METHODS
Characterization of Epoxy Carotenoids by Fast
Atom Bombardment Collision-Induced
Dissociation MS/MS
Takashi Maokaa, Yasuhiro Fujiwarab, Keiji Hashimotoc, and Naoshige Akimotod,*
a
Research Institute for Production Development, Kyoto 606-0805, bKyoto Pharmaceutical University, Kyoto
607-8414, Japan, cNagahama Institute of Bio-sciences and Technology, Shiga 526-0829, Japan, and
d
Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan
ABSTRACT: The characterization and structure of epoxy carote-
noids possessing 5,6-epoxy, 5,8-epoxy and 3,6-epoxy end groups
conjugated to the polyene chain were investigated using high-
energy fast atom bombardment collision-induced dissociation
MS/MS methods. In addition to [M – 80]+⋅, a characteristic fragment
ion of an epoxy carotenoid, product ions resulting from the cleav-
age of C–C bonds in the polyene chain from the epoxy end group,
such as m/z 181 (b ion) and 121 (c ion), were detected. On the other
hand, diagnostic ions of m/z 286 (e-H ion) and 312 (f-H ion) were
observed, not in the 5,6-epoxy or 5,8-epoxy carotenoid but in the
3,6-epoxy carotenoid. These fragmentation patterns can be used to
distinguish 3,6-epoxy carotenoids from 5,6-epoxy or 5,8-epoxy
carotenoids. The structure of an epoxy carotenoid, 3,6-epoxy-5,6-
dihydro-7′,8′-didehydro-β,β-carotene-5,3′-diol (8), isolated from
oyster, was characterized using FAB CID-MS/MS by comparing
fragmentation patterns with those of related known compounds.
Paper no. L9383 in Lipids 39, 179–183 (February 2004).
Carotenoids are the pigments responsible for many of the yel-
low, orange, red, and purple colors distributed throughout na-
ture. More than 600 different, but closely related, structures
have been identified from biological sources (1,2), all of
which are very labile. Therefore, there is a need to establish a
rapid and reliable method for analyzing them (2). Fast atom
bombardment collision-induced dissociation (FAB CID)-
MS/MS methods have widely been used for the structural
analysis of natural products. Van Breemen et al. (3) reported
the structural analysis of 17 carotenoids by positive ion FAB-
MS/MS using a two-sector mass spectrometer with linked
scanning at a constant B/E [ratio of magnetic (B) and electric
(E) fields held at a constant] and high-energy collisionally acti-
vated dissociation. Recently, we reported the relationship be-
tween structure and the product ions obtained from molecular
.
ions (M+ ) by using four-sector tandem MS with high-energy
FAB CID-MS/MS spectra of 28 carotenoids (4).
Here, we report the characterization by high-energy FAB
CID-MS/MS of epoxy carotenoids possessing 5,6-epoxy, 5,8-
*To whom correspondence should be addressed at the Graduate School of Phar-
maceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
E-mail: akimoto@pharm.kyoto-u.ac.jp
Abbreviations: FAB CID-MS/MS, fast atom bombardment collision-induced
dissociation MS/MS; NBA, nitrobenzyl alcohol.
Copyright © 2004 by AOCS Press 179
epoxy, and 3,6-epoxy end groups conjugated to the polyene
chain and the structural analysis of an epoxy carotenoid iso-
lated from the oyster Crassostrea gigas by using this method.
EXPERIMENTAL PROCEDURES
FAB CID-MS/MS. MS/MS spectra were recorded using a
JEOL JMS-HX/HX 110A four-sector tandem mass spectrom-
eter. A few micrograms of sample dissolved in benzene was
placed on a stainless-steel probe tip and added to 1–2 µL of
m-nitrobenzyl alcohol (NBA) as a matrix. The positive FAB
ionization was achieved by bombardment with 6 kV xenon
atoms at an atom gun current of 5 mA. The accelerating volt-
.
age in the source was 10 kV. The radical cation M+ was se-
lected as a precursor by MS1, and collided with argon in a
collision cell located in the third field-free region. Argon gas
pressure was adjusted to attenuate the intensity of the precur-
sor ion by 30%. The collision cell potential was 3 kV. The re-
sulting product ions were acquired by accumulating several
linked scans on MS2.
Sample preparation. The isolation and purification of epoxy
carotenoids were performed by routine procedures. Violaxan-
thin (5,6,5′,6′-diepoxy-5,6,5′,6′-tetrahydro-β,β-carotene-3,3′-
diol; 1), auroxanthin (5,8,5′,8′-diepoxy-5,8,5′,8′-tetrahydro-β,β-
carotene-3,3′-diol; 2), cycloviolaxanthin (3,6,3′,6′-diepoxy-
3,6,3′,6′-tetrahydro-β,β-carotene-5,5′-diol; 3), antheraxanthin
(5,6-epoxy-5,6-dihydro-β,β-carotene-3,3′-diol; 4), mutatoxan-
thin (5,8-epoxy-5,8-dihydro-β,β-carotene-3,3′-diol; 5), and cu-
curbitaxanthin A (3,6-epoxy-3,6-dihydro-β,β-carotene-5,3′-
diol; 7) were isolated from the pumpkin, Cucurbita maxima (5);
and diadinochrome (5,8-epoxy-5,8-dihydro-7′,8′-didehydro-
β,β-carotene-3,3′-diol; 6) was isolated from the tunicate,
Amaroucium pliciferum (6), according to methods previously
described. An epoxy carotenoid (8) was isolated from the oys-
ter, C. gigas, according to routine procedures. The structures of
these carotenoids are shown in Scheme 1. The acetone extract
of the edible parts (purchased commercially in that form, hence
weight of intact animal unknown) of C. gigas (10 kg) was parti-
tioned between n-hexane/ether (1:1) and aqueous NaCl. The or-
ganic layer was dried over Na2SO4 and then concentrated to dry-
ness. The residue was subjected to column chromatography on
silica gel using an increasing percentage of acetone in n-hexane.
Lipids, Vol. 39, no. 2 (2004)
180 METHODS

