A Review of the Collision Induced Dissociation Fragmentation and the Metabolism of Synthetic Cathinone Derivatives

A review of the collision induced dissociation fragmentation and the metabolism of synthetic cathinone derivatives

Nichola Cunningham

A thesis submitted in fulfilment of the requirements for the degree of Master of Forensic Science (Professional Practice)

The school of Veterinary and Life Sciences

Murdoch University

Supervisors: Dr John Coumbaros Associate Professor James Speers

Semester 1

2018

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 contributions by others, with whom I have worked is explicitly acknowledged.

Signed: Nichola Cunningham

Acknowledgments

Firstly, I would like to thank my supervisor John Coumbaros for all of your support, feedback, and advice throughout this project. You have been an invaluable help and I am grateful for all the time you invested in my work. To Bob Mead, thank you so much for all your help with this project. You have always been a support throughout the years and this was no exception; thank you for the time and feedback you provided me along the way. Finally, to my family and friends who have supported me through this, and the entire degree, thank you! I finally made it through!

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Table of Contents

Title page ...... i

Declaration ...... ii

Acknowledgments ...... iii

PART ONE

Literature Review ...... 1– 42

PART TWO

Manuscript ...... 43 – 66

PART ONE: Literature Review

A review of the metabolism and mass spectral fragmentation of synthetic cathinones

Abstract

Synthetic cathinones constitute one of the most commonly abused new psychoactive substances. The rapid development of synthetic cathinones creates a challenge for forensic laboratories to develop and maintain analytical methods for identifying and quantifying synthetic cathinones and their metabolites in illicit drug and toxicological samples. The characterisation of synthetic cathinone metabolites is therefore important for both developing an understanding of their pharmacokinetics and for identifying unique metabolites that could be used for identification. In this review, the current state of knowledge regarding the metabolism of synthetic cathinones will be discussed; focussing primarily on the human in vitro and in vivo metabolism, with the objective of providing a systematic overview of the currently known metabolic profiles. In addition to this, the mass-spectral dissociation fragmentation pathways of synthetic cathinones will be reviewed in an attempt to identify common fragmentation patterns that may assist in the identification of previously unidentified synthetic cathinones.

Keywords: Synthetic cathinones; Metabolism; Cathinone Fragmentation patterns; Novel psychoactive substances; Collision induced dissociation.

Table of Contents

Abstract ...... 2 List of Figures ...... 4 List of Tables ...... 4 Abbreviations ...... 5 CHAPTER ONE: Introduction ...... 6 1.1 Cathinone pharmacology and mechanism of action ...... 8 1.2 Chemical structure and classification of cathinone derivatives ...... 9 1.2.2 Methylenedioxy-N-alkylated derivatives...... 11 1.2.3 N-pyrrolidine substituted cathinone derivatives ...... 11 1.2.4 Methylenedioxy-N-pyrrolidine cathinone derivatives ...... 14 1.3 Analytical methods for the identification of cathinone derivatives ...... 14 1.4 Methods for metabolic profiling ...... 16 1.5 Overview of phase I and phase II metabolism...... 17 2.1 Metabolism of N-alkylated cathinone derivatives ...... 20 2.2 Metabolism of 3,4-methylenedioxy cathinone derivatives ...... 22 2.3 Metabolism of N-Pyrrolidine cathinone derivatives...... 24 2.4 Metabolism of 3,4-methylenedioxy-N-alkylated cathinone derivatives ...... 27 2.5 Summary of synthetic cathinone metabolism ...... 28 CHAPTER THREE: Cathinone fragmentation patterns ...... 29 3.1 General fragmentation patterns of synthetic cathinones ...... 29 3.2 Fragmentation patterns for N-pyrrolidinyl substituted cathinone derivatives...... 34 3.3 Summary of fragmentation ...... 35 CHAPTER FOUR: Conclusion ...... 36 REFERENCES ...... 37

List of Figures Page

Figure 1: Comparison of the natural cathinone structure (left) and the amphetamine structure (right) ...... 8

Figure 2: The generic structure of cathinone derivatives...... 9

Figure 3: The chemical structure of some common N-alkylated cathinone derivatives...... 10

Figure 4: Chemical structure of 3,4-methylenedioxy-N-alkylated cathinone derivatives (A) and the structurally related MDAs (B) ...... 11

Figure 5: Chemical structures of the N-pyrrolidine cathinone derivatives and 3,4-methylenedioxy-N-pyrrolidine cathinone derivatives ...... 13

Figure 6: A schematic of the major pathways of drug biotransformation ...... 18

Figure 7: Schematic representation of phase I cathinone metabolism...... 19

Figure 8: The proposed metabolic pathway of mephedrone in humans ...... 21

Figure 9: Proposed in vitro and in vivo metabolic pathway for methylone in humans...... 23

Figure 10: Proposed metabolic pathway for α-PVP based on metabolites observed in human urine and HLM ...... 25

Figure 11: A proposed metabolic pathway for PV9 in humans...... 26

Figure 12: The proposed metabolic pathway for MDPV in humans...... 28

Figure 13: A schematic representation of the postulated general CID fragmentation postulated of synthetic cathinones...... 32

Figure 14: A postulated fragmentation pattern of 3,4-methylenedioxy substituted cathinone derivatives ...... 33

Figure 15: The proposed generalised fragmentation pathways for pyrrolidinyl substituted cathinone derivatives...... 35

List of Tables

Table 1: Summary of the names, formulae and structures of the 39 investigated cathinone derivatives ...... 30

Abbreviations

NPS Novel psychoactive substances EU-EWS European Union Early Warning System EMCDDA European Monitoring Centre for Drugs and Drug Addiction UNODC United Nations Office on Drugs and Crime CID Collision-induced-dissociation DAT Dopamine transporter NAT Noradrenaline transporter SERT Serotonin transporter MDMA 3,4-methylenedioxymethamphetamine 4-MEC 4-methylcathinone 3,4-DMMC 3,4-dimethyl-methcathinone MDAs 3,4-methylenedioxyamphetamines MDEA 3,4-methylenedioxyethamphetamine MBDB 3,4-methylenedioxy-α-ethyl-N-methylphenethylamine MBDP 3,4-methylenedioxy-α-propyl-N-methylphenethylamine α-PPP α-pyrrolidinopropiophenone α-PVP α-pyrrolidinovalerophenone α-PBP alpha‐pyrrolidinobutyrophenone α-PHP Pyrrolidinohexanophenone α-POP α‐pyrrolidinooctanophenone MPPP 4-methyl-α-pyrrolidinopropiophenone MOPPP 4-methoxy-α-pyrrolidinopropiophenone α-PVT α-pyrrolidinopentiothiophenone MDPPP 3,4-methylenedioxy-α-pyrrolidinopropiophenone MDPBP 3,4-methylenedioxy-α-pyrrolidinobutiophenone MDPV 3,4-methylenedioxypyrolvalerone SWGDRUG Scientific Working Group for the Analysis of Seized Drugs GC-MS Gas chromatography-mass spectrometry LC Liquid-chromatography LC-MS Liquid-chromatography mass spectrometry HRMS High-resolution-mass-spectrometry methods UHPLC Ultra-high performance liquid chromatography TOF-MS Time-of-flight mass spectrometry UGTs UDP- glucuronosyltransferases PHH Primary human hepatocytes SULTs Sulfotransferases pHLC Pooled human liver cytostol pS9 Pooled human S9 fraction pHLM Pooled human liver microsomes DHMC Diydroxymethcathinone HMMC 4’-hydroxy-3’-methoxymethcathinone

CHAPTER ONE: Introduction

Novel psychoactive substances (NPS) constitute an evolving mixture of substances that have received considerable attention and public concern over the past few years. Also referred to as “legal highs” or “designer drugs”, NPS are designed to reproduce the effects of controlled illicit substances such as cannabis, hallucinogens and stimulants.1 NPS can be classified according to either their psychotropic effects, as stimulants, empathogens/entactogens, and hallucinogens, or the chemical family that they resemble as phenethylamines, tryptamines, synthetic cathinones and synthetic cannabinoids.2 Typically, these substances are analogues or chemical derivatives of already scheduled illicit substances such as cannabis, hallucinogens and stimulants. These analogues are synthesised with structural modifications making them structurally different and as such can circumvent existing legal restrictions.2, 3 As a result, some NPS can be marketed and sold as “legal highs”, increasing the widespread accessibility and availability of these substances.3

Over the last decade an exponential increase in the number of NPS identified and monitored by international bodies has been observed.1, 4, 5 Between 2005 and 2015, more than 560 NPS have been observed by the European Union Early Warning System (EU-EWS) of The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA).5 As of 2016, the United Nations Office on Drugs and Crime (UNODC) has reported the presence of over 643 NPS in 101 countries; an unparalleled globalisation rate.3, 6 In Australia, the most commonly abused NPS are the synthetic cannabinoids, synthetic cathinones, phenethylamines and tryptamines, with the synthetic cannabinoids and synthetic cathinones representing the greatest proportion of seized substances.7 Between 2015 and 2016 cathinone-type substances accounted for 33.3% of analysed seizure samples; a trend similar to that observed in some European countries.4, 8

Given the rate at which new cathinone derivatives are emerging on the drug market their legal status has become the subject of considerable legislation over recent years. A number of European countries and the USA have imposed regulations that target individual cathinone derivatives.9 In Australia legislation has been introduced that broadly states that criminal offences that apply for already existing scheduled drugs automatically apply to substances deemed to be structurally similar.1 However, despite such regulations new cathinone derivatives are continuously being produced in clandestine laboratories, creating a difficulty for legislation to stay current.3

The difficulty in attempting to regulate these substances lies in their rapid development, ease of synthesis and widespread availability, particularly through prominent advertisement and sale through online networks.3 Furthermore, the diversity of NPS in regards to both their psychotropic 6 effects and chemical structure creates challenges in the detection and identification these substances.3 Over the past few years the topic of synthetic cathinone derivatives has produced considerable research relating to the identification of new derivatives, their chemical behaviour and metabolism. This review will provide a systematic overview of the literature surrounding the chemistry, behaviour and the metabolism of synthetic cathinones focussing specifically on consolidating the current state of knowledge surrounding the human metabolic pathways of specific cathinone derivatives. Additionally, the structural characterisation of synthetic cathinones will be reviewed with a focus on the collision ionisation induced (CID) fragmentation patterns commonly encountered for synthetic cathinone derivatives. Knowledge of the fragmentation patterns of synthetic cathinones, and of fragment ions, could assist with the identification of previously unidentified metabolites and synthetic cathinones.

