2302956

BIOMEDICAL AND ENVIRONMENTAL MASS SPECTROMETRY, VOL. 18, 328-336 (1989)
Characterization of Epoxides of Polyunsaturated
Fatty Acids by Mass Spectrometry via
3-Pyridinylmethyl Esters
Michael Balazy and Alan S. Niest
University of Colorado Health Sciences Center, Division of Clinical Pharmacoiogy, Box C-237, Denver, Colorado 80262, USA
The isomeric epoxides of linoleic, arachidonic and docosahexaenoic acids were prepared by reaction with mchloroperoxybenzoic acid and, after separation by normal-phase high-performance liquid chromatography, were
esterified with 3-pyridylcarbinol via the unstable imidazolide generated by the reaction with 1 ,l'carbonyldiimidazole. The electron impact mass spectra of these derivatives showed a molecular ion and a sequence
of peaks with two characteristic abundant ions that resulted from formal cleavage of the carbon-carbon bonds at
the oxirane ring. Both these ions retained the ester group. This fragmentation pattern allowed the unequivucal
identification of the separate epoxide isomers.
INTRODUCTION
Oxygenation via cytochrome P450 linked monoxygenase is recognized as an important metabolic
pathway of polyunsaturated fatty acids such as linoleic,
arachidonic and docosahexaenoic acids. Among
several types of products of the NADPH-dependent
oxygenation, unsaturated epoxides have attracted attention because of their diverse biological actions, although
their physiological or pathophysiological role is unclear.
A variety of cell preparations has been reported to
produce these epoxides. For example, both expoxides of
linoleic acid have been found in incubates of leukocytes
from hyperoxic lung,* and one of them, 9,10-epoxy-12octadecenoic acid (leukotoxin), causes cardiac failure in
dogs and is a smooth muscle r e l a ~ a n t .Arachidonic
~
acid epoxides, epoxyeicosatrienoic acids (EETAs), are
found in microsomal preparations of rabbit liver4 and
kidney' and also in human
Isomeric EETAs
show a broad spectrum of biological activity including
inhibition of platelet aggregation and cyclooxygenase
a ~ t i v i t y ,inhibition
~
of vasopressin-stimulated osmotic
water f l o ~ relaxation
,~
of rabbit pulmonary artery,'
release of somatostatin," leutinizing hormone,' ' insulin
and glucagon,'2 and mobilization of microsomal
c a l ~ i u m . ' ~Epoxides derived from docosahexaenoic
acid, although not isolated, appear to be intermediate
products in the formation of vicinal diols that have been
extracted from rabbit liver microsomal incuba te^.'^
Structural differentiation of the regioisomeric
epoxides by electron impact (EI) or chemical ionization
(CI) mass spectrometry is difíicult with derivatives commonly used for gas chromatography (GC) such as methyl
or trimethylsilyl esters because of extensive fragmentation in the low-mass range and similarity of spectra.'j4
Several methods have been proposed to identify the
isomeric epoxides. Charge-exchange ionization with
'
t Author to whom correspondence should be addressed
0887-61 34/89/050328-09 $05.00
(C 1989 by John Wiley & Sons, Ltd.
carbon disulfide as the moderating reagent provides
m a s spectra of methyl arachidonate epoxides with
characteristic peaks at low masses and relatively weak
molecular ions.15 Another method involves fast atom
bombardment (FAB) desorption of lithiated fatty acid
epoxide molecular ions, followed by collisional activation.I6 Although pentafluorobenzyl esters of epoxides
produce an abundant M - 181 ion in negative ion Ci
and are useful for their quantification,6 the structural
information is lost because such derivatized isomeric
epoxides display similar spectra and are not well resolved by capillary GC.' Other methods are based on the
acidic aqueous hydrolysis of the epoxide with subsequent gas chromatography/mass spectrometry
(GC/MS) of the silylated vicinal dio1 or permethylation
of products after reaction with phenyltrimethylammonium hydroxide.'
We report here a rapid, mild and quantiative derivatization method for preparation of 3-pyridinylmethyl
(picolinyl) esters of epoxides derived from linoleic, arachidonic and docosahexaenoic acids. These picolinyl
esters provided very characteristic spectra that allowed
differentiation of the isomeric epoxides at nanogram
levels. A previously reported method for preparation of
picolinyl esters via fatty acid chlorides'8 could not be
used for derivatization of these epoxides because of
their instability in acidic solutions.
