Mechanism of the formation of a dehydrated ion by an unusual loss of oxygen at the 4′-carbonyl group of abscisic acid methyl ester in electron ionization mass spectrometry

Article abstract of DOI:10.1002/jms.878

Methyl ester of abscisic acid (ABA), a plant hormone, gives a dehydrated ion at m/z 260 in electron ionization mass spectrometry (EI-MS). This dehydrated ion had been considered to be derived only from the elimination of the tertiary hydroxyl group at C-1′. We found that 34% of the dehydrated ion was formed by elimination of the oxygen atom at the 4′-carbonyl group, and the remaining 66% by elimination of the 1′-hydroxyl group. This unusual elimination of the carbonyl oxygen was shown with [4′-¹⁸O]ABA methyl ester. Involvement of the 4′-carbonyl oxygen in dehydration was observed in methyl ester of phaseic acid (PA), a natural metabolite of ABA, but not in 1′-deoxy-ABA methyl ester or isophorone. This suggested that the 1′-hydroxyl group was necessary for the elimination of the 4′-carbonyl oxygen. ABA methyl esters labeled with stable isotopes showed that hydrogen atoms at the 1′-hydroxyl group and at C-4 or -5 or -3′ or - 5′ or -7′ were eliminated with the 4′-carbonyl oxygen. These results allow us to propose a formation mechanism of the dehydrated ion derived from the elimination of 4′-carbonyl oxygen and hydrogen atoms at C-4 and 1′-oxygen in ABA methyl ester as follows: first, ionization at the 1′-hydroxyl group occurs to give an ion radical, and the proton at the 1′-oxygen migrates to the 4′-carbonyl oxygen after the bond fission between C-1′-C-6′; second, migration of the proton at C-4 to the 1′-oxygen is followed by migration of the protons at C-5 and C-7′ to C-4 and C-5, respectively; finally, the proton at the 1′-oxygen migrates to the 4′-hydroxyl group, and H₂O at C-4′ is eliminated to give the dehydrated ion. Our findings point out that a dehydrated ion is not always derived from the elimination of a hydroxyl group. Copyright

Full text of DOI:10.1002/jms.878

JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2005; 40: 1035–1043
Published online 22 June 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.878
Mechanism of the formation of a dehydrated ion by an
unusual loss of oxygen at the 4ꢀ-carbonyl group of
abscisic acid methyl ester in electron ionization mass
spectrometry
Masahiro Inomata,1 Nobuhiro Hirai,2∗ Naohito Takeda3 and Hajime Ohigashi1
1
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
International Innovation Center, Kyoto University, Kyoto 606-8501, Japan
Faculty of Pharmacy, Meijo University, Nagoya 468-8503, Japan
2
3
Received 18 February 2005; Accepted 13 April 2005
Methyl ester of abscisic acid (ABA), a plant hormone, gives a dehydrated ion at m/z 260 in electron
ionization mass spectrometry (EI–MS). This dehydrated ion had been considered to be derived only from
the elimination of the tertiary hydroxyl group at C-1ꢀ. We found that 34% of the dehydrated ion was formed
by elimination of the oxygen atom at the 4ꢀ-carbonyl group, and the remaining 66% by elimination of the
1ꢀ-hydroxyl group. This unusual elimination of the carbonyl oxygen was shown with [4ꢀ-18O]ABA methyl
ester. Involvement of the 4ꢀ-carbonyl oxygen in dehydration was observed in methyl ester of phaseic acid
(PA), a natural metabolite of ABA, but not in 1ꢀ-deoxy-ABA methyl ester or isophorone. This suggested
that the 1ꢀ-hydroxyl group was necessary for the elimination of the 4ꢀ-carbonyl oxygen. ABA methyl esters
labeled with stable isotopes showed that hydrogen atoms at the 1ꢀ-hydroxyl group and at C-4 or -5 or -3ꢀ
or - 5ꢀ or -7ꢀ were eliminated with the 4ꢀ-carbonyl oxygen. These results allow us to propose a formation
mechanism of the dehydrated ion derived from the elimination of 4ꢀ-carbonyl oxygen and hydrogen atoms
at C-4 and 1ꢀ-oxygen in ABA methyl ester as follows: first, ionization at the 1ꢀ-hydroxyl group occurs to give
an ion radical, and the proton at the 1ꢀ-oxygen migrates to the 4ꢀ-carbonyl oxygen after the bond fission
between C-1ꢀ –C-6ꢀ; second, migration of the proton at C-4 to the 1ꢀ-oxygen is followed by migration of the
protons at C-5 and C-7ꢀ to C-4 and C-5, respectively; finally, the proton at the 1ꢀ-oxygen migrates to the
4ꢀ-hydroxyl group, and H2O at C-4ꢀ is eliminated to give the dehydrated ion. Our findings point out that
a dehydrated ion is not always derived from the elimination of a hydroxyl group. Copyright  2005 John
Wiley & Sons, Ltd.
KEYWORDS: electron ionization mass spectrometry; dehydrated ion; abscisic acid; phaseic acid
The EI mass spectrum of ABA methyl ester gives
fragment ions at m/z 260 [M ꢀ H2O]C, 246 [M ꢀ MeOH]C,
222, 205, 190, 162, 134, 125 and 112 in addition to a molecular
ion at m/z 278 (Fig. 1a). Gray et al. proposed the structures
and formation mechanism of these fragment ions on the
basis of an analysis of ABA methyl esters labeled with stable
isotopes.3 The fragment ions at m/z 190 and 125 with high
intensities are formed by loss of methanol at C-1 following
elimination of isobutene consisting of carbon atoms at C-60, -
80 and -90 from the molecular ion, and by cleavage of the intact
side chain at C-5–C-10, respectively. They suggested that the
dehydrated ion at m/z 260 was formed by elimination of
the tertiary hydroxyl group at C-10, but a hydrogen atom
eliminated with the hydroxyl group had not been identified.
