Development of Methods for the Detection of Novel Psychoactive Substances in Oral Fluid

Home , Benzedrone, BOH (drug), Buphedrone, Cathine, Clobenzorex, Ethcathinone

Development of methods for the detection of Novel Psychoactive Substances in Oral Fluid

Michelle Williams BSc (Physiology); MBD/MDR (Townsville) M Clin Tox (Florida)

A Thesis submitted in fulfilment of the requirements for the degree of

Doctor of Philosophy in Medicine

August 2018

This research was supported by an Australian Government Research Training Program (RTP) Scholarship

Declarations

STATEMENT OF ORIGINALITY

I hereby certify that the work embodied in the Thesis is my own work, conducted under normal supervision. The Thesis contains no material which has been accepted, or is being examined, for the award of any other degree or diploma in any university or other tertiary institution and, to the best of my knowledge and belief, contains no material previously published or written by another person, except where due reference has been made. I give consent to the final version of my Thesis being made available worldwide when deposited in the University’s Digital Repository, subject to the provisions of the Copyright Act 1968 and any approved embargo.

Michelle Williams

09/08/18

ii Statement of Authorship

I hereby certify that the work embodied in this Thesis contains published paper/s/scholarly work of which I am a joint author. I have included as part of the thesis a written declaration endorsed in writing by my supervisor, attesting to my contribution to the joint publication/s/scholarly work.

Thesis by publication

I hereby certify that this Thesis is in the form of a series of papers. I have included as part of the Thesis a written declaration from each co-author, endorsed in writing by the Faculty Assistant Dean (Research Training), attesting to my contribution to any jointly authored papers.

Michelle Williams

09/08/18

iii Acknowledgements

I would like to thank my supervisors Prof Jenny Martin and Dr Peter Galettis.

Jenny, you are an inspiration for all the women in science. Thank you for your guidance, support and willingness to take on my projects.

Peter, for always being right but having the patience to let me find out for myself. .

Dedication

To my fabulous Aunt Dr Brenda Graham, without her belief, positivity and occasional glass of red wine this work would not have been possible.

Included Publications

Williams, M., Taylor, P., Page, C., & Martin, J. (2014). Clinical research in synthetic cannabinoids-do we need a national approach? The Medical journal of Australia, 201(6), 317.

Schneider, J., Galettis, P., Williams, M., Lucas, C., & Martin, J. (2016). Pill testing at music festivals: can we do more harm? Internal medicine journal, 46(11), 1249-1251.

Williams, M., Martin, J., & Galettis, P. (2017). A Validated Method for the Detection of 32 Bath Salts in Oral Fluid. J Anal Toxicol, 41(8), 659-669.

Williams, M., Martin, J., & Galettis, P. (2018). A Validated Method for the Detection of Synthetic Cannabinoids in Oral Fluid. J Anal Toxicol, bky043-bky043. doi:10.1093/jat/bky043

Williams, M., Martin, J., & Galettis, P. (2018). Stability and Recovery of Novel Psychoactive Substances in Oral Fluid. Submitted August 2018 to Journal of Analytical Toxicology

Conference and Media Presentations

Andy Park (Writer). (2015). -Synthetic Drugs [Television]. In SBS Australia (Producer), The Feed.

Matt Wordsworth (Writer). (2016). Deadly and illegal synthetic drugs still available over the shop counter [Television]. In ABC Australia (Producer), The 7:30 Report. Australia.

Michelle Williams. (2016). Method for detection of cathinones in oral fluid. Paper presented at the The International Association of Forensic Toxicologists, Brisbane.

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Contents

Declarations ...... ii

Acknowledgements ...... iv

Dedication ...... v Included Publications ...... vi

Conference and Media Presentations ...... vii

Abstract ...... xi List of figures and tables ...... xiv

Glossary ...... xv

Chapter 1:- Introduction ...... 1 1.1 Introduction ...... 2

1.1.1 Workplace Drug Testing ...... 3

1.1.2 New Illicit Drugs ...... 5 1.2 Methods for literature review ...... 6

1.3 Toxic effects of NPS ...... 8

1.3.1 Case studies ...... 8 1.3.2 Drugs and Driving...... 14

1.3.3 Stability ...... 14

1.4 Analytical Methods for the detection of NPS ...... 15 1.4.1 Novel Psychoacive Substances ...... 19

1.5 Sample collection ...... 36

1.5.1 Process of sample collection ...... 36 1.6 Procedures and devices ...... 38

1.6.1 Properties of Oral Fluid ...... 41

1.7 Aims ...... 42 Chapter 2:- A National Approach to Synthetic drugs ...... 44

2 Chapter 2 Introduction ...... 45

2.1 Statement of Contribution ...... 46 2.2 Results ...... 51

2.3 Discussion ...... 52

2.4 Conclusion ...... 52 Chapter 3: Pill testing at Music Festivals ...... 53

3 Chapter 3 Introduction ...... 54

3.1 Statement of Contribution ...... 55

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Chapter 4 Methods for the detection of Cathinones in Oral Fluid ...... 61

4 Chapter 4 Introduction ...... 62

4.1 Statement of Contribution ...... 63

Chapter 5 Methods for the detection of synthetic cannabinoids in oral fluid ...... 76 5 Chapter 5 Introduction ...... 77

5.1 Statement of Contribution ...... 78

Chapter 6 Quantisal Validation data ...... 88 6 Quantisal validation ...... 89

6.1 Materials and methods ...... 89

6.2 Modifications to published methods ...... 89

6.2.1 Ion Suppression ...... 90

6.3 Validation data...... 93

6.4 Conclusion ...... 96 Chapter 7 Stability and Recovery ...... 98

7 Chapter 7 Introduction ...... 99

7.1 Statement of Contribution ...... 100 7.2 Abstract ...... 102

7.3 Introduction ...... 102

7.4 Materials and Methods ...... 104

7.4.1 Chemicals and reagents ...... 104

7.4.2 Preparation of internal standards, calibration solutions and quality controls ...... 106

7.4.3 Preparation of samples ...... 106 7.4.4 Validation of methods ...... 108

7.4.5 Data analysis ...... 109

7.5 Results and Discussion ...... 109 7.5.1 Cannabinoids ...... 109

7.5.2 Cathinones ...... 112

7.6 Results where the Quantisal test pad was used ...... 117 7.6.1 Adherence of drug to test pad ...... 124

7.6.2 Dilution effect of buffer ...... 126

7.6.3 Limits of study ...... 126 7.7 Conclusion ...... 126

Chapter 8 Discussion ...... 128

8 Discussion ...... 129 8.1 Challenges ...... 131

8.2 Limitations of Thesis ...... 132

8.3 Future directions ...... 133

8.4 Conclusion ...... 134

References ...... 135

Abstract

Novel psychoactive substances (NPS) are a group of drugs developed to evade legal and testing authorities whilst producing the effects similar to the drug they mimic. Two large groups of NPS are cathinones and synthetic cannabinoids. The first group, cathinones, or bath salts are named due to the crystalline appearance of the drug when purchased. This class of NPS originates from the naturally occurring alkaloid cathinone found in the Khat (Catha edulis) plant. Khat is native to Africa and Arabian countries and used, typically by chewing, for its stimulant effect. Cathinones, are analogues and variations of the original drug cathinone and some have functional groups similar to lysergic acid diethylamide (LSD) or Psilocybin imparting a hallucinogenic quality to some. The second group, synthetic cannabinoids has origins in legitimate research where some were synthesised as agonists of the endocannabinoid system for potential medical use. This research was abandoned in the 1970’s due to adverse effects noted by patients. More recently, these compounds have resurfaced on the illicit drug market as a legal alternative to cannabis. Synthetic cannabinoids are typically purchased in the form of a smokable blend with the original chemical having been dissolved in a solvent and sprayed onto plant material prior to packaging. There has also been an evolution of this class of NPS with significant deviation of the chemical structures from the original research.

Due to the novel nature and rapid evolution of these drugs, testing options have not been available for workplaces wishing to undertake drug testing as part of their safety management program. The existing standards AS/NZS 4308:2008 and AS 4760:2006 only allow for the detection and quantitation of traditional drugs in urine and oral fluid respectively. This lack of any testing options for these novel substances, combined with fragmented exposure reports mean that capturing the scope and depth of the NPS problem in Australia is difficult.

The specific aims of this PhD research were to: -

 Promote a cohesive, national approach to reporting and documentation of exposures to novel

psychoactive substances.

 Develop analytical methods for the detection of cathinones in oral fluid.

 Develop analytical methods for the detection of synthetic cannabinoids in oral fluid.

 Determine stability of novel psychoactive substances under varying conditions and time.

 Inform selection of collection devices by evaluating recovery from the Biophor and Quantisal

devices.

Outline of the Thesis

In the studies that follow, a national approach to the reporting of clinical exposures to NPS was promoted with clinicians and researchers demonstrating interest. Methods for the detection of cathinones and synthetic cannabinoids were developed and validated for use in neat oral fluid. These methods were modified and validated for use with a common collection device, Quantisal. The validated methods were applied to a 30-day study investigating the stability of 51 NPS over time in three storage conditions, room temperature, refrigerated and frozen. A comparison of the Quantisal device with and without the use of the test pad was also conducted to establish the effect of the test pad independent to the buffer.

