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研究生:何靖凱
研究生(外文):Jing-Kai Ho
論文名稱:萊克多巴胺在大鼠體內之藥物動力學與器官分佈探討
論文名稱(外文):Pharmacokinetics of Ractopamine and its Organ Distribution in Rats
指導教授:霍德義霍德義引用關係蔡東湖蔡東湖引用關係
指導教授(外文):Teh-Ia HuoTung-Hu Tsai
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:藥理學研究所
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:2014
畢業學年度:102
語文別:中文
論文頁數:80
中文關鍵詞:生體可用率高效液相層析串聯質譜儀藥物動力學萊克多巴胺器官分佈
外文關鍵詞:bioavailabilityHPLC-MS/MSpharmacokineticsractopamineorgan distribution
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萊克多巴胺為一種乙型受體素,可使動物的瘦肉比例上升,而被稱做所謂的瘦肉精。然而,有研究報告指出,萊克多巴胺對於心血管系統有潛在性的風險,基於此原因,萊克多巴胺在許多國家中被禁止使用。近年,是否要開放含有萊克多巴胺的肉品進入臺灣市場,已衍生成了一個政治議題,引起社會各界高度關注。然而,目前並沒有對於萊克多巴胺藥物動力學的詳細研究被發表。

本篇研究的實驗目的,首要是建立高效液相層析串聯質譜儀的測量方式,以定量萊克多巴胺於大鼠血液及器官樣品中的含量,並對於此分析方式加以確效。之後會利用該方法探討萊克多巴胺於大鼠體內的藥物動力學與器官分佈狀態。

根據美國食品藥物管理局的建議規範,方法確效包含了線性、精確度、準確度、基質效應、萃取回收率、以及安定性的評估。本實驗所建立的分析方式已符合美國食品藥物管理局之規範。

以1 mg/kg與10 mg/kg的劑量,靜脈給予萊克多巴胺之後,所得到的排除半衰期分別為118與165分鐘。而萊克多巴胺的口服生體可用率為2.99 %。以靜脈給藥之後,觀察到萊克多巴胺在大鼠體內的器官分佈程度由高至低分別為:腎、肺、脾、心、肝、肌肉、血漿、而濃度最低的是腦。

根據本研究之實驗結果,可發現萊克多巴胺具有高度組織分佈以及非線性藥物動力學的特性。推論該現象是因為萊克多巴胺於大鼠腎臟內的排除機制已達飽和,故呈現非線性排除所致。冀望此研究成果能做為衛生主管機關訂定萊克多巴胺相關政策標準的參考依據之一。

Ractopamine, a β-agonist, is applied to enhance the amount of lean meat in livestock. However, due to the potential risk to the cardiovascular system, ractopamine has been banned for use in food-producing animals in many countries. Importing ractopamine-containing meat has become a political issue in Taiwan. Nevertheless, pharmacokinetic studies of ractopamine have not been fully investigated. The aim of study is to develop a high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method to determine ractopamine. This validated method was used to investigate the pharmacokinetics and organ distribution of ractopamine in rats. Method validation includes linearity, accuracy, precision, matrix effect, recovery, and stability, as recommended by the U.S. Food and Drug Administration (US-FDA). The validation results met the US-FDA’s suggestion. After ractopamine administration, the average elimination half-lives of two intravenous doses were 118 and 165 min, respectively. The oral bioavailability of ractopamine was 2.99 %. After intravenous administration, the ractopamine levels in order from high to low were kidney, lung, spleen, heart, liver, muscle, plasma and brain. Nonlinear pharmacokinetics and high tissue distribution were found after ractopamine intravenous administration. These phenomena might be due to nonlinear elimination in the kidney. Our findings could be applied with reference to ractopamine-related policies.
目錄

