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研究生:林秀玲
研究生(外文):Hsiu-ling Lin
論文名稱:尿液樣本內愷他命代謝動力學及phaseⅡ代謝物
論文名稱(外文):The kinetics of ketamine metabolism and its phase Ⅱ metabolites in urine samples
指導教授:邱鐵雄邱鐵雄引用關係賴滄海賴滄海引用關係
學位類別:碩士
校院名稱:慈濟大學
系所名稱:藥理暨毒理學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:129
中文關鍵詞:phase Ⅱ 代謝物酸鹼水解氣相層析質譜儀液相層析-電噴灑-質譜儀愷他命
外文關鍵詞:LC-ESI-MSGC-MSacidic and basic hydrolysisketaminephase Ⅱ metabolites
相關次數:
  • 被引用被引用:2
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  • 下載下載:57
  • 收藏至我的研究室書目清單書目收藏:0
中文摘要
愷他命(Ketamine;K)化學性質與PCP有關,剛開始被設計為外科麻醉劑。這個藥物具有幻覺和麻醉性質,似乎會透過許多不同的路徑影響人群。因此可能在”rave party”內被使用,就為了達到他的分離效果。愷他命主要是在肝臟cytochrome P450代謝,經去甲基化及去氫化形成norketamine (NK)及Dehydronorketamine (DHNK)這兩種代謝物,其中NK會提供愷他命的藥理作用。在先前的研究,愷他命在代謝期間的共軛物性質未被確認。而此實驗的目的,就是經由氣相層析質譜儀和液相層析-電噴灑-質譜儀,確認愷他命的動力學、酸鹼水解後之濃度差異和共軛物之性質。
我們收集注射麻醉劑愷他命之外科病患的尿液(n=6),採尿時間最長為80個小時。在氣相層析質譜儀實驗,尿液樣本先鹽酸水解,之後利用液相-液相萃取(碳酸緩衝液和氫氧化鈉)及三氟醋酸酐衍生。愷他命、NK及DHNK在不同方法之偵測極限,個別為4-20 ng/mL, 2-6 ng/mL及4-20 ng/mL。三種化合物的定量極限,個別為6-20 ng/mL, 4-10 ng/mL及4-8 ng/mL。線性範圍極限,個別為3-4 ng/mL, 2-4 ng/mL及2-4 ng/mL。在不同濃度(40, 80, 160 ng/mL)及方法,愷他命、NK及DHNK的同日內及異日內之準確度(CV %)跟精密度(n=5),個別為0.8到9.8 %和-9.8到9.5。
在這個研究中,我們沒有偵測到明顯的愷他命共軛物的量,但是明顯的觀察到NK及DHNK共軛物的數量。在尿液排泄期間,愷他命及其代謝物至少有兩個高峰期。愷他命代謝物被排泄到尿液中的時間較長,特別是DHNK。酸水解後尿液樣品中NK的濃度明顯增加(n=4),酸鹼水解後DHNK的濃度明顯增加。愷他命、NK及DHNK的共軛型式,在尿液排泄型態上有個體差異。共軛物型式是利用固相萃取以及經由液相層析-電噴灑-質譜儀分析。在腎受損的情況,並沒有明顯干擾到尿液中norketamine (代謝物Ⅰ)濃度。Dehydronorketamine (代謝物Ⅱ)的排泄,在腎受損時會有明顯的變化。肝受損時,NK及DHNK並沒有明顯的變化。我們並沒有發現到NK-G及DHNK-G存在,但是這個共軛物可能以其他型式存在,此共軛型式應具備對酸鹼不穩定的特性。
將來調查共軛物型式之特性,則可以利用專一性消化酵素,確認愷他命代謝期間其他代謝共軛物的型式。
Ketamine (K) is related chemically to PCP, which was initially designed as a surgical anesthetic. The drug has hallucinogenic and analgesia qualities that seem to affect people in very different ways. Hence, it may be used at rave party for its dissociative effect. K is metabolized by demethylation and dehydrogenation in the liver cytochrome P450 to form norketamine (NK) and dehydronorketamine (DHNK). These two metabolites, especially NK, may contribute to the pharmacological effect of K. In previous studies, identities of the conjugates during K metabolism were not demonstrated. The aim of this study is to determine the kinetics of K, concentration variation after acidic and basic hydrolysis and characteristics of conjugates by GC-MS and LC-ESI-MS.
We collected surgical patient’s urine after injection of K as anesthesia(n=6). The longest time for sample collection was 80 hours. In the GC-MS experiment, urine samples were hydrolyzed with HCl, followed by liquid-liquid extraction (bicarbonate buffer and NaOH) and derivatization with trifluoroacetic anhydride. The limit of detection (LODs) of the different method for K, NK, and DHNK was 4-20 ng/mL, 2-6 ng/mL, and 2-6 ng/mL, respectively. The limit of quantification (LOQs) was 6-20 ng/mL, 4-10 ng/mL and 4-8 ng/mL, respectively for the 3 compounds. The limit of linearity range was 3-4 �慊/mL, 2-4 �慊/mL, and 2-4 �慊/mL, respectively. Within-run and between-run precision(% CV)and accuracy (n=5) for K, NK and DHNK at three different concentration (40, 80, and 160 ng/mL) and methods were 0.8 % to 9.8 % and -9.8 % to 9.5 %, respectively.
