臺灣博碩士論文加值系統

中文摘要
托美丁(TLM)和佐美酸(Z)為光敏感的非固醇類抗炎劑(NSAID)。首先將托美丁和佐美酸之甲醇溶液以Hanovia 200W的高壓汞燈照射下分別觀察到托美丁會分解成TLM-1至TLM-8八種光解產物 ; 而佐美酸會分解成Z-1至Z-4四種產物。除了利用製備型高壓液相層析儀來單離這些光解產物外並且以UV、IR、MS、NMR和LC-MS光譜法來一一解明光解產物的化學結構。
其次，為了要發展一個快速、敏感度高及準確的安定性指標的高壓液相層析法來定量托美丁和佐美酸的降解情況並利用其在標準溶液中、酸中、鹼中和光照情況下來作一波峰的純度確效。對托美丁的分析方法是用Inertsil ODS-3V管柱，移動相則採甲醇: 0.3% 醋酸為64: 36 (v/v) ; 而對佐美酸的定量亦用相同管柱，移動相為乙晴:甲醇: 0.3% 醋酸為2: 64: 34 (v/v/v)，UV偵測器則同樣設定為254 nm。發展出的分析方法的相關統計敘述包含有高壓液相層析系統規範、波峰純度完整以及光解產物和原料藥之間的解析度，及日內、日間差異分別在2.3%和4%以內，回收率亦在97.14%~101.73%之間。我們所建立的分析方法表現出良好的選擇性和特異性，適合用來作為托美丁(TLM)和佐美酸(Z)的光安定性的分析。
托美丁(TLM)和佐美酸(Z)在甲醇溶媒或是含水甲醇溶媒的光降解速率有顯著差距。托美丁(TLM)和佐美酸(Z)在甲醇溶液中的光降解動力學，若以一階次作圖時的k值相差將近10倍。而後再以光動力學反應較快之托美丁(TLM)以不同濃度、不同醇類溶媒、不同含水的甲醇溶液以期盼研究出托美丁(TLM)的光動力學模式。本研究發現托美丁(TLM)在高濃度呈現零階次動力學模式，至於三種醇類溶媒中，認為托美丁(TLM)的動力學和介電係數(dielectric constant)呈現反比的情況，可表示托美丁(TLM)的動力學可能是經由自由基反應途徑，而非離子化反應途徑。

Abstract
Tolmetin (TLM) and Zomepirac (Z) are used as photosensitizing nonsteroidal antiinflammatory drugs (NSAIDs). When TLM and Z were irradiated by a Hanovia 200W high pressure mercury lamp in methanol, a total of 8 photoproducts of TLM; TLM-1 to TLM-8 and 4 photoproducts of Z; Z-1 to Z-4 photoproducts were observed, respectively. Using preparative HPLC, photoproducts were isolated and their chemical structures were elucidated by UV、IR、MS、NMR and LC-MS spectroscopies.
Next, a rapid, sensitive, and accurate stability-indicating high perfor- mance liquid chromatographic assay method for determining the degra- dation of TLM and Z was developed and vali- dated under acidic, basic, or photo-irradiated conditions. The quantitation was monitored with an Inertsil ODS-3V column using a mobile phase of methanol- 0.3%acetic acid = 64:36 (v/v) for TLM and acetonitrile- methanol - 0.3%acetic = 2:64:34(v/v) for Z detector set at 254nm. The related satistics of the developed methods including the system criteria, peak integrity, and resolution among the parent drug and its degradation products were calculated. The C.V.(%) of intraday and interday tests were lower than 2.3% and 4% , respectively. The percent recovery were between 97.14% ~ 101.73 %. Our established HPLC assay method shows good selectivity and specificity which is suitable for the stability measurement of TLM and Z.
The rates of photodegradation between TLM and Z present significant difference, no mater the solvent is MeOH or 20% MeOH/H2O. When proceeding first order graphical analysis of these two compounds, we find that k of TLM is almost ten folds to B. After that, we use TLM, which possess more sensitive property to UV light. With different concentrations, alcohol solvents, and concentrations of 20% MeOH/H2O, to look forward to find out the exact photokinetic model of TLM. In our experimental model, the kinetic of TLM of high concentration is zero order. As for the results of using three alcoholic solvents, they showed an inverse proportional relationship between kinetics and dielectric constant of TLM. Hence we supposed that photokinetics of TLM might go through free radical reaction instead of ionic reaction.

