Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 427

Forcat et al. ( 2008 ) also described an efficient method for the rapid quan-
titative determination of ABA. Plant material was harvested into liquid nitrogen
and freeze dried, then 10 mg of powdered tissue was weighed into a new 2-mL
microfuge tube and extracted with 400 ʼ L of 10 % methanol containing 1 % ace-
tic acid to which internal standards had been added in a bead beater with 3-mm
tungsten beads for 2 min, placed on ice for 30 min then centrifuged at 13,000 g for
10 min at 4 °C. The supernatant was carefully removed and the pellet re-extracted
with 400 ʼ L of 10 % methanol containing 1 % acetic acid. Following a further
30 min incubation on ice, the extract was centrifuged and the supernatants pooled.
The two extractions resulted in 90-95 % recovery of the targeted analytes. The
extracts (50 ʼ L) were analyzed by HPLC-electrospray ionization/MS-MS using
an Agilent 1100 HPLC coupled to an Applied Biosystems Q-TRAP 2000 in the
MRM mode. The method requires minimal 10 mg freeze dried tissue and is highly
reproducible and can accurately measure ABA across the expected physiological
dynamic range. Moreover, it compares well with other methods that have more
complex extraction methods. The use of freeze dried material promotes ease of
handling and automation, and it is more convenient for scaling up extraction, espe-
cially dealing with multiple samples during a time course analysis.
A simple and fast protocol for ABA analysis developed by our group is as
follows: 50-200 mg of fresh plant tissue was well ground with a small glass pestle
in a 2-mL vial. Following the addition of 1 mL of 80 % methanol, homogenates
were well mixed in an ultrasonic bath and then kept at 4 °C overnight. After being
centrifuged at 15,200 g for 10 min, the supernatant was collected and then vacu-
umed to dryness in a Jouan RCT-60 concentrator. Dried extract was dissolved in
200 ﾵL of sodium phosphate solution (0.1 mol/L, pH 7.8) and later passed through
a Waters Sep-Pak C 18 cartridge. The cartridge was eluted with 1500 ﾵL of 80 %
methanol and the eluate was vacuumed to dryness again. After being dissolved
in 50 ﾵL of 10 % methanol and injected 5 ﾵL into the Shimadzu LC-MS/MS
8030 system in which an Acquity UPLC BEH column (2.1 mm I.D. 100 mm,
1.7 ʼ m) was used. The column temperature was set at 40 °C. Elution of the sam-
ples was carried out with 0.02 % aqueous acetic acid (solvent A) and acetonitrile
(solvent B), and a gradient mode [(min/%/%) for 0/90/10, 3/20/80; 5.0/20/80,
6.0/90/10] at a flow rate of 0.25 mL/min. MS/MS conditions were as follows: col-
lision energy − 18.0 eV, m/z 181/134.1. The mass spectrometer was set to MRM
mode using ESI in negative ion mode, with a nebulizing gas flow at 3L/min, a
drying gas flow at 15L/min, a desolvation temperature at 250 °C, a heat block
temperature at 480 °C. 2 H 6 -ABA was used as an internal standard. For ABA, the
ionization conditions (pre-bias voltages of 19 V for quadrupole 1 and 28 V for
quadrupole 3; collision energy of 10 eV; m/z of 263/153.2) were employed. While
for 2 H 6 -ABA, the ionization conditions (pre-bias voltages of 20 V for quadrupole
1 and 15 V for quadrupole 3; collision energy of 11 eV; m/z of 269/159.2) were
employed. The ABA concentrations were calculated according to calibration curve
which created by internal standard of deuterium labeled ABA. The method is
capable to analyze pg level ABA in about 100 mg of fresh plant samples.