Atmospheric pressure covalent adduct chemical ionization tandem mass spectrometry for double bond localization in monoene- and diene-containing triacylglycerols

Article abstract of DOI:10.1021/ac062055a

We report a method to elucidate the structure of triacylglycerols (TAGs) containing monoene or diene fatty acyl groups by atmospheric pressure covalent adduct chemical ionization (APCACI) tandem mass spectrometry using acetonitrile as an adduct formation reagent. TAGs were synthesized with the structures ABB and BAB, where A is palmitate (C16:0) and B is an isomeric C18 monoene unsaturated at position 9, 11, or 13 or an isomeric diene unsaturated at positions 9 and 11, 10 and 12, or 9 and 12. In addition to the species at m/z 54 observed in previous CI studies of fatty acid methyl esters, we also found that ions at m/z 42, 81, and 95 undergo covalent reaction with TAGs containing double bonds to yield ions at m/z 40, 54, 81, and 95 units greater than that of the parent TAG: [M + 40]⁺, [M + 54]⁺, [M + 81] ⁺, and [M + 95]⁺ ions. When collisionally dissociated, these ions fragment to produce two or three diagnostic ions that locate the double bonds in the TAG. In addition, ions [RCH=C=O + 40]⁺ and [RCH=C=O + 54]⁺ formed from collisional dissociation are of strong abundance in MS/ MS spectra, and collisional activation of these ions produces two intense confirmatory diagnostic ions in the MS³ spectra. Fragment ions reflecting neutral loss of an sn-1-acyl group from [M + 40]⁺ and [M + 54]⁺ are more abundant than those reflecting neutral loss of an sn-2-acyl group, analogous to previous reports for protonated TAGs. The position of each acyl group on the glycerol backbone is thus determined by the relative abundances of these ions. Under the conditions in our instrument, the [M + 40]⁺ adduct is at the highest signal and also yields all information about the double bond position and TAG stereochemistry. With the exception of geometries about the double bonds, racemic TAG isomers containing two monoenes or dienes and a saturate can be fully characterized by APCACI-MS/MS/MS.

Full text of DOI:10.1021/ac062055a

Anal. Chem. 2007, 79, 2525-2536
Atmospheric Pressure Covalent Adduct Chemical
Ionization Tandem Mass Spectrometry for Double
Bond Localization in Monoene- and
Diene-Containing Triacylglycerols
†
Yichuan Xu and J. Thomas Brenna*
Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853
We report a method to elucidate the structure of triacyl-
glycerols (TAGs) containing monoene or diene fatty acyl
groups by atmospheric pressure covalent adduct chemical
ionization (APCACI) tandem mass spectrometry using
acetonitrile as an adduct formation reagent. TAGs were
synthesized with the structures ABB and BAB, where A
is palmitate (C16:0) and B is an isomeric C18 monoene
unsaturated at position 9, 11, or 13 or an isomeric diene
unsaturated at positions 9 and 11, 10 and 12, or 9 and
spectrometry is a routine for lipid analysis; however, the complete
structural elucidation of lipids remains an important analytical
challenge.
Triacylglycerols (TAGs) are the most abundant dietary lipids.
They constitute the major form of energy and essential fatty acid
storage for plants and animals. TAGs are required at some minimal
levels within cells to support specialized functions. Alterations in
TAG synthesis and catabolism play important roles in obesity,
atherosclerosis, insulin release from pancreatic â cells, and
1-7
1
2. In addition to the species at m/z 54 observed in
alcohol-induced hepatic dysfunction, among many other functions.
previous CI studies of fatty acid methyl esters, we also
found that ions at m/z 42, 81, and 95 undergo covalent
reaction with TAGs containing double bonds to yield ions
at m/z 40, 54, 81, and 95 units greater than that of the
Considering that TAGs are made up of combinations of 3 fatty
acyl groups from any of 20-50 fatty acids present in cells, the
number of distinct molecular TAG species from biological extracts
may number in the thousands. TAGs have been extensively
studied by mass spectrometry, employing various ionization
+
+
+
parent TAG: [M + 40] , [M + 54] , [M + 81] , and
+
8,9
[
M + 95] ions. When collisionally dissociated, these ions
techniques, including electron ionization (EI), chemical ioniza-
tion (CI),1
0-12
atmospheric pressure chemical ionization (APCI),
13-19
fragment to produce two or three diagnostic ions that
locate the double bonds in the TAG. In addition, ions
20-22
23,24
desorption chemical ionization (DCI),
thermospray (TSP),
field desorption (FD),
+
+
25,26
fast atom bombardment (FAB),27-29 elec-
[
RCHdCdO + 40] and [RCHdCdO + 54] formed from
collisional dissociation are of strong abundance in MS/
MS spectra, and collisional activation of these ions
produces two intense confirmatory diagnostic ions in the
(
2) Goldberg, I. J. J. Lipid Res. 1996, 37, 693-707.
(3) Koyama, K.; Chen, G. X.; Wang, M. Y.; Lee, Y.; Shimabukuro, M.; Newgard,
C. B.; Unger, R. H. Diabetes 1997, 46, 1276-1280.
3
MS spectra. Fragment ions reflecting neutral loss of an
(4) Saudek, C. D.; Eder, H. A. Am. J. Med. 1979, 66, 843-852.
(5) Shimabukuro, M.; Ohneda, M.; Lee, Y.; Unger, R. H. J. Clin. Invest. 1997,
+
+
sn-1-acyl group from [M + 40] and [M + 54] are more
abundant than those reflecting neutral loss of an sn-2-
acyl group, analogous to previous reports for protonated
TAGs. The position of each acyl group on the glycerol
backbone is thus determined by the relative abundances
1
00, 290-295.
(6) Stanley, W. C.; Lopaschuk, G. D.; McCormack, J. G. Cardiovasc. Res. 1997,
34, 25-33.
(
(
(
7) Unger, R. H. Diabetes 1995, 44, 863-870.
8) Hites, R. A. Anal. Chem. 1970, 42, 1736-&.
9) Kallio, H.; Laakso, P.; Huopalahti, R.; Linko, R. R.; Oksman, P. Anal. Chem.
1989, 61, 698-700.
of these ions. Under the conditions in our instrument, the
+
(10) Demirbuker, M.; Blomberg, L. G.; Olsson, N. U.; Bergqvist, M.; Herslof,
[
M + 40] adduct is at the highest signal and also yields
B. G.; Jacobs, F. A. Lipids 1992, 27, 436-441.
all information about the double bond position and TAG
stereochemistry. With the exception of geometries about
the double bonds, racemic TAG isomers containing two
monoenes or dienes and a saturate can be fully character-
ized by APCACI-MS/MS/MS.
(
(
(
11) Marai, L.; Myher, J. J.; Kuksis, A. Can. J. Biochem. Cell Biol. 1983, 61,
840-849.
12) Cheung, M.; Young, A. B.; Harrison, A. G. J. Am. Soc. Mass Spectrom. 1994,
5
, 553-557.
13) Huang, A. S.; Robinson, L. R.; Gursky, L. G.; Profita, R.; Sabidong, C. G.
J. Agric. Food Chem. 1994, 42, 468-473.
14) Neff, W. E.; Byrdwell, W. C. J. Liq. Chromatogr. 1995, 18, 4165-4181.
15) Mottram, H. R.; Evershed, R. P. Tetrahedron Lett. 1996, 37, 8593-8596.
(
(
Lipidomics is the emerging field of systems-level analysis and
characterization of lipids and their interacting moieties. Mass
(16) Byrdwell, W. C.; Emken, E. A.; Neff, W. E.; Adlof, R. O. Lipids 1996, 31,
19-935.
9
(
17) Neff, W. E.; Byrdwell, W. C. J. Chromatogr., A 1998, 818, 169-186.
18) Neff, W. E.; List, G. R.; Byrdwell, W. C. J. Liq. Chromatogr. Relat. Technol.
1999, 22, 1649-1662.
(
*
To whom correspondence should be addressed. E-mail: jtb4@cornell.edu.
Phone: (607) 255-9182. Fax: (607) 255-1033.
(19) Byrdwell, W. C.; Neff, W. E. Rapid Commun. Mass Spectrom. 2002, 16,
300-319.
†
Present address: Barr Laboratories, Northvale, NJ.
(
1) Dhalla, N. S.; Elimban, V.; Rupp, H. Mol. Cell. Biochem. 1992, 116, 3-9.
(20) Laakso, P.; Kallio, H. Lipids 1996, 31, 33-42.
1
0.1021/ac062055a CCC: $37.00 © 2007 American Chemical Society
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2525
Published on Web 02/06/2007

