皮膚為人體最大器官，也是與外界環境的主要屏障，其中的汗腺（sweat gland）、皮脂腺（glandula sebace）主導著皮膚的分泌作用與排泄作用。汗腺的分泌具重要的生理意義，如散熱作用、電解質及激素的代謝等。一般臨床生物檢體樣品如血液、尿液等，樣品基質極為複雜，進行分析前須經繁複的前處理，不僅耗時且所費不貲。而由皮膚分泌的代謝物如汗液、脂質等，其基質效應少、具分析優勢，若能以體表所釋出的代謝物做為指標，可監測人體的生理活動外，也可應用於疾病的篩檢，故發展具快速檢測皮膚代謝物之技術為一大趨勢。然而因無較簡易的取樣及分析方法，故皮膚代謝物分析在臨床上的應用並不常見。若可發展簡易的皮膚代謝物取樣及分析方法，將有助於提升醫療檢驗之技術，並可應用在精準醫療（Precision Medicine）中進行健康評估。
　　大氣質譜法（Ambient Mass Spectrometry, AMS），為一種可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、只需極少量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）結合探針對生物體表進行取樣。將採樣探針置入一簡單的加熱源區域，依溫度控制將分析樣品進行熱脫附，再以電噴灑游離裝置所產生的帶電荷的溶劑液滴進行融合，經由離子-分子反應（Ion molecule reaction, IMR）或是電噴灑過程，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。
　　第一部分的研究使用TD-ESI/MS直接進行皮膚取樣，檢測由體內向外釋出的相關化合物及代謝物。實驗中使用金屬探針對人體皮膚表面進行取樣，使用熱脫附電噴灑游離質譜系統進行偵測，可檢測人體的皮脂訊號（Lipid profile），也可測得皮脂中的角鯊烯（Squalene）與膽固醇（Cholesterol）等化合物；而在部分受試者的皮膚樣本中也可觀察到常見塑化劑的訊號（鄰苯二甲酸酯鹽類：Dibutyl Phthalate （DBP）、Dimethyl Phthalate（DEHP）、Di-iso-nonyl Phthalate（DINP））。也針對人體飲用咖啡後進行皮膚檢測，測得咖啡因（Caffeine）及咖啡因代謝物（Paraxanthine）的訊號。人體服用藥物後，經代謝傳輸後也可由皮膚表面上檢測出。實驗中受試者服用普拿疼（Acetaminophen, Panadol）後對皮膚進行檢測，可測得明顯的藥物訊號；也檢測服用抗黴菌藥物的灰指甲（Onychomycosis）患者，透過皮膚檢測可測得療黴舒（Lamisil, Terbinafine）之訊號。
　　第二部分研究檢測藥物於全身皮膚的分佈情形與相關代謝之檢測。用於治療黴菌感染（灰指甲）的療黴舒經服用後，可檢測該藥物於人體不同部位的濃度分佈情形，並針對治療後殘留於皮膚上的藥物進行代謝監測。在結束服藥療程後持續進行監測，可測得藥物於皮膚中的代謝情形，於八週後皮膚所釋出的療黴舒訊號已低於儀器的偵測極限。另外，TD-ESI/MS除了可以檢測皮膚釋出的代謝物與藥物訊號，也可針對藥物的代謝情形進行即時監測。飲用咖啡後進行6小時的監測，觀察皮膚所釋出的咖啡因濃度變化，可明確觀察到隨時間的變化而有不同的咖啡因釋出量，由血液中的咖啡因濃度可推測咖啡因於體內的代謝變化進而影響皮膚表面的釋出量。直接透過皮膚取樣檢測方式，測定咖啡因於體內吸收後分佈釋放於全身皮膚表面的情況；更進一步繪製臉部皮膚咖啡因釋出的影像分布圖。

Skin, the largest organ in human beings, plays a key role in protecting the human body against different pathogens in the environment. Chemical and biological compounds on skin correspond with endogenous and exogenous components originating from underlying blood vessels, glandular secretions, and skin cells. Since these compounds are potentially useful in clinical diagnoses. Some of the metabolites on skin are potential disease biomarkers, however, current analytical techniques do not provide proper way to efficiently identify them on skin. In this study, a novel ambient mass spectrometric technique - thermal desorption electrospray ionization/mass spectrometry (TD-ESI/MS) was used to characterize the chemical and biological compounds including drugs, lipids, and metabolites released on skin.
