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博碩士論文 etd-0807112-003603 詳細資訊
Title page for etd-0807112-003603
論文名稱
Title
開發薄層層析電噴灑游離技術於快速檢測生化樣品中所含之化學物質
Developing a TLCspray technique to rapidly characterize small organic chemical in biological fluid
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
87
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-07-20
繳交日期
Date of Submission
2012-08-07
關鍵字
Keywords
質譜法、薄層層析電噴灑
Mass Spectrometry, TLCspray
統計
Statistics
本論文已被瀏覽 5665 次,被下載 489
The thesis/dissertation has been browsed 5665 times, has been downloaded 489 times.
中文摘要
血液尿液等生物樣品常需要經過繁複的樣品前處理才能使用質譜法分析。在質譜分析之前,使用離心、固相萃取以及液液萃取等樣品前處理方法能夠將干擾物質從生物樣品中移除,但是這些樣品前處理的經常需要大量的時間與複雜的步驟。薄層層析法 (Thin Layer Chromatography, TLC)是一種化學實驗室常用的樣品純化與分離方法。使用質譜法定性分析鑑定薄層層析片上的分析物是目前最有效的方式。由於分析物分子主要都是埋藏在薄層層析片中的矽粉層中,因此開發能將分析物自矽粉層中的分析物帶至質譜儀的採樣方法便極其重要。本研究所開發的薄層層析電噴灑(TLCspray)技術便是能分析薄層層析片上分析物的分析技術。薄層層析電噴灑技術的觀念與本實驗是先前開發的直接電噴灑探針 (Direct Electrospray Probe, DEP) 類似:沾附在金屬棒上的樣品溶液在高電場的影響下可產生電噴灑噴霧而得到分析物質譜訊號。薄層層析電噴灑技術中所使用的薄層層析片構型為具有尖端的長條薄層層析片,其中尖端的固定相粉末都以刀片刮除。分析時,樣品先被點在薄層層析片上,接著薄層層析片使用特製的固定架固定在質譜入口前。流動向流過樣品點之後帶著分析物流向薄層層析片的尖端,分析物與流動相溶液到達尖端後在高電場的影響下產生細緻的電灑噴霧而被游離。薄層層析片上的固定相可將血液樣品中複雜基質如血球與蛋白質滯留在原處而不會移動,固定相在此處能作為過濾器除去生物樣品中不需要的干擾。在本研究中使用安眠藥Zolpidem 與心血管疾病藥物Verpamil 作為樣品藥物,測試薄層層析電噴灑的靈敏度。使用薄層層析電噴灑分析血液樣品中的Zolpidem 定量極限為 50 ng/mL, 線性範圍 50-1000 ng/mL。薄層層析電噴灑分析血液樣品過程可在四分鐘之內完成。與商業化LCMS相比,雖然在靈敏度少了一個order,但薄層層析電噴灑質譜法可大幅縮短樣品前處理所需的時間。
Abstract
Analyzing the chemical compounds in biological fluid, such as human blood, by mass spectrometry, sample pretreatments are frequently needed. Those methods, including centrifugation, solid phase extraction (SPE) and liquid-liquid extraction, separate and remove the interferences in biological samples prior to mass analysis. The drawbacks of sample pretreatments mentioned above are time consuming and tedious operation procedures. Thin layer chromatography (TLC) is an analytical tool commonly used for sample purification and separation. To characterize the chemical compounds on TLC plate, coupling TLC with mass spectrometry seems to be a most efficient approach. Since the analytes are buried in the silica layer of TLC plate, it is important to develop a sampling method to transfer analytes from silica layer to mass analyzer. We develop a technique named “TLCspray” to analyzing the chemical compounds deposited on TLC plate. The concept of TLCspray is similar to the direct electrospray probe (DEP) which has been described by our group: droplets deposed on a metal coated rod can producing ESI plume while applying high voltage. The TLC plate used for TLCspray is cut into stripes with sharp end. Samples are applied on the TLC stripes, than mount onto a holder which fix TLC stripes in front of mass inlet. The mobile phase flow through sample spot, and carry the analyte to the edge of TLC stripe where the fine ESI plume is produced in the effect of electric field. Complex matrixes such as red blood cell (RBC) and proteins are stop on the original position by the silica layer which acts as filter to clean up the unwanted interferences in biological samples. Zolpidem, a drug used to treat insomnia, is chose to demonstrate the sensitivity of TLCspray. The LOQ of zolpidem is 50 ng/mL, and linear dynamic range is 50-1000 ng/mL in human blood. The analysis process of TLCspray for human blood can be finished within 4 minutes. Comparing to other sample pretreatment prior to mass analysis for biological samples, TLCspray can reduce the time needed for clean up the samples without loss the sensitivity.
目次 Table of Contents
目錄
謝 誌 i
Abstract ii
論文摘要 iv
目錄 v
圖目錄 viii
表目錄 xii
第一章 緒論 1
一、 前言 1
二、 薄層層析/質譜介面開發 4
1. 間接採樣(Indirect Sampling)薄層層析/質譜介面 4
2. 直接採樣(Direct Sampling)薄層層析/質譜介面 5
三、 大氣壓力游離源直接採樣(Direct sampling using ambient source)之薄層層析/質譜介面 5
1. 使用電噴灑游離法(Electrospray Ionization)為大氣壓力游離源之薄層層析/質譜介面 5
2. 使用大氣化學游離法(Atmospheric Pressure Chemical Ionization)之薄層層析/質譜介面 10
四、 大氣壓力質譜法(Ambient Mass Spectrometry, AMS) 13
1. 直接電噴灑探針(Direct Electrospray Probe, DEP) 16
2. 探針電噴灑游離法(Probe Electrospray Ionization, PESI) 18
3. 使用木質尖端產生電噴灑(Electrospray Ionization Using Wooden Tips) 19
4. 使用塑膠製尖端管道進行連續式電噴灑分析(Sequential Electrospray Analysis Using Sharp-Tip Channels Fabricated on a Plastic Chip) 19
5. 紙噴灑(Paper Spray Ionization, PSI) 21
6. 葉片噴灑游離法(Leaf spray) 22
五、 論文目標 23
第二章 實驗 24
一、 藥品與試劑 24
二、 儀器與材料 24
1. 微量注射幫浦(Syringe pump) 24
2. 高電壓電源供應器(High voltage power supply) 24
3. 質譜儀(Mass spectrometer) 24
4. 薄層層析片(TLC plate) 26
5. 掃描式電子顯微鏡(Scanning electron microscope, SEM) 26
6. 手術刀與刀片 26
7. 相機 26
8. 離心機 26
三、 TLCspray裝置設計 27
四、 TLC片切割方式 30
五、 以TLCspray 分析Zolpidem,Verapamil 標準品及生物樣品小分子 32
1. Zolpidem,Verapamil 檢量線 32
2. 分析生化樣品中的藥物分子 32
第三章 結果與討論 35
一、 使用SEM 檢視TLC片尖端表面構形 35
二、 TLCspray質譜資料處理方法 39
三、 薄層層析電灑 (TLCspray)參數探討 41
四、 TLCspray/MS分析對Zolpidem的靈敏度與線性範圍 49
五、 使用內標準品對藥物標準品進行定量 55
六、 分析人類血液中醫療藥物小分子 59
第四章 結論 65
第五章 參考文獻 66
參考文獻 References
第五章 參考文獻
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