3
0-35
+
trospray ionization (ESI),
ionization (MALDI).
and matrix-assisted laser desorption/
These techniques typically yield infor-
intermediate [C
4
H
5
N
2
] at m/z 81, which loses HCN to form the
3
6-38
+
(1-methyleneimino)-1-ethenylium (MIE, CH
m/z 54.46 MIE in turn reacts with carbon-carbon double bonds
in FAMEs to form a covalent adduct ion appearing at 54 mass
units greater than that of the parent FAME, [M + 54] . Isolation
2 2
dCdN dCH ) ion at
mation about the molecular weight, fatty acid carbon number,
degree of unsaturation, and regiospecificity, but do not reveal the
arrangement of double bonds, which is the single most significant
structural property determining biological activity. A rapid, in-
strumental method for analysis of the double bond position in
TAGs is required.
+
+
and collisional activation of the [M + 54] ions yields fragments
corresponding to bond cleavage at specific locations in FAMEs.
The two ions that are normally of greatest abundance contain
either the R- or ω-carbon atoms and have masses unique to the
positions of double bonds within the FAME. Early studies of this
reaction included double bond identification in monoene hydro-
Conventional approaches for determination of the double bond
position by mass spectrometry rely on an initial hydrolysis of fatty
acyl groups, followed by chemical derivatization to enhance the
yield of diagnostically useful fragment ions obtained upon colli-
sional dissociation. Single acyl groups are esterified with charge-
localizing groups such as 4,4-dimethyloxalone (DMOX) or picoli-
nyl esters with subsequent analysis of charge-remote fragmentation.
The disadvantages of these methods have been discussed39 and
include the need for derivatization chemistry prior to analysis and
generally low sensitivity.
carbons and polyene alcohols,4 as well as monoene FAMEs.
7,48
49
TAGs are neutral molecules that can be analyzed by high-
temperature gas chromatography (GC) but for lipidomics applica-
tions are more commonly analyzed from a liquid by ESI. ESI is a
soft ionization technique which excels at analysis of charged or
high proton affinity compounds. To adapt CACI to the liquid phase,
we investigated the use of APCI as a source for reagent ions for
covalent adduct formation with TAGs, similar to our gas-phase
FAME analysis. Conditions were first developed to produce
adequate levels of reagent ions for derivatization of neutral TAGs,
under conditions we refer to as atmospheric pressure covalent
adduct chemical ionization (APCACI). TAGs of the forms ABB
and BAB (sn-1,2,3) were synthesized, where B is a monoene or
diene fatty acyl group of one of several isomers and A is palmitic
acid (hexadecanoic acid, 16:0). The resulting ions were character-
ized for their ability to form diagnostically useful fragments by
single-, double-, and triple-stage mass spectrometry.
3
9-42
In previous work,
we have demonstrated a rapid technique
for double bond localization in fatty acid methyl esters (FAMEs)
based on chemical derivatization of neutral FAMEs in the gas
phase, which we recently termed “covalent adduct chemical
ionization” (CACI).4 The CI mass spectrum of CH
internal ionization ion trap includes ions at m/z 40 and 42, formed
by loss or gain of H, and an ion at m/z 54.
demonstrated that an ion/molecule reaction between [C
m/z 40) and neutral CH
3
3
CN in a 3D
4
4,45
It has been
+
2 2
H N]
(
3
CN results in the formation of an
(
21) Stroobant, V.; Rozenberg, R.; Bouabsa, E. M.; Deffense, E.; Dehoffmann,
E. J. Am. Soc. Mass Spectrom. 1995, 6, 498-506.
22) Anderson, M. A.; Collier, L.; Dilliplane, R.; Ayorinde, F. O. J. Am. Oil Chem.
EXPERIMENTAL SECTION
(
Chemicals. The following fatty acids were purchased from
Soc. 1993, 70, 905-908.
(
(
23) Lehmann, W. D.; Kessler, M. Biomed. Mass Spectrom. 1983, 10, 220-226.
24) Evans, N.; Games, D. E.; Harwood, J. L.; Jackson, A. H. Biochem. Soc. Trans.
Matreya, Inc. (Pleasant Gap, PA): 9c-octadecenoic acid (oleic
acid), 9c,12c-octadecadienoic acid, 9c,11t-octadecadienoic acid
1
974, 2, 1091-1093.
25) Sundin, P.; Larsson, P.; Wesen, C.; Odham, G. Biol. Mass Spectrom. 1992,
1, 633-641.
(rumenic acid), and 10t,12c-octadecadienoic acid. 11c-Octadecenoic
(
2
acid (vaccenic acid), 13c-octadecenoic acid, 1,3-dicyclohexylcar-
bodiimide, and 4-(dimethylamino)pyridine were purchased from
Sigma Chemical Co. (St. Louis, MO). 1-Palmitin and 2-palmitin
were obtained from Research Plus, Inc. (Manasquan, NJ). Thin
layer chromatography (TLC) plates were obtained from Analtech
Inc. (Newark, DE). The following TAGs were synthesized by
esterification of fatty acids with sn-1- or sn-2-monopalmitoylygly-
(
(
(
26) Kim, H. Y.; Salem, N. Anal. Chem. 1987, 59, 722-726.
27) Lamberto, M.; Saitta, M. J. Am. Oil Chem. Soc. 1995, 72, 867-871.
28) Hori, M.; Sahashi, Y.; Koike, S.; Yamaoka, R.; Sato, M. Anal. Sci. 1994,
1
0, 719-724.
(
29) Evans, C.; Traldi, P.; Bambagiottialberti, M.; Giannellini, V.; Coran, S. A.;
Vincieri, F. F. Biol. Mass Spectrom. 1991, 20, 351-356.
(
(
(
(
(
30) Duffin, K. L.; Henion, J. D.; Shieh, J. J. Anal. Chem. 1991, 63, 1781-1788.
31) Cheng, C. F.; Gross, M. L. Anal. Chem. 1998, 70, 4417-4426.
32) Hsu, F. F.; Turk, J. J. Am. Soc. Mass Spectrom. 1999, 10, 587-599.
33) Hvattum, E. Rapid Commun. Mass Spectrom. 2001, 15, 187-190.
34) Fard, A. M.; Turner, A. G.; Willett, G. D. Aust. J. Chem. 2003, 56, 499-
50
cerol as reported elsewhere: rac-glyceryl-1,3-9c-octadecenoate-
2-palmitate [(9-18:1/16:0/9-18:1)-TAG], rac-glyceryl-1-palmitate-2,3-
5
08.
9c-octadecenoate [(16:0/9-18:1/9-18:1)-TAG], rac-glyceryl-1,3-11c-
octadecenoate-2-palmitate [(11-18:1/16:0/11-18:1)-TAG], rac-glyceryl-
(
(
(
(
(
35) Segall, S. D.; Artz, W. E.; Raslan, D. S.; Ferraz, V. P.; Takahashi, J. A. Food
Res. Int. 2005, 38, 167-174.
36) Asbury, G. R.; Al-Saad, K.; Siems, W. F.; Hannan, R. M.; Hill, H. H. J. Am.
Soc. Mass Spectrom. 1999, 10, 983-991.
1
-palmitate-2,3-11c-octadecenoate [(11-18:1/16:0/11-18:1)-TAG],
rac-glyceryl-1,3-13c-octadecenoate-2-palmitate [(13-18:1/16:0/13-
37) Robins, C.; Limbach, P. A. Rapid Commun. Mass Spectrom. 2003, 17, 2839-
1
1
8:1)-TAG], rac-glyceryl-1-palmitate-2,3-13c-octadecenoate [(16:0/
3-18:1/13-18:1)-TAG], rac-glyceryl-1,3-9c,11t-octadecadienoate-
2
845.
38) Calvano, C. D.; Palmisano, F.; Zambonin, C. G. Rapid Commun. Mass
Spectrom. 2005, 19, 1315-1320.
39) Michaud, A. L.; Diau, G. Y.; Abril, R.; Brenna, J. T. Anal. Biochem. 2002,
2-palmitate [(9,11-18:2/16:0/9,11-18:2)-TAG], rac-glyceryl-1-palmi-
tate-2,3-9c,11t-octadecadienoate [(9,11-18:2/16:0/9,11-18:2)-TAG],
rac-glyceryl-1,3-10t,12c-octadecadienoate-2-palmitate [(10,12-18:2/
16:0/10,12-18:2)-TAG], rac-glyceryl-1-palmitate-2,3-10t,12c-octadeca-
3
07, 348-360.
(
(
40) Van Pelt, C. K.; Brenna, J. T. Anal. Chem. 1999, 71, 1981-1989.
41) Van Pelt, C. K.; Carpenter, B. K.; Brenna, J. T. J. Am. Soc. Mass Spectrom.
1
999, 10, 1253-1262.
(
42) Michaud, A. L.; Yurawecz, M. P.; Delmonte, P.; Corl, B. A.; Bauman, D. E.;
(46) Oldham, N. J. Rapid Commun. Mass Spectrom. 1999, 13, 1694-1698.
(47) Moneti, G.; Pieraccini, G.; Dani, F. R.; Turillazzi, S.; Favretto, D.; Traldi, P.
J. Mass Spectrom. 1997, 32, 1371-1373.
(48) Moneti, G.; Pieraccini, G.; Favretto, D.; Traldi, P. J. Mass Spectrom. 1999,
34, 1354-1360.
Brenna, J. T. Anal. Chem. 2003, 75, 4925-4930.
(
43) Lawrence, P.; Brenna, J. T. Anal. Chem. 2006, 78, 1312-1317.
44) Moneti, G.; Pieraccini, G.; Dani, F. R.; Catinella, S.; Traldi, P. Rapid Commun.
Mass Spectrom. 1996, 10, 167-170.
(
(
45) Moneti, G.; Pieraccini, G.; Favretto, D.; Traldi, P. J. Mass Spectrom. 1998,
(49) Oldham, N. J. S. A. Rapid Commun. Mass Spectrom. 1999, 13, 331-336.
(50) Jie, M.; Lam, C. C.; Yan, B. F. Y. J. Chem. Res., Synop. 1993, 141-141.
3
3, 1148-1149.
2526 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

