A Review of Synthetic Fentanyl Metabolism and the Metabolism of Select Synthetic Fentanyl Analogues

A review of synthetic fentanyl metabolism and the metabolism of select synthetic fentanyl analogues

Gerard Lee

A thesis submitted for the completion of a degree in Master of Forensic Science (Professional Practice)

The School of Veterinary and Life Sciences Murdoch University

Supervisors: Associate Professor James Speers (Murdoch) Associate Professor Bob Mead (Murdoch)

Semester 1, 2019

Declaration

I declare that this thesis does not contain any material submitted previously for the award of any other degree or diploma at any university or other tertiary institution. Furthermore, to the best of my knowledge, it does not contain any material previously published or written by another individual, except where due reference has been made in the text. Finally, I declare that all reported experimentations performed in this research were carried out by myself, except that any contribution by others, with whom I have worked is explicitly acknowledged.

Acknowledgements

I would like to thank my supervisor Bob Mead for his time guiding me and providing feedback on this endeavour. He has been a great help providing insights and advice from when I started university at Murdoch until now for which I am extremely grateful. To James Speers, thank you for helping me find a direction for this project when I started out. And finally, to my family and friends for their encouragement and support.

iii

Table of Contents

Title page...... I

Declaration...... Ii

Acknowledgements ...... iii

PART ONE

Literature Review...... 1-57

PART TWO

Manuscript ...... 58-87

Part One: Literature Review

A review of synthetic fentanyl metabolism and fragmentation patterns

of synthetic fentanyl analogues

Table of Contents

List of Figures ...... 3 List of Tables ...... 4 Abbreviations ...... 4 Abstract ...... 5 Chapter 1: Introduction ...... 6 1.1. Fentanyl pharmacology and mechanism of action ...... 9 1.2. Chemical structure of fentanyl and its derivatives ...... 10 1.2.1. Acetylfentanyl, structure and history……………………………………………………………..12 1.2.2. Acrylfentanyl, structure and history……………………………………………………………….14 1.2.3. Butyrfentanyl, structure and history………………………………………………………………15 1.2.4. Furanylfentanyl, structure and history…………………………………………………………..17 1.3. Methods of identifying fentanyl analogues ...... 19 1.4. Metabolism pathways of fentanyl and analogues ...... 22 1.5. Fentanyl phase I and II metabolites ...... 23 Chapter 2: Discussion ...... 27 2.1. Metabolism of Acetylfentanyl ...... 27 2.2. Metabolism of Acrylfentanyl ...... 31 2.3. Metabolism of Butyrfentanyl ...... 33 2.4. Metabolism of Furanylfentanyl ...... 36 2.5. Summary of fentanyl analogue metabolism ...... 39 Chapter 3: Fentanyl analogue fragmentation patterns ...... 41 3.1. Fragmentation patterns of fentanyl ...... 41 3.2. Fragmentation patterns of fentanyl analogues ...... 44 3.3. Summary of fragmentation patterns...... 46 Chapter 4: Conclusion ...... 47 References ...... 49

List of Figures Page

Figure 1: Synthesis of fentanyl and acetylthiofentanyl ...... 10

Figure 2: Possible modifications to the fentanyl molecule ...... 12

Figure 3: Comparison of acetylfentanyl and fentanyl ...... 13

Figure 4: Comparison of acrylfentanyl and fentanyl ...... 14

Figure 5: Comparison of fentanyl and butyrfentanyl ...... 16

Figure 6: Comparison of furanylfentanyl molecule to fentanyl ...... 18

Figure 7: Metabolic pathways and possible metabolites of fentanyl ...... 25

Figure 8: Proposed metabolic pathways of acetylfentanyl in human hepatocyte and human urine metabolites, with primary metabolites in enclosed boxes...... 29

Figure 9: Proposed metabolic pathways of acrylfentanyl in human hepatocyte and human urine metabolites, with major metabolites in enclosed boxes ...... 32

Figure 10: Proposed metabolism pathways of butyrfentanyl in vivo and in vitro ...... 35

Figure 11: Proposed metabolite structures for furanylfentanyl...... 37

Figure 12: Proposed metabolic pathways of furanylfentanyl in human hepatocyte and human urine metabolites, with major metabolites in enclosed boxes ...... 38

Figure 13: Proposed fragmentation pathway schematic for fentanyl and associated 4-ANPP analogues ...... 42

Figure 14: Piperidine ion ...... 43

Figure 15: Product ion of the four fentanyl analogues generated by cleavage b/c ...... 44

Figure 16: Product ion of the four fentanyl analogues generated by cleavage d ...... 45

Figure 17: Proposed fragmentation pathways for fentanyl analogues ...... 47

List of Tables Page

Table 1: Summary of the names, formula and mass-to-charge ratio of the investigated fentanyl analogues ...... 30

Abbreviations

EMCDDA European Monitoring Centre for Drugs and Drug Addiction GTP Guanine triphosphate GDP Guanine diphosphate cAMP Cyclic adenosine monophosphate ED50 Effective dose SWGDRUG Scientific Working Group for the Analysis of Seized Drugs GC-MS Gas chromatography-mass spectrometry LC-HRMS Liquid chromatography/high-resolution mass spectrometry LC-MS/MS Liquid chromatography tandem mass spectrometry m/z Mass to charge ratio LC-MS Liquid-chromatography mass spectrometry LC Liquid-chromatography ToF time-of-flight MS/MS Tandem mass spectrometry MRM Multiple reaction monitoring LC-QTOF Liquid chromatography quadrupole time of flight ESI Electrospray Ionisation CYP 3A4 Cytochrome P450 3A4 CYP 2D6 Cytochrome P450 2D6 HPLC-MS High performance liquid chromatography mass spectrometry iPS Induced pluripotent stem cells h-iPS-HEP Human induced pluripotent stem cell-derived hepatocytes h-PRM-HEP Human primary hepatocytes 4-ANPP 4-anilino-N-phenethyl-piperidine UHPLC Ultra-high-performance liquid chromatography QTOF Quadrupole time-of-flight EI-MS Electron ionisation mass spectrometry CID Collision-induced dissociation

Abstract

Fentanyl is a fast acting, potent synthetic opioid. It is also used as a narcotic of abuse posing a life-threatening hazard to everyone that comes into contact with it. The illicit production of new fentanyl analogues creates challenges as new analytical approaches have to be developed in order to identify the new analogues and their metabolites. Further understanding of metabolites and metabolic pathways would therefore improve the ability to identify new analogues. This review compiles knowledge on in vivo and in vitro metabolism of specific fentanyl analogues to identify common metabolic pathways and fragmentation patterns of key metabolites to assist in the identification of novel fentanyl analogues.

Keywords: Fentanyl; Fentanyl analogues; Fentanyl fragmentation patterns; Metabolism;

Synthetic opioid;

CHAPTER 1: Introduction

Fentanyl and its predecessor morphine belong to a family of compounds called opioids.

Derivatives of opioids have been used for recreational and medicinal purposes for thousands of years because of their pain relief and euphoric effects. These effects are achieved via the activation of opioid receptors which inhibit the release of neurotransmitters in areas of the nervous system associated with pain transmission.1 Side effects of opioid use include sedation, nausea, constipation, vomiting, dizziness, and respiratory depression.2 Ongoing exposure also results in tolerance and physical dependence. Fentanyl was first synthesised in 1960 by chemist

Paul Janssen, founder of the Belgian pharmaceutical firm Janssen Pharmaceutica. Dr Janssen’s goal at the time was to develop a compound that was a more effective analgesic than morphine, with fewer detrimental side effects and a higher therapeutic index.3 This was achieved by increasing the lipid solubility of the meperidine molecule through the addition or replacement of chemical groups, the result being lipid-soluble analgesics with increased potency and a faster rate of onset.3

Because of the high potential it has for abuse, fentanyl is a controlled substance.4 Whether it is from misuse or illicit use, a fentanyl overdose can generate severe respiratory depression and result in death.5 Exposure to fentanyl more often occurs through polydrug use than via solitary administration. In a comparative study in Massachusetts of fentanyl-related deaths between illicit and licit users found a higher likelihood of co-intoxication with illicit drugs such as cocaine or other opioids such as morphine, methadone and oxycodone.6 The European Monitoring

Centre for Drugs and Drug Addiction (EMCDDA) has reported a similar trend of co-intoxication

6 with illicit drugs and fentanyl as it is used as a more accessible and cheaper substitute than heroin.7

While some fentanyl analogues such as alfentanil, remifentanil and sufentanil were designed as anesthetics or analgesics for use in surgical procedures,8 many other fentanyl analogues have been produced illicitly. Legislation exists in many countries to define the status of analogues of controlled drugs. In the USA, analogues of controlled substance are defined as drugs with no accepted medical use and a high potential for abuse.9 Australia possesses similar legislation pertaining to analogues of controlled drugs which includes a list of structural modifications that constitute structural similarity.10 However, due to the reactive nature of legislation whenever an analogue is classified as a controlled substance, a new undefined derivative generally appears within a few months.11

Detection of fentanyl and its metabolites in the clinical setting is somewhat lacking as commonly used opiate immunoassays are used to detect morphine and cross react with molecules with similar structures and thus fail to detect synthetic opioids like fentanyl.12

Extensive research has been carried out on fentanyl and on some of its analogues to assess their relative potencies and to identify their metabolic pathways. However, the illicit production of new fentanyl analogues encompasses challenges in the identification of both the parent compound and its metabolites. This review provides an overview of the current state of knowledge of the metabolism of fentanyl and of the structure and metabolism of specific fentanyl analogues. The known mass spectral fragmentation patterns of fentanyl and its

7 metabolites will be used to predict the fragmentation patterns of the analogues and their likely biotransformation products. This will provide a resource which may aid in the detection of new analogues and their metabolites.