　　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 drugs and metabolites released from body and small molecules from external environment. A stainless steel probe was used as TD probe to scrap the skin to sample the chemical compounds. After that, 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. After analysis, the probe was cleaned by burning it with a flame. The total analysis time required less than 30 seconds.
　　The first study focus on skin screening, directly sampling and analysis analytes scraped on skin without any pretreatment. Detection of metabolites and related compounds released inside out to the skin surface. TD-ESI/MS is able to detect the lipid profile including diglycerides and triglycerides. Furthermore, squalene and cholesterol, common ingredients in lipid were detected. TD-ESI/MS is capable to detect the related compounds through the skin after taking medicine or eating food. Caffeine and the metabolites released on the skin of volunteers were detected. The drugs Panadol (Acetaminophen) and Lamisil (Terbinafine) were detected as well.
　　In secondary study, by means of detecting caffeine released from different sites of skin, and thus can explore the chemical metabolism in various parts of body. The signals mostly are found over the upper body. For metabolism profile of caffeine, the volunteer was sampled with metallic probes every 30 minutes after receiving single doses of coffee, then was subsequently characterized with TD-ESI/MS. The metabolism profiles of caffeine were rapidly obtained from the analytical results of the compounds on skin. In the volunteer who drank coffee, caffeine signal was gradually rise and decreased over the time; the subject without drinking coffee still keep the signal steady in all times, no upward or downward trend was observed. It is possible to determine metabolism profiles on skin by TD-ESI/MS. There was a similar tendency of signals (the model are commonly referred to as the ADME scheme). Determination of caffeine released over the skin surface and create 3D topographical maps of caffeine distributed on the human skin (face).

論文審定書.................................................................................................. i
論文公開授權書...........................................................................................ii
誌謝............................................................................................................iii
中文摘要......................................................................................................v
英文摘要....................................................................................................vii
目錄 ...........................................................................................................ix
圖目錄........................................................................................................xii
表目錄.......................................................................................................xv
第一章、緒論..............................................................................................1
　第一節、前言...........................................................................................1
　第二節、皮膚結構...................................................................................2
　第三節、臨床皮膚檢測方法.....................................................................4
　　一、汗液檢驗（Sweat Test）..............................................................4
　　二、皮膚貼布測試（Skin Patch Test）................................................6
　　三、皮膚切片（Skin Biopsy）.............................................................7
　第四節、大氣質譜法 （Ambient Mass Spectrometry）..........................9
　第五節、大氣質譜法於檢測人體皮膚之應用 ..........................................12
　　一、脫附電噴灑游離法Desorption electrospray ionization (DESI).....12
　　二、低溫電漿Low-Temperature Plasma (LTP) ..................................13
　　三、萃取電噴灑游離法Extractive electrospray ionization (EESI)......14
　　四、脫附大氣壓力化學游離法 Surface Desorption atmospheric pressure chemical ionization (DAPCI)......................................................15
　第六節、熱脫附電噴灑游離質譜法Thermal Desorption-Electrospray Ionization/Mass Spectrometry（TD-ESI/MS）.........................................17
第二章、實驗............................................................................................20
　第一節、實驗試劑與藥品.......................................................................20
　　一、 試劑 ..........................................................................................20
　　二、 藥品...........................................................................................20
　　　1.　標準品......................................................................................20
　　　2.　市售藥物及商品.......................................................................20
　　三、 試劑配製...................................................................................21