目錄……………………………………………………………………………I
圖目次………………………………………………………………………III
表目次…………………………………………………………………………V
中文摘要………………………………………………………………………VI
英文摘要……………………………………………………………………VII
第一章 緒論…………………………………………………………………1
1. 1 導言…………………………………………………………………1
1. 2 NSAID藥物光安定性研究近況……………………………………6
1. 3 研究背景及目的……………………………………………………9
第二章 實驗材料與方法……………………………………………………10
2. 1 材料與儀器………………………………………………………10
2. 1. 1 試藥…………………………………………………………10
2. 1. 2 儀器…………………………………………………………10
2. 2 實驗方法--光解產物單離………………………………………12
2. 2. 1 托美丁與佐美酸的樣品處理………………………………12
2. 2. 2 光解產物分離條件…………………………………………12
2. 2. 2. 1 製備型高壓液相層析系統分析條件………………12
2. 2. 2. 2 分析型高壓液相層析系統分析條件………………15
2. 2. 2. 3 高壓液相層析質譜儀系統分析條件………………17
2. 2. 3 結構確認……………………………………………………17
2. 2. 3. 1 高壓液相層析質譜儀………………………………18
2. 2. 3. 2 直譜儀………………………………………………18
2. 2. 3. 3 核磁共振儀…………………………………………18
2. 2. 3. 4 紅外線光譜儀………………………………………19
2. 2. 3. 5 紫外線光譜儀………………………………………19
2. 2. 4 虐待試驗……………………………………………………19
2. 2. 4. 1 托美丁………………………………………………19
2. 2. 4. 2 佐美酸…………………………………………………20
2. 2. 5 光安定動力學實驗步驟……………………………………20
第三章 結果與討論…………………………………………………………22
3. 1 虐待試驗…………………………………………………………22
3. 1. 1 托美丁之虐待試驗…………………………………………22
3. 1. 2 佐美酸之虐待試驗…………………………………………26
3. 2 檢量線之線性迴歸、精密度、準確度以及The Lack of Fit Test……………………………………………………………………………31
3. 2. 1 托美丁………………………………………………………32
3. 2. 2 佐美酸………………………………………………………34
3. 3 HPLC系統適合參數………………………………………………36
3. 3. 1 托美丁之HPLC系統適合參數………………………………38
3. 3. 2 佐美酸之HPLC系統適合參數………………………………39
3. 4 光解產物單離與結構鑑定………………………………………40
3. 4. 1 佐美酸的光解產物結構探討………………………………42
3. 4. 2 托美丁的光解產物結構探討………………………………56
3. 5 托美丁的光安定性試驗…………………………………………82
3. 5. 1 光安定性試驗結果與討論…………………………………82
參考文獻………………………………………………………………………91
圖目次
圖1. 1 托美丁和佐美酸化學結構式………………………………………………………………………………1
圖1. 2 NSAID抑制COX-1和COX-2的藥理徑…………………………………5
圖1. 3 托美丁(TLM)及其體內主要代謝產物………………………………7
圖1. 4 佐美酸(Z)及其體內主要代謝產物…………………………………7
圖1. 5 在PBS溶液中，以波長310-390 nm的磷燈(phosphor lamp)照射，於空氣中和氮中托美丁(TLM)的光降解產物…………………………………8
圖3. 1 以HPLC分析托美丁(TLM)標準溶液在20%甲醇溶液中(100μM)…22
圖3. 2 以HPLC分析托美丁(TLM)及其內部標準品(IS)美洒辛(IND)在20%甲醇溶液中(100μM)……………………………………………………23
