1. 本研究利用旋轉式中空纖維管液相微萃取法結合GC/MS同時對五種異構物作定性定量分析，定量部份使用電子游離法對異構物進行離子化，定性部份則是使用自身離子/分子反應法進行五種同分異構物的區別鑑定。此技術的偵測極限可達4~5μg/mL，相對回收率在97%以上，enrichment factor在25~35倍之間，校正曲線的線性關係亦在0.99以上。相較於其它微萃取法如一滴溶劑微量萃取法、中空纖維管液相微萃取法、固相微萃取法，本方法除了改善液相微萃取法的萃取效率，相較於固相微萃取法亦有相同的靈敏度和準確度。而且有操作方便、萃取快速、低成本、低汙染、高穩定性等優點；在真實樣品分析，在稀釋2倍的尿中作定量，回收率可達82%以上，在稀釋5倍的血漿中作定量，回收率達63%以上。此外使用自身離子/分子反應的方式，結合高次串聯質譜作多次碰撞活化解離，以快速區別五種分子量為120的同分異構物。

2. 本研究提供一個簡單且高效率的魚體內微量藥物的直接定量偵測方式且不需要複雜的前處理和純化步驟。首先，利用簡單的方式從魚體內取得初步的魚萃取液(使用1毫升的甲醇)；接著加入9毫升的水稀釋並使用一滴溶液微量萃取法(SDME)作預濃縮；最後使用AP-MALDI-MS對萃取後的2微升有機溶劑作分析，並用內標準法作定量消除在用AP-MALDI-MS游離時的非均相共結晶的誤差。此方法在水中和魚體組織的偵測極限分別為0.046μM和0.077μM；日內和日間精密度落在8.6%至9.5%之間；而且結果顯示在魚體實際分析時相對回收率可達92%以上。此方法結合SDME和AP-MALDI-MS來縮短在預濃縮、萃取和偵測時所花費的時間，並且可免使用任何層析的儀器。應用於魚類分析時，本方法証實可以快速且正確定量魚體內的藥物濃度，從魚體採樣後至分析完畢時間可控制在20分鐘以內，此定量方法可以大大的簡化在藥物動力學研究時消耗的時間和物力成本。

1. A rapid and dynamic liquid phase microextraction technique named Rotating hollow fiber - liquid phase microextraction (RHF-LPME) was developed to couple to GC/MS using electronic ionization (EI) under selective ion monitor (SIM) for quantitative analysis and using self-ion/molecule reaction (SIMR) in conjunction with tandem mass spectrometry for discrimination of five aromatic hydrocarbon isomers including cumene, propylbenzene, 2-ethyltoluene, 1,2,3-trimethylbenzene and 1,2,4-trimethylbenzene. 
The optimized parameters of this approach was: organic solvent toluene, extraction time 2min, stirring rate of stirrer 700rpm, rotating speed of motor driving rotator 250rpm, no addition of salt, pH of aqueous phase 6 and both rotator and stirrer were operated in reversed directions. The linear range of calibration curve of RHF-LPME was from 0.002 to 0.4 μg/mL; the R2 was 0.99 and the relative standard deviation (RSD) values were from 4.5 to 5.2%. Comparing to single drop microextraction (SDME), the RHF-LPME method improved the limit of detection (LOD) and enrichment factor (EF) more than one fold. The LODs of RHF-LPME (4-5 pg/mL) were closed to those of (2-3 pg/mL) solid phase microextraction (SPME) but SPME was seriously limited to high cost and peaks broaden.
 In addition, comparing to traditional dynamic liquid phase microextraction, this approach with the advantages of easy to operate, high reproducibility, extremely fast, high extraction efficiency and good reliability for analyzing biological samples.

2. A rapid, simple and efficient pharmacokinetic approach for the quantitative determination of trace amount of quinidine drug in fish has been demonstrated by combining single drop microextraction with AP-MALDI/Ion Trap Mass Spectrometry. This technique provide good correlation coefficient (>0.99) in work. The limits of detection of quinidine in water, and fish samples were 0.05 and 0.08μM, respectively.  The intraday and interday precision of water and fish samples with relative standard deviations ranging from 6.7% to 9.4% and 8.8% to 9.6%. Results also showed high relative recovery (>91.8%) of fish sample analysis in water. This method of combing SDME with AP-MALDI-MS analysis has proven to be a fast technique for direct preconcention of fish tissues.

