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標題: | 使用液相層析質譜儀定量生物檢體中之N-acetylcysteine與Perfluoroalkyl compounds Quantification of N-acetylcysteine and Perfluoroalkyl compounds in Biological Specimens by Liquid Chromatography - Mass Spectrometry |
作者: | Shu-Han Chang 張淑涵 |
指導教授: | 郭錦樺(Ching-Hua Kuo) |
關鍵字: | 乙?半胱胺酸,全氟碳化合物,管柱後灌流內標準品,液相層析/串聯式質譜儀,乳癌,淋巴液,血漿, N-acetylcysteine,perfluoroalkyl compounds,postcolumn infused-internal standard,liquid chromatography tandem mass spectrometry,breast cancer,perilymph,plasma, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 中文摘要(一)
N-acetylcysteine常作為黏液溶解劑或祛痰劑,也可作為解毒劑,治療Acetaminophen中毒引起的肝損傷。除此之外,近年來有研究指出N-acetylcysteine可能對於Cisplatin等抗癌藥物引起之耳毒性具有預防或治療效果。 本研究開發一簡單、快速、準確且靈敏之超高壓液相層析串聯式質譜儀(UHPLC-MS/MS)分析方法,並應用於測定天竺鼠內耳淋巴液中N-acetylcysteine藥品濃度。因天竺鼠內耳淋巴液樣品量少,方法建立需考量節省樣品,並同時達到良好的靈敏度,本方法經質譜條件最適化後,樣品處理取5 μL淋巴液樣品,加入含內標準品(N-acetylcysteine-15N)之甲醇溶液萃取,經振搖離心後,收集上清液用氮氣吹乾,再用10%甲醇水溶液回溶。液相層析條件選用Poroshell 120 EC-C18作為分析管柱,進樣體積10 μL,移動相使用0.1%甲酸添加於水與乙腈溶液做梯度分析,可在6分鐘內完成分析,質譜條件採用電灑法正離子搭配MRM模式分析,N-acetylcysteine定量離子對m/z 164→m/z 122、定性離子對m/z 164→m/z 76,可獲得最佳的偵測靈敏度及快速且準確的分析結果。 本方法確效結果顯示分析方法之線性範圍1~1000 ng/mL,準確度85.7~107.8%,同日間精密度RSD介於1.37~2.17%之間,異日間精密度RSD皆小於7.32%,定量極限0.5 ng/mL。本研究將開發方法應用於分析天竺鼠給藥後1至48小時之藥物濃度,經耳內注射2% N-acetylcysteine,內耳淋巴液濃度在給藥後1小時測得Cmax為64.6 ± 1.20 μg/mL。 中文摘要(二) 全氟碳化合物(Perfluoroalkyl compounds, PFCs)是化學合成之氟化碳化合物,廣泛使用在工業及各種生活商品上。其具高度化學穩定性之特性,並可能造成肝臟毒性、免疫毒性、生殖發育及神經毒性等,甚至有致癌的風險。近年來,關於這類化合物對環境及人體健康影響越來越被重視。 本研究開發一準確、經濟且靈敏之超高壓液相層析串聯式質譜儀(UHPLC-MS/MS)分析方法測定人體血漿中11種全氟碳化合物濃度,並使用管柱後灌流內標準品(postcolumn infused-internal standard, PCI-IS)結合基質校正因子(matrix normalization factor, MNF)分析策略,校正基質效應及不同基質間的差異,提高定量準確度。 本研究經樣品萃取溶劑、管柱選擇、移動相條件、質譜參數最佳化後,獲得良好的專一性及回收率,液相層析條件選用ACQUITY UPLC® HSS T3 1.8um, 2.1*100mm作為分析管柱,移動相用2 mM ammonium acetate添加於水與甲醇溶液做梯度分析,質譜條件採用電灑法負離子MRM模式分析,可在8.5分鐘內完成分析。本研究選用[13C7]-perfluoroundecanoic acid作為定量使用之柱後灌流內標準品,確效結果顯示本方法之線性範圍0.5~25 ng/mL,準確度大於83.3%,同日間精密度RSD小於9.47%,異日間精密度RSD小於17.15%。 本研究將開發方法應用於乳癌病人血漿中全氟碳化合物之定量分析,195個臨床血漿檢體包含96位乳癌患者與99位正常女性,比較兩組血漿中PFC濃度,未有顯著差異(p>0.05),依年齡分群,挑選出50歲以下之女性年輕族群,包含57位乳癌病患及52位正常女性,結果顯示,PFOS在乳癌患者較正常女性濃度高,有顯著差異(p=0.020),因此PFOS之毒性有可能與乳癌年輕族群之風險因子相關。 本研究提供一個經濟、準確之全氟碳化合物分析平台,可用於研究全氟碳化合物與相關疾病之風險性。 Abstract (Ⅰ) N-acetylcysteine is often used as a mucolytic agent or expectorant, but also as an antidote for the treatment of liver damage caused by acetaminophen poisoning. Several recent studies have found that N-acetylcysteine may have prophylactic or therapeutic effects against the toxic side effects relating to listening impairment caused by anti -cancer drugs such as cisplatin. This study developed a simple, rapid, accurate and sensitive analytical method using ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS / MS) for N-acetylcysteine quantification. The method was applied to determine N-acetylcysteine concentration in the inner ear lymphatic fluids of guinea pigs. As the volume of guinea pig inner ear lymphatic fluid is generally small, we needed to establish a method which is able to achieve good sensitivity using small volumes. After optimization of the parameters, the final method uses methanol containing N-acetylcysteine-15N, internal standard, as the extraction solution to extract 5 μL of lymphatic fluids . The extract was dried under nitrogen stream and reconstituted in 10% methanol solution. In terms of the liquid chromatographic conditions, we used a Poroshell 120 EC-C18 column and 10 μL as the injection volume. The mobile phases were composed of 0.1% formic acid in the aqueous phase (water) and organic phase (acetonitrile) flowing in a gradient form. The analysis time could be completed in 6 minutes. In the mass conditions, Multiple Reaction Monitoring (MRM) was used coupled with electrospray positive ionization. The quantitative ion pair used to monitor N-acetylcysteine was m/z 164 to m/z 122, and the qualifier ion pair was m/z 164 to m/z 76. This method is able to achieve good detection sensitivity and fast and accurate results. Validation results showed the linear range of the developed method is between 1 ng/mL and 1000 ng/mL, and the accuracy is in the range of 85.7 to 107.8%. The intra-day precision is in the range of 1.37 to 2.17%, and the inter-day precision is less than 7.32%, expressed as the relative standard deviation. The quantification limit is 0.5 ng/mL. The developed method was applied to quantify N-acetylcysteine concentration within 1 to 48 hours post dose in guinea pig inner ear lymphatic fluid. After intratympanic injection of 2% N-acetylcysteine, the perilymph detected the highest concentration of 64.6 ± 1.20 μg/mL at 1 hour post dose. Abstract (Ⅱ) Perfluoroalkyl compounds (PFCs) are chemically produced chemicals. Iit has thus been widely used on many products. Because of its durable characteristics, it is viewed as a persistent organic pollutant. Its effects on the environment and also on the human body have recently become a hot topic. Current literature has linked PFCs to liver, immune, reproductive, and neural toxicity, and it has also been found to be carcinogenic. This study aims to develop an accurate, economic and sensitive method to measure 11 PFCs in human plasma using UHPLC-MS/MS. The Postcolumn Infused-Internal Standard (PCI-IS) combined with Matrix Normalization Factor (MNF) method was applied to correct matrix effects caused quantification errors and provided the accurate quantification of these PFCs. The extraction solvent, column, mobile phase, gradient and various mass parameters were optimized to establish a method with the best sensitivity and selectivity for the selected PFCs, with the final method being run on a ACQUITY UPLC HSS T3 1.8 µm, 2.1*100 mm column, using mobile phases of either water or methanol, both with 2 mM ammonium acetate, and measured using electrospray ionization on negative mode collected with MRM. This analysis is complete within 8.5 minutes. [13C7]-perfluoroundecanoic acid was selected as the PCI-IS for PFCs quantification. The validation results revealed the developed method exhibited a linear range between 0.5 ng/mL and 25 ng/mL, and accuracies above 83.3% with repeatability RSD within 9.47% and reproducibility RSD within 17.2%. The developed method was applied to measure PFC levels in breast cancer patients. A total of 195 clinical subjects were used to evaluate the correlation between PFC and breast cancer risk, with 96 breast cancer patients and 99 healthy volunteers. The results showed no significant difference (p> 0.05) among the 11 PFC compounds in plasma between the two groups. We further analyzed the data by filtering individuals by age, selecting 57 breast cancer patients and 52 healthy volunteers. In the age filtered cohort, PFOS was found to be statistically significant (p = 0.020) between healthy subjects and breast cancer patients. The concentration of PFOS in breast cancer patients is higher than in healthy subjects. Thus, the toxicity of PFOS may be a possible risk factor associated with breast cancer in the younger age group. This study provides an economic and accurate method for the detection of multi-residue analysis of PFC, and it can be used to other studies to invesstigate the risk of PFCs and related diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/49391 |
DOI: | 10.6342/NTU201601279 |
全文授權: | 有償授權 |
顯示於系所單位: | 藥學系 |
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