DETECTION OF ILLICIT DRUG USE IN BLOOD: A VALIDATION STUDY OF SOLID PHASE EXTRACTION COUPLED WITH LIQUID CHROMATOGRAPHY AND TANDEM MASS SPECTROMETRY Latisha Pipes A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of SELECT ONE: May 2020 Committee: Jon Sprague, Advisor Phillip Gibbs Travis Worst © 2020 Latisha Pipes All Rights Reserved iii ABSTRACT Jon Sprague, Advisor With the increase of driving while under the influence of drugs and the legalization of marijuana there is a need to develop and validate solid phase extraction coupled with liquid chromatography tandem mass spectrometry methods using blood. The protocol that was followed was in the Clinical Lab Consulting Method Validation manual. The parameters that were tested were linearity/reportable range, limit of detection, and precision. The linearity/reportable range showed that 80/81 of the analytes had an average R2 value of 0.950 or higher. The exception was cannabidiol with an average R2 value of 0.932. The limit of detection showed that a cutoff of 1 ng/mL was sufficient for 80/81 analytes. THC requires a cutoff of 2 ng/mL. The precision showed issues with negative mode analytes such as cannabidiol and barbiturates. iv Dedicated to my parents, Mr. Michael Pipes and Mrs. Mary Pipes who have offered their love and support every step of the way through this research. Also, to my dog, Freckles Pipes for his constant companionship through the long nights of writing. v ACKNOWLEDGMENTS I would like to thank the National Highway Traffic Safety Administration for funding this research. I would like to thank all of my committee members for their knowledge and advice that they have passed down to me and for their patience through all of the setbacks that were encountered. I would like to thank my advisor, Dr. Jon Sprague, for encouraging me to build confidence in myself and my abilities. Also, for taking the time to teach me throughout this research project based on where I started to where I am today. For always taking the time to come in on weekends when issues would arise. Lastly, for going above and beyond for his students. I would also like to thank Dr. Phillip Gibbs, for mentoring me along every step of this research process. The knowledge that I have gained from him will help me to begin a career and for that I am grateful. I would like to thank Dr. Travis Worst, for calming me down multiple times when I felt overwhelmed and stressed. Also, for being a great professor and academic advisor. I would like to thank a fellow Graduate student, Amal Aburahma, for taking time out of her busy schedule to help me prepare my samples. Her help was greatly appreciated. I would also like to thank my fellow research partner, Nathan Bunch, for his support. Lastly, I would like to thank my parents for their constant encouragement, love, and support. Also, for calming me down late at night over the phone when the stress levels were high. vi TABLE OF CONTENTS Page CHAPTER 1. INTRODUCTION & BACKGROUND ......................................................... 1 1.1 Drug Abuse and the Legal System....................................................................... 1 1.2 Pharmacology, Toxicology, Pharmacokinetics and Tolerance of a Drug ........... 2 1.3 Blood .................................................................................................................... 7 1.4 Solid Phase Extraction ......................................................................................... 10 1.5 Precipitating Agent .............................................................................................. 12 1.6 Liquid Chromatography ....................................................................................... 15 1.7 Mass Spectrometry............................................................................................... 20 1.8 Example of an Optimization ................................................................................ 23 1.9 Internal Standards ................................................................................................ 25 CHAPTER 2. RESEARCH FOCUS ..................................................................................... 31 CHAPTER 3. MATERIALS AND EXPERIMENTAL DESIGN ........................................ 32 3.1 Materials .............................................................................................................. 32 3.2 Experimental Design ............................................................................................ 33 CHAPTER 4. RESULTS ....................................................................................................... 41 4.1 Linearity/Reportable Range ................................................................................. 41 4.2 Limit of Detection ................................................................................................ 45 4.3 Precision ............................................................................................................... 51 CHAPTER 5. DISCUSSION ................................................................................................. 65 5.1 Linearity/Reportable Range ................................................................................. 65 5.2 Limit of Detection ................................................................................................ 66 vii 5.3 Precision ............................................................................................................... 69 5.4 Overall Analysis................................................................................................... 70 CHAPTER 6. CONCLUSION............................................................................................... 73 REFERENCES ...................................................................................................................... 75 APPENDIX A. STANDARD ANALYTES .......................................................................... 80 APPENDIX B. INTERNAL STANDARDS ......................................................................... 81 APPENDIX C. CALIBRATION CURVE STOCK SOLUTION PREP GUIDE ................. 82 APPENDIX D. HIGH QC STOCK SOLUTION PREP GUIDE .......................................... 84 APPENDIX E. LOW QC STOCK SOLUTION PREP GUIDE............................................ 86 APPENDIX F. INTERNAL STANDARD STOCK SOLUTION PREP GUIDE ................. 88 APPENDIX G. CALIBRATION CURVE STOCK AND QC STOCK PREP GUIDE ........ 89 APPENDIX H. ARTIFICIAL SERUM CALIBRATORS AND QC PREP GUIDE ............ 90 APPENDIX I. BATCH FILE EXAMPLE............................................................................ 91 APPENDIX J. STANDARD ANALYTES WITH THEIR INTERNAL STANDARD ASSOCIATIONS ................................................................................................................... 92 APPENDIX K. STANDARD ANALYTE METHOD PARAMETERS ............................... 93 APPENDIX L. INTERNAL STANDARD METHOD PARAMETERS .............................. 94 viii LIST OF TABLES Table Page 1 Linearity/Reportable Range Results .......................................................................... 42 2 Limit of Detection Results ......................................................................................... 47 3 HiQC Precision Results ............................................................................................. 53 4 HiQC Spread of the Precision Results with Caffeine ................................................ 55 5 HiQC Spread of the Precision Results without Caffeine ........................................... 55 6 LoQC Precision Results ............................................................................................. 57 7 LoQC Spread of the Precision Results with Caffeine ................................................ 59 8 LoQC Spread of the Precision Results without Caffeine........................................... 59 9 SST Precision Results ................................................................................................ 61 10 SST Spread of the Precision Results with Caffeine ................................................... 63 11 SST Spread of the Precision Results without Caffeine .............................................. 63 ix LIST OF ABBREVIATIONS Abbreviation Meaning ACN Acetonitrile ADME Absorption Distribution Metabolism Excretion ANOVA Analysis of Variance AS Artificial Serum Avg R2 Average R2 BCI Bureau of Criminal Investigation CB Cannabinoid CCS Calibration Curve Stock CCSS Calibration Curve Stock Solution CE Collision Energy CO Cutoff CV% Coefficient of Variance Percentage Da Daltons DL Desolvation Line DOF Degrees of Freedom ESI Electrospray Ionization GC/MS Gas Chromatography Mass Spectrometry HiQC High Quality Control HiQC_SS High Quality Control Stock Solution HPLC High Performance Liquid Chromatography IPA Isopropyl Alcohol ISTD_SS Internal Standard Stock Solution LC Liquid Chromatography LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry LLOQ Lower Limit of Quantitation LOD Limit of Detection LOQ Limit of Quantitation LoQC Low Quality Control LoQC_SS Low Quality Control Stock Solution MRM Multiple Reaction Monitoring MS/MS Tandem Mass Spectrometry NAC Nucleus Accumbens NegQC Negative Quality Control NHTSA National Highway Traffic Safety Administration OF Oral Fluid OSHP Ohio State Highway Patrol PEEK Polyether Ether Ketone PFC Prefrontal Cortex PSI Pounds