1.1 Cathinone pharmacology and mechanism of action

Cathinone derivatives are analogues of the natural compound cathinone, a psychoactive stimulant found in the leaves of the Catha edulis plant commonly known as Khat.10 Due to its psychoactive effects, the practice of khat chewing has traditionally been used by many communities throughout the Horn of Africa, Yemen and in the Southwest Arabian Peninsula.10 The process of khat chewing involves chewing small bundles and shoots of khat to release the active constituents into the saliva before swallowing the plant material.11 While khat contains numerous non-cyclic nitrogen containing compounds it is believed that cathinone, a β-ketone phenylalkylamine, is the primary compound responsible for the psychoactive effects of khat.12 Cathinone can be considered a β-ketone analogue of amphetamine, which differs structurally from amphetamine by the presence of a β-ketone in the α-position of the side chain of the cathinone (Figure 1).13 As such, cathinone and its derivatives produce a distinct cluster of pharmacological effects similar to amphetamine and methamphetamine. Primarily these include central nervous stimulant and sympathomimetic effects including euphoria, excitation, agitation, and increased sensory stimulation.10, 14 Synthetic derivatives of cathinones are commonly sold through online vendors and are typically marketed under alternative names of “Bath salts”, “Research chemicals” and “Plant food”. These substances are primarily available in a crystalline or powder form and are commonly administered by snorting, smoking, orally ingested or in a less common method by intravenous injection.15

Figure 1: Comparison of the natural cathinone structure (left) and the amphetamine structure (right).16

As cathinone derivatives possess a β-ketone structure they are less lipophilic than amphetamine and therefore typically require higher doses to achieve equivalent effects to that of amphetamines.16, 17 Like amphetamines, cathinone and its derivatives exert effects by interacting with plasma membrane monoamine transporters such as dopamine transporter (DAT), noradrenaline transporter (NAT) and serotonin transporter (SERT).14 Cathinone derivatives can interact with monoamine transporters in two ways; as either monoamine transporter blockers or monoamine transporter substrates. Those acting as blockers exert their effects via the inhibition of monoamine re-uptake in 8 a similar fashion to cocaine or 3,4-methylenedioxymethamphetamine (MDMA).18 The monoamine transporter substrates act via the preferential inhibition of monoamine reuptake, resulting in a decreased clearance of neurotransmitters—a mechanism similar to amphetamines.18, 19 In vitro studies performed using cell and synaptosomal preparations have demonstrated differences in the pharmacology of different cathinone derivatives as a result of differences in their affinity towards the aforementioned transporters. Therefore the structure of the derivatives will have an influence on the pharmacology of the synthetic cathinones.

1.2 Chemical structure and classification of cathinone derivatives

The basic cathinone structure lends itself easily to modifications at several different locations resulting in a wide array of cathinone derivatives.20 These modifications include the addition of various substituents at the R3 position on the aromatic ring, variation in the α-carbon substituent and 16 N-alkylation at the R1 and R2 positions (Figure 2). Cathinone derivatives can be classified into four major groups based on their structure: N-alkylated compounds, methylenedioxy-substituted compounds, N-pyrrolidine compounds and 3,4-methylenedioxy-N-pyrrolidine cathinones.10

Figure 2: The generic structure of cathinone derivatives. The various points on the structure at which substituents are added are represented.16

1.2.1 N-alkylated cathinone derivatives

The N-alkylated cathinone derivatives are a cluster of related compounds characterised by substituents at the R1 and/or R2 sites or compounds with an alkyl or halogen substituent at a position 9 along the aromatic ring (R3). This group of cathinones contains some of the first derivatives generated for therapeutic purposes, namely dimethylpropion and diethylpropion, which are N- 10 methylated and N-ethylated at the R2 position respectively (Figure 3). Modifications in the length of the aliphatic chain can produce a range of derivatives that differ only by the number of carbons at the R4 position, such as buphedrone and pentedrone. Similarly, derivatives can differ by variation in the addition of methyl and ethyl groups at the R1 and R2 position (i.e. methylpropion, diethylpropion, ephedrone, ethcathinone etc.). Additionally, the insertion of halogen substituents at the aromatic ring, namely fluorine and chlorine, give derivatives such as flephedrone and bupropion (Figure 3); the latter of which has been investigated for potential therapeutic use. 9, 10, 16, 21

Figure 3: The chemical structure of some common N-alkylated cathinone derivatives. 4-MEC 4-methylcathinone, 3,4-DMMC 3,4-dimethyl-methcathinone.10

1.2.2 Methylenedioxy-N-alkylated derivatives

This group encompasses N-methylated and N-ethylated compounds which result from an alkylation at the R1 and R4 positions with the addition of a methylenedioxy group at the aromatic ring (Figure

4). Methylation at the R1 position produces methylone whilst addition of an ethyl group at the same site produces ethylone (Figure 4). Further ethylation and methylation of the R4 position results in butylone and pentylone respectively (Figure 4). Derivatives belonging to this group show structural similarity with 3,4-methylenedioxyamphetamines (MDAs) such as MDMA and MDEA (3,4- methylenedioxyethamphetamine).10

Figure 4: Chemical structure of 3,4-methylenedioxy-N-alkylated cathinone derivatives (A) and the structurally related MDAs (B). MDMA 3,4-methylenedioxymethamphetamine; MDEA 3,4-methylenedioxyethamphetamine; MBDB 3,4-methylenedioxy-α-ethyl-N- methylphenethylamine; MBDP 3,4-methylenedioxy-α-propyl-N-methylphenethylamine.10

1.2.3 N-pyrrolidine substituted cathinone derivatives

N-pyrrolidine cathinone derivatives are characterised by the presence of a pyrrolidinyl moiety at the nitrogen atom of the cathinone structure.22 While this group has the underlying natural cathinone structure, α-pyrrolidinopropiophenone (α-PPP) acts as the primary prototype for other cathinone derivatives within this group (Figure 5).16 For instance, α-PPP can be modified by extending the side chain at the R4 position to give α-pyrrolidinovalerophenone (α-PVP); a derivative in which the aliphatic chain is one carbon longer than α-PPP. The aliphatic chain of α-PVP can be shortened to give α-pyrrolidinobutyrophenone (α-PBP) or lengthened to give α‐pyrrolidinohexanophenone (α- PHP) and α‐pyrrolidinooctanophenone (α-POP) (Figure 5). In addition to chain substituents, α-PPP can be modified by the addition of a phenyl substituent at the R3 and R4 position of the aromatic

11 ring; with the addition of methyl and methoxy groups resulting in the generation of 4-methyl-α- pyrrolidinopropiophenone (MPPP) and 4-methoxy-α-pyrrolidinopropiophenone (MOPPP), respectively (Figure 5).23 Similarly, α-PVP can be modified by the addition of halogen, methoxy or methyl groups at the aromatic ring to give 4-fluoro α-PVP, 4-methoxy α-PVP, and 3,4-dimethoxy- α- PVP, respectively. Additional modification, such as the replacement of the phenyl ring with other aromatic rings (i.e. thiophene) can give other derivatives such as α-pyrrolidinopentiothiophenone (α-PVT).22 Interestingly, the high lipophilicity of the pyrrolidine ring has been suggested to increase the permeability of pyrrolidinyl substituted compounds through the blood-brain barrier, potentially increasing the potency and abuse potential.18, 24

Methylenedioxy Phenyl substitution Deletion

Shorter Longer side side chain chain

Phenyl substitution

Figure 5: Chemical structures of the N-pyrrolidine cathinone derivatives and 3,4-methylenedioxy-N- pyrrolidine cathinone derivatives. 3,4-MDPPP, 3,4-MDPBPand MDPV belong to the 3,4- methylenedioxy-N-pyrrolidine group of cathinone derivatives. Image adapted from22

1.2.4 Methylenedioxy-N-pyrrolidine cathinone derivatives

Structurally, 3,4-methylenedioxy-N-pyrrolidine cathinone derivatives resemble pyrrolidinyl derivatives with the addition of a methylenedioxy moiety at the 3,4 position of the aromatic ring (Figure 5). Derivatives belonging to this group include 3,4-methylenedioxy-α- pyrrolidinopropiophenone (MDPPP), 3,4-methylenedioxy-α-pyrrolidinobutiophenone (MDPBP), 3,4-methylenedioxypyrolvalerone (MDPV). The addition of a methyl or an ethyl group on the MDPPP

25 molecule at the R4 position gives the MDPBP and MDPV derivatives, respectively (Figure 5). Structurally, these derivatives resemble those of MPPP, MPBP, and pyrovalerone (belonging to the N-pyrrolidine derivatives); the difference being the methylenedioxy ring substitution.

1.3 Analytical methods for the identification of cathinone derivatives

The number of structurally related cathinone derivatives that exist and their continuous emergence on the drug market poses an analytical challenge for forensic chemistry laboratories. The analysis of seized samples in forensic laboratories is performed in accordance with the guidelines outlined by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG).26 This involves a combination of analytical techniques such as gas chromatography-mass spectrometry (GC-MS), liquid chromatography (LC), Fourier transform-infrared spectroscopy, and nuclear magnetic resonance spectroscopy for the identification of substances.27 The continuous emergence of newly- synthesised synthetic cathinones however, poses a challenge for toxicological forensic laboratories to develop and maintain reliable analytical methods for the qualitative and quantitative identification of compounds in biological matrices.

GC-MS analysis is a technique that involves the production of gas-phase ions using different ionisation techniques that are accelerated and separated in a mass analyser under a high vacuum according to the mass-to-charge-ratio (m/z).28 While conventional GC-MS methods have been used for drug analysis, traditional electron ionisation (EI) GC-MS suffers from extensive fragmentation in the ion source resulting in the absence of molecular ions.29 Additionally, it has been noted that to reliably detect some cathinone derivatives using GC-EI-MS methods derivatisation was necessary, a process which increases the cost and time of analysis.30 Recently, liquid-chromatography mass spectrometry (LC-MS) techniques have become increasingly used for drug analysis and metabolism studies, particularly when coupled to high-resolution-mass-spectrometry (HRMS) methods.28 The versatility of LC to be coupled with various analysers and the number of different analysers available

14 has resulted in the development of many analytical approaches for identifying metabolites. Among the most attractive methods is the use of ultra-high performance liquid chromatography (UHPLC) coupled with time-of-flight mass spectrometry (TOF-MS) and its quadruple analyser which separates ions according to velocity and the time-of-flight.27

Tandem mass spectrometry (MS/MS) is often applied for the structural elucidation of cathinone derivatives, the determination of fragmentation pathways, and to determine elemental compositions.28 This technique primarily consists of two mass analyser interfaces; the first which isolates a precursor ion and the second which separates the product ions.28 Of the various scan modes available for tandem MS, the product ion scan mode is the most utilised for the identification of compounds.31 In this mode, a precursor ion is chosen and the product ions (i.e. fragment ions) are determined from the CID. CID is used to facilitate the fragmentation of precursor ions using collisions between low and high energy accelerated ions and collision gas. When coupled with time- of-flight mass analysers the introduced ions are accelerated into a flight tube by an electric field. These ions are then separated on the basis of their velocities as they travel through the flight tube, in the free-drift region. An advantage of the QTOF-MS based methods is that they provide a broad mass range, high sensitivity and fast analysis time.9, 31 The combination of CID-MS with a high resolution mass spectrometer can provide information on the elemental composition of the molecule, the fragmentation patterns, and for the tentative identification of compounds without the need for reference standards—which are often unavailable for newly-emerged cathinones.28

Overall, LC-MS methods are advantageous as they are able to assess a broad range of carious compounds without derivatisation of the compound.32 Additionally, softer ionisation techniques such as electrospray ionisation (ESI) produces less fragmentation in the ion source, which results in more useful spectra with more molecular ions. In HRMS, the protonated molecule can be used to estimate the chemical structure of a compound and/or fragments and therefore the presence of molecular ions is important for structural elucidation.32 Recently, HRMS has been employed in a number of studies for the identification and structural characterisation of synthetic cathinones.31 Particularly, searching for common fragments using QTOF-MS is useful for investigating compounds that normally share common moiety for drugs of the same chemical family.31