Figure 1. APCI mass spectra of CH3CN. (A) N2 and (B) He were
used as the nebulizer and auxiliary gases. The spectra include ions
at m/z 40 and 42, formed by loss and gain of H, and at m/z 54, 81,
and 95, generated by ion/molecule reactions of acetonitrile.
Figure 2. APCACI-MS spectra of (A) (16:0/9-18:1/9-18:1)-TAG and
+
+
(B) (16:0/9,12-18:2/9,12-18:2)-TAG: MH , [M - RCO ] ions arising
2
+
+
+
from loss of fatty acid moieties, [M + 54] , [M + 40] , [M + 81] ,
and [M + 95] .
+
dienoate [(10,12-18:2/16:0/10,12-18:2)-TAG], rac-glyceryl-1,3-9c,-
1
2c-octadecadienoate-2-palmitate [(9,12-18:2/16:0/9,12-18:2)-TAG],
rac-glyceryl-1-palmitate-2,3-9c,12c-octadecadienoate [(9,12-18:2/16:
/9,12-18:2)-TAG]. Solvents were obtained from Aldrich Chemical
performed with the third quadrupole operated in linear ion trap
mode. The degree of collisional activation was adjusted through
variation of the collision energy.
0
Co. (Milwaukee, WI).
Instrumentation. An ABI MDS/Sciex QTRAP 2000 triple-
quadrupole linear ion trap mass spectrometer was employed in
the APCI positive ion mode. The data acquisition and processing
software was Analyst 1.4, and MS-1 masses were centroided to
reduce clutter in the spectrum. Helium, rather than the conven-
RESULTS AND DISCUSSION
APCI-MS of Acetonitrile. Figure 1 presents APCI mass
2
spectra of acetonitrile using (A) N and (B) He as the nebulizer
and auxiliary gases, respectively. They consist of five main ionic
+
+
species at m/z 40 [M - H] , 42 [M + H] , 54, 81, and 95. The
tional N
2
, was used as the nebulizer, auxiliary, and collision gas.
former three ions are in agreement with previous investigations
of acetonitrile CI.4
3-45
It was demonstrated that an ion/molecule
The heated pneumatic nebulizer probe conditions were 350 °C,
+
6
5
5 psi nebulizing gas pressure, 25 psi auxiliary gas pressure, and
0 psi curtain gas pressure. The needle current was set at 2.0 µA.
reaction between [C
2
H
2
N] (m/z 40) and neutral acetonitrile
+
results in the formation of a [C
which loses HCN to yield the [C
MIE. m/z 95 is likely to correspond to C
4
H
5
N
2
]
(m/z 81) intermediate,
+
The declustering potential was set at 90 V. Samples were delivered
into the ionization source with a syringe pump by infusion at a
flow rate of 25 µL/min of the TAG dissolved in chloroform/
acetonitrile (1/9) to a final concentration of 100 µg/mL. Data were
acquired for 1 min per spectrum. Under these conditions, a
continuous plasma discharge is observed in the region from the
quartz tube to the curtain plate. Chemically active adducts were
not detected under conventional APCI conditions; the plasma glow
was essential. This may have been due to the large ionization
volume within which the neutral TAG and active ions interacted
within the plasma; further experiments are required to clarify this
point. After approximately 300 h of analysis, we found that the
heater coil, though still functional, had cracked at a stress point
apparently due to metal degradation by the plasma and high
temperature.
3
H
4
N] m/z 54 ion, identified as
+
H
5 7
N
2
originating from
reaction of m/z 54 ions with neutral acetonitrile (m/z 41). The
m/z 54 ions are formed at about 20% and 70% abundances of the
+
[C
2
H
2
N] ions when using N
2
and He, respectively. In this work,
He was used as the nebulizer and auxiliary gas.
APCACI-MS of TAGs. Parts A and B of Figure 2 show the
single-stage MS of a TAG of the form ABB with a monoene or
diene as fatty acyl group B, 16:0/9-18:1/9-18:1 and 16:0/9,12-18:
+
+
2/9,12-18:2. The major ions are MH , [M - RCO
2
+
]
ions from
+
+
loss of fatty acid moieties, [M + 54] , [M + 40] , [M + 81] ,
+
+
and [M + 95] . [RCO] (ketene ions) derived from the fatty acyl
moieties themselves were also present at low relative abundance.
+
The MH ion intensity for the diene-containing TAG is about
double the intensity of the monoene-containing TAG compared
to the acyl and ketene neutral loss peaks, relative to the base peak.
TAG molecular species were directly ionized in the positive
ion mode by APCACI. Tandem mass spectrometry of TAGs was
performed by collisional activation with He gas. MS/MS/MS was
+
+
2 2
The [M + 40] species indicates that the [C H N] ion from
acetonitrile adds to the TAG, and analogous ions have been
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2527