The methods developed were validated according to the criteria set out by the National Association of

Testing Authorities (NATA). The criteria for validation are linearity, limit of detection, limit of quantitation, selectivity, imprecision, repeatability and measurement of uncertainty. Both methods were linear from 2.5ng/mL to 500ng/mL with cathinones having an accuracy of 85.3 - 108.4% of the target concentration and an imprecision of 1.9 – 14% CV. The synthetic cannabinoid analysis method demonstrated an accuracy of 90.5 – 112.5% and an imprecision of 4-14.7% CV. Stability analysis demonstrated best recovery when samples were refrigerated and the Quantisal buffer improved recovery in some, but not all compounds. The use of the Quantisal collection pad added concerns relating to volume of sample collected, dilution factor and retention of drug on the test pad in addition to drug stability and effect of the buffer.

xii

Collectively the methods developed in this Thesis allow for the analysis of a class of drug not previously detected by routine analysis along with advice on collection device. These methods have been adapted to a real-world situation and are established in a commercial pathology company. The ability to detect these drugs will assist in elucidating the scope of NPS use within Australia.

xiii

List of figures and tables

Figure 1.1 Structure of review ...... 8 Figure 1.2 Typical procedure for collection of oral fluid for drug testing ...... 37

Figure 1.3 Non-buffered collection systems ...... 38

Figure 1.4 Devices using a collection pad ...... 39 Figure 1.5 Buffered collection devices...... 39

Figure 6.1 Comparison of 1µL and 5µL injection at LOD (1ng/mL) synthetic cannabinoids ...... 91

Figure 6.2 Comparison between cathinone signal and cannabinoid signal at Low QC (3ng/mL) . 92

Figure 7.1 Rapid degradation of cathinone ...... 117

Figure 7.2 Recovery of representative cannabinoid and cathinone from samples applied to the collection pad ...... 125

Table 1.1 Specific search terms used for literature search ...... 6 Table 1.2 Toxic effects of NPS (primary drug of investigation in bold) ...... 12

Table 1.3 Analytical methods 2002 - 2005 ...... 23

Table 1.3 cont. Analytical methods 2006 - 2011 ...... 25 Table 1.3 cont. Analytical methods 2011 - 2018 ...... 27

Table 2.1 Survey responses...... 51

Table 6.1 Accuracy and imprecision parameters for cathinones...... 93 Table 6.2 Accuracy and imprecision parameters for cannabinoids...... 94

Table 6.3 Repeatability values of cathinones...... 95

Table 6.4 Repeatability values for synthetic cannabinoids ...... 96 Table 7.2 Recovery of synthetic cannabinoids when stored at room temperature ...... 109

Table 7.3 Recovery of cannabinoids at 4°C ...... 110

Table 7.4 Recovery of cannabinoids when at -20°C ...... 111 Table 7.5 Recovery of cathinones when stored at room temperature ...... 112

Table 7.6 Recovery of cathinones when stored at 4°C ...... 114

Table 7.7 Recovery of cathinones when stored at -20°C ...... 115 Table 7.8 Recovery of cannabinoids in all storage conditions when sample is applied to the Quantisal test pad...... 119

Table 7.9 Recovery of cathinones in all storage conditions when sample is applied to the Quantisal test pad. 121

xiv

Glossary

25B-NBOMe 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine

25C-NBOMe 2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine 25T4-NBOMe 2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-4-[(1-methylethyl)thio]- benzeneethanamine

2C-T-2 4-(ethylthio)-2,5-dimethoxy-benzeneethanamine 2C-T-7 2,5-dimethoxy-4-(propylthio)-benzeneethanamine

AB-005 [1-[(1-methyl-2-piperidinyl)methyl]-1H-indol-3-yl](2,2,3,3- tetramethylcyclopropyl)-methanone

AB-CHMINACA N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-(cyclohexylmethyl)-1H-indazole- 3-carboxamide AB-FUBINACA N-[(1S)-1-(aminocarbonyl)-2-methylpropyl]-1-[(4-fluorophenyl)methyl]-1H- indazole-3-carboxamide

AB-PINACA (S)-N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3- carboxamide AKB-48 1-pentyl-N-tricyclo[3.3.1.13,7]dec-1-yl-1H-indazole-3-carboxamide

AM-2201 [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone

Amp Amphetamine AS 4760:2006 Procedures for the specimen collection and the detection and quantitation of drugs in oral fluid AS/NZS 4308:2008 Procedures for the specimen collection and the detection and quantitation of drugs of abuse in urine

BE Benzoylecgonine

Bup Buprenorphine BZO Benzodiazepines

CBD Cannabidiol

CBN Cannabinol CE Cocethylene

Coc Cocaine

Cod Codeine Cut-off level The value at or above which the drug class, drug and/or metabolite is deemed to be non-negative in the case of screening tests and positive in the case of confirmatory testing. DI direct immersion

DMA 2,5 Dimethoxyamphetamine

DOB Dimethoxybromoamphetamine

DOET 4-ethyl-2,5-dimethoxy-α-methyl-benzeneethanamine

EDDP 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine primary metabolite of methadone use

EIA Enzyme Immunoassay

ELISA Enzyme Linked Immunoassay Eph Ephedrine

FFA Fenfluramine

FMA 2-Fluoromethamphetamine GC-M Gas Chromatography – Mass spectrometry

Her/6-AM Heroin

HMA 3-hydroxy-4-methoxy-amphetamine HMMA 3-hydroxy-4-methoxy-methamphetamine

HS Headspace

IT-MS Ion Trap Mass Spectrometry JWH-122 (4-methyl-1-naphthalenyl)(1-pentyl-1H-indol-3-yl)-methanone

JWH-18 (1-pentyl-1H-indol-3-yl)-1-naphthalenyl-methanone

JWH-19 (1-hexyl-1H-indol-3-yl)-1-naphthalenyl-methanone JWH-20 (1-heptyl-1H-indol-3-yl)-1-naphthalenyl-methanone

JWH-200 [1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl]-1-naphthalenyl-methanone

JWH-250 1-(1-pentyl-1H-indol-3-yl)-2-(2-methoxyphenyl)-ethanone JWH-251 2-(2-methylphenyl)-1-(1-pentyl-1H-indol-3-yl)-ethanone

JWH-73 (1-butyl-1H-indol-3-yl)-1-naphthalenyl-methanone

Ket/KT Ketamine LOD limit of detection

LOQ limit of quantitation

MALDI Matrix Assisted Laser Desorption/Ionisation MBDB Methylbenzodioxolylbutanamine

MDA Methylenedioxyamphetamine

MDAI 5,6-Methylenedioxy-2-aminoindane MDE Methylenedioxyethamphetamine

MDEA Methylenedioxyethylamphetamine

MDMA Methylenedioxymethylamphetamine MDPV Methylenedioxypyrovalerone

MEC 4-Methylethcathinone

Met Methamphetamine

xvi

Mop Morphine

MPBP 1-(4-methylphenyl)-2-(1-pyrrolidinyl)-1-butanone Mpy Methylphenidate

MTA 4-Methylthioamphetamine

Mtd Methadone MXE Methoxetamine

Nal Naloxone

NAPS-MS Nib Assisted Paper Spray – Mass Spectrometry NATA National Association of Testing Authorities

NCADA National Campaign Against Drug Abuse

NPS Novel Psychoactive Compounds OF oral fluid

OPI Opiates

PM Phenmetrazine PMA Para-Methoxyamphetamine

PMMA Paramethoxymethamphetamine pSi LDI-MS porous Silicon assisted Laser Desorption/Ionisation Mass Spectrometry PT Phentermine

QTOF Quadrupole Time of Flight

RCS-4 (4-methoxyphenyl)(1-pentyl-1H-indol-3-yl)methanone Rit Ritalinic acid

SAMHSA Substance Abuse and Mental Health Services Administration

Sco Scopolamine SPE Solid Phase Extraction

SPME Solid phase microextraction

STS-135 N-adamantyl-1-fluoropentylindole-3-Carboxamide TFMPP 1-[3-(trifluoromethyl)phenyl]-piperazine, dihydrochloride

THC Tetrahydrocannabinol

THC-COOH Carboxy THC – the major urinary metabolite of Cannabis TIC Total Ion Count

TMA 3,4,5-Trimethoxyamphetamine

UR-144 (1-pentyl-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)-methanone VAMS Volumetric absorptive microsampling device

XLR-11 (1-(5-fluoropentyl)-1H-indol-3-yl)(2,2,3,3-tetramethylcyclopropyl)methanone

xvii

Chapter 1:- Introduction

1.1 Introduction In 1985, following a special Premier’s conference on drugs the National Campaign Against Drug

Abuse (NCADA) was established. With four main aims of 1. education and training, 2. treatment and rehabilitation, 3. research and information and, 4. controls and enforcement. NCADA forms a framework for building healthy, safe and resilient Australian communities (1). This campaign evolved into the national drug strategy in 1991 and aims at preventing and minimising alcohol, tobacco and other drug related health, social and economic harms among individuals, families and communities.

However, for harm minimisation strategies to be enacted and effective, data on usage patterns, demographics and exposure risks must be known. For these data to be collected detection must be possible. Where the drugs in question are Novel Psychoactive Substances (NPS) there are several challenges faced in gathering these data. The research teams are often small and not well linked to a national plan, there is inconsistent access to healthcare systems, exposures are not always reported to the state-based poison information centres, which are not linked in a national approach and detection methods for this class of drug are limited (2).

A distracting issue within the NPS discussion is that of pill testing at music and dance festivals where mobile testing labs aim to minimise the harm of drug use. However, the rapidly changing nature of

NPS mean that any operator is unlikely to be able to unequivocally identify all possible compounds raising the question of whether this service is doing more harm than good(3).

The presence of these drugs in the workplace represents a major challenge as industries who undertake a drug testing program are typically high risk such as mining, transport or construction.

The core focus of this work is to develop methods for the detection of NPS for use in workplace drug testing programs.

Chapter 1:- Introduction

1.1.1 Workplace Drug Testing Workplace drug testing in Australia was first regulated by the introduction of the standard AS

4308:1995 – Recommended practice for the collection, detection and quantitation of drugs of abuse in urine (4) . This standard was revised in 2001 (5) and again in 2008 (6) to produce the current edition.

The interval between introduction of this standard and present testing has seen a number of major changes in both the testing methodologies and the landscape in which drug testing is performed.

The devices used for testing have been refined to allow for easier use along with incorporated adulteration and temperature test areas. The detection limits for each class of drug have decreased in line with the requirements of the standards and improved manufacturing technology.

Workplace drug testing was previously only used in very high risk workplaces such as underground mining, oil rigs and the like, however, has progressed to many everyday occupations such as construction, transport, councils and some elite schools perform testing on students. Simultaneous to this shift in tested industries, has been the desire to move to a less invasive matrix. This allows the donor freedom from employer interference in their off-work time whilst still maintaining a safe working environment.

Oral fluid has become the preferred matrix for such testing with one national testing company reporting an increase in oral fluid testing from 30% to 50% of their total tests performed between 2017 and the same period in 2018 (personal communication to author from company (commercial in confidence)). Oral fluid as a matrix is preferred by industrial unions as it is simpler to collect, does not require the use of bathroom facilities and can be collected under direct observation. The detection window for drugs in oral fluid is typically shorter than urine, enabling a greater correlation between recent use and possible impairment. This shorter detection window also affords the donor privacy to make lifestyle decisions surrounding drug use free from employer oversight.

Chapter 1:- Introduction

With the growing interest and use of oral fluid as a testing matrix there was need for the development of a Standard to match that published for urine testing. In 2006 the first AS 4760 – Procedures for the specimen collection and the detection and quantitation of drugs in oral fluid, was published (7).

There were a number of major differences between AS/NZS 4308:2008 and AS 4760:2006.

