Acetonitrile Covalent Adduct Chemical Ionization Mass Spectrometry

reacts across double bonds to yield molecular ions 54 mass units above ... methylene units from a three, four, or five C homoallylic system dissociate...
0 downloads 0 Views 160KB Size
Download PDF
Anal. Chem. 2006, 78, 1312-1317

Acetonitrile Covalent Adduct Chemical Ionization Mass Spectrometry for Double Bond Localization in Non-Methylene-Interrupted Polyene Fatty Acid Methyl Esters Peter Lawrence and J. Thomas Brenna*

Division of Nutritional Sciences, Savage Hall, Cornell University, Ithaca, New York 14853

Covalent adduct chemical ionization (CACI) using a product of acetonitrile self-reaction, (1-methyleneimino)1-ethenylium (MIE; CH2dCdN+dCH2), has been investigated as a method for localizing double bonds in a series of 16 non-methylene-interrupted fatty acid methyl esters (NMI-FAME) of polyenes with three and more double bonds. As with polyunsaturated homoallylic (methyleneinterrupted) FAME and conjugated dienes, MIE (m/z 54) reacts across double bonds to yield molecular ions 54 mass units above the parent analyte. [M + 54]+ ions of several 20- and 22-carbon FAME that include one double bond in the C2-C3 position separated by two to five methylene units from a three, four, or five C homoallylic system dissociated according to rules for the homoallylic system, with an additional fragment corresponding to cleavage between the lone double bond and the carboxyl group and defining the position of the lone double bond. Triene FAME with both methylene and ethylene interruption yielded characteristic fragments distinguishable from homoallylic trienes. Fragmentation of fully conjugated trienes in the MS-1 spectra yields ratios of [M + 54]+/[M + 54 - 32]+ (loss of methanol) near unity, which distinguishes them from homoallylic FAME having a ratio of 8 or more; collisionally activated dissociation of [M + 54]+ yields a series of ions, including some rearrangement products, indicative of double bond position. Unlike conjugated dienes, fully conjugated triene diagnostic ion signal ratios did not follow any pattern based on double bond geometry. Partially conjugated trienes behave similarly to monoenes and conjugated dienes, yielding [M + 54]+/[M + 54 - 32]+ of 2-3 and, permitting them to be assigned as partially conjugated FAME using the MS-1 spectrum. They yield unique MS/MS spectra with weaker but assignable fragment ions, along with a diagnostic fragment that locates the lone double bond and permits 6,10,12-octatrienoate to be distinguished from 6,8,12octatrienoate. The presence of a triple bond did not affect fragment formation in a methylene-interrupted yne-ene but did change fragments in a conjugated yne-ene. These data extend the principle of double bond localization by * To whom correspondence should be addressed. E-mail: [email protected]. Phone: (607) 255-9182. Fax: (607) 255-1033.

