Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 420

with mortar or tissue lyser. No matter what method is selected, the homogenizing
process of tissue must be kept at low temperature to minimize the ABA loss
caused by enzymatic metabolism and chemical degradation.
The extracting method should be based on the physicochemical properties of
ABA, and the ideal method would provide high extraction efficiency. Solvent
extraction is still the most widely used method for ABA extraction. Plant mate-
rial extracted in distilled water has also been reported the same extraction effi-
ciency as organic solvents for ABA in several plant tissues (Loveys and van Dijk
1988 ; Quarrie et al. 1988 ; Vernieri et al. 1989 ). However, most of the reports show
that the polarity of the extraction solvent should match closely that of the target
compound, and thereby, the ratio of organic solvent to water has been defined
according to the polarity of ABA (Fu et al. 2011 ). Many procedures for extrac-
tion have been developed and diverse solvents such as chloroform, methanol, etha-
nol, acetone, propanol, ethylacetate, and acetic acid have been used (Barkawi et al.
2010 ; Vine et al. 1987 ; Ross et al. 2004 ; Vilaró et al. 2006 ; Novák et al. 2008 ;
Engelberth et al. 2003 ). In the organic solvents, methanol has become the pre-
ferred solvent for its small molecular size and low molecular weight (Fu et al.
2011 ). In our practice of ABA analysis, 80 % methanol is applied for ABA extrac-
tion with good results (Li et al. 2012 ). The modified Bieleski's solvent (methanol/
formic acid/water 15:1:4) is a good choice for the simultaneous extraction of mul-
tiple classes of phytohormones, including auxins, CTKs, ABA, JAs, and a number
of related metabolites, because it blocks the enzymatic degradation of phytohor-
mones without extracting large quantities of lipids (Kojima et al. 2009 ; Izumi et al.
2009 ; Giannarelli et al. 2010 ).
21.2.2 Sample Purification
Determination of ABA can be hindered by hundreds of other abundant primary
and secondary metabolites co-extracted by organic solvents, the purification and
enrichment of ABA is crucial for the final assay. An increasing number of methods
and techniques have been adapted for plant sample purification by considering
both the physicochemical properties of ABA and the method types of analysis.
21.2.2.1 Liquid-Liquid Extraction
Liquid-liquid extraction (LLE) is based on the solubility difference of target
analytes between two liquid phases. The most classical extraction is performed
in separating funnels to extract analytes from an aqueous sample solution into a
nonpolar or less polar organic solvent. Many organic solvents, such as hexane,
diethyl ether, butanol, ethyl acetate, dichloromethane/isopropanol, and methyl-
ene chloride/1-propanol, have been used to purify phytohormones including ABA
(Schmelz et al. 2003 ; Duffield and Netting 2001 ; Blake et al. 2002 ; Durgbanshi