'
EXPERIMENTAL
Materials
Epoxides were synthesized by the reaction of metachloroperoxybenzoic acid (1.5 mEq, 50 mM) with linoleic.
arachidonic or docosahexaenoic acid (Nucheck) iri
methylene chloride for 8 h at room temperature as
d e ~ c r i b e d . After
~
the solvent was evaporated, the
epoxides were injected in hexane onto a highperformance liquid chromatography (HPLC) column
Receiued 16 Septemher 1988
Revised 22 Novemher 1988
329
EPOXIDES OF UNSATURATED FATTY ACIDS
a
AA
C
o
MCPBA
, i,
r
L Y
0 - 2
O
N
c,
a
m
u
c
a
n
ii
O
UI
n
a
O
10
20
retention
time
30
C m i n l
Figure 1. Normal-phase HPLC separation of epoxidec (EETA) synthesized from arachidonic acid (AA) and rn-chloroperoxybenzoic acid
(MCPBA) as described in the text.
(Altex, Ultrasphere Si, 250 x 4.6 mm, 5 pm particles)
and eluted with hexane-2-propanol-acetic
acid
(500:0.5:0.25 v/v/v) at a ñow rate of 1 ml/min with
ultraviolet (UV) detection at 200 nm. In some experiments 0.1 pCi of (l-14C)arachidonic acid was added to
the reaction mixture in order to quantify the epoxides
and to determine the yield of the subsequent derivatization.
tions of the sample dissolved in hexane were made with
a splitless injector. Mass spectra were recorded with a
VG Micromass 16 spectrometer interfaced with a VG
2000 data system. Operating conditions were : electron
energy, 70 eV; trap current, 200 pA; acceleration
voltage, 4 kV; scan 3 s per decade in exponential downfield mode. Some epoxide esters were also analyzed with
a 15 m DB-1 column interfaced with a VG 7070H mass
spectrometer and a Teknivent data system.
Derivatization
To 0.15-1.5 nmol of epoxide dissolved in 100 p1 dry
methylene chloride, 10 p1 of freshly prepared solution
(50 pg/ml) of 1,l'-carbonyldiimidazolewas added. After
1 min, 10 pl of 1% 3-pyridylcarbinol solution was
added along with 10 p1 of triethylamine. The reagent
solutions were prepared with dry methylene chloride.
After 10 min at 37°C the solution was washed with 1 N
sodium hydroxide saturated with carbon tetra~ h l o r i d e , ' washed
~
with water and evaporated under
nitrogen. The residue was redissolved in hexane-ethyl
acetate (3: 1) and eluted from a 2 cm silica CC4
(Mallinckrodt) column. Epoxide esters with N , N dimethylethanolamine, 1-methyl-3-piperidinemethanol
and 3-hydroxypyridine were prepared in the same way.
Al1 reagents were purchased from Aldrich. Analysis by
thin-layer chromatography (TLC) was performed on
silica gel G plates (Analtech) with hexane-ethyl acetate
(1 : 1 v/v) as eluent, and radioactivity was detected by
using a computerized Berthold scanner.
GC/MS
A 30 m DB-1 column (J&W Scientific) was temperature
programmed from 220 "C to 300 "C at 16 "C/min. Injec-
RESULTS AND DISCUSSION
Epoxides of the three unsaturated fatty acids-linoleic,
arachidonic and docosahexaenoic acids-were prepared
with good yield (6&70%) via the reaction with mchloroperoxybenzoic acid. The crude reaction mixture
was analyzed by normal-phase HPLC, and a representative chromatogram for the epoxides obtained from
arachidonic acid is shown in Fig. 1. The retention times
for al1 prepared epoxides are reported in Table 1. Each
of the separately collected epoxides was subsequently
derivatized in a two-step esterification. First, by reaction with 1,l'-carbonyldiimidazole the unsaturated
epoxy acid was converted into the reactive imidazolide
which, without isolation, was reacted with 3pyridylcarbinol in the presence of triethylamine. The
mild and basic conditions stabilized the epoxide
throughout the derivatization, and the pyridine ring in
the ester protected the epoxy group against acidic
impurities during chromatography. The radioactivity
added was quantitatively recovered in about 4 ml of
eluate from the silica column chromatography. The
crude reaction mixture applied directly to a TLC plate
showed a single radioactive peak corresponding to the
M. BALAZY AND A. S . NIES
330
Table 1. Normal-phase HPLC retention times (RT) for
epoxides synthesized from linoleic, arachidonic and
docosahexaenoic acids, and GC relative retention
times expressed as carbon numbers (CN) for the picolinyl esters of the epoxides
RTb (min)
Relative peak area
CNC
12.7
16.1
1 .o
1.3
27.8
14,15
11,12
8. 9
5. 6
11.4
12.2
16.2
24.4
3.1
1.4
1 .o
1.1
30.2
EDP
16,17
13,14
19,20
7, 8
10.8
11.7
13.5
17.1
1.1
1 .o
2.0
1.1
33.2
Epoxide"
EOE
12,13
9, 10
EET
EOE, epoxyoctadecenoic acid; EET, epoxyeicocatrienoic acid;
ED P. epoxydocosapentaenoic acid.