Significantly, was the difference in analyte tested where AS/NZS 4308:2008 specifically listed the metabolite, particularly of cannabis (THC-COOH), AS 4760:2006 specified the parent (THC). There were also target concentrations as opposed to cut-off concentrations allowing more flexibility with regard to devices reaching them and benzodiazepines as a family were omitted from the list of required drugs under AS 4760:2006.

The imprecise nature of AS 4760:2006, combined with poor industry and scientific knowledge led to an overwhelming amount of testing and collection devices available on the market with very few meeting both the analyte and target concentrations defined.

In the second decade of this Century, industry knowledge of accreditation, testing devices and methodologies grew with more laboratories offering confirmatory testing services. Throughout this time devices with excessively high target concentrations, a high failure to run rate or incorrect analytes were eliminated from the market. However, around 2015 when testing organisations attempted to gain accreditation to perform instant oral fluid in accordance with section 3 of AS

4760:2006, the weakness in the standard, particularly in comparison to the mature requirements of the instant testing section of AS/NZS 4308:2008 became apparent. This prompted the National

Association of Testing Authorities (NATA), the accreditation body to withdraw the option for accreditation of instant oral fluid testing. In response, representatives from industrial unions, pathology companies, testing entities and device manufacturers met and pressured Standards

Australia to allow revision of AS 4760:2006. At the time the desired outcomes for this new standard were specifically to clarify the requirements for instant test devices, collection and transport devices and to introduce clear cut-off concentrations for both screening and confirmatory testing.

Chapter 1:- Introduction

Meanwhile, as testing devices improved, individuals wishing to avoid detection came under increasing pressure to find alternative means to both continue drug use and produce negative tests in the workplace.

1.1.2 New Illicit Drugs During 2012 the first known incidence of the use of synthetic cannabinoids were discovered in the resource sector in Western Australia. Synthetic cannabinoids are one part of the group of drugs known as Novel Psychoactive Substances (NPS) with the other major group being bath salts or cathinones.

With the shift away from urine testing toward oral fluid testing there are well founded concerns that the technology is less advanced. Further, with the introduction of a completely new class of drugs, fundamental questions such as detection times, stability and recovery, parent vs. metabolite and appropriate cut-off concentrations are all unknown.

In order to understand the possibilities for improved testing in oral fluid, examination of current and potential methods for oral fluid is needed. Therefore, an investigation into the methods for the detection of NPS in oral fluid was undertaken. In preparation of this review it was noted that there were few manuscripts available addressing this topic specifically therefore manuscripts that address traditional drug detection in oral fluid were also reviewed and discussed. ‘Traditional drugs’, is a generic term here to indicate drugs other than NPS. These drugs include but are not limited to

Amphetamine (Amp) Methamphetamine (Met) Cocaine (Coc) Benzodiazepines (BZO) and Cannabis

(THC). In addition, this review examined the matrix specific properties of oral fluid. It also evaluated information about NPS gleaned from case reports and population studies.

Chapter 1:- Introduction

1.2 Methods for literature review Literature was selected for review by conducting a comprehensive search of five online databases

(PubMed, Embase, Web of Science, Biological abstracts and Cochrane) up to July 20 2018. The specific search terms are summarised in Table 1.1. Database specific strategies were employed e.g. symbol for truncation. Where drug or product names would have multiple variations, the name was truncated to maximise results and prevent transcription errors e.g. JWH rather than the individual drug names and ‘monster’, rather than ‘green monster’, ‘blue monster’ etc.

The parameters were that either a common or product name, along with a drug related term should appear in any field and oral fluid or saliva in the title or abstract.

Table 1.1 Specific search terms used for literature search

Matrix Oral fluid, saliva

Drug related Research chemicals, cathinone, synthetic cannabinoid, novel psychoactive, terms substituted amphetamine, designer drug, legal high, bath salt 2C, benzedrone, benzylpiperazine, buphedrone, butylone, BZP, cathinone, Cathinone dimethoxyamphetamine, DMBDB, DOB, DOET, Dragon FLY, Dragonfly, EABDI, drug ethcathinone, eutylone, flephedrone, FMA, IPV, MBDB, MDA, MDAI, MDDMA, common MDEA, MDPV, MEC, mephedrone, mephtetramine, methedrone, methylone, names MPBP, MTTA, naphyrone, NBOMe, NRG, pentedrone, PMA, PV8, PVP, PV9, pyrovalerone, TFMPP, TMA, trimethoxyamphetamine

2201 , ABICA, ADBICA, AKB 48, AM, AMB, APICA, CAF, CBL, CHMICA, Cannabinoid CHMINACA, CP, FUBINACA, HU 210, IMMA, JWH, JZL, KML, MMB, MN 25, PB drug 22, PINACA, PTI, PX, SDB, SER, STS 135, THJ, THPINACA, UR 144, URB, WIN, common XLR 11 names Arctic blast, Bayou ivory flower, bloom, Blue magic, blue silk, Bolivian bath, bonsai Cathinone winter boost, Ciginal, cloud 10, cloud 9, cotton cloud, dynamite, energizing product aromatherapy powder, euphoria, gold rush, hurricane charlie, ivory, lady bubbles, names lunar wave, mr. nice guy, mystic, ocean snow, pure white, red dove, route 99, scarface, snow day, snow leopard, tranquillity, vanilla sky, white, wicked, zoom

Alice in wonderland, Amsterdam high, angry birds, atomic, black diamond, bizarro, Cannabinoid bliss, demon, diablo, dream, fake weed, geeked up, genie, giant, godfather, good product times, green monster, joker, K2, klimax, kryptonite, matrix, nice guy, mojo extreme, names scooby snax, sense, sinsence, skunk, sky scraper, smacked, spice, synergy, voodoo, wet lucy, yucatan fire, zohai

Chapter 1:- Introduction

This yielded less than twenty results which is insufficient for a comprehensive review thus the parameters were widened for saliva or oral fluid to be present in any field. Common and product names were used in place of chemical names as the chemical names contain substantial punctuation, are subject to variation within the literature and are often not specified, particularly for cathinones.

Using these extended search terms, 645 individual records were identified, however, given the broad nature of the search 555 were not relevant as they covered topics such as nutrients affecting brain composition and behaviour, resin based dental sealants and DNA persistence on bite marks in food.

The remaining 90 manuscripts were available for review. However not all could be found in full text and are highlighted with an asterisk (*). These manuscripts were categorised according to the primary focus of the research into one of three groups: - methods, matrix or drug. Methods manuscripts outline procedures for the detection of drugs in oral fluid. Matrix manuscripts discuss the use of oral fluid as a matrix, often in comparison to another matrix such as urine or serum. Drug manuscripts include those case studies of intoxications with NPS, statistical analysis of drug use over time and those that investigate drug specific properties such as stability.

The primary purpose of this review is to evaluate detection methods for NPS in oral fluid. Due to the lack of knowledge of pharmacokinetics, dosage, effects, detection windows and analytical methods, many parallels are to be drawn from the field of traditional drug testing in oral fluid. Manuscripts that evaluated traditional drugs were therefore, not excluded on the basis of not being directly relevant to

NPS. The inclusion of these manuscripts provided more context to the evolution of testing and other data that are not available for specifically NPS. As the analysis of these areas were not the core search parameters there will undoubtedly be manuscripts not captured, this is represented by the incomplete circles for the major areas in Figure 1.1. It is important to highlight some reviews that each more thoroughly discuss one aspect of this work such as the analysis of oral fluid (8),the properties of cathinones (9, 10) or synthetic cannabinoids (11) or the utility of instant screening devices (12)

Chapter 1:- Introduction

Analytical Methods for detection of NPS in Oral Fluid Analytical Methods

Toxic Matrix Effects Properties

Figure 1.1 Structure of review

1.3 Toxic effects of NPS Twenty-seven manuscripts were categorised as having a major focus on the drug. These can be further grouped into case studies (12), prevalence reviews (14) and one on drug stability. All manuscripts are summarised in Table 1.2 where the primary drug of interest is noted in bold.

1.3.1 Case studies

Twelve manuscripts investigated the properties of a drug of interest to the researchers, five examined different cathinones. Two manuscripts each looked at Methoxetamine, synthetic opioids and synthetic cannabinoids with and additional manuscript evaluating drugs likely to cause psychosis.

Chapter 1:- Introduction

Mephedrone (4-Methyl-methcathinone, M-cat) is a synthetic stimulant similar to Cathinone. Wood et al. (2010) described seven patients who presented to their Emergency Department having consumed

Mephedrone. These individuals were all male aged between 16 and 36. Four of the patients had

Mephedrone only, with Cocaine, MDPV and Butylone also detected in the remainder. Six patients were discharged with a mean hospital stay of 12 hours. The seventh patient was admitted to ICU and following tonsillar herniation died (13). Methiopropamine is another of the cathinones investigated and is a structural analogue of Methamphetamine with some selective norepinephrine-dopamine reuptake inhibitor properties (14). This supports the proposal by Anne et al. (2015) that the fatality characterised, with a peripheral blood concentration of Methiopropamine at 38mg/L was due to cardiac arrhythmia leading to cardiovascular collapse (15).

The cathinones, Ephylone, 2C-B and alpha-PVP were also investigated by various groups. Krotulski et al. (2018) describe multiple exposures to Ephylone arising from death or driving under the influence investigations. There was considerable variation in blood concentrations ranging from 12-1200ng/mL, with one fatal case recording a blood concentration of 50,000 ng/mL (16). Papaseit et al. (2018) describe an observational study on the effects of 2C-B. The sixteen participants consumed their own drug and a number of vital and subjective effects were monitored along with concentrations of drug detected in oral fluid samples. Overall 2C-B appeared to have a hallucinogenic and euphoric effect similar to others that affect serotonin signalling (17). Pierluigi et al. (2018) discuss a case of a chronic alpha-PVP user who increased the daily dose from the typical 25-30mg dose to 300-400mg/day for 3-

4 days per week over a 5-6 month period. This patient had a psychiatric history and was prescribed antipsychotic medication. There was no mention of specific withdrawal symptoms other than a general lack of energy. This case also highlights the difficulty in discovering NPS use even when the patient is known to psychiatric services (18). The potential adverse effects of cathinone use can affect multiple organ systems with the effects on the central nervous system such as psychosis, hypomania, delusions and paranoia well known (19). By investigating psychosis as an end point to a series of drug exposures Vallersnes et al. (2016) were able to determine that psychosis was more frequent in Tryptamine exposure than those involving Methedrone, Mephedrone or MDMA (20)

Chapter 1:- Introduction

NPS can encompass novel variations on drugs such as opiates and benzodiazepines.

