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