人體內各種代謝物的濃度高低，可反映出個人的生理狀況，藉由檢測目標代謝物的含量，有助於判斷是否罹患相關疾病。皮膚為人體面積最大器官，其中廣布汗腺及皮脂腺具有調控、分泌、排泄及調節生理等功能，將各種自人體產生的代謝物由體內排出至皮膚表面。體表下也廣布自律神經。自律神經又分為讓各器官處於亢奮狀態的交感神經以及對各器官產生放鬆抑制作用的副交感神經，使情緒產生變化。故有極大的機會可從體表分析神經傳導物質。若能分析皮膚所釋出的化合物，解析化合物與人體疾病的相關性，有助於深入探討人體相關代謝及生理疾病，並應用於各種疾病快速篩檢診斷，此為臨床檢驗技術的一大突破。
　　大氣質譜法（Ambient mass spectrometry, AMS），是在大氣環境下進行分析的質譜應用技術，其所需分析時間短、不需經過多的樣品前處理即可進行分析，故有助於大批次樣品的篩檢，因此有許多大氣質譜技術陸續被開發出。本研究以熱脫附電噴灑游離質譜法（Thermal desorption electrospray ionization mass spectrometry, TD-ESI/MS）結合採樣探針對人類體表進行取樣，首先以酒精棉片擦拭預採樣部位，待至一段時間後以採樣探針輕刮取體表，再將採樣探針置入一加熱源區域，採樣探針上的分析物會進行熱脫附成為中性氣態分子，再以電噴灑游離裝置所產生的帶電荷溶劑液滴與中性氣態分子進行融合，經由離子-分子反應（Ion molecule reaction, IMR）生成帶電荷的分析物離子，最後經載流氣體送至質譜端進行偵測。
研究第一部分為最適化以熱脫附電噴灑游離質譜法鑑定體表代謝物的取樣方法。首先探討直接以金屬環探針進行取樣與經酒精棉片擦拭後再取樣的訊號差異，再來是酒精擦拭後到分析的最適化時間、以及金屬環探針對於體表採樣的長度最適化，由實驗結果發現，以擦拭酒精棉片等待10分鐘再以探針刮取皮膚1 cm訊號較佳。接著為了配合在醫院或是戶外採樣時能夠使受試者避免恐懼及方便取樣，所以將金屬環探針更換成棉花棒來進行採樣，採樣後再攜帶回實驗室進行分析。由於棉花棒的材質不同，可能對質譜訊號也會造成影響，因此本研究選用市面上三種不同材質（紙軸、竹軸及塑膠軸）的棉花棒來進行測試，並選用不同溶劑將棉花棒進行清洗，比較清洗前後的差異。由實驗結果得知，再經過分別以二次水、甲醇、乙腈等溶劑清洗後的紙軸棉棒較適合。
第二部分為以金屬環探針對皮膚直接取樣下，快速從質譜儀得到不同生化物種的訊號，在全掃描模式下，可直接偵測出尿素（urea）、胺基酸（amino acid）、游離脂肪酸（free fatty acid）、蠟質（wax ester）、三酸甘油脂（triglyceride）、角鯊烯（squalene）、膽固醇（cholesterol）、乳酸（lactic acid）等化合物；若以選擇性離子監測（Selected reaction monitoring, SRM）模式檢測，則可檢測出微量的胺基酸、肌酸酐（creatinine）、尿酸（uric acid）及其合成所需代謝物、角鯊烯代謝途徑所產生的代謝物、神經傳導物質（neurotransmitter）。在能快速得到這些生化物種的優勢下，本研究針對上述物種進行一系列的即時監控分析，藉由建立正常受試者的模型，以提供未來比對病患對照組的資料。以肌酸酐為例，肌酸酐及尿酸的含量每個成人會維持恆定狀態下，但其含量的多寡也會隨著不同年齡層而有所差異，由實驗結果發現，兒童皮膚的肌酸酐含量遠低於成人皮膚的釋出，該數據也符合文獻報導。因肌酸酐為腎臟疾病最具重要的生物指標，以期未來能透過正常受試者模型與腎臟病患做比較，提供臨床醫學另一項診斷工具。

The concentration of metabolites in the human body can reflect the physiological condition. By detecting the concentration of the target metabolite, it helps to diagnose the disease. The skin is the largest organ of the human body, which widely spread sweat glands and sebaceous glands have regulation, secretion, excretion and regulation of physiological functions. It will produce various of metabolites from body to skin surface. Under the skin also widely distributed autonomic nerve. The autonomic nerve contains a parasympathetic nerve that causes the organs to be in a state of excitement and a parasympathetic nerve that causes a relaxing effect on the organs. So there is the greatest opportunity to analyze neurotransmitters on skin. If we can analyze compounds released from the skin, resolving dependencies compound with human disease, it will help to study hard of human diseases related to metabolism and physiology, and applied to the diagnosis of various diseases, rapid screening, this is a major breakthrough in clinical testing technology.