1.4 Methods for metabolic profiling

For both ethical and safety reasons controlled drug studies are often not performed with illicit drugs, therefore several in vitro and in vivo approaches have been employed for studying the metabolism of synthetic cathinones. In vitro methods often utilise systems such as fractions of liver homogenate and cell cultures to assess metabolic pathways.33 A number of approaches use fractions of drug metabolising tissues such as pooled human liver cytostol (pHLC), pooled human S9 fraction (pS9) and pooled human liver microsomes (pHLM).34 pHLM are the most commonly used approach for metabolism studies as they contain both the primary drug-metabolising cytochrome enzymes and the primary conjugation enzymes UDP-glucuronosyltransferases (UGT).35 However, HLM fractions lack some enzymes such as N-acetyltransferases and glutathione-S-transferase, which restricts the metabolic pathways that can be studied.36

The pS9 fraction is a sub-cellular fraction prepared from collecting the supernatant of a centrifuged tissue homogenate. This contains both pHLC and pHLM and as such can provide simultaneous investigation of both phase I metabolites and phase II metabolites, which are mediated by non-CYP enzymes.35 However, methods using sub-cellular fractions often require the addition of cofactors that mediate oxidative metabolism (i.e. NADPH) or conjugation reactions (i.e. Uridine diphosphate glucuronic acid, S-adenosylmethionine), which are lost during the isolation process.35 As these cofactors are necessary to catalyse enzymatic reactions the inclusion or exclusion of specific cofactors may highlight the involvement of specific metabolism pathways. The cytosol is an isolated supernatant of the pS9 fraction which contains a group of drug-metabolizing enzymes and thus can be used for investigating specific routes of metabolism. Cytosol incubations are the simplest system of the three addressed methods and tend to be used to complement other microsomal based studies.35 Recently, primary human hepatocytes (PHH) have been used for investigating both phase I and phase II metabolites. PHH cultures are considered the most suitable approach for studying drug metabolism as they contain cofactors, metabolism enzymes and drug transporters that closely mimic in vivo conditions.37

In vivo drug metabolism studies include the analysis of metabolites in blood, tissues, and urine to assess unique human metabolites.38 In vivo animal or human models offer a method of analysis that provides insight of the combined effect of permeability, distribution, metabolism, and excretion and hence yield a greater understanding of drug pharmacokinetics.23, 38 The current trend for in vivo animal studies is the use of rats to study the metabolism of drugs of abuse. However, interspecies variation between the metabolic processes between humans and animals can result in differences in the metabolites formed.38 As such, in vitro assays in combination with samples taken from forensic 16 casework are used to collect metabolic profiling data with controlled animal studies treated as secondary information.34

1.5 Overview of phase I and phase II metabolism

Knowledge of the phase I and phase II metabolism of cathinone derivatives is crucial for developing both screening identification methods and toxicological risk assessments of these compounds.23 The behaviour of foreign substances within the body can be divided into four main phases; absorption, distribution, metabolism and excretion.39 The absorption of compounds into the body typically involves the dissolution of a lipid soluble substance into the lipid membrane via passive diffusion.40 The route of absorption will depend on the site of administration and the physiochemical characteristics of the molecules, particularly relating to their size, shape, lipid solubility, and polarity.41 Following absorption, substances will be distributed around the body via either the bloodstream or the lymphatic system. Distribution depends on the ability of the molecules to pass through the plasma membranes to the extracellular fluid by passive diffusion, active transport or facilitated diffusion.42 While some compounds absorbed into the body are eliminated intact, drugs that are well absorbed are generally lipophilic and require biotransformation or metabolism into water-soluble molecules to facilitate their excretion from the body.40, 41

Drug metabolism within a biological system can be divided into two phases.41 Phase I metabolism consists of functionalization reactions which introduce or reveal functional groups to a structure such as hydrogen (–OH), amine (-NH2), thiol (-SH) or carboxylic acid (–COOH). These introduced groups often act as the site of further conjugation reactions which occur in phase II metabolism (Figure 6).39 A number of reactions are responsible for phase I metabolism, the most common of which include oxidation, reduction and hydrolysis reactions. In humans, phase I reactions are catalysed by several bio-transforming enzymes primarily belonging to the cytochrome p-450 monooxygenase system; a collection of isoenzymes located primarily in the small endoplasmic reticulum (microsomal fraction) and liver cells.40 While majority of compounds will undergo some phase I metabolism prior to phase II some compounds which already contain an appropriate functional group can undergo phase II conjugation reactions independently of phase I reactions.43

Phase II metabolism consists of conjugation reactions that introduce an endogenous water-soluble group to a molecule at the functional groups previously introduced during phase I. Conjugation reactions typically render a molecule lipid soluble (less hydrophilic) and more polar and increases the molecular weight of the molecule. As a result, the membrane permeability of the molecule is 17 lower, which facilitates excretion from the body (Figure 6).41 The most common phase II reactions include glucuronidation, sulphation, and glutathione conjugation reactions. Glucuronidation is a process in which a glucuronide moiety is attached to a substrate to produce negatively charged hydrophilic glucuronides that can exit the cell via efflux transporters.44 Sulphation involves the transfer of a sulfonate group to a substrate containing a hydroxyl or amino group (i.e. the introduced functional group) in a reaction catalysed by human cytosolic sulfotransferases (SULTs). Similar to glucuronidation this results in possible inactivation of the substrate compound and increased water solubility of the substrate to facilitate removal from the body.45

Figure 6: A schematic of the major pathways of drug biotransformation. 40

Natural cathinone metabolises by two main pathways involving reduction and oxidation reactions. Cathinone undergoes reduction of the β-keto group to an alcohol to give norpseudoephedrine (cathine) and norephedrine (Figure 7).10 This metabolism has previously been shown to be stereoselective with the primary metabolite of the S-(−)-cathinone stereoisomer being

18 norephedrine, while the R-(+)-cathinone stereoisomer is primarily metabolised to R,R-(−)- norpseudoephedrine.12 Cathinone can also undergo oxidation reactions to give 1-phenyl-1,2- propanedione or alternatively it can undergo dimerization followed by oxidation to produce 3,6- dimethyl-2,5-diphenylpyrazine.12 However, in the human body cathinone is metabolised primarily to norephedrine.14, 46

Figure 7: Schematic representation of the phase I metabolism of cathinone. 46

As previously eluded to, the lipophilicity of the compound is the major factor that affects the absorption, distribution, metabolism and excretion of foreign compounds within the body; all of which will influence the biological activity of the compound.41 In human metabolism, genetic factors which result in non or less functional enzymes may affect the metabolism of a compound resulting in different responses to the toxicological reactions of compounds.40 All four phases of drug disposition (i.e. absorption, distribution, metabolism and excretion) could be subject to species variation which include, the preferred route of metabolism, the extent to which some compounds are metabolised, and the degree to which they undergo phase II conjugation reactions.41

CHAPTER TWO: Discussion

Characterising the metabolism of synthetic cathinones is crucial for both developing the understanding of their pharmacokinetics and for identifying targets that could be used for identification. Unique metabolites and an understanding of metabolic pathways can be incorporated into analytical approaches for identification and to assist in the development of urine screening methods. Over the past decade the metabolic profiles of synthetic cathinone derivatives have been documented by several research groups using a number of different methods. Various review articles published in recent years have provided a general overview of synthetic cathinones particularly focussed on their pharmacokinetics and analytical methods for metabolic profiling.9, 10, 23 This section will collate the findings of multiple publications to provide an overview of the metabolic pathways observed for a number of synthetic cathinones. For practical purposes, the metabolism of each of the synthetic cathinone groups will be discussed separately focussing primarily on the most well studied derivatives. As metabolism is likely to differ between species this review will primarily focus on studies assessing human in vitro and in vivo metabolism.

2.1 Metabolism of N-alkylated cathinone derivatives

The majority of compounds grouped as N-alkylated cathinone derivatives contain various alkyl or halogen substituents at the ring and side chain positions. Despite structural differences, common metabolic reactions and pathways have been identified for compounds within this group. One of the most well studied derivatives is mephedrone, the metabolism of which has been studied in rats or rat hepatocytes, in human liver microsomes, and using human urine.47-51 Recently, the in vivo human metabolism of mephedrone was evaluated using UHPLC coupled to QTOF-MS. This is one of few controlled studies in which urine samples were collected from two volunteers four hours after they ingested a known quantity (200 mg) of mephedrone.50 Six phase I and four phase II metabolites were identified; four of which (M2, M4, M8, M10) had not previously been reported in the literature. The proposed metabolic pathway for mephedrone is summarised in Figure 8.

Reduction

Hydroxylation

N-Demethylation Hydroxylation

Reduction

Oxidation

Figure 8: The proposed metabolic pathway of mephedrone in humans.50

The identified phase I metabolic reactions included N-demethylation, reduction of the β-ketone moiety, oxidation of both the C3 carbon and the aromatic ring, and hydroxylations in the C3 carbon.47, 50, 51 Overall, it has been suggested that mephedrone is metabolised via three main pathways. In both rat and human subjects mephedrone primarily undergoes N-demethylation to give the primary amine normephedrone (M1, Figure 8).47, 50 This structure is further transformed via β-ketone reduction to give 4′-methylnorephedrine (M5). Metabolites resulting from β-ketone reduction and N-demethylation have been suggested as the primary metabolites, both in vivo and in HLM incubations.51,49 Additionally, oxidation of the tolyl moiety of mephedrone has been observed to give the corresponding 4’-carboxymephedrone structure (M7, Figure 8) as previously reported.48, 51

In regards to phase II metabolism, four conjugated metabolites were identified (Figure 8) three of which were conjugated with glucuronic acid (M8, M9 and M10). Interestingly, Pozo et al50 was the first to identify a conjugate with succinic acid metabolite in human urine (M4, Figure 8).50 Few other researchers have investigated the phase II metabolism of mephedrone, however the metabolism of other N-alkylated cathinones has been investigated. In an experiment investigating the metabolism of 3,4-DMMC, urine samples were analysed both before and after hydrolysis to identify conjugated metabolites.52 The amount of 3,4-DMMC and its metabolites in the urine increased with hydrolysis suggesting that they were conjugated, likely with glucuronic acid.52

Similar metabolic pathways involving N-demethylation and β-ketone reduction have been described in both in vivo human and HLM studies for other N-alkylated derivatives such as 3,4-DMMC, buphedrone, 4-fluoromethcathinone and 4-methylbuphedrone.49, 52, 53 In a comprehensive study conducted by Uralets et al.49 the metabolic profiles of 16 synthetic cathinones detected in human urine samples were assessed.49 Of the 16 cathinones identified, nine cathinones were classified as N- alkylated substituents: pentedrone, 4-MEC, flephedrone, buphedrone, mephedrone, ethcathinone, DMMC, 4-Methylbuphedrone and N-ethylbuphedrone. Ring alkylated derivatives primarily underwent a ketone reduction to give the corresponding alcohol with a subsequent N-dealkylation to form N-dealkylated alcohols in similar pathways to that observed for mephedrone. As such, the abundant β-ketone reduced metabolites were proposed as potential markers for parent drug use. Interestingly, Uralets et al.49 noted that the parent drugs were less abundant that their metabolites, suggesting a high extent of metabolism. However, as samples were collected at unknown stages of urinary excretion, with unknown dosage and time of ingestion no determination could be made as to the longevity of the identified metabolites. Additionally, the authors only considered freely excreted non-conjugated metabolites and therefore it is possible that other metabolites were present but were undetected.