Hirai et al. revealed that about 65% of the dehydrated ion was
formed by elimination of the 10-hydroxyl group and 4- or 5-
hydrogen atoms bonded to a double bond, and proposed the
structure of the dehydrated ion.4 However, the eliminating
group giving the residual 35% of dehydrated ion was still
INTRODUCTION
A plant hormone, abscisic acid (ABA, 1) is a sesquiterpenoid
which induces tolerance to various forms of abiotic stress
including dehydration and low temperature, and regulates
seed dormancy and germination.1 ABA is produced not
only by higher plants but also by fungi,2 although the
physiological function of ABA in fungi is unknown.
6
8'
5'
9'
1'
2
OH4
7'
CO2R
O
3'
1: R = H
1-Me: R = Me
ŁCorrespondence to: Nobuhiro Hirai, International Innovation
Center, Kyoto University, Kyoto 606-8501, Japan.
E-mail: hirai@kais.kyoto-u.ac.jp
Copyright  2005 John Wiley & Sons, Ltd.

1036
M. Inomata et al.
Figure 1. EI mass spectra of methyl esters of ABA (a), [40-18O]ABA (b), PA (c) and [40-18O]PA (d). Relative intensities are not
corrected by natural 13C-, 2H- and 17O-isotopic abundance.
unknown. Recently, we found that 35% of the dehydrated
ion was formed by loss of the oxygen at the 40-carbonyl
group, in the course of labeling studies of ABA produced by
a fungus, Botrytis cinerea, with 18O2 and H2 18O.5 This paper
describes the unusual involvement of the 40-carbonyl oxygen
atom in the formation of the dehydrated ion, and proposes a
dehydration mechanism based on an analysis of compounds
labeled with stable isotopes.
the relative intensities of the ions were calculated from peak
areas. The mass spectra of [40-17O]ABA methyl ester, and
compounds 3 and 4 were given in the form of a bar graph.
The content of deuterium atom of [10-O-D,40-18O]ABA methyl
ester, and 18O contents of labeled ABA methyl esters and PA
methyl ester were calculated after correction by natural 13C-,
2H- and 17O-isotopic abundance. Percentages of dehydrated
ions derived from loss of 10-OH and 40-O in EI–MS of ABA
and PA methyl esters, and of hydrogen atoms involved in
dehydration in EI–MS of ABA methyl esters were shown as
the mean value š SD of three scans.
EXPERIMENTAL
General experimental procedures
1H and 17O NMR spectra were measured with a Bruker
Materials
1
ARX500 instrument (500 MHz for H and 68 MHz for 17O).
(šꢀ-ABA was purchased from Aldrich Chemical Co., Inc.,
Milwaukee, WI, USA. Isophorone was purchased from Wako
Pure Chemical Industries Ltd., Osaka, Japan. H2 18O (more
than 95 atom% 18O) was purchased from Nippon Sanso
Co., Kanagawa, Japan. H2 17O (48 atom% 17O), D2O and
CD3OD were purchased from Isotec Inc., Miamisburg, OH,
USA. Methyl esters of PA, [40-18O]PA,6 10-deoxy-ABA,7 [4-
D]ABA,4 [5-D]ABA4 and [30,50,50,70,70,70-D6]ABA4 which had
been prepared in our laboratory were used.
Chemical shifts were given with TMS (υ 0.00 ppm) as the
internal standard for 1H NMR analysis, and with H2O
(υ 0.00 ppm) as the external standard for 17O NMR analysis.
Direct EI mass spectra were measured with a JEOL JMS-600H
mass spectrometer, the temperature of the direct probe being
°
°
°
increased from 50 C to 450 C at a rate of 128 C/min. The
°
instrument was operated at a chamber temperature of 250 C,
an accelerating voltage of 3 kV, an ionization voltage of 70 eV,
and an ionization current of 300 µA in the positive ion mode,
the resolution being 1000 during the measurement. The
GC/EI mass spectrum of compound 4 was recorded on the
above mass spectrometer equipped with a Hewlett Packard
HP6890 instrument, using an HP-5 column (30 m length
ð0.32 mm i.d., 5% diphenyl-95% dimethylpolysiloxane, film
thickness 0.25 µm, Hewlett Packard Co., Wilmington, DE,
USA), and 1.0 ml/min of He flow. The parameters of
the mass spectrometer were the same as those for direct
electron ionization mass spectrometry(EI–MS). The oven
[4ꢀ-17O] and [4ꢀ-18O]ABA methyl ester
ABA (10 mg) was dissolved in 2 ml of MeOH, and an ether
solution of CH2N2 was added. After standing for 1 h at room
temperature, the solution was concentrated to give methyl
ester of ABA (10 mg). 1H NMR (CDCl3ꢀ : υ 1.02 (3H, s, H-90),
1.11 (3H, s, H-80), 1.93 (3H, d, J D 1.2 Hz, H-70), 2.01 (3H, d,
J D 1.0 Hz, H-6), 2.30 (1H, d, J D 17.1 Hz, H-50proRꢀ, 2.48 (1H,
d, J D 17.1 Hz, H-50proSꢀ, 3.71 (3H, s, 1-OCH3), 5.76 (1H, s, H-
2), 5.95 (1H, s, H-30), 6.16 (1H, d, J D 16.1 Hz, H-5), 7.87 (1H,
d, J D 16.1 Hz, H-4). EI–MS (probe) 70 eV: m/z (rel. int.): 278
[M]C (3), 260 (6), 246 (4), 222 (6), 205 (6), 190 (100), 162 (29),
134 (26), 125 (28), 112 (6), 91 (9). ABA methyl ester (10 mg)
was dissolved in a mixture of 50 µl of acetic anhydride and
°
temperature for GC was programmed to increase from 50 C
°
°
to 100 C at a rate of 5 C/min. ABA and phaseic acid (PA)
methyl esters and their labeled compounds (except for [40-
17O]ABA methyl ester) were measured in profile mode, and
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

Dehydration of abscisic acid methyl ester in EI–MS 1037
17
°
of the compositions of the dehydrated ions was difficult.