Methoxetamine (MXE) is a novel ketamine analogue with properties similar to both ketamine and phencyclidine such as dissociation, euphoria and hallucinations; as are the side effects of confusion, aphasia and time distortion (21). It produces a rapid antidepressant effect making it a possible future medication (22). However, the multiple intoxications and fatalities documented along with the ease of access via the internet make it a concern for abuse potential, particularly for young and vulnerable individuals (21, 22)

Novel opioid agonists discussed in the literature are U-50488 and mitragynine. Amin et al. (2017) compare the more unknown analogue U-50488 with known opioid agonists U-47700 and U-49900, the former leading to 46 deaths prior to DEA scheduling. The authors highlight the changing landscape of NPS and that control is a multi-level approach (23). Individuals are also consuming synthetic opioid analogues in an attempt to mitigate the effects of opioid withdrawal. Wright et al.

(2018) discuss one such drugs in driving case where the driver declared using mitragynine. The driver was stopped for drifting out of the lane and making abrupt movements. Upon evaluation by a

Drug Recognition Expert (DRE), the driver presented fidgety, with rapid and slurred speech and dilated pupils. Analysis of a blood sample collected three hours after the stop qualitatively confirmed the presence of mitragynine (24)

In the same manner that synthetic stimulants (cathinones) and synthetic opioids have been evaluated, two manuscripts investigated the effects of synthetic cannabinoids. Lam et al. (2017) describe a non- fatal case of exposure to AB-FUBINACA and ADB-FUBINACA. The patient consumed two drops of a liquid used in conjunction with e-cigarettes and within 30 minutes experienced drowsiness, agitation, confusion, palpitations and vomiting. In addition to the clinical presentation, this report describes the non-traditional method of consumption (e-cigarette) of NPS and that the instant urine drug test returned negative results for traditional drugs of abuse (25). Toennes et al. (2018) investigated the pharmacokinetic properties of JWH-018, one of the first marketed synthetic cannabinoids, following controlled administration. The concentrations of drug present in oral fluid decreased rapidly following

Chapter 1:- Introduction

administration with no metabolites detected above the limit of quantitation (LOQ). Collection of oral fluid was performed using the Quantisal system where little stability data is available (26).

Collectively, these manuscripts highlight the array of issues faced when working with NPS. Many substances are not defined in legislation or are subject to complicated analogue clauses. Therefore, the decision to define a specific substance as controlled is often led by clinical cases reporting harms

(13). This can be particularly difficult when the traditional methods of detection, such as instant drug tests provide negative results (25), there is significant inter-patient variability (16), the detection windows can be very short (26) or longer than traditional drugs (17). The approach to controlling these substances needs to be multifaceted (23). Individuals may also consume these drug due to the longer duration of action (21). The potential for tolerance leading to increasing dosages (18) as well as self-medication with NPS when access to traditional pharmaceuticals are denied (24) have also been documented .

Chapter 1:- Introduction

Table 1.2 Toxic effects of NPS (primary drug of investigation in bold) Year published Number of cases Drugs identified Symptoms of Intoxication Matrix concentrations Outcome Ref Key NPS in bold Mephedrone Not defined 2010 6 patients released Agitation, palpitations, chest pain, seizures, 7 (13) Cocaine, Butylone, headaches Analytical method Wood 1 fatality MDPV linearity 0.01-1mg/mL

Desired effects:- sensory deprivation, derealization, dissociation, euphoria, empathy, pleasant intensification of sensory experiences, 2012 mood enhancement, hallucinations. N/A Methoxetamine N/A N/A (21) Corazza Side effects:- confusion, psychomotor agitation, time distortion, aphasia, synaesthesia, depressive thoughts, insomnia, cognitive impairment.

2015 Proposed cause of death – fatal cardiac 38mg/L in peripheral 1 Methiopropamine Fatal exposure (15) arrhythmia. blood Anne

Tryptamines 2016 Release from 348 MDPV Psychosis N/A (20) hospital Vallersnes Multiple other drugs

2016 Methoxetamine Multiple exposures reviewed (22) Zanda

U-50488 2017 Animal models demonstrate diuresis and U-47700 N/A N/A (23) dysphoria Amin U-49900

Chapter 1:- Introduction

AB-FUBINACA - 2017 AB-FUBINACA 5.6ng/mL Somnolence, confusion, agitation, palpitations 1 released (25) and vomiting. Lam ADB-FUBINACA ADB-FUBINACA – 15.6ng/mL

12ng/mL – 1200ng/mL in blood 18 death 2018 26 death or DUID cases Ephylone investigations One case 50,000ng/mL (16) in blood Krotulski 5 oral fluid Multiple other drugs Outcome unknown for remainder 12.6ng/mL – 1377ng/mL in oral fluid

2018 Increase in heart rate and blood pressure, Max concentration in 16 2C-B euphoria, altered perception and mild oral fluid 4.19 ± (17) Papaseit hallucinations. 1.86ng/mL

2018 Stimulation, euphoria, energy, insomnia, sexual 1 Alpha-PVP arousal, panic attacks, tachycardia, N/A (18) Pierluigi hyperpyrexia, anxiety and delusions

JWH-018 2018 6 2.2-2036ng/mL (26) 10 metabolites of JWH- Toennes 018

2018 Leg tremors, clenching of hands and fingers, 1 Mitragynine fidgeting, exaggerated movements, slurred Qualitative unknown (24) Wright speech, dilated pupils.

Chapter 1:- Introduction

1.3.2 Drugs and Driving

Driving under the influence of drugs, alcohol or a combination has been proven to be a serious concern to the safety of road users with drugs more likely to be detected in drivers involved in an incident (27).

Six manuscripts reviewed the prevalence of drugs in drivers either randomly stopped or involved in incidents (28-33) with one involving boat captains (34). In all cases, the drugs detected were traditional drugs of abuse such as Cannabis and Amphetamine with testing performed in two stages.

The first stage is an immunoassay with confirmatory testing being a chromatographic technique. Two manuscripts investigated expanded panels of drugs in patients from corrective institutions, workplaces

(35) or opioid treatment programs (36). These panels include Benzodiazepines, Barbiturates,

Buprenorphine and Methadone however there were no NPS tested. In the US, the Substance Abuse and Mental Health Services Administration (SAMHSA) develop mandatory guidelines for workplace drug testing that outline the drugs to be tested, cut-off concentrations and analytical methods (37).

The equivalent regulations in Australia are AS/NZS 4308:2008 and AS 4760:2006.

One of the major challenges facing law enforcement is the speed at which NPS enter and exit the market with Mohr et al. (2018) describing multiple cases of Alpha -PVP one year and none the next

(38). Also there are no instant test kits available for use roadside (39) leading to an underestimation of NPS prevalence in not only the driving population (40) but the general population as well.

1.3.3 Stability

Miller et al. (2017) investigated the stability of 10 cathinones in preserved neat and buffered oral fluid

(41). Stability is a concern, particularly for oral fluid where parent drug is present and may degrade due to storage conditions or adsorption to the container walls (42). Reported losses of up to 100%

(41) are significant as a driver suspected of driving under the influence may return a negative test due to pre-analytical storage conditions.

Chapter 1:- Introduction

1.4 Analytical Methods for the detection of NPS

Analytical techniques for the detection of NPS in oral fluid reflect the established procedures used for the detection of traditional drugs of abuse (Table 1.3). In this case the first assessment is via immunoassay, either in the laboratory or via a screening device with confirmatory procedures carried out via a chromatographic technique in the laboratory.

One of the first manuscripts to discuss the detection of drugs of abuse in oral fluid was Gentili et al.

(2002) who applied a method developed for hair testing to the alternate matrices of blood, urine and oral fluid (43). This method required no sample preparation however the narrow linearity range may pose some difficulties in reporting above the highest calibrator or result in samples requiring multiple dilutions.

Mortier et al. (2002) described a method using Quadrupole Time of Flight (QTOF) for multiple drugs with a wider calibration range of 2-100ng/mL. This manuscript provides valuable information on the possibility of contamination of a sample by the collection device. Here, the polymer in the collection device, a syringe with a spongy plunger, liberates oligomeric combinations of its monomers into the sample. This is clearly visible on the QTOF TIC in full scan mode. However, this is only observable as generic suppression in MS-MS mode. The concern here is that the sample clean-up of SPE was not sufficient to eliminate the contamination (44).

The first evaluation of the on-site device Drugwipe along with the separate handheld reader Drugread was performed by Pichini et al. (2002). This laboratory based investigation demonstrated a proof of concept, in that the drug wipe is more likely to detect positive samples in the first hours following consumption of MDMA. The high cut-off concentration of 450ng/mL and improved performance when the samples were manually applied to the device were significant weaknesses (45). This work, and that of Fucci et al. (2003) demonstrated the first generation of devices becoming available for the on- site screening of drugs of abuse in oral fluid. Fucci et al. (2003) described the use of a buffered

Chapter 1:- Introduction

system for oral fluid collection and the detection of multiple drugs of abuse with an acceptably low

LOQ.

The novel application of direct immersion (DI) or headspace (HS) solid phase microextraction (SPME) is suitable for quantitation though the authors note that selection of a suitable internal standard is crucial (46). One of the challenges faced when analysing NPS is the availability of analytical standards, though manufacturers have more comprehensive offerings today it is commonplace for many analytes not to have matched internal standards. Given the requirement for matched internal standards, further investigation is required to evaluate the applicability of SPME for NPS.

Wood et al. (2003) first described a method for the detection of multiple amphetamine type drugs from non-buffered oral fluid in 2003 (47). This method was then further developed to include drugs from the opiate and cocaine classes. The collection device used was the intercept device that was FDA approved and was being widely used in the US. The presence of buffer in the intercept device prompted SPE as the clean-up method and the dilution of 1 oral fluid:2 buffer was arbitrarily accepted

(48).

The Toxiquick device is an onsite testing device evaluated by Biermann et al. (2004). The results were compared to blood concentrations and produced lower false negative than false positive results.

The exception to this was cannabis where the device was detecting the metabolite THC-COOH and demonstrated an accuracy of 64% (49). It was not until 2011 that Milman et al. (2011) evaluated the presence of THC-COOH in oral fluid post continuous infusion. This work determined that THC-COOH is excreted into oral fluid from plasma in the order of ng/L (50). Therefore, devices capable of detecting THC-COOH in the ng/mL range are unlikely to report a true positive result. Where results are correct this is likely to be from the cross reactivity of the device to THC deposited in the oral cavity during smoking.