1312 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

acetonitrile CACI-MS/MS to double bond structure in complex FAME found in nature. Double bond localization in fatty acid methyl esters (FAME) by purely mass spectrometric methods has been a major challenge. It has long been known that double bonds in FAME migrate during electron impact ionization,1 presumably driven by charges localized on double bonds. Effective methods thus rely upon conversion of the FAME to an ester specialized for localization of charge, so that electron impact spectra contain charge remote fragments representative of bond breakage across all carboncarbon bonds.2,3 Although effective for many fatty acids, difficulties arise due to double bond rearrangement during preparative reactions. Also, changes in chromatographic properties of derivatives compared to the original FAME make peak matching between chromatograms difficult or impossible. A preferable approach is analysis of FAME by applying a technique that locates double bonds directly. In a series of papers,4-7 we have described an alternative approach based on chemical derivatization of neutral FAME in the gas phase, which we have recently termed “covalent adduct chemical ionization” (CACI).8 Acetonitrile under chemical ionization conditions self-reacts to form the (1-methyleneimino)-1ethenylium (MIE; CH2dCdN+dCH2) ion at m/z 54. MIE in turn reacts across double bonds to form four- or six-membered heterocyclic species, depending on whether double bonds are initially homoallylic or conjugated, yielding adduct ions appearing at 54 mass units greater than the neutral, [M + 54]+. The [M + 54]+ ions may include isomers corresponding to reaction of MIE across all double bonds in two different orientations with respect (1) McCloskey, J. A. In Topics in Lipid Chemistry; Gunstone, F. D., Ed.; Logos Press: London, 1970; Vol. 1, pp 369-440. (2) Zhang, J. Y.; Yu, Q. T.; Liu, B. N.; Huang, Z. H. Biomed. Environ. Mass Spectrom. 1988, 15, 33-44. (3) Yu, Q. T.; Liu, B. N.; Zhang, J. Y.; Huang, Z. H. Lipids 1989, 24, 79-83. (4) Van Pelt, C. K.; Brenna, J. T. Anal. Chem. 1999, 71, 1981-1989. (5) Van Pelt, C. K.; Carpenter, B. K.; Brenna, J. T. J. Am. Soc. Mass Spectrom. 1999, 10, 1253-1262. (6) Michaud, A. L.; Yurawecz, M. P.; Delmonte, P.; Corl, B. A.; Bauman, D. E.; Brenna, J. T. Anal. Chem. 2003, 75, 4925-4930. (7) Michaud, A. L.; Diau, G. Y.; Abril, R.; Brenna, J. T. Anal. Biochem. 2002, 307, 348-360. (8) Brenna, J. T. In New Techniques and Applications in Lipid Analysis and Lipidomics; Mossoba, M. M., Kramer, J. K., Brenna, J. T., McDonald, R. E., Eds.; AOCS Press: Champaign, IL. In press. 10.1021/ac0516584 CCC: $33.50

© 2006 American Chemical Society Published on Web 01/12/2006

to the neutral FAME, yielding a possible total of 2x [M + 54]+ isomers for x double bonds. Isolation and collisional activation of the [M + 54]+ ions yields fragments characteristic of double bond position for all homoallylic FAME and for conjugated diene FAME. For the latter, intensities of ions in the MS/MS spectra are characteristic of double bond geometry as well, and recently we have observed that for cis/cis versus trans/trans double bond geometries information is present in the MS-1 spectral intensity of the [M + 54]+/MH+ (Lawrence and Brenna, 2004, unpublished). Homoallylic and conjugated FAME constitute the vast majority of polyunsaturated fatty acids found in mammalian tissue. In the terrestrial plant kingdom and in marine samples, fatty acids are found with complex double bond positions, commonly termed nonmethylene-interrupted (NMI) polyunsaturated fatty acids (PUFA). NMI-PUFA fall into several classes based on structure and source. Homoconjugated PUFA with a double bond distant by two or more methylene units are found primarily in plants; for example, the genus Asteraceae that includes the Chrysanthemum produces a wide range of such fatty acids.9 NMI-PUFA with three or more double bonds all in conjugation (“fully conjugated”) are found in various seed oils, such as pomegranate and tung.10 Isomers of homoallylic PUFA are created thermally and have been reported recently in cow’s milk.11 Our purpose in this study is to determine whether CACI-MS/MS spectra of a series of more common NMIFAME follow a discernible pattern for ion formation and to establish whether the method yields MS/MS spectra useful for unambiguously assigning double bond position in this class of molecules. EXPERIMENTAL SECTION Chemicals. All-cis FAME standards were obtained from Matreya, Inc. (State College, PA) as an equal weight mixture. Pure FAME samples were obtained from Larodan Lipids except for the partially conjugated 18:3, which was obtained as a mixture from Natural ASA (Hovdebygda, Norway), were purified by HPLC by P. Delmonte (FDA, Rockville, MD), and were supplied by D. Bauman (Cornell University, Ithaca, NY). Solvents were obtained from Aldrich Chemical Co. (Milwaukee, WI). Instrumentation. All analyses were performed on a Varian Star 3400CX gas chromatograph equipped with a 1078 split/ splitless injector, operated in splitless mode, coupled to a Varian Saturn 2000 3D quadrupole ion trap (Varian Inc., Walnut Creek, CA). A BPX70 capillary column (60 m × 0.32 mm × 0.25 µm; SGE Inc., Austin TX) was used for all analysis. The column temperature and injector parameters for both CIMS and CIMS/ MS analysis were as follows: Injector temperature was maintained at 250 °C in splitless mode with a purge at 0.85 min after injection; initial column temperature was 80 °C ramped up to 200 °C at 50 °C/min, held for 5 min, and then ramped to 220 °C at 4 °C/min for 12 min; total run time was 24.4 min. Ion trap parameters during analysis have been described in detail previously.4 Optimal [M + 54]+ formation is obtained by (9) Tsevegsuren, N.; Fujimoto, K.; Christie, W. W.; Endo, Y. Lipids 2003, 38, 573-578. (10) Hopkins, C. Y. In Topics in Lipid Chemistry; Gunstone, F. D., Ed.; John Wiley & Sons: New York, 1972; Vol. 3, pp 37-87. (11) Destaillats, F.; Trottier, J. P.; Galvez, J. M.; Angers, P. J. Dairy Sci. 2005, 88, 3231-3239.