附表目錄……………………………………………vii
附圖目錄……………………………………………viii
Abstract…………………………………………1
中文摘要…………………………………………2
第一章 緒論……………………………………4
第一節 萊克多巴胺介紹與相關研究…………4
一、乙型受體素藥理活性研究………………4
二、萊克多巴胺簡介與相關安全性評估………………5
三、萊克多巴胺相關分析及藥物動力學研究…………6
第二節 高效液相層析儀與方法確效簡介……………7
一、高效液相層析儀簡介………………………………7
1. 移動相………………………………………………7
2. 層析管柱……………………………………………8
3. 偵測裝置……………………………………………8
二、分析方法確效簡介………………………………10
第三節 藥物動力學……………………………………13
一、 藥物動力學概述……………………………13
二、藥物動力學基本定義……………………………17
1. 藥物動力學模式定義………………………………17
2. 線性與非線性藥物動力學…………………………20
三、藥物動力學參數介紹………………………………21
第二章 研究動機與目的………………………………26
第三章 實驗材料與方法………………………………27
第一節 實驗材料………………………………………27
一、化學試藥…………………………………………27
二、儀器設備……………………………………………27
三、實驗動物……………………………………………28
四、試藥配製……………………………………………29
五、應用軟體……………………………………………29
第二節 動物實驗………………………………………29
一、萊克多巴胺於大鼠內靜脈給藥實驗………………29
二、萊克多巴胺於大鼠內口服給藥實驗………………30
三、萊克多巴胺於大鼠內器官分佈實驗………………31
第三節 樣品處理………………………………………32
一、血液樣品處理………………………………………32
二、器官樣品處理………………………………………32
第四節 方法確效………………………………………33
一、HPLC-MS/MS分析條件………………………………33
二、線性、精確性與準確性評估………………………34
三、基質效應與萃取回收率……………………………35
四、穩定性評估…………………………………………36
第五節 數值統計………………………………………37
第四章 實驗結果………………………………………39
第一節 HPLC-MS/MS層析結果…………………………39
第二節 樣品處理………………………………………41
第三節 方法確效………………………………………42
一、線性、精確性與準確性評估………………………42
二、基質效應與萃取回收率……………………………43
三、穩定性評估…………………………………………44
第四節 萊克多巴胺之藥物動力學……………………45
第五節 萊克多巴胺之器官分佈………………………46
第五章 實驗討論………………………………………48
第一節 萊克多巴胺樣品製備與分析方式討論………48
第二節 萊克多巴胺之非線性藥物動力學……………49
第三節 萊克多巴胺之口服生體可用率………………49
第四節 萊克多巴胺於大鼠器官內之蓄積……………50
第六章 結論……………………………………………54
第七章 參考文獻………………………………………55
附表………………………………………………………61
附圖………………………………………………………70

附表目錄

Table 1.
Inter-day and intra-day assay precision (% RSD) and accuracy (% Bias) values of HPLC-MS/MS method for the determination of ractopamine in rat plasma and various organ homogenates.…………………………………………………………………61

Table 2.
Matrix effect (ME) and recovery (RE) data for ractopamine and nylidrin (IS) in rat plasma and organ homogenates.……………………………………………………64

Table 3.
The stability evaluation data for ractopamine in rat plasma, liver, heart, spleen, lung, kidney, brain, and muscle homogenates.……………………………………………67

Table 4.
Pharmacokinetic data of ractopamine in the rat blood after ractopamine administration. ………………………………………………………………………69

附圖目錄

Figure 1.
Chemical structures and the product ion mass spectra of (a) ractopamine (m/z 302.10) and (b) nylidrin (m/z 300.15) with electrospray positive-ion mode. ………………70

Figure 2.
HPLC-MS/MS chromatograms of (a) blank plasma, (b) blank plasma spike with ractopamine 5 ng/mL, (c) plasma sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (5-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ……………………………………………………………………………71

Figure 3.
HPLC-MS/MS chromatograms of (a) blank liver homogenate, (b) blank liver homogenate spike with ractopamine 5 ng/mL, (c) liver sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (5-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………72

Figure 4.
HPLC-MS/MS chromatograms of (a) blank heart homogenate, (b) blank heart homogenate spike with ractopamine 5 ng/mL, (c) heart sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (10-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………73

Figure 5.
HPLC-MS/MS chromatograms of (a) blank spleen homogenate, (b) blank spleen homogenate spike with ractopamine 5 ng/mL, (c) spleen sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (50-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………74

Figure 6.
HPLC-MS/MS chromatograms of (a) blank lung homogenate, (b) blank lung homogenate spike with ractopamine 5 ng/mL, (c) lung sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (50-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………75

Figure 7.
HPLC-MS/MS chromatograms of (a) blank kidney homogenate, (b) blank kidney homogenate spike with ractopamine 5 ng/mL, (c) kidney sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (50-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………76

Figure 8.
HPLC-MS/MS chromatograms of (a) blank brain homogenate, (b) blank brain homogenate spike with ractopamine 5 ng/mL, (c) brain sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.). Peak 1 : ractopamine, peak 2: nylidrin. ……………………………………………………………………………77

Figure 9.
HPLC-MS/MS chromatograms of (a) blank muscle homogenate, (b) blank muscle homogenate spike with ractopamine 5 ng/mL, (c) muscle sample collected at 45 min after ractopamine administration (1 mg/kg, i.v.) (10-time dilution). Peak 1 : ractopamine, peak 2: nylidrin. ………………………………………………………78

Figure 10.
Concentration versus time profile of ractopamine in rat plasma after drug administration (10 mg/kg, i.v.; 1 mg/kg, i.v.; and 10 mg/kg, p.o.) Data are expressed as means ± SD (n=6). ………………………………………………………………79

Figure 11.
Rat plasma, liver, heart, spleen, lung, kidney, brain, and muscle levels of ractopamine after administration of ractopamine (1 mg/kg, i.v.); data expressed as means ± SD (n=6). The ractopamine concentration units of plasma and organs are μg/mL and μg/g, respectively. …………………………………………………………………………80
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