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This Work Is Protected by Copyright and Other Intellectual Property Rights This work is protected by copyright and other intellectual property rights and duplication or sale of all or part is not permitted, except that material may be duplicated by you for research, private study, criticism/review or educational purposes. Electronic or print copies are for your own personal, non- commercial use and shall not be passed to any other individual. No quotation may be published without proper acknowledgement. For any other use, or to quote extensively from the work, permission must be obtained from the copyright holder/s. Analysis of cuticular hydrocarbons in forensically important blowflies using mass spectrometry and its application in Post Mortem Interval estimations A thesis submitted by Hannah Elizabeth Moore to Keele University For the degree of Doctor of Philosophy June 2013 Abstract Forensic entomology relies on accurate identification of forensically important blowflies to species level, preceded by ageing the Calliphorid specimens present on a cadaver to determine the post-mortem interval (PMI). The task of identifying blowflies based on morphological criteria can be challenging due to complex keys, limited diagnostic features in the immature stages and poor preservation of entomological samples. Therefore, the larvae will mostly be reared to adult flies to confirm identification, which is a time consuming process. This thesis presents work examining the cuticular hydrocarbon (CHC) profiles of three forensically important blowfly species in the UK, Lucilia sericata, Calliphora vicina and Calliphora vomitoria with two main aims to this study. Firstly to establish if CHC analysis could be used to determine whether the three species yield characteristic profiles, allowing for identification to be achieved. The second aim was to examine the hydrocarbon profiles over time to determine if chemical changes occurred at certain points in time, giving an indication of age. The CHCs were extracted from all life stages (empty egg cases, larvae, pupae, empty puparial cases and adult flies) and analysed using gas chromatography – mass spectrometry (GC-MS) and Direct-analysis-in-Real-Time MS (DART-MS). Statistical interpretation was applied in the form of principal component analysis (PCA) and preliminary Artificial Neural Networks (ANN). Results showed species-specific characteristics within the chromatograms in all life stages, meaning distinctions can be made between the three species, even in 1st instar larvae. Significant chemical changes were observed within the hydrocarbon profiles over time, hence accurate ageing could be established for larvae, empty puparial cases and adult flies. Early results show great potential to utilise this technique and to develop it into a highly useful identification and ageing tool. i Acknowledgements I would like to start by thanking my principal PhD supervisor, Dr Falko Drijfhout, for his endless guidance and support through my studies. He gave me this amazing opportunity and allowed me the freedom to choose my own research paths. He has been inspirational and I have learnt an enormous amount from his vast knowledge base. My thanks also go to… ACORN from Keele University, for funding part of my research. Dr Craig Adam, my second supervisor, who not only helped me with the statistical side of my research, but also improved my writing skills. Dr Peter Haycock, to whom I owe a great deal, for had he not given me a chance all those years ago, I would have never started my undergraduate studies at Keele. I hope my academic progress has given him the confidence to allow other students the same opportunities. Deena van Logchem for his help in the laboratory during his time at Keele. Dr Martin Hall, Dr Cameron Richards, Amoret Whittaker and Dr Andrew Hart, for their advice, inspiration and support. Dr John Butcher for his contribution on the artificial neural network results. On a personal note, I would like to thank him for being so patient with me over the last four years and for keeping a smile on my face. Everything is reachable with him by my side. The Winston Churchill Memorial Trust for awarding me the opportunity of a life time. ii To all the wonderful people I met and hosted me during my stay in America, whose passion and enthusiasm greatly assisted with the final stages of my writing on return to the UK. My fellow postgraduate friends, with a special mention to Sue Shemilt for casting her expert eye over some of my work. All of my fantastic friends away from the Keele bubble, for your belief in me. Victoria Gilby, for training my horse so I was able to continue to compete. Horses have been a huge release from my academic studies and perhaps we could all learn a lot more from their noble, trusting and forgiving nature. Finally my family, especially my parents, who never doubted my ability to succeed. My whole PhD experience has been one of complete enjoyment. I have always been highly passionate about my research and over the duration of my studies it has