Ambient mass spectrometry （AMS）owns many advantages such as rapid analysis, few or no pretreatment procedure. In view of this, there are more and more AMS techniques employing in many fields such as chemical analysis, biomedical, and forensics science. Especially in clinical detection, AMS has been used to directly characterize chemicals on skin. Techniques such as DESI, LTP, and DAPCI had been successfully demonstrated for rapidly characterizing chemicals on skin. In this study, a novel mass spectrometric technique, termed as thermal desorption electrospray ionization/mass spectrometry (TD-ESI/MS), was developed an easy sample method and quickly characterize compounds on skin surface including metabolites and neurotransmitters released from body. First, wipe the pre-sampling site with alcohol cotton sheet, wait until a period of time after TD probe to scrap the skin to sample the chemical compounds, and then the TD probe was inserted into a heated oven to thermally desorb the analytes for the following ionization in a ESI plume and characterization by mass spectrometry. Compared to other AMS techniques, TD-ESI/MS can perform remote sampling and do not limit to the shape, size, or distance of subjects. Most importantly, this method is noninvasive.
The first part of the study was the best method for analysis of skin sample by thermal desorption and electrospray ionization mass spectrometry. First explore the stainless steel probe directly to the sampling and alcoholic cotton wipe and then sampling the signal difference, followed by alcohol wipe to the analysis of the most appropriate time, and the stainless steel probe for the length of the surface sampling of the most appropriate. From the experimental results found the best signal that.to wipe the alcohol cotton sheet, wait 10 minutes and then probe the skin 1 cm. In order to sampling in the hospital and outside, stainless steel probe will fear. So the stainless steel probe into cotton swap for sampling, and then carried back to the laboratory for analysis. Because of the different material of the cotton swab, it may also affect the mass spectrometry signal. Therefore, in this study the cotton swap of three different materials (paper shaft, bamboo shaft and plastic shaft) for testing, and the different solvent to washed cotton Compare the signal of washed cotton swap whether or not. From the experimental results that, the cotton swap washed by the water, methanol, acetonitrile more suitable.
In secondary study focus on skin screening, directly sampling and analysis analytes scraped on skin. Detection of metabolites and related compounds released inside out to the skin surface. TD-ESI/MS detect the lipid profile including free fatty acid, squalene, cholesterol , wax ester, triglycerides. Furthermore, amino acid ,urea, creatinine ,uric acid and neurotransmitters were detected. It is fast to obtain these biochemical species. This study conducts a series of immediate monitoring and analysis of the above species, by establishing a model of normal subjects to provide information on future comparison with the patient control group. For example, creatinine and uric acid content of each adult will maintain a constant state, but its content will vary with the different age groups, the experimental results found that children's skin creatinine content far lower than the release of adult skin, the data also in suitable on the literature. Because creatinine is the most important biological indicator of kidney disease, with a view to the future through the normal subject model and kidney patients to compare, to provide another diagnostic tool for clinical medicine.

論文審定書 i
論文公開授權書 ii
誌謝 iii
中文摘要 iv
英文摘要 vi
目錄 viii
圖目錄 xi
表目錄 xiv
第一章、緒論 1
第一節、前言 1
第二節、皮膚結構 2
一、表皮（epidermis） 2
二、真皮（dermis） 3
三、皮下組織（subcutanous tissue） 4
四、皮膚附屬腺體（skin glands） 4
第三節、常見臨床生物檢體檢驗方法 5
一、 酵素結合免疫分析法（Enzyme-linked immunosorbent assay，ELISA） 5
二、 液相層析質譜法（Liquid chromatography mass spectrometry, LC/MS） 8
三、 氣相層析質譜法（Gas chromatography mass spectrometry, GC/MS） 9
第四節、大氣質譜法（Ambient mass spectrometry, AMS） 10
第五節、大氣質譜法於檢測體表代謝物之臨床應用 12
一、脫附電噴灑游離法（Desorption electrospray ionization, DESI） 12
二、萃取電噴灑游離法（Extractive electrospray ionization, EESI） 14
三、脫附大氣壓力化學游離法（Surface desorption atmospheric pressure chemical ionization, SDAPCI） 15
四、低溫電漿（Low-temperature plasma, LTP） 17
第六節、熱脫附電噴灑游離質譜法 Thermal Desorption-Electrospray Ionization/Mass Spectrometry（TD-ESI/MS） 18
第七節、 論文目標 20
第二章、實驗材料與方法 21
第一節、實驗試劑與藥品 21
一、試劑 21
二、標準品 21
三、試劑配製 22
四、採樣棉花棒 22
第二節、儀器與參數設定 23
一、熱脫附電噴灑游離質譜（Thermal desorption electrospray ionization mass spectrometry）系統 23
二、儀器參數設定 24
三、分析物參數設定 25
第三節、實驗設計 29
一、實驗受試者 29
二、TD-ESI/MS 分析流程 29
三、樣品分析與保存 30
第三章、結果與討論 32
第一節、建立以熱脫附電噴灑游離質譜法分析皮膚樣品之採樣方法 32
一、金屬探針 32
二、棉花棒 35
第二節、以熱脫附電噴灑游離質譜法鑑定體表代謝物及神經傳導物質 45
一、體表代謝物檢測 45
二、　體表釋放之神經傳導物質檢測 70
第四章、結論 73
第五章、 參考資料 74

［1］Hirokawa, T.; Okamoto, H.; Gosyo, Y.; Tsuda, T.; Timerbaev, A. R., Simultaneous monitoring of inorganic cations, amines and amino acids in human sweat by capillary electrophoresis. Anal Chim Acta 2007, 581, 83-88.