1.1 Fentanyl pharmacology and mechanism of action

Fentanyl belongs to the 4-anilidopiperidine class of opioids and was first synthesised by Janssen in 1960. Its generation was the culmination of research on the chemical features common to narcotics with morphine-like activity.13 It exhibits its effects by stimulating the “μ” opioid receptor to produce the desirable pharmacological effects common to other opioids14 including analgesia, euphoria, anxiolysis, feelings of relaxation and drowsiness.15 Undesirable side effects include nausea, dizziness, constipation, vomiting, tolerance, and respiratory depression. These effects can be manifest for up to 3 hours.2,16 Fentanyl derivatives and their precursors can be purchased in kilograms from both conventional websites and the dark web.17 Fentanyl illicitly extracted from transdermal patches, is commonly administered via oral ingestion, intravenous injection, or less commonly, via snorting.18

Fentanyl and its derivatives possess higher lipid solubility than morphine due to the presence of a benzene ring attached to the piperidine molecule.3,19 Like other opioids they exhibit their effects via the inhibition of nociceptive afferent neurons by inhibiting neurotransmitter release.1,20 The decrease in the duration of the calcium ion flux in the formation of an action potential21 is due to the activation of the opioid receptor. This results in the formation of guanine triphosphate (GTP) from guanine diphosphate (GDP) which decreases intracellular cyclic adenosine monophosphate (cAMP) levels via the inhibition of the enzyme adenylate cyclase responsible for cAMP production.1,22 While the mechanism of action does not differ greatly between fentanyl analogues, structural differences can influence potency and metabolic pathways. Therefore, the structure of these analogues will have a significant impact on the

9 metabolites produced. Not all analogues possess divergent metabolic pathways however and this can complicate the identification of a specific analogue when similar metabolic data is present.

1.2 Chemical structure of fentanyl and its derivatives

Approaches to the synthesis of fentanyl and its analogues have undergone significant modification since fentanyl was initially generated by Paul Janssen in 1960.13,23 The optimised synthetic pathway for producing fentanyl now involves a three-step process consisting of the alkylation of the commercially available 4-piperidone monohydrate hydrochloride base (12) using 2-(bromoethyl)benzene and caesium carbonate to create an alkylated piperidone (13);

The second step involves the reductive amination the alkylated piperidone with aniline, sodium triacetoxyborohydride and acetic acid to produce the 4-piperidineamine precursor (14); The final step in the process is the acylation of the 4-piperidineamine using propionyl chloride and

N,N-diisopropylethylamine to create fentanyl (Figure 1). 24

Figure 1. Synthesis of fentanyl and acetylthiofentanyl.24

Since its initial synthesis in the 1960s, the fentanyl nucleus has undergone numerous chemical modifications with a view to the generation of derivatives either for clinical or illicit purposes.

Clinical examples of fentanyl analogues include sufentanil, alfentanil, remifentanil and carfentanil. Illicit modifications, however, are far more numerous and are designed to not only preserve the pharmacological effects of fentanyl but also to circumvent law enforcement screening and legislation25. Modifications to the fentanyl molecule include changes to the aniline portion of the nucleus at R1 which involve the replacement of a hydrogen atom with a fluorine atom; insertion of methoxycarbonyl or methoxymethyl groups into the 4th position of the piperidine ring R2 or an additional methyl group introduced to the 2nd 3rd or 5th positions of the same piperidine ring; replacement of the piperidine ring R2 with a tropine, pyrrolidine or azepine ring and replacement of the phenyl group R5 with other aromatic heterocycles such as tetrazole, thiophene or a carbomethoxy group (Figure 2). 26 Among the fentanyl analogues four of the more prevalent compounds have been selected for discussion in this review.

Acetylfentanyl, Acrylfentanyl, Butyrlfentanyl, and Furanylfentanyl.

Figure 2: Possible modifications to the fentanyl molecule.

Various sites in the structure where modifications are possible.26

1.2.1 Acetylfentanyl, structure and history

Acetylfentanyl is an opioid of the phenylpiperidine class and possesses a single modification in which a phenylacetamide group replaces the a phenylpropanamide group of the fentanyl molecule (Figure 3).27 As this essentially involves the deletion of a methyl group from the propionyl side chain, acetylfentanyl is also known as desmethyl fentanyl. During the synthesis or degradation of fentanyl, acetylfentanyl can also be present as an impurity.28

Figure 3: Comparison of acetylfentanyl and fentanyl.

Removal of a methyl group from the phenylpropamide group in fentanyl creates

acetylfentanyl.27

Acetylfentanyl is synthesised from the same 4-piperidineamine precursor (14) used in the synthesis of fentanyl by dissolving it in methylene chloride and treating it with N,N- diisopropylethylamine; The cooled solution is reacted with acetic anhydride to generate acetylfentanyl; The organic (methylene chloride phase) is then washed with brine, saturated

24 with NaHCO3 and dried over Na2SO4 to isolate the acetylfentanyl in the form of a yellow oil.

Acetylfentanyl has one third of the potency of fentanyl and an LD50/ED50 ratio of 442:1. This compares with 1424:1 for morphine and 10163:1 for fentanyl in mice.32 It is not on the World

Health Organization’s list of essential medicines and does not have any known approved medical or industrial applications.27 It is primarily used illicitly and was associated with fourteen

13 overdose deaths in Rhode Island, USA between March and May, 2013.29 It has also been identified as either the sole cause of death or a component of comorbidity with other drugs in

26 cases of poisoning in Sweden between April and October, 2015,30 and in an overdose death in Western Australia in 2017.31

1.2.2 Acrylfentanyl, structure and history

Acrylfentanyl is an opioid of the phenylpiperidine class in which a propenamide group replaces the propanamide group in the fentanyl molecule 33 (Figure 4).11 Although its synthesis and pharmacological activity were described in 1981,34 acrylfentanyl re-emerged as a designer drug in 2016.11

Figure 4: Comparison of acrylfentanyl and fentanyl.

Alteration of the propanamide group to propenamide creates acrylfentanyl.11

Acrylfentanyl is synthesised from the same 4-piperidineamine precursor (14) used in the synthesis of fentanyl by acylation with acryloyl chloride in the presence of sodium carbonate.35

In vivo studies tp investigate the pharmacological activity of acrylfentanyl in mice demonstrated a 0.76:1 potency ratio (acrylfentanyl:fentanyl) and a 170:1 potency ratio when compared to morphine.34 While fentanyl’s duration of action at doses between 0.1 and 0.5mg/kg decreased considerably 60-70 minutes after administration, acrylfentanyl maintained its effects for 90 to

120 minutes after administration of the same dose.35 A dose of 50mg/kg generated convulsions after one hour post-administration and resulted in 60% lethality occasioned by respiratory depression. In comparison, the LD50 for fentanyl in mice is 62mg/kg.35,32

Acrylfentanyl is not contained in the World Health Organization’s list of essential medicines and has no known approved medical or industrial applications.33 Between April and December

2016, the illicit use of acrylfentanyl was implicated in 47 deaths in the European Union, 43 deaths in Sweden; 1 death in Denmark, and 3 deaths in Estonia. Acrylfentanyl was present in all post-mortem samples.36 In the USA, acrylfentanyl was responsible for at least 83 confirmed fatalities between 2016 and 2017.37

1.2.3 Butyrfentanyl, structure and history

Butyrfentanyl is an opioid of the phenylpiperidine class in which a butyramide group replaces the propanamide group in fentanyl38 (Figure 5).39 Synthesis of butyrfentanyl was first described

15 in 1961 by Janssen in a US Patent40 with the first appearance of illicit butyrfentayl in 2013 from a drug seizure by police in Poland.39

Figure 5: Comparison of fentanyl and butyrfentanyl.

Addition of a methyl group to the propanamide group creates butyrfentanyl.39

Butyrfentanyl is synthesised from the same 4-piperidineamine precursor (14) used in the synthesis of fentanyl via its acylation with butyryl chloride in the presence of sodium carbonate.40 In vivo studies to investigate the potency of n-butyrfentanyl in mice demonstrated a 1.5:1 potency ratio (butyrfentanyl:morphine) and a 0.03:1 potency ratio

(butyrfentanyl:fentanyl) with an effective dose (ED50) of 0.22 mg/kg.32 No experiments were conducted in the study to estimate its lethality. Butyrfentanyl is not listed as an essential medicine by the World Health Organization and has no known medical or industrial applications.38 Illicit butyrfentanyl use has caused at least 40 confirmed fatalities in the United

States with 38 cases in New York, 1 in Maryland, and 1 in Oregon.41 Reported illicit butyrfentanyl use in Europe consists of 3 cases of exposure in Sweden on May 2014 with 1 case of 4F-butyrfentanyl exposure in January 2015.39 This is due to factors such as butyrfentanyl’s lower potency compared to fentanyl, along with the findings resulting from self-report information.39

1.2.4 Furanylfentanyl, structure and history

Furanylfentanyl is an opioid of the phenylpiperidine class in which a heterocyclic furan ring replaces the ethyl group of fentanyl (Figure 6).30 Synthesis of furanylfentanyl was first described in a US patent in 198642 and illicit use of furanylfentanyl was reported in 2015.30,37

Figure 6: Comparison of furanylfentanyl molecule to fentanyl.

