DRUG DETECTION USING EOSIN Y AND COBALT THIOCYANATE PAPER ASSAYS Jeremy Canfield A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE May 2020 Committee: Jon Sprague, Advisor Sandip Agarwal Travis Worst ii ABSTRACT Jon Sprague, Advisor Eosin Y is a new color test that has potential for use in detecting illicit drugs that has not been studied extensively. In this study, a variety of drugs of abuse and fentanyl analogues were tested to determine which drugs will bind to Eosin Y and which functional groups are capable of binding and eliciting a color change. These drugs were then combined with common cutting agents and other drugs of abuse in order to determine the fentanyl detection limit in a drug mixture using an Eosin Y test strip. Additionally, cobalt thiocyanate was used to determine if the combination of cobalt thiocyanate and Eosin Y has the potential to uniquely identify fentanyl. Through the testing performed, the following conclusions were made: 1) Eosin Y is capable of detecting low amounts of fentanyl down to 1%, 2) Eosin Y binds to select tertiary amines to produce an orange to pink color change. While the cobalt thiocyanate assay detected 1% fentanyl in some of the mixtures, Eosin Y detected 1% fentanyl in all mixtures. Eosin Y has the ability to test for fentanyl and fentanyl analogues and can detect fentanyl in low amounts when mixed with cutting agents. iii This thesis is dedicated to my Papa, who unfortunately passed before he was able to see this and to see me graduate but I know is so proud of me and lives in my heart every day. iv ACKNOWLEDGMENTS I would like to thank first and foremost my Lord and Savior Jesus Christ. I know I would not be where I am today and would not be the man I am today without Him. I would like to thank my thesis advisor, Dr. Jon Sprague, for his guidance and encouragement throughout this entire process. This would not have been possible without him and all of his help and support. I would like to thank the other members of my committee, Dr. Travis Worst and Dr. Sandip Agarwal for providing insight and expertise in this field of research. Also for Dr. Worst’s guidance through selecting classes and my whole graduate school career. I would like to thank Michele Nagel and Samuel Fortener for their help with ordering supplies and for providing testing and assistance from the Ohio Bureau of Criminal Investigation. I would like to thank my friends and family who have supported me throughout this entire journey. Those who have provided wisdom, and words of encouragement throughout this entire 2 year journey and those who have shaped me into the person I am today. Lastly, I would like to thank my beautiful girlfriend Kristin Stobinski for all of her love and support and help throughout this project. Thank you for all that you do and for all of your help and encouragement. I love you. v TABLE OF CONTENTS Page BACKGROUND..………………………………………………………………………..... 1 SPECIFIC AIMS ………….……………………………………………………………….. 8 Aim I: Determine the compounds that react with Eosin Y paper assays…………… 8 Aim II: Determine if cobalt thiocyanate used in conjunction with Eosin Y can specifically and uniquely identify fentanyl………………………………….… 8 Aim III: Determine the effects cutting agents have on fentanyl reacting with the cobalt thiocyanate and Eosin Y test………………………………………………... 8 Preliminary results from testing already performed using Eosin Y and cobalt thiocyanate in paper assay form………………………………………..…… 9 EXPERIMENTAL DESIGN……………………………………….………………………. 10 Aim I: Determine the compounds that react with Eosin Y paper assays…………… 10 Aim II: Determine if cobalt thiocyanate used in conjunction with Eosin Y can specifically and uniquely identify fentanyl…………………….……………… 10 Aim III: Determine the effects cutting agents have on fentanyl reacting with the cobalt thiocyanate and Eosin Y test……………………………………………. 11 Chemicals used and suppliers……………………………………………………… 11 RESULTS………………….……………………………………….………………………. 13 Eosin Y individual compound results………………………………….…………… 13 Cobalt thiocyanate individual compound results……………………………...…… 13 Eosin Y mixture results…………………………………………….………….…… 13 Cobalt thiocyanate mixture results……………………………………………….… 14 vi DISCUSSION………………………………………………………………………………. 19 CONCLUSIONS……………….…………………………………………………………… 21 REFERENCES……………………………………………………………………………… 22 vii LIST OF FIGURES Figure Page 1 Cocaine binding to cobalt thiocyanate in a 2:1 ratio ................................................. 3 2 Potential mechanisms for fentanyl binding to Eosin Y ............................................. 5 3 Preliminary results ..................................................................................................... 