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 are necrophagous insects that are important forensically as they are the first to appear on a decedent. 50 g portions of ground pork were treated with ketamine and norketamine, the metabolite of ketamine, and the larvae were allowed to feed for approximately three days. Once a sufficient number of larvae were produced and collected, QuEChERS extraction was performed on the larval samples, and analyzed using gas chromatography-mass spectrometry (GC-MS) which used a selected ion monitoring (SIM) method programmed to detect both ketamine and norketamine.

The findings of this present study conclude that ketamine could successfully be detected using GC-MS. Analysis of the larval samples proved that both ketamine and

vi Texas Tech University, Caitlin M. Cranston, December 2020

norketamine could be extracted using QuEChERS, analyzed using GC-MS and be successfully detected. The benefits of this study were to show a drug, not commonly researched, could be extracted and detected from larval samples. This study also showed that entomotoxicology is a useful technique for forensic toxicology purposes and can be utilized during criminal investigations

Keywords: Entomotoxicology, Forensic Entomology, Forensic Toxicology,

Ketamine, QuEChERS, GC-MS

vii Texas Tech University, Caitlin M. Cranston, December 2020

LIST OF TABLES Table 1. Items purchased for the purpose of S. bullata rearing ...... 26 Table 2. S. bullata larvae collected on August 9, 2020 ...... 32 Table 3. S. bullata larvae collected on August 29, 2020 ...... 33 Table 4. Final larvae sample groups for ketamine analysis ...... 34 Table 5. Third preparation of ketamine calibration standards for GC-MS analysis...... 36 Table 6. Final preparation of ketamine calibration standards for GC-MS analysis...... 37 Table 7. Temperature Ramp for GC analysis ...... 40 Table 8. Calculations made to determine LOD and LOQ ...... 48 Table 9. Percent Recovery for Ketamine ...... 49 Table 10. Percent Recovery for Norketamine ...... 49 Table 11. GC-MS detector response for ketamine and norketamine ...... 50 Table 12. Final concentration calculations of ketamine from larvae extraction samples 51 Table 13. Final concentration calculations of norketamine from larvae extraction samples ...... 51 Table 14. Percent recovery of ketamine and norketamine from larvae extraction samples ...... 51

viii Texas Tech University, Caitlin M. Cranston, December 2020

LIST OF FIGURES Figure 1. Artwork that depicts the earliest account of maggots on a corpse [10] ...... 6 Figure 2. Analytical techniques categorized based on SWGDRUG guidelines. Category A refers to confirmatory analyses, while Categories B and C are preliminary analyses [26]...... 15 Figure 3. General set up of a GC-MS [27]...... 17 Figure 4. Simple block diagram of a mass spectrometer (left). A representation of a known mass spectra of ketamine (right) [29,30]...... 17 Figure 5. Chemical structures of PCP and Ketamine [31]...... 18 Figure 6. Oxidative metabolism of ketamine [34]...... 20 Figure 7. Instrumentation used for the detection of ketamine from larval samples. Agilent 6890N (right), Agilent 7683B Series autoinjector (top), Agilent 5975 MS (left)...... 29 Figure 8. Final larvae sample groups for ketamine analysis ...... 35 Figure 9. Steps taken to extract ketamine from S. bullata larvae samples for GC-MS analysis ...... 38 Figure 10. Chromatogram (left) and SIM mass spectra (right) as a result from the QuEChERS test extraction using spiked S. bullata pupae...... 44 Figure 11. GC-MS response to the concentrations of ketamine and norketamine ...... 46 Figure 12. GC-MS response to the concentrations of ketamine and norketamine, adjusted to strengthen r2 values ...... 47 Figure 13. Final calibration curve that shows GC-MS response of the concentrations of ketamine and norketamine ...... 47 Figure 14. Chromatograms for larvae sample group 7 (left) and larvae sample group 6 (right) ...... 53 Figure 15. Representative chromatogram of the 500 ppb ketamine/norketamine mix calibration standard. First peak is norketamine and the second peak is ketamine...... 54 Figure 16. SIM mass spectra for ketamine from larvae sample group 1 (left) and norketamine from larvae sample group 4 (right)...... 55

ix Texas Tech University, Caitlin M. Cranston, December 2020

CHAPTER I

INTRODUCTION 1.1 Research Objectives/Goals Entomology can be defined as the description, classification, and evolution of insects; entomology can also involve the study of insect metabolism, behavior, and genetics [1]. For the last 30 years, forensic entomology has played a vital role in criminal investigations [2]. The main use of insects forensically includes calculating postmortem interval, or time of death, of a deceased victim. Other forensic purposes of insects include proper species identification, growth rate and developmental history, behavior, and geographic distribution [3]. Entomological physical evidence can be seen in the form of fragmented insects that are located on clothing and other personal effects of the deceased victim. These insects could be used to link the victim, suspect, and crime scene to one another [3]. While forensic entomology typically focuses on determining the time of death of a victim, insects have also been shown to be useful for toxicological analyses.

In the United States, and other countries alike, there has been a rapid increase in drug use, which has also led to an increase in drug-related deaths. According to the

National Institute on Drug Abuse (NIDA), in 2018 alone, there was a total 67,300

Americans that died from a drug overdose whether it had been from prescription or illicit drugs [4]. According to the Centers for Disease Control and Prevention (CDC), opioids have contributed to most of the drug overdose deaths that have been reported, with

46,802 Americans dying from drug overdoses involving opioids in 2018 [5]. Other reported deaths include approximately 14,000 Americans dying from cocaine overdose in

2017, and approximately 10,000 Americans dying from psychostimulant overdose in

2017 [5]. Individuals that have died from drug overdoses are usually found weeks, if not

1 Texas Tech University, Caitlin M. Cranston, December 2020

months, after their death. During this time, their body has likely gone through advanced stages of decomposition and reached skeletonization depending on the climate and area of the body’s location. Therefore, without viable tissues available, conducting a confirmatory toxicology analysis to determine if the individual died from a suspected drug overdose would be difficult. In this situation, insects can be collected for such analysis.

In the early 1980s, a paper published by Beyer brought to light the idea that maggots could be used as an alternate specimen choice to perform toxicological drug analysis [6]. When conducting a typical toxicology analysis, tissues and fluids, such as bile, vitreous humor, blood, urine, and the liver, are collected during an autopsy, if applicable. The issue arises when a decedent has been discovered in an advanced state of decomposition and fluids are absent. Beyer’s paper on entomology in toxicology opened a whole new sub-area of forensic entomology, later termed entomotoxicology. While this area of forensics is a relatively new concept, it has been researched heavily, but hasn’t been employed as another standard within toxicology analysis.

According to the World Health Organization (WHO), the typical method of extraction/pretreatment of a drug from a specimen is by liquid-liquid extraction [7], however for the present study, QuEChERS will be utilized as the extraction method.

While this particular extraction method has not been utilized in entomotoxicology, it’s been proven to be useful in extracting drug analytes from various specimens [8].

QuEChERS stands for Quick, Easy, Cheap, Effective, Rugged, and Safe. It is a simple and inexpensive way to extract analytes from a specimen, and it is also relatively quicker than other extraction methods. The use of QuEChERS has gained popularity and is

2 Texas Tech University, Caitlin M. Cranston, December 2020

beginning to replace extraction methods such as liquid-liquid extraction, solid phase extraction, and protein precipitation [8].

This present study focuses on detecting the drug ketamine and its metabolite norketamine from insect specimens in order to further research in the field of entomotoxicology. The drug of interest will be extracted from larvae and analyzed using gas chromatography-mass spectrometry (GC-MS). This research will develop an analytical method of preparing the sample for instrumental analysis to obtain qualitative and quantitative data. This study will also help the field of forensic science by utilizing drugs that are becoming increasingly popular illicitly and medically.

1.2 History of Entomology Insects have been on earth since the Devonian period, around 400 million years ago. They have withstood varying instances of exterminations which has allowed them to evolutionarily adapt to the insects we encounter today [9]. Of the species that have been discovered in the animal kingdom, more than 75% are insects. Insects play a pivotal role within the cultures and interactions with humans; however, it wasn’t until the 17th century that entomology was considered a category within zoology [9]. Anton Von

Leeuwenhoek, who was interested in studying the details of organisms, utilized the microscope to study the morphology and organs of insects [9]. His studies led to the establishment of insects as model subjects for scientific research. Before the scientific revolution, it was commonly believed that bacteria, insects, and other organisms formed from spontaneous generation. In 1668, Francesco Redi disproved the spontaneous generation hypothesis. He stated that insects rise from eggs laid by fertilized females [9].

Further insect studies were done by Jan Swammerdam, who documented the anatomical make up of insects. These discoveries lead to the investigations done by Charles Darwin

3 Texas Tech University, Caitlin M. Cranston, December 2020

and later Carl Linnaeus. Linnaeus documented use of insect wings as a simplistic way to classify insects and also developed the binomial system of naming species [9].

Entomology became firmly a part of European zoological sciences in the 19th century.

In the United States, advancements in entomology occurred around the 1800s.

Thomas Say is credited with the developments of entomology within the United States.

His books American Entomology outlined the insects he studied in America, which later provided the baseline for American entomologists [9].

1.2.1 Forensic Entomology The earliest documented use of forensic entomology dates back to 13th century China which is outlined in Sung Tźu’s The Washing Away of Wrongs [10]. The case described a man found in a rice field suspected to have been murdered by a sickle.

Since the sickle is a common tool used by rice field workers, locating the suspect would be nearly impossible [10, 11]. The individuals conducting the investigation decided it would be best to line up all the workers from the rice field in which the victim was found and have them place their sickles in front of them. At first glance, all of the sickles looked relatively clean, however, after some time passed, flies began to gather around one of the sickles [10, 11]. It was determined later that the flies were attracted to the traces of blood and human tissues not easily visible to the naked eye. This account led to the confession of the murder by one of the rice field workers. The theory that certain flies are attracted to blood was later reinforced in by Leclercq and Lambert, after finding

Calliphora vomitoria on a corpse 6 hours postmortem, laying their eggs in blood rather than open wounds [10]. The earliest documented account of maggots on corpses dates back to 15th and 16th century artwork of the Middle Ages (Figure 1).

4 Texas Tech University, Caitlin M. Cranston, December 2020

In the 18th and 19th centuries, France and Germany were experiencing mass exhumations of corpses. Doctors during that time saw that these bodies had many kinds of insects inhabiting them [10]. In 1831, physicians Orfila and Lesueur published their observations during their involvement with these exhumations [11]. This publication ultimately established the relationship between specific insects and decomposing bodies.