2.2 Metabolism of 3,4-methylenedioxy cathinone derivatives

Methylenedioxy substituted derivatives are characterised by the presence of a 3,4-methylenedioxy group at the aromatic ring. The metabolism of some major 3,4-methylenedioxy substituted derivatives such as methylone, ethylone, butylone and pentylone have been characterised both in vitro and in vivo.36, 49, 54, 55 As these derivatives are structurally similar, it is unsurprising that comparable metabolic pathways have been observed. Metabolites resulting from demethylenation and O-methylation are the most abundant phase I metabolites observed in human urine and HLM.49, 54 In addition, keto-reduced and N-dealkylated products have been observed as minor metabolites, 22 suggesting these reactions occur to a lesser extent. The proposed metabolism and metabolites of methylone, as detailed in Figure 9, will be used to illustrate the common metabolic pathways of 3,4- methylenedioxy derivatives. The major metabolic pathway identified involves demethylenation of methylone to form the intermediate diydroxymethcathinone (DHMC). Subsequent O-methylation in the aromatic ring of DHMC, via catechol-O-methyl transferase, results in the formation of 4’- hydroxy-3’-methoxymethcathinone (HMMC) demethylenated metabolites (Figure 9). A number of studies have indicated that HMMC is the major metabolite detected both in human urine and in HLM suggesting it is a key metabolite for screening purposes.36, 54, 55 Other minor metabolites have been identified in in vitro HLM incubations including Nor-methylone and Dihydro-methylone (Figure 9).54 These are the result of minor metabolic pathways, that involve N-dealkylation and the reduction of the β-keto moiety that have been proposed to occur to a lesser extent (Figure 9). In a study assessing human urine, Uralets et al.49 proposed that the presence of the methylenedioxy group may hinder carbonyl reduction as a result of steric hindrance and thus less reduced metabolites are identified.49

N-dealkylation Hydroxylation

Reduction Demethylenation

O-OMethylation-Methylation

Figure 9: Proposed in vitro and in vivo metabolic pathway for methylone in humans. 54 Image adapted from 23

The phase I metabolic pathways observed for methylone are common to butylone, ethylone and pentylone with the major metabolites observed being the result of demethylation reactions.49, 54-56 Currently, there is limited knowledge regarding their phase II metabolism however glucuronides have been proposed as potential phase II metabolites. Zaitsu et al.57 investigated the potential conjugation pathways for the hydroxylated and β-keto reduced metabolites on the basis of enzymatic hydrolysis.57 After hydrolysis, increases in the urinary levels of 4-HMMC and 3-HMMC metabolites were observed as well as increases in the levels of unchanged compounds and β-keto reduced metabolites. This suggests potential glucuronidation of the alcohol group of these compounds that are excreted in the urine.55, 57 However, given the lack of knowledge surrounding phase II metabolism further in vitro and in vivo studies on phase II conjugates is required.

2.3 Metabolism of N-Pyrrolidine cathinone derivatives

The major metabolic pathways of the α-pyrrolidine derivatives have been investigated using both in vitro and in vivo methodologies.58-61 Given that N-pyrrolidine derivatives possess both the 3,4- methylenedioxy substituent and pyrrolidine structure the metabolism of these cathinone derivatives is more complex than those previously discussed. One of the most extensively studied derivatives of this group is α-PVP, the metabolism of which has been studied using in vitro methods with HLM58, 62 and in vivo using human urine.49, 61, 62 Two major metabolic pathways identified for α-PVP metabolism involve reduction and hydroxylation reactions. One pathway involves reduction of the β- ketone to give alcohol diastereomers (OH-α-PVP), which are subsequently conjugated to their respective glucuronide (Figure 10).61 Further hydroxylation and oxidation of the pyrrolidine ring gives the respective lactam structure (2”-oxo-α-PVP) (Figure 10, M3). Overall, OH-α-PVP and PVP lactam metabolites represent the two major metabolites of α-PVP.58, 61 In a recent toxicological analysis in an acute poisoning case, Grapp et al.63 identified un-metabolized α-PVP and its 2”-oxo-α-PVP metabolite in human urine, which was suggested as a potential candidate for identification of α-PVP in human urine.49, 63

Recently, Negreira et al.58 identified for the first time a metabolite proposed to be the result of a complex combination of reactions that involve a reduction of the α-PVP, followed by a hydroxylation of the pyrrolidine ring, ring opening, and an additional hydroxylation (M2,M5 Figure 10).58 In addition, two phase II glucuronide metabolites of M3 were identified (Figure 10) for the first time in any in vitro or in vivo study. However, the glucurorindated metabolites of M6, previously identified in other human urine studies, were not identified by Negreira et al.58, which could result from

24 differences in the methodologies employed.41 While extensive metabolic pathways for α-PVP have been identified for humans, Tyrkko et al.62 noted, in a study on human urine, that the α-PVP was the most abundant compound found in all human urine samples assessed.

Figure 10: Proposed metabolic pathway for α-PVP based on metabolites observed in human urine and HLM. M1: PVP-amine (N,N-bis-dealkyl-α-PVP), M2: Carboxylic acid (4-((1,3 or 4-Dihydroxy-1- phenylpentan-2-yl)amino)butanal), M3:PVP-Lactam (2”-oxo-α-PVP), M4:2”-hydroxy-α-PVP, M5: Carboxylic acid (1 or 2-Hydroxy-4-((1-oxo-1-phenylpentan-2-yl)amino)butanal) ,M6: 1-hydroxy- α-PVP. Image adapted from58

The major metabolic pathways identified for α-PVP, namely β-ketone reduction, hydroxylation and lactam formation, are common to other α-pyrrolidinophenones such as α-PBP, α-PHP and α-PVT. Alpha-PBP has been demonstrated to undergo oxidation at the 2”-position of the pyrrolidine ring to give 2”-oxo- α-PBP, with 2”-OH- α-PBP as an intermediate, followed by reduction to form OH-α- PBP.60 Of the 19 metabolites identified of α-PHP identified in human urine, dihydroxy-α-PHP was the most abundant metabolite identified; formed by the reduction of the β-ketone group, similar to other pyrrolidinophenones.64 In contrast to the other pyrrolidinophenones, α-PVT was hydroxylated at both the thiophene ring and at the pyrrolidine ring to form a lactam species.37, 65 Therefore, broadly speaking, lactam species and metabolites generated by reduction of the β-ketone group could present a possible target for the identification of pyrrolidine substituted cathinone derivatives.

There has been a recent focus on investigating the metabolism of the newly synthesised α- pyrrolidine derivatives, which possess longer side chains such as α-POP and 1-phenyl-2-(1- pyrrolidinyl)-1-heptanone (PV8). It was proposed that the metabolism of these longer chained derivatives may differ from that of α-PVP or α-PBP. Shima et al.66 examined the metabolites of PV9 in human urine using GC-MS and LC-HRMS. Based on the relative intensities of the protonated metabolites identified it was suggested that metabolites formed via oxidation (M9 and M10, Figure 11), were the most abundant PV9 metabolites. Metabolites formed by the reduction of ketone groups and oxidation of pyrrolidine rings (M1 and M6, respectively) represented minor metabolites. This suggests that the primary metabolic pathway involved oxidation of the alkyl chain at the terminal or penultimate carbon atoms (Figure 11), which differs from that observed for the other α- pyrrolidionphenones.58, 61, 62 However, this pathway has not been established by other studies. In addition, a greater number of metabolites were observed with the increased alkyl chain as there is a greater opportunity for metabolism.37 Therefore, it was proposed that the length of the alkyl chain influenced the metabolism of the PV9 compared with other pyrrolidine derivatives such as α-PVP and α-PBP. However, given that there is minimal published research on the metabolism of PV9 further research is required to further clarify the relationship between the alkyl chain and the metabolism.

Reduction Hydroxylation

Hydroxylation Reduction Oxidation

Oxidation Hydroxylation/ Oxidation Oxidation Hydroxylation

Hydroxylation

Reduction

Figure 11: A proposed metabolic pathway for PV9 in humans. The prominent pathways are denoted by the highlighted arrows. Circled metabolites indicate the major metabolites identified. Image adapted from52 26

2.4 Metabolism of 3,4-methylenedioxy-N-alkylated cathinone derivatives

The 3,4-methylenedioxy-N-alkylated derivatives are characterised by both an aromatic substituent and a pyrrolidine moiety and hence their metabolism differs from that of the previously discussed methylenedioxy group. MDPV, one of the most studied derivatives belonging to this group, is structurally similar to α-PVP with the addition of a methylenedioxy substitution. The metabolism of MDPV has been studied in vitro using human HLM, human cytosol, and S9 cellular fractions as well as in human urine studies.30, 49, 58, 59 The major route for MDPV metabolism involves O-demethylation of the 4’-methoxy group to produce a catechol group, namely 3,4 dihydroxy-pyrovalerone (3,4- catechol-PV). Subsequent O-methylation at the 3’ position of 3,4-catechol-PV gives 4-hydroxy-3- methoxy-pyrovalerone (4-OH-3-meO-PV).30, 58, 59 In addition to O-demethylation, Negreira et al.58 observed metabolites that indicated other minor metabolic pathways for MDPV in vitro. MDPV can undergo hydroxylation reactions at the pyrrolidine ring and side chain followed by O-demethylation and dehydration. Additional hydroxylation of the pyrrolidine ring and oxidation causes opening of the pyrrolidine ring, similar to that observed for methylenedioxy substituted derivatives, to produce the corresponding aldehyde group (M10, Figure 12).

In regards to phase II metabolites, both glucuronidated conjugates and sulphated conjugates were identified for M2, M3 and M6 metabolites catalysed by UGTs and SULTs and excreted in the urine.58 Of the metabolites identified only M2 was sulphated while M2, M3 and M6 were glucuronidated mainly at the site of hydroxylations and carboxylations of the pyrrolidinophenones (Figure 12). The metabolic pathway observed for MDPV has been demonstrated to be common to other 3’,4’- methylenedioxy-N-alkylated derivatives. The metabolisms of MDPBP and MDPPP have shown a similar pathway of demethylenation, side chain and ring hydroxylation, oxidative degradation of pyrrolidine ring and oxidative deamination, and combinations thereof.67, 68

Figure 12: The proposed metabolic pathway for MDPV in humans. Image adapted from58

2.5 Summary of synthetic cathinone metabolism

It has been demonstrated that synthetic cathinone derivatives undergo extensive phase I metabolism, oxidation, reduction, methylation, N-dealkylation, and hydroxylation reactions. The 3,4- methylenedioxy substituted cathinones are primarily metabolized by demethylation reactions; however N-dealkylation, O-methylation and reduction of the β-ketone have also been observed. For pyrrolidinyl substituted derivatives the major metabolites identified were the products of reduction and hydroxylation/oxidation reactions. For derivatives with the presence of a methyl group on the phenyl ring, hydroxylation and subsequent oxidation to a carboxylic acid was observed. Multiple biotransformation reactions can occur at the pyrrolidine ring structure, the primary reaction being hydroxylation of the pyrrolidine ring and dehydrogenation to give a lactam structure. Glucuronidation and sulphation were the primary phase II reactions observed for the majority of synthetic cathinones, however there has been considerably less research on phase II metabolic pathways and thus further research addressing phase II metabolism is crucial; particularly for developing an understanding of the pharmacokinetics of these compounds.