Whilst many authors have evaluated post extraction stability of drugs, few have applied this to the pre- analytical phase. Concheiro et al. (2005) developed a fluorescence detection method for four amphetamines. The stability component evaluates three storage conditions (room temp, refrigerated

Chapter 1:- Introduction

and frozen) when the drug is stored in neat oral fluid or in water over 10 weeks. There were losses in both sets however those stored in oral fluid showed greater losses, up to 38% for MDEA (51)

Immunoassay analysis has been a mainstay of laboratory operations for many years with some matrix dependent or requiring the use of radioactively labelled material. Enzyme-linked immunoassay

(ELISA) gained popularity due to the ability to screen multiple sample types for multiple drug classes

(52). The ability to adapt, to oral fluid, an ELISA designed to detect amphetamine in plasma was investigated by Laloup et al. (2005). The authors determined that the kit produced a cut-off concentration of 66ng/mL in oral fluid though noted that validation should still be performed (53). It is not clearly defined if the model of intercept used for this experiment used a buffer as described elsewhere (48) though it is possible that the manufacturers produced a buffered and non-buffered product.

Between 2006 and 2011 there were several manuscripts published on the detection of traditional drugs of abuse in oral fluid with the general focus being on simplifying sample preparation while maintaining low limits of quantitation and increasing the number of drugs analysed. Scheidweiler et al. (2006) produced a relatively quick 16 min GC-MS method however the complicated sample preparation and detection of only amphetamines is a drawback (54). Concheiro et al. (2007) built on their earlier fluorescence work and developed an LC-MS method for the detection of multiple drug classes and compared this to two on-site test devices. The on-site devices demonstrated good results for limited drug classes however THC and amphetamines remain problematic (55). Peters et al. (2007) investigated enantiomeric selectivity of amphetamines in neat oral fluid with a relatively simple sample preparation - direct derivatisation (56). Wilson et al. (2007) and Lowe et al. (2009) both investigated the Rapiscan collection system. Wilson et al. (2007) compared the device to a GC-

Chapter 1:- Introduction

MS confirmatory assay determining, for amphetamines it performed well (57). Lowe et al. (2009) used the Rapiscan buffer to develop a porous Silicon assisted Laser Desorption/Ionisation Mass

Spectrometry method (pSi LDI-MS) as a rapid screen (2s/sample) for multiple drug classes however the LOD is comparatively high at 200ng/mL (58).

Meng et al. (2010) described a method for small volume liquid extraction of amphetamines and although the method significantly reduced the amount of solvent used throughout the process (59) the requirement for 1mL of neat oral fluid would constitute the entire sample in many cases. Concerns regarding the type of collection device were noted by Rohrich et al. (2010) who compared the on-site test RapidSTAT to GC-MS however the collection stick contained no volume adequacy indicator and substantial variability in the collection volume was observed (60).

Blencowe et al. (2011) compared the performance of eight on-site devices where none of them reached the lowered benchmark of 80% sensitivity, specificity and accuracy across all drug classes

(61). This is supported by the work of Pehrsson et al. (2011) who evaluated two versions of the

DrugWipe device and found good performance for amphetamines however poor sensitivity for

Opiates, Cocaine and Cannabis (62). Tang et al. (2018) more recently evaluated the latest evolution of the DrugWipe 6S, Ora-Check and SalivaScreen. The updated DrugWipe increased the cut-off concentration for Amp and Met to 80ng/mL from 50ng/mL in the 5+ and lowered the cut-off concentration for THC and cocaine to 20ng/mL and 10 ng/mL respectively from 30ng/mL in the 5+.

Overall, none of the devices performed well across all drug classes and all performed particularly poorly for THC (63).

Chapter 1:- Introduction

1.4.1 Novel Psychoactive Substances In 2011 Coulter et al. (2011) described the first method for the detection of synthetic cannabinoids in oral fluid (64). Previous mentions of this class of drug had been made however only in urine where the parent drug was undetectable (65) or through high resolution accurate mass screening (66).

Rodrigues et al. (2013) outlined a procedure for the production of an ELISA kit which showed the greatest affinity for JWH-200 however due to the similarity of the molecules, was also able to detect a number of other structurally similar synthetic cannabinoids (67). Another option investigated by two groups was the potential for existing instant testing devices to detect NPS, particularly the cross reactivity of cathinones to the Amphetamine or Methamphetamine test area. de Castro et al. (2014) investigated the cross reactivity with the Dräger DrugTest 5000, an electronic lateral flow immunoassay device which has an accuracy for Amphetamines of 98.5% (68). This device has the added benefit of performing the interpretation of the test and displaying the results in plain text as either positive or negative on the screen. Whilst the detection windows and concentrations of cathinones present in a drug user’s oral fluid remain unknown, for context the screening cut-off concentration for Methamphetamine and Amphetamine under AS 4760:2006 are 50ng/mL. The lowest concentration of drug producing a positive result was 75ng/mL for Fluoromethamphetamine with only the highest evaluated concentration of 100,000ng/mL producing a positive result for MDPV

(69)

Nieddu et al. (2014) also evaluated cross reactivity of cathinones however used instant testing devices where the collection sponge and test strips were combined in a single unit. Oral fluid absorbs into the sponge, in turn flowing to the test area producing a result. Unfortunately, all bar two of the 39 cathinones evaluated had cross reactivity <1 on both devices (70). This work was continued by evaluating the cross reactivity of 30 cathinones to a commercial ELISA kit designed for Amphetamine and Methamphetamine, with similar results (71).

Chapter 1:- Introduction

Overall, the delay for production of ELISA kits allow for the evolution of these drugs to the point that the kit may be obsolete before it reaches the market. The delay makes this approach a realistic option only for large scale testing with a focus on deterrent rather than detection. Utilising instant test devices with a view of detecting NPS via a cross reactivity mechanism is only viable at very high doses when clinical indicators of drug use would likely be present. This was investigated by

Swortwood et al. (2014) who examined 16 different ELISA reagents for cross reactivity between traditional and novel amphetamine like compounds in serum. The cross reactivity was <4% for most compounds, when the target for the kit was Amphetamine or Methamphetamine, increasing for compounds such as MDA, MDMA, Ethylamphetamine and α-Methyltryptamine. They further evaluated the Randox Mephedrone/Methcathinone ELISA and found cross reactivity at concentrations as low as 150ng/mL (72). This manuscript was comprehensive analysis of ELSA kits available at the time of publications however the low cross reactivity and relatively high concentrations make the use of these kits for oral fluid limited.

Two manuscripts used novel approaches to the detection of NPS, Lee et al. (2012) described a qualitative method using nib assisted paper spray (73) and Peiro et al. (2016) used ion mobility spectrometry for the detection of MDVP (74). Both manuscripts are proof of concept using few drugs, however provide rapid methods for screening purposes. The need for bulky equipment means that the samples must still be transported to the laboratory but these methods should not be discounted.

Mercolini et al. (2016) used a traditional LC-MS/MS approach to the analysis however a novel approach to the collection by using a volumetric absorptive microsampling device (VAMS) (75). This approach would make the collection process extremely simple, however, further investigation is needed with regard to addition of the internal standard post collection and drying as well as the ability to decrease the detection limits. Ares et al. (2017) used a miniaturised version of SPE, microextraction by packed sorbent to detect 12 cathinones in combination with traditional drugs by

LC-MS/MS in a rapid 4 min run (76).

Chapter 1:- Introduction

The majority of manuscripts reviewed use LC-MS/MS as a quantitative method with one group producing two manuscripts on the use of GC-MS. Strano-Rossi-et al. (2012) produced the only method to contain both cathinones and synthetic cannabinoids in a single 15 minute run for screening of samples collected at the roadside (77).

Amaratunga et al. (2013) developed the fastest method of 3 minutes and used SPE as a sample clean-up (78). At the time of development the one published method for the detection of cathinones was a dilute-and-shoot and some compounds had a noticeable matrix effect, possibly affecting the

LOQ (77). Amaratunga et al. (2013) addressed this by using SPE however with no preconditioning, evaporation or reconstitution steps this allowed for rapid clean up followed by rapid analysis time with one of the lowest LOQ (78). de Castro et al. (2013) also used SPE as a clean-up step to concentrate 500µL into 100µL. With this concentration they achieved a lower LOQ of 0.1ng/mL for JWH-200, JWH-250, JWH-073 and JWH-

018 however the need for one injection for each ESI positive and negative mode makes complete analysis time consuming (79)

Kneisel et al. (2013) investigated the detection of the largest suite of cannabinoids with 30 included in the oral fluid method (80) adapted from their previous work in serum (81). This method was used to inform a stability study comparing glass and polypropylene tubes where the well-known phenomena of THC adherence to plastic containers was investigated for 11 synthetic cannabinoids (82). They determined this was the case and that samples should be shipped refrigerated to minimise adsorption

(82). The quantitative method was also applied to an experiment on preliminary detection windows following an oral 5mg dose of AM-2201 however concentrations in oral fluid were very low and close to the method LOD of 0.02ng/mL after four hours. They continued to evaluate the possible concentrations of synthetic cannabinoid deposited in the oral cavity during smoking. Detection windows far exceeding the oral dose led to the conclusion that synthetic cannabinoids are excreted into oral fluid at very low levels and higher values are likely the result of contamination during the smoking process (83). Huppertz et al. (2014) further advanced this work by developing a library matching method following liquid chromatography-quadrupole ion trap-mass spectrometry for 46

Chapter 1:- Introduction

synthetic cannabinoids (84). This method was developed in serum however the authors have previously applied a method developed for serum to oral fluid and state that the same could be applied here.

Amaratunga et al. (2014) investigated the feasibility of detecting the metabolites and pyrolysis products of two synthetic cannabinoids in oral fluid (85). Because cannabinoids are most often smoked there is the possibility of a donor producing a positive test due to passive exposure. This work demonstrated that for UR-144 the parent, metabolite and pyrolysis product are detectable in oral fluid (85).

Mohamed et al. (2016) developed a GC-MS/MS method for the detection of select cathinones (86).

They expanded upon this work in 2017 to simplify sample preparation by developing a one-step derivatisation process (87) and further again to evaluate different derivatising agents (88).