The compositions of the dehydrated ions were calculated
using those of methyl esters of [40-18O]ABA and [10-O-
D]ABA: this method was used to calculate compositions
of dehydrated ions of [4-D,40-18O]ABA, [5-D,40-18O]ABA and
[30,50,50,70,70,70-D6,40-18O]ABA methyl esters. The percentage
of [M C 3 ꢀ DH 18O]C ion in the dehydrated ion formed by
loss of 40-18O was calculated using the following equation:
200 µl of H2 O, and the solution was stirred at 45–50 C for
15 h. After the addition of 2 ml of toluene to the solution, the
mixture was concentrated to give [40-17O]ABA methyl ester
(10 mg). 17O NMR (CDCl3ꢀ : υ 541.9 (O-40). EI–MS (probe)
70 eV: m/z (rel. int.): 280 [M C 2]C (1), 279 [M C 1]C (3), 278
[M]C (2), 262 (1), 261 (4), 260 (7), 247 (7), 223 (5), 206 (5),
205 (6), 191 (100), 190 (87), 163 (26), 162 (29), 135 (21), 134
(28), 125 (49), 112 (13), 91 (13); the 17O content was calculated
from relative intensities of the molecular ions and found to
be 48%. In the same manner as described for the preparation
of [40-17O] ABA methyl ester, ABA methyl ester (9 mg) was
reacted with 45 µl of acetic anhydride and 180 µl of H2 18O,
and the reaction mixture was concentrated to give [40-18O]
ABA methyl ester (9 mg). EI–MS (probe) 70 eV: m/z (rel.
int.): 280 [M C 2]C (4), 278 [M]C (0.3), 262 (6), 260 (4), 248 (7),
224 (8), 207 (7), 192 (100), 190 (17), 164 (38), 162 (8), 136 (23),
134 (20), 125 (50), 112 (16), 91 (16).
25% ꢁpercent [M C 3 ꢀ DH 18O]C ion at m/z 260ꢀ
f6% ꢁpercent [M C 3 ꢀ H2 18O]C ion at m/z 261ꢀ
C25% ꢁpercent[M C 3 ꢀ DH 18O]C ion at m/z 260ꢀg
ð 100% D 81%
[4-D,4ꢀ-18O]ABA methyl ester
[4-D,40-18O] ABA methyl ester (2 mg) was prepared from
[4-D] ABA methyl ester (2 mg) containing 8% and 92%
deuterium at H-2 and H-4, respectively, with 15 µl of acetic
anhydride and 45 µl of H2 18O by the same method as that
described for the preparation of [40-18O]ABA methyl ester. 1H
NMR (CDCl3ꢀ : υ 1.02 (3H, s, H-90), 1.11 (3H, s, H-80), 1.93 (3H,
d, J D 1.2 Hz, H-70), 2.01 (3H, d, J D 0.9 Hz, H-6), 2.30 (1H, d,
J D 17.1 Hz, H-50proRꢀ, 2.48 (1H, d, J D 17.1 Hz, H-50proSꢀ, 3.71
(3H, s, 1-OCH3), 5.76 (92% of 1H, s), 5.95 (1H, s, H-30), 6.15
(1H, br.s, H-5), 7.88 (8% of 1H, d, J D 16.0 Hz). EI–MS (probe)
70 eV: m/z (rel. int.): 282 [M C 4]C (0.9), 281 [M C 3]C (4), 280
[M C 2]C (0.6), 279 [M C 1]C (0.6), 264 (0.6), 263 (2), 262 (4),
261 (2), 260 (2), 249 (6), 225 (8), 208 (4), 193 (100), 192 (72),
191 (12), 190 (8), 165 (20), 164 (23), 137 (18), 135 (15), 126 (33),
113 (8), 92 (6). The 18O content was calculated from relative
intensities of the molecular ions to be 88%. The deuterium
contents at H-2 and H-4 were unchanged, as confirmed by the
1H NMR spectrum. The percentages of [M C 3 ꢀ DH 18O]C
and [M C 3 ꢀ DHO]C ions in the dehydrated ions formed by
loss of 40-18O and 10-OH, respectively, were calculated using
the following equations:
EI mass spectra of methyl esters of PA and [4ꢀ-18O]
PA, and of compounds 3 and 4
PA methyl ester: EI–MS (probe) 70 eV: m/z (rel. int.): 294
[M]C (24), 276 (21), 263 (12), 177 (27), 167 (47), 163 (36),
154 (40), 139 (44), 135 (34), 125 (100), 122 (78), 121 (68). [40-
18O]PA methyl ester: EI–MS (probe) 70 eV: m/z (rel. int.):
296 [M C 2]C (46), 294 [M]C (3), 278 (7), 276 (26), 265 (18), 177
(28), 169 (40), 163 (43), 154 (48), 141 (52), 135 (38), 127 (91), 125
(60), 122 (100), 121 (74); the 18O content was calculated from
relative intensities of the molecular ions and found to be
94%. Compound 3: EI–MS (probe) 70 eV: m/z (rel. int.): 262
[M]C (2), 247 (1), 231 (7), 215 (4), 189 (4), 174 (40), 146 (100),
125 (100), 119 (24), 112 (19), 103 (13), 91 (25). Compound 4:
GC/EI–MS (tR 7.3 min) 70 eV: m/z (rel. int.): 138 [M]C (28),
123 (3), 95 (4), 82 (100), 67 (2), 54 (9).
[1ꢀ-O-D,4ꢀ-18O]ABA methyl ester
[40-18O] ABA methyl ester (5 µg) in a quartz capillary (for
a direct probe of mass spectrometer) was dissolved in
3 µl of CD3OD, and the solution was dried by heating.