［2］“Skin care” (analysis), Health-Cares.net, 2007.
［3］Proksch, E.; Brandner, J. M.; Jensen, J. M., The skin: an indispensable barrier. Exp Dermatol 2008, 17, 1063-1072.
［4］Lampe, M. A.; Burlingame, A. L.; Whitney, J.; Williams, M. L.; Brown, B. E.; Roitman, E.; Elias, P. M., Human Stratum-Corneum Lipids - Characterization and Regional Variations. J Lipid Res 1983, 24, 120-130.
［5］Björklund, H.; Dalsgaard, C.-J.; Jonsson, C.-E.; Hermansson, A., Sensory and autonomic innervation of non-hairy and hairy human skin. Cell and tissue research 1986, 243 (1), 51-57.
［6］Dummer, R., Precision medicine and skin cancer therapy: dealing with a moving target. Curr Opin Oncol 2014, 26, 182-183.
［7］Huang, M. Z.; Zhou, C. C.; Liu, D. L.; Jhang, S. S.; Cheng, S. C.; Shiea, J., Rapid Characterization of Chemical Compounds in Liquid and Solid States Using Thermal Desorption Electrospray Ionization Mass Spectrometry. Anal Chem 2013, 85, 8956-8963.
［8］Shiea, C.; Huang, Y. L.; Liu, D. L.; Chou, C. C.; Chou, J. H.; Chen, P. Y.; Shiea, J.; Huang, M. Z., Rapid screening of residual pesticides on fruits and vegetables using thermal desorption electrospray ionization mass spectrometry. Rapid Commun Mass Sp 2015, 29, 163-170.
［9］Lee, C. W.; Su, H.; Chen, P. Y.; Lin, S. J.; Shiea, J.; Shin, S. J.; Chen, B. H., Rapid identification of pesticides in human oral fluid for emergency management by thermal desorption electrospray ionization/mass spectrometry. J Mass Spectrom 2016, 51, 97-104.
［10］Montagna, W., The structure and function of skin. Elsevier: 2012.
［11］Hornbeck, P. V., Enzyme‐linked immunosorbent assays. Current protocols in immunology 1991, 2.1. 1-2.1. 23.
［12］Bruce, S. J.; Tavazzi, I.; Parisod, V.; Rezzi, S.; Kochhar, S.; Guy, P. A., Investigation of human blood plasma sample preparation for performing metabolomics using ultrahigh performance liquid chromatography/mass spectrometry. Analytical Chemistry 2009, 81 (9), 3285-3296.
［13］Xu, X.; Roman, J. M.; Issaq, H. J.; Keefer, L. K.; Veenstra, T. D.; Ziegler, R. G., Quantitative Measurement of Endogenous Estrogens and Estrogen Metabolites in Human Serum by Liquid Chromatography− Tandem Mass Spectrometry. Analytical chemistry 2007, 79 (20), 7813-7821.
［14］Xu, X.; Veenstra, T. D.; Fox, S. D.; Roman, J. M.; Issaq, H. J.; Falk, R.; Saavedra, J. E.; Keefer, L. K.; Ziegler, R. G., Measuring fifteen endogenous estrogens simultaneously in human urine by high-performance liquid chromatography-mass spectrometry. Analytical chemistry 2005, 77 (20), 6646-6654.
