Federal Office & Research Center for Agriculture, Institute for Agricultural Ecology, Vienna, Austria
We developed a method to elucidate the complete structure of triacylglycerols by means of high-energy collisional
activation tandem mass spectrometry (MS/MS). Both
ESI- and FAB-produced [M + NH4]+ and [M + met.]+ ions
(where met. ) Li, Na, and Cs) of triacylglycerols undergo
charge-remote and charge-driven fragmentations. We
emphasize the study of fragment ions from ESI-produced
[M + NH4]+ and [M + Na]+ ions and FAB-produced [M
+ Na]+ ions. ESI-produced [M + NH4]+ ions fragment
to produce four types of ions, [M + NH4 - RnCOONH4]+,
[RnCO + 128]+, [RnCO + 74]+, and RnCO+ ions, from
which the carbon number and the degree of unsaturation
of each acyl group are obtained. In addition, three series
of ions are produced by charge-remote fragmentations
(CRFs), and analysis of their patterns gives the position
and the number of double bonds on the acyl groups.
Information about the position of each acyl group on the
glycerol backbone, however, is not provided by collisionally activated dissociation of [M + NH4]+ ions. On the
other hand, ESI- and FAB-produced [M + Na]+ ions
fragment to form eight types of ions (named A-J ions)
that, like those produced by CRF, are highly structurally
informative. The absence of certain series members also
carries useful structural information. Interpretation of
these patterns enables one to obtain the number of
carbons, degrees of unsaturation, and location of double
bonds, as well as the positions of acyl groups on the
glycerol backbone.
Triacylglycerols (TAGs) play an important role in nutrition and
other biological processes. They are the primary means of energy
storage in animals and humans, and their hormonally controlled
hydrolysis and oxidation release energy to meet the energygeneration needs of organisms.1 A major source of TAGs is seed
oils, which are found as renewable agricultural raw materials;
these materials have applications in technology and nutrition.2
* 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; The Benjamin/Cummings
Publishing Co., Inc.: Redwood City, CA, 1990; pp 571-578.
S0003-2700(98)00519-8 CCC: $15.00
Published on Web 09/12/1998
Analytical Chemistry, Vol. 70, No. 20, October 15, 1998 4417
4418 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
Figure 1. CAD spectra of ESI-produced TAG [M + NH4]+ 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 peak
corresponding to an allylic cleavage is labeled with an.
4419
Figure 2. CAD spectra of ESI-produced [M + NH4]+ ions of 1-palmitoyl-2-oleoyl-3-stearoylglycerol (16:0/18:1/18:0, MW ) 860.8) obtained by
different instruments: (A) an MS/MS spectrum from a four-sector mass spectrometer and (B) a linked-scan spectrum from a two-sector mass
spectrometer. The peak corresponding to a vinyl cleavage is labeled with v.
4420 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
Figure 3. Comparison of CAD spectra of the [M + Na]+ ions of tristearoylglycerol (18:0/18:0/18:0, MW ) 890.8) with different ionization
methods: (A) with FAB and (B) with ESI.
Scheme 1
groups on the glycerol backbone, however, cannot be deciphered
because there is no apparent discrimination in the formation of
ions in the same group; that is, all three ions of each type, [M +
NH4 - RnCOONH4]+, [RnCO + 128]+, [RnCO + 74]+, or RnCO+,
are produced with nearly the same abundance (Figure 2B).
Although the ions that arise by loss of R2COONH4 (Figure 2A
and B) are slightly less abundant than those from losses of R1COONH4 and R3COONH4, we are not certain that this is always
true.
[M + NH4]+ Ions of TAG Isomers. From the above discussion,
we predict that CAD of [M + NH4]+ ions of positional TAG
isomers would give similar product ions, making it impossible to
distinguish them. We found this to be true for the fragmentations
of the [M + NH4]+ ion of two pairs of TAG isomers [1,2-dioleoyl3-stearoylglycerol (18:1/18:1/18:0) and 1,3-dioleoyl-2-stearoylglycerol (18:1/18:0/18:1) as well as 1,2-dipalmitoyl-3-oleoylglycerol
(16:0/16:0/18:1) and 1,3-dipalmitoyl-2-oleoylglycerol (16:0/18:1/
16:0)]. Product-ion spectra (not shown) of the isomers are nearly
identical.