RESULTS AND DISCUSSION

SCHEME 1
The fraction eluted with n-hexane/acetone (7:3) was further pu-
rified by HPLC on octadecylsilane with chloroform/acetonitrile
(1:9) to yield the epoxy carotenoid, 8 (0.5 mg). Details of the
isolation and structural elucidation of 8 including an NMR
study are reported in the literature (7).
In the positive-ion FAB MS spectra of carotenoids obtained
.
using m-NBA as a matrix, the radical cation [M]+ was predom-
inant rather than the protonated molecular ion [MH]+ (4). There-
.
fore, M+ was used as the precursor ion of CID-MS/MS for the
epoxy carotenoids investigated in the present study. Character-
istic product ions observed in epoxy carotenoids are listed in
Table 1. The nomenclature used to denote typical fragments re-
sulting from specific bond cleavages in the polyene chain is
shown in Scheme 2.
FAB CID-MS/MS spectra of symmetrical epoxy carotenoids.
Violaxanthin (1), auroxanthin (2), and cycloviolaxanthin (3) are
symmetrical epoxy carotenoids possessing the same molecular
formula, C40 H56O4. Violaxanthin (1), having a 3-hydroxy-5,6-
epoxy end group at both ends of the polyene chain, showed the
.
major product ions m/z 582 [M – 18]+ (elimination of water)
.
and 520 [M – 80]+ , a characteristic fragment ion observed in
the epoxy carotenoids (9,10). Furthermore, a series of product
ions resulting from the cleavage of C–C bonds in the polyene
chain, from the epoxy end group such as m/z 221 (c ion: attrib-
uted to cleavage between C10 and C11), 352 (g-H ion: attrib-
uted to cleavage between C14′ and C13′ accompanied by a hy-
drogen transfer to the C13′ side) and 419 (i ion: attributed to
cleavage between C9′ and C8′) was detected. However, no loss
.
of toluene [M – 92]+ was observed.
Auroxanthin (2), having a 3-hydroxy-5,8-epoxy (furan)
end groups on both sides of the polyene chain, showed almost
the same product ion patterns as 1. Furthermore, auroxanthin
exhibited additional product ions resulting from the cleavage
of single bonds in the polyene chain from the epoxy end
group, m/z 181 (b ion: attributed to cleavage between C8 and
C9), 247 (d ion: attributed to cleavage between C12 and C13),
287 (e ion: attributed to cleavage between C14 and C15), and
379 (h ion: attributed to cleavage between C11′ and C10′).
Cycloviolaxanthin (3), possessing a 3-hydroxy-3,6-epoxy
end group on both ends of the polyene chain, also showed m/z