After this procedure had been repeated twice more, the
sample was dissolved in a mixture of 3 µl of CD3OD and
2 µl of D2O. The solution was concentrated to about 1 µl
by heating, and the capillary was immediately introduced
into the ionization chamber of the mass spectrometer with
the direct probe. To reduce back-exchange by H2O, 1 µl of
D2O in the capillary was introduced twice to the ionization
chamber before analysis of the sample. Substitution of the
10-hydroxyl proton with a deuterium ion was confirmed
by the disappearance of the 10-hydroxyl proton signal at
2.18 ppm in an 1H NMR spectrum of a CDCl3 solution
of [40-18O] ABA methyl ester (9 mg/0.7 ml) after addition
of 20 µl of D2O. EI–MS (probe) 70 eV: m/z (rel. int.): 281
[M C 3]C (3), 280 [M C 2]C (1), 279 [M C 1]C (0.5), 263 (3),
262 (4), 261 (1), 260 (3), 248 (6), 225 (6), 208 (4), 193 (88),
192 (100), 191 (11), 190 (11), 165 (24), 164 (22), 136 (20), 134
(16), 125 (49), 112 (16), 91 (11). The deuterium content at the
10-hydroxyl group and 18O content at C-40 were calculated
from relative intensities of the molecular ions and found
to be 79% and 88%, respectively. Since [10-O-D,40-18O]ABA
methyl ester was labeled with deuterium and 18O, calculation
Percent [M C 3 ꢀ DH 18O]C ion in the dehydrated
ion formed by loss of 4’- 18
O
9% ꢁpercent [M C 3 ꢀ DH 18O]C ion at m/z 260ꢀ
f21% ꢁpercent [M C 3 ꢀ H2 18O]C ion at m/z 261ꢀ
C9% ꢁpercent [M C 3 ꢀ DH 18O]C ion at m/z 260ꢀg
D
ð 100% D 30%
Percent [M C 3 ꢀ DHO]C ion in the dehydrated
ion formed by loss of 1’-OH
45% ꢁpercent [M C 3 ꢀ DHO]C ion at m/z 262ꢀ
D
f24% ꢁpercent [M C 3 ꢀ H2O]C ion at m/z 263ꢀ
C45% ꢁpercent [M C 3 ꢀ DHO]C ion at m/z 262ꢀg
ð 100% D 65%
Preparation of [5-D,4ꢀ-18O]ABA methyl ester
[5-D,40-18O]ABA methyl ester (2 mg) was prepared from [5-
D]ABA methyl ester (2 mg) containing 95% deuterium at
H-5 with 15 µl of acetic anhydride and 45 µl of H2 18O by
the same method as that described for the preparation of
1
[40-18O]ABA methyl ester. H NMR (CDCl3ꢀ : υ 1.02 (3H, s,
H-90), 1.11 (3H, s, H-80), 1.93 (3H, d, J D 1.1 Hz, H-70), 2.01
(3H, s, H-6), 2.30 (1H, d, J D 17.1 Hz, H-50proRꢀ, 2.48 (1H, d,
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

1038
M. Inomata et al.
J D 17.1 Hz, H-50proSꢀ, 3.71 (3H, s, 1-OCH3), 5.76 (1H, s, H-2),
5.95 (1H, s, H-30), 6.16 (5% of 1H, d, J D 16.1 Hz), 7.87 (1H,
br.s, H-4). EI–MS (probe) 70 eV: m/z (rel. int.): 281 [M C 3]C
(3), 280 [M C 2]C (0.3), 279 [M C 1]C (0.7), 263 (3), 262 (1),
261 (3), 260 (0.7), 249 (4), 225 (6), 208 (4), 193 (100), 192 (20),
191 (36), 190 (6), 165 (24), 164 (13), 137 (18), 135 (18), 126
(38), 112 (10), 92 (7). The 18O content was calculated from
relative intensities of the molecular ions and found to be
82%, and the deuterium content at H-5 was unchanged. The
percentages of [M C 3 ꢀ DH 18O]C and [M C 3 ꢀ DHO]C ions
in the dehydrated ions formed by loss of 40-18O and 10-OH,
respectively, were calculated using the following equations:
Percent [M C 8 ꢀ DHO]C ion in the dehydrated
ion formed by loss of 1’-OH
23% ꢁpercent [M C 8 ꢀ DHO]C ion at m/z 267ꢀ
D
f45% ꢁpercent [M C 8 ꢀ H2O]C ion at m/z 268ꢀ
C23% ꢁpercent [M C 8 ꢀ DHO]C ion at m/z 267ꢀg
ð 100% D 34%
RESULTS AND DISCUSSION
C-40 of ABA methyl ester was labeled with 18O from H2 18O
by hydration under the acidic condition. Introduction of 18
O
into C-40 was confirmed by the NMR analysis of [40-17O]ABA
methyl ester which was prepared with H2 17O by the same
method as that used for the preparation of [40-18O]ABA
methyl ester. Typical 17O NMR chemical shift ranges are
ꢀ50 to C140 ppm for alkoxy oxygen in ester group, ꢀ40 to
C100 ppm for oxygen at hydroxyl group, C290 to C380 ppm
for carbonyl oxygen in ester group, and C470 to C615 ppm
for oxygen at ketone.8 The 17O NMR spectrum of the 17O-
labeled ABA methyl ester showed a broad singlet signal at
541.9 ppm assignable to the O-40, showing that oxygen only
at C-40 was labeled under the acidic condition.
