Drug Detection Using Eosin Y and Cobalt Thiocyanate Paper Assays

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.

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

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 N O

NCS Co SCN

O N O

Figure 1: Cocaine binding to cobalt thiocyanate in a 2:1 ratio (modified from reference

11)

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)

Figure 2: Potential mechanisms for fentanyl binding to Eosin Y, (modified from fluvoxamine binding to Eosin Y from reference 13)

Many drugs are “cut” with other substances termed adulterants and diluents for various reasons. Adulterants refer to a substance that is presumedly pharmacologically active and a diluent refers to a substance that is presumedly pharmacologically inactive. Adulterants are usually used for cutting to potentiate the effects of illicit drugs. Diluents are usually used for cutting to increase profits for sellers. By cutting drugs with diluents it leads to more available product and will lead to more drug sold for a dealer. Commonly, heroin and cocaine are cut with various adulterants and diluents. Common adulterants for cocaine include diphenhydramine, caffeine, levamisole, and benzocaine while lactose and mannitol have been seen as diluents.

Diphenhydramine is a common adulterant used in cutting heroin while lactose and mannitol have been used as diluents in cutting heroin. (8) These cutting agents can produce color changes similar to those seen by controlled substances when tested using presumptive color tests. Some cutting agents such as caffeine, quinine, and diphenhydramine have shown to give false-positive color tests for other substances. (5,7) In addition, heroin has been seen cut with fentanyl. Cutting heroin with fentanyl can increase the potency as well as increase the amount of drug available to sell. When fentanyl is mixed in with heroin, a smaller amount is required to produce the same high, thus increasing profits and amount of substance available to sell. (9)

When using color tests in the field, the general strategy is to use field testing kits which contain the chemicals necessary in pre-determined amounts. It is more beneficial to use multiple color tests to narrow down which controlled substance is present. As multiple drugs will react or not react with different color tests. (7) However, it has been shown that these chemical tests can also be spotted or printed on paper and used in a different fashion. The traditional tests use strong acids such as sulfuric acid (Marquis) which can present a danger to the tester. Printing paper test 7 strips can be used as a safer alternative as the chemicals have already dried by the time they are used. (6,10)

There is, therefore, a critical need to develop a safe and rapid way of detecting fentanyl.

Given that the prevalence of fentanyl is on the rise and due to its harmful exposure effects, it is important that fentanyl can be identified in a field setting safely and quickly. Using paper assay strips instead of the chemicals normally used in presumptive color tests will be a safer alternative and combining different color tests has the potential to uniquely identify fentanyl from all other controlled substances and cutting agents.

SPECIFIC AIMS Different drugs are able to interact with different chemical color tests. It has been shown that fentanyl will react with cobalt thiocyanate, Marquis’ test, and the Eosin Y test. It has also been shown that cutting agents and non-illicit drugs will also interact and give false positives for illicit substances due to the non-specificity of these color tests. (5-7)

Specifically, we aim to:

Aim I: Determine the compounds that react with Eosin Y paper assays

Eosin Y has been presented as a potential new color test for identifying drugs of abuse.

(6) However, very few drugs have been tested with this compound. We aim to identify other drugs and classes of drugs that are able to interact with Eosin Y in a paper assay form and produce a visible color change.

Aim II: Determine if cobalt thiocyanate used in conjunction with Eosin Y can specifically and uniquely identify fentanyl

Because fentanyl has been shown to react with cobalt thiocyanate and Eosin Y, we aim to see if these two tests can be used in conjunction to uniquely and specifically identify fentanyl.

Other drugs have been shown to react and produce a visible color change with cobalt thiocyanate and Eosin Y, but so far fentanyl has been the only one shown to interact with both. We hypothesize that fentanyl may be a unique compound that reacts with both tests producing a color change.

