第一部分研究：利用離子對逆向層析法（Ion-pair reversed-phase chromatography）結合感應耦合電漿質譜儀（Inductively coupled plasma mass spectroscopy）對魚油中之汞物種進行分析。以4.6 mm × 33 mm的C8逆相層析管柱，搭配2-巰基乙醇（2-mercaptoethanol）作為離子對試劑以及甲醇，以等位沖提方式針對無機汞、甲基汞及乙基汞等三種汞物種進行分離。在最適化條件下，三種汞物種可在6.5分鐘內完成分離，偵測極限個別為0.013、0.014與0.026 ng mL-1，訊號再現性之RSD小於2%。進行魚油樣品分析時，則在動相溶液(0.6% v/v 2-mercaptoethanol、2.5% v/v MeOH)中添加1% m/v Lipase、2% v/v Triton X-100以及1% v/v HNO3作為萃取試劑，並搭配微波輔助萃取的方式，於60℃ 下萃取魚油樣品5分鐘，萃取效率可達91%以上。最後將本方法應用於魚油樣品之汞物種分析。市售魚油樣品主要含無機汞與甲基汞兩種汞物種，汞物種的濃度分布情形隨魚油廠牌之不同而有所差異；儘管目前尚未有任何國家的衛生機構對魚油中的汞物種濃度制定規範，但是若以世界衛生組織(WHO)對魚肉中的甲基汞濃度規範而言，相較之下是低出許多的。
第二部分研究：利用離子對逆向層析結合冷蒸氣生成技術( cold vapor generation )結合ICP-MS對食用菇類樣品中之汞物種進行分析。冷蒸氣生成技術的使用可大幅提升汞物種的訊號靈敏度，並藉由相同的離子對試劑與甲醇之調控，以等位沖提方式將甲基汞及無機汞兩種汞物種進行分離。在最適化條件下，兩種汞物種可在6分鐘內完成分離，偵測極限分別為0.011與0.029 ng mL-1，訊號再現性之RSD小於3.5%。直接使用動相溶液(0.6% v/v 2-mercaptoethanol，6% v/v MeOH)對標準參考樣品NRCC DOLT-3以及市售菇類樣品進行方法準確度之驗證及分析。於市售菇類樣品中，無機汞為主要的汞物種，佔了汞物種濃度的八至九成，而甲基汞的濃度相較甚少。由於無機汞的毒性相較甲基汞低出許多，只要在正常攝取的情況下，可安全無虞的食用菇類。

We have presented our research results in two parts, a first parts of research which is explained by, based on HPLC-ICP-MS for the simultaneous determination of Methyl-Hg, Hg(II) and Ethyl-Hg in fish oils. The separation was performed using ion-pair reversed-phase liquid chromatography on a C8 column with 0.6% (v/v) 2-mercaptoethanol, and 2.5% (v/v) MeOH as mobile phase using an isocratic elution. Separation of the three mercury species was carried out in less than 6.5 min. The detection limits for Methyl-Hg, Hg(II) and Ethyl-Hg were found to be 0.013, 0.014 and 0.026 ng mL-1, respectively. Reproducibility was less than 2% RSD for 5 replicate injections. Mercury species were extracted from fish oils by using an extractant, containing 0.6% (v/v) 2-mercaptoethanol, 2.5% (v/v) MeOH, 1% (m/v) lipase, 2% (m/v) Triton X-100, and 1.0% (v/v) nitric acid were also used to microwave assisted extraction procedure. The extraction efficiency of mercury achieved more than 90% at 60 oC within 5 min. The research results reveal that different mercury species were presented which depends on different brands of fish oil. However, Methyl-Hg and Hg(II) species were found in fish oil samples. Despite the fact that, there was no regulation about mercury for fish oils so far, the concentration of mercury was quiet low as compared to fish standards.
Second parts of results fully demonstrate based on HPLC-CV-ICP-MS for the simultaneous determination of Methyl-Hg and Hg(II) in edible mushroom samples. The vapor generation techniques were hyphenated liquid chromatography to enhance the sensitivity of mercury species. The separation was performed on C8 column with 0.6% (v/v) 2-mercaptoethanol, and 6.0% (v/v) MeOH as mobile phase, the separation of the mercury species was carried out in less than 6 min. The detection limits were also optimized, in Methyl-Hg and Hg(II) were found to be 0.011, and 0.029 ng mL-1, respectively. Reproducibility was less than 3.5% RSD for 5 replicate injections. Mercury species were extracted from mushroom samples using mobile phase as an extractant, containing 0.6% (v/v) 2-mercaptoethanol, and 6.0% (v/v) MeOH. The accuracy of the method was validated by using NRCC DOLT-3 Fish Liver Certified Reference. The Methodology were developed and applied to investigate mercury speciation in edible mushroom samples and results reveals that, which is predominantly presented in Hg(II) species. The concentration of Methyl-Hg was extremely low compared with Hg(II) concentrations. Finally, The developed method were optimized and applied to investigate mercury speciation in fish oils and mushroom samples.