　　　1. 標準品.......................................................................................21
　　　2. 電噴灑溶液................................................................................21
　　四、 檢測化合物之結構式.................................................................21
　第二節、儀器設備.................................................................................22
　　一、 TD-ESI/MS 質譜系統................................................................22
　　二、 參數設定...................................................................................25
　　　1.　參數設定I（Triple Quadrupole Mass Spectrometer）.............25
　　　2.　參數設定II（Linear Ion Trap Mass Spectrometer）................26
　　　3.　目標化合物分析離子對............................................................27
　第三節、實驗設計.................................................................................28
　　一、 TD-ESI/MS分析流程.................................................................28
　　二、 實驗受試者................................................................................30
　　三、 藥物及食品服用.........................................................................30
　　　1.　咖啡飲用（Caffeine）..............................................................30
　　　2.　感冒藥服用（Acetaminophen）..................................................................................31
　　　3.　療黴舒用藥（Lamisil）............................................................31
　　四、 標準取樣流程............................................................................32
　　　1.　探針取樣 ..................................................................................32
　　　2.　樣品分析與保存.......................................................................32
第三章、結果與討論.................................................................................34
　第一節、分析皮膚上的代謝物並檢測由皮膚所釋出藥物訊號................34
　　一、 皮膚代謝物................................................................................34
　　二、 皮膚代謝物檢測.........................................................................34
　　　1.　脂質（Lipid）..........................................................................34
　　　2.　膽固醇（Cholesterol）............................................................36
　　　3.　角鯊烯（Squalene）...............................................................37
　　三、 檢測由皮膚釋出之藥物及相關化合物........................................40
　　　1.　普拿疼（對乙醯胺基酚）.........................................................40
　　　2.　療黴舒（特比萘芬）................................................................42
　　　3. 咖啡因.....................................................................................46
　　　4.　咖啡因代謝物...........................................................................50
　第二節、使用TD-ESI/MS檢測皮膚釋出的藥物於全身分佈與代謝.........59
　　一、 檢測藥物於全身皮膚之分佈情形...............................................59
　　　1.　使用TD-ESI/MS檢測療黴舒標準品 ..........................................59
　　　2.　模擬藥物（療黴舒）於皮膚釋出之檢測...................................62
　　二、 檢測皮膚表面的療黴舒釋出與分佈情形.....................................63
　　　1.　療黴舒於全身皮膚表面的分布..................................................63
　　　2.　療黴舒於全身皮膚表面的代謝..................................................66
　　三、 檢測皮膚表面的咖啡因釋出與分佈情形.....................................69
　　　1.　咖啡因於全身皮膚表面之檢測..................................................69
　　　2.　咖啡因於全身皮膚表面的分布影像..........................................70
　　　3.　咖啡因於臉部皮膚表面的分布影像 ..........................................71
　　四、 檢測咖啡因於皮膚表面的代謝...................................................76
第四章、結論............................................................................................79
第五章、參考文獻.....................................................................................80

[1] 檢驗醫學部臨床檢驗手冊2014 https://www.ntuh.gov.tw/labmed/default.aspx，國立臺灣大學醫學院附設醫院 檢驗醫學部
[2] 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.
[3] al-Tamer, Y. Y.; Hadi, E. A.; al, B., II, Sweat urea, uric acid and creatinine concentrations in uraemic patients. Urol Res 1997, 25, 337-40.
[4] “Skin care” (analysis), Health-Cares.net, 2007.
[5] Proksch, E.; Brandner, J. M.; Jensen, J. M., The skin: an indispensable barrier. Exp Dermatol 2008, 17, 1063-1072.
[6] 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.
[7] Dummer, R., Precision medicine and skin cancer therapy: dealing with a moving target. Curr Opin Oncol 2014, 26, 182-183.
[8] 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.
[9] 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.
[10] 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.
[11] 吳志賢醫師。http://drtonywu.pixnet.net/blog，中國醫學大學附設醫院台北分院
[12] A+醫學百科。http://cht.a-hospital.com/
[13] Bouwstra, J. A.; Groenink, H. W.; Kempenaar, J. A.; Romeijn, S. G.; Ponec, M., Water distribution and natural moisturizer factor content in human skin equivalents are regulated by environmental relative humidity. J Invest Dermatol 2008, 128, 378-88.