Table 1. MS-1 Ratios of [M + 54]+/[M + 54 - 32]+ for FAME with Homoallylic Double Bond Systems Reported Herea FAME

mean ( SD

5t,9c,12c 18:3 5c,9c,12c 18:3 2t,8c,11c,14c 20:4 5t,8c,11c,14c 20:4 2t,10c,13c,16c 22:5 4t,7c,10c,13c,16c,19c 22:6 2t,7c,10c,13c,16c,19c 22:6

12.9 ( 0.7 14.9 ( 1.6 21.7 ( 1.5 31.2 ( 0.7 14.3 ( 0.3 71.3 ( 4.6 38.4 ( 1.8

a These FAME have no conjugated double bonds and produce ratios greater than 10, consistent with previously reported homoallylic FAME.

adjusting the CI gas inlet valve to obtain an m/z 42 (MH) to 54 (MIE) ratio of ∼6 with the acetonitrile reservoir at ambient temperature. RESULTS AND DISCUSSION Positional and Geometric Homoallylic FAME Isomers. MS-1 spectra for all FAME reported here yielded the expected [M + 54]+ peak, and additional expected peaks corresponding to [M + 54 - 32]+, and [MH]+. We have previously shown that intensity ratios for MS-1 peaks are related to double bond position and geometry.6 Table 1 presents [M + 54]+/[M + 54 - 32]+ intensity ratios for two geometric (cis f trans) isomers of two homoallylic polyenes, 5t,8c,11c,14c-20:4 and 4t,7c,10c,13c,16c,19c22:6, and six other FAME with a homoallylic double bond system and an isolated double bond. The intensity ratio ranges from 12.9 to 71.3, with a mean of 29.4 ( 19.3 (SD). As shown in more detail below, homoallylic FAME exhibit higher values for this ratio than conjugated FAME. Figure 1 presents the MS-2 spectrum for collisional dissociation of 5t,8c,11c,14c-20:4 and 4t,7c,10c,13c,16c,19c-22:6. As with the much more common all-cis analogues,4 the spectra are dominated by two principal diagnostic ions. As introduced previously,4 we label the fragment containing the ester group as the R diagnostic ion and the fragment containing the methyl end of the molecule as the ω ion. For 20:4, the ion appearing at m/z 260 is the R diagnostic ion and at m/z 230 is the ω diagnostic ion. The analogous ions are found at m/z 326 and 268 for 22:6. All four diagnostic ions appear at the same masses as their all-cis analogues, and there is no discernible trend in intensities that could guide assignment of double bond geometry, as has been seen in conjugated FAME.6 Figure 2A shows the MS/MS spectrum of 2t,10c,13c,16c-22:4. The ω diagnostic ion is formed by bond cleavage between C12 and C13, while the R diagnostic ion is formed by cleavage between C15 and C16. This fragmentation mirrors diagnostic ion formation reported previously for homoallylic C22 trienes7 as might be expected for the homoallylic double bonds at C10-11/C13-14/ C16-17. In addition, a strong peak was observed at m/z 340, which is not present in triene FAMEs, and can be assigned as an ω fragment formed by cleavage between the C1-C2 bond, as shown. Similarly, Figure 2B shows diagnostic ions for the fragmentation of 2t,7c,10c,13c,16c-22:5 at m/z 230 and 286, indicating identical fragmentation to that of the homoallylic system of 22:4n-6 (adrenic acid) reported previously.7 The m/z 338 ion Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