1
mijmin
hexane-2-propanol-acetic
acid
Conditionc:
(500:0.5:0.25, v/v/v); column, Ultracphere Si, 250 x 4.6 rnm, 5
pm particles.
"Conditionc: 15 m DE-1 column programmed from 200°C by
15 "C/min with helium as a carrier gas at linear flow of 40 crn/e.
a
esterified epoxide ( R , = 0.37). No spot was observed for
underivatized epoxide at R, = 0.20. G C analysis produced single, symmetrical peaks for al1 expoxide derivatives studied, but the isomeric epoxides were not
resolved with our G C conditions.
It is possible that the described esterification could be
applied differently. The biological extract, containing
epoxides of polyunsaturated fatty acids, could be derivatized first, followed by separation of the picolinyl
esters by HPLC. Although we have not tried to analyze
the picolinyl epoxides by HPLC, we expect that a reasonable separation should be achieved, at least to the
same extent as the quite satisfactory separation we have
accomplished with the isomeric methyl arachidonate
epoxides (not shown). This procedure would be more
eficient, since only a single derivatization step would be
required, and might be particularly useful when only
small amounts of biologicai material are available. This
approach also would offer the interesting possibility
that a liquid chromatographic/mass spectrometric
system could be used for an on-line separation and
analysis of picolinyl epoxides. In our case, however, the
amount of prepared epoxides was suficient to allow us
to analyze the separate HPLC fractions in order to
provide the unambiguous standard spectrum for each
isomeric epoxide.
Spectra of arachidonate epoxides
Figure 2 shows the E1 m a s spectra of the picolinyl arachidonate epoxides. General features of these spectra in
the low-mass range were similar to those observed previously by Harvey for picolinyl esters of polyunsaturated fatty acids." The m a s spectra of these compounds
as well as of epoxides studied here showed abundant
ions at m/z 92 (base peak), 93, 108, 151 (resulting from
McLafferty rearrangement) and 164, indicating an analogous fragmentation pattern for these ions.18 A series
of ions was also produced by radical-induced cleavage
at each single carbon-carbon bond following a remote
hydrogen abstraction from the unsaturated chain.
However, the spectra of the epoxides showed severa1
distinct features that made them valuable for analytical
purposes.
The molecular ion (m/z 411) was observed in al1
spectra with a relative abundance of 1.5-8.0%. This ion
was found at the odd mass because the derivative contained one nitrogen atom. The other high-mass ion that
was frequently observed was M - 18, corresponding to
loss of a molecule of water-a typical neutral loss found
in the spectra of many epoxides.20.21Its abundance was
about the same as that of the molecular ion.
The most characteristic and diagnostic feature of the
spectra of the picolinyl epoxides studied was the presence of two approximately equally abundant ions
(called here a and b) resulting from the formal cleavage
of the carbon-carbon bonds located at the oxirane ring.
These ions are separated by 42 daltons. Both ions
retained that part of the molecule containing the 3pyridinylmethyl ester function. Ion type b also formally
retained the epoxide group.
This type of fragmentation suggested the involvement
of radical-induced cleavage from the remote pyridyl
group. Adopting a similar pattern of fragmentation to
that proposed by Harvey," the most plausible mechanism for the formation of ions a and b requires the
initial rearrangement of methylene hydrogen to give a
distonic cation in which the charge and radical sites are
separated. For example, in the picolinyl ester of 11,12EET the migration of allylic hydrogen at carbon 7 (Fig.
2(b); Scheme 1) to the high hydrogen afinity pyridine
ring results in movement of the site of the unpaired electron to that carbon. The cleavage of the bond between
carbons 10 and 11 yields the ion m/z 258 (type a). This
ion may have the structure of a terminally unsaturated
fatty acid picolinyl cation.