Substitution of a furan ring on the carboxamide moiety in place of propanamide generates

furanylfentanyl.30

Furanylfentanyl is synthesised from the same 4-piperidineamine precursor (14) used in the synthesis of fentanyl by acylation with 2-furoyl chloride.42 It has an ED50 of approximately 0.02 mg/kg in mice42 however, because the studies use different routes of administration and different assay methods, an exact potency ratio could not be ascertained. Furanylfentanyl is not recognised by the World Health Organization as an essential medicine nor does it possess any

18 known medical or industrial applications.43 Illicit use of furanylfentanyl was implicated in at least 128 confirmed fatalities in the United States between 2015 and 2016. Thirty-six of these casualties in Illinois, 41 in Maryland, 49 in North Carolina, one in New Jersey and one in Ohio.37

In Europe, 23 deaths were attributed to furanylfentanyl intoxication as confirmed by analysis of post-mortem samples between November 2015 and February 2017. Twelve of these deaths were in Sweden, 4 in Estonia, 4 in Germany and one death in each of Finland, Norway and the

United Kingdom.44

1.3 Methods of identifying fentanyl analogues

There are many fentanyl analogues containing minor structural modifications which are designed to generate a higher level of pharmacological activity. The constant emergence of novel fentanyl analogues poses an analytical challenge for forensic laboratories. The analytical techniques employed by forensic laboratories are usually compliant with the standards established by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG).45

According to SWGDRUG’s recommendations, at least two analytical techniques need be used as the minimum standard. The analytical techniques employed can include immunoassays, gas chromatography-mass spectrometry (GC-MS), liquid chromatography/high-resolution mass spectrometry (LC-HRMS), and liquid chromatography tandem mass spectrometry (LC-MS/MS).46

Due to the constant appearance of new and novel analogues, forensic laboratories have to

19 adapt their analytical methods to keep abreast of trends while maintaining the status quo in the identification of fentanyl analogues and their metabolites in biological specimens.

Immunoassays are a preliminary screening technique commonly used to identify presumptively, if there are drugs in biological specimens.46 Immunoassays employ antibodies that are specific to a protein ligand so as to bind with the target and produce an observable signal.48

While immunoassays can be automated with high throughput to deliver rapid sensitive and cost-effective results, due to structural differences between morphine and synthetic opiates such as fentanyl, the immunoassays used in routine clinical tests often do not cross react with the synthetic analogues.47 It is because of this that immunoassays are often complemented by mass spectrometric analysis to either confirm the presence of an opioid or to detect an analogue not revealed, immunologically.

GC-MS analysis involves the use of ionisation techniques to produce gas-phase ions that are fragmented and then detected in the form of an electrical signal based on its mass to charge ratio (m/z).46 While GC-MS analysis can perform untargeted data acquisition, the acquired mass-spectral data needs to be compared with an MS-library to establish identity.47 GC-MS methods are also incapable of analysing drugs that are polar, non-volatile, or thermally unstable. Consequently, extra procedures such as derivatisation are required which not only take time but increase the cost of the analysis.47 Furthermore, GC-MS techniques are typically characterised by a detection limit of approximately 2 ng/mL while toxicologically significant concentrations of fentanyl and its analogues can occur in body fluids at concentrations below 1

20 ng/mL.49 Due to advancements in liquid-chromatography mass spectrometry (LC-MS) techniques, which have rendered the approach more easily applicable to a wide range of analytes, LC-MS has been increasingly employed as the methodology of choice for the detection and quantification of drugs and their metabolites.50 LC-MS techniques circumvent many of the limitations that typify GC-MS and allow underivatised and thermally labile metabolites to be analysed readily. Of interest is the use of liquid chromatography (LC) in combination with time- of-flight (ToF) mass spectrometry which provides high mass accuracy as an aid in the identification of key metabolites.51

Tandem mass spectrometry (MS/MS) is often used to characterise fragmentation pathways and to determine elemental composition while compensating for LC’s poor chromatographic capacity.50 This is done by using two sequential quadrupole mass filters. The sample, subjected to controlled fragmentation, is transferred into the first mass filter where the parent ion is selected, while the product ion is selected, subsequently, by the second filter.50 For the identification and quantification of fentanyl compounds, a multiple reaction monitoring (MRM) mode is often used.46,50 In this approach, the two quadrupoles alternate between different static mode filter settings to quantify known compounds with high levels of sensitivity. Limits of detection in blood and urine using this method range from 0.01 to 0.5 ng/mL for novel opioid compounds including acetylfentanyl, butyrylfentanyl furanylfentanyl and the major metabolite norfentanyl.46 For discovering and identifying relevant compounds using a non-targeted approach, liquid chromatography quadrupole time of flight (LC-QTOF) is a valid option.51 The ions generated by the source are accelerated into a flight tube using an electric field. Rate of

21 movement is dependent upon weight and charge with smaller ions travelling faster than ions with a similar charge but greater molecular mass.50 The advantage of this method is its capacity to collect fragmentation and precursor data with high resolution which can then be used to characterise the structure of metabolites.51

LC-MS possesses many advantages over GC-MS in the identification of fentanyl and its analogues. This is due, in part, to the capacity of LC-MS to identify metabolites that are thermally labile, but which have not undergone derivatisation.51 Furthermore, LC-MS employs ionisation methods, such as electrospray ionisation (ESI), which have a lower dependence on volatilisation. This reduces unnecessary fragmentation and generates results that are easier to interpret.52 Characterisation of metabolites using MS/MS and QTOF-MS, enables generic patterns to be identified which can then be used to predict the fragmentation of novel fentanyl analogues.

1.4 Metabolism of fentanyl and analogues

Fentanyl is a widely used synthetic opioid with a broad spectrum of applications from general anaesthesia to chronic pain management. Because of this, rigorous testing has been carried out on animal and human subjects to define its kinetics; how it interacts with the body, and how it is excreted.53,54 Fentanyl undergoes rapid and extensive biotransformation predominantly in the liver, and its metabolites can be detected in the plasma within 2 minutes of administration.

Fentanyl has a short half-life with 98.6% of the dose being eliminated from the plasma within an hour. Less than 8% is excreted unchanged53 and its biotransformation in the liver and intestine is largely derived from the activity of cytochrome P450 3A4 (CYP 3A4).54

Studies of the metabolic fate of illicit fentanyl analogues in humans encompass ethical and safety issues and metabolic data can be best obtained from in vitro experimentation using human liver microsomes,55 supplemented with information derived from post mortem samples.56 Because of their structural similarities to the parent drug, acetylfentanyl, acrylfentanyl, butyrfentanyl and furanylfentanyl are metabolised via the same CYP3A4 pathway.57 Though butyrfentanyl is also metabolised by CYP2D6.58

1.5 Phase I and Phase II Metabolism of Fentanyl

Due to the rapid biotransformation of fentanyl and its analogues,53 identification of a specific analogue, used either medically or illicitly, relies upon detailed knowledge of its metabolic fate.58 Fentanyl was designed to be highly lipophilic, to enable it to transverse membranes and physiological gateways such as the blood brain barrier, rapidly.3 Excretion of xenobiotics in the urine requires drugs to be hydrophilic so the elimination of fentanyl via the kidney entails its biotransformation to more polar metabolites.16

The metabolism of xenobiotics often encompasses two stages.60 Phase I metabolism involves the modification of the parent molecule via reduction, oxidation, decarboxylation, deamination, dehalogenation or hydroxylation to, in most cases, render the drug less biologically active and to prepare it for the conjugation reactions of Phase II. Of these reactions, hydroxylation, catalysed by members of the cytochrome P450 super-family of enzymes, is particularly important. Introduction of the hydroxyl group often provides a conjugation site for the Phase II reactions.60 Phase II metabolism involves the conjugation of the drug, often after modification in a Phase I reaction, with either hydrophilic or hydrophobic groups to render it soluble in the urine for excretion via the kidneys or soluble in the bile for excretion in the faeces. Fentanyl metabolism, however, does not appear to involve Phase II conjugation reactions. Instead its metabolic modification involves only Phase I biotransformations.55

Mediated by the microsomal monooxygenase, cytochrome P450 3A4, located in the liver and small intestine,57 fentanyl is metabolized via several pathways (Figure 7).59

Figure 7: Metabolic pathways depicting possible metabolites of fentanyl. Derived from Iula.59

Among the metabolites, norfentanyl (2) is the most abundant. 99% of the fentanyl in the incubation mixture was converted to norfentanyl at a substrate concentration of 100 μM,55 with despropionylfentanyl (4) and hydroxyfentanyl (9) being minor products. All 3 metabolites are inactive.47 Hydroxynorfentanyl (3,10) is an example of a secondary metabolite generated by

25 further hydroxylation of norfentanyl (2) or by N-dealkylation of hydroxyfentanyl (9). For most of the hydroxylated compounds (3, 5, 7, 8, 9, and 10) the exact location of the added hydroxyl group is unknown.59 Instead, theoretical sites are shown. Many other theoretical metabolites are possible but none have been detected in vitro or in vivo studies.17,54,55,61 Consequently, these have not been documented.

CHAPTER 2: Discussion

Identifying the biochemical pathways associated with the metabolism of fentanyl analogues is important from both a forensic and clinical perspective as the identification of metabolites derived from specific analogues can be useful in helping to identify the analogue administered.

An increased understanding of the pathways involved, and the metabolites produced can act as a resource for the development of immunoassays designed to target specific analogues.