A few years later, Reinhard, a German doctor, used the information published by Orfila and Lesueur to develop a systematic approach to further study the relationship between insects and decomposing bodies [11]. Reinhard’s studies involved exhuming bodies and identifying the insects found on the bodies. This research by Reinhard solidified scientists' theories about certain insects inhabiting decomposing bodies. With this information, scientists were now interested in the succession of insects, and which insects arrived first to a decedent, their life cycles, and the relationship between insects and a crime [11].

In 1855, Bergeret d’Arbois, developed an insect succession model to determine postmortem interval of human remains, which is the model that is still used today [11].

The development of this model began at a residence in Paris, where a couple was remodeling their home, and uncovered the mummified remains of a child. Suspicions were placed on the couple, despite having just moved into the residence. Bergeret was in charge of the autopsy and observed several insect populations on the corpse. Using techniques that are typically employed today, (i.e., estimated insect lifecycles, the number of insects on the corpse, and successive colonization inferences), Bergeret concluded that the body was placed in the home around 1849 which was several years before the couple moved into the house [11].

5 Texas Tech University, Caitlin M. Cranston, December 2020

Following the work conducted by Bergeret, in 1894 veterinarian Jean Pierre

Megnin, researched the predictability of insect colonization in cadavers. He published a book which documented the eight waves of insect succession which is applied to criminal investigations. All of this research is what shaped modern forensic entomology that is frequently employed today in criminal investigations [11].

Figure 1. Artwork that depicts the earliest account of maggots on a corpse [10]

1.3 Entomotoxicology Within the field of entomology, there are three subdisciplines, urban, stored-products, and medico-legal. Entomotoxicology falls under the medico-legal category. Entomotoxicology is typically used to discover the presence or absence of toxins or drugs in various arthropods, usually flies and/or beetles [12]. Insects can be analyzed very easily after the samples are homogenized and an extraction process has been used [13]. These samples can then be analyzed using radioimmunoassay (RIA), gas chromatography (GC), thin layer chromatography (TLC), high performance liquid chromatography (HPLC), or gas chromatography-mass spectrometry (GC-MS) [13].

Entomotoxicology can also be used to determine postmortem interval, by examining how drugs or toxins affect the growth and development of insects through their life cycle.

6 Texas Tech University, Caitlin M. Cranston, December 2020

Entomotoxicology isn’t a discipline that is solely about the analysis of drugs or toxins; this discipline can also be used to analyze pesticides and other environmental contaminants [12]. While it is a useful discipline within forensic entomology, there has been a lack of research in entomotoxicology because of the difficulties in detecting the drug or toxin within the insect sample. For example, the detection of the drug of interest can only be done if the rate of absorption exceeds the rate of elimination. This is difficult to accomplish because some drugs will not bioaccumulate over the life cycle of the larvae

[12].

The first article that brought to light the concept of entomotoxicology was published by Beyer et al. in 1980 [6]. The authors discuss their experience of analyzing insect larvae that were collected from a corpse in an advanced stage of decomposition.

The presumptive cause of death was drug overdose [6]. Since there were no viable tissues available for toxicological analysis, the authors turned to insects that were present in various cavities of the decedent’s body. A bottle of medication was found next to the body that contained phenobarbital which was later determined to have belonged to the decedent. This was used as the known for comparison.

When the larvae were collected from the corpse, they were to be used by the medical examiner to determine the postmortem interval. However, since there was a lack of tissue left on the body for toxicology analysis, the authors concluded that the maggots would be useful in confirming the suspected phenobarbital overdose [6]. After running a series of tests using the larvae, phenobarbital was identified. A series of confirmatory tests, including gas chromatography coupled with mass spectrometry, yielded the same phenobarbital identification. It was concluded by consulting entomologists that the only

7 Texas Tech University, Caitlin M. Cranston, December 2020

way the drug could have been identified in the larvae, was by them feeding on tissues that contained the drug [6]. It was further stated that it would have been impossible for the larvae to have metabolically produced phenobarbital.

Furthering the idea that was established by Beyer et al. (1980), an article by

Derrick Pounder was published in 1991. According to the article, the increased interest in entomotoxicology practices stems from the absence of blood and suitable tissues for typical toxicological analysis on corpses that are in an advanced stage of decomposition

[14]. In most of these cases, insects and insect larvae are usually in abundance and sampling these insects and larvae is quite simple compared to sampling blood or tissues.

There are other advantages to using larvae for toxicology analysis, according to Pounder.

One advantage is that when performing an extraction method on the larvae, no emulsions are present, which is not always the case when extracting from human tissues [14].

Another advantage is that the chromatograms that are produced after running a toxicological analysis using larvae, will produce less endogenous peaks (noise) than using decomposed tissues [14]. This is due to the increase in chemicals being secreted within the body in the natural progression of decomposition. Pounder also found some disadvantages to using larvae for toxicological analysis. According to the article, researchers have found that it is not possible to correlate the drug concentration detected in the larvae with the blood concentration in the decedent [14]. This could pose difficulties when trying to corroborate cause of death for testimonial purposes.

Since the publication of the previous articles, there have been approximately 60 articles that have been published related to entomotoxicology [15]. Much like the rest of

8 Texas Tech University, Caitlin M. Cranston, December 2020

the disciplines in forensic science, entomotoxicology faces challenges in its validity as a source of forensic evidence which has not been strongly researched.

1.4 Drug Research in Forensic Science The oldest documented use of toxic substances dates back to 1500 BC in Egypt.

This was followed later by the Greeks as they studied toxins along with using them as a means of execution, which can be seen by the death of Socrates by hemlock [16].

Criminal use of poisoning became increasingly common as time went on, so much so that the Roman Empire passed the first law against poisoning in 82 BC [16]. However, this attempt at deterring the criminal use of poisoning, did not work out well and poisoning continued to increase. For females, poison was their weapon of choice as it did not require direct assault, and it was difficult to prove the use of poison in a murder trial as there was no scientific basis to identify a poison in the body at that time [16]. Around the end of the 18th and beginning of the 19th century, research into ways to identify poisons in the body began to emerge. Hermann Boerhaave was the first to discover a chemical technique to identify poisons by placing unknown substances on hot coals and distinguishing each substance by their odor [16].

Toxicology became an official science by the research conducted by Mathieu

Orfila. His book established the first attempt to classify poisons as corrosives, astringents, acids, narcotics, or putrefacient [16]. Orfila also performed experiments on animals, one of which showed that arsenic was distributed throughout the body after ingestion. Orfila was able to extract arsenic from organs other than the stomach. This experiment rejected previous theories that stated that poisons only act on specific body tissues [16]. This first toxicological break through was then followed up in the 1830s by James Marsh. Marsh created the ‘Marsh Test’ which was developed to detect minute amounts of arsenic.

9 Texas Tech University, Caitlin M. Cranston, December 2020

Since the development of these tests to detect arsenic, criminals began to use different means of poisoning that would be virtually undetectable. Examples include, vegetable alkaloids, morphine, strychnine, and atropine. However, these also became susceptible to detection by the 1850s [16]. During the 1850s, Jean Stas created a method that was able to extract nicotine from organs of a murder victim. This method, later coined the Stas method, became the primary extraction technique used for the analysis of nonvolatile organic substances. Both the Marsh Test and the Stas method are still used today by forensic toxicologists [16].

1.4.1 Entomotoxicology Drug Research and Case Studies Basing their study on the entomotoxicology research conducted by Beyer et al.

(1980), Gunatilake and Goff in 1989 focused on the detection of organophosphates from larvae that were feeding on decomposing remains. The remains were of a 58-year old individual that was found in the crawl space of their mother’s home. The body was in an advanced stage of decomposition. A bottle of malathion 50 was found located near the victim, which led to the assumption that cause of death was overdose. Tissue samples were collected and sent for toxicology analyses. Malathion was determined by gas chromatography coupled with nitrogen-phosphorous detection. Even though tissues were used for the toxicology analysis, the researchers were curious about whether the larvae found on the body would contain malathion as well. A large amount of Diptera larvae were observed by the medical examiner during the autopsy, samples of which were collected for PMI estimations. After a careful observation of the insects, third instar representative larvae and larvae located in the mouth cavity of the decedent were sent for analysis. Gas chromatography was utilized and detected 574 μg of malathion in 0.28 g of larvae (2050 μg/g). This particular study represented the first analysis of

10 Texas Tech University, Caitlin M. Cranston, December 2020

organophosphates which were detectable in larvae that were feeding on a decomposing body [17].

In 1990, a study conducted by Kintz et al., conducted toxicological analysis of larvae found on a severely putrefied cadaver. The authors reported conducting their research using the cadaver of a man who was found lying in his home. This man was known to be a chronic heroin abuser [18]. The entire corpse had been putrefied and covered with a mass of larvae, which were later identified to be belonging to the

Calliphoridae family. Some viable tissues were collected for confirmatory toxicology analysis, and a large sample of larvae were also collected for toxicology analysis. After performing an extraction method, a preliminary screening was conducted on the larval samples before liquid chromatography and gas chromatography were used. The initial screening detected barbiturates and opiates; both chromatography methods detected phenobarbital and morphine [18]. Overall, this study proved that toxicological analysis is still possible even if a body was found in a putrefied state, by using larval specimens found on the body.

Another study published in 1990, focusing on detecting opiates from blowfly larvae was conducted by Introna et al. The researchers wanted to know if using blowfly larvae was useful in fatal drug intoxication investigations. During this time in the research, blowflies were not being used for entomotoxicology purposes. In this study, the researchers reared larvae by depositing blowfly eggs on liver samples that were taken from victims that were positive for opiates. The larvae were grown until the third instar.

Control larvae were also reared from opiate-free liver samples. Radioimmunoassay was used for morphine analysis and a statistical analysis was performed on the data. Results

11 Texas Tech University, Caitlin M. Cranston, December 2020

indicated that opiate concentrations that were detected in the blowfly larvae correlated with the concentration that was found in the liver samples. This result allowed researchers to conclude that blowfly larvae are useful as a test material for toxicology analysis [19].