Percent [M C 3 ꢀ DH 18O]C ion in the dehydrated
ion formed by loss of 4’- 18
O
5% ꢁpercent [M C 3 ꢀ DH 18O]C ion at m/z 260ꢀ
f30% ꢁpercent [M C 3 ꢀ H2 18O]C ion at m/z 261ꢀ
C5% ꢁpercent [M C 3 ꢀ DH 18O]C ion at m/z 260ꢀg
D
ð 100% D 14%
Percent [M C 3 ꢀ DHO]C ion in the dehydrated
ion formed by loss of 1’-OH
9% ꢁpercent [M C 3 ꢀ DHO]C ion at m/z 262ꢀ
[40-18O]ABA methyl ester (Fig. 2) gave molecular ions
corresponding to [M C 2]C and [M]C at m/z 280 and 278,
respectively, in a 92 : 8 ratio (Fig. 1b, Table 1), indicating that
the 18O content was 92%. Its dehydrated ions [M C 2 ꢀ H2O]C
and [M C 2 ꢀ H2 18O]C/[M ꢀ H2O]C were observed at m/z
262 and 260, respectively, in a 62 : 38 ratio. This showed that
the dehydrated ion of ABA methyl ester was formed not
only by loss of the 10-hydroxyl group but also by loss of
the 40-carbonyl oxygen. The percentages of dehydrated ions
[M C 2 ꢀ H2O]C and [M C 2 ꢀ H2 18O]C were calculated to
be 67% and 33%, respectively, using the following equations:
D
f55% ꢁpercent [M C 3 ꢀ H2O]C ion at m/z 263ꢀ
C9% ꢁpercent [M C 3 ꢀ DHO]C ion at m/z 262ꢀg
ð 100% D 14%
[3ꢀ,5ꢀ,5ꢀ,7ꢀ,7ꢀ,7ꢀ-D6, 4ꢀ-18O]ABA methyl ester
[30,50,50,70,70,70-D6]ABA methyl ester (2 mg) containing 97%,
93% and 96% deuterium at H-30, H-50 and H-70, respectively,
was dissolved in a mixture of 15 µl of acetic anhydride
and 45 µl of H2 18O, and the solution was left at room
temperature for 16 h. Two milliliters of toluene was added,
and the solution was concentrated to give [30,50,50,70,70,70-
D6,40-18O]ABA methyl ester (2 mg). 1H NMR (CDCl3ꢀ : υ 1.01
(3H, s, H-90), 1.11 (3H, s, H-80), 1.89 (4.5% of 3H, br.s), 2.01
(3H, d, J D 1.1 Hz, H-6), 2.29 (7% of 1H, br.s), 2.46 (7% of
1H, br.s), 3.71 (3H, s, 1-OCH3), 5.76 (1H, s, H-2), 5.94 (3%
of 1H, br.s), 6.15 (1H, d, J D 16.2 Hz, H-5), 7.88 (1H, d,
J D 16.2 Hz, H-4). EI–MS (probe) 70 eV: m/z (rel. int.): 286
[M C 8]C (4), 285 [M C 7]C (1), 284 [M C 6]C (2), 283 [M C 5]C
(0.5), 268 (3), 267 (2), 266 (3), 265 (2), 264 (0.5), 254 (4), 228
(6), 213 (3), 196 (100), 195 (20), 194 (40), 193 (7), 168 (21),
167 (12), 166 (13), 165 (6), 140 (10), 139 (8), 138 (13), 125
(34), 112 (8), 95 (6). The 18O content was calculated from
relative intensities of the molecular ions and found to be
69%, and the deuterium contents at H-30, H-50 and H-70
were unchanged. The percentages of [M C 8 ꢀ DH 18O]C and
[M C 8 ꢀ DHO]C ions in the dehydrated ions formed by loss
of 40-18O and 10-OH, respectively, were calculated using the
following equations:
Percent [M C 2 ꢀ H2O]C ion
62% ꢁpercent dehydrated ion at m/z 262ꢀ
92% ꢁpercent molecular ion at m/z 280ꢀ
D
ð 100% D 67%
Percent [M C 2 ꢀ H2 18O]C ion
f38% ꢁpercent dehydrated ion at m/z 260ꢀ
ꢀ8% ꢁpercent [M ꢀ H2O]C ion at m/z 260ꢀg
D
92% ꢁpercent molecular ion at m/z 280ꢀ
ð 100% D 33%
These percentages in the mass spectra of the other scans
were also calculated, and the mean of dehydrated ions
[M C 2 ꢀ H2O]C and [M C 2 ꢀ H2 18O]C of three scans were
67 š 3% and 33 š 3%, respectively. This meant that 33% of
OD
OH
OH
CO2Me
CO2Me
-D,4'-18O]-1-Me
CO2Me
D
18O
Percent [M C 8 ꢀ DH 18O]C ion in the dehydrated
18O
18O
[4'-18O]-1-Me
[1'-
O
[4-D,4'-18O]-1-Me
ion formed by loss of 4’- 18
O
D
D
D
18O
5% ꢁpercent [M C 8 ꢀ DH 18O]C ion at m/z 265ꢀ
f27% ꢁpercent [M C 8 ꢀ H2 18O]C ion at m/z 266ꢀ
C5% ꢁpercent [M C 8 ꢀ DH 18O]C ion at m/z 265ꢀg
OH
CD3
OH
OH
D
CO2Me
O
CO2Me
18O
CO2Me
18O
D
[3',5',5',7',7',7'-D6,4'-18O]-1-Me
[5-D,4'-18O]-1-Me
[4'-18O]-2-Me
ð 100% D 16%
Figure 2. Labeled methyl esters of ABA and PA.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

Dehydration of abscisic acid methyl ester in EI–MS 1039
the dehydrated ion was derived from loss of the 40-carbonyl
oxygen and 67% from loss of the 10-hydroxyl group. The
difference between the relative intensities of the dehydrated
ions at m/z 262 and 260 can be used to distinguish the
18O-labeled position between C-10 and C-40 of ABA methyl
ester.5 If C-10 is labeled with 18O, the relative intensities of
the dehydrated ions at m/z 262 and 260 should be 33% and
67%, respectively.
