大氣壓力質譜法(Ambient mass spectrometry) 相對於傳統在真空中進行游離，擁有了許多的優勢，包含樣品前處裡簡單、分析時間短、更換樣品快速且容易，因此近年來有許多新式的大氣游離技術被研發出來。而在1999年所發表的Direct electrospray probe (DEP)是一套能方便使用，快速偵測，以及大幅減少樣品使用量的電噴灑游離源。其做法是在高電場下直接使液體樣品由金屬探針上直接產生電噴灑的方法，其可應用於快速游離大分子蛋白質樣品或小分子有機物。與傳統電噴灑比較，DEP不需使用毛細管或微量幫浦推送樣品，因此避免分析物殘留的問題，且可簡化電噴灑游離源的設計，對液態樣品直接進行分析。
利用DEP的直接游離之概念下做設計，本研究第一部分針對植物葉片組織利用簡單的裁切方式使其具有一尖端型態，並在組織上添加溶劑且直接施加高電壓於組織上; 在此組織中的成分會因裁切的關係而隨汁液滲漏出來，並在與溶劑混合後於尖銳處產生電噴灑現象而游離得到主成分訊號。於是藉此種方式可簡單且極快速的分析植物葉片在不同顏色、成長時期或病蟲害下的訊號差別，除此之外亦可應用於葉表面的殺蟲劑或內部之除草劑類農藥物殘留的分析。
為了改善第一部分實驗方法只能用於分析葉片組織之缺點，因此對於實驗方法改變了設計。第二部分研究利用細針做為游離/取樣探針，對於植物各部位做快速的取樣與分析其成分，而細針材質可為各式金屬，在本研究使用不鏽鋼材質，因其具有取得容易與堅硬度較強之特性。其做法為將兩根直徑為0.27 mm，尖端約為25 μm的不鏽鋼細針並排，使溶劑可藉由毛細現象通過兩針間的通道。藉由刺入植物之不同的部位，如:根、莖、葉及花…等，使液態汁液沾附在針間的通道處，而當溶劑沿著通道前進至末端後會與沾附在探針上的分析物混合，隨之在針上直接施加高電壓，因此溶液會在高電場的作用下往前帶送，並於不鏽鋼針的末端產生電噴灑游離，而可得到各部位分析物的質譜訊號。而除了採取植物液體樣品分析外，利用此方法也可對果皮上固體成分做分析，藉由SEM ( Scanning Electron Microscope )照影，可觀察到固態樣品沾附在針尖上，因此除了分析植物本身的成分外，也可成功的針對蔬果表面做取樣，藉此也減少來自樣品本身的基質成分干擾，最後應用此方法去對市售蔬果做分析，可快速且不用前處理而可成功的測得表面所殘留的殺菌劑和殺蟲劑類農藥的成分。

Ambient ionization mass spectrometry is a technique used for the analysis of samples under ordinary ambient conditions. It has many advantages, such as fast sample exchange, low operation time and few sample preparation which comparing with thus ion source operating in vacuum. Therefore, several different ambient ionization sources have been developed in the past decade.
Direct Electrospray Probe ionization mass spectrometry (DEP-MS) was developed by Shiea, J in 1999, which providing an effective means of analyzing not only large biomolecules, but also small organic and inorganic compounds. It can rapidly generate electrospray from a droplet which was deposited on a probe. The DEP technique provides a number of unique analytical features containing: (a) low sample consumption and sample switching is immediate; (b) the capillary and pump are unnecessary; and (c) the probe is low-cost and easy to construct and clean, and is free to maintenance.
The first part of experiment in this research is based on the ideal of DEP. The leaf tissues were cut into a triangular shape, 15 mm long and 8 mm wide at the base, is held by a crocodile clip with the apex facing the inlet of the mass spectrometer and at a distance of 2 cm or more from it. Electrospray solutions are added onto the tissue to mix with the leaf sap, and a high voltage (4.5-5.5 kV) is applied to the leaf tissue through the crocodile clip, and generate ions for MS analysis. This method has been demonstrated to be applicable for the analysis of a wide variety of plant sample, such as the leaves tissue in different color, different growth, diseased and pesticide residue.
