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系統識別號 U0026-1606201916282100
論文名稱(中文) 利用奈升流速超高效能液相層析串聯質譜進行單根毛髮的皮質醇分析
論文名稱(英文) Cortisol Analysis Using a Nanoflow Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry in Sensitive Single Hair Analysis
校院名稱 成功大學
系所名稱(中) 環境醫學研究所
系所名稱(英) Institute of Environmental and Occupational Health
學年度 107
學期 2
出版年 108
研究生(中文) 張志瑋
研究生(英文) Chih-Wei Chang
學號 S76051059
學位類別 碩士
語文別 英文
論文頁數 62頁
口試委員 指導教授-廖寶琦
口試委員-陳逸然
召集委員-陳皓君
口試委員-張耀仁
口試委員-吳欣怡
中文關鍵字 皮質醇  單根頭髮  奈升流速液相層析-串聯質譜  非金屬毛細管融塊 
英文關鍵字 Cortisol  Single hair  Nanoflow ultra-high performance liquid chromatography-tandem mass spectrometry  nonmetallic in-capillary frit 
學科別分類
中文摘要 皮質醇與疾病診斷及心理狀況相關,被認為是壓力指標,因此測量皮質醇的含量對於反應個人健康評估至關重要。生物流體中的皮質醇對於短期變異的測量是一個優勢,但對於觀察長期變化則具有挑戰性。內生性代謝物和環境化學物質在頭髮生長過程中會進入頭髮儲存,頭髮中皮質醇的縱向分佈能夠反映過去幾個月累積的皮質醇分泌,因此頭髮的分析是長期生物監測的合適樣品。然而,通過目前的超高效液相層析串聯質譜法(ultra-high performance liquid chromatography-mass spectrometry, UHPLC-MS) 檢測單根毛髮中微量的皮質醇仍是一項挑戰,所以對於有限或微量的頭髮分析,需要建立更靈敏的分析方法。本研究目的是開髮用於單發皮質醇分析的奈升流速液相層析串聯質譜(nanoflow UHPLC-MS3)系統,此系統由毛細管管柱構建,並使用串聯質譜(MS/MS和MS3)來增強在單根毛髮中皮質醇檢測的特異性和靈敏度且確定了此分析平台的方法確校。與MS / MS分析相比,藉由MS3的分析產生更多產物離子(product ion) 提供更多皮質醇的定性資訊,主要的產物離子在標準品與頭髮萃取物中的比例都是固定,因此 MS3 對於皮質醇的定性是有力的方法,而且通過 MS3分析的提取離子層析圖(extraction ion chromatogram, EIC)的訊號雜訊比(signal-to-noise ratio, S/N)增強了大約15倍,這表明MS3為皮質醇分析提供了更靈敏的方法,所以本平台的方法確校與頭髮樣本的定量分析都是使用MS3 的方法。與微孔UHPLC-MS3(microbore UHPLC-MS3)系統相比,通過信號強度,靈敏度,S/N 和偵測極限(limit of detection, LOD)評估該分析平台的靈敏度改善,nanoflow UHPLC-MS3的信號響應在1-ng皮質醇標準品下增加約3個數量級、校正曲線的斜率增加約500倍以及純溶液和毛髮萃取物中的皮質醇LOD分別降低約100倍和30倍,將此分析平台應用於頭髮中皮質醇的分析,頭髮樣本由國立成功大學醫院提供,將相同來源且同一段1-cm頭髮樣本分成單根頭髮與20根頭髮萃取進行皮質醇定量分析,並且以配對t檢定評估其皮質醇濃度的差異(n=6),單根1公分的頭髮和20根1公分頭髮皮質醇濃度分別為27.45 和 31.82 pg/mg hair,由配對t檢定分析的p值為0.66,因此單根頭髮和20根頭髮的皮質醇含量沒有統計上顯著差異。取單根6公分頭髮樣本切成1公分的片段進行分析 (n=9),單根6公分頭髮中皮質醇變化趨勢和血漿中的變化的趨勢一致,因此單根頭髮的分析可以反映過去一段時間皮質醇水平的變化。本研究在單根頭髮分析中提出了一種高靈敏度的皮質醇分析平台。在未來,使用一根頭髮的方法對於診斷或暴露標記物回顧性研究以及有力於發現新穎但低濃度的生物標記物。
英文摘要 Cortisol measured in saliva, blood, and urine is used to examine the hypothalamic–pituitary–adrenal (HPA) axis and psychological conditions, considered as stress marker. However, daily cortisol concentrations variations are likely to be affected by acute stress, the diurnal rhythm and pulsatile secretion. Cortisol measurements in saliva, blood and urine are challenging to reflect long-term level variations. Hair incorporated with cortisol during growth might be a suitable long-term biomonitoring sample. The longitudinal distribution of cortisol in the hair could reflect an accumulation of cortisol secreted over the past months. Liquid chromatography-tandem mass spectrometry (LC-MS) has emerged as an analytical method for hair cortisol analysis. Moreover, the use of LC-MS with higher sensitivity is an advantage for analyzed limited hair and lower cortisol levels. This study aims to develop a nanoflow UHPLC-MS3 system for a single hair cortisol analysis. This system constructed by capillary columns and the use of tandem MS (MS/MS and MS3) to enhance specificity and sensitivity for cortisol detection in a single hair, and method validation of this analytical platform was performed. The MS3 method could detect molecule with the use of the specific fragment pattern in the MS. The signal-to-noise ratio (S/N) of extracted ion chromatogram (EIC) by MS3 analysis was enhanced approximately 