ions. All of the MS/MS spectra show abundant [RCO
2
H + 54]+
at m/z 336, the adduct of monoene fatty acyl moieties and MIE.
+
MS/MS fragmentation of [M + 54] of TAGs also resulted in the
adducts of ketene (generated from fatty acyl groups) and m/z 54
+
+
ions [RCHdCdO + 54] , analogous to the [M + 54 - 32] loss
of methanol in the corresponding FAMEs reported previously.
+
The [RCHdCdO + 54] peak at m/z 318 is the base peak. The
+
+
spectra also contain ions [BB + 54] and [AB + 54] at m/z 657
and 631, reflecting neutral loss of 16:0 and 18:1, respectively, from
+
[
M + 54] .
+
Upon collisional activation, [M + 54] undergoes fragmentation
to yield diagnostic ions. These ions correspond to cleavage either
vinylic or allylic to the site of the erstwhile double bond, as
indicated in the structures in Figure 3. The 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,
are displayed in the structure of the TAG in each spectrum.
Consistent with our nomenclature for CACI of FAMEs, we define
the R diagnostic ion as containing the remaining glycerol lipid
and the ω diagnostic ion as containing the terminal methyl group
of the fatty acyl chain. The strongest R diagnostic ion is due to
allylic cleavage and appears 1 unit above the expected mass due
to H transfer from a neutral group to an ion. In contrast, the strong
ω diagnostic ions are observed vinylic and allylic to the former
site of the double bond and are 1 unit lower than those expected
from homolytic cleavage. Smaller satellite ions at (14 units were
also observed. The ω diagnostic ions for (16:0/9-18:1/9-18:1)-TAG,
(16:0/11-18:1/11-18:1)-TAG, and (16:0/13-18:1/13-18:1)-TAG ap-
pear at m/z 194-206, 166-178, and 138-150, respectively, while
the R diagnostic ions for these TAGs appear at m/z 814, 842, and
8
70, respectively. These spectra show that TAGs containing
isomeric monoenes can be differentiated on the basis of APCACI
collisional dissociation.
Abundant fragment ions in the m/z range from 50 to 110 were
also observed in the spectra in Figure 3. These include a series
of alkyl ions at m/z 57, 71, etc. and an unsaturated series at m/z
+
Figure 3. [M + 54] -based APCACI-MS/MS spectra of (A) (16:0/
9
1
-18:1/9-18:1)-TAG, (B) (16:0/11-18:1/11-18:1)-TAG, and (C) (16:0/
3-18:1/13-18:1)-TAG. Collisional dissociation of m/z 913 yields
diagnostic ions corresponding to cleavage either vinylic or allylic to
the site of the erstwhile double bond.
6
7, 81, 95, etc. Similar ion series were observed under ESI
32
condtions.
49
APCACI-MS3 of Monoene-Containing TAGs. An important
feature of unsaturated TAG fragmentation is the abundant [RCHd
reported in the acetonitrile CI mass spectra of monoene FAMEs.
The [M + 54] ion corresponds to the addition of [C
+
+
3 4
H N] to
+
CdO + 54] , the ketene adduct ion. The mass of these ions
the TAG, consistent with results obtained for FAMEs in our
4
3-47
suggests that they should undergo a similar charge-driven
previous papers.
+
+
rearrangement and fragmentation as [M + 54] of FAMEs to yield
We first focus interest on the [M + 54] species since it is by
far the best studied adduct ion for analysis of the position of double
bonds in the carbon chain. The mass spectra are typified by that
of rac-glyceryl-1,3-9-octadecenoate-2-palmitate, in which specific
ions are observed: MH at m/z 860, two [M - RCO
m/z 604 and 578, corresponding to loss of palmitate to yield [BB]
and loss of octadecenoate to give [AB] , respectively, [M + 54]
at m/z 913, [M + 40] at m/z 899, and [M + 81] at m/z 940.
Starting with [M + 54] , we discuss spectra arising from
collisional dissociation of all the adduct ions.
3
R and ω diagnostic ions. This hypothesis is supported by MS
+
experiments performed on abundant [RCHdCdO + 54] ion at
m/z 318. The resultant APCACI-MS/MS/MS spectra are displayed
+
+
+
in Figure 4. By analogy to the fragmentation of FAME [M + 54] ,
2
] ions at
+
each isomer yields fragments characteristic of the double bond
+
+
position and corresponding to allylic cleavage. The R diagnostic
+
+
+
ions of the m/z 318 ion, [RCHdCdO + 54] , where fatty acyl
+
groups are 9-18:1, 11-18:1, and 13-18:1, appear at m/z 220, 248,
and 276, respectively, at mass 1 unit higher than would be
expected from homolytic bond cleavage. The ω diagnostic ions
of corresponding m/z 318 ions appear at m/z 206, 178, and 150,
1 mass unit lower than expected from homolytic cleavage. Thus,
each of the three positional isomers yields a strong and unique
+
[
M + 54] . APCACI-MS/MS of Monoene-Containing TAGs.
Figure 3 presents the APCACI-MS/MS spectra of a series of three
ABB TAGs isomeric only in the position of the double bond on
the monoene group: (16:0/9-18:1/9-18:1)-TAG, (16:0/11-18:1/11-
8:1)-TAG, and (16:0/13-18:1/13-18:1)-TAG. Mass spectra are
generated by collisional dissociation of the isolated [M + 54]
3
1
APCACI-MS spectrum that permits unambiguous identification
+
of the double bond location.
2528 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