List
List……………………………………………………………………壹
List of figure and table….………………………………………………參
Additional work…………………………………………………伍

Chapter 1. Rotating hollow fiber-liquid phase microextraction coupled to GC/MS to determine isomers

1.1、Abstract…………………………………………………………1
1.2、Introduction…………………………………………………………1
1.3、Experimental section………………………………………………2
  1.3.1、Chemicals and materials…………………………………2
  1.3.2、Gas chromatography/mass spectrometry instrumentation………………………………………………………3
  1.3.3、Extraction procedures for the RHF-LPME experiments……………………………………………………………3
1.4、Results and discussion………………………………………………4
  1.4.1、Optimization for the RHF-LPME technique ……………………………………………………………………………4
  1.4.2、Effect of solvent selection……………………………4
  1.4.3、Comparison of the directions…………………………4
  1.4.4、The effect of extraction time…………………………5
  1.4.5、Probing the effect of stirring rate of the magnetic stirrer………………………………………………………5
  1.4.6、Probing the rotating speed of motor driven rotator…………………………………………………………………5
  1.4.7、The effect of salt addition and pH of aqueous phase……………………………………………………………………6
  1.4.8、Quantitative approach for the RHF-LPME technique………………………………………………………………6
  1.4.9、Method Evaluation………………………………………6
  1.4.10、A comparison in performance for RHF-LPME with other microextraction methods…………………………………7
  1.4.11、Application of the RHF-LPME Method for biological samples…………………………………………………8
  1.4.12、Isomer differentiation achieved by self-ion molecule reaction/tandem mass spectrometry…………………8
1.5、Conclusion………………………………………………………9
1.6、Acknowledgement………………………………………………10
1.7、Reference………………………………………………………21
1.8、Supporting material…………………………………………22

Chapter 2. A rapid quantitative method for pharmacokinetic approach from direct fish analysis by coupling single drop microextraction with AP-MALDI/MS.

2.1、Abstract…………………………………………………………33
2.2、Introduction……………………………………………………33
2.3、Experimental section…………………………………………34
2.3.1、Chemicals and materials…………………………………34
  2.3.2、Preparation of fish extracts…………………………35
  2.3.3、Single drop microextraction procedures……………35
  2.3.4、AP-MALDI/MS analysis…………………………………35
2.4、Result and discussion………………………………………36
  2.4.1、Optimization of SDME parameters……………………36
  2.4.2、Effect of selection of solvents……………………36
2.4.3、Effects of extraction time and pH of aqueous solution………………………………………………………………37
2.4.4、Salt concentration………………………………………37
2.4.5、Stirring rate and matrix concentration………………37
2.4.6、Quantitative approach for the SDME coupled to AP-MALDI-MS……………………………………………………37
2.5、Conclusion……………………………………………………38
2.6、Acknowldegment………………………………………………39
2.7、Reference……………………………………………………48
2.8、Supporting material………………………………………49




  

List of figure and table

Figure 1-1. Structures of five aromatic hydrocarbon isomers…………………………………………………………………11

Figure 1-2. Sketch diagram of rotating hollow fiber – liquid phase microextraction technique.………………………12

Figure 1-3. A total ion current chromatogram of a mixture of five aromatic hydrocarbon isomers.…………………………13

Figure 1-4. Self ion/molecule reaction spectra for a) cumene, b) propylbenzene c) 2-ethyltoluene, d) 1, 2, 3,-trimethylbenzene, e) 1, 2, 4-trimethylbenzene.……………14

Figure 1-5. MS/MS of (M+H)+ ion at m/z 121 for a) cumene, b) propylbenzene c) 2-ethyltoluene, d) 1, 2, 3,-trimethylbenzene, e) 1, 2, 4-trimethylbenzene………………15

Figure 1-6. MS3 of product ion at m/z 105 ([M-CH3]+) for a) cumene, b) 1, 2, 3,-trimethylbenzene, and c) 1, 2, 4-trimethylbenzene.……………………………………………………16

Figure 2-1. Structures of (a) quinidine and (b) cinchonidine (internal standard)………………………………40

Figure 2-2. Single drop microextraction apparatus…………41

Figure 2-3. An AP-MALDI-MS spectrum of quinidine and cinchonidine after SDME……………………………………………42

Table 1-1. Optimization parameters for the rotating hollow fiber - liquid phase microextraction technique……………17

Table 1-2. Linearity range, correlation coefficient, and relative standard 
deviation (RSD) for combining RHF-LPME with GC/MS to detect five hydrocarbon isomers in water……………………18

Table 1-3. Comparing RHF-LPME with SDME and SPME for limit of detections (LODs), enrichment factor (EF), and relative recovery (R).………………………………………………………19

Table 1-4. Application of RHF-LPME technique for analysis of biological Samples including plasma and urine.………20

Table 2-1. Optimization parameters for the proposed methods including the solvent selection, extraction time, salt addition and pH effect………………………………………43

Table 2-2. Linearity, correlation coefficient and LODs for the SDME coupled to AP-MALDI-MS…………………………………44

Table 2-3. Interday and intraday precisions…………………45

Table 2-4. Relative recovery and enrichment factor investigated for the SDME coupled to AP-MALDI-MS…………46

Table 2-5. Real sample analysis………………………………47


Additional work

Additional work……………………………………………52

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