Aim III: Determine the effects cutting agents have on fentanyl reacting with the cobalt thiocyanate and Eosin Y test

Fentanyl can be used as a cutting agent in other drugs but can also be cut with other adulterants and diluents. (9) We aim to see if when fentanyl is mixed with other drugs it can 9 create a false negative for presumptive color tests. In this situation, even though fentanyl would be present the color test would not indicate the presence of fentanyl.

Preliminary results from testing already performed using Eosin Y and cobalt thiocyanate in a paper assay form The following pictures show preliminary testing that has been done with Eosin Y and cobalt thiocyanate transferred to a paper assay form.

A. B. C. Figure 3: Preliminary results. (A) Comparison of control (left) and cocaine (right) reaction with cobalt thiocyanate in paper assay form; (B) Comparison of control (left) and fentanyl (right) reaction with Eosin Y test in paper assay form; (C) Comparison of control (left) and fentanyl (right) reaction with cobalt thiocyanate in paper assay form.

EXPERIMENTAL DESIGN Aim I: Determine the compounds that react with Eosin Y paper assays

To determine the compounds that reacted with Eosin Y, Eosin Y paper assay strips were used. These were nitrocellulose strips that had Eosin Y at a pH of 5 and a pH of 7 placed onto them in separate columns. These strips were made by Vuronyx Technologies. The following compounds were tested in a 2-3 mg amount to determine if there was a visible color change: oxycodone, methadone, heroin, morphine, fentanyl, benzylfentanyl, furanylfentanyl, o- fluorofentanyl, methamphetamine, methylone, cocaine, diazepam, boric acid, lactose, mannitol, levamisole, quinine, lidocaine, diphenhydramine, caffeine, and dopamine. The testing consisted of each compound being deposited on the strip and the strip immersed in water for 2 minutes. A control was also used where no sample was deposited and the strip was immersed in water for 2 minutes. Each strip was then allowed to dry for 3 minutes before a photograph was taken for comparison.

Aim II: Determine if cobalt thiocyanate used in conjunction with Eosin Y can specifically and uniquely identify fentanyl

To determine if fentanyl is a unique compound that reacts with Eosin Y and cobalt thiocyanate, Eosin Y and cobalt thiocyanate paper assay strips were used. The Eosin Y results were the results found from the testing done in Aim I. The cobalt thiocyanate strips contained cobalt thiocyanate with tosic acid at a pH of 1 in one column and Tris buffer at a pH of 8 in the other column. These strips were made by Vuronyx Technologies. The above-mentioned compounds (in Aim I) were tested with the cobalt thiocyanate strips to determine if the compounds react with cobalt thiocyanate. The color changes were compared to those from the

Eosin Y test to determine if fentanyl is the only compound that reacted with both. The testing 11 consisted of each compound being deposited on the strip and the strip immersed in water for 2 minutes. A control was also used where no sample was deposited and the strip was immersed in water for 2 minutes. Each strip was then allowed to dry for 3 minutes before a photograph was taken for comparison.

Aim III: Determine the effects cutting agents have on fentanyl reacting with the cobalt thiocyanate and Eosin Y test

To determine if cutting agents affect fentanyl reacting with both Eosin Y and cobalt thiocyanate, fentanyl was mixed with each compound tested in Aim I. Fentanyl was mixed with the following compounds: oxycodone, methadone, morphine, methamphetamine, cocaine, diazepam, boric acid, lactose, mannitol, levamisole, quinine, lidocaine, diphenhydramine, and caffeine. Fentanyl was deposited in the mixture at a concentration of 1%, 5%, and 10% with each compound listed above comprising the remaining 99%, 95%, and 90% of the mixture respectively. Approximately 1-2 mg of each mixture was tested with both Eosin Y and cobalt thiocyanate in paper assay form. The testing consisted of each mixture being deposited on the strip and the strip immersed in water for 2 minutes. Each strip was then allowed to dry for 3 minutes before a photograph was taken for comparison.