1313

Figure 1. CACI-MS-1 spectra of two rare geometric (cis f trans) isomers of common homoallylic polyene FAME. Spectra show diagnostic ions at m/z values identical to those in the all-cis analogues. (A) 5t,8c,11c,14c-20:4. (B) 4t,7c,10c,13c,16c,19c-22:6. In all figures, the principal diagnostic ions only are labeled with R or ω.

corresponds to the C1-C2 fragment, similar to that observed in Figure 2A. Homologous fragments are found for 2t,8c,11c,14c20:4 and 2t,7c,10c,13c,16c,19c-22:6, presented in Figures 2C and D, respectively. 5t,9c,12c-18:3 and 5c,9c,12c-18:3 are geometric isomeric trienes with an ethylene and a methylene interruption between consecutive double bonds. Panels A and B in Figure 3 show that the R diagnostic ion (m/z 234) results from fragmentation allylic to the methylene-interrupted double bonds, in contrast to that found for diene diagnostic ions, which were previously found to fragment vinylic to the double bonds.4,7 The ω ion (m/z 204) is formed from fragmentation internal to the double bond methylene-interrupted double bonds. We also find fragments at 14 Da higher mass than both the main R and ω ions, at m/z 248 and 218, respectively. The m/z 218 corresponds to cleavage between carbons β and γ on the carboxyl side of the homoallylic double bonds, while the m/z 248 is internal to the homoallylic double bonds. Another ion that can be rationalized in this spectrum is at m/z 272, and this fragment also arises from cleavage between carbons β and γ from the lone double bond. We have not previously noted fragments of this type as useful for diagnostic purposes in other FAME CACIMS/MS spectra. No ion intensity differences appear to distinguish the geometric isomers. Fully and Partially Conjugated 18:3. Previous work6 has shown the ratio of [M + 54]+/[M + 54 - 32]+ obtained from MS-1 spectra can be used as a diagnostic tool to distinguish between homoallylic 18:2 FAMEs and conjugated linoleic acids (CLA). The [M + 54]+/[M + 54 - 32]+ ratios for fully and partially conjugated 18:3 compared to homoallylic 18:3n-3 and 18:3n-6 are presented in Table 2. Mean intensity ratios are 0.81 ( 0.08, 2.46 ( 0.76, and 8.31 ( 0.03, respectively, for the three classes of double bond positions and permit unambiguous classification of octadecatrienes into one of these three groups based on whether the ratio values are 8. 1314 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

Figure 2. CACI-MS/MS spectra for [M + 54]+ ions from homoallylic FAME with a lone double bond in the C2-C3 position. (A) 2t,10c,13c, 16c-22:4 produces diagnostic ions expected for the triene homoallylic system, and an additional cleavage between C1 and C2 to yield an ω ion at m/z 340 to locate the lone double bond. (B) 2t,7c,10c,13c,16c-22:5 yields homologous fragment ions to those in (A), also locating the lone double bond. (C, D) Spectra for 2t,8c,11c,14c20:4 and 2t,7c,10c,13c,16c,19c-22:6, respectively, showing homologous fragments.