論文審定書……………………………………………………………………………………i
誌謝………………………………………………………………………………………….. ii
摘要…………………………………………………………………………………………...iii
Abstract…………………………………………………………………….......................iv
目錄…………………………………………………………………….............................v
圖目錄…………………………………………………………………….........................vii
表目錄…………………………………………………………………….........................ix

第一章　液相層析儀結合感應耦合電漿質譜儀於魚油樣品中汞物種分析之應用
壹、前言……………………………………………………………………………...…1
貳、實驗部分………………………………………………………………………...…3
一、儀器裝置…………………………………………………………………......……3
二、藥品與溶液的配製………………………………………………...………...……4
三、實驗過程………………………………………………………...…………...……7
參、結果與討論………………………………………………………………..….…..10
一、液相層析分離條件的探討………………………………………....…………….10
二、ICP系統操作條件的最佳化…………………………….......…………………..20
三、汞物種的分析……………………………………………..……………..……….21
四、萃取條件……………………………………………………....………………....27
五、真實樣品分析……………………………………………………....……….......33
肆、結論……………………………………………………………………………….45
伍、參考文獻………………………………………………………………………….46

第二章　液相層析冷蒸氣生成技術結合感應耦合電漿質譜儀於菇類樣品中汞物種
　　　　分析之應用
壹、 前言……………………………………………………………………………...51
貳、冷蒸氣形成原理……………………………………………………………….…52
參、實驗部分……………………………………………………………………….…53
一、儀器裝置……………………………………………………………………........53
二、藥品與溶液的配製...............................................................................55
三、實驗過程............................................................................................56
肆、結果與討論........................................................................................ 60
一、液相層析分離條件的探討.....................................................................60
二、冷蒸氣生成系統操作條件的最適化........................................................66
三、ICP系統操作條件的最佳化...................................................................70
四、汞物種分析.........................................................................................72
五、萃取條件............................................................................................78
六、真實樣品分析...........................................................................……....79
伍、結論........................................................................................……....86
陸、參考文獻..................................................………………………….........87
圖目錄
第一章 液相層析儀結合感應耦合電漿質譜儀於魚油樣品中汞物種分析之應用
圖1-1 HPLC-ICP-MS之系統裝置圖…………………………………………........6
圖1-2 樣品萃取流程圖………………………………………………………….….10
圖1-3 沖提液中2-mercaptoethanol濃度對汞物種滯留時間的影響…………….12
圖1-4 沖提液中2-mercaptoethanol濃度對汞物種滯留時間的影響…………….13
圖1-5 沖提液中MeOH濃度對汞物種滯留時間的影響…………………..………14
圖1-6 沖提液中MeOH濃度對汞物種滯留時間的影響…………………..………14
圖1-7 沖提液的pH值對層析分離的影響…………………………………..……..15
圖1-8 沖提液中pH值對汞物種滯留時間的影響…………………………..……..16
圖1-9 沖提液流速對汞物種滯留時間的影響………………………………….….17
圖1-10 沖提液流速對(a)汞物種滯留時間及(b)波峰積分面積之S/B的影響….…18
圖1-11 沖提液中MeOH濃度對汞物種滯留時間的影響………………………..…19
圖1-12 霧化氣體流速對汞元素分析訊號的影響………………………………….20
圖1-13 電漿輸出功率對汞元素分析訊號的影響………………………………….21
圖1-14 HPLC-ICP-MS 系統中所得到的汞物種之層析圖………………………..22
圖1-15 不同萃取方式對於魚油樣品中汞的萃取效率…………………………….27
圖1-16 於不同的萃取方法中添加0.5%硝酸對於魚油樣品中汞的萃取效率…...28
圖1-17 不同硝酸濃度對於魚油樣品中汞的萃取效率…………………………….30
圖1-18 不同硝酸濃度萃取魚油樣品中汞的物種之所得層析圖………………….31
圖1-19 溫度對於魚油樣品中汞的萃取效率……………………………………….32
圖1-20 萃取時間對於魚油樣品中汞的萃取效率………………………………….33
圖1-21 魚肝標準參考樣品(NRCC DOLT-3)萃取所得汞物種之層析圖………….34
圖1-22 美術館湖水層析圖………………………………………………………….36
圖1-23 後勁溪水層析圖…………………………………………………………….37
圖1-24 化學館飲水機飲用水層析圖……………………………………………….38
圖1-25 魚油樣品( Fish oil #1 )萃取後所得汞物種之層析圖………………..…...41
圖1-26 魚油樣品( Fish oil #2 )萃取後所得汞物種之層析圖……………..……...42
圖1-27 魚油樣品( Fish oil #3 )萃取後所得汞物種之層析圖…………..………...43

