(3.230.154.160) 您好!臺灣時間:2021/05/07 19:18
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果

詳目顯示:::

我願授權國圖
: 
twitterline
研究生:何欣頻
研究生(外文):Hsin-Pin Ho
論文名稱:液相層析串聯質譜術於人體血清中盤尼西林代謝物之定性分析
論文名稱(外文):Identification of Penicillin G Metabolites in Human Serum by Liquid Chromatography-Tandem Mass Spectrometry
指導教授:李茂榮李茂榮引用關係
指導教授(外文):Maw-Rong Lee
學位類別:碩士
校院名稱:國立中興大學
系所名稱:化學系所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
畢業學年度:97
語文別:英文
論文頁數:72
中文關鍵詞:串聯質譜盤尼西林G代謝物
外文關鍵詞:Tandem Mass SpectrometryPenicillin GMetabolites
相關次數:
  • 被引用被引用:0
  • 點閱點閱:101
  • 評分評分:系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔系統版面圖檔
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
盤尼西林G及其代謝產物導致人體過敏反應,為其用藥限制最主要的原因。至今對其代謝產物機制仍有許多不明白的地方,造成在臨床上無法確切和有效的預防過敏反應的產生。故本實驗將採用具有高選擇性及高靈敏度的數據依據性液相層析串聯質譜技術 (Data-dependent LC-MSn) 對人體血清中盤尼西林G及其微量代謝物進行鑑定分析,試圖找出目前尚未發現之微量代謝物,推測其代謝途徑,並藉由MassWorks軟體對推測結果進行精確分子量的測量。根據實驗結果成功檢測出七個盤尼西林G代謝產物,包括二個已知代謝物 (Penicilloate, C16H21N2O5S 和 Penilloate, C15H21N2O3S) 及五個未知代謝物 (C19H25N2O7S、C19H27N2O7S、C16H21N2O6S、C22H29N2O11S和C22H31N4O8S3)。本實驗同時證實Data-dependent LC-MSn於人體藥物代謝物偵測的可行性,所得之實驗結果將作為盤尼西林G微量代謝物於過敏反應機制的研究的參考依據。
Penicillin G (PCN G) was the first antibiotic found and widely applied for human bacterial disease; however, hypersensitivity reactions to penicillin are due to different metabolites formed in vivo when the antibiotic is administered. Several penicillin metabolites in human allergic reaction have been studied and assessed by using penicillin skin test. Unfortunately, the skin test still may give false-negative results and bring the risk of unsafety, such as anaphylactic reactions. Currently, LC-MS and LC-MS/MS were applied to characterize drug metabolites, but they remain time-consuming processes. Frequently, the data obtained are insufficient to locate the site of metabolism on a candidate molecule. Data-dependent LC-MSn is a powerful tool to provide large amounts of the necessary structural information regarding each analyte in one chromatographic run, thereby allowing for a more detailed characterization of the metabolites. Furthermore, it is highly sensitive and selective to detect trace metabolites even in a complex matrix. In the study, Data-dependent LC-MSn was utilized to identify trace metabolites of PCN G in human serum. From the results, in addition to the known metabolites, such as penicilloate and penilloate, the trace unknown metabolites of PCN G metabolites were successfully identified. The structures were determined as the proposed unknown metabolites have been further confirmed by using MassWorks.