TABLE 1
Fragment Ions Obtained by FAB CID-MS/MS of Epoxy Carotoids
1 2 3 4 5 6 7 8
M (m/z) 600 600 600 584 584 582 584 582
M – 18 582 582 582 566 566 564 566 564
M – 33 — — — — — 531 — 531
M – 80 520 520 520 504 504 502 504 502
M – 92 — — — 492 492 490 492 490
b — 181 181 181 181 181 181 181
c 221 221 221 221 221 221 221 221
d — 247 247 — — — — —
e — 287 286 (–H) — — — 286 (–H) 286 (–H)
f — — 312 (–H) — — — 312 (–H) 312 (–H)
g 352 (–H) 352 (–H) — 352 (–H) 352 (–H) 352 (–H) — —
h — 379 379 — — — — —
i 419 419 419 — — — — —
j — — 445 — — — — —
b′ — — — 404 (–H) 404 (–H) 402 (–H) — —
c′ — — 379 — — — 364 (+H) 362 (+H)
x′ — — — — — — 416 414

Lipids, Vol. 39, no. 2 (2004)

 METHODS
SCHEME 2
.
520 [M – 80]+ . The sequential product ions obtained by cleav-
age of all single bonds in the polyene chain except for cleavage
between C14′ and C13′ (g-H ion; m/z 352) were observed in cy-
cloviolaxanthin (3). Notably, m/z 286 (e-H ion: attributed to
cleavage between C14 and C15 and transfer of a hydrogen to the
C15 site) was detected, which was not observed in 5,6-epoxy or
5,8-epoxy carotenoids. On the other hand, m/z 352 (g-H ion) was
not observed for 3. These fragment patterns characterize the 3,6-
.
epoxy carotenoid. No loss of toluene [M – 92]+ was observed
in 2 or 3.
FAB CID-MS/MS spectra of asymmetrical epoxy carotenoids.
Antheraxanthin (4), having a 3-hydroxy-5,6-epoxy end group
and a 3-hydroxy-β-end group on each side of the polyene chain,
.
showed the major product ions m/z 566 [M – 18]+ , 504 [M –
+. +.
80] , and 492 [M – 92] with elimination of a neutral molecule
of toluene from the polyene chain (8). Furthermore, a series of
product ions resulting from the cleavage of C–C bonds in the
polyene chain from the epoxy end group side such as m/z 181 (b
ion), 221 (c ion), and 352 (g-H ion) were obtained. In addition
to these ions, 4 exhibited the diagnostic ion m/z 404 (b′ + H ion:
loss of the C1 to C8 moiety from the molecule by cleavage be-
M-80
M-18 (m/z 504)
Relative abundance
(m/z 566)
FIG. 1. Fast atom bombardment collision-induced dissociation (FAB CID)-MS/MS spectrum of M+. of mutatoxanthin (5).
181
tween C8 and C9 accompanied by a hydrogen transfer to the C8
site), which could provide structural information on the 3-
hydroxy-β-end group side of the molecule.
Mutatoxanthin (5), having a 3-hydroxy-5,8-epoxy (furan)
end group and a 3-hydroxy-β-end group on both ends of the
polyene chain, showed almost the same product ions as that
of 4 as shown in Figure 1.
Diadinochrome (6), a 7′,8′-didehydro analog of 5, also
showed almost the same fragmentation pattern—m/z 580 [M –
. . .
18]+ , 502 [M – 80]+ , 490 [M – 92]+ , 181 (b ion), 221 (c ion),
and 352 (g-H ion)—as 5, as shown in Figure 2. On the other
. .
hand, 6 showed [M – 33]+ (loss of CH3 and H2O from M+ ), a
fragment ion characteristic of carotenoids possessing an
acetylenic group at 7,8 and/or 7′,8′ in the polyene chain such as
alloxanthin, diatoxanthin, and halocynthiaxanthin (4). Moreover,
the shift down of b′ + H ion (m/z 402) by 2 mass units compared
with 5 agreed with the presence of an acetylenic group at the 3-
hydroxy-β-end group side (7′,8′ position) in 6.
Cucurbitaxanthin A (7), possessing a 3-hydroxy-3,6-epoxy
end group and a 3-hydroxy-β-end group, has the same molecu-
lar formula, C40 H56 O3, as 4 and 5 and also exhibited the prod-
. . .
uct ions m/z 582 [M – 18]+ , 504 [M – 80]+ , 492 [M – 92]+ ,
181 (b ion), and 221 (c ion) as shown in Figure 3. However, no
g-H ion was observed. On the other hand, 7 showed the diag-
nostic ions m/z 416 (x′ ion: cleavage of C=C bond between C7
and C8) and 364 (c′ + H ion). These fragmentation patterns can
be used to distinguish a 3,6-epoxy carotenoid from a 5,6-epoxy
or 5,8-epoxy carotenoid.
Application of FAB CID-MS/MS to the structural analysis
of the epoxy carotenoid isolated from oyster. After character-
izing CID-MS/MS fragments of known epoxy carotenoids, we
.
M+
M-18
(m/z 566)
.
[M-80]+
M-92 (m/z 492)
.
[M-80]+
566
m/z
Lipids, Vol. 39, no. 2 (2004)
182 METHODS

