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Complete Structural Elucidation of Tryacyl Glicerols

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determinacao de glicerideos
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  Complete Structural Elucidation of Triacylglycerols by Tandem Sector MassSpectrometry Changfu Cheng and Michael L. Gross* Department of Chemistry, Washington University, St. Louis, Missouri 63130  Ernst Pittenauer* Federal Office & Research Center for Agriculture, Institute for Agricultural Ecology, Vienna, Austria  Wedevelopedamethodtoelucidatethecompletestruc-tureoftriacylglycerolsbymeansofhigh-energycollisionalactivation tandem mass spectrometry (MS/MS). BothESI-andFAB-produced[M + NH 4 ] + and[M + met.] + ions(wheremet. ) Li,Na,andCs)oftriacylglycerolsundergocharge-remote and charge-driven fragmentations. WeemphasizethestudyoffragmentionsfromESI-produced[M +  NH 4 ] + and[M +  Na] + ionsandFAB-produced[M +  Na] + ions. ESI-produced [M  +  NH 4 ] + ions fragmenttoproducefourtypesofions,[M + NH 4 - R n  COONH 4 ] + ,[R n  CO  +  128] + , [R n  CO  +  74] + , and R n  CO + ions, fromwhichthecarbonnumberandthedegreeofunsaturationofeachacylgroupareobtained. Inaddition,threeseriesof ions are produced by charge-remote fragmentations(CRFs), andanalysisoftheir patternsgivesthepositionand the number of double bonds on the acyl groups.Informationaboutthepositionofeachacylgroupontheglycerolbackbone,however,isnotprovidedbycollision-allyactivated dissociation of[M  +  NH 4 ] + ions. On theother hand, ESI- and FAB-produced [M  +  Na] + ionsfragmentto form eighttypes of ions (named A -  J ions)that,likethoseproducedbyCRF,arehighlystructurallyinformative. Theabsenceofcertainseriesmembersalsocarries useful structural information. Interpretation of these patterns enables one to obtain the number of carbons,degreesofunsaturation,andlocationofdoublebonds, as well as the positions of acyl groups on theglycerol backbone. Triacylglycerols(TAGs) play an important rolein nutrition andother biological processes. They aretheprimary meansof energystorage in animals and humans, and their hormonally controlledhydrolysis and oxidation release energy to meet the energy-generation needsof organisms. 1 A major sourceof TAGsisseedoils, which are found as renewable agricultural raw materials;these materials have applications in technology and nutrition. 2 Because triacylglycerols are important in biology, manyanalytical methodsinvolving massspectrometry (MS) havebeenused to determine their structures; the MS methods includeelectron ionization (EI), 3 - 5 chemical ionization (CI), 3,6 - 18 desorp-tion chemical ionization (DCI), 19 - 21 fast atom bombardment(FAB), 22 - 24 field desorption (FD), 25,26 thermospray (TSP), 27,28 electrospray ionization (ESI), 29 - 31 and atmospheric pressure * Corresponding author.Tel.314-935-4814;Fax 314-935-7484;E-mail mgross@wuchem.wustl.edu.(1) Mathews,C.K.;van Holde,K.E.in  Biochemistry  ;TheBenjamin/ CummingsPublishing Co., Inc.: Redwood City, CA, 1990; pp 571 - 578.(2) Barrett, L. W.; Sperling, L. H.; Murphy, C. J.  J. Am. Oil Chem. Soc.  1993 , 70  , 523 - 534.(3) Murphy,R.C.In  Handbook of Lipid Research 7: MassSpectrometryof Lipids;  Snyder F., Ed.; Plenum Press: New York, 1993; p 213.(4) de Mirbuker, M.; Blomberg, L. G.; Olsson, N. U.; Bergqvist, M.; Herslof,B. G.; Jacobs, F. A.  Lipids  1992 ,  27  , 436 - 441.(5) Kallio, H.; Laasko, P.; Huopalathi, R. Linko, R. R.; Oksman, P.  