Figure 4 shows MS/MS spectra of fully conjugated 18:3 isomers derived from collisional activation of [M + 54]+: four geometric isomers of 9,11,13-18:3 (trans-trans-trans (ttt), ctt, ttc, ctc) and two isomers of 8,10,12-18:3 (ttt, ttc). R and ω diagnostic ions at masses predicted by cleavage vinylic to the double bond system are in most cases of slightly higher intensity than surrounding ions and are observed at m/z 190 and 290 for the 9,11,13 isomers and at m/z 204 and 276 for the 8,10,12 isomers. Unlike homoallylic or conjugated FAME,4,6 the expected diagnostic ions are accompanied by several ions corresponding to multiples of (14 Da. These peaks imply that there is some allylic cleavage that accompanies the slightly more dominant vinylic cleavage. The ions appearing at multiples of 14 above the mass of the principal diagnostic ions can be explained by cleavage allylic to the double bond system. However, ions at multiples of 14 mass units below the principal diagnostic ions cannot be explained from previously established mechanisms for the [M + 54]+ ions.5,12 In the 9,11,13-18:3 spectra, the R ion at m/z 290 appears with ions at m/z 276 and, the cases of ctt and ctc, m/z 262. Similarly, the ω (12) Michaud, A. L.; Lawrence, P.; Adlof, R.; Brenna, J. T. Rapid Commun. Mass Spectrom. 2005, 19, 363-368.

Figure 3. CACI-MS/MS spectra for geometric isomers of 18:3 with ethylene and methylene interruption. (A) 5t,9c,12c-18:3 produces diagnostic ions allylic to the homoallylic diene. Another set of ions of weaker intensity is observed due to cleavage β-γ and shown with dashed lines. No reproducible fragments are found vinylic or allylic to the lone double bond; however, a weak ion is found at m/z 272. (B) 5c,9c,12c-18:3 yields a spectrum similar to that in (A). Table 2. MS-1 [M + 54]+/ [M + 54 - 32]+ Ratios for Fully and Partially Conjugated, and Homoallylic Octadecatrienoic Acid (18:3) Isomers 18:3

[M + 54]+/[M + 54 - 32]+ (mean ( SD)

Fully Conjugated 8t,10t,12t 8t,10t,12c 9c,11t,13c 9c,11t,13t 9t,11t,13t 9t,11t,13c mean FC ) 0.81 ( 0.08

0.93 ( 0.03 0.88 ( 0.02 0.77 ( 0.05 0.75 ( 0.03 0.76 ( 0.03 0.76 ( 0.02

Partially Conjugated 6c,8t,12c 2.99 ( 0.09 6c,10t,12c 1.92 ( 0.08 mean PC ) 2.46 ( 0.76 Homoallylic 18:3n-3 (9c,12c,15c) 18:3n-6 (6c,9c,12c) mean homoallylic ) 8.31 ( 0.03

8.29 ( 0.47 8.33 ( 0.86

ion at m/z 190 is accompanied by ions at m/z 176 in spectra of three isomers (ttt, ctt, ctc) and m/z 162 for two isomers (ttt, ctc). In the 8,10,12-18:3 MS-2 spectra, both geometric isomers yield fragments at 14 and 28 Da below the principal R and ω ions at m/z 276 and 204, respectively. Clearly then, a different structure exists here for the [M + 54]+ and thus also a different fragmentation mechanism. Our previous mechanistic studies of CLA (conjugated 18:2’s) provide evidence that the [M + 54]+ ion contains a double bond that has migrated from the conjugated position to the six-membered ring, leaving the hydrocarbon chains on either side of the ring saturated.12 Collisional activation in those

