Study on the Techniques of Simultaneous Analysis of Various Phytohormones by Gas Chromatography-Mass Spectrometry（GC-MS）
Date Issued
2011
Date
2011
Author(s)
Yang, Tzu-Yin
Abstract
The techniques of simultaneous analysis of phytohormones refer to a series of procedures that begin with extraction of various phytohormones simultaneously,
followed by comprehensive derivatization, adequate purification and concentration, and the final quantification and qualification via instrumental analysis that possess high sensitivity and selectivity. In this study, derivatization protocols and sample pre-treatment methods for phytohormone standards were studied by using GC-MS as the analytical tool.
Three derivatization reagents, Trimethylsilyldiazomethane （ TMSD ）,
N,O-bis(trimethylsilyl)trifluoroacetamide,（BSTFA）, N-Methyl-N-(tert-butyldimethylsilyl)
trifluoroacetamide（MTBSTFA）, were applied to react with four phytohormone standards, indole-3-acetic acid（IAA）, abscisic acid（ABA）, jasmonic acid （JA）, and salicylic acid（SA）under 25、60、80 or 100℃ for 30, 60, or 90 min followed by GC-MS analysis. Results showed that four phytohormones were successfully derivatized by the three derivatization reagent. However, TMSD would trimethylsilylate and methylate IAA, ABA, and SA at the same time causing problems in qualification; while BSTFA and MTBSTFA would trimethysilylate and tert-butyldimthylsilylate four phytohormones, respectively. Furthermore, derivatization products of MTBSTFA yield
stronger signal responses with no need for higher reaction temperature or longer reaction time. Hence, MTBSTFA was considered to be the recommended derivatization reagent and the optimum reaction condition was 25℃for 30 min.
For concentration and purification of the phytohormone derivatives, two extraction techniques, solid phase microextraction（SPME）and vapor phase extraction（VPE）,
were compared based on the concentration effect and the ease of handling. Although both SPME and VPE displayed proper concentration ability on phytohormonederivatives, the former method took more time on adsorption and desorption, which is more time-consuming than the latter one. In addition, VPE performed steadily during 1~5 min extraction time period with high recoveries（96.9~119.5 %）of TBDMS derivatives of four phytohormones. Consequently, the VPE method was considered as the recommended concentration method and the optimum extraction condition was 2 minutes at 200℃ per sample.
In this study, two important steps during the simultaneous analysis of phytohormones: derivatization and sample pre-treatment were studied. Optimum conditions for these two steps in analyzing four phytohormone standards were
established. The results showed enhanced detectability of target analytes and should be very useful in the further development of a complete procedure for simultaneous
analysis of plant hormones in plant tissues.
Subjects
Phytohormones
simultaneous analysis
derivatization
solid phase microextraction (SPME)
vapor phase extraction (VPE)
gas chromatography-mass spectrometry (GC-MS)
Type
thesis
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