The second abundant ion was found at m/z 300 (type
b) and may originate from radical abstraction of hydrogen at carbon 10 followed by a rearrangement in the
oxirane ring and cleavage of the bond CI2-Cl3
(Scheme 1). This ion might have the structure of an
even-electron aldehyde. Such an aldehyde ion may
readily lose a neutral carbon monoxide, also producing
an ion at m/z 272 (type c).
In the spectra of derivatives of 5,6-EET and 8,9-EET,
in addition to ions a and b, peaks at mfz 208 and 248
were found, respectively. Corresponding ions (type d),
possibly of the structure of an aldehyde (Scheme 2)
might result from carbon-carbon and carbon-oxygen
bond cleavage within the epoxide ring. These ions were
not observed in the two other epoxides. The relative
preference of formation of ion type d versus c reflects
the differences in the bond polarities between CO- and
C-C bonds in the epoxide ring that are additionally
influenced by the inductive effect of the ester group in
the derivative of $6-, and 8,9-EET. In these two epoxide
esters the ion at m/z 300 corresponded to the M - 11 1
fragement resulting from clevage of CI3-C,, bond.
The ion M - 111 has been observed in E1 spectra of
33 1
EPOXIDES OF UNSATURATED FATTY ACIDS
92
1O0
l
O
340
70
60
50
40
30
20
10
O
50
1O 0
150
200
250
300
350
400
450
400
450
M / Z
90
O
-1
""11 I
70
50
1O0
150
200
250
M
/
300
350
Z
of the double bonds certainly contributed to the shortsevera1 silyl ether and methyl ester derivatives of lipoxyening of the distance between the hydrogens and the
genase metabolites of arachidonic acid, e.g. 12-HETEZ2
nirogen by bending the unsaturated chain towards the
or LTB423 and also in the picolinyl derivative of arachiester group. This also implies that some hydrogens are
donic acid.
more favored for abstraction than others.
The formation of distonic cations, as exemplified in
The relations between the thermochemical and
Scheme 1, followed by fragmentation to ions a and b,
stereochemical factors has a clear impact on the spectra
comprises two factors. The first, thermochemical, indistudied. For example, although the BDE of the allylic
cates that the hydrogen(s) of the lowest C-H bond dishydrogens at C , and C in the picolinyl derivative of
sociation energy (BDE) will be most easily abstracted as
a radical by the positively charged picolinyl g r o ~ p . ~ ~11,12-EET (Fig. 2(b)) should be the same and lower
than that of hydrogens at C , , the abstraction of hydroSecond, a certain spatial arrangement (stereochemical
gen from the more remote C,, did not occur (the ion
factor) is also necessary for hydrogen abstraction. It was
found at m / z 354 was very small). Rather, an abstraction
shown that in the primary, unbranched alkylamine radof the closer hydrogen at C , actually took place
icals the energy barrier for hydrogen shift decreased
producing ion b (m/z 258), aithough the BDE of the
with increasing distance, and the 1 3 shift was the most
C,-H bond was less favorable. Thus the stereochemif a v o ~ r a b l e In
. ~ ~the compounds studied here the epoxy
cal factor contributed to the higher abundance of ion a
group lowered the dissociation energy of the adjacent
over ion b in the epoxides studied: the distonic cation
carbon-hydrogen methylene bonds, and the 2 geometry
,
M. BALAZY AND A. S. NIES
332
92
1O 0
90
80
70
60
50
40
30
20
1 0
O
50
I
1 O8
I
164
151
1
218
354
395411
L +
1 O0
150
250
200
350
300
400
450
M / Z
92
1
O0
208
90
80
70
60
50
40
30
20
10
O
50
1O 0
150
250
200
M
/
300
350
400
450
Z
Figure 2. Mass spectra (70 eV) of picolinyl esters of epoxides originated from arachidonic acid. About 400 ng of epoxide was derivatized
to obtained each spectrum. Fragments formally corresponding to ions a and b are schematically indicated in the chemical structure.
resulting in ion a was produced by abstraction of a
hydrogen closer to the picolinyl group. Also in the spectrum of the picolinyl derivative of 5,6-EET (Fig. 2(d))
the peaks a and b were very small, reíiecting the very
unfavorable conformation for hydrogen abstraction,
especially for ion a ( m / z 178).