［15］Niessen, W. M., Liquid chromatography-mass spectrometry. CRC Press: 2006.
［16］Harkey, M. R.; Henderson, G. L.; Zhou, C., Simultaneous quantitation of cocaine and its major metabolites in human hair by gas chromatography/chemical ionization mass spectrometry. Journal of analytical toxicology 1991, 15 (5), 260-265.
［17］Zlatkis, A.; Bertsch, W.; Lichtenstein, H.; Tishbee, A.; Shunbo, F.; Liebich, H.; Coscia, A.; Fleischer, N., Profile of volatile metabolites in urine by gas chromatography-mass spectrometry. Analytical chemistry 1973, 45 (4), 763-767.
［18］Dunn, W. B.; Broadhurst, D.; Begley, P.; Zelena, E.; Francis-McIntyre, S.; Anderson, N.; Brown, M.; Knowles, J. D.; Halsall, A.; Haselden, J. N., Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry. Nature protocols 2011, 6 (7), 1060-1083.
［19］Watson, J. T.; Sparkman, O. D., Gas chromatography/mass spectrometry. Wiley Online Library: 2008.
［20］Huang, M.-Z.; Yuan, C.-H.; Cheng, S.-C.; Cho, Y.-T.; Shiea, J., Ambient ionization mass spectrometry. Annual review of analytical chemistry 2010, 3, 43-65.
［21］Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306 (5695), 471-473.
［22］Shiea, J.; Huang, M. Z.; HSu, H. J.; Lee, C. Y.; Yuan, C. H.; Beech, I.; Sunner, J., Electrospray‐assisted laser desorption/ionization mass spectrometry for direct ambient analysis of solids. Rapid Communications in Mass Spectrometry 2005, 19 (24), 3701-3704.
［23］Cheng, C.-Y.; Yuan, C.-H.; Cheng, S.-C.; Huang, M.-Z.; Chang, H.-C.; Cheng, T.-L.; Yeh, C.-S.; Shiea, J., Electrospray-assisted laser desorption/ionization mass spectrometry for continuously monitoring the states of ongoing chemical reactions in organic or aqueous solution under ambient conditions. Analytical chemistry 2008, 80 (20), 7699-7705.
［24］Razunguzwa, T. T.; Henderson, H. D.; Reschke, B. R.; Walsh, C. M.; Powell, M. J., Laser-Ablation Electrospray Ionization Mass Spectrometry (LAESI®-MS): Ambient Ionization Technology for 2D and 3D Molecular Imaging. Ambient ionization mass spectrometry. Royal Society of Chemistry, Cambridge 2014, 462-481.
［25］Chang, D.-Y.; Lee, C.-C.; Shiea, J., Detecting large biomolecules from high-salt solutions by fused-droplet electrospray ionization mass spectrometry. Analytical chemistry 2002, 74 (11), 2465-2469.
［26］Chang, D.-Y.; Lee, C.-C.; Shiea, J., Detecting large biomolecules from high-salt solutions by fused-droplet electrospray ionization mass spectrometry. Analytical chemistry 2002, 74 (11), 2465-2469.
［27］Na, N.; Zhao, M.; Zhang, S.; Yang, C.; Zhang, X., Development of a dielectric barrier discharge ion source for ambient mass spectrometry. Journal of the American Society for Mass Spectrometry 2007, 18 (10), 1859-1862.
［28］Whitson, S. E.; Erdodi, G.; Kennedy, J. P.; Lattimer, R. P.; Wesdemiotis, C., Direct probe-atmospheric pressure chemical ionization mass spectrometry of cross-linked copolymers and copolymer blends. Analytical chemistry 2008, 80 (20), 7778-7785.
［29］Fayad, P. B.; Prévost, M. l.; Sauvé, S., Laser diode thermal desorption/atmospheric pressure chemical ionization tandem mass spectrometry analysis of selected steroid hormones in wastewater: method optimization and application. Analytical chemistry 2009, 82 (2), 639-645.
［30］Harper, J. D.; Charipar, N. A.; Mulligan, C. C.; Zhang, X.; Cooks, R. G.; Ouyang, Z., Low-temperature plasma probe for ambient desorption ionization. Analytical chemistry 2008, 80 (23), 9097-9104.
［31］Chen, H.; Venter, A.; Cooks, R. G., Extractive electrospray ionization for direct analysis of undiluted urine, milk and other complex mixtures without sample preparation. Chemical Communications 2006, (19), 2042-2044.
［32］Chen, H.; Yang, S.; Wortmann, A.; Zenobi, R., Neutral desorption sampling of living objects for rapid analysis by extractive electrospray ionization mass spectrometry. Angewandte Chemie International Edition 2007, 46 (40), 7591-7594.
