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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 陳家揚(Chia-Yang Chen) | |
dc.contributor.author | Shu-Yen Liu | en |
dc.contributor.author | 劉書雁 | zh_TW |
dc.date.accessioned | 2021-06-07T23:44:38Z | - |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2021 | |
dc.date.submitted | 2021-02-08 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/16732 | - |
dc.description.abstract | 化學物質的應用隨著工業及日常所需日漸增加,人體可能藉由不同途徑暴露多種化學品,進而對人體產生危害。標的性分析方法針對預先選擇的化合物進行定量,但此種方法無法得知未知化學物的暴露。若透過非標的性生物偵測,則能在不預設待測物下進行檢測,釐清人體暴露的化學物。 本研究利用超高液相層析搭配飛行時間串連式質譜儀,分析彰化大二林地區自2005年至2014年蒐集的186個血清樣本,包含罹患直腸癌及腺腫之樣本。以全離子模式採集訊號並進行後端比對資料庫,以鑑定化學物質之種類。針對篩查出之化學物質使用ToxPi軟體進行排序,以樣本中檢出頻率及訊號強度,連結生物活性、每日容許攝取量及致癌性或致突變性作為排序依據,建立優先化學品清單。樣本共計檢出296中化合物,多數化合物檢出率低於10%,檢出率大於90%之化合物共計3個,分別為ethotoin抗癲癇藥物、cinnamic acid食品添加劑桂皮酸和phenylalanine人體必需胺基酸。根據優先化排序結果,選擇六種化學物進行定量,包含兩種阻燃劑tris(2-butoxyethyl)phosphate和tributylphosphate、塑化劑diethyl phthalate、兩種藥物methyltestosterone和ethotoin、農藥8-hydroxyquinoline。 除了優先化的化學物,亦選擇可能誘發直腸癌之化學物進行定量。多環芳香烴是經由燃燒不完全產生,其脂溶性容易被哺乳類腸道吸收。根據毒理學及流行病學顯示多環芳香烴具有生殖和致癌毒性,可能誘發消化道癌症和乳癌,因此本研究也納入分析8種多環芳香烴和1-羥基焦腦油,並完成分析方法開發。分析物使用Waters ACQUITY UPLC I-Class PLUS極致液相層析搭配Waters Xevo TQ-XS串聯式質譜儀,優先化合物和1-羥基焦腦油使用Waters CORTECS C18 (30 × 2.1 mm, 1.6 μm),游離介面為正電灑游離,有機動相為甲醇,水性動相為0.1%甲酸水溶液,採梯度流析,層析時間共7.1分鐘。8種多環芳香烴則以正大氣壓力化學游離,並使用Agilent RRHD PAH (50 × 3.0 mm, 1.8 μm),有機動相為乙腈,水性動相為5-mM甲酸銨水溶液,採梯度流析,層析時間共8.1分鐘。質譜儀以多重反應偵測模式進行離子監測。標準品校正迴歸線之線性範圍為0.2-1,000 ng/mL,其判定係數達到0.990以上。定性極限範圍為0.029-17.5 ng/mL,定量極限範圍介於0.098-42.0 ng/mL。 在定量分析中,進行血清前處理方法優化以提升多環芳香烃之萃取效率,在進行Ostro 96-well plate萃取時,以250微升含有1%甲酸之乙腈和100微升甲醇進行沖提,並添加二甲基亞碸20微升避免多環芳香烃在濃縮過程中蒸發,優化結果顯示化合物訊號能提高1.09-1.59倍。血清之基質效應和萃取效率範圍分別為56.1-132%和42.5-90.6%。同日和異日之回收率介於70-140%,而變異係數大都小於20%。 本研究定量分析之樣本共93個血清,包含29個直腸癌病例、33個腺腫病例、31個健康樣本。優先化學物中,tris(2-butoxyethyl)phosphate、tributylphosphate和diethyl phthalate在樣本中皆有檢出。多環芳香烴中benzo[a]anthracene在所有樣本中皆有檢出,且幾何平均濃度高(97.0 ng/mL, n = 93)。濃度資料數值搭配問卷資料進行中位數差異和複回歸分析,並訂定p-value < 0.05有顯著影響。結果顯示tris(2-butoxyethyl)phosphate、tributylphosphate、diethyl phthalate和 benzo[a]anthracene濃度在不同健康狀態組別並無顯著差異。tris(2-butoxyethyl)phosphate濃度和暴露二手菸及雞蛋食用頻率呈正相關。tris(2-butoxyethyl)phosphate與牛奶攝取頻率成顯著負相關,而diethyl phthalate和tributylphosphate濃度則和牛奶攝取頻率呈正相關。benzo[a]anthracene與茶攝取頻率呈正相關。本研究結合篩查及定量分析方法,有助於降低篩查時偽陽性偵測率,能更瞭解潛在暴露物及其人體內在濃度,以提供更清楚的人體暴露情形。 | zh_TW |
dc.description.abstract | Humans may be exposed to chemicals through diet, consumer products or environmental matrixes. Target analysis quantifies pre-selected chemicals; therefore, it could not illustrate the exposure of unknown chemicals. In contrast, non-target analysis can identify unknown contaminants without prior information to chemicals and can clarify chemical exposure in humans. In this study, 186 serum samples including colorectal and adenoma cases from Erlin area at Changhua county collected between the years of 2005 and 2014 were analyzed at all ions MS/MS mode with a UHPLC-QTOF MS. The spectra were matched with libraries. Identified chemicals were prioritized using ToxPi software based on the information of their detected frequencies, peak abundance, bioactivity data, acceptable daily intake and carcinogenicity. A Total of 296 chemicals were identified in samples and most detection frequencies of the chemicals were below 10%. There were three compounds with detection frequencies above 90% which were ethotoin (anticonvulsant drug), cinnamic acid (a flavoring agent) and phenylalanine (an amino acid). According to