+
Figure 5. [M + 54] -based APCACI-MS/MS spectra of m/z 909
+
Figure 4. [M + 54] -based APCACI-MS/MS/MS spectra of (A) (16:
from (A) (16:0/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,-
2-18:2)-TAG, and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. Cleavages
directly before the first double bond and immediately after the second,
as shown in the structures, give rise to diagnostic ions.
0
/9-18:1/9-18:1)-TAG, (B) (16:0/11-18:1/11-18:1)-TAG, and (C) (16:
/13-18:1/13-18:1)-TAG. The [ketene + 54] ions at m/z 318 derived
from MS/MS of [M + 54] of monoene TAGs were selected and
collisionally dissociated (m/z 913 f m/z 318 f products) to yield a
pair of diagnostic ions. Each of these pairs of diagnostic ions is unique
to a particular isomer.
1
+
0
For TAGs containing homoallylic dienes, collisional dissocia-
+
tion of the [M + 54] ion also proceeds via bond cleavage at a
APCACI-MS/MS of Diene-Containing TAGs. In Figure 5 we
present the APCACI-MS/MS spectra of TAGs containing 18:2 fatty
acyl groups, two with conjugated double bonds, and the acyl group
of linoleic acid, with a homoallylic (methylene-interrupted) set of
position vinylic to the site of the original double bond. For (9,12-
18:2/16:0/9,12-18:2)-TAG, cleavage directly before the first double
bond and immediately after the second gives rise to the diagnostic
ions at m/z 190 and 838, as indicated in Figure 5C. As with the
TAG containing monoenes, the R ions appear 1 unit above the
expected mass while the ω ions appear at masses 1 unit lower
than those expected from homolytic cleavage.
+
double bonds. The spectra show abundant [M + 54] at m/z 909,
+
+
+
[
RCO
2
H + 54] at m/z 334, [ketene + 54] at m/z 316, [BB] at
+
m/z 652 or 653, and [AB] at m/z 628 or 629 as well as two
diagnostic ions.
3
APCACI-MS of Diene-Containing TAGs. Figure 6 shows the
For TAGs containing conjugated double bonds, C-C cleavage
occurs vinylic to the erstwhile double bond, producing R and ω
diagnostic ions. Parts A and B of Figure 5 show MS/MS spectra
of TAGs containing two conjugated 18:2 bonds, the 9,11 and 10,-
APCACI-MS/MS/MS spectra of a series of TAGs containing
+
dienes. The [ketene + 54] ions at m/z 316 derived from MS/
+
MS of [M + 54] of the TAG were selected and collisionally
3
dissociated to yield MS spectra. In Figure 6A,B, ketene derived
1
9
2 isomers, respectively. The R and ω diagnostic ions for (16:0/
,11-18:2/9,11-18:2)-TAG appear at m/z 824 and 190, respectively,
from a TAG containing a 9,11-18:2 conjugated fatty acyl group
yields m/z 190 and 232 diagnostic ions and can readily be
distinguished from ketene derived from a TAG containing a 10,-
12-18:2 conjugated fatty acyl group, where m/z 176 and 246 are
the diagnostic ions. The APCACI-MS/MS/MS spectrum of ketene
derived from (16:0/9,12-18:2/9,12-18:2)-TAG is shown in Figure
6C. The diagnostic ions are m/z 190 and 246. These ions originate
while the analogous ions for (16:0/10,12-18:2/10,11-18:2)-TAG
occur at m/z 838 and 176, respectively. As with the TAG
containing the monoenes, the R ions appear 1 unit above the
expected mass while the ω ions appear at masses 1 unit lower
than those expected from homolytic cleavage.
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2529

+
Figure 7. [M + 40] -based APCACI-MS/MS spectra of m/z 899
for (A) (16:0/9-18:1/9-18:1)-TAG, (B) (16:0/11-18:1/11-18:1)-TAG, and
(C) (16:0/13-18:1/13-18:1)-TAG. Specific cleavages at the allylic
carbons shown in the structures at the top of each spectrum give
rise to a pair of diagnostic ions.
+
Figure 6. [M + 54] -based APCACI-MS/MS/MS spectra of (A) (16:
0
/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,12-18:2)-TAG,
and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. The [ketene + 54] ions
derived from [M + 54] of diene TAGs (m/z 909 f 318 f products)
+
+
yield two fragments characteristic of the double bond position.
m/z 57, 71, etc. and an unsaturated series at m/z 67, 81, and 95,
similar to those observed under ESI condtions.32
The spectra contain two diagnostic ions, with dominant
cleavage allylic to the former site of the double bond, as indicated
in the structures in Figure 7. Again, the R ions appear 1 unit above
the expected mass, thus implying hydrogen transfer from the
neutral group to the ion, while ω ions appear at masses 1 unit
lower than those expected from homolytic cleavage, indicating
the reverse. For (16:0/9-18:1/9-18:1)-TAG, the ion appearing at
m/z 800 is the R diagnostic ion and that at m/z 192 is the ω
diagnostic ion. The analogous ions are found at m/z 828 and 164
for (16:0/11-18:1/11-18:1)-TAG and at m/z at 856 and 136 for (16:
from C-C bond cleavage at a position that is vinylic to the site of
the former double bonds, and as with the TAG containing
monoenes, the R ions appear 1 unit above the expected mass while
ω ions appear at masses 1 unit lower than those expected from
homolytic cleavage. These spectra show fragments analogous to
those presented in Figure 4.
+
[
M + 40] . APCACI-MS/MS of Monoene-Containing TAGs.
+
The APCACI-MS/MS spectra of [M + 40] adducts of (16:0/9-
1
1
8:1/9-18:1)-TAG, (16:0/11-18:1/11-18:1)-TAG, and (16:0/13-18:
/13-18:1)-TAG are shown in Figure 7. The base peaks obtained
0
/13-18:1/13-18:1)-TAG, respectively. Satellite ions at (14 units
for all three TAGs are ions at m/z 304 arising from the adducts
are also observed at lower intensity.
+
of ketene and m/z 40 ions [RCHdCdO + 40] . Unlike the [M +
3
APCACI-MS of Monoene-Containing TAGs. The results pre-
+
5
4] MS/MS spectra of Figure 3, no loss of an acyl group is
+
sented above show that [RCHdCdO + 40] ions at m/z 304, the
+
+
observed from the [M + 40] ion. The other expected products
for all TAGs are m/z 643 and 617 arising by neutral loss of 16:0
and 18:1, respectively, from the [M + 40] complex. Abundant
adduct of ketene from the fatty group and C H N , are generated
2
2
+
in MS/MS. The generation of [RCHdCdO + 40] creates the
+
3
opportunity to analyze the product ions in MS to confirm the
fragment ions in the m/z range from 50 to 110 were also observed
in the spectra in Figure 7. These include a series of alkyl ions at
double bond location in unsaturated fatty acyl substituents,
indicated in the MS/MS data. Diagnostic ions produced from
2530 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