Anal. Chem. 1989 ,  61  , 698 - 700.(6) Johansson,A.;Laakso,P.;Kallio, H. Z. Lebensm. Unters. Forsch. 1997 , 204  ,308 - 315.(7) Evershed, R. P.  J. Am. Soc. Mass Spectrom.  1996 ,  7  , 350 - 361.(8) Manninen, P.; Laakso, P.; Kallio, H.  Lipids  1995 ,  30  , 665 - 671.(9) Manninen,P.;Laakso,P.;Kallio,H. J. Am. Oil Chem. Soc. 1995 , 72  ,1001 - 1008.(10) Kallio, H.; Rua, P.  J. Am. Oil Chem. Soc.  1994 ,  71  , 985 - 992.(11) Cheung,M.;Young,A.B.;Harrison,A.G. J. Am. Soc. MassSpectrom. 1994 ,5  , 553 - 557.(12) Laakso, P.; Kallio, H.  J. Am. Oil Chem. 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Soc.  1995 ,  72  , 867 - 871.(23) Hori, M.; Sahashi, Y.; Koike, S.; Yamaoka, R.; Sago, M.  Anal. Sci.  1994 , 10  , 719 - 724.(24) Evans, C.; Traldi, P.; Bambigiotti-Alberti, M.; Gianelli, V.; Coran, S. A.;Vincieri, F. F.  Biol. Mass Spectrom.  1991 ,  20  , 351 - 356.(25) Lehmann, W. D.; Kessler, M.  Biomed. MassSpectrom.  1983 ,  10  , 220 - 226.(26) Evans,N.;Games,D.E.;Harwood,J.L.;Jackson,A.H.  Biochem. Soc. Trans. 1974 ,  2  , 1091 - 1092.(27) Sundin, P.; Larsson, P.; Wese´n, C.; Odham, G.  Biol. MassSpectrom.  1992 , 21  , 633 - 641.(28) Kim, H.-Y.; Salem, N., Jr.  Anal. Chem.  1987 ,  59  , 722 - 726. Anal. Chem.  1998,  70,  4417 - 4426 S0003-2700(98)00519-8 CCC: $15.00 © 1998 American Chemical Society  Analytical Chemistry, Vol. 70, No. 20, October 15, 1998   4417 Published on Web 09/12/1998  chemical ionization (APCI). 32 - 38 Although all these techniquesare able to provide the molecular weight of the intact moleculeand, when combined with tandem mass spectrometry, someinformation about the acyl groups, it has not been possible toperform acompletestructural determination with massspectrom-etry alone.A complete determination includes elucidating the structureof each acyl group (carbon number, degree of unsaturation,position of branchesor doublebonds,etc.) and locating thethreeacyl groups on the glycerol backbone ( sn  -1,  sn  -2, or  sn  -3). Fortheformer,charge-remotefragmentations(CRFs) can play arolebecause this technique has proven very useful for the structuralelucidation of a variety of long-chain molecules, including fattyacids and their esters, fatty alcohols, surfactants, phospholipids,and peptides. 39 - 59 CRFs are gas-phase dissociations that areanalogous to thermal processes, 39 and they involve losses of C n  H 2 n  + 1 and C n  H 2 n  + 2 from precursor ions. Therearetwoproposedmechanismsfor reactionsalonganalkyl chain, 40 - 49 but that subjectisnot within thescopeof thisarticle. In arecent exampleutilizingCRFs, the structural elucidation of sulfoquinovosyl, monogalac-tosyl, and digalactosyl diacylglycerols was carried out by Kim etal.; 60 the structure of the sugar head and the two acyl groups(including double-bond positions) can bedetermined completely.Sofar,CRFsfor determiningthestructuresof triacylglycerolshavenot been systematically applied,although interesting resultshavebeen demonstrated by previous researchers. 