圖3. 3 以HPLC分析托美丁(TLM)在60oC酸性(0.2N HCl)之20%甲醇溶液中(100μM)狀態下三天………………………………………………………23
圖3. 4 以HPLC分析托美丁(TLM)在60oC鹼性(0.2N NaOH)之20%甲醇溶液中(100μM)狀態下三天……………………………………………………24
圖3. 5 以HPLC分析托美丁(TLM)在20%甲醇溶液中(100μM)照光1.5小時……………………………………………………………………………24
圖3. 6 以HPLC分析佐美酸(Z)標準溶液在20%甲醇溶液中(100μM)……26
圖3. 7 以HPLC分析佐美酸(Z)及其內部標準品(IS)Butyl paraben(BP)在20%甲醇溶液中(100μM)……………………………………………………26
圖3. 8 以HPLC分析佐美酸(Z)在60oC酸性(0.2N HCl)之20%甲醇溶液中(100μM)狀態下三天………………………………………………………27
圖3. 9 以HPLC分析佐美酸(Z)在60oC鹼性(0.2N NaOH)之20%甲醇溶液中(100μM)狀態下三天…………………………………………………………27
圖3. 10 以HPLC分析佐美酸(Z)在20%甲醇溶液中(100μM)照光40小時…28
圖3. 11 托美丁(TLM)溶於甲醇溶液之濃度檢量線(n=6)…………………32
圖3. 12 佐美酸(Z)溶於甲醇溶液之濃度檢量(n=6)………………………34
圖3. 13 非固醇類藥物在甲醇溶液中廣被接受之反應機轉………………41
圖3. 14 以HPLC分析佐美酸在甲醇溶液中(1mM)照光3天後之產物………42
圖3. 15 以HPLC分析佐美酸在甲醇溶液中(10mM)照光14天後之產物……42
圖3. 16 佐美酸(Z)之質譜圖………………………………………………47
圖3. 17 佐美酸(Z)之質譜斷裂圖…………………………………………48
圖3. 18 佐美酸(Z)之LC-MS圖………………………………………………49
圖3. 19 光解產物Z-1之LC-MS圖……………………………………………50
圖3. 20 光解產物Z-2之LC-MS圖……………………………………………50
圖3. 21 光解產物Z-3之LC-MS圖……………………………………………51
圖3. 22 光解產物Z-4之LC-MS圖……………………………………………51
圖3. 23 佐美酸(Z)在酸虐待實驗的酸產物之LC-MS圖……………………52
圖3. 24 佐美酸(Z)酸試驗和光解實驗中推判之產物結構式……………54
圖3. 25 佐美酸(Z)在甲醇溶液中可能之光解反應途徑…………………55
圖3. 26 以HPLC分析托美丁在甲醇溶液中(1mM)照光10小時之層析圖…56
圖3. 27 圖3.25等比例放大，可發現分裂為兩支根波峰TLM-1、TLM-2…56
圖3. 28 以HPLC分析型管柱分析，當移動相為--水:甲醇為80:20(不含酸)時，TLM-1、TLM-2波峰可被分離…………………………………………57
圖3. 29 以HPLC分析托美丁在甲醇溶液中(10mM)照光4天後之產物……57
圖3. 30 圖3.28依比例放大…………………………………………………58
圖3. 31 托美丁(TLM)之質譜圖圖…………………………………………65
圖3. 32 托美丁(TLM)之質譜斷裂圖………………………………………66
圖3. 33 托美丁(TLM)之LC-MS圖……………………………………………67
圖3. 34 以HPLC單離光解產物TLM-1之LC-MS圖……………………………68
圖3. 35 以HPLC單離光解產物TLM-2之LC-MS圖……………………………69
圖3. 36 光解產物TLM-3之LC-MS圖…………………………………………71
圖3. 37 光解產物TLM-4之LC-MS圖…………………………………………71
圖3. 38 光解產物TLM-5之LC-MS圖…………………………………………72
圖3. 39 光解產物TLM-7之LC-MS圖…………………………………………72
圖3. 40 托美丁(TLM)在酸虐待實驗的酸產物之LC-MS圖…………………73
圖3. 41 托美丁(TLM)酸試驗和光解實驗中推判之產物結構式…………75
圖3. 42 托美丁(TLM)在甲醇溶液中可能之光解反應途徑………………76
圖3. 43 含有Carbonyl group的光反應途徑………………………………77