+
Figure 8. [M + 40] -based APCACI-MS/MS/MS spectra of m/z 899
+
Figure 9. [M + 40] -based APCACI-MS/MS spectra for m/z 895
f m/z 304 f products for (A) (16:0/9-18:1/9-18:1)-TAG, (B) (16:0/
1-18:1/11-18:1)-TAG, and (C) (16:0/13-18:1/13-18:1)-TAG. The
ketene + 40] ions at m/z 304 derived from MS/MS of [M + 40] of
monoene TAGs were selected and collisionally dissociated to yield
a pair of diagnostic ions. Each of these pairs of diagnostic ions is
unique to a particular isomer.
of (A) (16:0/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,12-
8:2)-TAG, and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. Cleavages di-
rectly before the first double bond and immediately after the second,
as shown in the structures, give rise to diagnostic ions.
1
1
+
+
[
APCACI-MS/MS of Diene-Containing TAGs. Figure 9 shows
the APCACI-MS/MS spectra of TAGs containing 18:2 fatty acyl
+
collisional dissociation of these ions correspond to C-C bond
cleavage adjacent to either allylic site of the double bond and
produce the R diagnostic ion containing the ketene group and
the ω diagnostic ion containing the terminal methyl group.
groups derived from collisional activation of [M + 40] ions. The
+
common products observed for all TAGs are [M + 40] at m/z
895, [RCHdCdO + 40] at m/z 302, and [BB] at m/z 639 and
[AB] at m/z 615 arising, respectively, by loss of C16:0 and C18:2
+
+
+
+
Diagnostic ions analogous to those observed in Figure 4 for the
from the [M + 40] ions.
+
[
RCHdCdO + 54] ions are observed. The R diagnostic ion
Spectra for TAGs containing 9,11-CLA and 10,12-18:2 isomers
are presented in parts A and B, respectively, of Figure 9. The R
and ω diagnostic ions, at masses predicted by cleavage vinylic to
the double bond, are observed at m/z 810 and 176 for (16:0/9,-
11-18:2/9,11-18:2)-TAG and m/z 824 and 162 for (16:0/10,12-18:
2/10,11-18:2)-TAG. For (9,12-18:2/16:0/9,12-18:2)-TAG, vinylic
cleavage is also observed to give rise to the diagnostic ions at
m/z 176 and 824, as indicated in Figure 9C. As with the TAG
containing monoenes, the R ions appear 1 unit above the expected
mass while the ω ions appear at masses 1 unit lower than those
expected from homolytic cleavage.
appears at mass 1 unit higher than would be expected from
homolytic bond cleavage, while the ω diagnostic ion appears at 1
mass unit lower than expected from homolytic cleavage. Figure
8
0
shows MS/MS/MS spectra of (16:0/9-18:1/9c-18:1)-TAG, (16:
/11-18:1/11-18:1)-TAG, and (16:0/13-18:1/13-18:1)-TAG. The R
+
diagnostic ions resulting from the [RCHdCdO + 40] ion at m/z
304, where fatty acyl groups are 9-18:1, 11-18:1, and 13-18:1, appear
at m/z 206, 234, and 262, respectively. The ω diagnostic ions of
the corresponding ions at m/z 304 appear at m/z 192, 164, and
3
+
1
36. MS of [RCHdCdO + 40] is dominated by diagnostic ion
3
fragments that stand out more clearly than diagnostic ions in MS/
MS (Figure 7) to unequivocally confirm the double bond location.
APCACI-MS of Diene-Containing TAGs. As with TAGs contain-
ing monoene groups, collisional activation of the [ketene + 40]
+
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2531

+
Figure 10. [M + 40] -based APCACI-MS/MS/MS spectra of (A)
(
16:0/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,12-18:2)-
+
TAG, and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. The [ketene + 40]
+
ions at m/z 302 derived from [M + 40]+ of diene TAGs (m/z 895 f
Figure 11. [M + 81] -based APCACI-MS/MS spectra of (A) (16:
0
/9-18:1/9-18:1)-TAG (B) (16:0/11-18:1/11-18:1)-TAG, and (C) (16:
3
02 f products) yield two fragments characteristic of the double bond
0/13-18:1/13-18:1)-TAG. Specific cleavages at the allylic carbons
position.
shown in the structures at the top of each spectrum give rise to a
pair of diagnostic ions. Ions at m/z 194 (A), m/z 166 (B), and m/z
+
1
38 (C) correspond to ω diagnostic ions of [M + 54] , which appears
ions at m/z 302 derived from MS/MS of [M + 40]+ of TAGs
containing conjugated dienes produces a pair of diagnostic ions
resulting from C-C bond cleavage at a position that is vinylic to
either site of the conjugated diene unit. A single hydrogen atom
is transferred to the charged fragment during R diagnostic ion
formation, while a single hydrogen atom is transferred to the
at m/z 913 in all spectra.
homolytic cleavage. In all three spectra, an ion occurring at m/z
274 is also apparent. This ion represents a neutral loss of 28 Da
from the [ketene + 40] ion, likely to be either CO or ethylene.
[M + 81] . APCACI-MS/MS of Monoene-Containing TAGs.
Figure 11 presents results from CAD of [M + 81] . The spectra
+
+
neutral fragment during ω diagnostic ion formation. CAD of
+
+
[
RCHdCdO + 40] , where R is derived from 9,11-18:2, yields R
diagnostic ions at m/z 218 and ω diagnostic ions at m/z 176, as
shown in Figure 10A, whereas that of [RCHdCdO + 40] of 10,-
are dominated by ions at m/z 345 arising from the adducts of
ketene and m/z 81 ions [RCHdCdO + 81] . Each spectrum
+
+
1
2-18:2 gives ions at m/z 232 and 162, respectively (Figure 10B).
consists of products at m/z 684 and 658 arising by neutral loss of
+
Figure 10C shows that CAD of the [RCHdCdO + 40] ion, a
ketene containing a 9,12-18:2 fatty acyl group, yields an R
diagnostic ion at m/z 232 and an ω diagnostic ion at m/z 176
originating from C-C bond cleavage at a position that is vinylic
to each double bond. As with the TAG containing conjugated
dienes, the R ions appear 1 unit above the expected mass while
ω ions appear at masses 1 unit lower than those expected from
palmitic acid (16:0) and octadecenoic acid (18:1), respectively,
+
from [M + 81] .
Diagnostic ions corresponding to cleavage adjacent to either
allylic site of the double bond were observed. For (16:0/9-18:1/
9-18:1)-TAG (Figure 11A), the ion appearing at m/z 841 is the R
diagnostic ion and that at m/z 233 is the ω diagnostic ion.
Similarly, parts B and C of Figure 11 show diagnostic ions for
2532 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