the results of ToxPi ranking list and toxicological references, six compounds were chosen for targeted quantitative analysis, including two flame retardants tris(2-butoxyethyl)phosphate and tributylphosphate, diethyl phthalate, diethyl phthalate, two drugs methylestosterone and ethotoin) and one pesticide (8-hydroxyquinoline). Besides prioritized compounds, chemicals that may induce colorectal cancer were included for quantification. Polycyclic aromatic hydrocarbons (PAHs), resulting from incomplete combustion of organic matters, are lipid soluble and thus are easily absorbed from gastrointestinal tracts of mammals. PAHs possess reproductive toxicity and carcinogenicity and may induce digestive tract cancer and breast cancer. Therefore, eight PAHs and 1-hydroxypyrene (1-OHP) were also chosen as analytes. A method was developed for quantifying 14 analytes in serum; prioritized chemicals and 1-OHP were determined using ultra-performance liquid chromatograph coupled with tandem mass spectrometer (UPLC-MS/MS) at positive electrospray ionization (ESI+). Eight PAHs were analyzed in serum using UPLC-MS/MS at positive atmospheric pressure chemical ionization (APCI+) mode. Two different chromatographic conditions were applied to separate analytes. Regarding the chromatography, the ESI+ used a Waters CORTECS C18 column (30 × 2.1, 1.6 μm) with mobile phases of (A) 0.1% formic(aq) and (B) methanol, and the APCI+ utilized an Agilent RRHD PAH column (50 × 3.0 mm, 1.8 μm) with mobile phases of (A) 5-mM ammonium formate(aq) and (B) acetonitrile. The linear ranges of calibration curves were from 0.2 to 1,000 ng/mL with the correlation of determination (r2) greater than 0.990. LODs were 0.029-17.5 ng/mL and LOQs were 0.098-42.0 ng/mL. A sample preparation applied on serum samples was optimized for improving the extraction efficiency of PAHs. During extractions of samples, 250-μL of 1% formic acid in acetonitrile and 100-μL methanol were eluted from Ostro 96-well plate and 20-μL dimethyl sulfoxide was added to extract to prevent evaporation of PAHs. The optimal results showed 1.09 to 1.59-fold increase in signal intensities of PAHs. The matrix effects and extraction efficiencies of the 14 analytes ranged from 56.1 to 132% and 42.5 to 90.6%, respectively. The recoveries of inter- and intra-day were 70-140% and most coefficient of variation (CV%) were below 20%. In this study, totally 93 serum samples were conducted on the target analysis, including subjects of 29 colorectal cancer (CRC) patients, 33 adenoma patients and 31 healthy controls. For the prioritized chemicals, tris(2-butoxyethyl)phosphate, tributylphosphate and diethyl phthalate were detected in all serum samples. For PAHs and 1-OHP, only benzo[a]anthracene was detected in all samples with a GM concentration of 97.0 ng/mL. Kruskal-Wallis test and multiple linear regression were conducted with the analytes levels and questionnaire data. Statistical significance was defined as p < 0.05. The median levels of tris(2-butoxyethyl)phosphate, tributylphosphate, diethyl phthalate and benzo[a]anthracene have no significant differences among colorectal cancer, adenoma and control groups. The levels of tris(2-butoxyethyl)phosphate were positively associated with secondhand smoke and weekly egg consumption. The levels of tris(2-butoxyethyl)phosphate showed significant negative association with weekly milk consumption; however, the levels of diethyl phthalate and tributylphosphate have positive association with weekly milk consumption. The levels of benzo[a]anthracene were positively associated with weekly tea consumption. A combination of suspect screening method and quantification for prioritized chemicals could reduce false positives detected rates in suspect screening, determine the levels of potential chemicals in human bodies. Furthermore, applying the combination of screening and quantification methods could get a better understanding of chemicals’ potential risk for humans. | en |