CHAPTER THREE: Cathinone fragmentation patterns

Knowledge of fragmentation pathways is essential for both the identification and structural characterisation of synthetic cathinones. As some synthetic cathinones are produced by only small modifications in their chemical structure the resultant derivatives possess shared structural moieties. As a result, some derivatives can show similar fragmentation pathways and common fragment ions. Determination of common fragmentation pathways could assist in the detection and identification of cathinone derivatives and assist researchers to predict the formation of fragment ions for derivatives of similar structure. A number of articles have been published that have illustrated the fragmentation pathways for selected cathinone derivatives.69-75 Majority of these publications used high resolution methods that incorporated electrospray ionisation and CID to assist in structural elucidation. This section will provide an overview of the current knowledge of the fragmentation pathways of synthetic cathinones focussing on a number of key publications that have assessed general fragmentation pathways.

3.1 General fragmentation patterns of synthetic cathinones

The fragmentation patterns of synthetic cathinones are considered to be characterised by the molecule side-chain; with the base peak of the spectra largely dependent on the number of carbon atoms substituted at the R3, R4 and R5 position. As such, different cathinone derivatives with similar side chains (i.e. those with identical number of carbon atoms in R3 and R5 positions) can produce base peaks with identical m/z values.71 Cathinone compounds that fall into the same class have been shown to fragment via similar pathways with some common primary product ions.69 To establish the general fragmentation pathways of synthetic cathinones, Fornal69 investigated the CID fragmentation pathways of 39 protonated cathinone derivatives using reverse-phase HPLC coupled Q-TOF-MS equipped with a collision cell for ion fragmentation. Based on their chemical structure each of the assessed cathinones was divided into six classes and the fragmentation patterns of each were investigated (Table 1). Overall four general fragmentation pathways were identified for the range of cathinones investigated (Figure 13).

Table 1: Summary of the names, formulae and structures of the 39 investigated cathinone derivatives. Substituents: methyl (CH3); Et, ethyl (C2H5); Pr, propyl (C3H7); MeOx, methoxy (CH3O); Pyr, pyrrolidinyl (C4H8NH); DMe, dimethyl (two CH3); MeDOx, methylenedioxy (–O–CH2–O). 69

The loss of water (Pathway 1) resulted in the formation of the highly abundant dehydrated [M-

+ H2O+H] ion which is characterised by a loss of 18.01060 Da (H2O) from the precursor molecular ion (Figure 13).69, 70, 73 The loss of water is proposed to occur as a result of the affinity of the carbonyl’s oxygen for the hydrogen on the α-carbon, which results in a hydrogen shift form the amine to the protonated oxygen of the ketone.73 As this hydrogen shift requires physical proximity between the amine and the oxygen, longer carbon chains between these structure have been suggested to limit the migration of hydrogen and hence the loss of water.76 Therefore, the loss of water is mostly expected for cathinone derivatives in which the amine group is a primary or secondary amine that possesses at least one hydrogen atom.69, 76 It was noted that the water loss was most abundant for classes IA and IIIA, which are comprised mainly of primary aliphatic amines and methylenedioxy

+ substituted primary aliphatic amines. No water loss (i.e. an absence of [M-H2O+H] ions) was noted for classes IIIB, IV and V (Table 1).

The second common fragmentation pathway identified was the cleavage of the CH-NR3R4 bond + (Pathway 2) resulting in the loss of an amine (NHR3R4) and production of a [NR3R4+H] ion (Figure 13). This pathway was proposed to be characteristic to all cathinones (with the exception of BMDP);

+ however, the highest abundance of the characteristic [NR3R4+H] ion was observed for classes IB, II, 69 IIIB, IV and VA (Table 1). The introduction of different R1 and R2 substitutions influenced the + proportion of [NR3R4+H] ions detected for specific cathinones. The introduction of a 4-methyl substituent at the phenyl ring of the structure (i.e. mephedrone, methedrone, MPPP etc.) was

+ associated with an increase in the formation of [NR3R4+H] ions, while the introduction of a methoxy + substituent at the same position was associated with a decrease in the intensity of [NR3R4+H] ion 69 formation. For compounds with the same R1 substituent (i.e. 4-me, 4-meox, 3,4-mediox) the abundance of ion formation showed dependence on the length of the R2 substituent such that increases in the length of the R2 substituent were associated with a decreased occurrence of + [NR3R4+H] ions.

Figure 13: A schematic representation of the postulated general CID fragmentation pathways of synthetic cathinones. 69

A key feature of the fragmentation of cathinone derivatives was the formation of an immonium + cation [R2CH2CH=NR3R4] as a result of the cleavage of the CO-CHCH2R2 bond (Pathway 3a). The formation of immonium cations was observed for all cathinone derivatives assessed, which is in agreement with previous publications.70, 74 The cumulative relative intensity of immonium ions in the spectra was highest for cathinones belonging to classes IIIB and IV, while the abundance was comparatively for those cathinones belonging to classes IIA and VA (Table 1). Additionally it was noted that immonium cations were generated at higher collision energies (20 eV and 40 eV) unlike

+ + the other observed ions ([NR3R4+H] and [M-H2O+H] ), which were observed at lower collision energies.69 A secondary pathway (Pathway 3b) was identified that involved the formation of an oxonium ion and the loss of an amine R2(CH2)2NR3R4 (Figure 13). However, a low abundance of this ion was observed across all six cathinone classes, suggesting it is not a favourable pathway. The final pathway identified (Pathway 4) was the loss of CH4O2 (48.0211Da), which is attributed to the 32 methylenedioxy group (Figure 13). This pathway was common to cathinones of classes IIIA and VI (both methylenedioxy substituted derivatives).

The fragmentation of 3,4-methylenedioxy substituted derivatives has been studied in detail in a separate study by Fornal70 in which LC-ESI-Q/TOF was used to investigate the CID fragmentation pathways of methylone, butylone, pentylone, MDPBP, MDPV and BMDP. The major pathway observed was loss of CH4O2 from the methylenedioxy group, that was accompanied by the formation of phenyloxazole cations (Cation B, Figure 14).69, 70, 73 Additionally the formation of methylenedioxybenzoyloxonium ions (Cation D), allyldioxybenzoyloxonium ions (cation C), and immonium ions (Cation E) was also observed for 3,4-methylenedioxy substituted derivatives (Figure 14).70 Interestingly, the assessed MDPV and MDPBP compounds, both of which contain a pyrrolidinyl group had different fragmentation mechanisms compared to the other compounds assessed. Neither fragment ion A nor fragment ion B were observed for these compounds at any assessed collision level. This was proposed to be the result of steric hindrance from the pyrrolidinyl moiety, 70 which prevents the removal of the CH4O2. This observation is consistent with the findings of 69 Fornal where no CH4O2 loss or and fragment ion formation was observed for MDPV, 3,4-MDPPP or 3,4-MDPBP.

Figure 14: A postulated fragmentation pattern of 3,4-methylenedioxy substituted cathinone derivatives. (a) Fragment formation inhibited for MDPBP and (b) Fragment formation inhibited for BMPP. 70

3.2 Fragmentation patterns for N-pyrrolidinyl substituted cathinone derivatives

To investigate the generalised fragmentation routes of pyrrolidinyl substituted derivatives 13 substituted cathinones of varying alkyl chain length (ranging from one to seven carbons) and various

72 aromatic ring substituents (i.e. H, Cl, F, CH3, OCH, O(CH2)2 to (CH2)4) were selected. The two major fragmentation routes observed for pyrrolidinyl substituted derivatives were: the cleavage of the CH- 72 N(CH2)4 bond resulting in the loss of an amine moiety, and cleavage of the CO-CHN bond. The loss of the amine resulted in the formation of product ion A (Figure 15); which is suggested to be characteristic of all pyrrolidinyl substituted derivatives. Further dissociation of CO and the alkyl chain from product ion A was suggested to result in the production of product ion C (Figure 15), another highly abundant ion in the spectra of most of the assessed cathinones.72 However, for the formation of product ion B and C an alkyl substitution (R2 substitution) of at least two carbon atoms was required. Cleavage of the CO-CHN bond resulted in the formation of benzoyl ions (ion D) and immonium cations (Ion G); similar to that observed by Fornal69 (Figure 15). Lastly, loss of the alky chain from the protonated molecule was observed for most of the pyrrolidinyl substituted cathinones which resulted in the formation of radical ion F (Figure 15). However, this ion was observed at a relatively low intensity especially with the introduction of O(CH2)2 or (CH2)4 substitutions on the phenyl moiety.72

Figure 15: The proposed generalised fragmentation pathways for pyrrolidinyl substituted cathinone derivatives. 72

3.3 Summary of fragmentation

The literature has demonstrated a degree of predictability within the fragmentation of synthetic cathinones. Synthetic cathinones that belong to the same group (as denoted by their chemical structure) fragment via similar pathways, producing characteristic fragment ions that can be used to assist in the classification of cathinone derivatives. The generalised fragmentation pathways described in this review can be treated as a framework to assist in the categorisation of unknown compounds into a specific group of cathinones. These fragmentation pathways could assist in not only classifying compounds according to class but could also highlight unique peaks that can be used to identify potentially viable structures in the identification of unknown compounds.

CHAPTER FOUR: Conclusion

The aim of this review was to provide a comprehensive and up-to-date overview of the knowledge surrounding the metabolism and mass-spectral fragmentation of synthetic cathinones. A major problem in the analysis of synthetic cathinones is that a large number of them are structurally similar, which makes identification difficult; especially in consideration of the lack of reference standards for newly synthesised synthetic cathinones. While a substantial amount of information is known about the metabolism of synthetic cathinones, a considerable number of cathinone metabolites have yet to be characterised. Further studies are therefore necessary to not only establish the metabolic profiles of newly synthesised cathinone derivatives, but to also incorporate the identified metabolites into identification and quantification methods. While a number of publications have proposed potential metabolites to be used as detection markers, there is a lack of validation studies to confirm the consistent presence of such metabolites in a range of biological matrices. Another limitation of the current knowledge of the metabolism of synthetic cathinones stems from a lack of controlled studies in humans. Studies using human urine were often performed using samples with unknown dosage amount, time of ingestion, and with samples collected at unknown urinary phases; each of which has an impact on the metabolites observed.

Knowledge of mass spectral fragmentation patterns is a crucial tool for the identification of synthetic cathinone structures. This review focussed on the characteristic fragmentation patterns identified for cathinone derivatives using high-resolution LC-ESI/QTOF-MS techniques. While an abundance of information regarding CID fragmentation pathways currently exists, most of the information is scattered across a number of publications; many of which focus on the identification of an individual cathinone. While the publications discussed in this review have attempted to collate this information by establishing generic fragmentation pathways, further evaluation and examination of the literature would be beneficial to produce a consolidated resource that summarises the fragmentation data obtained for synthetic cathinones.

