612
A. NETTING AND R. O. LIDGARD
9
chromatographed immediately on a Chiracel OD column
(
4.6 ð 250 mm) from Daicel Chemical Industries (Tokyo,
ꢀ1
Japan) using propanzol–hexane (1 : 9) at 0.8 ml min
.
The fractions containing (C)-, (2E) and (ꢀ)-MeABA were
collected separately and immediately saponified with 60%
°
KOH–EtOH (1 : 2) at 40 C for 30 min. After acidification
and dilution with water, the (C)-, (2E)- or (ꢀ)-ABA was
extracted three times with diethylether, the latter being
dried over Na SO . The ABA enantiomers/isomers were
2
4
chromatographed using four developments on thin layers
using the conditions mentioned above and the appropriate
bands removed. After extraction from the silica the ABA
Figure 1. Structure and numbering system for abscisic acid
ABA).
(
enantiomers/isomers were finally purified and quantified
separated by multiple development on a silica gel 60
TLC plate (Merck, Kilsyth, Victoria, Australia) with
toluene–ethyl acetate–acetic acid (60 : 30 : 4) contain-
2
by HPLC as described above. Further [sc- H ]ABA could
4
2
be obtained by isomerizing the (2E)-[sc- H ]ABA in sun-
4
light and separating the two isomers by TLC. Aliquots
0
ing butylated hydroxytoluene. The band containing [1 -
2
of the resulting [sc- H ]ABA were converted to PFB[sc-
1
8
4
O]ABA was scraped off and extracted with ethanol,
2
2
H ]ABA (see below) or to Me[sc- H ]ABA with CH N
0
18
4
4
2
2
the latter being evaporated. The desired [1 - O]ABA was
purified and quantified by high-performance liquid chro-
matography (HPLC) (HP 1090, Hewlett-Packard, Wald-
bronn, Germany) on a Zorbax SB-C18 column (9.4 ð
for GC/MS.
2
[
Ring- H]ABA. ABA (0.1 mg) was dissolved in ¾1 M
2
2
NaO H in H O (1.0 ml), allowed to stand overnight at
2
2
50 mm, 5 µm; Activon Scientific, Thornleigh, NSW,
room temperature and then acidified with oxalic acid.
Australia) using MeOH–0.2% aqueous acetic acid (7 : 3)
Dilution with water, extraction with diethylether and
ꢀ
1
at 2 ml min . Diode-array detection was used with the
2
evaporation gave the desired [ring- H ]ABA (principally
6
pilot wavelength at 254 š 2 nm. An aliquot was methy-
0 2
0 2
0 2
[
3 - H, 5 - H , 7 - H ]ABA). Again, an aliquot was
methylated to give Me[ring- H ]ABA.
2
3
0
18
lated with CH N to give Me[1 - O]ABA and the PFB
2
2
2
6
ester prepared as described below.
0
18
Automated synthesis of pentafluorobenzyl esters
[
4 - O]ABA. ABA (0.5 mg) was suspended in 50 µl of
18
18
H2 O (97% O; Novachem, South Yarra, Australia) with
µl of acetic acid to give a final pH of 3.5. The mixture
was incubated in the dark at room temperature for 72 h.
Evaporation under N gave [4 - O]ABA and methylation
with diazomethane resulted in Me[4 - O]ABA. Incuba-
tion of 0.5 mg of MeABA with the same mixture of H
and acetic acid gave Me[4 - O]ABA.
1
The following procedure was completed on a PrepStation
SPE Module (Hewlett-Packard, Wilmington, DE, USA).
The appropriately labelled ABA was taken up in 10 µl
of dimethylacetamide (DMA)–tetramethylammonium
hydroxide (TMA-OH) (5 : 1). This was followed by 10 µl
of DMA–PFB bromide (10 : 3) whereupon the total was
mixed for 1 min. Water–butanol–hexane (2 : 1 : 10) was
added, mixed and the hexane layer extracted and evap-
orated to give PFBABA. A silica column (0.1 g) was
washed with hexane and the PFBABA was transferred
to it using hexane. The column was then washed with
dichloromethane and the PFBABA eluted with 0.15 ml
ethyl acetate, which was evaporated. Full details of the
methods for extracting ABA and its precursors from
plant material, for their derivatizations on the PrepStation
and for their quantification by GC/MS will be reported
elsewhere.
0
18
2
0
18
18
O
2
0
18
[2
(
2
H]MeABA. ABA (100 µg) was dissolved in C H OH
0.2 ml) and diazomethane (5 ml) that had been dis-
tilled from CH O H (1 ml) was added. After standing
for 20 min the diazomethane was evaporated to give the
desired [ H ]MeABA. Any Me[5 - H]ABA produced as a
by-product of this procedure was back-exchanged by incu-
bation for 30 min at room temperature in MeOH–H O
pH 9.0) (1 : 1).
3
2
3
2
0 2
1
2
(
2
2
2
[
Side-chain- H]ABA. [Side-chain- H]ABA ([sc- H ]ABA)
4
was prepared by an adaption of the method described
previously. Approximately 10 mg of ABA (Sigma-
4
Gas chromatography/mass spectrometry
Aldrich, Castle Hill, NSW, Australia) were methylated
with CH N and, after evaporation and drying under vac-
2
2
A ThermoQuest (Austin, Tx, USA) GCQ gas chromato-
graph/mass spectrometer operating in the electron capture
negative ionization mode was used. The multiplier gain
was set at 100 V above the optimum obtained by tun-
ing in the electron impact mode (typically 1300–1400 V).
Methane was used as the reagent gas for all the experi-
ments reported here.
2
uum, dissolved in dry (molecular sieve) CH O H (2.5 ml).
3
2
Then 0.6 M NaOCH in CH O H was prepared from Na
pieces and dry CH O H and added to the MeABA to give
3
3
2
3
a 0.3 M solution of NaOCH . After flushing with N this
3
2
was stored in the dark for ¾2 months. After an equal vol-
2
ume of H O had been added the MeABA was extracted
2
immediately three times with hexane. If H O had been
A standard split/splitless injector was used in the split-
less mode and 1 µl of sample containing ¾500 pg of ana-
lyte for MeABA or ¾100 pg for PFBABA was injected.
2
used there was a possibility that deuterium in the Me[sc-
2
H ]ABA could have exchanged out owing to the presence
4
2
°
of MeOH from the MeO H–H O equilibrium. Since the
For MeABA samples, the injector temperature was 200 C
2
2
H tends to exchange out of MeABA on standing, the
and the column (BPX5, 220 µm ð 25 m, from SGE, Ring-
°
hexane solution was dried over Na SO , evaporated and
wood, Victoria, Australia) was held at 80 C for 1 min
2
4
Copyright 1999 John Wiley & Sons, Ltd.
J. Mass Spectrom. 34, 611–621 (1999)
Netting