cases induces back-migration of the double bond from the leaving group. We cannot establish with the present data the structure(s) of the [M + 54]+ in fully conjugated triene FAME, but a structure similar to our previous structure for the [M + 54]+ of conjugated systems could conceivably lose a fully saturated fragment. Further studies are required to establish the structure and mechanism involved in this ion chemistry. Although the unimolecular decomposition chemistry of the fully conjugated [M + 54]+ ions does not lead to two strong diagnostic ions, as with all other [M + 54]+ ions previously reported, the ion series that is generated is sufficient to distinguish between positional isomers, at least as a fingerprint until mechanistic work can be undertaken. The fully conjugated nature of the double bond system is established by MS-1 intensity ratios, as noted above. In MS-2, the 9,11,13-18:3 isomers yield R ions at m/z 290 and 304 and, in some cases, at m/z 276, and 262. The ω ions appear at m/z 190 and 204 and, in some cases, at m/z 176 and 162. The 8,10,12-18:3 isomers produce R ions at m/z 276, 290, 262, and 248, and ω ions at m/z 204, 218, 190, and 176. Although these ion series overlap, the m/z 218 and 248 ions are unique to 8,10,12-18:3, while m/z 304 and in two spectra, m/z 162, is unique to 9,11,13-18:3. Figure 5 shows MS-2 spectra for diagnostic ions from two partially conjugated 18:3’s. Diagnostic fragment yield is poor compared to nonspecific background ions as for other FAME that we have previously reported. Using the information from the MS-1 spectrum showing these to be 18:3 FAMEs of partially conjugated character, three specific diagnostic ions can be assigned to determine structure. Two diagnostic ions appear at masses that are similar to those previously reported for CLA6 and correspond to fragmentation about the conjugated diene system. For 6,10,12-18:3, the R diagnostic ion, now containing the lone double bond, is found at 2 Da lower mass than for the corresponding diene. Bond cleavage around the lone double bond shows the formation of a ω ion, and there is no evidence of an R fragment from bond cleavage between the lone double bond and the conjugated diene system. The 6,8,12-18:3 produces fragments consistent with this pattern. Previous MS/MS data reported for CLA6 have shown the ratio of R/ω diagnostic ions to be a determinate of double bond geometry. The two partially conjugated 18:3 contain cis/trans (6c,8t,12c) and trans/cis (6c,10t,12c) conjugated systems, but inspection of the diagnostic ions in Figure 2 shows that they are in a ratio near 1 for both FAME, and thus, geometry cannot be established via these intensity ratios. Similarly, we examined R/ω diagnostic ion ratios for fully conjugated 18:3, as shown in Figure 1. Ratios were all close to 1 and independent of double bond geometry. FAME with a Triple Bond. We present data from triple bondcontaining FAME, one which is homoallylic to a double bond and the other with the two unsaturated bonds arranged in conjugation. Figure 6A shows the MS/MS spectrum of the homoallylic methyl crepenynate, demonstrating that diagnostic ions are formed by vinylic cleavage on either side of the double bond system. This MS/MS behavior is analogous to that for homoallylic dienes4 and is evidence that the triple bond does not perturb formation of analogous [M + 54]+ structures or fragments.5 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

1315

Figure 4. CACI-MS/MS spectra obtained upon collisional dissociation of [M + 54]+ ion at m/z 346 for six fully conjugated 18:3 isomers. (A) MS-2 spectra for [M + 54]+ ions of 9t,11t,13t-18:3, showing principal diagnostic ions at m/z 190 and 290 and additional ions at multiples of 14 Da adjacent to the principal diagnostic ions. (B-D) MS-2 spectra of 9c,11t,13t-18:3, 9t,11t,13c-18:3, 9c,11t,13c-18:3, respectively. (E, F) MS-2 spectra of 8t,10t,12t-18:3 and 8t,10t,12c-18:3. Both yield principal diagnostic ions at m/z 204 and 276.

Figure 5. CACI-MS/MS spectra for partially conjugated 18:3. (A) 6c,10t,12c-18:3 produces relatively weak diagnostic ions corresponding to cleavage about the conjugated diene and a slightly stronger ω ion at m/z 276 for vinylic cleavage at the lone double bond. Relative ion count (RIC) ) 921, (B) 6c,8t,12c-18:3 produces similar diagnostic ions about the conjugated diene, and lone double bond, yielding an R ion; RIC ) 951.