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47. Meyer MR, Wilhelm J, Peters FT, Maurer HH. Beta-keto amphetamines: studies on the metabolism of the designer drug mephedrone and toxicological detection of mephedrone, butylone, and methylone in urine using gas chromatography–mass spectrometry. Analytical and Bioanalytical Chemistry. 2010;397(3):1225-33.

48. Khreit OIG, Grant MH, Zhang T, Henderson C, Watson DG, Sutcliffe OB. Elucidation of the Phase I and Phase II metabolic pathways of (±)-4'-methylmethcathinone (4-MMC) and (±)-4'- (trifluoromethyl)methcathinone (4-TFMMC) in rat liver hepatocytes using LC-MS and LC-MS2. Journal of Pharmaceutical and Biomedical Analysis. 2013;72:177-85.

49. Uralets V, Rana S, Morgan S, Ross W. Testing for Designer Stimulants: Metabolic Profiles of 16 Synthetic Cathinones Excreted Free in Human Urine. Journal of Analytical Toxicology. 2014;38(5):233-41.

50. Pozo ÓJ, Ibáñez M, Sancho JV, Lahoz-Beneytez J, Farré M, Papaseit E, et al. Mass spectrometric evaluation of mephedrone in vivo human metabolism: identification of phase I and phase II metabolites, including a novel succinyl conjugate. Drug metabolism and disposition: the biological fate of chemicals. 2015;43(2):248-57.

51. Pedersen AJ, Reitzel LA, Johansen SS, Linnet K. In vitro metabolism studies on mephedrone and analysis of forensic cases. Drug testing and analysis. 2013;5(6):430-8.

52. Shima N, Katagi M, Kamata H, Matsuta S, Nakanishi K, Zaitsu K, et al. Urinary excretion and metabolism of the newly encountered designer drug 3,4-dimethylmethcathinone in humans. Forensic Toxicology. 2013;31(1):101-12.

53. Helfer AG, Turcant A, Boels D, Ferec S, Lelièvre B, Welter J, et al. Elucidation of the metabolites of the novel psychoactive substance 4‐methyl‐N‐ethyl‐cathinone (4‐MEC) in human urine and pooled liver microsomes by GC‐MS and LC‐HR‐MS/MS techniques and of its detectability by GC‐MS or LC‐MSn standard screening approaches. Drug Testing and Analysis. 2015;7(5):368- 75.

54. Pedersen AJ, Petersen TH, Linnet K. In vitro metabolism and pharmacokinetic studies on methylone. Drug metabolism and disposition: the biological fate of chemicals. 2013;41(6):1247-55.

55. Katagi M, Zaitsu K, Shima N, Kamata H, Kamata T, Nakanishi K, et al. Metabolism and forensic toxicological analyses of the extensively abused designer drug methylone. TIAFT Bulletin. 2010;40(1):30-6.

56. Zaitsu K, Katagi M, Tsuchihashi H, Ishii A. Recently abused synthetic cathinones, α- pyrrolidinophenone derivatives: a review of their pharmacology, acute toxicity, and metabolism. Forensic Toxicology. 2014;32(1):1-8.

57. Zaitsu K, Katagi M, Kamata HT, Kamata T, Shima N, Miki A, et al. Determination of the metabolites of the new designer drugs bk-MBDB and bk-MDEA in human urine. Forensic science international. 2009;188(1-3):131-9.

58. Negreira N, Erratico C, Kosjek T, van Nuijs ALN, Heath E, Neels H, et al. In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol. Analytical and Bioanalytical Chemistry. 2015;407(19):5803-16.

59. Strano-Rossi S, Cadwallader AB, de la Torre X, Botrè F. Toxicological determination and in vitro metabolism of the designer drug methylenedioxypyrovalerone (MDPV) by gas chromatography/mass spectrometry and liquid chromatography/quadrupole time-of-flight mass spectrometry. Rapid communications in mass spectrometry : RCM. 2010;24(18):2706-14.

60. Matsuta S, Shima N, Kamata H, Kamata T, Kakehashi H, Nakano S, et al. Metabolism of the designer drug α-pyrrolidinobutiophenone (α-PBP) in humans: Identification and quantification of the phase I metabolites in urine. Forensic Science International. 2015;249:181-8.

61. Shima N, Katagi M, Kamata H, Matsuta S, Sasaki K, Kamata T, et al. Metabolism of the newly encountered designer drug α-pyrrolidinovalerophenone in humans: identification and quantitation of urinary metabolites. Forensic Toxicology. 2014;32(1):59-67.

62. Tyrkkö E, Pelander A, Ketola RA, Ojanperä I. In silico and in vitro metabolism studies support identification of designer drugs in human urine by liquid chromatography/quadrupole-time- of-flight mass spectrometry. Analytical and Bioanalytical Chemistry. 2013;405(21):6697-709.

63. Grapp M, Sauer C, Vidal C, Müller D. GC–MS analysis of the designer drug α- pyrrolidinovalerophenone and its metabolites in urine and blood in an acute poisoning case. Forensic science international. 2016;259:e14-e9.

64. Paul M, Bleicher S, Guber S, Ippisch J, Polettini A, Schultis W. Identification of phase I and II metabolites of the new designer drug α‐pyrrolidinohexiophenone (α‐PHP) in human urine by liquid chromatography quadrupole time‐of‐flight mass spectrometry (LC‐QTOF‐MS). Journal of Mass Spectrometry. 2015;50(11):1305-17.

65. Swortwood MJ, Carlier J, Ellefsen KN, Wohlfarth A, Diao X, Concheiro-Guisan M, et al. In vitro, in vivo and in silico metabolic profiling of α-pyrrolidinopentiothiophenone, a novel thiophene stimulant. Bioanalysis. 2016;8(1):65-82.

66. Shima N, Kakehashi H, Matsuta S, Kamata H, Nakano S, Sasaki K, et al. Urinary excretion and metabolism of the α-pyrrolidinophenone designer drug 1-phenyl-2-(pyrrolidin-1-yl)octan-1- one (PV9) in humans. Forensic Toxicology. 2015;33(2):279-94.

67. Meyer MR, Mauer S, Meyer GMJ, Dinger J, Klein B, Westphal F, et al. The in vivo and in vitro metabolism and the detectability in urine of 3’,4’‐methylenedioxy‐alpha‐ pyrrolidinobutyrophenone (MDPBP), a new pyrrolidinophenone‐type designer drug, studied by GC‐MS and LC‐MSn. Drug Testing and Analysis. 2014;6(7-8):746-56.

68. Springer D, Staack RF, Paul LD, Kraemer T, Maurer HH. Identification of cytochrome P450 enzymes involved in the metabolism of 3′,4′-methylenedioxy-α-pyrrolidinopropiophenone (MDPPP), a designer drug, in human liver microsomes. Xenobiotica. 2005;35(3):227-37.

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PART TWO: Manuscript

A review of the collision induced dissociation fragmentation and the metabolism of synthetic cathinones

*Tables 3-5 referenced in text have been removed from this document for copyright reasons. A whole version of the thesis is available upon request.

A review of the collision induced dissociation fragmentation patterns and the metabolism of synthetic cathinone derivatives

Nichola Cunningham1, Dr John Coumbaros2

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

2 ChemCentre, Perth, WA

Abstract

The emergence and propagation of synthetic cathinones on the designer drug market is an evolving worldwide problem. Synthetic cathinones encompass a diverse range of compounds with varying structural features and substituted functional groups. This variability, in combination with their widespread availability, poses a challenge for forensic and toxicology laboratories to develop and maintain analytical methods for the detection and identification of synthetic cathinone derivatives and their associated metabolites. The aim of this dissertation was to consolidate the current knowledge regarding the metabolism and the collision induced dissociation spectral fragmentation of synthetic cathinone derivatives. This will involve the collation and discussion of the m/z values observed for characteristic fragment ions of each class of cathinone derivative, in addition to some characteristic higher-order fragment ions; with the objective of developing a combined resource from which general conclusions regarding the fragmentation patterns of synthetic cathinones can be drawn. The main fragmentation pathways for cathinone derivatives and the primary CID fragment ions could assist in the identification of newly-synthesised cathinone derivatives and facilitate the development of detection methods for synthetic cathinones.

Keywords: Synthetic cathinones; Metabolism; Cathinone Fragmentation patterns; Novel psychoactive substances; Collision induced dissociation.

1.0 Introduction

The continual emergence of novel psychoactive substances (NPS), also known as “designer drugs” or “legal highs” is a growing worldwide problem. NPS constitute an evolving mixture of modified substances that are designed to reproduce the effects of other illicit substances such as cannabis, hallucinogens and stimulants.1 Synthetic cathinones constitute a class of NPS that has experienced an increased emergence on the drug market over the last decade. A report published by the Australian Criminal Intelligence Commission suggested that cathinone-type substances accounted for 33.3% of analysed seizure samples between 2015 and 2016, which is in line with the trends observed in European countries.2, 3 Synthetic cathinones are derivatives or analogues of the β-keto amphetamine cathinone; the major psychoactive stimulant component of the psychoactive plant Catha Edulis or Khat.4 These derivatives possess numerous structural modifications and substitutions creating a diverse range of compounds. Synthetic cathinones are primarily marketed and sold through online networks; often in packaging with minimal composition information.5 Therefore, analytical approaches for the detection and identification of these compounds are crucial to ascertain the structure of new cathinone compounds and to assist in toxicological risk assessment. However, the structural diversity of cathinone derivatives available on the drug market and the lack of available reference standards or database libraries poses a challenge for forensic laboratories to develop and maintain analytical methods for the detection of these substances and their metabolites.6

Various spectrometric techniques have been employed to assist in the identification and structural characterisation of synthetic cathinone compounds.7 Spectrometric methods such as gas- chromatography mass spectrometry (GC-MS) and liquid-chromatography mass spectrometry (LC- MS), coupled to high resolution mass spectrometry (HRMS) are commonly published methodologies for the identification and structural characterisation of synthetic cathinones.8 The incorporation of collision induced dissociation (CID) fragmentation with a high resolution mass spectrometer can provide crucial information regarding both the composition of the molecule, and in identifying the fragmentation pathways and the primary metabolites of specific synthetic cathinones. Furthermore, HRMS methods can be useful for the identification of compounds without the need for reference standards; which are often unavailable for newly synthesised synthetic cathinones.9 While an abundance of information regarding the fragmentation of synthetic cathinones exists, the information is often scattered across multiple publications; the majority of which focus on the structural characterisation of an individual derivative or a few selected cathinone derivatives. As

45 such there is a lack of consolidated information that compares the similarities and differences of the pathways observed within the literature for specific cathinone derivatives.

The aim of this dissertation is to provide a comprehensive review of the mass spectral fragmentation characteristics of synthetic cathinone derivatives and the metabolism of these substances. This will involve comparing the published data, specifically the mass-to-charge ratio (m/z) values observed throughout the literature for specific cathinone fragment ions with the objective of producing a consolidated resource. A review of the literature was conducted, focussing on literature that discussed the characterisation of mass-spectral fragmentation pathways of cathinone derivatives, the identification of metabolic pathways, and identification of metabolites.