24,31 In this article, we report the fragmentations, including CRFs,of triacylglycerolscharged with ammonium and alkali metal ionsand formed under ESI and FAB conditions. Interpretation of theMS/ MSdataallowsoneto characterizecompletely thestructureof these molecules; that is, their molecular weights, the carbonnumber and the degree of unsaturation of each acyl group, theposition of double bonds along acyl groups, and the positions of acyl groups on the glycerol backbone. No effort, however, wasmade to determine chirality of TAGs by MS/ MS. EXPERIMENTAL SECTION All synthetic triacylglycerols, including tristearoyl-, trilinole-noyl-,1,2-dipalmitoyl-3-oleoyl-,1,3-dipalmitoyl-2-oleoyl-,1,2-dioleoyl-3-stearoyl-, 1,3-dioleoyl-2-stearoyl-, and 1-palmitoyl-2-oleoyl-3-stearoylglycerols, were purchased from Sigma (St. Louis, MO)and used without further purification. All matrix and solventcompounds, including 3-nitrobenzyl alcohol, NaI, NaOAc, NH 4 I,NH 4 OAc,CsI,LiI,CH 3 OH,and CHCl 3 ,wereobtained from eitherSigma or Aldrich (Milwaukee, WI).ESI-MS/ MSexperimentswerecarried out with aZAB-T four-sector tandem massspectrometer manufactured by VGAnalytical(Manchester, UK), equipped with a VG electrosprary source(Micromass, Manchester, UK). 61 A triacylglycerol and the ap-propriate salt (NH 4 OAc, NaOAc, etc.) were dissolved in CHCl 3 and CH 3 OH (7:3 volume ratio). The final concentration of thetriacylglycerol was10  µ M,and thoseof NH 4 OAcand NaOAcwere10and 1  µ M,respectively (low concentration of NaOAc wasusedbecause high concentration of sodium salt makes the signalunstable). 29 The solution was infused in the continuous mode ata flow rate of 10  µ L/ min through a Harvard model 22 syringepump(Harvard Apparatus,South Natick,MA). Thespray needlewasmaintained at 8000V,and thecounter electrode(pepper pot)potential was5000V. Thesamplingcone,skimmer lens,skimmer,hexapole, and ring electrode were 4200, 4160, 4150, 4150, and4120 V, respectively. Nitrogen was used separately as both bathand nebulizer gaswith flow ratesof 400and 12L/ h, respectively.The bath gas temperature was maintained at 80  ° C. Argon wasused asthecollision gas,and sufficient gaswasadded toattenuatethe main beam by 60%. The collision cell was floated to 2 kV(resulting in  E  LAB ) 2keV). Theproduct ionswereanalyzed withMS2; approximately 40 15-s scans at a mass resolving power of  ∼ 1000 (full width at half-maximum) were taken.ESI-linked scan ( B/ E   )  constant) experiments were carriedout with aFinnigan MAT 95Sdouble-focusing instrument (Finni-gan MAT, Bremen, Germany), fitted with a second-generationatmospheric pressure ionization (API) source. The samplesolution (described above) was continuously infused with aHarvard Apparatus 22 syringe pump at aflow rate of 50  µ L/ min.Thesourceconditionsfor producingabundant precursor ionswereasfollow: sheath gaspressure,6bar N 2 ;temperatureof theheatedcapillary, 250  ° C; capillary exit voltage, - 45 V; tube lens voltage, + 55V; skimmer voltage, - 2.5V; and rf octapole, - 5V. Scansof product ionsformed by collisionally activated dissociation (CAD) (29) Duffin, K. L.; Henion, J. D.; Shieh, J. J.  Anal. Chem.  1991 ,  63  , 1781 - 1788.(30) Myher, J. J.; Kuksis, A.; Geher, K.; Park, P. W.  Lipids  1996 ,  31  , 207 - 215.(31) Pittenauer,E.;Aichinger,T.;deHueber,K.;Bailer,J.  Proceedingsof the44th ASMS Conferenceon MassSpectrometryand Allied Topics  ,Portland,OR,May12 - 16, 1996; p 928.(32) Neff, W. E.; Byrdwell, W. C.  J. Am. Oil Chem. Soc.  1995 ,  72  , 1185 - 1191.(33) Byrdwell, W. C.; Emken, E. A.  Lipids  1995 ,  30  , 173 - 175.(34) Neff, W. E.; Byrdwell, W. C.  J. Liq. Chromatogr.  1995 ,  18  , 4165 - 4181.(35) Byrdwell, W. C.; Neff, W. E.  J. Liq. Chromatogr.  1996 ,  18  , 2203 - 2225.(36) McIntyre, D.; Fischer, S.  Proceedingsof the 44th ASMS Conference on Mass Spectrometry and Allied Topics  , Portland, OR, May 12 - 16, 1996; p 289.(37) Byrdwell, W. C.; Emken, E. A.; Odlof, R. O.  Lipids  1996 ,  31  , 919 - 935.