Bugs and Drugs: Ketamine Detection from Necrophagous Insects using Gas Chromatography-Mass Spectrometry by Caitlin M. Cranston, B.A. A Thesis In Forensic Science Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Approved Todd A. Anderson, Ph.D. Chair of Committee Paola A. Tiedemann, Ph.D. Steven M. Presley, Ph.D. Mark Sheridan Dean of the Graduate School December, 2020 Copyright 2020, Caitlin M. Cranston Texas Tech University, Caitlin M. Cranston, December 2020 ACKNOWLEDGMENTS First, I would like to thank the members of my committee, Dr. Anderson, Dr. Tiedemann, and Dr. Presley, who have encouraged me through this whole process despite the multiple setbacks due to the pandemic and adaptations that had to be made for me to successfully complete this project. I am extremely grateful for Dr. Anderson for accepting the role as my thesis advisor and immediately being on board with my strange, yet interesting research topic. I am also thankful for Dr. Tiedemann for providing all the forensic knowledge that helped me with deciding on a research topic, and ultimately helping me realize my true potential within forensic science. I would like to thank Dr. Presley for teaching a seminar class on forensic entomology and his vast knowledge of entomology which helped me immensely with my research. Second, I would like to thank the members of my lab group for always being a massive ball of sunshine whenever we have lab meetings or get togethers. I will be forever grateful for the support I’ve received from this group. I would like to thank Seenivasan Subbiah for helping me understand the GC-MS, giving me advice when things seemed to be going wrong, and always being there to answer any and all questions I had. I would also like to thank Nicole Dennis; we may not have worked together often, but the friendship we developed over the short time we’ve known each other I will cherish; us introverts have to stick together. Lastly, but certainly not least, I would like to thank my friends and family for sticking by me throughout my time in graduate school. If it wasn’t for their support, I definitely would not have made it to this point. Thanks to Yaireth Castro, Maureen Oliva, Elizabeth Lewis, and Deanna Beatty for always being there to cheer me up when I’m at ii Texas Tech University, Caitlin M. Cranston, December 2020 my lowest point and for the constant encouragement. I would like to thank my mom and dad for helping me get to this point in my life; they have shaped me who I am today and I am incredibly grateful for the constant love, support, and guidance they give me no matter what situation I’m dealing with. I need to also thank my two cats, P and Moose, for never leaving my side and giving me all the love, especially when I’m working. To all my pets back home, I’ll be back soon to play and cuddle. I hope everyone I mentioned knows that I will be forever grateful to have you in my life, I could not have achieved this goal without y’all! iii Texas Tech University, Caitlin M. Cranston, December 2020 TABLE OF CONTENTS ACKNOWLEDGMENTS ............................................................................................... ii LIST OF TABLES ..........................................................................................................vii LIST OF FIGURES ........................................................................................................ .ix CHAPTER I ...................................................................................................................... 1 INTRODUCTION ............................................................................................................. 1 1.1 Research Objectives/Goals ....................................................................................... 1 1.2 History of Entomology ............................................................................................. 3 1.2.1 Forensic Entomology ......................................................................................... 4 1.3 Entomotoxicology ..................................................................................................... 6 1.4 Drug Research in Forensic Science .......................................................................... 9 1.4.1 Entomotoxicology Drug Research and Case Studies ....................................... 10 1.5 Typical Toxicology Methods .................................................................................. 14 1.6 Gas Chromatography – Mass Spectrometry ........................................................... 15 1.7 Ketamine ................................................................................................................. 18 1.7.1 Illicit Use .......................................................................................................... 18 1.7.2 Pharmacokinetics and Toxicity ........................................................................ 19 1.7.3 Entomotoxicology Research ............................................................................ 21 1.8 Thesis Study Objective ........................................................................................... 22 CHAPTER II ................................................................................................................... 24 METHODOLOGY AND DATA ANALYSIS .............................................................. 24 2.1 Materials ................................................................................................................. 24 2.1.1 Insects .............................................................................................................. 24 2.1.2 Insect Rearing Materials .................................................................................. 24 2.1.3 Human analogue .............................................................................................. 28 2.1.4 Instrumentation ................................................................................................ 28 2.1.5 Chemicals ......................................................................................................... 29 2.2 Methods .................................................................................................................. 30 2.2.1 Experimental Procedure ................................................................................... 30 2.2.2 Preparation of Ketamine Stock and Working Solutions .................................. 35 2.2.3 Calibration Curve ............................................................................................. 35 2.2.4 Extraction of Ketamine .................................................................................... 37 2.2.5 Gas Chromatograph-Mass Spectrometer Parameters ...................................... 39 iv Texas Tech University, Caitlin M. Cranston, December 2020 2.2.6 Data Analysis ................................................................................................... 40 CHAPTER III ................................................................................................................. 42 RESULTS AND DISCUSSION ..................................................................................... 42 3.1 Preliminary Tests on Ketamine ............................................................................... 42 3.2 Preliminary Extraction Tests .................................................................................. 42 3.3 Calibration Curve .................................................................................................... 45 3.3.1 GC-MS Method Optimization ......................................................................... 45 3.4 LOD and LOQ ........................................................................................................ 48 3.5 Results from Extraction Procedure ......................................................................... 49 CHAPTER IV .................................................................................................................. 56 CONCLUSION ............................................................................................................... 56 REFERENCES ................................................................................................................ 60 v Texas Tech University, Caitlin M. Cranston, December 2020 ABSTRACT In the 1980s, research into a subdiscipline within entomology began to emerge. This was later termed entomotoxicology. Since the initial publication in 1980 by Beyer et al., approximately 60 articles have been published relating to entomotoxicology. Using insects as an alternative means for toxicology analysis can help with cause of death determination when viable tissues are not available. With the increase in drug use in the United States, and globally, there has also been an increase in drug related deaths. These victims are usually not found in a timely fashion which can eventually lead the body being in an advanced stage of decomposition. Having an alternative tissue source for toxicology analysis will be beneficial for forensic and criminal investigation purposes. This present study determined if the drug ketamine could be extracted from larvae that had been exposed to it through decomposition tissues containing said drug. This experiment was accomplished by rearing Sarcophaga bullata flies which would subsequently produce larvae used for analysis. S. bullata flies