Research into the metabolism of fentanyl analogues is often reactive rather than proactive because an analogue must appear before its metabolism can be studied. Several reports have appeared in the literature which have provided a generic overview of fentanyl, its pharmacokinetics, and its metabolism.16,35,47 Consequently, using these studies as a basis, the following section will focus on the metabolism of the more common fentanyl analogues in an attempt to predict the likely metabolites derived from these compounds. As the metabolism of fentanyl analogues often differs between species, wherever possible, human in vivo and in vitro studies will be prioritised over animal studies.

2.1 Metabolism of Acetylfentanyl

Due to its structural similarity to fentanyl, it would be reasonable to assume that acetylfentanyl primarily follows the same oxidative N-dealkylation pathway described for fentanyl. Multiple studies on the metabolism of acetylfentanyl have produced mixed results.61,62,63 In a study performed by Melent’ev et al. in 2015, urine obtained from individuals who had consumed

27 acetylfentanyl was provided by the Chelyabinsk Regional Bureau of Forensic Medical

Examination. Samples were subjected to derivatisation in preparation for GC-MS analysis.62

Following SWGDRUG recommendations which require at least 2 analytical methods to be employed,45 high performance liquid chromatography mass spectrometry (HPLC-MS) as well as

GC-MS was performed on the samples. The major metabolic pathway identified in this study involved hydroxylation of the phenylethyl moiety, in contrast to the main N-dealkylation pathway previously described for fentanyl.62

In a later study published in 2017 by Watanabe et al., acetylfentanyl was incubated with human hepatocytes in vitro.61 Urine samples were also obtained from acetylfentanyl overdose cases

(which were confirmed by analysis blood samples) to provide an in vivo viewpoint of acetylfentanyl metabolism. In this study, 32 metabolites were identified and their pathways of production elaborated (Figure 8).61

Figure 8: Proposed pathways of acetylfentanyl metabolism, with primary metabolites shown

enclosed in boxes (after Watanabe et al.61)

In the hepatocytes sample, seven different metabolites were detected (A3, A4, A6, A18, A24,

A26, A30) with noracetylfentanyl (A3) being the most abundant. This was generated by N- dealkylation of the piperidine nitrogen resulting in the removal of the phenethyl moiety. The next most abundant metabolites were (A24) which was hydroxylated at the ethyl linker, and the dihydrodiol metabolite (A4) generated by the modification of the phenylethyl ring.61 In the urine samples, the most abundant metabolite was the hydroxymethoxy derivative (A26) formed by dihydroxylation followed by methylation at the meta position. Noracetylfentanyl

(A3) and the hydroxylated metabolite (A24) were also significant. These results support the

29 findings of Melent’ev et al. in that the nor-metabolic pathway is not the main metabolic route.62

In another study performed in 2018 by Kanamori et al. the capacity of human iPS cell-derived hepatocytes (h-iPS-HEP) to metabolise fentanyl and acetylfentanyl was compared with that of human primary hepatocytes (h-PRM-HEP).63 Supernatants derived from the incubation mixtures were analysed by LC-ion trap MS in both scan and product ion analysis mode. Metabolites of acetylfentanyl identified in both cell types included the main metabolite nor-acetylfentanyl, and the minor metabolites 4’-hydroxy-acetylfentanyl, hydroxyacetylfentanyl, and β-hydroxyacetylfentanyl. Conversely, 4’-hydroxy-3’-methoxy-acetylfentanyl was only detected in the h-

PRM-HEP medium.63 Though the pathways of acetylfentanyl metabolism were clarified in general terms by Melent’ev et al., the exact structures of the hydroxylated metabolites was not elucidated.62 Furthermore, the study did not investigate in vivo biotransformation via the analysis of urine or blood derived from acetylfentanyl users.63 Consequently the results should be interpreted cautiously because of potential differences between in vitro and in vivo pathways. While β-hydroxy-acetylfentanyl was detected in both h-iPS-HEP and h-PRM-HEP samples, it has not, thus far, been detected in vivo.61,62

2.2 Metabolism of Acrylfentanyl

Acrylfentanyl differs from fentanyl in that it contains an unsaturated double bond in the amide group.33 Apart from a review of its general properties64, only two primary studies have been published on acrylfentanyl, one designed to investigate its analgesic properties and another on its metabolic fate.35,61 Because of the structural similarities btween fentanyl and acrylfentanyl it is likely that the metabolism of the latter mirrors that of the parent compound in that the oxidative N-dealkylation pathway is predominant.

In the study conducted by Watanabe et al.61, human hepatocytes were incubated with acrylfentanyl in vitro in an attempt to elucidate its metabolic fate.61 Urine was also obtained from overdose victims in an attempt to correlate in vivo and in vitro results. A total of 14 metabolites were identified, and the suggested pathways by which these are derived are illustrated in Figure 9.61

Figure 9. Proposed pathways of acrylfentanyl metabolism as determined from human

hepatocyte studies in vitro and from the analysis of human urine generated in vivo. Major

metabolites are enclosed in boxes.61

Among the 14 metabolites identified, eight were derived from hepatocyte metabolism (B1, B2,

B6, B9, B10, B11, B13, B14). Of these, the most abundant were noracrylfentanyl (B1) formed by

N-dealkylation of the piperidine nitrogen, and two hydroxylated metabolites (B9) which was generated by hydroxylation at the ethyl link, and (B13), formed by hydroxylation within the piperidine ring.61 In the hydrolysed urine samples, noracrylfentanyl (B1) was found to be the major metabolite, followed by the hydroxylated metabolite (B9), and a dihydroxy metabolite

(B8) which contained a hydroxyl group at the ethyl link, and another on the adjacent phenyl group.61 However the quantification must be interpreted with caution as three glucuronides

(B3, B4, and B5) were also identified, which would have contributed to the quantification of the free mono (B8 and B9) and dihydroxylated (B11) metabolites.61 While Phase I and Phase II metabolites were identified in this study, further in vivo and in vitro studies are required to confirm the results.

2.3 Metabolism of Butyrfentanyl

Butyrfentanyl’s differs from fentanyl in that it contains an extra methyl group in the amide side chain converting it from a propanamide to a butyramide moiety.38 Because of its structural similarity to fentanyl, it is likely that butyrfentanyl will be metabolised in like fashion by N- dealkylation. Two studies have been reported on the metabolism of butyrfentanyl.56,58 Staeheli et al.56, analysed post mortem samples derived from a case of butyrfentanyl intoxication using

LC-MS/MS, and LC-HRMS. Hydroxybutyrfentanyl was found to be the major metabolite, while carboxybutyrfentanyl, desbutyrfentanyl and norbutyrfentanyl were found to be present in smaller amounts.56 Metabolites were in highest concentration in the liver, urine and kidney.

Hydroxybutyrfentanyl was the most abundant metabolite in every tissue except blood nine hours after death and its concentration was still greater than that of carboxybutyrfentanyl 28 hours after death.56 Desbutyrfentanyl and norbutyrfentanyl, on the other hand were generally much lower in concentration but were slightly more abundant in the kidney and liver.56

In another in vitro study performed by Steuer et al., using human liver microsomes (HLM) the metabolic fate of butyrfentanyl was investigated and the results compared with those derived from the in vivo study of Staeheli et al. 58,56 Using LC-QTOF and HRMS analysis, the potential metabolic pathways suggested by the authors are illustrated in Figure 10.58

Figure 10. Proposed metabolic pathways of butyrfentanyl in vivo and in vitro.58

Identification of the metabolites detected in vitro led to the postulation of 6 possible interacting pathways involving (a) hydroxylation of either the phenethyl moiety, the piperidine ring, or the butanamide side chain (b) N-oxide formation (c) elimination of butyraldehyde to form desbutyrfentanyl (d) N-dealkylation to form norbutyrfentanyl (e) oxidation of hydroxylated metabolites to the corresponding carboxylic acids, and (f) dihydroxylation and methylation of the phenethyl-hydroxy metabolite.58 The main metabolites detected in vitro were alkyl-hydroxy butyrfentanyl (M25), norbutyrfentanyl (M10), and phenethyl-hydroxy butyrfentanyl (M30). However, in vivo, norbutyrfentanyl (M10) was detected at very low levels, while carboxybutyrfentanyl (M14), alkyl-hydroxy butyrfentanyl (M25), and carboxy-pehenthyl- hydroxy-butyrfentanyl (M13) were more prominent.58 Though these findings differs from those reported for fentanyl metabolism,55 for which the nor-metabolite is the primary metabolic product, they are compatible with the findings of Staeheli et al. who reported concentrations of norbutyrfentanyl, much lower than those of carboxybutyrfentanyl and hydroxybutyrfentanyl.56

2.4 Metabolism of Furanylfentanyl

Furanylfentanyl is the most structurally variant analogue amongst the fentanyl derivatives addressed in this review. It contains a heterocyclic furanyl ring instead of the ethyl group that is common to the other analogues.43 Because of this, it is unlikely that furanylfentanyl will be subjected to the same N-dealkylation pathway described for the analogues discussed earlier.

Two studies have been published on the metabolic fate of furanyl fentanyl and the metabolites generated.61,65 In the study conducted by Goggin et al., 500 urine samples testing positive for

36 heroin metabolites were screened for the presence of furanylfentanyl via LC-MS/MS.