Table of Contents
謝誌 i
中文摘要 ii
Abstract iii
Table of contents iv
List of figures vi
List of tables ix
Abbreviations x
Chapter1 Background of Penicillin G
1.1 Penicillin G 1
1.1.1 Introduction to Penicillin G 1
1.1.2 Penicillin Allergic Reactions 1
1.1.3 Metabolism of Penicillin G 3
1.2 Analytical Methods for Penicillin Metabolite Analysis 7
Chapter 2 Introduction to Mass Spectrometry in Metabolisms
2.1 Liquid Chromatography-Mass Spectrometry (LC-MS) 8
2.2 Atmospheric Pressure Ionization 9
2.2.1 Electrospray Ionization 9
2.3 Mass Analyzer 10
2.3.1 Ion Trap Mass Analyzer 12
2.4 Tandem Mass Spectrometry (MS/MS) 16
2.4.1 Scan Modes in Ion Trap Tandem Mass Spectrometry 16
2.4.2 Data-Dependent MSn Acquisition Method 17
2.5 Accurate Mass Measurement by MassWorksTM Software 18
2.6 Aims of the Thesis 18
Chapter 3 Experimental 20
3.1 Chemical and Reagents 20
3.2 Sample Pretreatment 20
3.3 LC-MS/MS Analysis 21
3.3.1 HPLC System 21
3.3.2 MS System 21
3.4 MassWorksTM Experiment 24
Chapter 4 Results and discussion 27
4.1 LC-MS/MS Analysis of Available Standards 27
4.1.1 Fragmentation of Penicillin G and Penicilloate 27
4.1.2 LC-MS analysis 30
4.1.3 Pretreatment Validation 30
4.2 In vivo Metabolic Analysis 33
4.3 Metabolite Identification 46
4.3.1 Identification of M1 (penicilloate, m/z 353) 46
4.3.2 Identification of M2 (penilloate, m/z 309) 48
4.3.3 Identification of M3 (m/z 425) 51
4.3.4 Identification of M4 (m/z 427) 51
4.3.5 Identification of M5 (hydroxylated metabolite, m/z 369) 54
4.3.6 Identification of M6 (glucuronide metabolite, m/z 529) 54
4.3.7 Identification of M7 ( m/z 575) 57
4.4 PCN G Metabolite Pathway 62
4.5 Real Sample Analyses 64
4.6 Accurate Mass Measurement 64
Chapter 5 Conclusions 67
Reference 68

List of Figures
Figure 1. Structure of the beta-lactam ring and penicillins that have this ring in their structure 2
Figure 2. β-Lactam ring changes with metabolism 5
Figure 3. Schematic representation of an electrospray ionization 11
Figure 4. Main components of a 3D QIT and the characteristic ion trajectory in the trap 13
Figure 5. A schematic diagram of a linear ion-trap mass analyzer 15
Figure 6. The general flow of MassWorks calibration and its elemental composition determination process 26
Figure7. Spectra from MSn analyses of the PCN G 28
Figure 8. Spectra from MSn analyses of the penicilloate. 29
Figure 9. Mass ion chromatograms obtained from standard penicilloate by using data-dependent MSn scans. 31
Figure 10. Mass ion chromatograms obtained from the treated blank sample and treated spiked sample by using data-dependent MSn scans. 32
Figure 11. Extracted ion chromatograms of m/z 160 in the product ion MS/MS scans. 34
Figure 12. Mass ion chromatograms produced by extracting neutral-loss of m/z 159 in product ion MS/MS scans 35
Figure 13. Total ion chromatograms of serum samples by using data-dependent MSn screen 36
Figure 14. Extracted ion chromatograms of m/z 335 by using data-dependent MSn screen 38
Figure 15. Extracted ion chromatograms of m/z 353 by using data-dependent MSn screen 39
Figure 16. Extracted ion chromatograms of m/z 309 by using data-dependent MSn screen 40
Figure 17. Extracted ion chromatograms of m/z 425 by using data-dependent MSn screen 41
Figure 18. Extracted ion chromatograms of m/z 427 by using data-dependent MSn screen 42
Figure 19. Extracted ion chromatograms of m/z 369 by using data-dependent MSn screen 43
Figure 20. Extracted ion chromatograms of m/z 529 by using data-dependent MSn screen 44
Figure 21. Extracted ion chromatograms of m/z 575 by using data-dependent MSn screen 45
Figure 22 Mass ion chromatograms extracted from an administrated serum sample obtained after administration of PCN G 47
Figure 23. The sequential MSn spectra obtained for M1 (m/z 353) 49
Figure 24. Product ion (MS2) spectra of [M+H]+ of M2 m/z 309 50
Figure 25. The sequential MSn spectra obtained for M3 (m/z 425) 52
Figure 26. The sequential MSn spectra obtained for M4 (m/z 427) 53
Figure 27. The sequential MSn spectra obtained for M5 (m/z 369) 55
Figure 28. The sequential MSn spectra obtained for M6 (m/z 529) 56
Figure 29. Mass spectra of M7 58
Figure 30. Mass spectrum of [M+2H]2+ at m/z 288 obtained by using zoom scan 59
Figure 31. MSn spectra of M7 at [M+2H]2+ 60
Figure 32. MSn spectra of M7 at [M+H]+ 61
Figure 33. Total ion chromatograms of the three human serum samples by using data-dependent LC-MSn method 65
List of Tables
Table 1. Classification of allergic penicillin reactions 4
Table 2. ESI-MS conditions 21
Table 3. Chromatographic and mass spectrometric data of PCN G and its metabolites found. 37
Table 4. Accurate mass measurements of penicillin G metabolites 66
Reference
[1]A. D. Deshpande, K. G. Baheti,N. R. Chatterjee, Degradation of β-lactam antibiotics, Current Science, 87 (2004) 1684.
