以氣相層析質譜儀同步分析多種植物荷爾蒙技術之研究

Abstract

植物荷爾蒙的同步分析技術，是指同時將植物體中之多種植物荷爾蒙萃取出來，經衍生及純化濃縮後，以具有高靈敏度之分析儀器以將分析物進行定性和定量的分析技術。本試驗以氣相層析質譜儀（gas chromatography-mass spectrometry, GC-MS）作為分析儀器，利用植物荷爾蒙標準品，探討衍生化反應和衍生物純化及濃縮之最適條件。 以吲哚乙酸（indole-3-acetic acid, IAA）、離層酸（abscisic acid, ABA）、茉莉酸（jasmonic acid, JA）及水楊酸（salicylic acid, SA）四種植物荷爾蒙標準品為材料，利用三甲基矽烷重氮甲烷（Trimethylsilyldiazomethane, TMSD）、N,O-雙三甲基矽烷基三氟乙醯胺（N,O-bis(trimethylsilyl)trifluoroacetamide, BSTFA）及N-第三丁基 二甲基矽基-N- 甲基三氟乙醯胺（ N-Methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide,MTBSTFA）為衍生劑，以GC-MS 觀察在25、60、80 和100℃不同衍生溫度下，反應時間分別為30、60 和90 分鐘時各植物荷爾蒙之衍生情形。四種植物荷爾蒙經TMSD、BSTFA 或MTBSTFA 衍生後，皆能於不同滯留時間下產生相對應之成分峰。TMSD 不僅會將IAA、ABA 及SA 進行甲基酯化，亦會產生該分析物之三甲基矽烷化產物，較不利於定性；而BSTFA 及MTBSTFA 兩種衍生劑則分別可將四種植物荷爾蒙衍生為其三甲基矽烷化及丁基二甲基矽烷化之產物；其中以MTBSTFA 作為衍生劑時，所產生的衍生物以GC-MS 分析其反應量較強又穩定，加上衍生反應進行時不需另外提高反應溫度，且在較短的時間內即可完成衍生。因此選擇MTBSTFA 作為此技術之所使用的衍生試劑，最適條件為於25℃下反應30 分鐘。 在樣品前處理部分利用固相微萃取（solid phase microextraction, SPME）及氣相萃取（vapor phase extraction, VPE）兩種方式，將濃縮效果、操作便利性作為考量因子，探討於此分析技術上應用之可能性。雖SPME 及VPE 對樣品皆有濃縮效果並也都能成功吸附衍生產物，但SPME 在操作上較為耗時，每個樣品吸附時間約需10 分鐘，且有樣品脫附不完整的情形，容易影響分析結果。相較之下，以VPE 萃取各植物荷爾蒙TBDMS 衍生物時，萃取時間1~5 分鐘皆有穩定的反應量，且回收率高（96.9~119.5 %）能夠有效在短時間內達到濃縮純化樣品的效果。因此在樣品前處理方面，選擇以VPE 作為濃縮純化之工具，最適萃取條件為於200℃下萃取2 分鐘。 本研究確立了可運用於植物荷爾蒙同步分析技術中之衍生劑之種類和衍生反應條件，以及適當的樣品純化及濃縮方式及條件，以有效增加微量植物荷爾蒙於儀器中的偵測訊號，提高分析物的可偵測性，將有助於未來植物荷爾蒙之研究。

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.