圖3. 44 佐美酸(Z)3D立體分子模型模擬圖………………………………79
圖3. 45 圖3.42向紙面翻轉45度……………………………………………79
圖3. 46 托美丁(TLM)可能所產的共振反應………………………………80
圖3. 47 托美丁(TLM)和佐美酸(Z)於甲醇中之光降解連結圖……………83
圖3. 48 托美丁(TLM)和佐美酸(Z)於甲醇中之光降解零階迴歸作圖……83
圖3. 49 托美丁(TLM)和佐美酸(Z)於甲醇中之光降解一階迴歸作圖……84
圖3. 50 不同托美丁(TLM)濃度於甲醇溶液中之光降解連結圖…………85
圖3. 51 不同托美丁(TLM)濃度於甲醇溶液中之光降解零階迴歸作圖…85
圖3. 52 不同托美丁(TLM)濃度於甲醇溶液中之光降解一階迴歸作圖…86
圖3. 53 托美丁(TLM)於不同醇類溶媒中之光降解連結圖………………87
圖3. 54 托美丁(TLM)於不同醇類溶媒中之光降解零次迴歸作圖………87
圖3. 55 托美丁(TLM)於不同醇類溶媒中之光降解一次迴歸作圖………88
圖3. 56 托美丁(TLM)溶於不同含水量的甲醇溶液中的光降解連結圖…89
表目次
表1. 1 在細胞中以NSAID藥物去抑制COX-1和COX-2一半濃度所需的藥物濃度(mmol/L)的比值…………………………………………………………4
表2. 1 佐美酸(Z)光解產物之製備型HPLC分析條件……………………13
表2. 2 佐美酸(Z)光解產物的滯留時間…………………………………13
表2. 3 托美丁(TLM)光解產物之製備型HPLC分析條件…………………14
表2. 4 托美丁(TLM)光解產物的滯留時間………………………………14
表2. 5 佐美酸(Z)之分析型HPLC分析條件………………………………15
表2. 6 佐美酸(Z)光解產物的滯留時間…………………………………15
表2. 7 托美丁(TLM)之分析型HPLC分析條件……………………………16
表2. 8 托美丁光(TLM)解產物的滯留時間………………………………16
表2. 9 托美丁與佐美酸LC-MS的質譜分析條件…………………………17
表3. 1 托美丁(TLM)經虐待試驗剩餘百分比……………………………25
表3. 2 托美丁(TLM)峰面積以254/270 nm作一比值……………………25
表3. 3 以ANOVA統計托美丁(TLM)在254/270 nm的峰面積比值…………25
表3. 4 佐美酸(Z)經虐待試驗剩餘百分比………………………………28
表3. 5 佐美酸(Z)峰面積以254/270 nm作一比值………………………29
表3. 6 以ANOVA統計佐美酸(Z)在254/270 nm的峰面積比值……………29
表3. 7 托美丁(TLM)檢量線變異分析……………………………………32
表3. 8 托美丁(TLM)日內與日間確校試驗………………………………33
表3. 9 佐美酸(Z)檢量線變異分析………………………………………34
表3. 10 佐美酸(Z)日內與日間確校試驗…………………………………35
表3. 11 托美丁(TLM)之HPLC系統適合參數………………………………38
表3. 12 佐美酸(Z)之HPLC系統適合參數…………………………………39
表3. 13 佐美酸(Z)和Z-3之NMR數據比較…………………………………43
表3. 14 佐美酸(Z)和Z-4之NMR數據比較…………………………………45
表3. 15 托美丁(TLM)和TLM-3之NMR數據比較……………………………59
表3. 16 托美丁(TLM)和TLM-7之NMR數據比較……………………………61
表3. 17 托美丁(TLM)和TLM-8之NMR數據比較……………………………63
表3. 18 托美丁(TLM)和佐美酸(Z)相似之光解產物及酸催化產物………77
表3. 19 托美丁(TLM)和佐美酸(Z)於甲醇中之光降解的零階和
一階作圖方程式………………………………………………………………84
表3. 20 不同托美丁(TLM)濃度於甲醇溶液中之光降解的零階和
一階作圖方程式………………………………………………………………86
表3. 21 托美丁(TLM)於不同醇類溶媒中之光降解的零階和
一階作圖方程式………………………………………………………………88
表3. 22 三種醇類溶媒的介電係數…………………………………………90

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