Chapter 1:- Introduction

Table 1.3 Analytical methods 2002 - 2005 Screening method Confirmatory Year of Syn Sample Detection Additional Traditional drugs Cath Sample collection method Validation Ref Publication Can Cut-off preparation range comments Quant/Qual concentration (ng/mL) Quant

No sample LOQ, LOD 2002 MDA, MDMA, MDE, Swab and buffer (not 2-24ng/mg Primarily for preparation for Headspace Precision, (43) MBDB specified) detection in hair Gentili OF samples SPME-GC-MS accuracy 0.5-4ng/mL

15 min run Test tube for validation samples 200µL OF LOD, LOQ,

precision, Contamination Amp, Met, Cod, Mop, Quant 2-100ng/mL 2002 Syringe device with Evaporation and accuracy, of sample from Coc, BE, MDEA, . Quadratic fit (44) spongy plunger for reconstitution in extraction collection device Mortier MDMA Q-TOF genuine samples 200µL recovery, matrix observed 28 min run SPE effect, linearity

Direct wiping to DrugWipe 2002 Above be avoided, use MDMA Plastic tube (45) 450ng/mL of hand Pichini DrugRead photometer

Quant 10- 1000ng/mL EDDP, Coc, CE, HS-SPME For most LOD, LOQ, Amp, MDMA, Met, Cozart Rapiscan 2003 No sample or analytes reproducibility, THC, CBD, CBN, 1mLOF:3mL buffer (46) preparation DI-SPME- sensitivity, Fucci MDEA, MBDB ratio (89) THC, CBD, linearity

Coupled to CBN qual GC-MS only 18 min run LOD, LOQ,

Quant linearity, 2003 MDMA, MDA, Amp, Protein 0.5/1- Plastic tube LC-MS/MS precision, (47) Met, Eph, MDEA precipitation 500ng/mL Wood 6 min run variation,

sensitivity

Chapter 1:- Introduction

Toxiquick cut- No sample off preparation for concentration Toxiquick® Mop-35 Limited 2004 Amp, MDMA, MDE, Cotton bud THC-COOH- methodology (49) THC, Her, Mtd, Mop 20 published Biermann Protein THC-75 precipitation for Coc/BE -50, EIA AMPs-500 LOD, LOQ, 1mL linearity, LLE Evaluation of 2005 MDMA, MDA, MDEA, Fluorescence 10- precision, Not specified Evaporation pre-analytical (51) MBDB 12 min run 250ng/mL recovery, Reconstitution in stability Concheiro selectivity, 200µL stability Optimal cut- MDMA, MDA, MDEA, 2005 ELISA off MBDB, Met, Amp, Intercept concentratio (53) Eph, PMA, Mpy, Rit n calculated Laloup at 66ng/mL 250µL OF buffer mix LOD, LOQ, Multiple 2005 Amp, Met, MDA, Intercept Quantitative selectivity, reconstitution MDMA, Coc, BE, SPE LC-MS/MS 2-200ng/mL stability, (48) techniques Mop, 6-AM, Cod 1:2 OF:buffer mix Evaporation and 20 min run recovery matrix Wood evaluated reconstitution in effects 1mL

Chapter 1:- Introduction

Table 1.3 cont. Analytical methods 2006 - 2011 Screening method Year of Confirmatory Syn Sample Detection Additional Publicatio Traditional drugs Cath Cut-off Sample preparation method Validation Ref Can collection range comments n concentration Quant/Qual (ng/mL)

400µL

Filtration LOD, LOQ, recovery, SPE accuracy, Quantitative 2006 Amp, Met, MDMA, Polypropylene Evaporation 5/25- precision, GC/MS-EI MDA, MDEA, HMMA, tubes Reconstitution 250/500/100 dilution (54) Scheid- HMA Derivatisation 0ng/mL integrity, weiler 16 min run sensitivity, Organic layer specificity, transferred to stability autosampler vials 3µL inj vol

OraLab Mop-40 Coc 20 Amp/Met 50 LOD, LOQ, THC 100 100µL OF linearity, 6-AM, Amp, Met, SPE Quant LC- 2007 Polypropylene 1/2- recovery, MDA, MDMA, MDEA, MS (55) tubes 250ng/mL precision, MBDB, Coc, BE, THC Dräger Drug test 200µLOF 17 min run Concheiro accuracy , OPI 40 LLE for THC matrix effect Coc 5 Amp 50 Met 10 THC 20

LOQ, 5-250 ng/mL selectivity, Quantitative MDA 2007 Amp, Met, MDA, Polypropylene 50µL OF linearity, Enantiomeric GC-NICI-MS 25- (56) MDMA, MDEA, tube LLE recovery, selectivity Peters 16 min run 1250ng/mL precision, other drugs stability

Chapter 1:- Introduction

Rapiscan device Quantitative Cut-off GC-MS concentration 20 min run Full validation MDMA, Met, Amp, Rapiscan 45ng/mL Met, 2007 2/5- LOQ, LOD, data for GC- MDMA and MDA, MDEA, collection Rapiscan (57) Wilson MBDB, 180ng/mL linearity, MS assay not MBDB system immuno- 1500ng/mL available assay 5 min MDEA, incubation 30,000ng/mL Amp Amp, Met, MDA, MDMA, MDEA, diazepam, Coc, Rapiscan Qualitative 2009 200-1200 alprazolam, collection LLE for THC only pSi LDI-MS (58) ng/mL Lowe clonazepam, system 2s run flunitrazepam THC LOD, linearity, Quantitative 2010 Amp, Met, MDA, 1ml OF 10- reproducibility Sponge stick GC-MS (59) MDMA SVLE 1000ng/mL recovery Meng 24 min run

Rapid STAT 0.27mL OF buffer mix 2010 device 1-10 ng/mL LOD, LOQ, No volume Amp, Met, MDMA, Manufacturer Rapid STAT SPE GC-MS THC accuracy, adequacy (60) Rohrich MDEA, MDA, THC states 250µL diluted in buffer Evaporation 31 min run 20-500 precision, indicator in collected Derivatisation ng/mL Amps recovery collector 1mL buffer reconstitution in 50µL BIOSENS Cozart DDS 806 DrugWipe 5 Dräger DrugTest DRUID cut- Amp, THC, Coc, OPI, 5000 off 2011 BZO various (61) Blencowe Oralab 6 concentratio

OrAlert n Oratect RapidSTAT

Chapter 1:- Introduction

Table 1.3 cont. Analytical methods 2011 - 2018 Screening method Confirmatory Year of Traditional Sample Sample Detection Additional Cath Syn Can method Validation Ref Publication drugs collection Cut-off preparation range comments Quant/Qual concentration (ng/mL) Quantitative Recovery by LC-MS/MS JWH-018 vortex and 12 min run LOQ Stability JWH-250 compression +ve Selectivity experiments JWH-073 JWH-18 Imprecision 2011 Quantisal over 7 days at CP47497 1ml SPE JWH-250 0.5-100µg/L Ion (64) system room temp Coulter C8 Evaporation JWH-73 Suppression and CP 47497 and -ve Matrix refrigerated HU-210 reconstitution CP47497 C8 effects in 50µL CP 47497 HU-210

Comparison with 2011 DrugWipe concentrations (62) Pehrsson 5/5+ obtained in blood by GC- MS

3mL 1:3 OF: Salivette LOD, LOQ, buffer dilution Selectivity, Amp, Met, SPE linearity, MDMA, Evaporation 2012 Quantisal Quantitative GC- recovery, MDA, PT, Biochip array derivatisation 5-200ng/mL (90) MS precision, Choi FFA, PM, accuracy, KT Reconstitution Polypropylene freeze-thaw volume not tube stability specified

Predates o-, m- and p- analytical Chloro- reference 2012 Qualitative amphetamine Direct No sample material NAPS-MS LOD (73) Lee o-, m- and p- expectoration preparation availability

Fluoro- Useful for amphetamine rapid screening

Chapter 1:- Introduction

Benzylpiperazine, JWH-200, LOD, Methylone, 5,6- AM 694, specificity, MDAI, JWH-250, Combines LOQ 1- ion fenproporex, 4- JWH-073, Qualitative both synthetic 2012 20ng/mL suppression/ MEC, JWH-018, Dräger No sample LC-MS/MS cannabinoids linear from enhancemen (77) Strano- Mephedrone, CP47497, DCD5000 preparation and stimulants LOQ to t, linearity, Rossi Furfenorex, JWH-019, 15 min run in single 1000ng/mL memory MBDB, 4-MA, HU-210, method effect, MTA, MDPV, JWH-122, stability Clobenzorex Nabilone Analytical Mephedrone, Linearity, samples were MDPV, carryover diluted 1:3 in 2013 400µL SPE Flephedrone, accuracy, Quantisal Amaratunga (equiv 100µL Quantitative LC- Methedrone, precision, Buffer without Quantisal neat OF) MS/MS LOQ Methylone, sensitivity, the use of the (78) system 1-500ng/mL Butylone, selectivity, collection pad, 50µL elution 3 min run Ethylone, PVP, matrix effect, genuine 50µL dilution Methcathinone, recovery, samples were Pyrovalerone stability collected using pad. LOD, LOQ, JWH-200, Analytical selectivity, Two injections JWH-250, specimens linearity, required for JWH-073, collected using 500µL SPE Quantitative LC- imprecision, complete JWH-018, Salivette. Evaporation MS/MS LOQ 0.1- 2013 accuracy, analysis. THC, Genuine and 8 min pos 2.5ng/mL to (79) extraction Preparation of de Castro HU-211, samples reconstitution 200ng/mL recovery, genuine CP47497, collected using in 100µL 6 min neg Quantisal samples not CP47497- Quantisal recovery and specified C8 system. matrix effect. JWH-007, LOD, LOQ, JWH-015, selectivity, JWH-018, accuracy, AB-001, imprecision, Method was JWH-019, 333mL LLE matrix effect, applied to JWH-020, Quantitative LC- Evaporation LOQ 0.15- recovery and preliminary 2013 JWH-073, Dräger MS/MS 12 min and 3ng/mL to process stability (82) (80) JWH-081 DCD5000 run (81) Kneisel reconstitution 6-120ng/mL efficiency, and detection JWH-122, in 100µL autosampler window study MAM- stability, (83) 2201. carryover JHW-200, and fit for JWH-203, purpose. JWH-210,

Chapter 1:- Introduction

JWH-250, JWH-251, JWH-307, JWH-387, JWH-398, JWH-412, AM-694, AM-1220, AM-2201, AM-2233, CRA-13, Methanand amide, RCS-4, RCS-4 ortho isomer, RCS-8, WIN- 48,098, WIN- 55,212-2

