Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 426

and low background (Tarkowski et al. 2009 ). Selective ion monitoring (SIM) mode,
in which only specific characteristic ions of the target compound are measured,
provides higher sensitivity and selectivity than monitoring all ions simultaneously,
a data acquisition mode named full scan. A better alternative is the multiple reac-
tions monitoring (MRM) mode with higher selectivity based on the tandem mass
spectrometry (MS/MS) technique, which can be performed either in time or space,
corresponding to triple quadrupole mass spectrometry (QQQ MS) and ion trap
mass spectrometry (IT MS), respectively. In MRM, a precursor mass ion is selected
in the first-stage quadrupole and then fragmented in the collision cell to yield diag-
nostic product ions filtered by the third-stage quadrupole. A signal is detected only
when the selected precursor ion passes the first-stage quadrupole and the selected
product ion passes the third-stage quadrupole; thus each ionized compound gives a
distinct precursor-to-product ion transition in the MRM mode, which is diagnostic
for the presence of a particular compound in an extract (Fu et al. 2011 ).
Although chromatographic methods have high separation efficiency, direct
loading of crude extracts onto chromatographic columns would cause separa-
tion efficiency deteriorating, irreparable column damage and fouling, and MS
signal suppressing, and thus, purification and enrichment of the crude extracts
is necessary. Overall, advances of highly sensitive MS techniques have greatly
reduced the amount of plant material required and overcome the low detectability
of ABA. In recent years, LC-MS/MS becomes the most accurate technique for
ABA analysis (López-Carbonell and Jáuregui 2005 ; Fletcher and Mader 2007 ;
Pan et al. 2008 , 2010 ; Ma et al. 2008 ; Izumi et al. 2009 ; Balcke et al. 2012 ; Yu
et al. 2013 ).
Since the metabolomics is of more and more importance in the mechanism
and cross-talk research of ABA (Forcat et al. 2008 ; Kanno et al. 2010 ); Pan et al.
( 2008 ) developed a rapid and sensitive method for simultaneous quantification of
multiple classes of phytohormones including ABA and some related metabolites
in 50-100 mg of fresh Arabidopsis leaves without purification or derivatization.
After being frozen in liquid nitrogen, the leaves were ground into powder, and
500 ʼ L of 1-propanol/H 2 O/concentrated HCl (2:1:0.002, vol/vol/vol) with internal
standards were added, followed by agitation for 30 min at 4 °C. One milliliter of
CH 2 Cl 2 was added, followed by agitation for another 30 min and then centrifu-
gation at 13,000 g for 5 min. After centrifugation, two phases were formed and
plant debris was in the middle of two layers. The lower layer was concentrated
and re-solubilized in 0.3 mL of MeOH and then 25 ʼ L was injected to column for
analysis. The mixtures of standard compounds were separated by reversed-phase
HPLC and analyzed by tandem mass spectrometry (RP-HPLC/ESI-MS/MS) in
the MRM mode. The identities of phytohormones in the crude plant extracts were
confirmed by analysis of product ion fragments obtained by the hybrid triple quad-
rupole/linear ion trap mass spectrometry, operating in the information dependent
acquisition (IDA) mode. A variety of synthetic, isotopically labeled, or modified
compounds were selected as internal standard. The extraction efficiencies of the
phytohormones in this study ranged from 85 to 98 % in one round of extraction.
By this method, one person can easily analyze 80 samples in 6 h.