[2]J. P. Hou,J. W. Poole, beta-lactam antibiotics: Their physicochemical properties and biological activities in relation to structure, Journal of Pharmaceutical Sciences, 60 (1971) 503.
[3]H. C. Neu, ß-Lactam Antibiotics: Structural Relationships Affecting in Vitro Activity and Pharmacologic Properties, Reviews of Infectious Diseases, 8 (1986) S237.
[4]N. A. Rosário,A. S. Grumach, Allergy to beta-lactams in pediatrics: a practical approach, Jornal de Pediatria, 82 (2006) S181.
[5]F. Sáchez-Sancho, E. Perez-Inestrosa, R. Suau, M. I. Montañez, C. Mayorga, M. J. Torres, A. Romano,M. Blanca, Synthesis, characterization and immunochemical evaluation of cephalosporin antigenic determinants, Journal of Molecular Recognition, 16 (2003) 148.
[6]J. Frumin,J. C. Gallagher, Allergic Cross-Sensitivity Between Penicillin, Carbapenem, and Monobactam Antibiotics: What Are the chances?, The Annals of Pharmacotherapy, 43 (2009) 304.
[7]J. N. Franklin Adkinson, MD, M. David Essayan, M. Rebecca Gruchalla, H. Haggerty, T. Kawabata, D. Sandler, L. Updyke,M. Neil H. Shear, Task Force Report:Future research needs for the prevention and management of immune-mediated drug hypersensitivity reactions, The Journal of Allergy and Clinical Immunology, 109 (2002) 461.
[8]W. J. Pichler, Pharmacological interaction of drugs with antigen-specific immune receptors: the p-i concept, Current Opinion in Allergy and Clinical Immunology, 2 (2002) 301.
[9]A. T. Nagao-Dias, P. Barros-Nunes, H. L. L. Coelho,D. Solé, Allergic drug reactions, Jornal de Pediatria, 80 (2004) 259.
[10]R. Y. Lin, A perspective on penicillin allergy, Archives of Internal Medicine, 152 (1992) 930.
[11]W. J. Pichler, Drug Hypersensitivity Reactions: Classification and Relationship to T-Cell Activation, Karger, Basel, 2007.
[12]M. E. Arroliga,L. Pien, Penicillin allergy: Consider trying penicillin again, Cleveland Clinic Journal of Medicine, 70 (2003) 313.
[13]S. Roland,M. L. M., Systemic reactions to antibiotics, Immunology and Allergy Clinics of North America, 21 (2001) 679.
[14]A. I. Neugut, A. T. Ghatak,R. L. Miler, Anaphylaxis in the United States, Archives of Internal Medicine, 161 (2001) 15.
[15]M. Blanca, C. Mayorga,M. J. Torres, Side-chain-specific reactions to beta-lactams: 14 years later, Clinical and Experimental Allergy: Journal of the British Society for Allergy and Clinical Immunology, 32 (2002) 192.
[16]F. Silviu-Dan, S. Mcphillips,R. J. Warrington, The frequency of skin test reactions to side-chain penicillin determinants, The Journal of Allergy and Clinical Immunology, 91 (1993) 694.
[17]G. O. Solley, G. J. Gleich,R. G. V. Dellen, Penicillin allergy: clinical experience with a battery of skin-test reagents, The Journal of Allergy and Clinical Immunology, 69 (1982) 238.