9 4 Eosin Y results ........................................................................................................... 17 5 Cobalt thiocyanate results .......................................................................................... 18 viii LIST OF TABLES Table Page 1 Results of Eosin Y and cobalt thiocyanate testing for each compound and mixture along with type of amine in compound ........................................................ 15 1 BACKGROUND Opioid abuse has increased in the last decade, resulting in increased overdoses and deaths due to opioids. (1) One of these opioids that has seen an increase is fentanyl which is a synthetic opioid that acts as an agonist at the µ opioid receptor. (2) Currently being manufactured around the world illicitly by many labs, fentanyl has seen a drastic increase in the amount of legal seizures. By 2015, the amount of fentanyl legal seizures were eight times higher than in 2006. In addition to being used pharmaceutically as a pain medication, fentanyl is also being added as an adulterant to other street drugs such as heroin and other opioids. (3) Because fentanyl and other drugs can harm based on exposure, many harm reduction agencies and law enforcement agencies are attempting to develop methods and procedures to presumptively and safely identify drugs without risk to the person collecting, confiscating, or testing these drugs. (4) There are many different methods and techniques to identify unknown substances. These include instrumentation methods such as mass spectrometry, Raman spectroscopy and infrared spectrometry. In addition, there are presumptive color tests and microcrystalline tests. Generally, instrumentation methods are more discriminatory, but are also more expensive and have limited field applications. Instrumentation methods also require more knowledge to perform the tests and for interpretation of results. Color tests are less discriminatory but can be useful to identify substances in a field setting and tend to be inexpensive and much more rapid than instrumentation methods. Color tests can also be interpreted easily in the field by persons who may not have a scientific background. Color tests are usually a first step in characterizing an unknown substance. Once an idea is formed about the identity of an unknown substance, then further instrumental testing is done to confirm the identify a substance. (4) 2 There are currently many different presumptive tests to identify drugs of abuse. These include Marquis test, cobalt thiocyanate test, Eosin Y test and Duquenois-Levine test among others. These tests rely on color changes that occur in the presence of certain controlled substances and functional groups present in chemical compounds. Many of these tests have been extensively tested and validated to determine which substances result in a color change. The cobalt thiocyanate test has been shown to react with tertiary protonated amines. When cobalt thiocyanate interacts with cocaine or fentanyl a blue color change occurs. Cobalt thiocyanate has also been demonstrated, through our preliminary testing, to give a false-positive blue color change for diphenhydramine, an anti-histamine that is not a controlled substance. (5,7) It has been postulated that cocaine binds in a 2:1 ratio with cobalt thiocyanate and forms the following structure to give a color change. (11) 3 O O O N O NCS Co SCN O N O O O Figure 1: Cocaine binding to cobalt thiocyanate in a 2:1 ratio (modified from reference 11) 4 Eosin Y test has been shown to react with hydromorphone, fentanyl, and cocaine resulting in a purple or pink change depending on the chemical structure of the interacting agent. (6) While Eosin Y has not been extensively studied in the chemistry and drug identification industry, it has been used in the past as a biological stain. Eosin is a negatively charged, acidic dye. Given these properties it is possible that Eosin Y is reacting with basic functional groups, such as the tertiary amine groups in fentanyl, hydromorphone, and cocaine to give the color change, however the actual mechanism is not known. (12) The structure of Eosin Y binding to another drug, fluvoxamine has been postulated. This binding is based on a primary amine binding with an oxygen group on Eosin Y. Given that fentanyl has two tertiary amines, the possibility exists that one of these two amine groups is binding to the same oxygen group of Eosin Y in the manners shown below. (13) 5 Figure 2: Potential mechanisms for fentanyl binding to Eosin Y, (modified from fluvoxamine binding to Eosin Y from reference 13) 6 Many drugs are “cut” with other substances termed adulterants and diluents for various reasons. Adulterants refer to a substance that is presumedly pharmacologically