15-fold comparing to MS/MS analysis. Thus, the MS3 was employed in method validation and hair cortisol analysis. The sensitivity improvement of this analytical platform was evaluated by the signal intensity, sensitivity, S/N and limit of detection (LOD), comparing to microbore UHPLC-MS3 system. Comparing to microbore UHPLC-MS3, the signal response and sensitivity of nanoflow UHPLC-MS3 were increased by about 3-order of magnitude at 1-ng cortisol standard and about 500-fold respectively, and the LOD of cortisol in neat solution and hair extract by nanoflow UHPLC-MS3 was improved about 100- and 30-fold respectively. This analytical platform was applied for hair cortisol analysis. Hair samples were provided from National Cheng Kung University Hospital, and cortisol levels were evaluated in a single hair and 20 hairs by pair samples t-test. The average of cortisol level in a single hair and 20 hairs was closed (27.45 vs. 31.82 pg/mg hair), and there was no significant difference between a single hair and 20 hairs (p=0.66). The trends of cortisol level in hair was closed to plasma during pregnancy, so one hair could reflect the variation of cortisol level over the past period. This study presented a highly sensitive analytical platform for cortisol in a single hair analysis. In the future, this approach using only a single hair is powerful for diagnosis, exposure markers retrospective studies, and novel but low abundant biomarker discovery.
論文目次 摘要…………………………………………………………………………………………I
Abstract…………………………………………………………………………………...IV
致謝…………………………………………………………………………………….…VI
Content…………………………………………………………………………………..VII
List of tables………………………………………………………………………………IX
List of figures……………………………………………………………………………..IX
Abbreviation……………………………………………………………………………...XI
Chapter 1. Introduction…………………………………………………………………..1
1.1 Cortisol for human health assessment…………………………………………1
1.2 Hair can be as long-term biomonitoring………………………………………6
1.3 Major analytical approaches for hair analysis………………………………..8
1.4 Nanoflow LC-MS for a single hair analysis………………………………….12
Chapter 2. Objective…………………………………………………………………….14
Chapter 3. Material and method……………………………………………………….15
3.1 Study design……………………………………………………………………15
3.2 Chemical and apparatus………………………………………………………17
3.3 Experiment section…………………………………………………………….17
3.3.1 Sample preparation…………………………………………………………17
3.3.2 MS/MS and MS3 parameter for metabolites analysis…………………….17
3.3.3 Microbore UHPLC-MS3……………………………………………………18
3.3.4 Nanoflow UHPLC-MS3……………………………………………………..18
3.3.5 Tunnel frit capillary column fabrication…………………………………..19
3.3.6 Data processing……………………………………………………………...20
Chapter 4. Results and Discussions…………………………………………………….22
4.1 Development of a sensitive nanoflow LC-MS platform……………………..22
4.2 Method validation of the nanoflow UHPLC-MS3 analytical platform……..31
4.3 Sensitivity improvement of nanoflow UHPLC-MS3 comparing to microbore UHPLC-MS3…………………………………………………………………………...36
4.4 Applied the highly sensitive platform for a single hair analysis……………40
Chapter 5. Conclusion and Prospects…………………………………………………..44
References………………………………………………………………………………...46
Appendix 1. Sensitivity improvement of DEHP metabolites detection by nanoflow UHPLC-MS/MS system………………………………………………………………….57
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