STRUCTURAL AMD ANALYTICAL STUDIES BY TANDEM MASS SPECTROMETRY A Thesis submitted by TRACEY MADDEN for the degree of Doctor of Philosophy in the University of London Faculty of Science Department of Pharmaceutical Chemistry The School of Pharmacy University of London ProQuest Number: U556261 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest U556261 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 CONTENTS CHAPTER 1: INTRODUCTION 1.1 Tandem Mass Spectrometry ................... 1 REFERENCES ................................. 4 CHAPTER 2: THEORY 2.1 Mass Spectrometry ......................... 5 2.2 Formation of the Molecular Ion ............ 8 2.2.1 Vertical and Adiabatic Ionisation Potentials ......................... 9 2.2.2 Ionisation Efficiency Curve .......... 9 2.2.3 Appearance Energy ................... 10 2.3 Quasi Equilibrium Theory ................. 14 2.4 Fragmentation ............................ 16 2.4.1 Stevenson's Rule .................... 17 2.5 Rearrangement ............................ 19 2.6 Ion Stability ............................ 20 2.6.1 Stable Ions ......................... 20 2.6.2 Unstable Ions ....................... 21 2.6.3 Metastable Ions ..................... 21 2.6.3.1 Kinetic Energy Release ......... 22 2.6.3.2 Metastable Peak Shapes ........ 27 2.7 Collisions ............................... 31 2.7.1 Gas pressure ........................ 33 2.7.2 Scattering .......................... 35 Charge Exchange .................... 38 Neutralisation ................ 38 Ionisation .................... 41 Collision Techniques in Mass Spectrometry 44 Collision Induced Dissociation ..... 44 Neutralisation-Reionisation ........ 50 Collisionally-Induced Dissociative Ionisation ....................... 57 Neutralised Ion Beam Spectroscopy ... 59 Charge Stripping ................... 62 Charge Inversion (Charge Reversal) .. 63 REFERENCES .............................. 65 3: INSTRUMENTATION Ionisation Techniques ................... 69 Electron Impact .................... 70 Chemical Ionisation ................ 74 Fast Atom Bombardment .............. 80 Field Ionisation ................... 84 Field Desorption ................... 87 Analysers ............................... 88 Magnetic Sector .................... 88 Electrostatic Analyser ............. 89 Quadrupole Mass Analyser ........... 90 Detectors ............................... 94 Focal Plane Detection .............. 94 ii 3.3.2 Faraday Cup (Faraday Cage) ......... 94 3.3.3 Electron Multiplier ................ 95 3.3.4 Photomultiplier .................... 95 3.3.5 Scintillation Detector ............. 96 3.4 Instrument Geometry ....................... 98 3.5 Scanning Regimes ........................ 101 3.5.1 Linked Scanning .................... 101 3.5.1.1 Daughter Ion Scans (B/E) 101 3.5.1.2 Parent Ion Scans (B2/E) 103 3.5.1.3 Constant Neutral Loss Scan .... 104 3.5.2 MIKES Scan ......................... 104 3.6 VG ZAB-2F ............................... 107 3.6.1 Cell Voltage ....................... Ill 3.6.2 Deflector Voltage ................. 113 3.7 Data Acquisition and Recording......... 115 3.7.1 UV Oscilloscope .................... 115 3.7.2 Data System ........................ 117 REFERENCES... ........................... 121 CHAPTER 4: EXPERIMENTAL 4.1 Hydroxyquinoline Isomers ................. 123 4.1.1 Metastable Peak Shapes ............ 123 4.1.1.1 Introduction .................. 123 4.1.1.2 Experimental ................. 126 4.1.1.3 Results and Discussion ....... 126 4.1.2 High Energy CID Spectra ........... 131 iii 4.1.2.1 Introduction 131 4.1.2.2 Experimental 131 4.1.2.3 Results and Discussion ....... 132 4.1.3 Deuterium Labelling ............... 154 4.1.3.1 Introduction 154 4.1.3.2 Experimental 154 4.1.3.3 Results and Discussion ....... 156 4.2 Isomer Differentiation by NRMS ......... 163 4.2.1 Introduction ...................... 163 4.2.2 Experimental ...................... 163 4.2.3 Results and Discussion ............ 165 4.3 Fragmentation of Tertiary Butylbenzene .. 188 4.3.1 Introduction....................... 188 4.3.2 Establishing the Major Fragments of t-butylbenzene .................. 188 4.3.2.1 Experimental 188 4.3.2.1.1 High Energy CID/MIKES .... 189 4.3.2.1.2 B/E and B2/E Linked Scans 189 4. 3. 2.1.3 NRMS 18 9 4.3.2.2 Results and Discussion ....... 201 4.3.3 Structure of the C7H7+ Ion ........ 196 4.3.3.1 Introduction 196 4.3.3.2 Experimental 200 4.3.3.3 Results andDiscussion ......... 201 4.4 C2H5NO'k Isomers - Structural Information From MS/MS ........................... 