In 1991, an article by Nolte et al. used insect larvae to detect cocaine from a decomposed body. The case starts with the discovery of a corpse that had been nearly completely skeletonized in a wooded area. The corpse belonged to a 29-year-old male that had a history of drug use through intravenous means, that had been reported missing

September of the previous year. The body was found partially embedded in the ground and snow, and no paraphernalia was found in the area around the body. The head and chest were completely skeletonized, and the arms were nearly skeletonized. Both legs, however, were still mostly intact with decomposed tissue [20]. There were several maggots located on and around the body. Many were dead, some were partially decomposed, some partially intact, and some were quite large. Pupal cases were also found on and within the body cavity of the corpse. Both muscle and larvae were collected, and toxicological analysis was conducted on those samples for cocaine.

Radioimmunoassay (RIA) was used to screen the samples for cocaine, which produced a positive result for both the muscle tissue and larvae. This was later confirmed with gas chromatography and gas chromatography-mass spectrometry [20]. Although, cocaine was detected in both samples, quantification of the cocaine could not be done on the muscle samples because of the interference of chemicals that were produced in tissues during decomposition, which is why larvae samples were proven to be a viable alternative for toxicology analysis [20].

12 Texas Tech University, Caitlin M. Cranston, December 2020

While most of the drug related research was done in the 1990s, more entomotoxicological research is slowly making its way to present times. For instance, in

2014, a study was conducted in which Magni et al. developed a gas chromatography coupled with mass spectrometry (GC- MS) method for methamphetamine detection in

Calliphora vomitoria insects. The procedure the researchers used in their study proved capable in detecting methamphetamine in the insects. Compared to the technique (RIA) used by Goff et al. in a previous study which yielded a weak methamphetamine detection,

GC-MS was more sensitive as it was able to detect methamphetamine in immature stages of the C. vomitoria. The insects used for this detection technique were feeding on livers that contained 5 ng/mg or 10 ng/mg of methamphetamine. The researchers concluded that

GC-MS was a more useful technique for entomotoxicology analyses because it was able to detect minute concentrations of methamphetamine in fly larvae [21].

Another study was conducted in 2017, which utilized entomotoxicology in a maternal filicide-suicide by fire case. A large fire was reported, and once the firefighters extinguished the fire, a car was discovered with three bodies inside. All of the bodies had been severely burned and some recent insect activity was discovered. Although there were some viable tissues available on the body for a toxicological analysis, larvae were analyzed to corroborate the drugs that were detected from toxicological analysis in 2 of the 3 bodies [22]. The results were as follows: the bodies that were recovered were proven to be a mother and her two kids, a son and daughter. The larvae that were collected from the children were positive for diazepam, oxazepam, and nordiazepam; the larvae that were collected from the mother however were negative for drugs. This evidence supported the theory that the children were sedated before death [22]. This

13 Texas Tech University, Caitlin M. Cranston, December 2020

article overall concluded that even after an event, such as a fire, larvae samples can still be used as an alternative media for toxicology analysis.

It should also be mentioned that some studies focusing on entomotoxicology, failed to detect drugs or poisons from larvae samples. In an article by Amendt et al.

(2011), they concluded that just because a drug was not detected from the larvae that was analyzed does not necessarily mean that the drug was not present in the tissues of the decedent. The drugs and/or toxins that are detected from the larvae were a result of the substance having a higher absorption rate than its metabolism in the decedent prior to death [23].

1.5 Typical Toxicology Methods When conducting a typical toxicology analysis, the samples that are usually collected include, blood, vitreous humor, liver, stomach contents, urine, bile, hair, muscle, and/or other organs like kidney’s or lung contents when applicable. These samples are usually collected depending on the type of death case [24]. Once these samples are collected, they are sent from the medical examiner to a laboratory for toxicology analysis. A series of tests will be conducted in order to establish if there is a substance in the sample such as over-the-counter medications, prescription medications, illicit drugs, alcohol, and other toxicants such as carbon monoxide, pesticides, metals, inhalants, etc. [24]. Tests range from screening methods such as immunoassays and color tests to confirmatory tests like gas chromatography or liquid chromatography coupled with mass spectrometry. Quantitative analysis is usually conducted as well in order to determine how much of the substance was found in the sample that was analyzed.

The Scientific Working Group has specific guidelines and recommendations that should be followed when conducting certain analyses. For example, there are specific

14 Texas Tech University, Caitlin M. Cranston, December 2020

groups for toxicology (SWGTOX) and for the analysis of seized drugs (SWGDRUG).

According to the SWGDRUG guidelines, analysis of a drug must undergo multiple

uncorrelated techniques for the analysis to be valid and accurate [25]. Analytical

techniques that are used are separated into three different categories which are listed in

the Figure 2.

Laboratories must follow these guidelines when conducting certain analyses;

when a technique is utilized from category A, at least another method must be used from

another category. If a technique from category A is not used, three other methods must be

used from category B. When utilizing coupled techniques, such as gas chromatography-

mass spectroscopy, these are considered two separate methods as long as data from each

method is documented in a report [25].

Figure 2. Analytical techniques categorized based on SWGDRUG guidelines. Category A refers to confirmatory analyses, while Categories B and C are preliminary analyses [26].

1.6 Gas Chromatography – Mass Spectrometry In forensic science, gas chromatography-mass spectrometry (GC-MS) is

considered the gold standard for confirmatory tests, especially for drug analyses. The

reason being is that any analyte that is volatile at 300-500°C, does not decompose at

15 Texas Tech University, Caitlin M. Cranston, December 2020

those temperatures, and has a molecular weight at or below 600amu, can be analyzed by

GC-MS [27].

How a GC-MS works is as follows. The analysis begins with the gas chromatograph (GC) where the sample is volatized. When the autosampler injects the unknown sample into the GC, the instrument heats up, vaporizes the sample, and then sends it through the capillary column where the analytes of the sample get separated [28].

The analytes are pushed through the column using an inert gas, such as argon, helium, or nitrogen; the analytes will interact with the solid phase of the column resulting in them eluting from the column at different times, also called retention times [28]. The general set up of a GC can be seen in Figure 3.

Once analyzed by the GC, the analytes will then travel to the mass spectrometer

(MS) which is shown in the Figure 4. The sample is first ionized, which can be done by a number of ionization sources. Most commonly used ion sources are either a chemical or electrical ionization. Once the analytes are ionized, they then pass through the mass analyzer, typically an ion trap or quadrupole, where they are separated based on their mass to charge ratio (m/z) [28]. Once this separation takes place, the detector will arrange the ions based on their mass to charge ratio, also known as the mass spectrum (Figure 4).

The peaks correspond with the compounds of the analyte, and the heights of the peak correspond with the quantity of the compound [28]. Researchers are able to identify and quantify the compounds that make up the sample by utilizing a library of known mass spectra.

16 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 3. General set up of a GC-MS [27].

Figure 4. Simple block diagram of a mass spectrometer (left). A representation of a known mass spectra of ketamine (right) [29,30].

17 Texas Tech University, Caitlin M. Cranston, December 2020

1.7 Ketamine Ketamine (C13H16CINO, 2-(ortho-chlorophenyl-2-methylaminocyclohexanone) is structurally and pharmacologically similar to phencyclidine (PCP) (Figure 5) and is commonly used in medicine as a safer surgical anesthetic. It is more well known as a sedative/tranquilizer used in veterinary practices. With its exclusive use in medical and veterinary practices, it has been established that ketamine provides short term anesthesia and sedation. However, due to its psychoactive effects, ketamine has become more popular as a recreational drug; and because of its ease of access, has led to ketamine being increasingly used in drug facilitated sexual assaults. Despite ketamine being used illicitly, some clinics are using ketamine for treatment research. Most of the current research is focusing on ketamine being used as a treatment for depression and post- traumatic stress disorder.

Figure 5. Chemical structures of PCP and Ketamine [31].

1.7.1 Illicit Use In 1965, the first account of ketamine being used illicitly for recreational purposed was documented by Professor Edward Domino, who described the drug as a

‘dissociative anesthetic’ [32]. These dissociative properties are favorable amongst the club drug enthusiasts because of how the drug distorts sight and perception of the user as well as producing illusions. Increased recreational use of ketamine began around the

18 Texas Tech University, Caitlin M. Cranston, December 2020

1970s and was seen being used worldwide. For example, in Argentina, ketamine was being used for therapy purposes, but other areas were using the drug for ‘mind exploration’ and ‘new age spiritualism’ [32]. This illicit use of ketamine began expanding well through the 1980s and 1990s, where experimental uses started becoming more common. Specifically, the popularity of ketamine use came from the publication of two books ‘Journeys into the Bright World’ by Marcia Moore and Howard Alltounian, and

‘The Scientist’ by John Lilly; where the authors outlined their personal use of the drug

[32]. As mentioned previously, ketamine increased in illicit use because of its ease of access. In the 1980s, ketamine was readily available in capsules, powder, tablets, crystals, liquids and other injectable solutions found in the illegal drug markets [32]. Ketamine has often been found used as a club drug, where party goers often confuse ketamine with ecstasy. This trend of use increased throughout the 1990s and can be seen being used in present times. The most rapid increase of illicit ketamine use was in the 1990s in Hong

Kong, China. This led to China regulating ketamine in the 2000s, where it remains a

Schedule I drug today [32]. In the United States in 1995, because of its high abuse potential and continued medical usage, ketamine was listed as a Schedule III drug by the

Drug Enforcement Agency (DEA).

1.7.2 Pharmacokinetics and Toxicity Ketamine can be administered by several routes including, intravenously, intramuscularly, nasal insufflation of the powder form, smoking, or orally when in the tablet form [33]. Ketamine is a lipid soluble molecule, and in its hydrochloride form, is water soluble. Once absorbed, it is rapidly distributed through the central nervous system

[34]. Ketamine has a pKa of 7.5 and has a large volume of distribution, about 3-5 L/kg, and has a plasma protein binding of about 30% [33]. Ketamine metabolizes into

19 Texas Tech University, Caitlin M. Cranston, December 2020

norketamine by N-demethylation and can also undergo dehydrogenation to form dehydronorketamine (Figure 6). These ketamine metabolites can reach the same concentration in the blood as the parent drug. Ketamine has a relatively short half-life, approximately 2-4 minutes, and in humans, it has a longer half-life of approximately 2-4 hours [34].

Figure 6. Oxidative metabolism of ketamine [34].

As mentioned previously, ketamine can produce hallucinogenic effects and can additionally produce effects such as gastrointestinal distress, irrational behavior, and blurry vision [33]. More severe effects include seizures and cardiac arrythmias. In relation to drug facilitated sexual assaults, ketamine can induce central nervous system depression which can impair speech, thought processes, and can produce amnesia. These effects are produced because of how ketamine noncompetitively binds with the NMDA

(N‐methyl‐D‐aspartate) receptor [33].