2.0 Structure and chemistry of synthetic cathinones

Synthetic cathinone derivatives possess the common structure of the natural compound cathinone. The structure of cathinone closely resembles that of amphetamine, which is differentiated by the presence of a ketone functional group at the β-amino chain attached to the phenyl ring (Figure 1).10 Given this structural similarity, cathinone and its synthetic derivatives produce effects similar to those of amphetamines such as central nervous system effects, including increased sensory stimulation, euphoria, excitation, and agitation.11 Synthetic cathinones are primarily available in crystalline and powder forms that can be administered by snorting, smoking, oral ingestion, and less commonly via intravenous injection.12

Figure 1: Comparison of the natural cathinone structure (Left) and the amphetamine structure (Right). Source: Kelly11

The natural structure of cathinone lends itself easily to modifications at several locations on the molecule. The addition of various substitutions on the cathinone molecule results in the generation of a large number of synthetic cathinone derivatives that can be classified based on their chemical

4 structures. Synthetic cathinones are formed by either ring substitution at the R1 position, variation 10 in the α-carbon substituent (R2), or N-alkylation at the R3 and R4 positions (Table 1). Based on these substitutions, synthetic cathinones can be categorised into four chemical families: N-alkylated derivatives, methylenedioxy-substituted derivatives, N-pyrrolidine derivatives, and 3,4- methylenedioxy-N-pyrrolidine derivatives.4 Table 1 displays a list of the N-alkylated and methylenedioxy synthetic cathinones arranged by their chemical substituents at each of the substitution points.10 Table 2 represents the chemical and substituents of the pyrrolidine substituted cathinone derivatives.

Table 1: The chemical structures of the N-alkylated and methylenedioxy substituted cathinone derivatives. 3,4-DMMC, 3,4-dimethyl-methcathinone; 4-MEC, 4- methylcathinone; DMMC, Dimethylmethcathinone; BMDP, 3,4-methylenedioxy-N-benzyl cathinone.

Substituents: CH3, methyl; C2H5, ethyl; C3H7, propyl; OCH3, methoxy. 47

The N-alkylated cathinones are a group of derivatives characterised by the presence of substituents at the R3 and R4 sites, and/or compounds with an alkyl or halogen substituent at the R1 position (Table 1). The methylenedioxy cathinone derivatives are characterised by the addition of a methylenedioxy substituent at the aromatic ring (R1) (Table 1). Pyrrolidinyl cathinone derivatives are characterised by the presence of a pyrrolidine ring at the nitrogen, instead of an amine or N-methyl amine substitute (Table 2). The 3,4-methylenedioxy-N-pyrrolidine cathinone derivatives are characterised by the presence of both a methylenedioxy substitution at the R1 position and the presence of a pyrrolidinyl substituent at the nitrogen atom (Table 2). Similarity between the chemical structures of synthetic cathinones, in addition to their increasing numbers, poses an analytical challenge for the detection and identification of specific cathinones, particularly if the analytical technique used does not provide any information relating to the parent compound.

Table 2: The chemical structures of pyrrolidinyl substituted cathinone derivatives. α-PPP, α- pyrrolidinopropiophenone; α-PHP, α-pyrrolidinohexanophenone ;α-PVP,α-pyrrolidinovalerophenone; α-PBP, α- pyrrolidinobutyrophenone; α-PEP, α-pyrrolidinoenanthophenone; α-; α-POP, α-pyrrolidinooctanophenone; MPPP, 4-methyl-α-pyrrolidinopropiophenone; MOPPP, 4-methoxy-α-pyrrolidinopropiophenone; MPBP, 4'- Methyl-α-pyrrolidinobutiophenone; MDPPP, 3,4-methylenedioxy-α-pyrrolidinopropiophenone ; MDPBP,3,4- methylenedioxy-α-pyrrolidinobutiophenone; MDPV, 3,4-methylenedioxypyrolvalerone.

Substituents: CH3, methyl; C2H5, ethyl; C3H7, propyl; OCH3, methoxy. 48

3.0 Metabolism of synthetic cathinones

Characterisation of both the in vivo and in vitro synthetic cathinone metabolites is important for identifying unique metabolite targets that can be incorporated into analytical approaches for the identification of compounds and in the development of urine screening methods.13

3.1 N-alkylated cathinone derivatives

Primary metabolic pathways of N-dealkylation and β-ketone reduction have been described in both in vivo human urine and HLM studies for N-alkylated cathinone derivatives.14, 15 A recent comprehensive study conducted by Uralets et al.16, summarised the metabolic profiles of 16 synthetic cathinones that were detected in human urine samples. Nine of the cathinones identified were classified as N-alkylated substituents, which included: pentedrone, 4-MEC, flephedrone, buphedrone, mephedrone, ethcathinone, DMMC, 4-Methylbuphedrone and N-ethylbuphedrone. These derivatives were proposed to be metabolised via N-dealkylation and ketone reduction to give the corresponding alcohol; with subsequent N-dealkylation resulting in N-dealkylated alcohol metabolites (Figure 2).16-18

For those cathinone derivatives with a 4-methyl group hydroxylation and carboxylation of the 4- methyl group was observed, while those with a 4-methoxy-substitution (i.e. methedrone) O- demethylation is a commonly observed metabolic pathway in vitro.15, 19, 20 Uralets et al.16 noted that the parent drugs were less abundant than their metabolites, suggesting a high extent of metabolism. However, urine samples were collected at unknown stages of urinary excretion, with unknown dosage and time of ingestion and therefore no determination can be made about the longevity of the metabolites. Additionally, the authors only considered freely excreted non-conjugated metabolites and therefore it is possible that other metabolites were present but were undetected. In regards to phase II metabolism, glucuronic conjugates have been identified both in vitro and in human urine.21 In a study investigating mephedrone, Pozo et al.15 identified glucuronide conjugates

15 within human urine samples. In addition, Pozo et al. was the first to identify a conjugate with succinic acid metabolite in human urine.

Figure 2: The proposed metabolic pathway of mephedrone in humans. Image adapted from

Pozo et al.15.

3.2 Pyrrolidine-substituted cathinone derivatives

The metabolism of selected pyrrolidine substituted cathinone derivatives has been studied extensively using both in vitro methodologies using pS9, pooled human liver microsomes (pHLM) and liver cytosol, and within human urine samples.17, 18, 20, 22-24 The metabolic pathways of pyrrolidine derivatives primarily involve the reduction of the ketone group to the corresponding alcohol, hydroxylation of the pyrrolidine ring to generate a corresponding pyrrolidone, and hydroxylation of the aliphatic side chain (Figure 3).20 Recently, Manier et al.13 examined the in vitro metabolism of the pyrrolidine derivatives α-PBP, α-PVP, α-PHP, α-PEP, and α-POP using PS9 and pHLM incubations. All of the assessed compounds were shown to undergo hydroxylation reactions and the formation of lactam groups (Figure 3); findings that are consistent with other publications assessing in vitro metabolism of α-pyrrolidinophenones.18, 20 The majority of compounds such as α-PBP, α-PHP, α-PEP, and α-POP are hydroxylated at the pyrrolidine ring, resulting in the formation of mono- and dihydroxy metabolites. Interestingly, α-PBP undergoes hydroxylation solely at the pyrrolidine ring while α-PHP, α-PEP, and α-POP have shown additional hydroxylation at the alkyl chain and mono 50 and bis-hydroxylation.13 In contrast to other α-pyrrolidinophenones, α-PVT undergoes hydroxylation reactions at both the thiophene ring and the pyrrolidine ring to form mono- and dihydroxy metabolites.25 Additional hydroxylation at the aliphatic side chain of α-PEP and α-POP has also been observed to produce oxo and carboxy metabolites respectively.13 Shima et al.17 proposed that the main metabolic pathways of pyrrolidine cathinone derivatives will differ markedly based on the length of the alkyl chain. For compounds with longer chains such as α-POP and α-PV8 (Table 2), metabolites formed by oxidation reactions have been proposed as the primary metabolites for PV9, while metabolites formed via hydroxylation or reduction of the ketone group represent minor metabolites.13, 17, 18

Figure 3: Proposed metabolic pathway for α-PVP based on metabolites observed in human urine and HLM. Image adapted from Negreira et al.20

3.3 Methylenedioxy substituted cathinone derivatives

Methylenedioxy cathinones such as methylone, butylone, ethylone, and pentylone have been characterised both in vitro and in vivo.16, 19, 26, 27 As these derivatives are structurally similar, it is unsurprising that comparable metabolic pathways have been observed. These compounds primarily undergo demethylenation reactions both in vitro and in vivo; with metabolites resulting from demethylenation and O-demethylenation among the most common metabolites identified (Figure 4). Additionally, the N-dealkylation and β-ketone reduction reactions have also been observed for the methylenedioxy substituted derivatives at a lesser extent than was observed for N-alkylated cathinone derivatives.16 This may be a consequence of steric restrictions resulting from the presence of the methylenedioxy substitution at the aromatic ring.28

N-dealkylation Hydroxylation

Reduction Demethylenation

O-OMethylation-Methylation

Figure 4: Proposed in vitro and in vivo metabolic pathway for methylone in humans. Image adapted from Pedersen et al. 26

4.0 Proposed fragmentation patterns of cathinone derivatives

An understanding of the CID fragmentation pathways for synthetic cathinones is crucial for both the identification and structural characterisation of synthetic cathinones and unknown metabolites. The fragmentation patterns of numerous synthetic cathinones have been investigated by several research groups; producing an abundance of published research.29-34 The fragmentation of synthetic cathinones is considered to be dependent on the functional group attached to the molecule side chain, and the number of carbon atoms substituted at the R3, R4, and R5 positions. As such, compounds with a similar structure have been suggested to fragment via similar fragmentation pathways and produce common fragment ions.32 The cleavage of functional groups from the chemical structure is associated with the production of distinct peaks on a resultant mass spectrum; the m/z values of which differ based on the absence or presence of substituents. Therefore, different fragmentation patterns for synthetic cathinones can be anticipated based on the chemical structure of the compound, and can be used to differentiate between compounds.

General CID fragmentation pathways that apply to a range of cathinone derivatives have been explored in a number of publications.29, 30, 33 Fornal29 investigated the CID fragmentation patterns and primary product ions of 39 synthetic cathinone derivatives using quadruple time-of-flight mass spectrometry (Q-TOF) with electrospray ionisation and CID. Four main dissociation pathways have been observed across a range of synthetic cathinone derivatives. The primary product ions identified are the result of the loss of water, the loss of an amine, production of an immonium ion, production 29 of an oxonium ion, and the loss of the CH4O2 moiety (Figure 5). The elimination of H2O from the + protonated molecule results in the production of a [H2O+H] ion which is characterised by a loss of

18.01060Da (H2O) from the precursor molecular ion (Figure 5, Pathway 1). The cleavage of the CH- + NR3R4 bond results in the loss of an amine (NHR3R4) producing [NR3R4H] ions (Figure 5, pathway 2).

Cleavage of the CO-CHCH2R2 bond results in the production of immonium ions, the m/z value of which will depend on the R2, R3 and R4 substituents. The production of immonium ions is dominant in most cathinones suggesting this as a major fragmentation pathway. A secondary pathway that involves the formation of an oxonium ion (R2(CH2)2NR3R4) has also been observed (Figure 5, pathway 3B); however these ions have been observed at low abundance indicating it is not a primary fragmentation pathway.29 The final generalised fragmentation pathway is ascribed to the loss of a

CH4O2 (48.0211 Da) from the molecule, that is attributed to the methylenedioxy group.