205 4.4.1 Introduction....................... 205 iv 4.4.2 Experimental ...................... 206 4.4.2.1 Unimolecular MIKES ........... 207 4.4.2.2 High Energy CID/MIKES..... .... 208 4.4.2.3 B/E Linked Scans ............. 208 4. 4.2. 4 NRMS ......................... 209 4.4.3 Results and Discussion ............ 209 4.4.3.1 Unimolecular MIKES ........... 209 4. 4. 3.2 High Energy CID/MIKES..... .... 214 4.4.3 B/E Linked Scans ............. 218 4. 4. 3. 4 NRMS ......................... 221 4.5 Factors Affecting Immonium Ion Production in the High Energy CID Spectra of Peptides ............................. 22 6 4.5.1 Introduction ...................... 226 4.5.2 Peptide analysis .................. 228 4.5.3 Experimental ...................... 234 4.5.4 Results and Discussion ............ 236 4.5.4.1 C-terminal Effect ............ 236 4.5.4.2 N-terminal Effect ............ 240 4.5.4.3 Substitution of Single Residues 242 4.5.4.4 Immonium Ions from Cationated Species .................... 245 4.5.4.5 Excess Protons at Ionisation .. 247 4.5.4.6 Adding a Basic Residue to the Sequence ................... 24 9 REFERENCES ............................. 254 v CHAPTER 5: SUMMARY AND CONCLUSIONS 256 APPENDICES Appendix I : Calculating an Average, Weighted by the Uncertainty in Individual Results ................................ 261 vi LIST OF FIGURES Fig 1 Schematic of a 'tandem' mass spectrometer Fig 2 Schematic of a mass spectrometer Fig 3 Ionisation efficiency curve Fig 4 Relationship between appearance potential, 80 and kinetic shift Fig 5 Illustration of Stevenson's rule in relation to the fragmentation of M1+- Fig 6 Origins of T§ and T® Fig 7 Measuring the peak with at 20% of the peak height Fig 8 Examples of non-gaussian peaks shapes (a) flat-topped (b) dished topped Fig 9 Example of a composite peak Fig 10 Variation in the average number of collisions suffered by a molecule with increasing gas pressure vii Fig 11 Simple model of elastic collision between rigid spheres Fig 12 Simplified model of elastic collision between rigid spheres Fig 13 Arrangement of collision cells and deflector plate for NRMS Fig 14 Deflection of ions using a voltage on the second collision cell Fig 15 Collision between an ion emerging from collision cell 1 and gas streaming back from collision cell 2 Fig 16 NRMS using soft neutralisation and ionisation gases Fig 17 NRMS using a hard neutralisation gas and a soft ionisation gas Fig 18 NRMS using a soft neutralisation gas and a hard ionisation gas Fig 19 Arrangement of deflector and collision cell for CIDI viii 20 Arrangement of deflector and collision cell for NIBS 21 Alternative arrangement of collision cells for NIBS 22 Electron impact source 23 Fast atom bombardment source 24 Field ionisation source 25 Magnetic sector 26 Electrostatic sector 27 Quadrupole assembly 28 Schematic of a Faraday cup 29 Schematic of an electron multiplier 30 Schematic of a photomultiplier 31 Schematic of the VG ZAB-2F 32 Detail of the 2FFR ix Fig 33 Detail of the two detectors Fig 34 Composite peak for unimolecular CO loss from 2-hydroxyquinoline = quinolin-2(1H)-one Fig 35 Composite peak for unimolecular CO loss from 4-hydroxyquinazoline Fig 36 Dished-topped peak for unimolecular CO loss from 2-hydroxyquinoxalinol Fig 37 Composite peak for unimolecular CO loss from quinoline N-oxide Fig 38 Partial CID spectra of the [M-CO]+' ions from (a) 2-hydroxyquinoline and (b) quinoline N- oxide (Vc=0) . Fig 39 Partial CID spectra of the [M-CO]+- ion from (a) 2- hydroxyquinoline and (b) the M+> ion from indole (Vc=0) Fig 40 Partial CID spectra of the [M-CO]+‘ ions from (a) 3- and (b) 4-hydroxyquinoline (Vc=0) Fig 41 Partial CID spectra of the [M-CO]+- ions from (a) 5- and (b) 8-hydroxyquinoline (Vc=0) x 42 Partial CID spectra of the [M-CO]+* ion from (a) 2-hydroxyquinoline and (b) the M+- ion from indole (Vc=1.6kV) 43 Partial CID spectra of the [M-CO]+- ions from (a) 3- and (b) 4-hydroxyquinoline (Vc=1.6kV) 44 Partial CID spectra of the [M-CO]+‘ ions from (a) 5- and (b) 8-hydroxyquinoline (Vc=l. 6kV) 45 Partial CID spectrum of the [M-CS]+> ion from (a) 2-mercaptoquinoline (Vc=0) 46 Partial CID spectra of the [M-CS]+- ions from (a) 3- and (b) 4-mercaptoquinoline (Vc=0) 47 Partial CID spectrum of the [M-CS]+* ion from (a) 8-mercaptoquinoline (Vc=0) 48 Possible structures for the [M-CO]+* ions from the hydroxyquinoline isomers 49 Partial CID spectrum of the [MD-CO]+‘ ion from (a) 2-hydroxyquinoline 50 Partial CID spectra of the