Ketamine has a wide therapeutic index, i.e. it is very rare for a ketamine overdose to result in death; the average dose of ketamine intravenously is approximately 1-4.5 mg/kg. The LD50 (lethal dose in 50% of the test group) is approximately 4.2 g for a 70 kg

20 Texas Tech University, Caitlin M. Cranston, December 2020

human [35]. The Acute ketamine toxicity can lead to cardiovascular effects; however, injury is a more common occurrence. The reason being is because of ketamine’s dissociative properties, this can induce aggression and/or agitation, and make the user believe that they can do almost anything without suffering injury in the process [36]. It should also be mentioned that this acute toxicity, could be caused by the mixing of substances, because ketamine is commonly used in conjunction with ethanol and/or other recreational drugs. Chronic ketamine toxicity can lead to neuropsychiatric symptoms that are similar to that of schizophrenia [36]. Other adverse effects of ketamine can be seen in the following cases. The first case is of a 11-month-old child developing prolonged apnea and respiratory depression after being administered ketamine intramuscularly for a surgical procedure [37]. The child recovered in approximately 90 minutes after being intubated and put under ventilation. Other studies/cases of ketamine toxicity focused on urological pathology. A study of several long-term recreational ketamine users showed several dysuria symptoms, and other lab tests showed that several of the user’s bladders had thickened walls, perivascular stranding, and extreme ulcerative cystitis. Abstaining from ketamine use reduced these effects which proves that chronic use of ketamine causes toxicity to the urinary system [36, 38].

1.7.3 Entomotoxicology Research When it comes to entomotoxicology, research related to ketamine is not a particular drug of interest. Since most commonly abused drugs include opiates, amphetamines, cocaine, etc., only a couple entomotoxicology studies have used ketamine as their analyte of interest. An article by Magni et al. in 2018, claimed to be the first to develop and validate an analytical method, high performance liquid chromatography- mass spectrometry/mass spectrometry (HPLC-MS/MS), that is suited to detect ketamine

21 Texas Tech University, Caitlin M. Cranston, December 2020

in insects [39]. The researchers based their methodology on a previous article in which they used GC-MS for methamphetamine detection in insects [21]. In this method development article, Magni et al. (2018) collected C. vomitoria flies and performed a laboratory rearing of the insects using ketamine spiked beef livers. After a rearing period, larvae (2nd and 3rd instars), pupae, pupae casings, and adult flies were collected, and an extraction method was performed to prepare the samples for HPLC-MS/MS analysis. As a result, ketamine was detected in all the samples except for the adult flies. This result was then validated by following a set of international standards [39]. This article ultimately outlined an experimental procedure in which ketamine can be extracted and detected from insects.

The other study that used ketamine as their analyte of interest was from 2014.

However, this study focused on how ketamine effects the development of blowflies, not the detection of the drug from the insects. The researchers of this study concluded that deaths that were a result of ketamine intoxication would delay the growth of larvae when using regression analysis for PMI estimations. The ketamine from the body, combined with lower temperatures, significantly suppressed development of the larvae. While ketamine has an effect on the development of insects, the researchers found no dose- dependent relationship between concentration of the drug and inhibition of larvae development [40].

1.8 Thesis Study Objective The main purpose of this study was to determine if ketamine can be extracted from larvae that have been feeding on decomposing tissues and be detected using gas chromatography-mass spectrometry. This study will act as a continuation of the research already performed in relation to entomotoxicology with the analyte of interest being a

22 Texas Tech University, Caitlin M. Cranston, December 2020

drug that has not been heavily researched. The extraction method of choice for this study will be QuEChERS, which stands for “quick, easy, cheap, effective, rugged, and safe.”

This extraction method is becoming increasingly popular, especially in forensic sciences, because it decreases the time needed for sample prep. This decrease in sample preparation allows for more analyses to be conducted in a given day, thus reducing the ever-growing backlogs in laboratories. While QuEChERS is not the typical method used in toxicology, it has been used, although rarely, in some entomotoxicology studies.

In this present study, I hypothesize that it is possible to extract ketamine from the larvae collected from the decomposing subject to show another source that is useable for toxicology analysis. A previous study that utilized GC-MS to successfully detect methamphetamine from various larval stages of Calliphora vomitoria will be used as a baseline for this study [21]. As mentioned earlier, ketamine has a relatively short half-life, and rapidly metabolizes into norketamine [33, 34]. Because of this, I hypothesize that there will be a higher concentration of norketamine, the metabolite of ketamine extracted from the insect. Due to the current pandemic situation, this study had to be converted into a strict laboratory experiment. This included having to rear insects in the laboratory, where temperatures and environmental conditions were controlled for. This controlled laboratory experiment also required ketamine and norketamine to be injected into tissue samples by hand in order to replicate a real overdose scenario. That being said, the second hypothesis will be affected by this.

Statistical analysis will also be conducted to further determine if ketamine can be detected from larval samples.

23 Texas Tech University, Caitlin M. Cranston, December 2020

CHAPTER II

METHODOLOGY AND DATA ANALYSIS 2.1 Materials 2.1.1 Insects Sarcophaga Bullata fly larvae and pupae were ordered from Ward’s Science

(Rochester, New York). Larvae were purchased to immediately begin the feeding process with a treated food supplement, and pupae were purchased to perform a laboratory rearing of larvae. Both larvae and pupae were placed in a fridge located at TIEHH laboratory 120B maintaining a temperature of approximately 4ºC until needed as stated by the care and handling guidelines provided by Ward’s Science. The 100 count larvae and pupae will be equally separated into treatment and control groups, the remaining larvae and pupae (if any) will be used for any further preliminary tests.

S. bullata fly larvae and pupae were purchased to mimic the species of fly that would first arrive at a decomposing corpse [41]. These flies, also known as flesh flies, are important forensically as they have unique characteristics that help determine postmortem interval estimations [41]. S. bullata are also responsible for the initial destruction phase of decomposition. Laboratory rearing of S. bullata will allow for better control of environmental conditions that could prevent, postpone, or unintentionally kill the insects.

Controlling these factors will allow for the treatment of the food supplement to be the only variable in the study.

2.1.2 Insect Rearing Materials The following items were purchased and acquired for the purpose of rearing the S. bullata pupa in order to have larvae emerge much like how they would in an actual decedent. Mesh enclosures were first purchased from Amazon but proved to be ineffective because the right conditions could not be maintained within the fume hood

24 Texas Tech University, Caitlin M. Cranston, December 2020

where the rearing was taking place in TIEHH laboratory 120B. Table 1 showcases the remaining materials that were purchased for insect rearing.

25 Texas Tech University, Caitlin M. Cranston, December 2020

Table 1. Items purchased for the purpose of S. bullata rearing

Item Purchased From Reason One aquarium was used for pupae/adults S. Bullata flies, and 5-gallon aquarium (2) Walmart the other aquarium was for the larvae that was also purchased. One lamp was used for each aquarium to maintain the right 5.5” Dome Lamp (2) PetSmart temperature and supplement the light for the daytime hours.

60W Daylight Heat Blubs purchased for each of the lamps; for temperature and PetSmart Lamp Bulb (2) light purposes Habitat Screen Cover Covers purchases for each aquarium so the flies would not PetSmart (2) escape, and for the heat lamps to sit on Timers were set according to the guidelines outlined for S. Day/Night Timer (2) PetSmart bullata rearing from Ward’s Science. The flies/larvae required daylight for 16 hours and ‘night’ for 8 hours. Mini Hygrometer Purchased for each aquarium to maintain temperature and Amazon Thermometer (2) humidity for the insects.

28oz Spray Bottle Walmart Purchased to maintain humidity levels in each aquarium

Hermit Crab Sponges Sponges used for the water dish so the flies could acquire Amazon (4) water without drowning themselves

Vermiculite Amazon Used for substrate purposes in the aquariums

Topsoil Tractor Supply Co. Used for substrate purposed in the aquariums

26 Texas Tech University, Caitlin M. Cranston, December 2020

Table 1 Continued

Clear Plastic Square Used to hold the pupae when placed in the enclosure, was Amazon Containers also used to hold meat and larvae in the enclosures Used to make a 1:1 ratio as a food supplement for the flies as Powdered Milk & Sugar United Supermarkets recommended by the guideline provided by Ward’s Science Used for the flies drinking water and used to spray the Bottled Water Walmart enclosures to maintain humidity

27 Texas Tech University, Caitlin M. Cranston, December 2020

2.1.3 Human analogue For experimental purposes, specifically for forensics and decomposition research, pigs are typically used because they serve as the closest representation to humans anatomically [42]. However, given the circumstances with the current pandemic, which added some time constraints with the current research, IACUC approval was not possible to acquire to execute a full experiment with the use of pigs. Since this present experiment was converted to be conducted solely in a laboratory setting, ground pork was utilized instead, to replicate a human analogue decedent. Smithfield ground pork was purchased from Walmart. The pork was 85% lean and 25% fat. Since this pork was store bought, it is unknown whether this had been previously treated with antibiotics or other additives before packaging. These additives could have caused some interference with the experiment, i.e. interfered with the larvae feeding process and/or interfered with the production of larvae from adult flies.

Initially, the pork was stored in the freezer until it was ready to be used. To accurately represent a decomposing decedent, the pork was set out to thaw and left out for several days to begin the decomposition process. Each time the pork was portioned out, the remaining meat would be placed back into the freezer and the ‘thawing process’ would be repeated. Once portioned out, the pork was either treated with a dose of ketamine and norketamine or left untreated. Plastic weight boats were utilized to portion out and accurately weigh the pork.

2.1.4 Instrumentation GC-MS was used for this experiment to detect the target analyte from the larval samples. The instrument used is an Agilent 6890 N gas chromatograph equipped with an

Agilent 7683B Series autoinjector, coupled with an Agilent 5975 Inert Mass Selective

28 Texas Tech University, Caitlin M. Cranston, December 2020

Detector (Figure 7). The column used in the GC was a capillary column (30m x 250 µm,

DB5). The instrumentation utilized for extraction purposes was a Beckman Allegra™ 6R

Centrifuge, a Biotage TurboVap® LV nitrogen evaporator, a Scientific Industries, INC. vortex, a VWR® 200 Homogenizer, and 0.2 µm nylon filters with syringes. Other miscellaneous items used included GC vials and caps, crimper and decrimper, glass test tubes, and pipettes.