[18]A. Basomba, A. Peláez, I. G. Villalmanzo,A. Campos, Allergy to penicillin unsuccessfully treated with a haptenic inhibitor (benzyl-penicilloyl-N2-formil-lysine; BPO-Flys), Clinical Allergy, 8 (1978) 341.
[19]J. E. Erffmeyer,M. S. Blaiss, Proving penicillin allergy, Postgarduate Medicine, 2 (1990) 33.
[20]J. A. Anderson, in P. Lieberman (Editor), Current Clinical Practice: Allergic Diseases: Diagnosis and Treatment, 3th, Humana Press, Totowa, 2007, p. 295.
[21]P. J. Bousquet, H. B. Co-Minh, B. Arnoux, J. P. Daures,P. Demoly, Improtance of mixture of minor determinants and benzylpenicilloyl poly-L-lysine skin testing in the diagnosis of β-lactam allergy, Journal of Allergy and Clinical Immunology, 115 (2005) 1314.
[22]A. K. Chandel, L. V. Rao, M. L. Narasu,O. V. Singh, The Realm of Penicillin G acylase in β-lactam antibiotics, Enzyme and Mecrobial Technology, 42 (2008) 199.
[23]M. Cole, M. D. Kenig,V. A. Hewitt, Metabolism of Penicillins to Penicilloic Acids and 6-Aminopenicillanic Acid in Man and Its Significance in Assessing Penicillin Absorption, Antimicrobial Agents and Chemotherapy, 3 (1973) 463.
[24]J. R. Everett, K. R. Jennings, G. Woodnutt,M. J. Buckingham, Spin-echo 1H N.M.R spectroscopy: a new method for studying penicillin metabolism, Journal of the Chemical Society. Chemical Communications (1984) 894.
[25]W. J. J. Krauwinkel,N. J. Volkers-Kamermans, Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction, Journal of Chromatography B, 679 (1996) 129.
[26]M. L. Vestal, Tandem Mass Spectrometric Studies of the Fragmentation of Penicillins and Their Metabolites, Biomedical and Environmental Mass Spectrometry, 16 (1988) 381.
[27]K. Kobayashi, K. Sato, Y. Mizuno,Y. Katsumata, Capillary High-Performance Liquid Chromatography-Fast Atom Bombardment Mass Spectrometry of 24 cephem Antibiotics, Journal of Chromatography B, 677 (1996) 275.
[28]M. Scandola, G. Tarzia, G. Gaviraghi, D. Chiarello,P. Traldi, Mass Spectrometric Approaches in Structural Characterization of Cephalosporins, Biomedical and Environmental Mass Spectrometry, 18 (1989) 851.
[29]A. F. Casy, C. Cryer,E. M. A. Ominde, Mass Spectrometry of β-lactam antibiotics with special reference to ionization by fast atom bombardment (FAB), Journal of Pharmaceutical and Biomedical Analysis, 7 (1989) 1121.
[30]M. P. Barbalas, F. W. McLafferty,J. L. Occolowitz, Targeted Class Analysis of β-lactam Antibiotics by Tandem Mass Spectrometry, Biomedical Mass Spectrometry, 10 (1983) 258.
[31]J. L. Gower, G. D. Risbridger,M. J. Redrup, Positive and negative ion fast atom bombardment mass spectra of some penicilloic acids, The Journal of Antibiotics, 37 (1984) 33.
[32]D. M. Holstege, B. Purchner, G. Whitehead,F. D. Galey, Screening and Mass Spectral Confirmation of β-lactam Antibiotic Residues in Milk Using LC-MS/MS, Journal of Agricultural and Food Chemistry, 50 (2002) 406.
[33]D. N. Heller, M. L. Smith,O. A. Chiesa, LC/MS/MS Measurement of Penicillin G in Bovine Plasma, Urine, and Biopsy Samples Taken From Kidneys of Standing Animals, Journal of Chromatography B, 830 (2006) 91.
[34]Y. Kazakevich,R. Lobrutto, HPLC for pharmaceutical scientists, Jonh Wiley and Sons, 2007.
[35]W. Paul,H. Steinwedel, A New Mass Spectrometer Without Magnetic Field, Zeitschrift für Naturforschung, 8a (1953) 448.
