Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 422

the sorbents, spherical particles of hydrophobic silica bonded with octadecyl chain
(C 18 ) are the most widely used for the sample pre-purification. When the SPE col-
umn is conditioned with weak acid solution and the plant extracts' pH is adjusted
with weaker acid, the acidic ABA will remain the neutral form and can be trapped
on the C 18 column after sample loading. Then after, ABA is eluted with the corre-
sponding acidic aqueous solution of methanol or ethanol (Fu et al. 2011 ).
With the progress of analysis techniques, high-performance liquid chroma-
tography (HPLC) has also been used for sample pre-purification. HPLC can be
regarded as a special solid-phase extraction technique and can enrich the tar-
get hormones efficiently and remove interfering compounds significantly.
Fractionation by HPLC is more accurate and reliable than that by other methods.
However, HPLC has disadvantages such as longer running time, more organic sol-
vents consumption and higher cost compared to ordinary SPE methods.
Solid-phase microextraction (SPME) is a more developed SPE technique which
allows simultaneous extraction and enrichment of analytes from different sample
matrices (Prosen and Zupancic-Kralj 1999 ; Ouyang and Pawliszyn 2006 ). Since
SPME was introduced in the early 1990s, it has been widely applied in combina-
tion with GC/HPLC to the sampling and analysis of food, aroma, forensic, environ-
mental, and pharmaceutical samples (Kataoka et al. 2000 ; Ouyang and Pawliszyn
2006 ). SPME was also used for the purification of phytohormones and plant
growth regulators including ABA, IAA, indole-3-butyric acid (IBA), and NAA in
plant samples. SPME was capable of selective extraction of phytohormones with
high recovery and was simpler and faster than other procedures for phytohormonal
extraction (Liu et al. 2007 ). As a sampling and sample preparation method, it elim-
inates the need for solvents and combines sampling, isolation and enrichment in
one step, and is suitable for plant samples of small quantities. However, application
of SPME is still limited because of the commercial availabilities of fiber coatings
and special fiber desorption chamber to couple with the GC/HPLC systems.

21.2.2.3 Immunoaffinity Purification

Immunoaffinity purification methods are based on antibody-antigen (Ab-Ag)
recognition, and this specific interaction can provide extremely selective enrich-
ment of the sample and thus greatly enhance the sensitivity of the analysis
(Tarkowski et al. 2009 ). Several papers have reported on the immunoaffinity chro-
matography (IAC) purification and immunoaffinity gel (IAG) purification of ABA,
the results suggest that the method is useful for quantifying ABA in plant material
and represents the advantage of a short-time sample preparation with a high accu-
racy and capability. (Va

ková et al. 1998 ; Hauserová et al. 2005 ; Hradecká et al.
2007 ; López-Carbonell et al. 2009 ).
Immunoaffinity purification methods purify analytes according to struc-
tural similarities, so the setup for a suitable affinity system requires a highly
specific anti-ABA antibody. However, as small organic molecules, ABA and
its metabolites are haptens, which caused problems for specific recognition by

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