(38) Mottram, H. R.; Evershed, R. P.  Tetrahedron Lett.  1996 ,  37  , 8593 - 8596.(39) Adams, J.; Gross, M. L.  J. Am. Chem. Soc.  1989 ,  111  , 435 - 440.(40) Jensen, N. J.; Tomer, K. B.; Gross, M. L.  J. Am. Chem. Soc.  1985 ,  107  ,1863 - 1868.(41) Cordero, N. N.; Wesdemiotis, C.  Anal. Chem.  1994 ,  66  , 861 - 866.(42) Contado, M. J.; Adams, N. J.; Jensen, N. J.; Gross, M. L.  J. Am. Soc. Mass Spectrom.  1991 ,  2  , 180 - 183.(43) Cheng, C.; Pittenauer, E.; Gross, M. L. submitted to  J. Am. Soc. Mass Spectrom.  1998 ,  9  , 840 - 844.(44) Claeys, M.; Van den Heuvel, H.; Claereboudt, J.; Corthout, J.; Pieters, L.;Vlietinck, A. J.  Biol. Mass Spectrom.  1993 ,  22  , 647 - 653.(45) Claeys, M.; Van den Heuvel, H.  Biol. Mass Spectrom.  1994 ,  23  , 20 - 26.(46) Claeys, M.; Nizigiyimana, L.; Van den Heuvel, H.; Derrick, P. J.  Rapid Commun. Mass Spectrom.  1996 ,  10  , 770 - 774.(47) Griffiths, W. J.; Yang, Y.; Lindgren, J. A.; Sjo¨vall, J.  Rapid Commun. Mass Spectrom.  1996 ,  10  , 21 - 28.(48) Wysocki, V. H.; Bier, M. E.; Cooks, R. G.  Org. Mass Spectrom.  1988 ,  23  ,627 - 633.(49) Wysocki, V. H.; Ross, M. M.  Int. J. Mass Spectrom. Ion Processes   1991 , 104  , 179 - 211.(50) Gross, M. L.  Int. J. Mass Spectrom Ion Processes  1992 ,  118/ 119  , 137 - 165.(51) Jensen, N. J.; Gross, M. L.  Mass Spectrom. Rev.  1987 ,  6  , 497 - 536.(52) Adams, J.  Mass Spectrom. Rev.  1990 ,  9  , 141 - 186.(53) Tomer, K. B.; Crow, F. W.; Gross, M. L.  J. Am. Chem. Soc.  1983 ,  105  ,5487 - 5488.(54) Adams, J.; Gross, M. L.  J. Am. Chem. Soc.  1986 ,  108  , 6915 - 6921.(55) Adams, J.; Gross, M. L.  Anal. Chem.  1987 ,  59  , 1576 - 1582.(56) Jensen, N. J.; Tomer, K. B.; Gross, M. L.  Anal. Chem.  1985 ,  57  , 2018.(57) Deterding, L. J.; Gross, M. L.  Anal. Chim. Acta   1987 ,  200  , 431.(58) Crockett, J. S.; Gross, M. L.; Christie, W. W.; Holman, R. T.  J. Am. Soc.Mass Spectrom  .  1990 ,  1  , 183 - 190.(59) Jensen, N. J.; Hass, G. W.; Gross, M. L.  Org. Mass Spectrom  .  1992 ,  27  ,423 - 427.(60) Kim, Y. H.; Yoo, J. S.; Kim, M. S.  J. Mass Spectrom.  1997 ,  32  , 968 - 977.(61) Gross, M. L. Tandem Mass Spectrometry: Multisector Magnetic Instru-ments. In  Methods in Enzymology  ; McCloskey, J. A., Ed.; Academic Press:San Diego, CA, 1990; Vol. 193, pp 131 - 153. 4418  Analytical Chemistry, Vol. 70, No. 20, October 15, 1998   in the first field-free region were taken with a grounded gascollision cell ( E  LAB  )  4.75 keV) with sufficient argon added toattenuate the main beam by 70 - 80%. All data were acquired intheprofilemode(scanspeed10s/ 100u) andrepresent anaverageof 20 scans.All FAB-MS/ MSexperiments were carried out with the four-sector tandem instrument. Inatypical experiment,asmall amount( ∼ 1  µ g) of a triacylglycerol was mixed with approximately 1  µ Lof matrix (3-nitrobenzyl alcohol saturated with NaI) on the FABtip and bombarded with ahigh-energy ( ∼ 25 keV) Cs + ion beam.The ions were accelerated by 8 kV, selected by the first stage(MS1) at a mass resolving power of   ∼ 1500, and collided withhelium in the collision cell between MS1 and MS2 with a 70%main beam attenuation. The collision cell was floated to 4 kV( E  LAB ) 4keV). Theproduct ionsweredetected with MS2usingapproximately 30 15-s scans at amass resolving power of  ∼ 1000(full width at half-maximum). To investigate the fragmentationof the first generation of product ions, ESI source CAD-MS/ MSexperiments were carried out with the four-sector mass spec-trometer; MS/ MS/ MSexperiments under FAB conditions werecarried out with the Kratos triple-analyzer mass spectrometer, 62 following procedures described elsewhere. 