Oraltwist LC-MS For Coc and Confirmation Amp, Met, 2013 EviteC Mop performed on MDMA, SmartClip GC-MS for blood (91) Matz- Coc, Mtd, - 2 variants remainder THC not opoulous Mop, THC confirmed Drugwipe

JWH-200, JWH-018, 0.1-5ng/mL JWH-073, JWH-200 Synthetic oral JWH-022, Quantisal fluid used for 2013 ELISA (67) Rodrigues AM-2201, system Dilution validation AM-2232, experiments AM-1220

Chapter 1:- Introduction

LOD, LOQ, linearity, XLR-11, 400µL SPE carryover, Samples were UR-144 (equiv 100µL accuracy, kept at room Quantitative LC- + Quantisal neat OF) precision, temperature 2014 MS/MS 5-100ng/mL (85) Amaratunga metabolite system sensitivity, prior to 4.5 min run + pyrolysis 50µL elution selectivity, analysis product 50µL dilution matrix effect, recovery, stability LOD, LOQ, selectivity, linearity, Calibrators and imprecision, Methylone, QC – Salivette 500µL SPE accuracy, Methedrone, Drager Evaporation Quantitative LC- matrix effect, 2014 Mephedrone, 0.2- Authentic DrugTest and MS/MS Quantisal (69) mCPP, TFMPP 200ng/mL de Castro samples 5000 reconstitution 10 min run and 4-FMA Quantisal in 75µL extraction 4-FMC system recovery, dilution integrity and stability AB-001, AKB-48, AKB-48 5- F. AM- 1220, AM1220- azepane isomer, 1mL serum AM-1248, Qualitative AM-2201, LLE LC-MSn AM-2232, 2014 LOD 0.1- AM-2233, (84) Evaporation With library 0.5ng/mL Huppertz AM-694, and matching APICA, reconstitution 12 min run Cannabpip in 25µL eridethano ne, CRA- 13, JWH-007, JWH-015, JWH-018, JWH-019, JWH-020, 30

Chapter 1:- Introduction

JWH-022, JWH-073, JWH-081, JWH-122, JWH-182, JWH-200, JWH-203, JWH-210, JWH-250, JWH-251, JWH-307, JWH-370, JWH-387, JWH-398, JWH-412, MAM- 2201, Methanan damide, RCS-4, RCS-4 C4, RCS-4 ortho isomer, RCS-8, STS-135, UR-144, UR-144 isomer, WIN- 48.098, WIN- 55.212-2, XLR-11, XLR-11 isomer

, PMA, PMMA, 2,5- Screen Device cut-off DMA, DOB, DOC, Multidrug OFD Quantitative for DOET, DOI, immunoassay at amphetamine 2014 DOM, DON, set cut-off point and (70) Nieddu DOPR, 2C-B, 2C- by visualisation methampheta I, 2C-N, 2C-M, of a line mine is 2C-T, 2C-T-2, 2C- 50ng/mL 31

Chapter 1:- Introduction

T-4, 2C-T-5, 2C- T-7, 2C-T-13, 2C- GIMA One Step T-17, ALEPH, Multi-Line ALEPH-2, Screen Test ALEPH-5, OFD ALEPH-7, ALEPH-8, ALEPH-13, ALEPH-17, TMA, TMA-2, TMA-3, TMA-6, MDIP, MDBZ, MDCPM, BOH, BOB, BOD, BOHD

LOD, LOQ, precision, Methylone, 10µL Volumetric absolute Ethylone, evaporated Quantitative LC- 2016 absorptive 10- recovery, Butylone, and MS/MS (75) microsampling 500ng/mL matrix effect, Mercolini Mephedrone, 4- reconstituted 15 min device accuracy, MEC, MDPV in 100µL selectivity and stability Cathine, Cathinone, Methcathinone 500µL and Ephedrine LLE Cotton pad Evaporation Quantitative GC- LOD, LOQ, 2016 collection device derivatisation 2- MS imprecision, (86) (no brand evaporation 2000ng/mL Mohamed 12.5 min accuracy specified) and reconstitution in 50µL

2,5-DMA, DOC, DOET, DOI, DON, DOPR, 2C-N, 2C- 2016 T, 2C-T-2, 2C-T-5, ELISA kits Nieddu 2C-T-7, 2C-T-13, Not specified targeted for (71) 2CT-17, ALEPH, Amp, Met ALEPH-2, ALEPH-5, ALEPH-7,

Chapter 1:- Introduction

ALEPH-13, ALEPH-17, TMA, TMA-2, TMA-3, TMA-6, MDIP, MDBZ, MDCPM, BOH, BOB, BOD, BOHD Direct Semi LOD 500µL LLME 2016 expectoration IONSCAN- quantitative Ion 40- 22ng/mL MDPV dilution 5:1 (74) unbuffered LS Mobility 1000ng/mL LOQ 73 Peiro chloroform plastic tube Spectrometry ng/mL LOQ, LOD, 80µL OF specificity, buffer mix MALDI-QqQ- recovery, LLE 2016 Quantisal MS/MS 5- matrix MDMA Evaporation (92) system 2000ng/mL effects, Poetzsch and 10s run accuracy, reconstitution precision, in 50µL stability

100µL OF buffer mix 5- LOQ, LOD, Met, DDS collection SLE Quantitative 1250ng/mL accuracy, 2016 MDMA, system 1ml Evaporation LC/MS MDMA, met precision, (93) Rositano THC OF:3ml Buffer and 5 min run 1-112ng/mL matrix reconstitution THC effects, in 80µL linearity

MDPV, Mephedrone, Methylone, 300µL pre- Variable 6-AM, Pentedrone, LOD, LOQ, treated, LOQ Mop, Coc, Ethylone, linearity,

CE, BE, Butylone, precision, 2017 MEPS, 250ng/mL Mtd, Ethylcathinone, Salivette LC-MS/MS trueness, (76) evaporation Mephedrone Ares EDDP, Ethylcathinone 4min run recovery and and Bup, Nal, Ephedrine matrix reconstituted ULOQ Sco metabolite, effects in 50µL 500ng/mL Pyovalerone, Flephedrone, Methylephedrine,

Chapter 1:- Introduction

400µL Quantisal/oral LOD, LOQ, fluid imprecision, Primarily a accuracy, stability paper Cathinone, Quantisal 300µL extraction though the Methcathinone, 1:3mL oral oralese/oral efficiency, method Buphedrone, fluid:buffer ratio fluid Quantitative LC- process developed is 2017 Mephedrone, 4- MS/MS 1-250ng/mL efficiency, fully validated (41) MEC, MDPV, Miller and oral 100 µL neat 14 min run matrix effect, Methylone, eze1:2mLoral oral fluid carryover, Small sample Naphyrone, PVP, fluid:buffer ratio SPE dilution volume used Ethylcathinone evaporation integrity and for and autosampler experiments reconstitution stability in 100µL

Specificity, Amp, Met, 500µL sensitivity, Evaluation of 4-Met, MDEA, Cotton pad LLE Quantitative GC- 2.5-5ng/mL linearity, different MDA, Cathinone, collection device Evaporation 2017 MS to precision, derivatising (87) MDMA Methcathinone, (no brand and Mohamed 17min run 1000ng/mL accuracy, agents Eph, Mephedrone. specified) reconstitution extract conducted (88) fenethylline in 50µL stability

LOQ, linearity, Amp, Met, Quantitative LC- matrix Matrix 2018 MDA, 900µL Glass tube MS/MS 0-500ng/mL effects, independent (94) MDEA, SURPAS Accioni 15 min run time precision, method MDMA accuracy, recovery

Chapter 1:- Introduction

400µL oral LOQ, fluid buffer mix accuracy,

Drug Wipe precision, Evaporation Opi, Met, 6S extraction and 0.5/1/5- 2018 Coc, THC, Quantisal Quantitative LC- efficiency, reconstitution 200/500/150 (63) MDMA, system Ora-Check MS matrix effect, Tang in 100µL 0ng/mL Ket, carryover,

SalivaScreen dilution 25 fold dilution integrity and for some stability. analytes

Mtd, Bup, Pregabalin, Fentanyhl, Protein Amp, precipitation Qualitative 2018* MDMA, IT-MS (95) Plecko Coc, Hybrid SPE Acetylcode phospholipid Library matching ine, extraction Nordiazep am

Coc, CE, 2018* MDMA, MDA, 0.5µg/L PMA, PMMA CE-FD (96) Sarr- MDEA, 50mg/L Reismaa Amp, Met, THC, CBD

Chapter 1:- Introduction

1.5 Sample collection

1.5.1 Process of sample collection

The process for the typical analysis of an oral fluid sample is outlined in Figure 1.2

The testing performed in steps one and two typically uses an immunoassay technique where an antibody antigen conjugate is formed, producing a line in a negative sample. The presence of drug prevents line formation in a non-negative sample. The exception to this is the DrugWipe where a line indicates a non-negative test. Second level screening is optional, at the requesting authorities’ discretion and may or may not involve the use of a different testing device or matrix. Where a second level screening test is performed this may involve the use of a machine such as the Dräger DrugTest

5000 or Cozart DDS2. These devices perform mixing of the sample with buffer and display the results in plain text on the screen. This eliminates human error in the interpretation of the visibility of the line. An example of this process is the testing performed by Australian Police where the first screen uses the DrugWipe, followed by the Rapiscan (29) or its successor the DDS (93).

Urine is provided in larger quantities and multipurpose devices are available for sample collection, on- site testing and transportation to the laboratory where necessary. Oral fluid is provided in much smaller quantities with the screening and confirmatory tests often requiring separate collections.

Therefore, devices are typically either designed for on-site testing and consume the entirety of the sample, or, for sample collection and transport to the laboratory.

It is important to note that a sample may proceed directly from collection to laboratory based screening though this is less common than on-site screening.

Devices used for on-site screening have been described in detail along with brief descriptions for use and the cut-off concentrations for traditional drugs (61). Devices used for collection and transport only have not been similarly described and will be discussed in more detail further on in this review.

Chapter 1:- Introduction

Step 5 is quantitative confirmatory testing where the results are deemed positive if above a predefined cut-off concentration or negative if below.

Upon receipt of confirmatory results, the requesting authority can take action including legal prosecution or disciplinary procedures in the workplace. The sample is also placed into long-term storage. Storage duration may be determined by the standard to which testing is conducted, legislation, or the laboratory. The interval between a non-negative onsite test and laboratory confirmation can be a stressful time for the donor as they are often stood down from work or have their drivers licence revoked. Transport of samples can take 1-3 days particularly in a country as geographically dispersed as Australia where screening may be performed in a remote area and confirmatory testing only in a major centre. The expectation, by requesting authorities, is for the laboratory to issue results as quickly as possible. This leads to the need for simpler sample preparation and faster analytical run times while still maintaining a low LOQ.