[36]R. E. March,J. F. J. Todd, Quadrupole Ion Trap Mass Spectrometry, Wiley-Interscience, New Your, 2005.
[37]E. d. Hoffmann,V. Stroobant, Mass Spectrometry-Principles and Applications, John Wiley & Sons Ltd., London, 2007.
[38]J. Yinon, Advances in Forensic Applications of Mass Spectrometry, CRC PRESS, New York, 2003.
[39]M. C. McMaster, LC/MS: a practical user''s guide, John Wiley and Sons (2005) 165.
[40]J. E. McClellan, S. T. Quarmby,R. A. Yost, Parent and Neutral Loss Monitoring on a Quadrupole Ion Trap Mass Spectrometer: Screening of Acylcarnitines in Complex Mixtures, Analytical Chemistry, 74 (2002) 5799.
[41]M. L. Salomonsson, U. Bondesson,M. Hedeland, In Vitro Formation of Phase I and II Metabolites of Propranolol and Determination of Their Structures Using Chemical Derivatization and Liquid Chromatography-Tandem Mass Spectrometry, Journal of Mass Spectrometry: JMS, 44 (2009) 742.
[42]Y. Sawada, K. Akiyama, A. Sakata, A. Kuwahara, H. Otsuki, T. Sakurai, K. Saito,M. Y. Hirai, Widely Targeted Metabolomics Based on Large-Scale MS/MS Data for Elucidating Metabolite Accumulation Patterns in Plants, Plant & Cell Physiology, 50 (2009) 37.
[43]S. Bourcier,Y. Hoppilliard, Use of diagnostic neutral losses for structural information on unknown aromatic metabolites: an experimental and theoretical study, Rapid Communications in Mass Spectrometry, 23 (2009) 93.
[44]L. L. Lopez, D. M. Drexler, I. Mylchreest,J. C. Schwartz, in Proceedings of the 47th ASMS Conference on Mass Spectrometry and Allied Topics, Dallas, TX, 1999.
[45]J. V. Johnson, R. A. Yost, P. E. Kelly,D. C. Bradford, Tandem-in-Space and Tandem-in-Time Mass Spectrometry (MS/MS): Triple Quadrupoles and Quadrupole Ion Traps, Analytical Chemistry, 62 (1990) 2162.
[46]Y. Wang,H. Prest, Accurate Mass Measurement on Real Chromatographic Time Scale with a Single Quadrupole Mass Spectrometer, Chromatography, 27 (2006) 135.
[47]M. Gu, Y. Wang, X. G. Zhao,Z. M. Gu, Accurate Mass Filtering of Ion Chromatograms for Metabolite Identification Using a Unit Mass Resolution Liquid Chromatography/Mass Spectrometry System, Rapid Communications in Mass Spectrometry, 20 (2006) 764.
[48]B. J. Goolsby,J. S. Brodbelt, Characterization of β-lactams by Photodissociation and Collision-activated Dissociation in a Quadrupole Ion Trap, Journal of Mass Spectrometry: JMS, 33 (1998) 705.
[49]A. Tevell, U. Bondesson, K. Törneke,M. Hedeland, Identification of Some New Clemastine Metabolites in Dog, Horse, and Human Urine With Liquid Chromatography-Tandem Mass Spectrometry, Rapid Communications in Mass Spectrometry, 18 (2004) 2267.
[50]M. Holčapek, L. Kolářová,M. Nobilis, High-Performance Liquid Chromatography-Tandem Mass Spectrometry in The Identification and Determination of Phase I and Phase II Drug Metabolites, Analytical and Bioanalytical Chemistry, 391 (2008) 59.
[51]H. Lioe,R. A. J. O. Hair, Comparison of Collision-induced Dissociation and Electron-induced Dissociation of Singly Protonated Aromatic Amino Acids, Cystine and Related Simple Peptides Using A Hybride Linear Ion Trap-FT-ICR Mass Spectrometer, Analytical and Bioanalytical Chemistry, 389 (2007) 1429.
[52]C. S. Wright, Evaluation of Penicillin Allergy in an Allergy Immunology Practice, Journal of Allergy and Clinical Immunology, 119 (2007) S67.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
系統版面圖檔 系統版面圖檔