Approximately 10% returned a positive result.65 Urine samples containing furanylfentanyl were then analysed by ultra-performance liquid chromatography time of flight mass spectrometry

(UPLC-TOF-MS) before and after incubation with glucuronidase to detect and identify Phase I and Phase II metabolites. Among the metabolites identified was a fentanyl precursor 4-anilino-

N-phenethyl-piperidine (4-ANPP) detected as the sulfate conjugate (M1, Figure 11) and a free dihydrodiol metabolite (M2, Figure 11).65

Figure 11: Proposed metabolites of furanylfentanyl.65

Among the 51 positive results for furanylfentanyl, 41 samples contained free 4-ANPP; 42 contained its sulfate conjugate (M1) and 44 contained the dihydrodiol metabolite (M2).

However, only 4 samples contained the N-dealkylated metabolite, furanylnorfentanyl, which was present at concentrations 10-100-fold lower than those found for the corresponding N- dealkylated product typical of furanylfentanyl metabolism.65

In another study conducted by Watanabe et al., human hepatocytes were incubated with furanylfentanyl to investigate its metabolic fate in vitro.61 These results were correlated with those obtained from the analysis of urine samples acquired from overdose victims with a view to identifying differences between the in vivo and in vitro biotransformation pathways.

Fourteen metabolites were identified overall, and their metabolic relationships are illustrated in

(Figure 12).61

Figure 12. Proposed pathways of furanylfentanyl metabolism as determined from human

hepatocyte studies in vitro and from the analysis of human urine generated in vivo. Major

metabolites are enclosed in boxes61.

Incubation of hepatocytes with furanylfentanyl resulted in the formation of almost every metabolite displayed in Figure 12 except the glucuronide conjugate (D3). The most abundant

38 metabolites were 4-ANPP (D14), produced by amide hydrolysis; the nor-metabolite (D6) formed by N-dealkylation of the piperidine nitrogen and the dihydrodiol metabolite (D10) formed by epoxidation of the furan ring followed by hydration.61 In the urine samples the most abundant metabolites were 4-ANPP (D14), the dihydrodiol metabolite (D10), and the hydroxylated sulfate conjugate (D5). This is generated by a sequential Phase I and Phase II reaction. 4-ANPP is hydroxylated in a Phase I reaction to generate hydroxy 4-ANPP which is then conjugated with phosphoadenosine phosphosulfate in a Phase II reaction.61 These findings support those of

Goggin et al. in that N-dealkylation to produce a nor-metabolite is not the major pathway of furanylfentanyl metabolism.65

While both studies indicated that the main metabolic pathway of furanylfentanyl involves amide hydrolysis to generate 4-ANPP and also involves production of a dihydrodiol metabolite, there is uncertainty as to the site(s) of sulfate conjugation. Goggin et al., propose that sulfation is associated with the hydroxyl groups attached to the furan ring, while Watanbe et al. propose

61,65 that sulfate conjugation is associated with the hydroxyl group of hydroxylated 4-ANPP.

Further in vivo and in vitro experimentation is required to ascertain the site or sites of sulfate conjugation.

2.5 Summary of fentanyl analogue metabolism

The Phase I pathways associated with the metabolism of fentanyl and of selected “fentalogues” have been described. These involve N-dealkylation, hydroxylation, oxidation, dihydrodiol

39 formation, dihydroxylation and methylation reactions. While N-dealkylation to generate a nor- metabolite is the primary pathway associated with fentanyl metabolism, this biotransformation does not apply to all of its analogues. Though acetylfentanyl and acrylfentanyl are metabolised primarily by this pathway, butyrfentanyl and furanylfentanyl are metabolised by alternative routes. Instead of being subjected to N-dealkylation, butyrfentanyl primarily undergoes hydroxylation, or hydroxylation/oxidation reactions. In furanylfentanyl metabolism, reactions tend to be focused on the furan ring and involve amide hydrolysis, or epoxidation and hydration. While fentanyl is not known to participate in any significant Phase II reactions, its analogues, acrylfentanyl and furanylfentanyl undergo sulfation and glucuronidation. However, further research is required to elucidate the extent to which fentanyl analogues participate in

Phase II conjugation reactions and whether detection of these metabolites in the urine will assist in the identification of an administered analogue.

CHAPTER 3: Fentanyl analogue fragmentation patterns

Knowledge of fragmentation pathways is very important in the identification and characterisation of fentanyl analogues. Though many fentanyl analogues possess only minor structural modifications when compared to fentanyl, itself these differences can generate vastly different effects. However, because of the structural similarities, the mass-spectrometric fragmentation patterns and hence the ions produced are often similar or even identical. While this can render a distinction between similar analogues difficult, the identification of commonalities in the fragmentation pathway can be used to predict the structure of an unknown analogue based upon the m/z values of the ions generated. Literature is already available on the mass-spectrometric fragmentation patterns generated by a range of fentanyl analogues.46,58,61,62,65 In most publications, liquid chromatography coupled to a time of flight system was used to identify ions formed from the metabolites. The aim of this section is to collate the published fragmentation data relevant to fentanyl and to the four named fentanyl analogues discussed in this review.

3.1 Fragmentation patterns of fentanyl

As fentanyl and its analogues undergo similar mass-spectral cleavages, the ions generated from the fragmentation of fentanyl itself can help to predict the ions generated by its analogues and their metabolites. Because the structural modifications that characterise the four relevant fentanyl analogues are associated with the amide region, ions generated from elsewhere in these molecles should reflect the corresponding fragmentations seen for fentanyl itself. This is

41 supported by the fact that fentanyl analogues often generate ions with m/z values identical to those produced by fentanyl despite the precursor ions having different masses.46,66 Noble et al. investigated the fragmentation of fifty 4-ANPP fentanyl analogues using ultra high-performance liquid chromatography (UHPLC) followed by HR-MS performed in an orthogonal accelerator

QTOF.66 Four fragmentation pathways characteristic of fentanyl itself, were found to be applicable to the 4-ANPP fentanyl analogues investigated (Figure 13).66

Figure 13: Proposed fragmentation pathway of fentanyl and associated 4-ANPP analogues.66

The first pathway involves the removal of the amide group via cleavage of the nitrogen- carbonyl carbon bond (cleavage a, Figure 13)66 resulting in the generation of 4-ANPP (m/z

281.2025).61 While this C-N bond cleavage was characteristic of more than fifty percent of the investigated compounds, the cation signal intensity was very low in all cases, rendering it unsuitable as a means of identification of fentanyl analogues particularly when present at low concentrations.66

The second pathway involves the cleavage between the piperidine ring and the adjacent nitrogen (cleavage b, Figure 13). In molecules that had no substitutions in the piperidine ring, a carbocation with m/z 188.1431 is formed, while in molecules that lacked a phenylethyl tail

(norfentanyl, acetylnorfentanyl) the same fragmentation resulted in an m/z peak of 84.0805

66 corresponding to the unsubstituted piperidine ring (Figure 14). Further fragmentation (b1,

Figure 13)involving cleavage between the piperidine nitrogen and the adjacent carbon

66 generated a phenylethyl ion (C8H9; m/z 105.0699). This fragmentation was common to all 4-

ANPP fentanyl analogues, so the presence of this ion proved to be characteristic of this group.

Figure 14: Piperidine ion. Adapted from Noble et al.66

The third pathway is characterised by the presence of the second product derived from cleavage b (see b/c, Figure 13). For fentanyl itself, the product is an ion with m/z 150.0913

(Figure 13), while for acetylfentanyl, acrylfentanyl, butyrfentanyl and furanylfentanyl, ions of m/z 136.0757, 148.0757, 146.0964, and 188.0706 are generated respectively.66 For acetylfentanyl, acrylfentanyl and furanylfentanyl, the m/z value of these ions simply reflect the structural differences between these analogues and fentanyl itself at R1, Figure 15. However, it appears that the corresponding ion generated from butyrfentanyl is particularly susceptible to dehydration such that an ion with a lower m/z value is generated via the type of transformation shown in c1, Figure 13.66

Figure 15: Product ion of the four fentanyl analogues generated by cleavage b/c

Structural differences between analogues at R1 alter the m/z value accordingly.67

The fourth pathway corresponds to a degradation of the piperidine ring with cleavages on both sides of the carbon to nitrogen bonds in the ring (see d Figure 13). Fifty percent of the targeted analogues generated product ions via this fragmentation pathway.66 Fentanyl itself generated an ion with m/z 216.1383 (Figure 13) while acetylfentanyl, acrylfentanyl, butyrfentanyl and furanylfentanyl generated ions with m/z 202.1226, 214.1223, 230.1539, and 254.1176 respectively (Figure 16).66

Figure 16: Product ion of the four fentanyl analogues generated by cleavage d

Structural differences between analogues at R1 alter the m/z value accordingly.67

3.2 Fragmentation patterns of fentanyl analogues

A separate study was conducted into the fragmentation of fentanyl analogues by Qian et al.67 in which GC electron ionisation mass spectrometry (EI-MS) and UPLC-Q-TOF MS were used in collision-induced dissociation (CID) mode. The main fragmentation route identified was the cleavage of the piperidine ring from the amide moiety.61,66,67 This resulted in the formation of a product ion of m/z 188.1434 (E, Figure 17). It was suggested that this fragmentation was characteristic of fentanyls that do not possess piperidine or phenylethyl modifications.