The fragmentation of the picolinyl derivatives of arachidonate epoxides seemed to occur through distonic
cations formed by the rearrangement of allylic hydrogens at both sides of the double bond adjacent to the
epoxide ring.
Spectra of linoleate and docosahexaenoate epoxides
The general features of these spectra were similar to
that observed for the arachidonate epoxides. In both
picolinyl linoleate epoxides the intensity of the M - 18
peak (m/z 369) was about five time greater than that of
the molecular ion peak. In contrast, the molecular ion
was more abundant in picolinyl docosahexaenoate
epoxides. Again, two characteristic ions of type a and h
were observed (Figs 3 and 4) that helped to indicate the
elution order of these epoxides from HPLC. The ion at
m/z 164 was not abundant in the docosahexaenoate
epoxides, refiecting the presence of the delta 4 double
bond that prevented the loss of the allylic radical after
cleavage between carbons C , and C,. The epoxidation
of docosahexaenoic acid yielded very small amourits of
the 4 3 and 10,ll isomers, and these epoxides were riot
characterized as picolinyl esters.
The picolinyl derivative of 13,14-epoxydocosapentaenoic acid (Fig. 3) showed a sequence of abundant ions
333
EPOXIDES OF UNSATURATED FATTY ACIDS
..
H
m/z
248 d
O
0 H
m/z 258
H
a
H
O
O
f-'.
m/z 208 d
Scheme 2
H
H
-
0
-
C8HlS
"
H
m/z 300
b
Scheme 1
beginning with an ion at m/z 164 and separated from
subsequent ions by 40 daitons, which corresponds to a
difference of a -CH=CH-CH,unit. Therefore,
these fragment ions indicate the position of the double
bonds in the unsaturated chain. A gap of 42 daltons was
observed only between ions m/z 284 and 326, which
indicates the presence of the oxirane ring. This spectrum
therefore showed that there were three double bonds
between the ester group and oxirane ring, namely delta
4. 7 and 11. The other two double bonds were located
at C,, and CL,.The ion at m/z 406 resulted from the
loss of a C2H, radical from the molecular ion (m/z 439,
indicating that the terminal double bond was at C , , . A
small ion at m/z 417 originated from the loss of H 2 0 .
Thus the sequence of ions demonstrated the location of
the double bonds and the oxirane ring in this compound and other epoxides studied. Interestingly, the
intensities of the ion peaks in the major sequence followed approximate Gaussian-type distribution, as compared to about equal intensities in the spectrum of the
picolinyl ester of docosahexaenoic acid. * Obviously,
introduction of an epoxy group changed the BDE of
adjacent carbon-hydrogen bonds favoring abstraction
by the pyridinyl group, thereby changing the otherwise
random hydrogen abstraction.
The fragmentation pattern of a compound can be elucidated by several methods. One is based on the study
of spectra of isotopically labeled analogs and/or appropriate derivatives. We expected that the similarity
between the spectra of various amino esters of epoxides
would indicate a common origin of fragmentation. To
verify this assumption the esters of 12,13-epoxy-9-octadecenoic acid with several substituted amino alcohols
92
P
r326
'O0
1
l
o
284
284
244
I
1
326
366
435
406
d - L
50
1 O0
150
200
250
300
350
J L
400
M / Z
Figure 3. Mass spectrum of picolinyl ester of 13.1 4-epoxydocosapentaenoic acid.
4 50
M. BALAZY AND A. S . NIES
334
A
1
O0
0
-
90
O
274
-
316
- O T
a7 o0 h.
tu,
60
164
-
274
z
c
w
z
w
2
4
w
IL
50
1O0
150
200
250
300
350
400
450
M / Z
1 o9
B
1O 0
90
80
h.
tvi
70
60
c
W
z
50
W
t
3
40
W
<z
30
355
20
10
O
50
1O 0
150
200
were prepared. Mass spectra were compared with that
for the picolinyl derivative (Fig. 4).