[14] Harding, C. R.; Watkinson, A.; Rawlings, A. V.; Scott, I. R., Dry skin, moisturization and corneodesmolysis. Int J Cosmet Sci 2000, 22, 21-52.
[15] Clar, E. J.; Fourtanier, A., Pyrrolidone carboxylic acid and the skin. Int J Cosmet Sci 1981, 3, 101-13.
[16] Marks, James G; Miller, Jeffery. Lookingbill and Marks' Principles of Dermatology (4th ed.). Elsevier Inc., 2006
[17] Coleman RA, Lee DP. Enzymes of triglyceride synthesis and their regulation. Progress in Lipid Research. 2004, 43: 134–76.
[18] Seia, M.; Costantino, L.; Paracchini, V.; Porcaro, L.; Capasso, P.; Coviello, D.; Corbetta, C.; Torresani, E.; Magazzu, D.; Consalvo, V.; Monti, A.; Costantini, D.; Colombo, C., Borderline sweat test: Utility and limits of genetic analysis for the diagnosis of cystic fibrosis. Clin Biochem 2009, 42, 611-6.
[19] Mishra, A.; Greaves, R.; Massie, J., The limitations of sweat electrolyte reference intervals for the diagnosis of cystic fibrosis: a systematic review. Clin Biochem Rev 2007, 28, 60-76.
[20] Johns Hopkins Cystic Fibrosis Center. Diagnosis: Testing: Sweat Test, http://www.hopkinscf.org/what-is-cf/diagnosis/testing/sweat-test/
[21] Barnes, A. J.; Smith, M. L.; Kacinko, S. L.; Schwilke, E. W.; Cone, E. J.; Moolchan, E. T.; Huestis, M. A., Excretion of methamphetamine and amphetamine in human sweat following controlled oral methamphetamine administration. Clin Chem 2008, 54, 172-80.
[22] 馬偕紀念醫院皮膚科。過敏原檢查，http://www.mmh.org.tw/taitam/derma/service/id-allergen.html
[23] 台北市立聯合醫院癌症防治研究發展中心。皮膚癌，http://website.tpech.gov.tw/cancer/
[24] Web MD. Punch Biopsy, http://www.webmd.com/melanoma-skin-cancer/punch-biopsy
[25] Venter, A.; Nefliu, M.; Graham Cooks, R., Ambient desorption ionization mass spectrometry. TrAC Trends Anal. Chem. 2008, 27, 284-290.
[26] Huang, M. Z.; Yuan, C. H.; Cheng, S. C.; Cho, Y. T.; Shiea, J., Ambient ionization mass spectrometry. Annu. Rev. Anal. Chem. 2010, 3, 43-65. 
[27] Carroll, D. I.; Dzidic, I.; Stillwel.Rn; Horning, M. G.; Horning, E. C., Subpicogram Detection System for Gas-Phase Analysis Based Upon Atmospheric-Pressure Ionization (Api) Mass-Spectrometry. Anal Chem 1974, 46, 706-710.
[28] John B.; Fenn. J. P., Electrospray Ion Source. Another Variation on the Free-Jet Theme. Analytical chemistry 1984, 88, 4451–4459.
[29] Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G., Mass spectrometry sampling under ambient conditions with desorption electrospray ionization. Science 2004, 306, 471-473.
[30] Harper, J. D.; Charipar, N. A.; Mulligan, C. C.; Zhang, X. R.; Cooks, R. G.; Ouyang, Z., Low-Temperature Plasma Probe for Ambient Desorption Ionization. Anal Chem 2008, 80, 9097-9104.
[31] Chen, H. W.; Venter, A.; Cooks, R. G., Extractive electrospray ionization for direct analysis of undiluted urine, milk and other complex mixtures without sample preparation. Chem Commun 2006, 2042-2044.
