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研究生:呂守正
研究生(外文):LU, SHOU-CHENG
論文名稱:利用液相層析串聯質譜儀建立和驗證人血清中尿毒症毒素硫酸吲哚酚和對甲酚基硫酸鹽的方法
論文名稱(外文):Establishment and validation for uremic toxin indoxyl sulfate and p-cresol sulfate in human serum by using LC-MS/MS method
指導教授:劉俊仁劉俊仁引用關係
指導教授(外文):LIU, JUN-JEN
口試委員:李麗花楊沂淵劉俊仁
口試委員(外文):LEE, LI-HUAYANG, YI-YUANLIU, JUN-JEN
口試日期:2023-07-03
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系碩士在職專班
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2023
畢業學年度:111
語文別:中文
論文頁數:47
中文關鍵詞:液相層析串聯質譜儀硫酸吲哚酚甲酚基硫酸鹽
外文關鍵詞:LC-MS/MSindoxyl sulfatep-cresol sulfate
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目的:慢性腎臟病患者腎功能的惡化和尿毒素具有相當高的相關性,因為尿毒素會誘發炎症反應與增加氧化壓力,導致腎絲球硬化與腎間質纖維化,加重腎功能的衰退。依據2021年台灣腎病年報指出台灣透析發生率及盛行率居高不下,2019年臺灣透析盛行患者數共86,840 人,相較2015年上升11.7 %。近年來有許多研究證明硫酸吲哚酚(indoxyl sulfate,以下簡稱IS)和對甲酚基硫酸鹽(p-cresol sulfate,以下簡稱pCS)對於早期發現腎臟病有直接相關。本研究旨在利用液相層析串聯質譜儀建立和驗證人血清中硫酸吲哚酚和對甲酚基硫酸鹽的方法供臨床檢驗使用。
方法:樣品經蛋白質沉澱處理後注射於層析儀,接著利用電噴灑方式進入偵測器,並以多重反應監控模式偵測質荷比,透過固定相滯留時間之長短,於層析圖呈現之波峰面積進行定量,每一檢體分析時間為5.5分鐘。
結果:(1)線性分析(Linearity):IS與pCS的線性相關係數皆大於0.995;(2)精密度分析(Precision):within day變異係數皆小於3.6%,between day變異係數皆小於6.8%;(3)專一性分析(Specificity):IS與pCS其保留時間(Retention Time)分別為1.76±0.02與2.15±0.01可以很清楚地在分析圖譜上分別開來;(4)基質效應分析(Matrix effect):IS為86.9 %~110.5 %,pCS為95.6 %~101.0 %;(5)最低定量濃度(Limit of quantitation):IS與pCS均可達到0.001 ppm, 其中IS訊號雜訊比為143,pCS訊號雜訊比為391。
結論:我們驗證了一種快速,靈敏的液相層析串聯質譜儀的檢驗方法,可用於定量測定血清中IS和pCS的濃度,將可提供臨床上早期發現腎臟病的可能性。
Purpose:The deterioration of renal function in patients with chronic kidney disease has a high correlation with uremic toxins, because uremic toxins can induce inflammation and increase oxidative stress, leading to glomerulosclerosis and renal interstitial fibrosis, aggravating the decline of renal function.According to the 2021 Taiwan Kidney Disease Annual Report, the incidence and prevalence of dialysis in Taiwan remain high. In 2019, the number of patients with dialysis in Taiwan was 86,840, an increase of 11.7% compared to 2015.In recent years, many studies have proved that indoxyl sulfate (hereinafter referred to as IS) and p-cresol sulfate (hereinafter referred to as pCS) are directly related to the early detection of kidney disease. This study aims to establish and validate a method for indoxyl sulfate and p-cresol sulfate in human serum using liquid chromatography tandem mass spectrometry for clinical testing.
Method:The sample was injected into the chromatograph after protein precipitation, and then entered the detector by electrospray, and the mass-to-charge ratio was detected by the multiple reaction monitoring mode. The retention time of the stationary phase was displayed in the chromatogram. The peak area was quantified, and the analysis time for each sample was 5.5 minutes.
Results:(1) Linearity analysis:the IS and pCS linear correlation coefficients were both greater than 0.995;(2) Precision analysis:the intra-day coefficients of variation were all less than 3.6%, and the inter-day coefficients of variation were all less than 6.8%;(3) Specificity analysis:The retention time of IS and pCS is 1.76±0.02 and 2.15±0.01 respectively, which can be clearly separated on the analysis spectrum; (4) Matrix effect analysis:IS was 86.9 %~110.5 %, pCS was 95.6 %~101.0 %;(5) The Limit of quantitation:IS and pCS can reached 0.001 ppm, of which the signal-to-noise ratio of IS was 143, and the signal-to-signal ratio of pCS was 391.
Conclusions:We validated a rapid and sensitive liquid chromatography tandem mass spectrometry method for the quantitative determination of IS and pCS concentrations in serum, which will provide the possibility of early detection of renal disease clinically.
壹、 背景
1.1 腎臟病概況………………………………………………………...第06頁
1.2 腎臟病篩檢與診斷...........................................................................第07頁
1.2.1 硫酸吲哚酚(indoxyl sulfate)..................................................第08頁
1.2.2 對甲酚基硫酸鹽(p-cresol sulfate).........................................第09頁
1.3 質譜儀…………………………………………………...………...第11頁
1.3.1 質譜儀原理............................................................................第11頁
1.3.2 液相層析串聯質譜儀(LC-MS/MS)......................................第12頁
1.3.3 電噴灑游離法(ESI)...............................................................第13頁
1.3.4 質量分析器(Mass analyzer)..................................................第13頁
1.4 研究動機與目的…………...............................................................第15頁
貳、 研究材料與方法
2.1 藥品與試劑………….......................................................................第16頁
2.2 標準品溶液配置…………………………………………………...第16頁
2.2.1 1000 μg/mL儲存溶液(stock solution)配置………………...第16頁
2.2.2 混合標準溶液之配置………………….…………………...第17頁
2.3 實驗器材與儀器設備…...................................................................第18頁
2.3.1 實驗器材…….…...................................................................第18頁
2.3.2 儀器設備…….…...................................................................第18頁
2.3.3 液相層析條件.…...................................................................第18頁
2.4 標準曲線製備……………………………………………………..第20頁
2.5 樣品前處理………………………………………………………..第20頁
2.6 資料分析…………………………………………………………..第20頁
參、 分析與結果
3.1 層析圖…………………………...…………………………………第21頁
3.2 進樣體積測試...................................................................................第25頁
3.3 標準曲線製作……………...............................................................第29頁
3.4 精密度驗證…..................................................................................第31頁
3.5 基質效應…………………...............................................................第33頁
3.6 最低定量濃度……………...............................................................第36頁
3.7 準確度驗證………………...............................................................第39頁
肆、 討論與結論……………………….........................................................第41頁
伍、 參考文獻………….................................................................................第45頁
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