第二章　液相層析冷蒸氣生成技術結合感應耦合電漿質譜儀於菇類樣品中汞物種
　　　　分析之應用
圖2-1 HPLC-CV-ICP-MS之系統裝置圖...............................................................54
圖2-2 標準參考樣品及真實樣品之萃取流程圖.....................................................59
圖2-3 實驗流圖...............................................................................................60
圖2-4 沖提液組成對層析分離的影響..................................................................61
圖2-5 沖提液中2-mercaptoethanol 濃度對層析分離的影響...................................63
圖2-6 沖提液中甲醇濃度對層析分離的影響........................................................64
圖2-7 沖提液流速對層析分離的影響..................................................................65
圖2-8 冷蒸氣系統中NaBH4濃度對汞物種(a)分析物波鋒面積訊號及(b)波峰高
度之S/B的影響.................................................................................................67
圖2-9 冷蒸氣系統中HNO3濃度對汞物種(a)分析物波峰面積訊號及(b)波峰高度
之S/B的影響....................................................................................................69
圖2-10 CV-ICP-MS系統中，霧化氣體流速對汞元素分析訊號的影響......................71
圖2-11 CV-ICP-MS 系統中，電漿輸出功率對汞元素分析訊號的影響.....................72
圖2-12 HPLC-CV-ICP-MS系統中所得到的汞物種之層析圖...................................75
圖2-13 萃取液中2-mercaptoethanol的濃度對萃取效率之影響...............................79
圖2-14 魚肝標準參考樣品(NRCC DOLT-3)萃取所得汞物種之層析圖......................81
圖2-15 菇類樣品( mushroom #1 )萃取後所得汞物種之層析圖...............................83
圖2-16 菇類樣品( mushroom #2 )萃取後所得汞物種之層析圖...............................84
表目錄
第一章 液相層析儀結合感應耦合電漿質譜儀於魚油樣品中汞物種分析之應用
表1-1 微波消化升溫程式…………………………………………………………...9
表1-2 微波輔助萃取升溫程式……………………………………………………...9
表1-3 汞物種分析在HPLC-ICP-MS系統的最適化條件………………………...23
表1-4 三種汞物種的滯留時間及訊號再現性…………………………………….23
表1-5 HPLC-ICP-MS測定三種汞物種之校正曲線及其偵測極限……………...24
表1-6 分離時間與偵測極限之比較…….…….…………………………………..25
表1-7 添加硝酸前後之比較……………………………………………………….29
表1-8 HPLC-ICP-MS測定魚肝標準參考樣品NRCC DOLT-3中各個汞物種之含量及回收率………………………………………………………………........................….35
表1-9 利用HPLC -ICP-MS測定水樣中汞物種之含量…………………………..39
表1-10 以HPLC-ICP-MS測定魚油樣品中之汞物種……………………………..44

第二章　液相層析冷蒸氣生成技術結合感應耦合電漿質譜儀於菇類樣品中汞物種
　　　　分析之應用
表2-1 微波消化升程式........................................................................................59
表2-2 不同酸試劑對汞物種訊號之比較.................................................................68
表2-3 汞物種分析在HPLC-CV-ICP-MS系統的最適化條件.......................................74
表2-4 二種汞物種的滯留時間及訊號再現性...........................................................75
表2-5 HPLC-CV-ICP-MS測定二種汞物種之校正曲線及其偵測極限..........................76
表2-6 分離時間與偵測極限之比較........................................................................77
表2-7 HPLC-CV-ICP-MS測定魚肝標準參考樣品NRCC DOLT-3中各個汞物種
之含量及回收率...........................................................................................80
表2-8 以HPLC-CV-ICP-MS測定菇類樣品中之汞物種...............................................85

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