Figure 7. Instrumentation used for the detection of ketamine from larval samples. Agilent 6890N (right), Agilent 7683B Series autoinjector (top), Agilent 5975 MS (left).

2.1.5 Chemicals The drug of interest used for this experiment was ketamine (C13H16CINO, 2-

(ortho-chlorophenyl-2-methylaminocyclohexanone), which was obtained from Sigma

Aldrich. Norketamine (C12H14CINO, N-desmethylketamine), was also purchased from

Sigma Aldrich. Norketamine, which is the metabolite of ketamine, was utilized to attempt to replicate the biotransformation that occurs in the body after ingesting ketamine. The internal standard that was used was Naphthalene-d8.

29 Texas Tech University, Caitlin M. Cranston, December 2020

The chemical used for dilution purposes, calibration standards preparation, and the reconstitution of the sample’s contents after nitrogen evaporation, was methylene chloride. Methylene chloride was utilized because it was the solvent of choice that Magni et al. (2014) used for their experiment [21]. The QuEChERS salts that were used for sample extraction was composed of 4000mg of magnesium sulfate and 1000mg of sodium chloride. Acetonitrile and Milli Q water was also used to perform the sample extraction.

2.2 Methods 2.2.1 Experimental Procedure Rearing larvae from S. bullata pupae took several tries before finally acquiring enough larvae for extraction and detection of ketamine/norketamine. The first round of insect rearing involved the purchase of 100 S. bullata pupae, separating them into a controlled and treatment group, and placing them in an enclosure located in the insectary.

The environmental conditions and basic rearing information was acquired from Byrd

(1995) [43]. The insectary was to be kept at a temperature of 26-30°C, at 75-85% relative humidity, and have a 12:12 photoperiod. One enclosure was to be dedicated for the control fly group and another enclosure was to be used for the treatment fly group. On

July 20, 2020, at approximately 1030 hours, 25 pupae were placed in a clear plastic container that had a layer of vermiculite added; this container was placed in the treatment group enclosure. Another 25 pupae were placed in a clear plastic container that had a layer of vermiculite added; this container was placed in the control group enclosure.

Another 25 pupae were separated and placed into a 50-mL falcon tube which were later used for testing the extraction method. The pupae were checked on periodically for the next few days, and adult S. bullata flies began emerging on July 22, 2020. A plastic

30 Texas Tech University, Caitlin M. Cranston, December 2020

container filled halfway of water and a sponge was placed into each enclosure at approximately 0930 hours. On July 25, 2020 at approximately 1146 hours, pork was divided into three 50 g portions for the treated and control fly groups. The pork portions given to the treatment fly group were spiked with 50 µL of ketamine standard (1000 ppm) and 25 µL of norketamine standard (1000 ppm) and mixed by hand to homogenize the drugs within the pork as thoroughly as possible. At this time, it was also noted that some of the flies have already began to die. On July 27, 2020, all of the S. bullata flies had perished without larvae being produced. The following day all of the flies, remaining pupae, pupae casings, and pork were collected placed in labeled 50-mL falcon tubes and stored in the freezer located in TIEHH laboratory 120B. The suspected cause of death of the flies was the temperature in the insectary reaching temperatures as high as 40°C in the afternoon/evenings. Rearing was then tried again in the fume hood located in TIEHH laboratory 120B.

On August 5, 2020 at approximately 1118 hours, a new order of 100 S. bullata were separated and placed into the mesh enclosures set up in the fume hood. 30 pupae were placed into a clear plastic container that had a layer of vermiculite and was placed into the enclosure for the treatment group. Another 30 pupae were placed into a clear plastic container that had a layer of vermiculite and was placed into the enclosure for the control group. On August 6, 2020 at approximately 1000 hours, three portions of pork approximately 50 g each were measured out and treated each with 50 µL of ketamine and

25 µL of norketamine. Each tray of treated pork then had 20 larvae added to them from the order of 100 S. bullata larvae. These trays were then placed in another enclosure set up in the fume hood. Another portion of meat that was approximately 50 g was left

31 Texas Tech University, Caitlin M. Cranston, December 2020

untreated and about 15 larvae were added to the tray and placed in another enclosure set up in the fume hood. On August 9, 2020, the pupae were transferred to a 5-gallon aquarium that was lined with paper towels and vermiculite. A habitat screen cover was placed on the aquarium and a heat lamp connected to a timer was placed on the cover.

The timer was set to keep the lamp on for 16 hours and turn the lamp off for 8 hours. This and other rearing conditions were followed by Ward’s Science [44]. Larvae and pork were collected the same day and placed into labeled 15-mL and 50-mL falcon tubes

(respectively) and placed in the freezer until further analysis took place. The number of larvae collected, and their weights, were recorded and can be seen in Table 2.

Table 2. S. bullata larvae collected on August 9, 2020

Larval group Number of Larvae Weight Treatment 1 9 0.7379g Treatment 2 5 0.1874g Treatment 3 3 0.3224g Control 6 0.7277g

On August 14, 2020, adult S. bullata flies began to emerge. A plastic weight boat was filled with water with a sponge and placed in the aquarium. Another plastic weight boat was filled with a 1:1 ratio of powdered milk and sugar and also placed aquarium, as recommended for a food supplement by Ward’s Science [44]. On August 16, 2020, four portions of pork (50 g) were measured out with three being treated with ketamine and norketamine and one portion left untreated. These were placed into the fly aquarium in hopes of larvae being deposited. More meat was portioned out on August 19th, to replace the meat that had dried out. Another aquarium, screen cover, heat lamp, timer and thermometer/hygrometer were also placed in the fume hood for another round of larval rearing that day. On August 26, 2020, four portions of pork (50 g each) were measured

32 Texas Tech University, Caitlin M. Cranston, December 2020

out; three were spiked with ketamine and norketamine and one was left untreated. This was placed in the larvae aquarium which was lined with paper towel and a mixture of vermiculite and topsoil. Another smaller portion of meat, approximately 25 g, was also placed in the larvae aquarium, this was treated with a higher spike of ketamine and norketamine (100 µL and 50 µL respectively) to increase the chance of the instrument detecting the drug within the larvae. Each portion of meat had approximately 20 larvae placed in the tray. The larvae were left to feed on the pork for approximately three days.

However, within two days larvae from treatment group 2 and the control group had to be collected because they began to migrate from their food source. The remaining larvae were collected the next day, however most of the larvae had migrated from their food source so it was unknown if they came from a treated meat portion or from the control meat portion. Table 3 shows the total larvae, their respected group, and weight collected on August 29, 2020.

Table 3. S. bullata larvae collected on August 29, 2020 Larval Group Number of Larvae Weight Treatment 1 10 1.29g Treatment 2 13 1.6363g Treatment 3 3 0.2167g Treatment 4 3 0.4304g Control 19 1.825g Unknown (due to 56 7.0225g migration)

On September 9, 2020, all of the adult S. bullata flies had died, without any larvae being deposited on the pork that was provided. However, after further inspection there were larvae located under one of the trays that contained a portion of treated pork which had turned upside down when being placed into the aquarium. These larvae were

33 Texas Tech University, Caitlin M. Cranston, December 2020

collected and placed on another portion of pork which was treated with 50 µL of ketamine and 25 µL of norketamine to see if they would grow over the next three days.

The remaining flies, pupae, and pupae casings were collected and placed into labeled 50- mL falcon tubes; these were stored in the freezer in TIEHH laboratory 120B. On

September 12, 2020 and September 14, 2020, the remaining group of larvae had been collected. 49 larvae (4.2549g) were collected on September 12, and 13 larvae (0.7529g) were collected on September 14.

For extraction purposes, the larvae that were collected over the course of approximately four weeks were combined so that each sample had about 2-3 g of larvae, which is necessary for QuEChERS extraction. Table 4 and Figure 8 show the finalized sample groups which extraction would be performed and tested with GC-MS.

Table 4. Final larvae sample groups for ketamine analysis

Sample # Which group larvae came from Weight # of larvae 1 Treatment 5 2.3149g 33 2 Treatment 5 2.6441g 29 3 Treatment 1 & Unknown 2.6219g 21 4 Treatment 2 & Unknown 2.7195g 19 Treatment 3, 4, 2 (8/9), 1 (8/9), & 5 2.4820g 27 Unknown 6 Control 2.5814g 19 7 Unknown 2.2481g 17 8 Unknown 2.0860g 17

34 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 8. Final larvae sample groups for ketamine analysis

2.2.2 Preparation of Ketamine Stock and Working Solutions A stock solution of ketamine and norketamine was prepared by taking 10 µL of the ketamine standard (1000 ppm) and 10 µL of the norketamine standard (1000 ppm) and adding methylene chloride to make a 10 mL solution. This created a 1 ppm stock solution which was used for the calibration curve and LOD and LOQ determination. A stock solution of naphthalene-d8 was also prepared. This was done by taking 1mL of

4000 ppm naphthalene-d8 and adding 9 mL of methylene chloride to make a 400 ppm solution. This solution was then further diluted by taking 25 µL of the 400 ppm solution and adding methylene chloride to make 10 mL. This created a 1 ppm stock solution which was used as the internal standard for the calibration curve.

2.2.3 Calibration Curve Several calibration curves were prepared in order to test the capabilities of the

GC-MS to detect ketamine and norketamine. Standards 1, 5, 10, 25, 50, 100 ppb of ketamine and norketamine were prepared, however only the 100 ppb gained a response

35 Texas Tech University, Caitlin M. Cranston, December 2020

by the GC-MS. With several failed attempts of creating a working calibration curve standard, an article by Lin and Lua (2005) who were using GC-MS to screen for ketamine, showed a cutoff level of 100 ppb and prepared a calibration of 0, 100, 200, and

500 ng/mL [45]. This calibration curve created can be seen in Table 5. While this calibration curve was effective, a stronger calibration curve was created which was used for the remainder of the study for calculation purposes. The final calibration curve utilized standards that were 50, 100, 250, 500, and 1000 ppb and can be seen in Table 6.

To determine the reliability and validity of the calibration curve, an r2 value and its associated equation was obtained. This equation was used to determine the concentration of ketamine and norketamine extracted from the larval samples. A methylene chloride blank was also utilized for the larval sample analysis and the calibration curve analysis to prevent carryover between samples, and to prevent other contaminants that could remain from other analyses conducted using the instrument.

Table 5. Third preparation of ketamine calibration standards for GC-MS analysis.