In this study, we did not detect significant amount of conjugated K, but significant amount of conjugated NK and DHNK was observed. Ketamine and its metabolites have at least two peaks phase in urine excretion. Keteamine metabolites were excreted in urine for a long time, especially DHNK. NK concentration in urine samples were significantly increased after acid hydrolysis (n=4). DHNK concentration was significantly increased after acid and base hydrolysis. The excretion pattern of conjugated form of K, NK, and DHNK was different among individual patients. Conjugated metabolites were extracted by solid phase extraction and analyzed by LC-ESI-MS. The urine concentration levels of norketamine (metabolite I) did not alter significantly in states of renal impairment. The kinetics of the other metabolite (II), dehydronorketamine, did change significantly in renal impairment. The kinetics of NK and DHNK, did not change significantly in liver impairment. We have not positively identified the existence of NK-G and the DHNK-G, but the conjugates may exist in other form. This type of conjugates should have the characteristics of acid and base-lability.
In the future, identity of the conjugated form will be investigated with specific enzyme digestion to determine the type of conjugated metabolites in ketamine metabolism.
前言………………………………………………………………………………………………1
壹、緒論…………………………………………………………………………………………2
一、背景介紹……………………………………………………………………………………2
二、化學結構……………………………………………………………………………………4
三、藥理作用……………………………………………………………………………………5
1.認知功能………………………………………………………………………………………5
2.行為反應………………………………………………………………………………………5
3.心理層面………………………………………………………………………………………6
四、藥物動力學…………………………………………………………………………………7
五、濫用情形……………………………………………………………………………………9
六、氣相層析及液相層析質譜儀………………………………………………………………10
1.氣相層析………………………………………………………………………………………10
2.液相層析………………………………………………………………………………………11
3.質譜儀…………………………………………………………………………………………11
3-1.電子撞擊游離法(Electron Impact;EI)……………………………………………12
3-2.電噴灑游離法(Electrospray Ionization;ESI)……………………………………12
3-3.四極注(Quadrupole)……………………………………………………………………12
3-4.離子阱(Ion Trap)………………………………………………………………………13
七、研究主題……………………………………………………………………………………14
1.動機……………………………………………………………………………………………14
2.目的……………………………………………………………………………………………14
貳、實驗方法……………………………………………………………………………………15
一、尿液樣本……………………………………………………………………………………15
二、試劑…………………………………………………………………………………………15
三、設備…………………………………………………………………………………………16
四、分析步驟……………………………………………………………………………………17
1.氣相層析質譜儀………………………………………………………………………………17
1-1.水解及萃取…………………………………………………………………………………17
1-2.衍生…………………………………………………………………………………………17
1-3.儀器設定……………………………………………………………………………………18
1-4.標準曲線……………………………………………………………………………………18
1-5.校正曲線設定………………………………………………………………………………19
1-6.定性離子及定量離子之選擇………………………………………………………………19
1-7.離子交互作用………………………………………………………………………………19
1-8.偵測極限、定量極限及範圍極限…………………………………………………………19
1-9.精密度及準確度之評估……………………………………………………………………20
1-10.回收率……………………………………………………………………………………20
2.液相層析質譜儀………………………………………………………………………………20
2-1.萃取…………………………………………………………………………………………20
2-2.實驗步驟……………………………………………………………………………………21
2-3.儀器設定……………………………………………………………………………………21
參、實驗結果……………………………………………………………………………………23
一、氣相層析質譜儀……………………………………………………………………………23
1.標準層析及質譜圖……………………………………………………………………………23
2.離子交互作用…………………………………………………………………………………23
3.定性及定量離子之選擇………………………………………………………………………23
4.標準曲線及校正曲線之測定…………………………………………………………………24
5.偵測極限、定量極限及範圍極限……………………………………………………………24
6.精密度及準確度之評估………………………………………………………………………25
7.回收率…………………………………………………………………………………………25
8.尿液樣本中ketamine之代謝動力學評估……………………………………………………25
8-1.Patient 1…………………………………………………………………………………25
8-2.Patient 2…………………………………………………………………………………26
8-3.Patient 3…………………………………………………………………………………27
8-4.Patient 4…………………………………………………………………………………27
8-5.Patient 5…………………………………………………………………………………28
8-6.Patient 6…………………………………………………………………………………29
二、液相層析質譜儀……………………………………………………………………………30
1.萃取……………………………………………………………………………………………30
2.標準品分析結果………………………………………………………………………………30
3.陰性尿液及空白尿液樣本分析結果…………………………………………………………31
4.尿液樣本分析結果……………………………………………………………………………31
肆、討論…………………………………………………………………………………………33
一、尿液樣本取得………………………………………………………………………………33
二、氣相層析分析方法建立的困難……………………………………………………………33
1.水解及萃取方法………………………………………………………………………………33
2.內標準品………………………………………………………………………………………34
3.回收率…………………………………………………………………………………………35
4.尿液樣品分析…………………………………………………………………………………35
三、液相層析分析方法建立的困難……………………………………………………………35
伍、結論…………………………………………………………………………………………36
陸、參考文獻……………………………………………………………………………………37
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