+
Figure 12. [M + 81] -based APCACI-MS/MS spectra for m/z 936
of (A) (16:0/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,12-
+
18:2)-TAG, and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. Cleavages di-
Figure 13. [M + 95] -based APCACI-MS/MS spectra for m/z 954
rectly before the first double bond and immediately after the second,
as shown in the structures, give rise to diagnostic ions.
of (A) (16:0/9-18:1/9-18:1)-TAG, (B) (16:0/11-18:1/11-18:1)-TAG, and
(C) (16:0/13-18:1/13-18:1)-TAG. Specific cleavages at the allylic
carbons shown in the structures at the top of each spectrum give
rise to a pair of diagnostic ions.
the fragmentation of (16:0/11-18:1/11-18:1)-TAG at m/z 869 and
2
05 and for the fragmentation of (16:0/13-18:1/13-18:1)-TAG at
m/z 343, and [BB]+ at m/z 679 and [AB]+ at m/z 655 arising,
+
m/z 897 and 177, respectively. The R ions appear 1 unit above
the expected mass, while ω ions appear at masses 1 unit lower
than those expected from homolytic cleavage. Interestingly, in
respectively, by loss of 16:0 and 18:2 from the [M + 81] ions.
+
+
The [M + 54] ion at m/z 909 and [RCHdCdO + 54] at m/z
316 were also observed, similar to those observed in Figure 11.
In parts A and B of Figure 12, APCACI-MS/MS spectra of TAGs
containing 9,11 and 10,12 18:2 isomers are presented, respectively.
The R and ω diagnostic ions at masses predicted by cleavage
vinylic to the former site of the double bond are observed at m/z
851 and 217 for (16:0/9,11-18:2/ 9,11-18:2)-TAG and m/z 865 and
203 for (16:0/10,12-18:2/10,12-18:2)-TAG. Figure 12C shows the
APCACI-MS/MS spectrum of (16:0/9,12-18:2/9,12-18:2)-TAG. The
cleavage vinylic to the former double bond gives rise to the
diagnostic ions at m/z 217 and 865. As usual, the R ions appear 1
unit above the expected mass, while ω ions appear at masses 1
unit lower than those expected from homolytic cleavage.
+
these spectra, we also found [M + 54] diagnostic ions at m/z
+
9
13 resulting from CAD of [M + 81] . The latter ion may
+
dissociate to [M + 54] , which appears in the spectrum at m/z
9
13, and further dissociate to an ω diagnostic ion. The [RCHd
+
CdO + 54] ions at m/z 318 were also detected. The ω ions
derived from [M + 54] for (16:0/9-18:1/9-18:1)-TAG, (16:0/11-
+
1
8:1/11-18:1)-TAG, and (16:0/13-18:1/13-18:1)-TAG are at m/z 194,
+
1
66, and 138, respectively. R ions derived from [M + 54] may
be present at low levels but are isobaric with satellite ions of R
+
derived from [M + 81] .
APCACI-MS/MS of Diene-Containing TAGs. Figure 12 shows
+
the APCACI-MS/MS spectra for collisional dissociation of [M +
[M + 95] . APCACI-MS/MS of Monoene-Containing TAGs.
+
8
1] of TAGs containing 18:2 fatty acyl groups. The ions observed
Figure 13 shows the APCACI-MS/MS spectra of (16:0/9-18:1/9-
18:1)-TAG, (16:0/11-18:1/11-18:1)-TAG, and (16:0/13-18:1/13-18:
+
+
for all TAGs are [M + 81] at m/z 936, [ketene + 81] ion at
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2533

+
Figure 15. [M + 40] -based APCACI-MS/MS spectra of m/z 899
for (A) (16:0/9-18:1/9-18:1)-TAG and (B) (9-18:1/16:0/9-18:1)-TAG.
Fragment ions reflecting neutral loss of the sn-2 fatty acyl group are
less abundant than the corresponding ions reflecting such losses of
either the sn-1 or sn-3 fatty acyl group.
As shown in parts A anc B of Figure 14, the R and ω diagnostic
ions for (16:0/9,11-18:2/9,11-18:2)-TAG are observed at m/z 865
and 231, respectively, while the analogous ions for (16:0/10,12-
1
8:2/10,11-18:2)-TAG occur at m/z 879 and 217, respectively. For
(16:0/9,12-18:2/9,12-18:2)-TAG, the R and ω diagnostic ions occur
at m/z 879 and 231, respectively, as indicated in Figure 14C. The
R ions appear 1 unit above the expected mass, while the ω ions
appear at masses 1 unit lower than those expected from homolytic
cleavage.
+
Figure 14. [M + 95] -based APCACI-MS/MS spectra for m/z 950
of (A) (16:0/9,11-18:2/9,11-18:2)-TAG, (B) (16:0/10,12-18:2/10,12-
8:2)-TAG, and (C) (16:0/9,12-18:2/9,12-18:2)-TAG. Cleavages di-
rectly before the first double bond and immediately after the second,
as shown in the structures, give rise to diagnostic ions.
1
APCACI-MS3 performed on ketene adduct peaks for the
+
+
[M + 81] and [M + 95] ions yielded low signal-to-noise ratio
+
+
spectra, even though the [M + 81] and [M + 95] intensities
+
+
+
were similar to those of [M + 40] and [M + 54] that produced
high-quality spectra.
1
)-TAG derived from collisional activation of [M + 95] . The base
peaks obtained for all three TAGs are ions at m/z 357 arising from
Positions of Acyl Groups on the Glycerol Backbone. The
relative intensities of neutral loss of fatty acyl groups or ketenes
from TAGs have long been known to be related to their relative
positions (sn-2 vs sn-1,3) in conventional ESI-MS/MS analysis. In
APCACI-MS/MS, the neutral loss of a fatty acyl group from [M
the adducts of ketene (generated from the fatty acid group) and
m/z 95 ions [RCHdCdO + 95] . The other ions for all TAGs are
+
m/z 698 and 672 caused by neutral loss of 16:0 or 18:1,
+
respectively, from the [M + 95] ion. Diagnostic ions were formed
by allylic cleavage of the double bond, which enables assignment
of the double bond position. The R and ω ions are observed at
m/z 855 and 247 for (16:0/9-18:1/9-18:1)-TAG, at m/z 883 and
+
+
+ 40] of a TAG yields [M + 40 - RCO
2
] , while the neutral loss
+
of a fatty acyl group from [M + 54] of a TAG yields [M + 54 -
+
2
19 for (16:0/11-18:1/11-18:1)-TAG, and at m/z 911 and 191 for
2
RCO ] . We tested whether intensity ratios would yield similar
(
16:0/13-18:1/13-18:1)-TAG. The R ions appear 1 unit above the
regiospecific information for these ions.
+
expected mass, while ω ions appear at masses 1 unit lower than
those expected from homolytic cleavage.
The MS/MS spectra of [M + 40] of ABB- and BAB-type TAGs
containing 16:0 and 9-18:1 fatty acyl groups are given in Figure
15. The ions at m/z 643 and 617 reflect neutral losses of
substituent 16:0 and substituent 9-18:1, respectively. The results
indicate that an ABB TAG produces a [BB + 40] /[AB + 40]
ratio of 1.68 ( 0.08, whereas a BAB TAG gives 1.06 ( 0.07. Figure
APCACI-MS/MS of Diene-Containing TAGs. Collisional activa-
+
tion of [M + 95] ions of TAGs containing dienes produces the
+
+
+
+
[RCHdCdO + 40] ion at m/z 357 and [BB] at m/z 694 and
+
[
AB] at m/z 670 caused, respectively, by loss of 16:0 and 18:2
+
+
from the [M + 95] ions, as presented in Figure 14. The R and ω
diagnostic ions appear at masses predicted by cleavage vinylic to
the double bonds.
16 shows similar data for the [M + 54] adducts. The ions at
m/z 657 and 631, formed by neutral loss of 16:0 or 18:1,
+
+
respectively, yield [BB + 54] /[AB + 54] ratios of 1.69 ( 0.09
2534 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007