Ketamine/ Naphthalene-d8 Methylene Concentration Norketamine Total (1 ppm) Chloride Mix (1 ppm) 100 ppb 100 µL 25 µL 875 µL 1000 µL 150 ppb 150 µL 25 µL 825 µL 1000 µL 300 ppb 300 µL 25 µL 675 µL 1000 µL 350 ppb 350 µL 25 µL 625 µL 1000 µL 500 ppb 500 µL 25 µL 475 µL 1000 µL

36 Texas Tech University, Caitlin M. Cranston, December 2020

Table 6. Final preparation of ketamine calibration standards for GC-MS analysis.

Ketamine/ Naphthalene-d8 Methylene Concentration Norketamine Total (1 ppm) Chloride Mix (1 ppm) 50 ppb 50 µL 25 µL 925 µL 1000 µL 100 ppb 100 µL 25 µL 875 µL 1000 µL 250 ppb 250 µL 25 µL 725 µL 1000 µL 500 ppb 500 µL 25 µL 475 µL 1000 µL 1000 ppb 1000 µL 0 µL 0 µL 1000 µL

2.2.4 Extraction of Ketamine Over the course of several weeks, larvae were collected after their allotted three- day feeding period on their treated or untreated pork food source (Tables 2-4). When collecting the larvae, it was noted that there was a pinkish tint of the intestinal tract of each larva. This indicated proof that each larva did ingest an amount of the pork which was treated with ketamine and norketamine. After collection, the larvae were placed in the freezer to ensure euthanasia. Once the number of larvae and their weights were recorded (Table 4), QuEChERS extraction was performed (Figure 9).

37 Texas Tech University, Caitlin M. Cranston, December 2020

2-3g of larvae added to a The samples were then The samples were then 50mL falcon tube; 2.5mL homogenized; then 5g of centrifuged at 3000rpm for 5 of milli-Q water was added QuEChERS salts were minutes. 5-6mL of and; 10mL of ACN was added to each sample supernatant were taken and added pipetted into glass vials

these were then filtered The samples were then twice using a 0.2 µm samples were then evaporated to dryness via nylon filter connected to a adjusted to 1 mL with nitrogen; this took syringe into a 2 mL glass methylen chloride approximately 2-3 hours. GC vial

Figure 9. Steps taken to extract ketamine from S. bullata larvae samples for GC-MS analysis

38 Texas Tech University, Caitlin M. Cranston, December 2020

Extraction was performed by placing each larvae sample into a labeled 50 mL falcon tube. 2.5 mL milli-Q water was added to each sample and vortexed for 30 seconds.

10 mL of acetonitrile was then added to each sample and vortexed for 30 seconds. The samples were then taken to lab 120B where they were homogenized using a VWR® 200

Homogenizer. Five grams of QuEChERS salts were added to each sample, immediately shaken and then vortexed for 30 seconds. The samples were then centrifuged at 3000 rpm for approximately 5 minutes; 5-6 mL of the supernatant was pipetted into glass vials where they were then taken to lab 120B to be evaporated by nitrogen. The evaporation was done until the samples were dry which took approximately 2-3 hours. Once dry, 1 mL methylene chloride was added to each vial to reconstitute the sample, and vortexed for 5-10 seconds. The samples were then filtered twice using a 0.2 µm nylon filter connected to a syringe into 2-mL glass GC vials. These samples were then stored in the freezer until the GC-MS was available for use.

2.2.5 Gas Chromatograph-Mass Spectrometer Parameters The GC utilizes the front inlet where the injection was set to splitless mode with an injection volume of 1 µL. The initial temperature was set to 100°C which was held for

2 minutes. There was a solvent delay of 2.50 minutes. The temperature ramp increased at

20°C/min until 250°C was reached and this was held for 5.50 minutes. The total run time was 15 minutes per sample; this does not include the time it takes for the instrument to return to baseline temperatures between each sample injection (Table 7).

The MS uses electron ionization to ionize the samples once they pass through the

GC. Using selected ion monitoring, the ions for ketamine are 180, 182, and 209 m/z, the

39 Texas Tech University, Caitlin M. Cranston, December 2020

ions for norketamine are 166, 168, and 195 m/z, and the ions for naphthalene-d8 are 136,

108, 137. The GC-MS data was obtained using ChemStation software.

Table 7. Temperature Ramp for GC analysis

Ramp Rate Final Temp Hold Time 100°C 2.00 min 1 20°C 250°C 5.50 min Total run time 15.00 min

2.2.6 Data Analysis When testing the ability of using larval samples as a means for toxicology analysis, each 50 g meat portion, except for the ones that remined untreated as a control, was spiked with 50 µL ketamine standard (1000ppm) and 25 µL norketamine standard

(1000ppm). The final concentration of the meat portion should be approximately 1 ppm.

The amount of ketamine and norketamine that should be extracted from the larval samples is unknown, because it is not known whether the larvae will also undergo some kind of metabolism of the drug that the pork has been treated with.

The following parameters were obtained from this experiment: detection limit

(LOD), quantification limit (LOQ), coefficient of variation (COV), relative standard deviation (RSD), absolute standard deviation (s), and percent recovery. The equation used for LOD and LOQ is as follows:

$ LOD = 3.3 &% $ LOQ = 10 &%

Where $ represents the standard deviation of the response, and S represents the slope of the calibration curve.

40 Texas Tech University, Caitlin M. Cranston, December 2020

COV and RSD was calculated using the following equations:

) COV = ⁄* X 100 ) RSD = ⁄*

The absolute standard deviation is calculated using the following equation:

Percent recoveries were also calculated to attempt to determine how much ketamine and norketamine was extracted from the larval samples. The percent recovery equation is as follows:

Percent Recovery = !"#!$#"%&' ')$* !+,!&,%)"%-+, X 100% ./0&!%&' ')$* !+,!&,%)"%-+,

41 Texas Tech University, Caitlin M. Cranston, December 2020

CHAPTER III

RESULTS AND DISCUSSION

3.1 Preliminary Tests on Ketamine For the development of a working GC-MS method for analysis, both ketamine and norketamine standards were diluted from 1000 ppm to 10 ppm. This was done by taking 10 µL of the standard and adding 900 µL of methylene chloride. This new 10 ppm stock was then injected into the GC-MS using a full scan mode already loaded onto the instrument. The first ketamine injection was done with a run time of 30 minutes, at that time it was noticed that ketamine eluted fairly quick, at around 13 minutes. The second ketamine injection was done, still using full scan mode, but the run time was shortened to

15 minutes, which was used for the remainder of the analyses that were conducted. The results of this shorter run showed that ketamine eluted around 8.614 minutes. Using full scan mode, we were able to see the major ions that are detected for ketamine which were

180, 182, and 209. A full scan method was also performed for norketamine under the already adjusted method that was used for ketamine. It was shown that norketamine elutes close to when ketamine elutes, at around 8.410 minutes. The major ions detected for norketamine was 166, 168, and 195. These ions for both ketamine and norketamine were added to a selected ion monitoring method and for the remainder of the sample analysis, this method was used.

3.2 Preliminary Extraction Tests While the rearing process of the S. bullata flies was happening, a QuEChERS extraction test was performed using the pupae left over from the first attempted rearing in

July. 25 pupae were transferred from the vial it was packaged in to a labeled 50-mL falcon tube. Approximately 10 µL of a 50 ppm concentration ketamine solution was

42 Texas Tech University, Caitlin M. Cranston, December 2020

injected into each pupa. After each pupa was injected, they were placed in the freezer until extraction took place. The following day, QuEChERS extraction was performed as shown in Figure 9. The pupae extraction was then analyzed using GC-MS where it was shown that the ketamine eluted at 8.625 minutes, with fairly high response (Figure 10).

43 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 10. Chromatogram (left) and SIM mass spectra (right) as a result from the QuEChERS test extraction using spiked S. bullata pupae.

44 Texas Tech University, Caitlin M. Cranston, December 2020

3.3 Calibration Curve 3.3.1 GC-MS Method Optimization During the time that the rearing of S. bullata flies, calibration standards were being tested. Several iterations of calibration curve standards were tested and only one calibration test resulted in a r2 value that was close to >0.995.

In the first attempt, the calibration samples were prepared from 1 to 100 ppb (6 different levels) in methylene chloride as diluent. The observation from the GC-MS with

SIM mode only detected the 50 and 100 ppb concentration levels. In addition, during the preliminary method setup trials, we did not use norketamine and internal standards. With this in mind, new calibration samples were prepared. This had internal standard and a mix of ketamine and norketamine. However, when these calibration samples were analyzed, the GC-MS using SIM mode did not recognize a high enough response to quantify.

To try and improve the detector’s response, a change in solvent was tried. We received the ketamine and norketamine neat stock solutions made in a methanol solvent.

Methanol was used in exchange for methylene chloride. However, this solvent change did not change or improve the GC-MS response. An increase in concentration of the calibration samples was prepared based on a previous article [45]. This was analyzed, however, the r2 value for either ketamine or norketamine was not close to 0.995 (Figure

11). The r2 value needs to be as close to 0.995 as possible because this represents little variation between the observed data and the fitted values. Lower r2 values are not always an issue however, this just means that there is variation within the data that cannot be explained. A third attempt at improving the calibration curve was removing two of the calibration samples (150 and 300 ppb) and using a linear trendline to generate the r2 value and equation. Doing this strengthened the r2 values for both ketamine and norketamine

45 Texas Tech University, Caitlin M. Cranston, December 2020

(Figure 12). However, this left only a three-point calibration curve which is not ideal for method validation purposes. Having more points of calibration will strengthen the validation and reliability of the analysis and will improve the calculations that are performed using the calibration curve. The fourth and final calibration curve, created by preparing standards at 50, 100, 250, 500, 1000 ppb, was utilized in place of the third calibration and was used for the LOD, LOQ, and percent recovery calculations (Figure

13).