However, dissociation of the piperidine ring from the phenethyl moiety was also observed, generating an ion of m/z 105.0699 (D, Figure 17). This ion, was second only to E in abundance in the spectra of the analogues analysed.66 Degradation of the piperidine ring resulted in the generation of product ions B and F (Figure 17) and these products were detected in all spectra.67 Product ion G (Figure 13) was also observed with an m/z value dependent upon modifications to the amide group as described by Nobel et al.66 (Figure 17). Removal of the amide group by scission of the nitrogen carbonyl bond resulted in the generation of ion A

(Figure 17). However, this fragmentation was not found to represent a common pathway. It was only observed for butyrfentanyl and was detected at very low intensity.66,67

Figure 17: Proposed fragmentation pathways for fentanyl analogues.67

3.3 Summary of fragmentation patterns

The fragmentation of fentanyl and the selected four analogues is well documented in the literature across multiple publications.56,58,61,62,66 The m/z values expected from the fragmentation of fentanyl and the four analogues discussed in this review were collated in

(Table 1). This is to provide a quick resource that represents a blueprint of ions to look for when attempting to identify the presence of one of the discussed analogues.

Table 1: Summary of the names, formula and mass-to-charge ratio of the investigated fentanyl analogues.56,58,61,62,66

No. Parent molecule Parent molecular formula Mass-to-charge ratio m/z

1. Fentanyl C22H28N2O 105.0699 134.0964 150.0913 188.1434 216.1383 281.2025 2. Acetylfentanyl C21H26N2O 91.0542 119.0490 151.0758 234.1487 3. Acrylfentanyl C22H26N2O 103.0541 121.0646 204.1384 4. Butyrfentanyl C23H30N2O 105.0694 188.1436 281.2023 5. Furanylfentanyl C24H26N2O2 105.0697 188.1433

CHAPTER 4: Conclusion

This review was conducted for the purpose of providing a more comprehensive resource on the current state of knowledge surrounding the metabolism of fentanyl and some of its more common analogues along with their mass-spectral fragmentation profiles. The structural similarities between fentanyl and many of its analogues provide a unique challenge as they also result in similar mass spectral peaks complicating identification. Further tests and studies are required to identify metabolic and fragmentation pathways in newer more heavily modified analogues while also consolidating knowledge on currently known analogues and their metabolites for greater ease of identification via current analytical methods. For every new analogue that is created and distributed, a new publication is published to elaborate on its potency, dosage limitations and potential routes of metabolism. However, validation studies to verify the consistency of these publications to make certain that the proposed metabolites are indeed produced in biological matrices are often rare; as lack of repeat studies lowers reliability because data obtained has yet to be reproduced. Another issue with the experimental results is the source of the data itself. Due to ethical reasons, scientists cannot test live humans with substances that have no medical relevance. Thus data on many of fentanyl’s analogues are obtained from animal studies which have different metabolic pathways from humans, human hepatocytes that provide an accurate in vitro representation of liver metabolism but do not always show the same metabolic pathways as in vivo metabolism, or human urine samples obtained forensically or medically with often no data on the amount ingested, time of

49 ingestion, or time of sample collection which all have an effect on the type and quantity of metabolites observed.

Identification of fentanyl analogues is often done by mass spectrum analysis and thus data on mass-spectral fragmentation patterns are essential. The fragmentation and metabolic pathways identified in this review were most often done using HPLC-ESI coupled with QTOF MS with a focus on CID fragmentation pathways. There is no shortage of data on said fragmentation pathways however said data is widespread across many different publications that often publish data on a single analogue with publications that publish data on multiple analogues simultaneously in the minority. While this review attempts to consolidate the information on known metabolic and fragmentation pathways, future efforts to collate the current literature would prove useful in summarising the fragmentation patterns of fentanyl analogues and their metabolites.

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63. Kanamori T, Togawa IY, Segawa H, Yamamuro T, Kuwayama K, Tsujikawa K, et al. Metabolism of Fentanyl and Acetylfentanyl in Human-Induced Pluripotent Stem Cell- Derived Hepatocytes. Biological and Pharmaceutical Bulletin 2018; 41(1):106-14

64. Ujvary I, Jorge R, Christie R, Le Ruez T, Danielsson HV, Kronstrand R. et al. Acryloylfentanyl, a recently emerged new psychoactive substance: a comprehensive review. Forensic Toxicology 2017; 35(2):232-43

65. Goggin MM, Nguyen A, Janis GC. Identification of Unique Metabolites of the Designer Opioid Furanyl Fentanyl. Journal of Analytical Toxicology 2017; 41(5):367- 75

66. Noble C, Dalsgaard PW, Johansen SS, Linnet K. Application of a screening method for fentanyl and its analogues using UHPLC-QTOF-MS with data-independent acquisition (DIA) in MSE mode and retrospective analysis of authentic forensic blood samples. Drug Testing and Analysis 2017; 10(4):651-62

67. Qian ZH, Li P, Zheng H, Liu CM. Mass Fragmentation Characteristics of Fentanyl Analogues. Journal of Mass Spectrometry Society 2018; 39(5):584-92

Part Two: Manuscript

A review of synthetic fentanyl metabolism and the metabolism

of select synthetic fentanyl analogues

A review of synthetic fentanyl metabolism and the metabolism of select synthetic fentanyl analogues

Gerard Lee1, Bob Mead1

1 Murdoch University, School of Veterinary and Life Sciences, Perth WA.

Abstract

Fentanyl is a fast acting, potent synthetic opioid. The distribution of illicit fentanyl derivatives is a widespread problem across multiple continents. The detection and identification of novel fentanyl analogues poses a challenge to forensic laboratories due to minor modifications made to the fentanyl nucleus. This publication collates the known mass-spectral fragmentation patterns of prevalent fentanyl analogues. This is to identify common patterns that can be used in the structural characterisation of novel fentanyl derivatives and assist in the development of better detection methods for fentanyl analogues.

Keywords: Fentanyl; Fentanyl analogues; Fentanyl fragmentation patterns; Metabolism;

Synthetic opioid;

1.0 Introduction

Fentanyl and its derivatives belong to the family of compounds called opioids. Naturally occurring opiates and synthetic opioids have been used, medically for their anaesthetic and analgesic properties but have also been abused, illicitly, because of their euphoric effects.

Opioids activate opioid receptors which inhibit the release of neurotransmitters in the nervous system specifically in areas related to pain transmission.1 Fentanyl was first synthesised by Paul

Janssen in 1960 in an attempt to generate an opioid which was not only more effective than morphine as an analgesic but which possessed a higher therapeutic index and displayed fewer adverse side effects.2 Though fentanyl has delivered substantial medical benefits, it has also been widely abused. Mortality data from the National Vital Statistics System (NVSS) in the

United States reveals that 70,237 deaths were caused by drug overdose in 2017.3 Synthetic narcotics including fentanyl and fentanyl analogues were the most significant causative group, being implicated in 28,466 deaths. This represented a 45% increase over the previous year.3,4

Fentanyl analogues are derivatives of fentanyl which, possess subtle modifications and substitutions so as to create a wide variety of compounds. Often these modifications are designed to circumvent attempts at detection. Illicit fentanyl derivatives and their precursors can be purchased in kilogram quantities from both conventional websites and the dark web.5

To counteract this, analytical techniques capable of detecting and characterising new analogues together with processes which provide a toxicological risk assessment are essential. Because the structural alterations typical of fentanyl analogues are often minimal, characterisation of new analogues represents an analytical challenge.6 For isomeric forms, separation by liquid

60 chromatography (LC) or gas chromatography (GC) is required prior to mass spectrometric analysis as isomers cannot be distinguished by mass spectrometry (MS) alone.7

Common methodologies used to identify and characterise fentanyl analogues include gas chromatography-mass spectrometry (GC-MS), liquid chromatography tandem mass spectrometry (LC-MS/MS) and liquid chromatography high resolution mass spectrometry (LC-

HRMS).8 The integration of collision induced dissociation (CID) fragmentation with mass spectrometry can not only help to elucidate the structure of the analogue but can also provide information on its pathways of metabolism. For identifying compounds without a known standard, liquid chromatography quadrupole time of flight mass spectrometry (LC-QTOF-MS) is a viable option for novel analogues that have been newly synthesised.9 Though information on the fragmentation patterns of fentanyl analogues exists, the data is distributed across many publications many of which focus on the characterisation of a single analogue or on the most recent analogues that have emerged. There is a need, therefore, for a unified resource that compares the fragmentation pathways of known fentanyl analogues which can then be used to assist in the identification of novel compounds.

This publication consolidates the mass spectral fragmentation patterns of specific fentanyl derivatives and their metabolites in a way that allows the data to be extrapolated to novel analogues to assist in their identification.

2.0 Structure of fentanyl and analogues

Fentanyl analogues often possess minor modifications which render them structurally similar to the original fentanyl molecule. Whether the new analogues are generated for medical or for illicit purposes, the aim is to produce drugs that have similar or more profound pharmacological effects (analgesia, euphoria, anxiolysis, relaxation and drowsiness) than fentanyl itself.11

Fentanyl obtained illicitly is often administered via oral ingestion or intravenous injection, while less commonly administered via snorting.12

Modifications can be made at several positions on the fentanyl molecule while preserving its pharmacological effects. These additions and substitutions result in many potential analogues being produced which results in law enforcement taking on a reactive role when it comes to new derivatives.13 Modifications to fentanyl can occur in several positions on the molecule itself via a substitution at the phenyl ring R1, substituents into the piperidine ring R2, alterations to

10 the phenylethyl group at R3, R4 and R5, or changes to the propionyl group (Figure 1).