Introduction of a functional ester group containing
nitrogen which was separated by three or four carbons
from carboxylic oxygen strongly directed the fragmentation of the epoxide. A general similarity can be noticed
among the spectra in Fig. 4. In particular, ions a and b
were present but usually in lower relative abundance
when compared to the picolinyl derivative. These derivatives also showed quite extensive fragmentation in the
low-mass range, reducing the utility of these derivatives
for structural determination. The reduced abundance of
ions a and b was due in part to the lower hydrogen
aEnity of the ionized tertiary aliphatic or alicyclic
amine esters (Fig. 4(c) and (d)) relative to the ionized
3-pyridinylmethyl e ~ t e r . *However,
~
these ions were
also of low relative abundances in the spectrum of the
250
300
350
400
4 50
3-pyridyl ester (Fig. 4(b)) as compared to the 3pyridinylmethyl ester (Fig. 4(a)), although hydrogen
afinities of both ester groups should be similar. Most
probably, the missing methylene group in the 3-pyridyl
derivative did not permit the proper conformation for
hydrogen migration, similar to the mechanism that has
been proposed for the interpretation of the spectrum of
2-pyridinylmethyl ester of stearic acid.26 Mass spectra
of both picolinyl linoleate epoxides displayed additional
fragment ions of abundances comparable to those of
fragments a and b. Such fragment ions are: a + 14 (m/z
288) and b + 14 (m/z 330) in the spectrum of picolinyl
12,13-epoxyoctadecenoate (Fig. 4(a)), and also a - 14
( m / z 220) and a - 28 (m/z 206) in the spectrum of picolinyl 9,10-epoxyoctadecenotate(not shown). This fragmentation pattern is very similar to that of picolinyl
derivatives of monounsaturated acids, e.g. picolinyl
335
EPOXIDES OF UNSATURATED FATTY ACIDS
81
C
1O0
90
80
K
70
60
50
W
L
'4
40
W
349
[L
30
20
10
O
50
1O 0
150
200
250
300
350
400
450
M / Z
110
O
80 -
6:
70
336
I
-
CH3
128
60
-
50
-
W
t
5
40 -
W
n
30
10
zo
336
4
50
294
389 4 0 7
I
1O0
150
200
250
300
350
I
400
450
M / Z
Figure 4. Mass spectra of: (a) 3-pyridinylmethyl (picolinyl), ( b ) 3-pyridy1, ( c ) N,N-dirnethylaminoethyl and ( d ) 1 -methyl-3-piperidinemethyl esters of 12.13-epoxyoctadecenoic acid.
oieate,26 and can be explained in the same way as the
fragmentation of picolinyl arachidonate epoxides.
El spectra of amino and picolinyl esters of linoleate
epoxide confirmed the formation of ions a and b and
provided additionai evidence in favor of the proposed
fragmentation pattern. Picolinyl esters produced more
intense peaks in the high-mass range and, therefore,
were more suitable for qualitative m a s spectrometric
analysis of these substances. Analogs of these fatty acids
isotopicaily iabeied with deuterium or "0, should
confirm the mechanism leading to the production of
t hese ions.
Fragmentation in E1 spectra of epoxides is usuaiiy
controiied by a cleavage following electron abstraction
from the epoxide oxygen.20*2'There is no evidence that
this mechanism was significant in the derivatives
studied. The picolinyl function, with a lower ionization
potentiai than the epoxy group, inhibited this mode of
fragmentation, and changed the site of primary ionization to the pyridine ring, which directed subsequent
fragmentation.
It is interesting to note the difference between the
mass spectra of picolinyl derivatives of epoxides
described here and cyclopropane fatty acids discussed
by Harvey.,' The latter spectra are very diagnostic for
the determination of the position of the cyclopropane
ring, but in a different way when compared to the
spectra of the epoxides studied here. Ions that formaily
336
M. BALAZY AND A. S. NIES
correspond to epoxide ions a and b are relatively small
in cyclopropane spectra; in contrast, ions corresponding
formally to ion type c and c + 14 in epoxides are very
abundant.
We conclude that the unique advantage of the picolinyl esters for mass spectroscopic analysis can be
extended to another group of compounds, the epoxides.
However, because of the instability of epoxides in acidic
environments, the previously reported derivatization
procedure had to be modified by initially preparing an
imidazolide, which was permitted to react with 3pyridylcarbinol. This reaction was particularly suitable
for epoxide derivatization because of mild and basic
conditions. Mass spectrometry of the 3-pyridinylmethyl
esters of unsaturated epoxy fatty acids produced characteristic ions for each regio-isomer because of preferred
cleavage of the carbon bonds at the oxirane ring. This
method should provide a useful tool for the mass spectrometric analysis of epoxides of unsaturated fatty acids
that are attracting increasing attention in studies of biological systems.
Acknowledgements
The mass spectrometry experiments were conducted at the NIH Mass
Spectrometry Center, University of Colorado. This work was supported by Public Health Service grants HL21308 and RROl152.
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