Calibration Curve

1400000 y = 2224.3x + 50485 1200000 R² = 0.7102 1000000 800000 600000 400000 y = 1553.2x + 78436

Detector Response R² = 0.5805 200000 0 0 100 200 300 400 500 600 Concentration (ppb)

Ketamine Norketamine Linear (Ketamine) Linear (Norketamine)

Figure 11. GC-MS response to the concentrations of ketamine and norketamine

46 Texas Tech University, Caitlin M. Cranston, December 2020

Calibration Curve 1200000 y = 2414.6x - 152070 R² = 0.9925 1000000

800000

600000 400000 y = 1903x - 176772 R² = 0.9933 Detector Response 200000

0 0 100 200 300 400 500 600 Concentration (ppb)

Ketamine Norketamine Linear (Ketamine) Linear (Norketamine)

Figure 12. GC-MS response to the concentrations of ketamine and norketamine, Figure 12. GC-MS response to the concentrations2 of Ketamine and Norketamine, adjusted to strengthen r values2 adjusted to strengthen r values

Figure 13. Final calibration curve that shows GC-MS response of the concentrations of ketamine and norketamine

47 Texas Tech University, Caitlin M. Cranston, December 2020

3.4 LOD and LOQ The limit of detection (LOD) and quantitation limit (LOQ) was generated by using the calibration standard, from the fourth calibration curve, that was the lowest concentration with the highest response. It was observed that the 100 ppb standard had the highest response, which was then ran approximately 10 times, and the replicated data was then used to calculate the mean and standard deviation, which was then used to calculate LOD and LOQ. This data can be seen in Table 8. For ketamine, the LOD was calculated to be 58.13 ppb and the LOQ was calculated to be 193.76 ppb. For norketamine, the LOD was calculated to be 82.51 ppb and the LOQ was calculated to be

275.04 ppb. The LOD represents the smallest amount of analyte that can be detected at a known confidence level, and the LOQ represents the lowest concentration of analyte that can be measured with a defined precision and accuracy for a particular method.

Percent recovery was calculated for ketamine and norketamine utilizing the calibration standards from the fourth calibration curve. These values can be seen in Table

9 and Table 10.

Table 8. Calculations made to determine LOD and LOQ Replication Ketamine (ppb) Norketamine (ppb) 1 116.2 102.75 2 94.73 55.45 3 67.43 45.58 4 84.78 117.51 5 85.17 75.24 6 71.25 50 7 115.21 84.71 Mean 90.68 75.89 STDEV (±) 19.38 27.50

48 Texas Tech University, Caitlin M. Cranston, December 2020

Table 8 Continued

LOD 58.13 82.51 LOQ 193.76 275.04

Table 9. Percent Recovery for Ketamine

Expected Concentration Observed Concentration % Recovery 50 ppb 50.3143 ppb 100.63 100 ppb 105.7817 ppb 105.78 250 ppb 274.5275 ppb 109.81 500 ppb 482.8095 ppb 96.56 1000 ppb 872.1680 ppb 87.22

Table 10. Percent Recovery for Norketamine

Expected Concentration Observed Concentration % Recovery 50 ppb 48.6262 ppb 97.25 100 ppb 107.4935 ppb 107.49 250 ppb 221.6306 ppb 88.65 500 ppb 468.2243 ppb 93.64 1000 ppb 1129.5698 ppb 112.96

3.5 Results from Extraction Procedure The main purpose of this present study was to determine if ketamine or norketamine can be detected from an extraction of larval specimens using GC-MS.

According to the analysis of the larval samples, ketamine and norketamine was able to be extracted and detected by the instrument (Table 11).

49 Texas Tech University, Caitlin M. Cranston, December 2020

Table 11. GC-MS detector response for ketamine and norketamine

Ketamine Response Norketamine Response Larval Composite Larval Composite Detector Response Detector Response group Group 1 151638 1 49234 2 150499 2 279289 3 489272 3 22208158 4 0 4 22888672 5 374094 5 1685624 6 150553 6 581000 7 6123260 7 1968445 8 2998248 8 1284555

Much like previous entomotoxicology research, it is difficult to quantitate the

concentration of the drug from the larvae and correlate that with the concentration of the

drug in the body. The same can be said for this laboratory-controlled experiment. The

pork that was used as the larvae feeding source was treated with known amounts of

ketamine and norketamine and mixed by hand to homogenize the drug sample within the

meat as best as possible. The concentration of drug within the meat should have been

approximately 1 ppm. However, because the meat was homogenized by hand, there is the

chance that some sections of the pork could have been untreated. This could be the reason

for such low response from the instrumental analysis, such as seen in sample 4 of the

ketamine response. An attempt was made to calculate the concentration of ketamine and

norketamine utilizing a template from Microsoft Excel which can be seen in Table 12 and

Table 13. Despite the homogenization issues with the treated meat, an attempted to

calculate the percent recovery of ketamine and norketamine from the larval samples.

Utilizing the 1 ppm concentration that the pork was estimated to be treated with, the

calculations were performed using Microsoft Excel which can be seen in Table 14.

50 Texas Tech University, Caitlin M. Cranston, December 2020

Table 12. Final concentration calculations of ketamine from larvae extraction samples

Dilution Conc in Final conc Sample Area 1 Conc Factor ppb in ppm 1 151638 515.09 0.67 345.11 0.35 2 150499 511.22 0.67 342.52 0.34 3 489272 1661.99 0.67 1113.53 1.11 4 0 0 0.67 0 0 5 374094 1270.75 0.67 851.40 0.85 6 150553 511.41 0.67 342.64 0.34 7 6123260 20799.87 0.67 13935.91 13.94 8 2998248 10184.63 0.67 6823.70 6.82

Table 13. Final concentration calculations of norketamine from larvae extraction samples Dilution Conc in Final conc Sample Area 1 Conc Factor ppb in ppm 1 49234 76.28 0.67 51.11 0.05 2 279289 432.71 0.67 289.92 0.29 3 22208158 34408.09 0.67 23053.42 23.05 4 22888672 35462.44 0.67 23759.84 23.76 5 1685624 2611.61 0.67 1749.78 1.75 6 581000 900.17 0.67 603.11 0.6 7 1968445 3049.80 0.67 2043.37 2.04 8 1284555 1990.22 0.67 1333.45 1.33

Table 14. Percent recovery of ketamine and norketamine from larvae extraction samples Ketamine Norketamine Sample % recovery Sample % recovery 1 35 1 5 2 34 2 29 3 111 3 2305 4 0 4 2376 5 85 5 175 6 34 6 60 7 1394 7 204 8 682 8 133

51 Texas Tech University, Caitlin M. Cranston, December 2020

When analyzing the chromatograms that were produced from the GC analysis of the larval extraction samples, the peaks were not as clean as what was expected as compared to peaks of a standard ketamine and norketamine mix (Figures 14 and 15).

There could be several factors that could cause the chromatogram to look like this. For example, during the sample extraction process, when the sample was reconstituted from nitrogen evaporation, the samples were not “cleaned” enough when filtering through the

0.2 µm nylon filter. The reason for not filtering the sample multiple times, aside from the two filtrations that were performed, is because we did not want to risk the drug analytes being filtered out of the sample. There could be a chance that the some of the drug analytes had been filtered out, which would also contribute to the low response. Other factors could be caused by carry over or the column being contaminated by unknown substance from other analyses

When searching through the chromatogram from each sample analysis, the SIM mass spectra was checked based on the known retention time for both ketamine and norketamine (Figure 16). This was done to confirm that the drug of interest had been detected from the larvae samples. They were then searched using the NIST catalog of known analytes to further confirm the detection.

52 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 14. Chromatograms for larvae sample group 7 (left) and larvae sample group 6 (right)

53 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 15. Representative chromatogram of the 500 ppb ketamine/norketamine mix calibration standard. First peak is norketamine and the second peak is ketamine.

54 Texas Tech University, Caitlin M. Cranston, December 2020

Figure 16. SIM mass spectra for ketamine from larvae sample group 1 (left) and norketamine from larvae sample group 4 (right).

55 Texas Tech University, Caitlin M. Cranston, December 2020

CHAPTER IV

CONCLUSION

Forensic entomology is a very useful tool in criminal investigations, with an impressive historical background. Entomology is most often used to classify insects that have been found on a decedent. The classification of insects and knowledge of their life cycles are used to calculate postmortem interval (PMI), which is the estimated time of death of a victim. Insects can also be used as corroborative evidence which can link a suspect to the scene of a crime. Entomology research has delved into the use of insects for toxicological evidence as well, called entomotoxicology. With the increase of drug use within the United States alone, this has caused an increase in drug related deaths.

While traditional toxicology analysis is most common in determining the cause of death of these victims, there are some cases where these decedents are not located in a sufficient amount of time. For these cases, tissues commonly used for toxicology analyses are not available due to the bodies undergoing advance stages of decomposition.

This sparked interest in researchers in determining another tissue sample that could be used for toxicology purposes.

This study examined if ketamine could be detected from larval samples, using

QuEChERS as the extraction method, and GC-MS for analysis. QuEChERS extraction was used because it has been proven effective for forensic purposes, and, most importantly, is a relatively quick extraction method. Ketamine was the analyte of choice for this experiment, because of its recent gain in popularity for recreational use as well as for other medical treatments, such as depression and post-traumatic stress disorder.

Although ketamine isn’t a drug that is typically screened for, with the increase in use

56 Texas Tech University, Caitlin M. Cranston, December 2020

recreationally and its abuse potential, ketamine could definitely be seen as one of the drugs that caused a death by overdose. It was hypothesized that ketamine could be detected from the larval samples exposed to pork that had been treated with ketamine.

Because ketamine has a short half-life, norketamine was also studied as the main metabolite of ketamine. It was also hypothesized that there would be a higher rate of detection for norketamine, however this could not be accurately tested due to the current pandemic situation. Preliminary findings proved that the extraction method was a good way to extract ketamine from pupae that had been injected with a known amount of ketamine. Other tests conducted by running standards of both ketamine and norketamine proved that the instrument could in fact detect the drugs.

When conducting the analysis, the results proved that ketamine and norketamine could be extracted from larval samples and detected using GC-MS. However, it was difficult to determine how much ketamine and norketamine the larvae injected from their food source. This is one limitation to the study. Not much research has studied entomotoxicology in depth, meaning that most statistical analysis has not been conducted in order to compare and quantitate the amount of drug ingested from the insect to the amount and concentration of the drug in the body. It is also unknown whether the insect also metabolized drugs the same way as humans. This would also affect quantitating the amount of analyte that is calculated from the insect. A way to improve the quantification of the amount of drug that was extracted and detected from the larval samples would be to take the same amount of pork that was used for the experiment, spike it with the same amounts of ketamine and norketamine used in the study, perform the extraction method on the pork, and then analyze that to see if any of the drug could be detected. This

57 Texas Tech University, Caitlin M. Cranston, December 2020

analysis could help with the validation of the extraction method and improve the statistical analyses that were calculated.