Figure 1: Possible modifications to the fentanyl molecule.10

Clinical examples of fentanyl analogues include sufentanil, alfentanil, remifentanil and carfentanil. Among the numerous illicit fentanyl analogues four of the more prevalent compounds have been selected for analysis to identify common metabolic pathways that can be applied to newly discovered fentanyl analogues (Figure 2).14 Acetylfentanyl, acrylfentanyl and butyrfentanyl contain minor modifications in the form of deletions, alterations and additions to the phenylpropanamide group respectively while furanylfentanyl replaces the ethyl group with a furan ring.14,15,16,17

Figure 2: Structures and chemical formulae of fentanyl and select analogues with year of

appearance. Image adapted from Jannetto et al.18

3.0 Fentanyl metabolism

Metabolism of fentanyl occurs mainly in the liver and intestine via a specific enzyme within the cytochrome P450 superfamily, cytochrome P450 3A4 (CYP 3A4).19 The main metabolic reaction catalysed by this enzyme is N-dealkylation to produce norfentanyl.20 Therefore identification, in vivo and in vitro, of metabolites generated from fentanyl derivatives by similar reactions may assist in the identification of an administered analogue.

3.1 Metabolism of Acetylfentanyl

The primary metabolic pathway of acetylfentanyl in vivo involves hydroxylation, and dihydroxylation followed by methoxylation, while N-dealkylation constitutes a minor

64 pathway.21,22 However, in vitro incubation of acetylfentanyl with human iPS cell-derived hepatocytes (h-iPs-HEP) generated, via N-dealkylation, nor-acetylfentanyl as the main metabolite while hydroxyacetylfentanyl and 4’-hydroxy-3’-methoxy-acetylfentanyl were generated as minor products.22,23 A comprehensive study conducted by Watanabe et al.22 identified a total of 32 metabolites that were generated by a combination of N-dealkylation, hydroxylation, dihydroxylation, carbonylation, methylation, and amide hydrolysis. Some of the products were then further metabolised by the Phase II pathways, glucuronidation and sulfation (Figure 3).

Hydroxylation of acetylfentanyl is most likely to occur at the phenethyl moiety but hydroxylation followed by methylation can also occur on the phenyl ring as well.21,22 Watanabe et al.22 reported significant amounts of the unmetabolised drug in all samples indicative of a slow rate of metabolism.

Figure 3: Proposed biotransformation of acetylfentanyl as determined from metabolic studies in in human hepatocyte and analysis of urinary metabolites, (primary metabolites shown in enclosed boxes). Adapted from Watanabe et al.22

3.2 Metabolism of Acrylfentanyl

The metabolism of acrylfentanyl was reported in a single study by Watanabe et al.22 in an attempt to elucidate its metabolic fate in vitro, human hepatocytes were incubated with 66 acrylfentanyl, while urine obtained from overdose victims was analysed to assist in the determination of its biotransformation in vivo. Watanabe et al.22 found that, as for fentanyl, the most dominant metabolic pathway in both in vivo and in vitro was N-dealkylation resulting in the generation of noracrylfentanyl. Minor products included a hydroxylated metabolite, a dehydroxylated metabolite, and a dihydroxylated methylated metabolite.22 In total, Watanabe et al.22 identified 14 metabolites produced by a single reaction or by a combination of N- dealkylation, amide hydrolysis, hydroxylation, dihydroxylation, dihydrodiol formation, or dihydroxylation with methylation. Hydroxylated products were then potentially converted to glucuronides via Phase II glucuronidation (Figure 4). Three Phase II glucuronides were detected which could all be hydrolysed to generate the most abundant minor metabolites.22

Hydroxylation and subsequent methylation was more likely to occur at the phenethyl moiety.22

Watanabe et al.22 also reported a significant amount of the unchanged drug in all samples indicative of a slow rate of metabolism.

Figure 4: Proposed biotransformation of acrylfentanyl as determined from metabolic studies in human hepatocyte and analysis of urinary metabolites (primary metabolites shown in enclosed boxes). Adapted from Watanabe et al.22

3.3 Metabolism of Butyrfentanyl

The metabolism of butyrfentanyl has been studied both in vivo and in vitro using post-mortem samples to assess in vivo biotransformation and human liver microsomes (HLM) to evaluate its biotransformation in vitro.24,25 The main metabolite identified both in vivo and in vitro was hydroxybutyrfentanyl. Minor metabolites detected in vivo included carboxybutyrfentanyl, desbutyrfentanyl and norbutyrfentanyl while in vitro norbutyrfentanyl and several structural isomers of hydroxybutyrfentanyl were the predominant products.24,25 In the in vitro study conducted by Steuer et al.25, 24 metabolites were reported generated from a range of interacting pathways that included hydroxylation, N-oxide formation, elimination of butyraldehyde, N-dealkylation, oxidation of hydroxy metabolites and dihydroxylation followed by methylation (Figure 5).

It was found that hydroxylation of butyrfentanyl was more likely to occur at the phenethyl moiety, piperidine ring or at the butanamide side chain.25 Analysis of tissues, post mortem, indicated that the concentration of butyrfentanyl was highest in the liver, kidney and urine and that the concentrations of the minor metabolites carboxybutyrfentanyl and norbutyrfentanyl decreased slowly over time.24 No phase II metabolites were identified in either study.24,25

Figure 5: Proposed metabolic pathways of butyrfentanyl in vivo and in vitro.25

3.4 Metabolism of Furanylfentanyl

The metabolism of furanylfentanyl has been studied in vivo (Watanabe et al.22; Goggin et al.26) and in vitro (Watanabe et al.22). In both studies, the primary biotransformation in vivo was found to be amide hydrolysis which generated 4-anilino-N-phenethyl-piperidine (4-ANPP).22,26 A hydroxylated sulfate metabolite and a dihydrodiol metabolite were identified as minor products in both in vivo studies.22,26 Watanabe et al.22 detected a nor-metabolite in vitro but its concentration in vivo was much lower.26 In total, 14 metabolites were identified by Watanabe et al.22 which were produced either by a single reaction or by a combination of hydroxylation, amide hydrolysis, N-dealkylation, dihydrodiol formation, oxidative N-dealkylation and reduction of the keto group, furanyl ring opening and carboxylation. Hydroxylated products could then enter into Phase II sulfation or glucuronidation reactions (Figure 6).

This included 4-ANPP which after hydroxylation at the N-phenyl moiety, was converted to a sulfate conjugate. Goggin et al.26 identified a similar sulfate conjugate in the majority of urine samples that had screened positive for furanylfentanyl.

Figure 6: Proposed biotransformation of furanylfentanyl as determined from metabolic studies in human hepatocyte and analysis of urinary metabolites (primary metabolites shown in enclosed boxes) Adapted from Watanabe et al.22

4.0 Mass Spectrometric Fragmentation patterns of Fentanyl

Knowledge of mass spectrometric fragmentation patterns of fentanyl, fentanyl analogues and their metabolites are a useful aid in determining which parent drug has been administered.

Many fentanyl analogues possess only minor structural changes and, as a consequence, generate similar fragmentation patterns to the parent drug. Hence, it is often possible to predict the structure of a novel analogue by comparing the fragmentation pattern of the analogue with that of fentanyl itself. Any alteration to the parent molecule is likely to produce ions with unique mass to charge ratios which reflect these minor structural differences.

Extensive research has been conducted on the mass spectrometric fragmentation of fentanyl analogues.8,21,22,24,25 and the CID fragmentation pathways that apply to fentanyl have been 72 documented thoroughly in several publications.8,27 Noble et al.27 investigated the fragmentation patterns of fentanyl and 50 other fentanyl analogues via LC-QTOF-MS. This was performed using a screening method to identify analogues without the use of reference standards on compounds that shared a 4-anilidopiperidine core and thus had similar fragmentation patterns. Interpretation of fragment ions led to the proposal of four fragmentation pathways. The product ions identified were a result of the loss of the amide group, the separation of the phenethyl group from the amide moiety, and the degradation of the piperidine ring (Figure 7).27 Elimination of the amide group generated the 4-ANPP molecule with m/z 281.2025 (C19H24N2) (Figure 7, pathway a). Cleavage of the bond between the piperidine ring and the amide moiety resulted in the phenylethyl piperidine ion with m/z

188.1434 (C13H18N) (Figure 7, pathway b) and further fragmentation generated a phenylethyl group 105.0699 (C8H9) (Figure 7, pathway b1). This fragment was commonly observed for all targeted analogues in this study which suggests that this cleavage represents a major fragmentation pathway. A phenylpropanamide ion was also observed with m/z 150.0913

(C9H12NO) (Figure 7, pathway c) which was sometimes further modified by the loss of the carbonyl oxygen via dehydration to generate a product ion m/z 134.0964 (C9H11N) (Figure 7, pathway c1). Another ion, m/z 216.1383 (C14H18NO) was generated by degradation of the piperidine ring. I was found that 50% of the compounds analysed were fragmented in this fashion (Figure 7, pathway d).27

Figure 7: Proposed fragmentation pathway for fentanyl and associated 4-ANPP analogues.

Adapted from Noble et al.27

5.0 Summary of synthetic fentanyl analogue fragmentation ions

The mass to charge ratio of the product ions of the studied fentanyl analogues and their metabolites is collated from multiple sources. To ensure a reliable comparison, mass spectral data obtained from similar methodologies was used. The majority of studies employed LC-

QTOF-MS to amass fragmentation data after CID or electrospray ionisation (ESI).22,24-28,67 The exception to this was the study conducted by Melent’ev et al.21 which utilised high performance liquid chromatography mass spectrometry (HPLC-MS) instead.

5.1 Fragmentation pattern of acetylfentanyl

Acetylfentanyl shares many structural similarities with fentanyl but is metabolised somewhat differently with hydroxylation being the main pathway. In the study by Watanabe et al.22, the main metabolites were generated via hydroxylation, N-dealkylation, and dihydroxylation followed by methylation. In the investigation conducted by Melent’ev et al.21, the mass spectra of acetylfentanyl and its major metabolites were studied and structures of the main fragment ions, proposed (Figure 8).