Chemicals used and suppliers

The following compounds were acquired from Sigma-Aldrich (St. Louis, MO): boric acid, caffeine, cocaine (hydrochloride salt), diazepam, diphenhydramine (hydrochloride salt), dopamine (hydrochloride salt), lactose, levamisole (hydrochloride salt), lidocaine (hydrochloride salt), mannitol, quinine (sulfate salt), and methadone (±hydrochloride salt). The following compounds were acquired from Cayman Chemical (Ann Arbor, MI): morphine (sulfate hydrate salt), fentanyl (hydrochloride salt), benzylfentanyl( hydrochloride salt), o-fluorofentanyl 12

(hydrochloride salt), furanylfentanyl (hydrochloride salt), heroin (hydrochloride salt), methamphetamine (± hydrochloride salt), and methylone (hydrochloride salt).

RESULTS Eosin Y individual compound results

The results for the Eosin Y testing of the individual compounds are listed in Table 1 and displayed in Figure 4. The control had an orange color. Fentanyl, benzyl fentanyl, furanyl fentanyl, o-fluorofentanyl, diphenhydramine, quinine and methadone all gave a positive response turning the Eosin Y strip pink. Lactose, mannitol, caffeine, oxycodone, boric acid, cocaine, dopamine, methylone and diazepam were all negative with a color similar to that of the control.

Lidocaine, levamisole, morphine, and methamphetamine all had color changes but were not the same pink color seen with the positive responders. The colors exhibited by these compounds were brighter shades of orange or faded shades of pink which were ruled a negative response.

Cobalt thiocyanate individual compound results

The results for the cobalt thiocyanate testing of the individual compounds are listed in

Table 1 and displayed in Figure 5. The control had a red color. Fentanyl, benzyl fentanyl, lidocaine, diphenhydramine, levamisole, oxycodone, quinine, methadone, morphine, methamphetamine, cocaine, and methylone all gave a positive response turning the cobalt thiocyanate strip blue. Furanyl fentanyl, o-fluorofentanyl, lactose, mannitol, boric acid, dopamine, and diazepam were all negative with a color similar to that of the control.

Eosin Y mixture results

The results for the Eosin Y testing containing mixtures of compounds are listed in Table

1 as well as shown in Figure 4. Only the 1% mixture results are shown in Figure 4. When 1% fentanyl was mixed with the following compounds: oxycodone, methadone, morphine, methamphetamine, cocaine, diazepam, boric acid, lactose, mannitol, levamisole, quinine, lidocaine, diphenhydramine, and caffeine, there was a positive response indicated by a pink color change with the Eosin Y strip when compared with the control and the cutting agent compound. 14

All 5% and 10% fentanyl mixture results that were tested also gave positive responses for Eosin

Y when compared to the control test strip and the cutting agent when tested individually.

Cobalt thiocyanate mixture results

The results for the cobalt thiocyanate testing of the fentanyl mixtures are listed in Table 1 as well as shown in Figure 5. There was a positive color change when compared with the control and the cutting agent tested individually for the following 1% fentanyl mixtures: lidocaine, diphenhydramine, levamisole, oxycodone, methadone, morphine, methamphetamine, and cocaine. There was no observed color change when 1% fentanyl was mixed with lactose, mannitol, caffeine, quinine, boric acid, and diazepam. The results for the 5% and 10% fentanyl mixtures can be found in Table 1.

Table 1. Results of Eosin Y and cobalt thiocyanate testing for each compound and mixture along with type of amine in compound. + indicates a positive pink color change, - indicates no pink color change. NT indicates mixture was not tested. N/A indicates compound does not have an amine functional group.