M-18
(m/z 564)
Relative abundance

M-80 M-92 (m/z 490)

M-18 (m/z 502)
(m/z 564)

m/z
+.
FIG. 2. FAB CID-MS/MS spectrum of M of diadinochrome (6). For abbreviation see Figure 1.

used this method for the structural analysis of an epoxy
carotenoid (8) isolated from the oyster, C. gigas. Compound 8
was found to have a molecular ion at m/z 582.4080, compatible
with the formula C40H54O3 by high-resolution EI-MS. The CID
spectrum of 8 (Fig. 3) showed product ions typical of a 3,6-
.
epoxy carotenoid, i.e., m/z 502 [M – 80]+ , 181 (b ion), 221 (c
ion), and 286 (e-H ion). Furthermore, 8 showed m/z 431 [M –
33]+, a product ion characteristic of acetylenic carotenoid (4).

M-18
(m/z 566)
M-80
(m/z 504)
M-92 (m/z 492)
M-18
(m/z 566)
Relative abundance

m/z
+.
FIG. 3. FAB CID-MS/MS spectrum of M of cucurbitaxanthin A (7). For abbreviation see Figure 1.

Lipids, Vol. 39, no. 2 (2004)

 METHODS
M-80
(m/z 502)
Relative abundance
M-18
(m/z 564)
m/z
FIG. 4. FAB CID-MS/MS spectrum of M+. of an epoxy carotenoid, 3,6-epoxy-5,6-dihydro-7′,8′-didehydro-β,β-
carotene-5,3′-diol (8), isolated from oyster. For abbreviation see Figure 1.
Moreover, as well as diadinochrome (6), the 2-mass unit shift
down of the x′ ion at m/z 414 and c′ + H ion at m/z 362 compared
with 7 clearly indicated the presence of an acetylenic group at
the 7′,8′ position in the polyene chain. Therefore, the planar
structure of 8 was postulated to be 3,6-epoxy-5,6-dihydro-7′,8′-
didehydro-β,β-carotene-5,3′-diol as shown in Figure 4. This
finding was supported by NMR analysis (7).
In conclusion, high-energy FAB CID-MS/MS was used to
clarify the relationship between the structure and product ions
of epoxy carotenoids possessing 5,6-epoxy, 5,8-epoxy, and 3,6-
epoxy end groups conjugated to the polyene chain. In addition
.
to [M – 80]+ , a fragment ion characteristic of an epoxy
carotenoid, product ions resulting from cleavage of C–C bonds
in the polyene chain from the epoxy end group such as m/z 181
(b ion) and 121 (c ion) were detected. On the other hand, the
diagnostic ion m/z 286 (e-H ion) was observed, not in the 5,6-
epoxy or 5,8-epoxy carotenoid, but in the 3,6-epoxy carotenoid.
Furthermore, no g-H ion was detected in 3,6-epoxy carotenoid.
These fragmentation patterns can be used to distinguish a 3,6-
epoxy carotenoid from a 5,6-epoxy or 5,8-epoxy carotenoid. The
structure of an epoxy carotenoid (8) isolated from oyster was
characterized using FAB CID-MS/MS by comparing fragmen-
tation patterns with those of related known compounds. There-
fore, high-energy FAB CID-MS/MS is a useful method for the
structural analysis of carotenoids.
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183
M-18
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M-92 (m/z 490)
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[Received September 15, 2003, and in revised form December 24,
2003; revision accepted January 12, 2004]
Lipids, Vol. 39, no. 2 (2004)