The second part of experiment is improved from the first research. Electrospray was produced via a metal probe which binding two fine stainless steel needle (0.27 mm diameter) together. This probe plays the role of sampling and ionization. An amount of liquid sample was loaded onto the probe when the tip of probe touched the plant tissue, then electropray solution are introduced by syringe pump into the probe and applied high voltage on the probe to produce electrospray ionization. Therefore, direct rapid analysis was achieved using this probe interfaced and this method can direct profile of phytochemicals in a section of plant tissue in different regions, including root, stem, leaf, outer and inner part of fruit. In addition, using this probe, solid samples are directed sampling equally. The solid materials can be observed on the tip of probe by scanning electron microscope (SEM). It can obtain reasonable strong ion signals rapidly for agrochemicals deposited on the surface of vegetables or fruits peel.

論文審定書………………………………………………………………………………i
誌謝……………………………………………………………………………………...ii
中文摘要……………………….………………………………………...…....…..........iii
英文摘要………………………………………………………………………………...v
目錄…………………………………………………………………...……………..…vii
圖目錄……………………………………………………………………….………….ix
表目錄……………………………………………………………................................xiii
壹、 序論……………………………………………………………………….........…..1
一、 前言…………………………………...…………………....................................1
二、 大氣壓力游離技術 (Atmospheric Pressure Ionization Technique)……….........4
1、 大氣壓力化學游離法 (Atmosphere Pressure Chemical Ionization)…..…...4
2、 電噴灑游離法 (Electrospray Ionization, ESI)……………………….....…..5
三、大氣游離質譜技術 (Ambient Ionization Mass Spectrometry )………….….......8
A. 直接脫附/游離技術
1、 脫附電噴灑游離法 (Desorption Electrospray Ionization, DESI)…….........8
2、 即時直接分析法 (Direct Analysis In Real Time, DART)……….................9
3、 電噴灑輔助雷射脫附游離法 (Electrospray-assisted Laser Desorption
Ionization, ELDI)……………………………………………...….……….10
B. 直接游離技術
4、 直接電噴灑游離探針 (Direct Electrospray Probe, DEP)………..…….....11
5、 Probe Electrospray Ionization, PESI……………………………...….….…16
6、 Paper Spray Ionization, PSI……………………………………..…..…..….17
四、 論文目標…………………………………………………...………...…..…….18
貳、 實驗………………………………………………………………….…..….....….19
一、 實驗藥品與試劑……………………………………………………........….…19
二、 儀器裝置…………………………………………………………………...…..22
三、 藥品配製…………………………………………………………………....….24
參、 結果與討論………………………………………………..………………...……25
第一部份 : 直接電噴灑游離法對於植物內部組成之分析………………...…….25
第二部份 : 利用直接電噴灑游離探針分析植物不同部位 ……………...……...42
肆、 結論…………………………………………………………….…….…………...67
伍、 參考文獻…………………………………………………………….……...….…68

1. http://nobelprize.org/nobel_prizes/chemistry/laureates/1922/aston-bio.html
2. Dempster, A. J. “The Ionization and Dissociation of Hydrogen Molecules and the Formation of H3” Phil. Mag. 1916, 31, 438
3. Munson, M. S.; Field, F. H. “Chemical Ionization Mass Spectrometry. I. General Introduction.” J. Am. Chem. Soc. 1966, 88, 2621
4. Posthumus, M.; Kistemker, P. G.; Meuzelaar, H. L. C.; Braum, M. C. “Laser Desorption Mass Spectrometry of Polar Nonvolatile Bio-organic Molecules” Anal. Chem. 1978, 50, 985
5. Barber, M.; Bordoli, R. S.; Elliott, G. J.; Sedgwick, R. D.; Tyler, A. N. “Fast Atom Bombardment of Solids as An Ion Source in Mass Spectrometry” Natrue. 1981, 293, 270
6. Pachuta, S. J.; Cooks, R. G. “Mechanisms in Molecular SIMS” Anal. Rev. 1987, 87, 647