PA (2) is a natural metabolite of ABA, and formed by
80-hydroxylation and isomerization of ABA in plants.9 PA
methyl ester gives an [M]C ion at m/z 294 and an [M ꢀ H2O]C
ion at m/z 276 (Fig. 1c, Table 1). Dehydration by loss
of a carbonyl oxygen was observed in PA methyl ester
also. [40-18O]PA methyl ester (Fig. 2) showing molecular
ions [M C 2]C and [M]C at m/z 296 and 294, respectively,
in a 94 : 6 ratio (Fig. 1d, Table 1), gave dehydrated ions
[M C 2 ꢀ H2O]C and [M C 2 ꢀ H2 18O]C/[M ꢀ H2O]C at m/z
278 and 276, respectively, in a 20 : 80 ratio. This showed that
PA methyl ester gave the dehydrated ion derived from loss of
the 40-carbonyl oxygen in addition to that derived from loss
of the 10-hydroxyl group. The percentages of dehydrated
ions [M C 2 ꢀ H2O]C and [M C 2 ꢀ H2 18O]C derived from
the molecular ion [M C 2]C were calculated in the same
manner as that described for [40-18O]ABA methyl ester to be
21 š 2% and 79 š 2%, respectively. This result indicated that
the dehydrated ion of PA methyl ester was mainly formed
by loss of the 40-carbonyl oxygen, and dehydration by loss of
the 10-hydroxyl group was minor.
and 4 did not show dehydrated ions at m/z 244 and 120,
respectively. This finding revealed that the 10-hydroxyl group
was necessary for dehydration by loss of the 40-carbonyl
oxygen of ABA and PA methyl esters. The 10-hydroxyl
hydrogen may be eliminated with the 40-carbonyl oxygen
as water. We examined this possibility with [10-O-D,40-
18O]ABA methyl ester (Fig. 2). [10-O-D,40-18O]ABA methyl
ester showed a dehydrated ion [M C 3 ꢀ DH 18O]C at 25%
of all the dehydrated ions derived from the molecular
ion [M C 3]C at m/z 281 (Table 2). The percentage of
[M C 3 ꢀ DH 18O]C ion in the dehydrated ions derived from
[M C 3]C ion by loss of 40-18O was 81% (for the calculation,
see the experimental section). This finding showed that
one hydrogen atom of water derived from the 40-carbonyl
oxygen was the 10-hydroxyl hydrogen, confirming that the
10-hydroxyl group was necessary for dehydration by loss of
the 40-carbonyl oxygen.
OH
O
CO2R
CO2Me
O
O
O
2: R = H
3
4
2-Me: R = Me
Another hydrogen atom involved in dehydration by loss
of the 40-carbonyl oxygen seemed to be derived from C-4
or -5 or -30 or -50 or -70, since these hydrogen atoms were
eliminated to give the dehydrated ion of ABA methyl ester.4
[4-D,40-18O], [5-D,40-18O] and [30,50,50,70,70,70-D6,40-18O]ABA
methyl esters (Fig. 2) were prepared, and their EI–MS
were analyzed. Relative intensities and compositions of
molecular and dehydrated ions of these methyl esters are
summarized in Table 2. [4-D,40-18O]ABA methyl ester gave
the dehydrated ion [M C 3 ꢀ DH 18O]C at m/z 260 at 9% of all
the dehydrated ions derived from the molecular ion [M C 3]C
ABA and PA have a tertiary hydroxyl group at C-10.
In order to obtain clear evidence of the elimination of the
40-carbonyl oxygen in the absence of the tertiary hydroxyl
group, we examined EI mass spectra of 10-deoxy-ABA methyl
ester (3) and isophorone (4) which do not possess a tertiary
hydroxyl group at the position corresponding to C-10 of
ABA and PA methyl esters. Unexpectedly, compounds 3
Table 1. Relative intensitiesa and compositionsb of molecular and dehydrated ions of nonlabeled and 18O-labeled ABA and
PA methyl esters
Nonlabeled ABA methyl ester
[40-18O]ABA methyl ester
m/z
Ion species
[M]C
rel. int. (%)
3.4
Comp. (%)
100
m/z
Ion species
rel. int. (%)
Comp. (%)
278
280
278
262
260
[M C 2]C
3.4
0.3
5.6
3.5
92
8
62 (67)c
30 (33)c
8
[M]C
260
[M ꢀ H2O]C
6.1
100
[M C 2 ꢀ H2O]C
[M C 2 ꢀ H2 18O]C
[M ꢀ H2O]C
Nonlabeled PA methyl ester
[40-18O]PA methyl ester
m/z
Ion species
rel. int. (%)
24.1
Comp. (%)
100
m/z
Ion species
rel. int. (%)
Comp. (%)
294
[M]C
296
294
278
276
[M C 2]C
45.7
3.1
6.4
94
6
20 (21)c
74 (79)c
6
[M]C
276
[M ꢀ H2O]C
21.1
100
[M C 2 ꢀ H2O]C
[M C 2 ꢀ H2 18O]C
[M ꢀ H2O]C
25.6
a Relative intensities were corrected by natural 13C-, 2H- and 17O-isotopic abundance.
b Compositions of relative intensities of each ion group.
c Compositions of dehydrated ions [M C 2 ꢀ H2O]C and [M C 2 ꢀ H2 18O]C.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

1040
M. Inomata et al.
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

Dehydration of abscisic acid methyl ester in EI–MS 1041
Table 3. Percentages of hydrogen atoms involved in dehydration by loss of the 40-carbonyl oxygen and 10-hydroxyl group in EI–MS
of ABA methyl ester
Percentage of hydrogen atoms involved in each dehydrated ionb
Eliminating groups
involved in dehydration
Order of
hydrogen
Percenta,b
10-OH
4-H
5-H
30,50,70-H6
Others (%)
Total (%)
40-Carbonyl oxygen
33 š 3%
First
Second
81 š 0%
–
–
–
19%
41%
–
100%
100%
117%
–
–
–
–
30 š 1%
68 š 3%
–
14 š 1%
14 š 2%
–
15 š 1%
35 š 1%
–
10-Hydroxyl group
Total
67 š 3%
–
–
100%
–
a Percentage of each dehydrated ion in the total dehydrated ions.
b Percentages shown are the mean value š SD of three scans.