The MS/MS spectrum of the conjugated methyl santalbate is presented in Figure 6B. Diagnostic ions are found for allylic 1316 Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

cleavage, in contrast to that found previously for conjugated dienes, which produce vinylic cleavage products.6 The likely structure for [M + 54]+ ions formed from conjugated dienes is a six-membered ring, in part because formation of a four-membered ring found for homoallylic double bonds is sterically hindered. The triple bond may impose an additional steric constraint that alters either the [M + 54]+ structure or its available dissociation pathways. Further studies are required to investigate this point. FAME with homoallylic double bonds that also included an isolated double bond in the C2-3 position gave CACI-MS/MS diagnostic ions expected from the homoallylic systems, plus another ω ion of considerable abundance corresponding to cleavage between the C1 and C2. Octatrienes of methylene and ethylene interruption produced a unique allylic and β-γ cleavage pattern. In the cases of some conjugated polyene FAME, our data indicate that fragmentation in acetonitrile CACI-MS/MS is less pronounced than for homoallylic and CLA. However, the several pieces of information available in MS and MS/MS spectra in all cases allow unambiguous assignments. Through formation of MH, [M + 54]+, and [M + 54 - 32]+ MS-1 spectra establishes the identity of a compound as a FAME of a particular molecular weight, which gives its number of C atoms and degree of unsaturation (number of double and triple bonds). The ratio of [M + 54]+/[M + 54 - 32]+ establishes whether the PUFA is

Figure 6. CACI-MS and MS/MS spectra (insets) for triple bond-containing FAME isomers. (A) Methyl crepenynate, 9c-octaen-12-ynoate, a homoallylic species, produces two strong diagnostic ions corresponding to cleavage allylic to the unsaturated system. In MS-1, [M + 54]+/[M + 54 - 32]+ ) [346]+/[314]+ ) 8.3. (B) Methyl sanatalbate, 11t-octaen-9-ynoate, a conjugated species, yields strong fragment ions allylic to the unsaturated system. In MS-1, [M + 54]+/[M + 54 - 32]+ ) 3.9.

homoallylic, partially, or fully conjugated. MS/MS then yields several ions that are diagnostic of double bond structure. [M + 54]+ from fully conjugated FAME produce a series of fragment ions that include unique ions at the extremes. The two examples of partially conjugated FAME [M + 54]+ to which we had access had relatively poor yields of diagnostic ions but did produce unique spectra with peaks that could be rationalized on the basis of structure. Importantly, these spectra do not contain ions that would lead to assignment as another FAME and thus contain negative evidence to point to the weaker ions as diagnostic. Two triple bond/double bond FAME yield strong diagnostic ions that can easily distinguish them from isomeric trienes and locate the sites of unsaturation. CONCLUSION The work presented here, together with previous reports, shows that acetonitrile CACI-MS/MS is an effective method to characterize double bond position in monoene FAME,4,13 all homoallylic FAME,4,7,14 and conjugated diene FAME6,12 and here (13) Oldham, N. J.; Svatos, A. Rapid Commun. Mass Spectrom. 1999, 13, 331336. (14) Van Pelt, C. K.; Huang, M. C.; Tschanz, C. L.; Brenna, J. T. J. Lipid Res. 1999, 40, 1501-1505.

has been extended to non-methylene-interrupted polyenes of three or more double bonds. The various polyene FAME presented in this report are obtainable in pure form commercially and generally are derived from plant oils. Diagnostic ion signals are of variable strength in these FAME but, within the constraints imposed by the molecular masses in MS-1 and by the known fragmentation of common FAME, can be assigned with confidence. FAME of more complex double bond structure are present at trace levels in natural samples and also can be generated by chemical treatment of more common polyunsaturated FAME. It is left to future work to establish whether this approach can determine double bond structure in FAME of truly arbitrary structure. ACKNOWLEDGMENT This work was supported by NIH Grant GM71534. We thank Pierluigi Delmonte and Dale Bauman for the gift of the partially conjugated 18:3’s and an anonymous reviewer for many insightful comments.

Received for review September 16, 2005. Accepted December 15, 2005. AC0516584

Analytical Chemistry, Vol. 78, No. 4, February 15, 2006

1317