Another major limitation is that this study was not conducted in a way to accurately replicate a decedent being found of a suspected overdose. All factors, including the amount of drug used to treat the pork, environmental conditions, potential contaminations, etc. were all controlled for as this was strictly a laboratory experiment.

Since all these factors were being controlled for, a larval mass was not accomplished through this experiment. When a larval mass is achieved, certain chemicals are secreted from the larvae which aid in the decomposition of tissue, and also acts as an attractant so the larvae will less likely migrate from the tissue before they are able to go through the three instar stages. It should also be mentioned that proper decomposition was not accomplished in this study, by this it is meant that the autolysis stages of decomposition was not seen. What was accomplished is the putrefaction stages of decomposition, which was seen during the slow decay of the pork when it was being left out during the

‘thawing procedure.’ In order to accurately represent the decomposition part of the study

(including the faunal succession of insects), live pigs would have to be utilized, injected with an amount of ketamine, allowed time for the drug to begin metabolizing, and then perform euthanasia. Since the pandemic began, getting the proper approval to do such an experiment would not have been given in a decent amount of time to conduct the full decomposition and insect rearing.

Another limitation could be from the extraction method that was used. Even though in preliminary tests proved that QuEChERS can successfully extract ketamine from spiked pupae, it is not the typical method of extraction used for toxicology analysis.

58 Texas Tech University, Caitlin M. Cranston, December 2020

If this study was to be replicated, using other extraction methods, such as liquid-liquid extraction, solid-phase extraction, or protein precipitation, should be utilized to accurately represent typical toxicology extraction/pretreatment used for drug analyses from a specimen. Also, it should be mentioned that during the ‘clean up’ portion of

QuEChERS, nylon filters were utilized to filter the samples after being reconstituted from evaporation. The samples were filtered twice because after the first filtration, they were not completely clean. A suggestion to improve this part of the extraction method and make it less likely that the analyte of choice gets filtrated out of the sample is to use

PTFE filters which can be used for samples using methylene chloride as a solvent.

This study can ultimately help with forensic investigations by providing another sample that can be used for toxicology analysis. When a body is discovered, there are enough insects colonizing the corpse that multiple analyses could be performed in the instance that other organs or tissues are not available. Since there are not many present studies that focus on entomotoxicology, hopefully this study will open the door for more research into this discipline to strengthen it and potentially allow it to be more utilized within forensic toxicology.

59 Texas Tech University, Caitlin M. Cranston, December 2020

REFERENCES

1. Insects and Humans. In: Global Encyclopedia. Global Industries, Inc., 1988; 193

2. Goff ML, Lord WD. Entomotoxicology: A New Area for Forensic Investigation. The American Journal of Forensic Medicine and Pathology 1994;15(1):51–7.

3. Byrd JH, Castner JL, editors. Forensic entomology: the utility of arthropods in legal investigations. Boca Raton: CRC Press, 2010

4. https://www.drugabuse.gov/drug-topics/trends-statistics/overdose-death-rates (accessed [August 17, 2020]).

5. https://www.cdc.gov/drugoverdose/data/statedeaths.html (accessed [August 17, 2020]).

6. Beyer JC, Enos WF, Stajić M. Drug Identification Through Analysis of Maggots. Journal of Forensic Sciences 1980;25(2):411–2.

7. https://www.who.int/ipcs/publications/training_poisons/basic_analytical_tox/en/in dex5.htht#:~:text=2%20Solvent%20extraction,widely%20used%20in%20analytic al%20toxicoltox (accessed [August 17, 2020]).

8. Schmidt ML, Snow NH. Making the case for QuEChERS-gas chromatography of drugs. TrAC Trends in Analytical Chemistry 2016 Jan;75:49-56.

9. Smith EH, Kennedy GG. History of Entomology. In: Resh VH, Carde RT, editors. Encyclopedia of Insects. 2nd ed. Elsevier Science & Technology, 2009;449-457.

10. Benecke M. A brief history of forensic entomology. Forensic Science International 2001 Aug;120(1-2):2-14.

11. https://www.thoughtco.com/forensic-entomology-early-history-1300-1901- 1968325 (accessed [September 7, 2020]).

12. Wallace DR. Evolution of Forensic Entomotoxicology. Toxicology and Forensic Medicine 2017;SE1:1-4.

13. Introna F, Campobasso CP, Goff ML. Entomotoxicology. Forensic Science International 2001;120(1-2):42-47.

14. Pounder DJ. Forensic entomo-toxicology. Journal of the Forensic Science Society 1991;31(4):469–72.

60 Texas Tech University, Caitlin M. Cranston, December 2020

15. Silva EITD, Wilhelmi B, Villet MH. Forensic entomotoxicology revisited- towards professional standardization of study designs. International Journal of Legal Medicine 2017 May;131(5):1399-1412.

16. http://pubsapp.acs.org/subscribe/archive/tcaw/13/i09/pdf/904chronicles.pdf? (accessed [August 22, 2020]).

17. Gunatilake K, Goff LL. Detection of Organophosphate Poisoning in a Putrefying Body by Analyzing Arthropod Larvae. Journal of Forensic Sciences 1989;34(3):714–6.

18. Kintz P, Tracqui A, Mangin P. Toxicology and fly larvae on a putrefied cadaver. Journal of Forensic Science Society 1990 July;30(4):243-246.

19. Introna F, Dico CL, Caplan, YH, Smialek JE. Opiate Analysis in Cadaveric Blowfly Larvae as an Indicator of Narcotic Intoxication. Journal of Forensic Sciences 1990 Jan;35(1):118-122.

20. Nolte KB, Pinder RD, Lord WD. Insect Larvae Used to Detect Cocaine Poisoning in a Decomposed Body. Journal of Forensic Sciences 1992 July;37(4):1179-1185.

21. Magni PA, Pacini T, Pazzi M, Vincenti M, Dadour IR. Development of a GC-MS method for methamphetamine detection in Calliphora vomitoria L. (Diptera: Calliphoridae). Forensic Science International 2014 May;241:96-101.

22. Bugelli V, Papi L, Fornaro S, Stefanelli F, Chericoni S, Giusiana M et al. Entomotoxicology in burnt bodies: a case of maternal filicide-suicide by fire. International Journal of Legal Medicine 2017 June;131(5):1299-1306.

23. Amendt J, Richards CS, Campobasso CP, Zehner R, Hall MJR. Forensic entomology: applications and limitations. Forensic Science, Medicine and Pathology 2011 Jan;7(4):379-392.

24. http://www.forensicsciencesimplified.org/tox/how.html (accessed [August 23, 2020]).

25. https://swgdrug.org/Documents/SWGDRUG%20Recommendations%20Version %207-1.pdf (accessed [August 22, 2020]).

26. https://spectra-analysis.com/wp-content/uploads/2019/06/AppNote030- ForensicAnalysisDrugs-1.pdf (accessed [September 28, 2020]).

27. Stauffer E. Gas Chromatography-Mass Spectrometry. In: Houck MM, editor. Materials Analysis in Forensic Science. Elsevier Inc, 2016:115-122.

61 Texas Tech University, Caitlin M. Cranston, December 2020

28. https://www.thermofisher.com/us/en/home/industrial/mass-spectrometry/mass- spectrometry-learning-center/gas-chromatography-mass-spectrometry-gc-ms- information.html (accessed [September 27, 2020]).

29. Smith RW. Mass Spectrometry. In: Houck MM, editor. Materials Analysis in Forensic Science. Elsevier Inc, 2016:123-128.

30. https://www.caymanchem.com/product/9001961/(s)-ketamine-(hydrochloride) (accessed [September 28, 2020]).

31. Ho JH, Dargan PI. Arylcyclohexamines (Ketamine, Phencyclidine, and Analogues). Critical Care Toxicology 2016;1-46.

32. http://ketamine.com/history-of-ketamine/ (accessed [September 27, 2020]).

33. Jenkins AJ. Hallucinogens. In: Levine B, editor. Principles of Forensic Toxicology. 4th edition. Washington DC, 2013: 387-389.

34. Peltoniemi MA, Hagelberg NM, Olkkola KT, Saari TI. Ketamine: A Review of Clinical Pharmacokinetics and Pharmacodynamics in Anesthesia and Pain Therapy. Clinical Pharmacokinetics 2016;55(9):1059–77.

35. https://www.ncbi.nlm.nih.gov/books/NBK541087/?report=classic (accessed [September 27, 2020]).

36. Kalsi SS, Wood DM, Dargan PI. The epidemiology and patterns of acute and chronic toxicity associated with recreational ketamine use. Emerging Health Threats Journal 2011;4(1):1-10.

37. Jonnavithula N, Kulkarni DK, Ramachandran G. Prolonged apnea with intramuscular ketamine: a case report. Pediatric Anesthesia 2008;18(4):330–1.

38. Shahani R, Streutker C, Dickson B, Stewart RJ. Ketamine-Associated Ulcerative Cystitis: A New Clinical Entity. Urology 2007;69(5):810–2.

39. Magni PA, Pazzi M, Droghi J, Vincenti M, Dadour IR. Development and validation of an HPLC-MS/MS method for the detection of ketamine in Calliphora vomitoria (L.) (Diptera: Calliphoridae). Journal of Forensic and Legal Medicine 2018;58:64–71.

40. Lu Z, Zhai X, Zhou H, Li P, Ma J, Guan L et al. Effects of Ketamine on the Development of forensically important Blowfly Chrysomya megacephala (F.) (Diptera: Calliphoridae) and its Forensic Relevance. Journal of Forensic Sciences 2014;59(4):991-996.

62 Texas Tech University, Caitlin M. Cranston, December 2020

41. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6197121/ (accessed [September 29, 2020]). 42. Matuszewski S, Hall MJR, Moreau G, Schoenly KG, Tarone AM, Villet MH. Pigs vs. People: the use of pigs as analogues for humans in forensic entomology and taphonomy research. International Journal of Legal Medicine 2020; 134:793- 810.

43. Byrd JH. Laboratory Rearing of Forensic Insects. In: Byrd JH, Castner JL, editors. Forensic Entomology: The Utility of Arthropods in Legal Investigations. Boca Raton: CRC Press, 2010;121-142.

44. https://www.wardsci.com/www.wardsci.com/images/Sarchophoga_bullata.pdf (accessed [July 16, 2020]).

45. Lin HR, Lua AC. A fast GC-MS screening procedure for ketamine and its metabolites in urine samples. Journal of Food and Drug Analysis 2005;13(2):107- 111.

63