Principles and Practice of ABA Analysis - Abscisic Acid: Metabolism, Transport and Signaling - page 425

Although immuno-based methods are excellent for the detection of ABA
because of their high selectivity and sensitivity, the specificity of antibodies
restricts the applications of the method in the simultaneous detection of multiple
classes of phytohormones. On the other hand, the immuno-based methods are less
dependent on large equipment and are more amenable to miniaturization, they
are more portable and suitable for in situ screening of target hormones in plant
tissues (Bai et al. 2010 ). Compared with the existing chromatographic methods,
though the established immuno-based methods are not as sensitive as LC-MS/MS,
even LC-MS combined with solid-phase extraction, they offer the advantages of
fast detection, simplicity in operation because no complex purification steps are
required prior to analysis, thus are frequently used today.
21.2.3.2 Chromatographic Methods
Gas chromatography (GC) was the earliest chromatographic method used for
quantitative analysis of ABA (Lenton et al. 1968 ; Davis et al. 1968 ). As ABA is
not a volatile compound, it needs to be derivatized to increase the volatility and
also to improve the thermal stability prior to GC-based analysis (Fu et al. 2011 ).
After the application of mass spectrometry (MS) detector, GC with MS (GC-MS)
was more reliable and specific for identification and quantification of ABA (Li
et al. 1992 ; Mller et al. 2002 ). Although GC-based methods provide high resolu-
tion and low limit of detection, they are generally not used currently to analyze
ABA because they are labor-intensive and costly, more and more studies turn to
use alternative techniques such as HPLC or LC-MS.
HPLC, which enables rapid, high-resolution purification of ABA from extracts,
can provide relatively reliable identification and quantification of ABA by UV
absorbance without derivatization when using a UV detector. Therefore, HPLC
coupled with UV detector was a popular tool used for ABA analysis in a long
period of time. However, it is inadequate for the accurate measurement of ABA
only based on the retention time and UV or fluorescence spectroscopy because
many components may be present in one peak with a complicated matrix. Owing
to the introduction of MS detector, HPLC combined with MS detector (LC-MS),
especially with tandem MS detectors (LC-MS/MS), overcomes the disadvantages
of HPLC with UV detector and become a more powerful and more commonly
applied method for ABA determination. Ultra-performance liquid chromatography
(UPLC), which can achieve higher resolution, higher sensitivity and more rapid
separation than traditional HPLC, has also been employed to combine with tan-
dem MS detectors (UPLC-MS/MS) for ABA analysis in recent years (Mller and
Munn￩-Bosch 2011 ; Fu et al. 2012 ; Balcke et al. 2012 ; Niu et al. 2013 ).
MS is an analytical technique that measures the mass-to-charge ratio ( m/z ) of
charged particles. The analytes are ionized in the ion source, and then formed an
ion beam to pass through the ion guide system, followed by analysis with a mass
analyzer based on the m / z ratio (Fu et al. 2011 ). Electrospray ionization (ESI) is the
most common ion source used in MS systems because of its fairly high sensitivity