7. Cotter, R. “Plasma Desorption Mass Spectrometry” Anal. Chem. 1988, 60,781A
8. Yamashita, M.; Fenn, J. B. “Electrospray Ion Source. Another Variation on the Free-Jet Theme” J. Chem. Phys. 1984, 88, 4451
9. Karas, M.; Hillenkamp, F. “Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10,000 Daltons” Anal. Chem. 1988, 60, 2299
10. Horning, E. C.; Carroll, D. I.; Dzidic, I.; Haegele, K. D.; Horning, G.; Stillwell, R. N. “Atmospheric Pressure Ionization (API) Mass Spectrometry. Solvent-Mediated Ionization of Sample Introduced in Solution and in A Liquid Chromatography Effluent Stream” J. Chromatogr. 1974, 12, 725
11. Carroll, D. I.; Dzidic, I.; Stillwell, R. N.; Haegele, K. D.; Horning, E. C. “Atmospheric Pressure Ionization Mass Spectrometry: Corona Discharge Ion Source for Use in Liquid Chromatography-Mass Spectrometry-Computer Analytical System” Anal. Chem. 1975, 47, 2369
12. Dole, M.; Mack, L. L.; Hines, R. L. “Molecular Beams of Macroions” J. Chem. Phys. 1968, 49, 2240
13. Mack, L. L.; Kralic, P.; Rheude, A.; Dole, M. “Molecular Beams of Macroions Part II” J. Chem. Phys. 1970, 52, 4977
14. Wong, S. F.; Meng, C. K.; Fenn, J. B. “Mutiple Charging Inelectrospray Ionization for Poly(ethylene glycols)” J. Chem. Phys. 1988, 92, 546
15. Meng, C. K.; Mann, M.; Fenn, J. B. “Of Protons or Proteins – A Beams A Beam for A That” Z. Phys. D. 1988, 10, 361
16. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. “Mass Spectrometry Sampling Under Ambient Conditions with Desorption Electrospray Ionization” Science, 2004, 306, 471
17. Cody, R. B.; Laramee, J. A.; Durst, H. D. “Versatile New Ion Source for the Analysis of Materials in Open Air Under Ambient Conditions” Anal. Chem. 2005, 77, 2297
18. Cody, R. B.; Laramee, J. A.; Nilles, J. M.; Durst, H. D. “Direct Analysis in Real Time (DART) Mass Spectrometry” JEOL News. 2005, 40, 8
19. Shiea, J.; Huang, M. Z.; Hsu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J. “Electrospray-Assisted Laser Desorption Ionization Mass Spectrometry for Direct Ambient Analysis of Solids” Rapid Commun. Mass Spectrom. 2005, 19, 3701
20. Huang, M. Z.; Hsu, H. J.; Wu, C. I.; Lin, S. Y.; Ma, Y. L.; Shiea, J. “Applying Electrospray-Assisted Laser Desorption/Ionization Mass Spectrometry to the Direct Characterization of Chemical Components in Solids” J. Am. Soc. Mass Spectrom. 2006, 21, 1767
21. Hong, C. M.; Lee, C. T.; Lee, Y. M.; Kuo, C. P.; Yuan, C. H.; Shiea, J. “Generating Electrospray from Solutions Pre-deposited on a Copper Wire” Rapid Commun. Mass Spectrom. 1999, 13, 21
22. Kuo, C. P.; Shiea, J. “Application of Direct Electrospray Probe To Analyze Biological Compounds and To Couple to Solid-Phase Microextraction To Detect Trace Surfactants in Aqueous Solution” Anal. Chem. 1999, 71, 4413
23. Kuo, C. P.; Yuan, C. H.; Shiea, J. “Generation of Electrospray From a Solution Pre-deposited on the Optical Fibers Coiled With a Platinum Wire” J. Am. Soc. Mass Spectrom. 2000, 11, 464
24. Jehn, J.; Shiea, J. “Electrospray ionization from a droplet deposited on a surface- modified glass rod” Rapid Commun. Mass Spectrom. 2003, 17, 1709
25. Jehn, J.; Lin, C-H.; Shiea, J. “Electrospray from Nanostructured Tungsten Oxide Surfaces with Ultralow Sample Volume” Anal. Chem. 2005, 77, 8170
26. 鄭靜月“開發與蛋白質體學有關的新質譜游離技術” 國立中山大學化學研究所博士論文, 2002.