[M + NH4]+ Ions of TAGs Obtained by Linked Scanning. Figure
2B is a product-ion spectrum obtained by linked scanning (B/E
) constant) with a two-sector mass spectrometer. The quality of
the spectrum shows that linked scanning serves well in the
analysis of TAGs because it provides almost the same structural
information as does tandem four-sector mass spectrometry (Figure
2A). Nevertheless, it is difficult to locate a double bond by
interpreting the product-ion spectrum from linked scanning
because CRF ions are no longer singlets, owing to the inability to
select a single precursor ion. For example, the position of the
double bond is easily located in Figure 2A, but not in Figure 2B.
4421
Figure 4. Comparison of CAD spectra of the [M + Na]+ ions of 1-palmitoyl-2-oleoyl-3-stearoylglycerol (16:0/18:1/18:0, MW ) 860.8) with
different ionization methods: (A) with FAB and (B) with ESI.
Scheme 2
4423
Figure 5. Comparison of CAD spectra of FAB-produced [M + Na]+ ions of TAG isomers: (A) 1,3-dipalmitoyl-2-oleoylglycerol (16:0/18:1/16:0,
MW ) 832.7) and (B) 1,2-dipalmitoyl-3-oleoylglycerol (16:0/16:0/18:1, MW ) 832.7). A peak corresponding to an allylic cleavage is labeled with
a.
Table 2. Formulas for Calculating m/z Values for
Various Fragments from the [M + Na]+ Ion of
Triacylglycerols (M123 ) TAG + Na]+
A12 ) M123 - R3COOH + 72
A13 ) M123 - R2COOH + 72
A23 ) M123 - R1COOH + 72
B12 ) M123 - R3COOH
B13 ) M123 - R2COOH
B23 ) M123 - R1COOH
C12 ) M123 - R3COONa
C13 ) M123 - R2COONa
C23 ) M123 - R1COONa
D1 ) M123 - R2COOH - R3COOH + 132
D2 ) M123 - R1COOH - R3COOH + 132
D3 ) M123 - R1COOH - R2COOH + 132
E1 ) M123 - R2COOH - R3COOH + 74
E3 ) M123 - R1COOH - R2COOH + 74
F1 ) M123 - R2COOH - R3COOH + 60
F3 ) M123 - R1COOH - R2COOH + 60
G1 ) M123 - R2COOH - R3COOH + 2
G3 ) M123 - R1COOH - R2COOH + 2
J2 ) M123 - R1COOH - R3COOH - 12
the B ions, allows one to calculate the carbon number and the
degree of unsaturation of each acyl group. For example, the mass
difference between the precursor and the B ions is 284 u (Figure
3), indicating that each acyl group has 18 carbons and no double
bonds. This is verified by the peak pattern corresponding to ions
formed via CRF where no interruption is observed. Therefore,
the complete structure of the TAG is established to be 18:0/18:
0/18:0.
4424 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
Figure 6. Comparison of CAD spectra of ESI-produced [M + Na]+ ions of TAG isomers: (A) 1,2-dioleoyl-3-stearoylglycerol (18:1/18:1/18:0,
MW ) 886.6) and (B) 1,3-dioleoyl-2-stearoylglycerol (18:1/18:0/18:1, MW ) 886.8). A peak corresponding to a vinyl cleavage is labeled with
v.
4425
CONCLUSIONS
In addition to undergoing charge-remote fragmentations, ESIand FAB-produced [M + cat.]+ ions of triacylglycerols fragment
to produce characteristic ions. The CAD of ESI-produced [M +
NH4]+ or [M + Na]+ ions and of FAB-produced [M + Na]+ ions
of TAGs is the basis of an analytical tool to obtain detailed
structural information of triacylglycerols, including carbon numbers, degree of unsaturation of each acyl group, and the position
ACKNOWLEDGMENT
This research was supported by NIH Center for Research
Resources (Grant No. 2P41RR00954).
4426 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998