63 RESULTS AND DISCUSSION Fragmentation of triacylglycerols under EI and CI conditionshas been studied extensively by early researchers. 3 EI of TAGsproduces abundant [M  -  R n  COO] + , [R n  CO  +  128] + , [R n  CO  + 74] + , and RCO + ions, whereas CI of TAGs gives abundant [M  + H  - R n  COOH] + , [R n  CO + 74] + , and RCO + ions (where  n   ) 1, 2,and3). Generationof theseionsallowsonetocalculatethecarbonnumber and the degree of unsaturation of acyl groups. Bothmethods, however, cannot locate the position of double bonds inacyl groups or distinguish the  sn  -1,  sn  -2, or  sn  -3 substituents onthe glycerol backbone. 3 In the presence of an ammonium or alkali metal salt, ESI andFAB of TAGs produce abundant [M  +  cat.] + ions, where cat. ) Li,Na,Cs,or NH 4 . Duffin et al. 29 investigated thefragmentationsof ESI-produced [M  +  NH 4 ] + ions of TAGs under low-energycollisional activation (CA),and their resultsenableonetocalculatereadily the length and double-bond number of each acyl group.Detailed structural information on each acyl groupand itslinkageto the glycerol backbone, however, was not obtainable with low-energy collisional activation. Upon high-energy activation, ESI-and FAB-produced [M  + cat.] + ionsof TAGsfragment through anumber of reaction channels. In addition to the same ions thatwere produced by low-energy CA, more structurally diagnosticproduct ions are produced by high-energy CA, including ionsformed by charge-remote fragmentations. FragmentationofESI-ProducedTAG [M  +  NH 4 ] + Ions. Collisional activation of ESI-produced [M  + NH 4 ] + ionsfrom TAGgives product ions from both charge-remote and charge-drivenchemistry. Interestingly,themajority of theCRF ionsareformedby losses of C n  H 2 n  + 1 , indicating that the products are, at leastinitially, long-chain distonic ions (Figure 1). 64,65 Our previous (62) Gross, M. L.; Chess, E. K.; Lyon, P. A.; Crow, F. W.; Evans, S.; Tudge, H. Int  .  J. Mass Spectrom. Ion Phys.  1982 ,  42  , 243 - 254.(63) Burinsky,D.J.;Cooks,R.G.;Chess,E.K.;Gross,M.L. Anal. Chem  . 1982 , 54  , 295 - 299. (64) Hammerum, S.  Mass Spectrom. Rev  .  1988 ,  7  , 123 - 193. Figure1.  CAD spectra of ESI-produced TAG [M + NH 4 ] + ions with identical acyl groups: (A) tristearoylglycerol (18:0/18:0/18:0, MW ) 890.8)and (B) trilinolenoylglycerol (18:3/18:3/18:3, MW  )  872.8). The position of a double bond is indicated by the “ ) ” sign, whereas the peakcorresponding to an allylic cleavage is labeled with “a n  ”. Analytical Chemistry, Vol. 70, No. 20, October 15, 1998   4419  studies showed that the internal energy of the precursor ionsdetermines the nature of the products formed by CRF. 43 Never-theless, the pattern of the peaks corresponding to the CRF ionsprovidessufficient information tolocatethedoublebond. A vinylcleavage at the side proximal to the charge produces an ion byloss of C n  H 2 n  - 3 , indicating the position of the double bond. Inaddition to ions formed by CRFs, CA of the [M  + NH 4 ] + ions of TAG produces [M  +  NH 4 -  R n  COONH 4 ] + , [R n  CO +  128] + , [R n  -CO  +  74] + , and R n  CO + ions, presumably by charge-drivenprocesses. If the compositions we assigned are correct, theseionsshould bestructurally diagnostic. Totest,weobtained CADspectraof [M  + NH 4 ] + ions of TAGs with identical and differentacyl groups. [ M   +  NH  4   ]   + Ions of a TAG Containing Identical Acyl Groups. Collisional activation of the[M  + NH 4 ] + ionsof aTAGcontainingidentical acyl groups produces three series of CRF ions withidentical  m   /   z  valuesand four ionswith  m   /   z  valuescorrespondingto compositions of [M  +  NH 4  -  RCOONH 4 ] + , [RCO  +  128] + ,[RCO + 74] + ,and RCO + . For example,CAD of theESI-produced[M  + NH 4 ] + ionsof tristearoylglycerol (18:0/ 18:0/ 18:0) givesriseto [M  +  NH 4 -  RCOONH 4 ] + at  m   /   z   607, [RCO  +  128] + at  m   /   z  395, [RCO + 74] + at  m   /   z  341, and RCO + at  m   /   z  267(Figure1A),whereas that of trilinolenoylglycerol (18:3/ 18:3/ 18:3) gives cor-responding ionsat  m   /   z  595,389,335,and 261,respectively (Figure1B). In both cases, a typical pattern of CRF ions is generated,and one can readily see that the former TAG contains saturatedacyl groups and the latter has three double bonds on each acylgroup. The array of peaks corresponding to ions of CRFs of theformer showsnointerruptions,whereasthat of thelatter containsthree interruptions. Further, one can unmistakably locate thedoublebondson thealkyl chain of thelatter (they areat C9,C12,and C15) by identifying and interpreting the three ions formedby allylic cleavages, a 1 , a 2 , and a 3 , and the three gaps associatedwith those ions (Figure 1B). [ M   +  NH  4   ]   + Ions of a TAG Containing Three Different Acyl Groups.  Expectedly, CAD of ESI-produced [M  +  NH 4 ] + ions of TAGs containing three different acyl groups produces threedifferent members of each of the ion types: [M  +  NH 4  -  R n  -COONH 4 ] + , [R n  CO  +  128] + , [R n  CO  +  74] + , and R n  CO + ions(where  n   ) 1, 2, and 3). For example, CAD of ESI-produced [M +  NH 4 ] + ions of 1-palmitoyl-2-oleoyl-3-stearoylglycerol (16:0/ 18:1/ 18:0) produces [M  +  NH 4  -  R n  COONH 4 ] + ions at  m   /   z   607,579, and 577; [R n  CO +  128] + ions at  m   /   z   395, 393, and 367; [R n  -CO + 74] + ionsat  m   /   z  341, 339, and 313; and R n  CO + ionsat  m   /   z  267, 265, and 239 (Figure 2B). From the  m   /   z   values of theseions, one can readily identify that the intact molecule containstwo saturated acyl groups (C16:0 and C18:0) and one monoun-saturated (C18:1) acyl group. In addition, three series of CRFions are formed, although they are often not distinguishablebecausethey overlapwith each other when theneutralslost fromthe precursor ion are identical. But when a CRF ion is formedby vinyl cleavageat thesideproximal to thecharge(labeled with“v” in Figure 2), a doublet is produced, and the onset of thedoublet allows one to conclude that the position of the doublebond isat C9. Therefore, thestructuresof thethreeacyl groupsare elucidated by combining the information obtained from bothcharge-remote and charge-driven ions (to be 16:0, 18:0, and 18:1 ∆ 9 respectively). The linkage information of the three acyl (65) Stirk, K. M.; Kiminkinen, L. K. M.; Kenttamaa, H. I.  Chem. Rev  . 1992 ,  92  ,1649 - 1665. Figure 2.  CAD spectra of ESI-produced [M + NH 4 ] + ions of 1-palmitoyl-2-oleoyl-3-stearoylglycerol (16:0/18:1/18:0, MW ) 860.8) obtained bydifferent instruments: (A) an MS/MS spectrum from a four-sector mass spectrometer and (B) a linked-scan spectrum from a two-sector massspectrometer. The peak corresponding to a vinyl cleavage is labeled with “v”. 4420  Analytical Chemistry, Vol. 70, No. 20, October 15, 1998 
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