Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 419

have been described to analyze ABA in plants, and significant technical advances
on quantifying ABA are achieved (Weiler 1979 ; Blintsov and Gusakovskaya 2006 ;
Forcat et al. 2008 ; Hou et al. 2008 ; Su et al. 2013 ). Because of the good separation
and qualitative abilities, chromatographic methods coupled with mass spectrom-
etry (MS), especially with tandem MS, have become the most powerful tools for
ABA analysis from complex plant samples.
Accurate quantitative and qualitative analysis of ABA is difficult because of its
instability and low physiological concentrations in plants (in the ng/g fresh weight
range) (Weiler 1979 ). One of the challenges is to enrich these low-level ABA from
crude plant extracts in which large amounts of interfering substances are present.
In addition, the limited quantity of plant tissue available for ABA quantification is
another challenge. Consequently, the analytic methods must be extremely selective
and sensitive (Fu et al. 2011 ). Even the advanced tandem MS systems are used,
many random errors that bring about inaccurate and imprecise analysis of ABA are
still unavoidable. This chapter reviews the principles and practice of ABA analysis
by focusing on the recent advances.
21.2 Sample Preparation
The determination of trace ABA in complex biological samples often requires
extensive sample preparation techniques prior to analysis, whether quantitative
or qualitative. Sample preparation is thus of great importance in the rapid sep-
aration and ultra-sensitive analysis of trace ABA, and more sample preparation
techniques, including sampling, extracting, purifying, and concentrating meth-
ods, have been developed to analyze ABA (Bai et al. 2010 ; Fu et al. 2011 ). For
the analysis of ABA, sample preparation generally comprises several procedures,
including sampling, freeze-drying, comminuting, homogenization, exhaustive
extraction from the matrix, and subsequent removal of co-extracted interferences
by several cleanup steps prior to instrumental analysis. Such a preliminary sam-
ple preparation is often coupled with ABA loss, but the loss can be estimated and
corrected by recovery analysis of the sample preparation processes using isotope-
labeled ABA.
21.2.1 Sample Extraction
The concentrations and distribution of phytohormones including ABA in plant tis-
sues are affected by temperature, watering regime, air humidity, light intensity,
and many other environmental conditions (Peleg and Blumwald 2011 ; Alcázar
and Parker 2011 ; Qaderi et al. 2012 ; Susawaengsup et al. 2011 ). Therefore, it is
important to freeze the collected fresh tissues in liquid nitrogen immediately after
detaching the tissues from plants, and then, the tissues are ground into fine powder