dc.description.provenance | Made available in DSpace on 2021-06-07T23:44:38Z (GMT). No. of bitstreams: 1 U0001-0502202112120400.pdf: 4024856 bytes, checksum: 716e06443e71b09219c6bcec2d1b7ba5 (MD5) Previous issue date: 2021 | en |
dc.description.tableofcontents | 中文摘要 i Abstract iii Contents vii List of Figures x List of Tables xii Chapter 1 Introduction 1 1.1 Biomonitoring 1 1.2 High-resolution mass spectrometry (HRMS) and non-targeted analysis 2 1.3 Prioritization of chemicals 4 1.4 Polycyclic aromatic hydrocarbons 6 1.5 Colorectal cancer and Adenoma 8 1.6 Objectives 9 Chapter 2 Material and Methods 11 2.1 Reagents and Materials 11 2.2 Sample Collection 13 2.3 Sample preparation 13 2.3.1 Sample preparation for suspect screening 14 2.3.2 Sample preparation for target analysis 14 2.4 Instrument analysis 15 2.4.1 UHPLC QTOF MS 16 2.4.1.1 Mass spectrometric condition 16 2.4.1.2 Chromatographic conditions 16 2.4.1.3 Data processing 17 2.4.2 UPLC Xevo TQ-XS MS/MS 19 2.4.2.1 Mass spectrometric condition 19 2.4.2.2 Chromatographic conditions 20 2.5 Chemical prioritization criteria 20 2.5.1 Prioritization criteria 21 2.5.2 Parameters for prioritization 22 2.5.2.1 Empirical data 22 2.5.2.2 External information 22 2.6 Method validation 24 2.6.1 Suspect screening: Quality assurance and quality control 24 2.6.2 Target analysis 25 2.6.2.1 Matrix effect and extraction efficiency 25 2.6.2.2 Accuracy and precision 26 2.6.2.3 Identification and quantification 26 2.6.2.4 Quality assurance and quality control 27 2.6.2.5 Statistical analysis 27 Chapter 3 Results and Discussion 29 3.1 Suspect screening results 29 3.2 Prioritization and confirmation 30 3.3 Optimization of sample preparation 34 3.4 Chromatography 36 3.4.1 Selection of columns and mobile phases 36 3.4.2 Adjustment of UPLC gradient 37 3.5 Method validation 38 3.5.1 Matrix effect and extraction efficiency 38 3.5.2 Accuracy and precision 39 3.6 Identification and quantification 39 3.7 Real samples for target analysis 40 3.8 Limitations 42 Chapter 4 Conclusions 45 Reference 47 Figures 57 Tables 75 Attachment: IRB approval 97 | |
dc.language.iso | en | |
dc.title | 使用極致液相層析/串聯式質譜術篩查及定量血清中環境污染物 | zh_TW |
dc.title | Screening and Quantitation of Environmental Pollutants in Serum Using Ultra–performance Liquid Chromatography/Tandem Mass Spectrometry | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 陳保中(Pau-Chung Chen),陳鑫昌(Hsin-Chang Chen) | |
dc.subject.keyword | 血清,生物偵測,優先化,極致液相層析/串聯式質譜術,多環芳香烴, | zh_TW |
dc.subject.keyword | Serum,Biomonitoring,Prioritization,Ultra–performance liquid chromatography/tandem Mass Spectrometry,Polycyclic aromatic hydrocarbons, | en |
dc.relation.page | 98 | |
dc.identifier.doi | 10.6342/NTU202100572 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2021-02-08 | |
dc.contributor.author-college | 公共衛生學院 | zh_TW |
dc.contributor.author-dept | 環境與職業健康科學研究所 | zh_TW |
顯示於系所單位: | 環境與職業健康科學研究所 |
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