bond geometry in the specific case of conjugated dienes.42
Mechanistic and structural work indicates that the MIE ion can
add to the double bond in a parallel or an antiparallel manner,
which determines subsequent fragmentation and H transfer.41 In
all cases studied to date, H transfer is symmetric with respect to
the ion or neutral loss: in the case of monoenes and dienes, the
H transfer is to the ion, whereas H transfer is to the neutral group
with polyenes for both the R and ω ions. Here we observe that H
transfer is always to the R ion and away from the ω ion. This result
has no analytical consequences but may reflect a mechanism or
structure different from that found for FAME [M + 54]+.
Cleavage sites of TAG adducts follow a clear pattern, similar
+
to that observed for FAME [M + 54] ions. Cleavage is allylic to
the site of the erstwhile double bond of monoene-containing TAGs
+
+
for all R ions and for ω ions from [M + 40] , [M + 81] , and
+
[
[
M + 95] . Cleavage is both allylic and vinylic for ω ions from
M + 54] , and [M + 81] dissociates to yield ω ions at the same
masses as [M + 54] . For diene-containing TAGs, vinylic cleavage
is observed for both R and ω ions.
+
+
+
+
The [M + 40] ion appears to be the best choice for analysis.
The intensity of this adduct is slightly greater than that of [M +
54]+ in the MS mode. More importantly, the MS intensity for
the ketene fragmentation is most intense of all the adducts, about
+
3
Figure 16. [M + 54] -based APCACI-MS/MS spectra of m/z 913
for (A) (16:0/9-18:1/9-18:1)-TAG and (B) (9-18:1/16:0/9-18:1)-TAG.
Fragment ions reflecting neutral loss of the sn-2 fatty acyl group are
less abundant than the corresponding ions reflecting such losses of
either the sn-1 or sn-3 fatty acyl group.
+
double that of the [M + 54] ketene ion. Finally, the [M + 40]
adduct yields two strong diagnostic ions in MS/MS, both due to
+
allylic cleavage, whereas [M + 54] yields an extra ω ion due to
+
+
+
Table 1. [BB + 40] /[AB + 40] and [BB + 54] /[AB +5
vinylic cleavage which slightly complicates the spectrum.
+
4
1
]
Ratios in BAB- and ABB-Type TAGs, Where A Is
6:0 and B Is a Monoene or Diene Unsaturated Fatty
Acyl Substituent, Respectively
SUMMARY
APCACI using acetonitrile as an APCI reagent has been shown
to be an effective method to obtain detailed structural information
of TAGs, including the location of double bonds along acyl groups
and the position of acyl groups on the glycerol backbone. In
addition to ions at m/z 40 and 54, ions at m/z 81 and 95 were
observed. These ions appear to form a charged covalent adduct
across carbon-carbon double bonds in TAGs containing unsatur-
ated fatty acyl groups, analogous to those observed for FAMEs.
+
+
Collisionally activated dissociation of the [M + 40] , [M + 54] ,
[
+
+
M + 81] , and [M + 95] ions yields two or three diagnostic
ions similar to those observed previously in FAME analysis.
With few exceptions, fragmentation behavior is similar for all
four ions. For monoenes, the R ions containing the glycerol moiety
yield strong fragments at the site allylic to the erstwhile double
bond. For the ω ions containing the terminal methyl group of the
fatty acyl chain, fragmentation is primarily allylic to the former
and 1.08 ( 0.12 for TAGs of the forms ABB and BAB, respectively.
Table 1 presents similar data in summary form for six different
TAGs, yielding highly reproducible and consistent ratios.
We have published extensively on the reaction and fragmenta-
tion of MIE (m/z 54) with FAMEs using a gas chromatography
introduction into an internal ionization 3D ion trap. This gas-phase
implementation of CACI is able to fully determine double bonds
in all homoallylic (methylene-interrupted) FAMEs including those
at very low concentration. It also determines the double bond
position and geometry in conjugated diene FAMEs and in many
FAMEs of unusual double bond structure. In FAME CACI spectra,
+
+
site of the double bond for [M + 40] and [M + 95] and both
vinylic and allylic for [M + 54]+ and [M + 81] . For dienes,
cleavage is vinylic to the site of the former double bond for all
adducts and for both R and ω ions. In all cases of monoenes and
dienes, a H is transferred from the neutral group to the R ions,
but is transferred away from the ω ions.
+
+
40, 49
the major adduct ion is [M + 54] ,
with smaller amounts of
+
+
+
The fragmentations of ketene adduct [RCHdCdO + 54] or
[M + 40] . [M + 54] has proven most useful for locating double
+
[RCHdCdO + 40]
+
in MS
3
yields two strong diagnostic ions that
bonds. Collisional dissociation of [M + 54] yields two diagnostic
ions that are indicative of the double bond position for homoallylic
FAMEs, and the ion intensities provide information on the double
locate the position of double bonds along fatty acyl chains. Finally,
the ratios of [BB + 40] to [AB + 40] and [BB + 54] to [AB +
Analytical Chemistry, Vol. 79, No. 6, March 15, 2007 2535
39
+
+
+

+
5
4] enable determination of the fatty acyl group regiospecificity
a volume distant from the metal heater and other parts vulnerable
to degradation with extended plasma exposure.
on the glycerol backbone. It is left to future work to establish
whether this approach can determine the structures of other
glycerolipids.
The extended plasma discharge observed with the present
APCI ion source appears to be essential for production of sufficient
reagent ions for reaction. While it is possible to run the source
under these conditions for an extended period, a redesign of the
source is probably required to contain the reactive plasma within
ACKNOWLEDGMENT
This work was supported by NIH Grant GM071534.
Received for review November 2, 2006. Accepted
December 21, 2006.
AC062055A
2536 Analytical Chemistry, Vol. 79, No. 6, March 15, 2007