developed, changed direction and reshaped itself into a fascinating project which I am enormously proud to present. iii List of Content Chapter 1: Introduction 1 1 Background 1 1.1 Stages of decomposition 4 1.2 The life cycle of the blowfly 5 1.3 Factors influencing PMI 6 1.4 New developments in forensic entomology 10 1.5 Cuticular Hydrocarbons (CHC) 11 1.5.1 Chemical analysis of cuticular hydrocarbons 13 1.5.2 Cuticular hydrocarbons in (forensic) entomology 13 1.6 Rationale of Research 15 References 17 Chapter 2: Techniques and Data Analysis 23 2 Introduction 23 2.1 Chromatography 24 2.2 Gas Chromatography (GC) 28 2.2.1 Instrumentation 28 2.2.1.1 Split/Splitless injector 29 2.2.1.2 Column 31 2.2.1.3 Column temperature 33 2.2.2 Chromatogram 34 2.3 Mass Spectrometry (MS) 36 2.3.1 Instrumentation 36 2.3.1.1 Ion Source 37 2.3.1.2 Mass Analyser 39 2.3.1.3 Ion Detector 40 2.4 Gas Chromatography - Mass Spectrometry (GC-MS) 41 GC-MS data analysis and identification of cuticular 2.5 44 hydrocarbons 2.6 New MS developments 48 2.7 Multivariate analysis: Introduction 49 2.7.1 Principal Component Analysis (PCA) 49 References 52 Chapter 3: Methods and Materials 55 3 Fly rearing: 55 L. sericata 55 C. vicina 56 C. vomitoria 56 3.1 Larvae rearing 57 3.2 Sample Preparation 57 iv 3.2.1 Hydrocarbon extraction 58 Empty egg cases 58 Larvae 59 Puparia 61 Puparial cases 61 Adult fly 62 3.3 Column Chromatography 62 Chemical Analysis: Gas Chromatography - Mass 3.4 63 Spectrometry 3.5 Methylthiolation 64 3.6 Solvents 66 3.7 Principal Component Analysis 67 References 69 Chapter 4 Species identification using Cuticular Hydrocarbons 70 4 Introduction 70 4.1 Aims and Objectives 71 4.2 Egg cases 72 4.2.1 Results and Discussion for egg cases 74 4.3 Larvae 85 4.3.1 Results and Discussion for larvae 87 4.4 Puparia 97 4.4.1 Results and Discussion for Puparia 98 4.5 Puparial cases 106 4.5.1 Results and Discussion for puparial cases 109 4.5.2 Blind Puparial cases Results and Discussion 116 4.6 Adult Flies 120 4.6.1 Results and Discussion for adult flies 122 4.7 Overall Conclusion 131 References 133 Age Determination of Immature Life Stages Using Cuticular Chapter 5: 137 Hydrocarbons 5 Introduction 137 5.1 Aims and Objectives 140 5.2 Egg cases 140 5.2.1 Results and Discussion for egg cases 141 5.2.2 General Discussion for ageing egg cases 152 5.3 Larvae 153 5.3.1 Results and Discussion for larvae 155 5.3.2 General Discussion for ageing larvae 205 References 208 v Age Determination of the Later Life Stages Using Cuticular Chapter 6: 212 Hydrocarbons 6 Introduction 212 6.1 Aims and Objectives 213 6.2 Puparia 214 6.2.1 Results and Discussion for puparia 215 6.2.2 General Discussion for ageing puparia 224 6.3 Puparial Cases 225 6.3.1 Results and Discussion for puparial cases 226 6.3.2 General Discussion and Conclusion for ageing puparial cases 242 6.4 Adult Flies 244 6.4.1 Results and Discussion for adult flies 245 6.4.2 General Discussion and Conclusion for ageing adult flies 277 Overall Conclusion for ageing all six life stages of three 6.5 278 forensically important blowflies using CHC’s References 280 Future Developments: Direct Analysis in Real Time and Chapter 7: 282 Artificial Neural Networks 7 Introduction 282 7.1 Aims and Objectives 285 7.2 Direct Analysis in Real Time 286 7.2.1 Instrumentation 286 7.2.1.1 DART Ion Source 286 7.2.1.2 Mass Analyser 289 7.2.2 Materials and Method for DART 290 7.3 DART Results 292 7.3.1 DART Discussion and Conclusion 298 7.4 ANN 299 7.4.1 Materials and Methods for ANN 300 7.4.2 ANN Results 302 7.4.3 ANN Discussion and Conclusion 304 References 306 Chapter 8: Summary, Conclusion and Future Work 309 Identification 310 Ageing 312 Conclusion 315 Future Work 317 References 319 Appendices 320 vi List of Figures Figure 1.1 The life cycle of the blowfly 6 Summary of the relationships of insect development (PMImin), time since colonization (TOC), period of insect Figure 1.2 activity (PIA), ecological succession and maximum PMI 8 (PMImax). The shaded regions represent windows of prediction associated with estimates of the time since death Figure 1.3 Isomorphen diagram of the L. sericata 9 Figure 1.4 Linear long chain cuticular hydrocarbons 12 Figure 2.1 Diagram showing the five mechanisms of chromatography 25 Figure 2.2 A schematic diagram of a Gas Chromatograph 29 Figure 2.3 Diagram of a split/splitless injector 30 Figure 2.4 A schematic diagram of a packed column 31 Figure 2.5 Example chromatogram of an alkane standard solution 35 Figure 2.6 Diagram showing the components that make up a MS 37 Figure 2.7 Diagram illustrating the process of electron ionisation 38 Figure 2.8 A schematic diagram of a quadrupole analyser 40 Figure 2.9 GC chromatogram of two alkane standards 42 Figure 2.10 Mass spectrum of heneicosane 43 Figure 2.11 Mass spectrum of docosane 43 Figure 2.12 Mass spectrum of an 11+13-Methylpentacosane 46 Figure 2.13 Mass spectrum of 2-Methyltetracosane 47 Figure 2.14 Mass spectrum of 4-Methyltriacontane 48 Figure 3.1 Column chromatography 63 Figure 3.2 Basic reaction scheme of 7-heptacosene with DMDS.
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