Amine Cobalt Compound Eosin Y Type Thiocyanate Boric Acid N/A - - Lactose N/A - - Mannitol N/A - - Dopamine 1° - - Methamphetamine 2° - + Methylone 2° - + Lidocaine 2°/3° - + Diphenhydramine 3° + + Caffeine 3° - - Quinine 3° + + Levamisole 3° - + Diazepam 3° - - Methadone 3° + + Oxycodone 3° - + Cocaine HCl 3° - + Morphine 3° - + Fentanyl 3° + + Benzylfentanyl 3° + + O-fluorofentanyl 3° + - Furanylfentanyl 3° + - 1% Fentanyl/99% Lactose 3°/N/A + - 5% Fentanyl/95% Lactose 3°/N/A + + 10% Fentanyl/90% Lactose 3°/N/A + + 1% Fentanyl/99% Mannitol 3°/N/A + - 5% Fentanyl/95% Mannitol 3°/N/A + - 10% Fentanyl/90% Mannitol 3°/N/A + + 1% Fentanyl/99% Caffeine 3°/3° + - 5% Fentanyl/95% Caffeine 3°/3° + - 10% Fentanyl/90% Caffeine 3°/3° + + 1% Fentanyl/99% Lidocaine 3°/2°/3° + + 16

5% Fentanyl/95% Lidocaine 3°/2°/3° + + 10% Fentanyl/90% Lidocaine 3°/2°/3° + + 1% Fentanyl/99% Diphenhydramine 3°/3° + + 5% Fentanyl/95% Diphenhydramine 3°/3° + + 10% Fentanyl/90% 3°/3° + + Diphenhydramine 1% Fentanyl/99% Boric Acid 3°/N/A + - 5% Fentanyl/95% Boric Acid 3°/N/A + - 10% Fentanyl/90% Boric Acid 3°/N/A + - 1% Fentanyl/99% Levamisole 3°/3° + + 5% Fentanyl/95% Levamisole 3°/3° + + 10% Fentanyl/90% Levamisole 3°/3° + + 1% Fentanyl/99% Quinine 3°/3° + - 5% Fentanyl/95% Quinine 3°/3° + + 10% Fentanyl/90% Quinine 3°/3° + + 1% Fentanyl/99% Oxycodone 3°/3° + + 5% Fentanyl/95% Oxycodone 3°/3° + + 10% Fentanyl/90% Oxycodone 3°/3° NT NT 1% Fentanyl/99% Methadone 3°/3° + + 5% Fentanyl/95% Methadone 3°/3° + + 10% Fentanyl/90% Methadone 3°/3° NT NT 1% Fentanyl/99% Morphine 3°/3° + + 5% Fentanyl/95% Morphine 3°/3° + + 10% Fentanyl/90% Morphine 3°/3° NT NT 1% Fentanyl/99% 3°/2° + + Methamphetamine 5% Fentanyl/95% 3°/2° + + Methamphetamine 10% Fentanyl/90% 3°/2° NT NT Methamphetamine 1% Fentanyl/99% Cocaine 3°/3° + + 5% Fentanyl/95% Cocaine 3°/3° + + 10% Fentanyl/90% Cocaine 3°/3° NT NT 1% Fentanyl/99% Diazepam 3°/3° + - 5% Fentanyl/95% Diazepam 3°/3° + - 10% Fentanyl/90% Diazepam 3°/3° NT NT

Figure 4: Eosin Y results. Abbreviations correspond in the following way: Con=control,

Fen=fentanyl, Ben=benzylfentanyl, Fur=furanylfentanyl, Off=o-fluorofentanyl, Lac=lactose,

Lev=levamisole, Mor=morphine, Man=mannitol, Oxy=oxycodone, Ma=methamphetamine,

Caf=caffeine, Qui=quinine, Coc=cocaine, Lid=lidocaine, Mdn=methadone, Dia=diazepam,

Dip=diphenhydramine, Ba=boric acid, Dop=dopamine, Myn=methylone

Figure 5: Cobalt thiocyanate results. Abbreviations correspond in the following way:

Con=control, Fen=fentanyl, Ben=benzylfentanyl, Fur=furanylfentanyl, Off=o-fluorofentanyl,

