z 316 and 344, arise from the loss of methanol (32 amu) from (M + 54)+. The diagnostic ions for the dienes are 1 amu higher than expected for homolytic cleavage.
Figure 6. CI-MS/MS spectrum of trans-18:2ω6. The diagnostic ions, m/z 192 and 278, are the same as for the cis isomer as shown in Figure 5A. The spectrum of trans-18:2ω6 has a significantly more abundant m/z 150 ion than the cis isomer, which may aid in differentiating the isomers.
In Figure 13 the CI-MS/MS spectra are displayed for four other unknowns in the Y79 retinoblastoma cell extract. The (M + 54)+ at m/z 398 and the diagnostic ions in Figure 13a at m/z 270 and 286 uniquely identify the FAME as 22:5ω6. Similarly, the diagnostic ions in Figure 13b at m/z 228 and 328 identify the FAME as 22:5ω3. The spectrum in Figure 13c reveals the (M + 54)+ ion at m/z 426 and the diagnostic ions at m/z 270 and 314, both consistent with an assignment as 24:5ω6. The compound in the spectrum in Figure 13d is identified as 24:6ω3 using the diagnostic ions m/z 268 and 354 and the (M + 54)+ ion at m/z 424. 1986 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
Figure 7. CI-MS/MS spectra of 9,11- (A) and 10,12-octadecadienoic acid methyl ester (B). A mixture of all stereoisomers of these two conjugated 18:2 FAME was analyzed simultaneously. The 9,11 conjugated FAME has diagnostic ions m/z 264 and 192, while the 10,12 conjugated FAME has ions m/z 178 and 278. In both of these cases, one of the diagnostic ions predominates. The cleavage sites and expected masses based on homolytic cleavage of the diagnostic ions are provided in the structure.
DISCUSSION In the CI-MS mode, acetonitrile CI produces abundant MH+ ion of FAME which is confirmed by both (MH - 32)+ and (M + 54)+ ions. In addition to their masses, the relative intensities of these ions are characteristic of the varying degrees of unsaturation in methylene-interrupted FAME, thereby providing a rapid confirmation of the degree of FAME unsaturation up to hexaenes. Thus, MS-I spectra are highly characteristic of FAME structure; however, they do not show fragment ions that permit further structural characterization. CI-MS/MS must be executed on the (M + 54)+ ion to deduce the locations of the double bonds. In the mass spectrum of acetonitrile, the (M - H)+ ion at m/z 40 and (MH)+ ion at m/z 42 appear, along with an additional ion at m/z 54, at about 15% the intensity of the (MH)+ ion under optimal tuning conditions. The structure of this ion has been reported to be CH2d+NdCdCH2 (1-methyleneimino-1-ethenylium),48-54 and it is formed when acetonitrile reacts with itself. We hypothesize that it is this ion that adds to neutral FAME to produce the (M + 54)+ product, as recently reported for unsaturated hydrocarbons.54 (M + 54)+ Ions. The structure of the (M + 54)+ ion is not readily predictable from the structures of the reagent ion and analyte neutral; however, the preceding data supply several clues. A peak corresponding to a loss of 54 amu is never observed in CI-MS/MS spectra of the (M + 54)+ unless it is can be assigned to a diagnostic ion. The (M + 54)+ ion is only observed for analytes with double bonds, and fragmentation is observed either adjacent or allylic to double bonds. As unsaturation increases, the intensity of the (M + 54)+ ion in both MS-I and MS/MS spectra
Figure 8. CI-MS/MS spectra of (A) cis-18:3ω6, (B) cis-18:3ω3, and (C) trans-18:3ω3. The diagnostic ions for the cis-18:3ω6 isomer, m/z 190 and 234, and the cis-18:3ω3 isomer, m/z 148 and 276, are characteristic of double-bond location. These ions are produced by cleavage on either side of the internal double bond as indicated in the structures. The base peak, m/z 314, is the (M + 54 - 32)+ ion. The cis-18:3ω3 isomer (B) and the trans-18:3ω3 isomer (C) produce the equivalent diagnostic-ion masses. However, the m/z 134 ion is more abundant in the trans-18:3ω3 spectrum than in the cis-18:3ω3 spectrum. All diagnostic ions are 1 amu lower than expected on the basis of homolytic cleavage.
increases, consistent with the increased probability that a reagent ion will react with a double bond. We conclude that the m/z 54 ion adds covalently to the unsaturated FAME, and further, that the (M + 54)+ peak represents a mixture of isomeric ions resulting from addition of 1-methyleneimino-1-ethenylium across all double bonds in the analyte. Thus, MS/MS spectra resulting from collisional dissociation are a superposition of fragments from the competing low-energy dissociation channels of the (M + 54)+ isomers. Several features of the (M + 54)+ fragmentation emerge from a careful consideration of the CI-MS/MS spectra. As the structures in the figures show, the two major monoene diagnostic ions result from cleavage at the allylic carbons on the side remote from the double bond. In contrast, FAME with two or more double bonds yield diagnostic ions by cleavage adjacent to a double bond in the original molecule. For polyunsaturates, a single fragmentation of one isomer dominates to produce the R ion and likewise for the ω ion, despite the many isomeric (M + 54)+ ions present. The figures also indicate that the molecular weight (MW) of the predominant diagnostic fragment of monoene and diene FAME is 1 amu higher than the MW expected from simple homolytic cleavage. On the other hand, the MW for the diagnostic
Figure 9. CI-MS/MS spectrum of (A) cis-20:3ω3 and (B) cis-20: 3ω6. The locations of the double bonds are identified by the diagnostic ions, m/z 148 and 304 and m/z 190 and 262, respectively. These ions are produced from cleavage on either side of the internal double bond. The (M + 54)+ ion, m/z 374, and the (M + 54 - 32)+ ion, m/z 342, are also present.
