A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/104986 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications Electron Paramagnetic Resonance Techniques for Pharmaceutical Characterization and Drug Design by Claudio Vallotto Thesis Submitted to the University of Warwick for the degree of Doctor of Philosophy Department of Chemistry August 2017 Contents Title page .................................................................................................................................... i Contents ..................................................................................................................................... ii List of Figures ........................................................................................................................... ix Acknowledgments .................................................................................................................... xv Declaration and published work ............................................................................................. xvi Abstract ................................................................................................................................... xix Abbreviations and symbols ...................................................................................................... xx Chapter 1 Principles of EPR ...................................................................................................... 1 1.1 Uses of EPR ..................................................................................................................... 1 1.2 The Zeeman effect ........................................................................................................... 2 1.3 The spin Hamiltonian ....................................................................................................... 3 1.4 The g-factor ...................................................................................................................... 4 1.5 Hyperfine interaction ....................................................................................................... 4 1.6 Multi-frequency EPR ....................................................................................................... 6 1.7 Spin trapping .................................................................................................................... 8 1.7.1 Spin trapping reaction ............................................................................................... 8 1.7.2 Choice of spin trapping agent ................................................................................... 8 1.8 References ...................................................................................................................... 10 Chapter 2 Sterilization of Pharmaceuticals .............................................................................. 13 2.1 Sterilization in the pharmaceutical industry ................................................................... 13 2.1.1 Dry heat sterilization ............................................................................................... 13 2.1.2 Steam sterilization ................................................................................................... 14 2.1.3 Ethylene oxide sterilization ..................................................................................... 14 2.1.4 Filtration sterilization .............................................................................................. 14 2.2 Radiation sterilization .................................................................................................... 14 2.2.1 Ionising radiation .................................................................................................... 15 ii 2.2.2 γ-irradiation sterilization ......................................................................................... 15 2.2.3 γ-sterilization of pharmaceuticals ........................................................................... 16 2.3 EPR in the detection of pharmaceutical degradants ....................................................... 17 2.4 Pharmaceutical excipients .............................................................................................. 17 2.5 Motivation of study ........................................................................................................ 19 2.6 Thesis outline ................................................................................................................. 20 2.7 References ...................................................................................................................... 21 Chapter 3 Experimental Methods ............................................................................................ 24 3.1 Experimental .................................................................................................................. 24 3.1.1 Materials.................................................................................................................. 24 3.2 Crystal growth ................................................................................................................ 24 3.2.1 Deuteration .............................................................................................................. 24 3.3 γ-irradiation .................................................................................................................... 25 3.3.1 Room temperature irradiation ................................................................................. 25 3.3.2 γ-irradiation in liquid nitrogen ................................................................................ 25 3.3.3 Removal of quartz background signal ..................................................................... 26 3.4 X-irradiation ................................................................................................................... 26 3.5 Instrumentation .............................................................................................................. 27 3.5.1 The EPR spectrometer ............................................................................................ 27 3.5.1.1 W-band EPR .................................................................................................... 29 3.5.1.2 mm-wave EPR ................................................................................................. 29 3.5.2 Low temperature ..................................................................................................... 30 3.5.3 Spin trapping ........................................................................................................... 31 3.5.3.1 Phosphate buffer solution ................................................................................. 32 3.5.3.2 Sample preparation .......................................................................................... 33 3.5.4 ENDOR ................................................................................................................... 33 3.5.5 1H NMR ................................................................................................................... 33 3.5.6 ESI-MS analysis ...................................................................................................... 34 3.5.7 XRF ......................................................................................................................... 34 iii 3.6 Quantitative EPR ........................................................................................................... 34 3.6.1.1 Quantification of persistent radicals in the solid state ...................................... 35 3.6.1.2 Quantification of spin adducts ......................................................................... 36 3.7 EPR simulations ............................................................................................................. 37 3.8 References ...................................................................................................................... 38 Chapter 4 Irradiation of L-histidine Free Base ........................................................................ 40 4.1 Introduction .................................................................................................................... 40 4.1.1 Use of L-histidine in parenteral formulations ......................................................... 40 4.1.2 Scavenging properties of histidine .......................................................................... 41 4.1.3 Solid state radicals .................................................................................................. 41 4.1.4 Spin trapped L-histidine radicals ............................................................................ 41 4.2 Experimental .................................................................................................................. 42 4.2.1 Instrumentation ....................................................................................................... 42 4.2.2 Sample preparation