Figure 5: A schematic representation of the postulated CID fragmentation pathways for the generation of primary product ions of protonated synthetic cathinones. Source: Fornal29

Each of these major fragmentation pathways results in the formation of distinct fragment ions, the m/z values of which will differ depending on the different functional group substitutions. However,

+ the formation of some of these fragment ions (i.e. immonium cations or [M-CH4O2] ions) are not unique to cathinone derivatives. For instance, cations formed from the loss of the amine are also common among phenethylamines.35

5.0 Summary of the observed m/z values for fragment ions of synthetic cathinone derivatives.

The published m/z data of primary fragment ions obtained from multiple publications will be collated and compared. To allow comparisons between articles the mass spectral m/z values compared were generated using comparable methodologies. In each of the selected articles the mass-spectral fragmentation of the selected cathinone derivatives were investigated using high- performance-liquid-chromatography quadruple time-of-flight-mass spectrometry (HPLC-QTOF-MS) following collision induced dissociation and electrospray ionisation techniques.29-31, 33 The discussed fragment ions were generated using fixed collision energies at 10, 20 and 40 eV; with the exception of Qian et al.33 where the mass spectra were acquired at sweeping collision energies set at 35±45 eV.

5.1 Fragmentation patterns of N-alkylated cathinone derivatives

The characteristic fragment ions listed and described in Table 3 suggest that the majority of N- alkylated cathinone compounds primarily yield dehydrogenated ions, ions resulting from the loss of amine, and immonium cations. Of the range of fragment ions identified in the collected data, 29 + Fornal suggested that the formation of the [M-H2O + H] ions were among the most abundant identified for the N-alkylated class of cathinones. In addition, Fornal29 proposed that the cumulative relative intensity of these ions increased in proportion to the length of the R1 substituent. The loss of water is proposed to result from a hydrogen shift from the α-carbon to the protonated oxygen of the 34 ketone; a shift that requires physical proximity between the amine and the oxygen. Therefore, longer carbon chains between these structures have been suggested to limit the migration of hydrogen and, as such, the loss of water from molecules has been proposed to be indicative of cathinone derivatives in which the amine group is a primary or secondary amine that possesses at least one hydrogen atom.31

The m/z values for a number of the discussed fragment ions were similar between different cathinone derivatives, indicating that derivatives of similar structure produced similar fragment ions. + This was specifically observed for fragments formed as a result of amine cleavage [M-NHR3R4+H] and immonium cations (Table 3). Three distinct clusters of m/z values could be observed for the [M-

+ NHR3R4+H] fragment ions with m/z values of approximately 161.0961 (C11H13O), 133.0649 (C9H9O+), and 147.0808 (C10H11O) (Table 3). Similarly for the produced immonium cations, m/z values of approximately 86.09 and 72.08 were observed across a number of the assessed derivatives. Fornal29 noted that the relative abundance of immonium ions in the spectra was significantly lower than

55 those of other characteristic product ions.29 However, it is worth noting that the data collated in Table 3 are limited to that published in Fornal29 and therefore, in regards to trends in the fragmentation, only limited conclusions can be drawn.

5.2 Fragmentation patterns of 3,4-methylenedioxy substituted cathinone derivatives

Fragment ions associated with the loss of the neutral group CH4O2 (48.0211) have been proposed as the primary fragment ion and fragmentation pathway for methylenedioxy cathinone derivatives.29, 30

For [M-CH4O2] fragment ions m/z values of 174.0911 (C11H12NO) and 188.1069 (C12H14NO) were consistently observed for butylone, ethylone, pentylone and eutylone, respectively (Table 4). Given that the loss of the CH4O2 is unique of methylenedioxy substituted cathinones, the observation of this fragment ion could be useful to facilitate identification. However, while this fragment ion represents a characteristic of methylenedioxy, additional fragments ions have also been identified that can identify the structural features of methylenedioxy substituted cathinone derivatives.29, 30 The fragment ions corresponding to the loss of water, the loss of amine, and immonium ion formation, which were observed for the N-alkylated derivatives, have also been observed in methylenedioxy substituted cathinone derivatives (Table 4).29, 30 However, Fornal29 noted that these were present at lower abundances in methylenedioxy derivatives than was observed for the N-

+ alkylated derivatives. For N-methyl derivatives at a low collision energy (10 eV), [M-H2O] and [M- + C4H2O] were the most abundant fragment ions observed for these compounds, while at higher collision energies (20 eV and 40 eV) the [C9H9N]+/ [C9H10N]+ ions formed from the elimination of 30 H20, methanedial and C-alkyl were the most abundant fragment ions observed. Therefore, consideration of the collision energies used must be considered during the analysis of mass spectrums.

In addition to the discussed general fragment ions, a number of characteristic higher-order product ions have been identified for methylenedioxy cathinone derivatives (Ion 5 and Ion 6, Table 4). For methylone, butylone, and pentylone characteristic fragment ions with m/z values of 147.0437

+ + + (C9H7O2) , m/z 161.0597 (C10H9O2) , and m/z 175.0751 (C11H11O2) have been observed, that 29 correspond to the loss of formaldehyde and amine (NHR3R4) from the protonated molecular ions. The emergence of these fragment ions has been proposed to be formed after the loss of water,

31 CH4O2, and methylamine ions and occur at higher collision energies.

Interestingly, the N-benzyl derivative BMDP (structure shown in Table 1) exhibited a different CID fragmentation profile from that observed for the other methylenedioxy substituted cathinone derivatives. For BMDP, the most abundant fragment ion observed in the literature was the stable

31 tropylium cation (m/z value 91.0542, [C7H7]) (Table 4). It has been proposed that the presence of the benzyl group (i.e. a group of atoms capable of forming the tropylium ion) results in a shift in the fragmentation to produce immonium ions.30, 31 As such, the BMDP spectra is characterised by a base peak of tropylium cations while the other characteristic ions observed for other cathinone derivatives are present only at lower collision energies.

For those compounds which contain both the 3,4-methylenedioxy structure and the pyrrolidine + moiety (i.e. 3,4-MDPPP, 3,4-MDPBP, MDPV), neither the dehydrogenated ions ([M-H2O] ) or + phenyloxazole ions ([M-CH4O2] ) have been observed (Table 4). It has been proposed that the presence of the pyrrolidine structure in these molecules restricts the 1,5 hydrogen shifts from the 30 pyrrolidine group to the carbonyl group and hence the elimination of the CH4O2 is impossible. + Similarly, the absence of [M-H2O] ions also suggests that the mobility of the hydrogen on the amino group could be restricted. Given that the loss of water has been proposed to involve the migration of hydrogen from the amine group, it is suggested that fragment ions resulting from the loss of water would not occur, or fragment ions would occur at low abundances when an amine group is a tertiary amine.29

5.3 Fragmentation patterns of pyrrolidinyl substituted cathinone derivatives

The two major fragmentation routes observed for pyrrolidinyl substituted derivatives are the cleavage of the CH-N(CH2)4 bond resulting in the loss of an amine moiety, and cleavage of the CO- CHN bond.33 Based on the identified fragment ions it can be determined that pyrrolidinyl substituted cathinone derivatives undergo elimination of the pyrrolidine ring via α-cleavage of the amine group,

+ resulting in the formation of (NHR3R4 +H ) ions and immonium cations; the m/z values of which differ depending on the chemical structure of the compound. The m/z values for the fragment ions observed are relatively consistent between each of the articles published, with only minor differences observed between values (Table 5). However, in regards to the observation of fragment ions resulting from the loss of water, the data published by Manier et al.13 did not report any [M-

+ 29 33 H2O] fragment ions, a finding that differs to the data published by both Fornal and Qian et al. . Fornal29 observed that the cumulative relative abundance of the immonium ions observed suggested that immonium cations represent a primary product ion for pyrrolidine substituted cathinone 57 derivatives. The cumulative relative abundance of the characteristic fragment ions is dependent on the collision energies applied. At higher collision energies (i.e. 20 eV and 40 eV) immonium ions are the most abundant fragment ions, while at lower collision energies other characteristic fragment

+ + 29 ions, [NR3R4+H] and [M-H2O+H] , were the most abundant observed.

Fragment ions between m/z 70.648 and 72.0809 have been observed for all pyrrolidine compounds, with the exception of MPBP and MOPPP which showed m/z values of 133.101 and 179.1055 respectively (Table 5). Manier et al13 proposed that these fragment ions are the result of hydroxylation of the pyrrolidine ring that results in the removal of an oxo group from the pyrrolidine ring, and the production of a fragment ion (C4H8N). The observation of fragment ions which represent the mono and dihydroxy metabolites corresponds to the metabolic pathways and metabolites observed in in vitro and in vivo metabolism studies.13, 20 Additional fragmentation ions corresponding to the formation of a tropylium ion (m/z 91.0542, [C7H7]) have consistently been observed for a number of pyrrolidine cathinone derivatives including α-PBP, α-PVP, α-PHP, α-PEP, and α-POP.13, 33 However, variation in the m/z values was observed for the other pyrrolidine derivatives assessed with the different structural features, especially relating to the length of the alkyl chain (Table 5).

6.0 Conclusion

While a substantial amount of information is known about the metabolism of synthetic cathinones, a considerable number of cathinone metabolites have yet to be characterised. Therefore, further research investigating the metabolism of newly-synthesised cathinone derivatives is necessary. While a number of metabolites for specific cathinone derivatives have been identified as potential markers of parent drug use, further work is required to incorporate the identified metabolites into quantification and drug screening methods for detection. The identification of common metabolic pathways among synthetic cathinones is crucial particularly for the detection or the characterisation of unknown metabolites identified in samples. For instance, in situations in which product ions are detected but unable to be correctly identified, an understanding of the common cathinone metabolism could aid in identifying the structure. Therefore an understanding of the metabolites generated for a range of cathinone derivatives, as well as their characteristic mass-spectral features (i.e. m/z values), could assist in potentially identifying similar, or potentially likely candidates for the unknown compound.

The fragment ions and the neutral losses described in this review are the most commonly observed fragment ions for cathinone derivatives that could facilitate the identification of the structural features unique to specific cathinone derivatives. Although cathinone derivatives share a common core structure, comparison of the CID mass spectra has indicated that different functional group substitutions can lead to different fragmentation pathways. For instance, the production of ions associated with the cumulative loss of C4HO2 suggests the loss of a methylenedioxy substitution. Therefore, the presence or absence of this specific fragment ion can serve as an indicator for identifying the specific class of cathinone compounds. However, it is worth noting that some of the fragment ions observed for synthetic cathinones are not unique to cathinones, as some are present in other substances. As such, the fragment ions identified in the current literature can be treated only as a foundation for structural identification, especially if only a limited number of fragment ions are available for analysis. Additionally, as only a selection of generalised fragment ions were discussed within this review, additional work that encompasses and compares a broader range of synthetic cathinone fragment ions would be beneficial. Further investigation of the fragmentation patterns for synthetic cathinone derivatives would be beneficial in order to identify additional fragment ions and to further assess the mechanism in which these compounds undergo fragmentations and re-arrangements. A greater understanding of these processes could be applied for the structural characterisation of cathinone derivatives and assist in the identification of newly- synthesised cathinones; especially when no reference standards are available.

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