at m/z 281, and this ion consisted of 30% of the dehydrated
ions derived from loss of 40-18O (for the calculation, see
the experimental section). This result showed involvement
of 4-hydrogen in dehydration by loss of the 40-carbonyl
oxygen. The dehydrated ion [M C 3 ꢀ DHO]C at m/z 262
corresponded to the dehydrated ion formed by loss of the
10-hydroxyl group and deuterium atom at C-4. This ion
accounted for 65% of the dehydrated ions derived from the
loss of 10-OH, confirming the involvement of 4-hydrogen
in the dehydration by loss of the 10-hydroxyl group.4 [5-
D,40-18O]ABA methyl ester showed the dehydrated ion
[M C 3 ꢀ DH 18O]C at m/z 260 in 5% of all the dehydrated
ions derived from the molecular ion [M C 3]C at m/z
281, and the percentage of [M C 3 ꢀ DH 18O]C ion in the
dehydrated ions derived from loss of 40-18O was 14%
(for the calculation, see the experimental section). This
result showed that the involvement of 5-hydrogen in the
dehydration by loss of the 40-carbonyl oxygen was small.
Nine percent of the dehydrated ion [M C 3 ꢀ DHO]C in
all the dehydrated ions derived from the molecular ion
[M C 3]C was detected at m/z 262. The [M C 3 ꢀ DHO]C
ion was produced at 14% in the dehydrated ions derived
from loss of 10-OH. [30,50,50,70,70,70-D6,40-18O]ABA methyl
ester gave the dehydrated ion [M C 8 ꢀ DH 18O]C at m/z
265 in 5% of all the dehydrated ions derived from the
molecular ion [M C 8]C at m/z 286, and the percentage of
[M C 8 ꢀ DH 18O]C ion in the dehydrated ions formed by loss
of 40-18O was 16% (for the calculation, see the experimental
section). This result showed that 30,50,70-hydrogen atoms
were partially involved in dehydration by loss of the 40-
carbonyl oxygen. [30,50,50,70,70,70-D6,40-18O]ABA methyl ester
showed 23% of [M C 8 ꢀ DHO]C ion at m/z 267 of all the
dehydrated ions derived from the molecular ion [M C 8]C,
revealing that 30,50,70-hydrogen atoms were eliminated with
the 10-hydroxyl group also. [M C 8 ꢀ DHO]C ion accounted
for 34% of the dehydrated ions derived from loss of 10-OH.
We could not examine the contribution of each hydrogen
atom at C-30, -50 or -70 to the dehydration because of the
difficulty with selective deuteration at these carbons.
water was derived from the hydrogen atom at C-4 or -5 or
-30 or 50 or 70, and the sum of the percentages of the second
hydrogen atoms was 59%. The origin of the residual 19% of
the first hydrogen and 41% of the second hydrogen atoms
could not be identified in this experiment. The hydrogen
atom eliminated with the 10-hydroxyl group as water was
derived from C-4 or -5 or -30 or 50 or 70 as well as the second
hydrogen eliminated with the 40-carbonyl oxygen. The sum
of percentages of hydrogen atoms eliminated with the 10-
hydroxyl group was 117%. This would be due to analytical
errors, and the sum should be close to 100%, suggesting that
other hydrogen atoms were not involved in dehydration by
loss of the 10-hydroxyl group.
We propose the formation mechanism of the dehydrated
ion derived from loss of the 40-carbonyl oxygen, and 10-
hydroxyl hydrogen and 4-hydrogen on the basis of the
above results (Fig. 3). The first step of the fragmentation is
ionization at the 10-hydroxyl group to give the ion radical 5.
The proton at 10-oxygen of 5 should migrate to 40-carbonyl
oxygen, so that it may be lost with the 40-carbonyl oxygen.
This migration needs the proximity of these two groups,
and seems to proceed by a similar mechanism to that in
dehydration by loss of the 40-hydroxyl groups in EI–MS of
10,40-trans- and 10,40-cis-diol-ABA methyl esters.4 The bond
fission between C-10-C-60 of the ion radical 5 gives 6, and
bond rotation at C-30-C-40 of the ion radical 6 forms 7. In the
ion radical 7, 10-hydroxyl hydrogen is close to 40-oxygen, and
the proton migrates from the 10-hydroxyl group to 40-oxygen
to give the ion radical 8. Successively, migration of double
bonds in 8 forms the ion radical 9. Migration of protons from
C-5 to C-4 and from C-70 to C-5 following proton migration
from C-4 to 10-oxygen gives the ion radical 10. A proton
at 10-oxygen of the ion radical 10, which is derived from
C-4, migrates to 40-oxygen to give the ion radical 11, and
then H2O at C-40 of 11 is eliminated to give the dehydrated
ion 12. Cyclization of the ion radical 12 by attack of an
unpaired ion at C-60 to C-10 gives the dehydrated ion 13
with an ABA-like skeleton. The dehydrated ion 13 may be
converted to other resonance forms. Dehydration by loss
of the 40-carbonyl oxygen with 10-hydroxyl hydrogen and
5-hydrogen seems to occur by a similar mechanism to that
with 10-hydroxyl hydrogen and 4-hydrogen. If the bond at
C-5-C-10 of the ion radical 9 is rotated, the distance between
5-hydrogen and 10-oxygen becomes closer. Migration of a
proton at C-5 to 10-oxygen and of a proton at C-70 to C-5
Percentage of hydrogen atoms involved in dehydration
by loss of the 40-carbonyl oxygen and 10-hydroxyl group
are summarized in Table 3. The first hydrogen atom
eliminated with the 40-carbonyl oxygen as water was 10-
hydroxyl hydrogen, and the percentage was 81%. The second
hydrogen atom eliminated with the 40-carbonyl oxygen as
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

1042
M. Inomata et al.
•
•
•+
+
1-Me
OH
OH
+
CO2Me
CO2Me
O
OH
CO2Me
CO2Me
O
O
5
6
7
H
H
•
•
•
+
+
+
OH
H
OH
O
O
CO2Me
OH
O
CO2Me
OH
8
9
10
•
•
−H2O
CO2Me
O•
+
Others
+
OH2
CO2Me
O
CO2Me
+
11
12
13
Figure 3. Dehydration by loss of the 40-carbonyl oxygen with 10-hydroxyl hydrogen and 4-hydrogen.