［33］Chen, H.; Zheng, J.; Zhang, X.; Luo, M.; Wang, Z.; Qiao, X., Surface desorption atmospheric pressure chemical ionization mass spectrometry for direct ambient sample analysis without toxic chemical contamination. Journal of mass spectrometry 2007, 42 (8), 1045-1056.
［34］Huang, M.-Z.; Zhou, C.-C.; Liu, D.-L.; Jhang, S.-S.; Cheng, S.-C.; Shiea, J., Rapid characterization of chemical compounds in liquid and solid states using thermal desorption electrospray ionization mass spectrometry. Analytical chemistry 2013, 85 (19), 8956-8963.
［35］Camera, E.; Ludovici, M.; Galante, M.; Sinagra, J.-L.; Picardo, M., Comprehensive analysis of the major lipid classes in sebum by rapid resolution high-performance liquid chromatography and electrospray mass spectrometry. Journal of lipid research 2010, 51 (11), 3377-3388.
［36］Rebollido‐Fernandez, M. M.; Castiñeiras, D. E.; Dolores Bóveda, M.; Luz Couce, M.; Cocho, J. A.; Fraga, J. M., Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism. Rapid Communications in Mass Spectrometry 2012, 26 (18), 2131-2144.
［37］Ceglarek, U.; Kortz, L.; Leichtle, A.; Fiedler, G. M.; Kratzsch, J.; Thiery, J., Rapid quantification of steroid patterns in human serum by on-line solid phase extraction combined with liquid chromatography–triple quadrupole linear ion trap mass spectrometry. Clinica Chimica Acta 2009, 401 (1), 114-118.
［38］Esteban, N. V.; Yergey, A. L.; Liberato, D. J.; Loughlin, T.; Loriaux, D. L., Stable isotope dilution method using thermospray liquid chromatography/mass spectrometry for quantification of daily cortisol production in humans. Biomedical & environmental mass spectrometry 1988, 15 (11), 603-608
［39］Piraud, M.; Vianey‐Saban, C.; Petritis, K.; Elfakir, C.; Steghens, J. P.; Morla, A.; Bouchu, D., ESI‐MS/MS analysis of underivatised amino acids: a new tool for the diagnosis of inherited disorders of amino acid metabolism. Fragmentation study of 79 molecules of biological interest in positive and negative ionisation mode. Rapid Communications in Mass Spectrometry 2003, 17 (12), 1297-1311.
［40］Lu, W.; Kimball, E.; Rabinowitz, J. D., A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites. Journal of the American Society for Mass Spectrometry 2006, 17 (1), 37-50.
［41］Sharma, K.; Singh, R.; Giri, S.; Rajagopal, S.; Mullangi, R., Highly sensitive method for the determination of adenosine by LC‐MS/MS‐ESI: method validation and scope of application to a pharmacokinetic/pharmacodynamic study. Biomedical Chromatography 2012, 26 (1), 81-88.
［42］Hou, H.; Xiong, W.; Zhang, X.; Song, D.; Tang, G.; Hu, Q., LC-MS-MS measurements of urinary creatinine and the application of creatinine normalization technique on cotinine in smokers’ 24 hour urine. Journal of analytical methods in chemistry 2012, 2012.
［43］Rebollido‐Fernandez, M. M.; Castiñeiras, D. E.; Dolores Bóveda, M.; Luz Couce, M.; Cocho, J. A.; Fraga, J. M., Development of electrospray ionization tandem mass spectrometry methods for the study of a high number of urine markers of inborn errors of metabolism. Rapid Communications in Mass Spectrometry 2012, 26 (18), 2131-2144.
［44］Carlsson, A.; Waters, N.; Holm-Waters, S.; Tedroff, J.; Nilsson, M.; Carlsson, M. L., Interactions between monoamines, glutamate, and GABA in schizophrenia: new evidence. Annual review of pharmacology and toxicology 2001, 41 (1), 237-260.
［45］Tseng, S.-P.; Lu, C.-C.; Liao, P.-C.; Chou, C.-H.; Sheu, H.-M.; Tsai, J.-C., Detection and distribution of endogenous steroids in human stratum corneum. Dermatologica Sinica 2014, 32 (1), 19-24.
［46］Kaufman, J. M.; Greene, M. L.; Seegmiller, J. E., Urine uric acid to creatinine ratio—a screening test for inberited disorders of purine metabolism: Phosphoribosyltransferase (PRT) deficiency in X-linked cerebral palsy and in a variant of gout. The Journal of pediatrics 1968, 73 (4), 583-592.