Figure 10. CI-MS/MS spectrum of cis-22:6ω3. The diagnostic ions, m/z 268 and 326, which result from designated cleavages, locate the positions of the double bonds. In addition to the diagnostic ions, (M + 54)+, m/z 396, and (M + 54 - 32)+, m/z 364, are also abundant.
ions of FAME with polyenes is 1 amu lower than the MW expected for homolytic bond cleavage. The cleavage that gives rise to the diagnostic ions occurs between two double bonds in these cases, so that the nascent ion and neutral both contain a double bond. In these molecules, cleavage of an aliphatic neutral also is observed, such that the ion retains all the double bonds; in these cases the ion MW is observed to be 1 amu higher than the expected value, similar to the monoenes and dienes as might be expected. An example of a triene in which both sorts of ions are observed is in Figure 8b where the diagnostic ions, m/z 148 and Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
1987
Figure 11. Total-ion chromatogram of an extract of Y79 retinoblastoma cells. Six different ion-preparation files were used so that CI-MS/MS spectra of the (M + 54)+ ion could be obtained for the various FAME present in the extract in a single chromatogram. Changes in ion-preparation files are noted along the time axis by ([). The peaks labeled A-G were identified by CI-MS/MS as detailed in the text. No useful FAME diagnostic ions were detected for the peak at 30 min, which apparently results from chemical noise.
Figure 13. CI-MS/MS spectra of later eluting FAME identified in the Y79 extract. (A) was identified as 22:5ω6 by the diagnostic ions m/z 270 and 286. (B) had the same (M + 54)+ ion, m/z 398, and (M + 54 - 32)+ ion, m/z 366, as (A), but had the different diagnostic ions of m/z 228 and 328 and thus was identified as 22:5ω3. (C) was identified as 24:5ω6 from its (M + 54)+ ion of m/z 426 and its diagnostic ions of m/z 270 and 314. In a similar manner, (D) was found to be 24:6ω3 with a (M + 54)+ ion of m/z 424 and diagnostic ions m/z 268 and 354. Table 2. Overlaps between the Major Diagnostic Ions for Monenes to Hexaenes number of double bonds
overlap between diagnostic fragment ions
1 2 3 4 5 6
Figure 12. CI-MS/MS spectra of four unknown FAME present in the biological extract. (A) was identified as 20:3ω9 by the diagnostic ions m/z 220 and 232. From the diagnostic ions, m/z 190 and 262, (B) was identified as 20:3ω6. (C) was identified as 20:4ω6 from the (M + 54)+ ion, m/z 372, and also the diagnostic ions, m/z 230 and 260.
276, are both 1 amu low, while m/z 190, resulting from bond breakage adjacent to a double bond between carbons 8 and 9, similar to diene bond breakage, is 1 amu high. Thus we can conclude that the presence of a third double bond, yielding a 1988 Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
(M + 54)+ with 2 double bonds, opens an alternative reaction channel that is favored in most cases. As shown in Figures 8-10, 12, and 13, for polyene FAME the diagnostic ions result from cleavages directly before the second double bond in the chain from either end of the molecule. This results in overlaps between the two major diagnostic ions common to all FAME of the same degree of unsaturation, as listed in Table 2. Even though the monoene and diene FAME appear to fragment via different mechanisms than the polyunsaturated FAME, the
Table 3. Graphical Summary of the Characteristics of the (M + 54)+ Ion Fragmentationa
a The sites of cleavage as well as the direction of proton transfer between the neutral and the ion are shown for each of the R and ω ions in the monoene, diene, conjugated diene, and polyene cases.
diagnostic ions for these compounds also give rise to characteristic overlaps, as displayed in Table 2. Table 3 summarizes in graphical form the characteristics of the (M + 54)+ ion fragmentation derived from the data presented here. Though there is considerable information as to the fragmentation properties and hydrogen transfer, there is not sufficient information to unambiguously assign a general structure for the (M + 54)+ ion on the basis of these data. Plausible mechanisms include simple addition to the double bond or cycloadditions; however, several structures are possible, many of which lead to fragmentation capable of explaining the observations. Further experiments are required to distinguish among the possibilities. CONCLUSION These data indicate that acetonitrile as a CI reagent coupled with MS/MS provides a rapid means to determine double-bond position in unsaturated FAME. The (M + 54)+ ion observed in CI-MS FAME spectra is a superposition of isomers corresponding to reaction across each double bond, and their intensity relative to the MH+ ion is sensitive to the degree of unsaturation. CI-MS/ MS performed on the isolated (M + 54)+ ion results in two diagnostic ions: ω ions include the hydrocarbon end of the
molecule and R ions include the methyl ester end. Location of double bonds is demonstrated for FAME with up to six double bonds. The method is conveniently applied to FAME mixtures using an inexpensive benchtop ion trap mass spectrometer. Further studies to deduce structures and the fragmentation mechanisms are currently in progress which may aid in extending the method to locating double bonds in molecules other than FAME. ACKNOWLEDGMENT We thank Barry Carpenter and Fred McLafferty for helpful comments. This work was supported by NIH Grant GM49209. Note Added in Proof. Oldham and Svatosˇ (Rapid Commun. Mass Spectrom. 1999, 13, 331-336) reported a study of the addition of the m/z 54 ion to a series of monounsaturated FAME to yield (M + 54)+ ions. Fragmentation of this parent yields the diagnostic ions reported in the present paper. Received for review December 15, 1998. Accepted February 24, 1999. AC981387F
Analytical Chemistry, Vol. 71, No. 10, May 15, 1999
1989