O
O
O
+
•
•
−H2O
•
O •
Others
9
+
+
+
OH2
OH
H
14
15
16
13
Figure 4. Dehydration by loss of the 40-carbonyl oxygen with 10-hydroxyl hydrogen and 70-hydrogen.
forms the ion radical 10, and the subsequent pathway is
the same as that shown in Fig. 3. The contribution of each
hydrogen atom at C-30, -50 or -70 to dehydration by loss
of the 40-carbonyl oxygen is unknown, as described above.
However, 70-hydrogen is likely to be eliminated with the 40-
carbonyl oxygen, since the distance between 40-oxygen and
by changes of the skeleton, including bond fission and rota-
tion. This would be the first case for dehydration by loss of
the carbonyl oxygen in nonaromatic and non-quinone-type
compounds. We should remind ourselves that the presence
of a dehydrated ion does not always mean the presence of a
hydroxyl group and sometimes means the presence of a car-
bonyl group. The positive CI mass spectrum of ABA methyl
ester gives a dehydrated ion at m/z 261 [MH ꢀ H2O]C as the
base peak in addition to a molecular ion [MH]C at m/z 279.14
The oxygen involved in the dehydration of ABA methyl ester
has been shown to be derived from the 10-hydroxyl group,
not from the 40-carbonyl oxygen. The 10-hydroxyl group
would be lost with a proton derived from a reagent gas to
give the dehydrated ion in positive CI-MS. Dehydration by
loss of the 40-carbonyl oxygen of ABA methyl ester would be
a fragmentation characteristic of EI–MS.
0
°
7 -hydrogen decreases if a 180 rotation occurs around the
C-20-C-30 bond of 9. The structure of the ion radical 14 formed
by this bond rotation is shown in Fig. 4. After migration of
a proton at C-70 to 40-oxygen in 14 to form the ion radical
15, elimination of the H2O molecule from 15 gives the ion
radical 16. The ion radical 16 is cyclized by the attack of an
unpaired ion at C-60 to C-10 to give the ion radical 13, and
then the ion may be converted to other resonance forms. The
possibilities of migration of protons in the ion radicals 9 and
14 and of existence of positive electric charge on the double
bonds in the ion radicals 12, 13, and 16 appear to be small.
However, these structures and structural exchanges could
not be avoided to explain our experimental results. In these
pathways of dehydration, the 10-hydroxyl group is not only
a source of a proton for H2O but also a mediator for the
proton migration from C-4 or -5 to 40-carbonyl oxygen. This
dehydration mechanism may apply to dehydration by loss
of the 40-carbonyl oxygen of PA methyl ester.
CONCLUSIONS
Detection of a dehydrated ion in EI–MS is often used to prove
the occurrence of a hydroxyl group in organic compounds,
and a dehydration mechanism derived from the elimination
of a hydroxyl group is usually proposed to explain the
formation of the dehydrated ion. However, the dehydrated
ion is not always derived from the elimination of a hydroxyl
group, and involvement of carbonyl oxygen in dehydration
is not restricted to aromatic and quinone-type compounds.
We have shown here that 34% of the dehydrated ion of ABA
methyl ester is derived from the elimination of the 40-carbonyl
oxygen and hydrogen atoms at the 10-hydroxyl group, and
mainly at C-4. For PA methyl ester, the percentage of the
dehydrated ion derived from elimination of the 40-carbonyl
oxygen is up to 79%. There are many natural compounds
having carbonyl and hydroxyl groups. We should consider
the involvement of a carbonyl group in the formation of
dehydrated ions, and analyze any compound labeled with
18O at carbonyl oxygen when involvement of carbonyl
oxygen in dehydration is plausible. We have proposed a
Dehydration in EI–MS usually occurs with elimina-
tion of a hydroxyl group,10 so dehydration by loss of the
carbonyl oxygen is an interesting fragmentation. Dehy-
dration by loss of the carbonyl oxygen is known to
occur in some aliphatic ketones and acyclic aldehyde,
and in o-methoxybenzaldehyde, o-methoxyacetophenone, o-
methoxybenzophenon, and peri-methoxyquinone.11,12 As the
mechanism for dehydration of these aromatic compounds,
migration of a proton at a methoxy group to the carbonyl
oxygen by a proximity effect is also suggested.13 In these
compounds, the hydrogen atom of the methoxy group and
oxygen atom of the carbonyl group are close enough to cause
a proximity effect. For ABA methyl ester, the proximity effect
for loss of the carbonyl oxygen as water may be achieved
Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043

Dehydration of abscisic acid methyl ester in EI–MS 1043
7. Todoroki Y, Hirai N, Koshimizu K. Synthesis and biological
activity of 10-deoxy-10-fluoro- and 80-fluoroabscisic acids.
Phytochemistry 1995; 40: 633.
mechanism for dehydration derived from the elimination
of the 40-carbonyl oxygen in ABA methyl ester, but more
examples may be necessary to find a rule for dehydration
derived from the elimination of carbonyl oxygen.
8. Baumstark AL, Boykin DW. 17O NMR spectroscopy:
applications to structural problems in organic chemistry. Adv.
Oxygenated Process 1991; 3: 141.
9. Zeevaart JAD. Abscisic acid metabolism and its regulation.
In Biochemistry and Molecular Biology of Plant Hormones,
Hooykaas PJJ, Hall MA, Libbenga KR (eds). Elsevier Science:
Amsterdam, 1999; 189.
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Copyright  2005 John Wiley & Sons, Ltd.
J. Mass Spectrom. 2005; 40: 1035–1043