(a)

(b)

(c)

Figure 8: Mass spectral data of the metabolites of acetylfentanyl with proposed molecular

structures of the ions generated.21

The mass spectrum of acetylfentanyl is shown in Figure 8(a). Acetylfentanyl is known to have a m/z ratio of 323, with a precursor ion m/z 188 (C13H18N) formed via the cleavage of the

8,22,27 piperidine ring from the amide moiety. The other proposed ion with m/z 134 (C9H11N) is likely to result from the degradation of the piperidine ring. Diagnostic product ions formed by

22 acetylfentanyl as observed by Watanabe et al. were m/z 188 (C13H18N), 132 and 105 (C8H9).

The m/z 132 fragment can potentially be explained by the formation of a phenethyliminomethylium ion.22

Mass spectrometric data derived from acetylfentanyl subsequent to hydroxylation within the phenyl ring is shown in Figure 8(b). Hydroxylated acetylfentanyl has an m/z ratio of 339, and generates diagnostic product ions of m/z 84 (C5H10N), 103 (water loss fragment), 121

22 (hydroxyphenethyl), and 204. This supports the finding of Melent’ev et al. in that cleavage of a piperidine ring from the m/z 204 (hydroxyphenethylpiperidinyl) ion generates the hydroxyl ethylbenzene m/z 121 ion.21,27

Mass spectrometric data derived from acetylfentanyl subsequent to dihydroxylation and methylation is shown in Figure 8(c). Hydroxymethoxy acetylfentanyl has an m/z ratio of 369, and generates diagnostic product ions with m/z 91.0542, 119.0490, 151.0758, and 234.1487.22

This supports the findings of Melent’ev et al. as both ions are formed from the same bond cleavage as above with the methanol group accounting for the difference in mass.22,27

5.2 Fragmentation pattern of acrylfentanyl

Acrylfentanyl shares many structural similarities with fentanyl and is also metabolised in a similar manner. In the study conducted by Briendahl et al.29, the mass spectral fragmentation

77 patterns of acrylfentanyl were studied with the mass spectrum of the precursor and product ions shown in (Figure 9).30

Figure 9: Mass spectra of the precursor and product ions of acrylfentanyl. Source: Qian et al.30

As observed from the above mass spectra, acrylfentanyl has an m/z of 335 while producing product ions 188.1437 and 105.0697.22,67 These ion fragments correspond with the piperidine and phenethyl tail. While these results are to be expected due to the minor structural changes between acrylfentanyl and fentanyl at the piperidine ring and phenethyl tail, the fact of the matter remains that these product ions are identical to that of fentanyl making differentiation extremely difficult.

In the study by Watanabe et al.22, N-dealkylation was the major metabolic pathway mirroring that of fentanyl. Acrylnorfentanyl is generated via this pathway and has a m/z of 231.1503

22 (C14H18N2O) and fragmentation product ion of m/z 84.0804. This is due to the cleavage of the amide moiety from the piperidine ring, the m/z 84.0804 ion being the piperidine ring. This ion is also generated from acetylnorfentanyl and norfentanyl. And thus, it is difficult to use this pathway exclusively for differentiation. Alternate pathways of metabolism include the hydroxylated metabolite hydroxyacrylfentanyl with a precursor m/z of 351.2075 and product ions of m/z 103.0541, 121.0646, and 204.1384.22 The latter product ion values m/z 204 and 121 correspond with the results found in Melent’ev et al.21 where a hydroxyl group is attached to the product ions generated by cleavage between the amide moiety and piperidine ring, and further cleavage between the piperidine ring and phenethyl moiety respectively. The m/z 103 product ion is likely due to the loss of a water molecule during ionisation as it is only present when hydroxylation takes place at the ethyl linker, the m/z 105 ion being formed instead when hydroxylation takes place at the N-phenyl or acryl moiety.22 The other metabolite produced from acrylfentanyl metabolism is the dihydroxylated metabolite hydroxylated at the ethyl linker and phenethyl moiety. Ions formed by the dihydroxylated metabolite are the precursor ion m/z

367.2028, with product ions m/z 84.0807, 91.0541, 119.0485, 137.0597, and 220.1330.22

5.3 Fragmentation pattern of butyrfentanyl

Butyrfentanyl shares many structural similarities with fentanyl but is metabolised differently with hydroxylation being the main pathway. The main fragment ions found by Noble et al.27 are

79 illustrated in (Figure 10). In a study conducted by Staeheli et al.24, the fragment ions of the most abundant hydroxybutyrfentanyl peak were 188.1436, 105.0694 and 281.2023. The first two fragment ions are generated by cleavage of the piperidine ring from the amide moiety followed by further cleavage between the phenylethyl and piperidine ring.27 The third is a result of amide cleavage eliminating the butyraldehyde group.25 Steur et al.25 found similar fragment ions as

Staeheli et al.24 and minor fragments m/z 132.0813, 134.0969, 146.0969, and 230.1544 formed via degradation of the piperidine ring. Fragment ions of butyrfentanyl that are identified in multiple publications are also common to other fentanyl analogues making identification difficult. The unique minor fragments formed as a result of piperidine ring degradation lacks reproducibility in other literature.24,27

Figure 10: Mass spectral data at high collision energy for butyrfentanyl. Adapted from Noble

et al.27

5.4 Fragmentation pattern of furanylfentanyl

Furanylfentanyl possesses some structural similarities with fentanyl but its main metabolic pathway is amide hydrolysis. In a study conducted by Labutin et al.28, the fragmentation patterns of furanylfentanyl were studied, its mass spectra shown in (Figure 11).

Figure 11: Mass spectra from dissociation of furanylfentanyl. Adapted from Labutin et al.28

The precursor ion for furanylfentanyl has an m/z of 375.2076 and has the main fragment ions of m/z 105.0694, 134.0955, 188.1436 and 254.1181. The 188.1436 and 105.0694 ions are a commonly occurring fragmentation across multiple analogues.27 The 254.1181 fragment ion is generated as a result of the degradation of the piperidine ring containing the furan moiety, while the 134.0955 ion is generated from the degradation of the piperidine ring on the phenylethyl moiety.28

In the study conducted by Watanabe et al.22 on the metabolism of furanylfentanyl, the most common metabolic pathway was amide hydrolysis. This produced 4-ANPP with an m/z of

281.202 which possessed an intact phenethylpiperidine moiety generating fragment ions m/z

188.1433 and 105.0697.22 The dihydrodiol metabolite formed by the epoxidation and hydration of the furan ring generated product ions m/z 188.1440 and 105.0698.22 The sulfate metabolite formed from 4-ANPP hydroxylation and subsequent conjugation with a sulfate group generated product ions of 188.1438 and 105.0698.22 The similar fragmentation pathway of all three metabolites can complicate the identification of metabolites when analysing a furanylfentanyl sample; the intact dihydrodiol metabolite is considered the best target.22

In the study conducted by Goggin et al.26, three metabolites were identified in urine samples.

The main metabolic pathway is amide hydrolysis which generates 4-ANPP with m/z 281.2019

(C19H25N2), and fragment ions with m/z 188.1442 (C13H18N), 134.0974 (C9H12N), and 105.0712

26 (C8H9). Hydroxylation and sulfation of the 4-ANPP metabolite generates a sulfate metabolite with initial m/z 377.1534 (C19H25N2O4S) and unique fragment ion m/z 297.1976 (C19H25N2O)

82 formed by the hydrolysis of the sulfate metabolite. Epoxidation and hydration of the furan ring generates the dihydrodiol metabolite with initial m/z 409.2124 (C24H28N2O4) and unique

26 fragment ions m/z 303.1324 (C16H19N2O4), and 148.1140 (C10H14N). These are generated via the loss of the phenylethyl tail, and the degradation of the piperidine ring respectively.

6.0 Conclusion

Despite the vast amount of literature that exists on the metabolism and structure of fentanyl and analogues, the synthesis of novel fentanyl analogues results in the emergence of uncharacterised fentanyl analogues. Further research is required on the metabolism of these analogues. The metabolites and fragmentation pathways of existing known fentanyl analogues should be incorporated into screening methods to increase the detection speed of these analogues. Knowledge of common fragmentation pathways can also be used in the characterisation of unknown analogues accelerating the identification of the structure via shared product ions.

The fragment ions and fragmentation pathways identified in this review represent the more common product ions for the fentanyl analogues acetylfentanyl, acrylfentanyl, butyrfentanyl and furanylfentanyl. These product ion values can be used to identify structurally unique features in these analogues and of novel analogues that share these features. Many fentanyl analogues possess a similar nucleus to the parent fentanyl molecule with only minor changes to the structure. The presence or absence of key product ions can provide valuable insights on the structure of a novel fentanyl analogue. For example, the presence of ions that correspond with

83 the loss of C13H18N suggests the formation of a phenylethyl piperidine ion, meaning no modifications have been made to the phenylethyl tail. Care should be taken as not all product ions are unique and structurally similar compounds will produce similar product ions. Thus, the ions identified in the literature should serve as a guideline when it comes to structural identification. This review is not complete and further experimentation is highly recommended on more novel compounds to establish a wider range of potential metabolic and fragmentation pathways. More information on the metabolism and mass spectral patterns of analogues with more diverse alterations will allow for faster identification of novel fentanyl analogues even in the absence of any reference standards.

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