Lac=lactose, Lev=levamisole, Mor=morphine, Man=mannitol, Oxy=oxycodone,

Ma=methamphetamine, Caf=caffeine, Qui=quinine, Coc=cocaine, Lid=lidocaine,

Mdn=methadone, Dia=diazepam, Dip=diphenhydramine, Ba=boric acid, Dop=dopamine,

Myn=methylone

DISCUSSION

In the present study, we found that certain compounds elicit the characteristic pink color change when exposed to Eosin Y that indicates a positive response when compared to the control. To date, only two compounds (hydromorphone and fentanyl) have been demonstrated to react with Eosin Y with a paper assay (6). Here, we found that the following compounds also react with Eosin Y: fentanyl, benzylfentanyl, furanylfentanyl, o-fluorofentanyl, methadone, diphenhydramine and quinine. Structurally, these compounds all contain a tertiary amine. Not all tertiary amine containing compounds tested displayed a positive Eosin Y interaction; namely morphine, oxycodone, cocaine, diazepam, levamisole, lidocaine, and caffeine. The lack of an

Eosin Y interaction with these compounds may be the result of steric hindrance based on the nitrogen being placed within the phenanthrene ring or the close to a phenyl substitution (14).

Exposure of the Eosin Y paper to morphine, lidocaine, methamphetamine and levamisole did yield a color change but not of the same pink intensity as the other positive responders. The color change observed with morphine, lidocaine, methamphetamine and levamisole was more characteristic of orange mixed with pink. While color changes can be subjective, there is a difference in the color that was observed by the positive responders (fentanyl, benzylfentanyl, furanylfentanyl, o-fluorofentanyl, methadone, diphenhydramine and quinine) and the other compounds that were tested. Therefore, morphine, lidocaine, methamphetamine and levamisole were detemed to be negative responders. The other compounds that did not react with Eosin Y either contained no amine group or a primary or secondary amine group.

The testing of 1% fentanyl mixed with 99% of various compounds showed that the Eosin

Y paper assay can detect 1% fentanyl. The cobalt thiocyanate paper assay was not consistent in the same manner that the Eosin Y paper assay was in this regard. While the cobalt thiocyanate 20 assay detected 1% fentanyl in some of the mixtures, Eosin Y detected 1% fentanyl in all mixtures. Given that methadone, diphenhydramine and quinine gave a false positive in the Eosin

Y paper assay there is difficulty in determining if the color change is due to the presence of fentanyl or from the adulterant drug. However, all the other drugs that tested negative or did not display a positive pink color change when tested without the presence of fentanyl then displayed the positive pink color change once 1% fentanyl was added to the drug. This is an indication that

Eosin Y can detect fentanyl in low amounts.

The sensitivity of detecting 1% fentanyl is important as only a couple milligrams or less can cause an overdose and a couple milligrams or less would most likely be the amount found in seized drug samples (15). Therefore, a test should be sensitive enough to detect low levels of fentanyl. Based on the results, Eosin Y is capable of detecting fentanyl at 1% levels while cobalt thiocyanate is not, thus making it a better choice for a color test to screen for fentanyl in compounds. While fentanyl is not the only compound that will give a color change, this test can still assist in narrowing down a drug sample during testing and provide an idea of what could possibly be in a drug sample. Eosin Y does have some false positives such as diphenhydramine and quinine. This is typical of color change tests as they are usually not specific due to the color change resulting from reaction with certain functional groups which can be present in many compounds. Although a positive Eosin Y test alone does not indicate the presence of fentanyl, this test can inform an analyst that a potentially harmful substance (fentanyl or an analogue) is present and to proceed with caution. Eosin Y could be used in together with other color tests and instrumentation to identify a drug sample.

CONCLUSIONS

Based on the collective findings and results from this study, the following conclusions can be drawn about using Eosin Y in a paper assay form:

1. Eosin Y can detect fentanyl in amounts as low as 1% in mixtures of drugs, which makes

it a better screening test than cobalt thiocyanate for fentanyl.

2. The color change of Eosin Y is due to binding to select tertiary amines, but not all tertiary

amines are capable of binding to Eosin Y.

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