27. Hiraoka, K.; Nishidate, K.; Mori, K.; Asakawa, D.; Suzuki, S. “Development of probe electrospray using a solid needle” Rapid Commun. Mass Spectrom. 2007, 21, 3139
28. Chen, C. L.; Nishidate, K.; Saito, Y.; Mori, K.; Asakawa, D.; Takeda, S.; Kubota, T.; Terada, N.; Hashimoto, Y.; Hori, H.; Hiraoka, K. “Application of probe electrospray to direct ambient analysis of biological samples” Rapid Commun. Mass Spectrom. 2008, 22, 2366
29. Yu, Z.; Chen, L. C.; Suzuki, H.; Ariyada, O.; Erra-Balsells, R.; Nonami, H.; Hiraoka, K. “Direct Profiling of Phytochemicals in Tulip Tissues and In Vivo Monitoring of the Change of Carbohydrate Content in Tulip Bulbs by Probe Electrospray Ionization Mass Spectrometry” J. Am. Soc. Mass Spectrom. 2009, 20, 2304
30. Yoshimura K.; Chen, L. C.; Asakawa, D.; Hiraoka, K.; Takeda, S. “Physical properties of the probe electrospray ionization (PESI) needle applied to the biological samples” J. Mass Spectrom. 2009, 44, 978
31. Yu, Z.; Chen, C. L.; Erra-Balsells, R.; Nonami, H.; Hiraoka, K. “Real-time reaction monitoring by probe electrospray ionization mass spectrometry” Rapid Commun. Mass Spectrom. 2010, 24, 1507
32. 平岡 賢三. “探針エレクトロスプレーの開發と应用” BUNSEKI. KAGAKU. 2010, 59 , 95
33. Liu, J.; Wang, H.; Manicke, N. E.; Lin, J-M.; Cooks, R. G.; Ouyang, Z “Development, Characterization, and Application of Paper Spray Ionization” Anal. Chem. 2010, 82, 2463
34. Wang, H.; Liu, J.; Cooks, R. G.; Ouyang Zheng. “Paper Spray for Direct Analysis of Complex Mixtures Using Mass Spectrometry” Angew. Chem. Int. Ed. 2010, 49, 877
35. Jackson, A. U.; Tata, A.; Wu, C.; Perry, R. H.; Haas, G.; West, L.; Cook, R. G. “Direct analysis of Stevia leaves for diterpene glycosides by desorption electrospray ionization mass spectrometry” Rapid Commun. Mass Spectrom. 2009, 134, 867
36. Geuns. J. M. C. “Molecules of Interest Stevioside” Phytochemistry. 2003, 64, 913
37. Wang, X.; Plomely, J. B.; Newman, R. A.; Cisneros, A. “LC/MS/MS Analyses of an Oleander Extract for Cancer Treatment” Anal. Chem. 2000, 72, 3547
38. 鄭統隆,施怡如,曾東海,賴永昌,吳明哲. “甘藷花青素與多酚含量之研究” 台灣農業研究, 2008, 57(1), 33
39. 施怡如,鄭統隆,曾東海,王強生. “有色水稻中花青素的種類及其功能探討” 中華農業研究, 2004, 53, 221
40. “農藥管理與安全使用” 台灣農家要覽-農作篇, 517
41. “市售農產品藥物殘留農藥監測” 藥物食品檢驗局調查研究年報, 2009, 27 : 143
42. Garcia-Reyes, J. F.; Jackson, A. U.; Molina-Diaz, A.; Cooks, R. G. “Desorption Electrospray Ionization Mass Spectrometry for Trace Analysis of Agrochemicals in Food” Anal. Chem. 2000, 72, 3547
43. “Determination of Fungicides in Fruits and Vegetables by Time-of Flight and Ion Trap LC/MS” Agilent Technologies, Inc. 2005
44. 侯麗雪 “Analysis of Carbendazim and Public Health Pesticides” 朝陽科技大學應用化學系碩士論文, 2000.
45. Wang, L.; Wen, X. G.; Wang, G.; Li, S.; Jiang, J. “Degradation of methamidophos by Hyphomicrobium species MAP-1 and the biochemical degradation pathway” Biodegradation. 2010, 21, 513
46. Chang, D.; Lee, C.; shiea, J. “Detecting Large Biomolecules from High Salt Solutions by Fused-Droplet Electrospray Ionization Mass Spectrometry ”Anal. Chem. 2002, 74, 2465


47. Robb, D. B.; Covey, T. R.; Bruins, A. P. “Atmospheric Pressure Photoionization: An Ionization Method for Liquid Chromatography Mass Spectrometry” Anal. Chem. 2000, 72, 3653
48. Nemes, P.; Barton, A. A.; Li, Y.; Vertew, A. “Ambient Molecular Imaging and Depth Profiling of Live Tissue by Infrared Laser Ablation Electrospray Ionization Mass Sperctrometry” Anal. Chem. 2008, 80, 4575