[32] Chen, H. W.; Wortmann, A.; Zhang, W. H.; Zenobi, R., Rapid in vivo fingerprinting of nonvolatile compounds in breath by extractive electrospray ionization quadrupole time-of-flight mass spectrometry. Angew Chem Int Edit 2007, 46, 580-583.
[33] Chen, H. W.; Zenobi, R., Direct analysis of living objects by extractive electrospray mass ionization spectrometry. Chimia 2007, 61, 843-843
[34] Chen, H. W.; Zheng, J.; Zhang, X.; Luo, M. B.; Wang, Z. C.; Qiao, X. L., Surface desorption atmospheric pressure chemical ionization mass spectrometry for direct ambient sample analysis without toxic chemical contamination. J Mass Spectrom 2007, 42, 1045-1056.
[35] 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, 8956-63.
[36] 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 Spectrom 2015, 29, 163-70.
[37] 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.
[38] 陳芃妤。以大氣質譜法大規模篩檢環境中塑化劑之物品，碩士論文，國立中山大學，2015。
[39] Thermo Fisher Scientific Inc. LTQ XL™ Linear Ion Trap Mass Spectrometer, https://www.thermofisher.com
[40] Porter, A. M. W., Why do we have apocrine and sebaceous glands? J Roy Soc Med 2001, 94, 236-237.
[41] 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.
[42] Mollmann, C.; Muller-Fahrnow, W.; Spyra, K., Person undergoing rehabilitation-management-categories (RMK) - A need and efficiency-oriented classification approach for the medical rehabilitation - Adaptation on the addiction rehabilitation. Nervenarzt 2007, 78, 176-176.
[43] 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, 83-8.
[44] 國立編譯館主編(88年版) 國民中學生物教科書。
[45] 橫田貴史。深海鯊魚的氧效果，正義出版社：台北，2005。
[46] Kelly, G. S., Squalene and its potential clinical uses. Altern Med Rev 1999, 4, 29-36.
[47] 朱洪法。精細化工常用原材料手冊，金盾出版社：北京，2003。
[48] 顧翼東、夏炎。化學詞典，上海辭書出版社：上海，2003
[49] Aghababian, Richard V. Essentials of Emergency Medicine, Jones & Bartlett Publishers: US, 2010.
[50] Ahmad, Jawad. Hepatology and Transplant Hepatology: A Case Based Approach, Springer: US, 2010.
[51] Hinz, B.; Cheremina, O.; Brune, K., Acetaminophen (paracetamol) is a selective cyclooxygenase-2 inhibitor in man. Faseb J 2008, 22, 383-390.
[52] Newton, R.; Broughton, L. J.; Lind, M. J.; Morrison, P. J.; Rogers, H. J.; Bradbrook, I. D., Plasma and salivary pharmacokinetics of caffeine in man. Eur J Clin Pharmacol 1981, 21, 45-52.
[53] The Pharmacogenetics and Pharmacogenomics Knowledge Base (Caffeine).
[54] Weimann, A.; Sabroe, M.; Poulsen, H. E., Measurement of caffeine and five of the major metabolites in urine by high-performance liquid chromatography/tandem mass spectrometry. Journal of mass spectrometry : JMS 2005, 40, 307-16.
[55] PharmGKB。Caffeine Pathway, Pharmacokinetics. https://www.pharmgkb.org/
[56] Ptolemy, A. S.; Tzioumis, E.; Thomke, A.; Rifai, S.; Kellogg, M., Quantification of theobromine and caffeine in saliva, plasma and urine via liquid chromatography-tandem mass spectrometry: A single analytical protocol applicable to cocoa intervention studies. J Chromatogr B 2010, 878, 409-416.
[57] 療黴舒仿單，衛署藥輸字第019320號
[58] James Ku CG Character Art. http://www.jameskuart.com/
[59] Newton, R.; Broughton, L. J.; Lind, M. J.; Morrison, P. J.; Rogers, H. J.; Bradbrook, I. D., Plasma and salivary pharmacokinetics of caffeine in man. Eur J Clin Pharmacol 1981, 21, 45-52.