• Initial test performed on worksite or roadside 1 • Example devices-DrugWipe, RapidSTAT, Oratect

• Second level screening test performed on different device 2 • Example devices-Dräger DrugTest 5000, Cozart DDS2

• Collection of sample for confirmatory testing 3 • Example devices-Quantisal, Salivette

• Transport to Laboratory 4

• Confirmatory testing 5

• Results and actions taken 6

• Storage of sample 7

Figure 1.2 Typical procedure for collection of oral fluid for drug testing.

Chapter 1:- Introduction

1.6 Procedures and devices Devices for the collection of oral fluid to be transported and subsequently tested in the laboratory come in two main variants, buffered and non-buffered. Non-buffered devices such as the UltraSal-2 or RapidEASE (Figure 1.3) collect oral fluid by direct expectoration into the tube or attached mouthpiece. Where a duplicate sample is required, the UltraSal-2 automatically splits the sample into two aliquots. The main difference between these two devices is in composition material. The

UltraSal-2 is made from plastic whereas the RapidEASE is borosilicate glass. Both systems have the advantage of collecting neat oral fluid which allows the laboratory to determine the exact volume collected. There may be stability concerns or adsorption of drug to the walls of polypropylene tubes

(82). Another concern with neat oral fluid collection is the small sample volume produced by the donor. Whilst the device is capable of holding up to 9ml the minimum sample volume is 1 mL/tube, and this can be difficult for some donors to provide.

Figure 1.3 Non-buffered collection systems. UltraSal 2 (97) (left) and Biophor RapidEASE (98) (right)

Some devices use a collection pad to help facilitate collection (Figure 1.4).

The salivette device does not employ a volume indicator but is designed so that the collection pad when saturated is inserted into the inner tube contained within the primary tube. Neat oral fluid is then collected in the conical bottom of the primary tube following centrifugation. The Dräger DCD

5000 collection device consists of a collection pad with volume indicator at 400µL which is then placed into a transport tube. There is some discrepancy on the use of isopropanol as a buffer with the Dräger DCD device, as Hall et al. (2015) do this for the analysis of traditional drugs of abuse (99)

Chapter 1:- Introduction

yet Kneisel et al. (2013) eject the pad into the salivette device, centrifuge and analyse the neat oral fluid (80).

Figure 1.4 Devices using a collection pad. Salivette (100) (left) and Dräger DCD 5000 (101)(right)

To enhance stability of the drug during transport and provide the laboratory with a greater sample for analysis, buffered collection devices are available (Figure 1.5). These devices, such as Oral-EZE or

Quantisal have two components, the collection pad and the transport tube containing the buffer solution. Both devices collect 1mL±10% of neat oral fluid onto the pad which in the Oral-EZE is ejected into 2ml or in Quantisal inserted (stick and all) into 3ml of buffer solution.

Figure 1.5 Buffered collection devices. Oral-EZE (102) (left) and Quantisal (103) (right)

The advantages of a buffered system are that there is more sample available for analysis and that the buffer stabilises the analytes in solution preventing or lessening degradation and adsorption to the

Chapter 1:- Introduction

tube, however, few studies are available to support the latter. The disadvantages to a buffered system are that there is a source of error in the amount of oral fluid collected onto the test pad with

1mL ±10% added to 3mL ± 10% of buffer in the case of Quantisal. Where the device uses a collection pad there is also the possibility of drug remaining on the collection pad rather than partitioning into the buffer. Coulter et al. (2011) addressed this for synthetic cannabinoids in sample preparation however a 15 minute vortex step followed by compression of the pad is somewhat labour intensive for a recovery of 55% - 74% (64). de Castro et al. (2014) investigated the recovery of eight cathinones with the use of the Quantisal test pad following 24 hours refrigeration with recoveries ranging from 79.6% to 107.7% (69).

Testing for drugs of abuse is a well established field with a large body of literature available for urine as the testing matrix (104, 105). The use of oral fluid is less established but has been used for traditional drugs for a number of years (106). One advantage of oral fluid is that the drug detection window is shorter than in urine, particularly for cannabis. This shorter detection window makes it more suited to the detection of impairment due to recent drug use. This is of interest for workplaces or police who aim for improved safety however, this benefit is negated if the sample must be sent to the laboratory for testing. Therefore, the primary benefit of oral fluid is for the test to be conducted on- site or roadside where a decision can be made from the results.

Chapter 1:- Introduction

1.6.1 Properties of Oral Fluid The ability to detect drugs or pharmaceuticals in oral fluid has been known for many years however itwas considered an alternative or adjunct matrix to the more common urine or blood (107, 108).

Technology and analytical procedures were less advanced than they are currently and to gain the sensitivity required for oral fluid analysis complicated sample preparation steps were required. This limited the number of drugs that could be analysed, usually to a chemically similar group.

As oral fluid gained acceptance more research was performed comparing plasma or serum concentrations to those found in oral fluid. Using MDMA as an example compound, many researchers found a high degree of intra- and inter-subject variability (109-111) following a controlled dose. This prevents the ability to calculate accurate plasma concentrations from those analysed in oral fluid (112). Using oral fluid as a screen followed by confirmatory plasma or serum analysis was a developing area (113, 114), particularly for roadside testing.

The properties of oral fluid as a matrix are also variable with much to be learned regarding the regulation and pathology of mucin secretion (115). However, known, age related changes in oral fluid composition have been described (116). Furthermore, medical conditions such as periodontal disease may promote bacterial and compositional changes in the saliva (117).

These same matrix associated challenges exist when analysing oral fluid for NPS and knowledge around detection window, excretion rates from plasma to saliva and typical oral fluid concentrations remain an area of much research interest (118, 119).

Chapter 1:- Introduction

1.7 Aims The emergence of NPS has posed several challenges in a variety of areas. Clinical presentations are similar to traditional drugs however without definitive testing, treatment options are predominantly supportive. Difficulties in categorising the drugs and rapidly changing chemical structures mean that legislation is also difficult.

Analytically, NPS are challenging due to unknown pharmacokinetics, the lack of analytical standards and the frequency of new drugs emerging and withdrawing from the market. The unregulated nature also provides little consistency with variation in the composition of the same branded product over time.

Some parallels can be drawn from the relatively mature area of traditional drug testing in either urine or oral fluid. From traditional drug testing it is known that some drugs are excreted into oral fluid whereas others are deposited in the oral cavity during smoking. The detection window in oral fluid is typically shorter than that of urine and the parent drug rather than the metabolite is more likely to be detected.

Parallels can also be drawn regarding collection device. Collection devices have potential to create multiple sources of error and to contaminate the sample. Therefore care should be taken when selecting such a device.

Chapter 1:- Introduction

Overall, whilst some general comparisons can be drawn from traditional drug testing very little remains known specifically about the analysis of NPS. Therefore, the specific aims of this Thesis are to: -

 Promote a cohesive, national approach to reporting and documentation of exposures to novel psychoactive substances.  Develop analytical methods for the detection of cathinones in oral fluid.  Develop analytical methods for the detection of synthetic cannabinoids in oral fluid.  Determine recovery of novel psychoactive substances under varying storage conditions and time.  Inform selection of collection devices by evaluating recovery from the Biophor and Quantisal devices.

Chapter Two discusses plans to promote a national approach to the collection of clinical and analytical data.

Chapter Three investigates the distracting issue of mobile pill testing at music and dance festivals and how, in the context of the rapidly changing landscape of NPS, this may not be minimising harm.

Chapters Four and Five describe the validated methods for the detection of cathinones and synthetic cannabinoids in neat oral fluid. The modifications to these methods making them applicable to samples collected in Qantisal Buffer, along with validation data are outlined in Chapter Six.

Chapter Seven uses these methods to investigate the stability of NPS in a number of storage conditions as well as the recovery from the Quantisal collection system.

Chapter 2:- A National Approach to Synthetic drugs

2 Chapter 2 Introduction The initial part of this Thesis was to establish the scope of synthetic cannabinoid use and establish a group of clinicians, researchers and other parties to share information. This manuscript only address synthetic cannabinoids as bath salts were unknown at the time of publication. Unlike Europe and the

USA, Australia has neither a monitoring system for drugs of abuse nor a national database of calls to poison control centres. The European Monitoring Centre for Drugs and Drug Addiction (EMCDDA) issues an annual report detailing drug trends and new and emerging drugs detected throughout

Europe. The American Association of Poison Control Centres (AAPCC) provides advice for consumers and clinicians on toxicity, drug interactions and pill identification. Data collected by these agencies provides valuable information on any emerging drug trends that pose a risk to the health of the population.

Chapter 2:- A National Approach to Synthetic drugs

2.1 Statement of Contribution

This is a co-author statement attesting to the candidate’s contribution to the publication listed below.

I attest that Research Higher Degree candidate Michelle Williams contributed to the publication listed below by providing the concept and writing the manuscript.

Williams, M., Taylor, P., Page, C., & Martin, J. (2014). Clinical research in synthetic cannabinoids-do we need a national approach? The Medical journal of Australia, 201(6), 317.

Chapter 2:- A National Approach to Synthetic drugs

This statement explains the contribution of all authors in the article listed above.

Author contribution percentage and description of contribution to the article listed above.

Author Contribution (%) Description of Signature Date contribution to article

Michelle Williams 60 Provided concept 07/08/18 Wrote the manuscript

Paul Taylor 5 Corrected the Retired manuscript Uncontactable

Colin Page 10 Provided clinical Uncontactable perspective Corrected the manuscript

Jennifer Martin 25 Provided concept 07/08/18

Wrote the manuscript

Michelle Williams

07/08/18

ADRT Signature: Date: 9/8/18

ADRT Name: Derek Laver

47 Chapter 2:- A National Approach to Synthetic drugs

Chapter 2:- A National Approach to Synthetic drugs

2.2 Results Between August 2014 and February 2015 fourteen individuals responded to the manuscript. The responses are summarised in Table 2.1.

Table 2.1 Survey responses Have you seen Have you heard an increase in Clinical/Research Date responded Sector of Synthetic presentations in interest drugs before the last 12 months (No) 09-Feb-15 Researcher Forensic Toxicology 21-Jan-15 Student 13-Nov-14 Clinician Mental Health Yes Yes (15) Mental Health Synthetic Cannabis 31-Oct-14 Researcher Use by people admitted to mental health services 03-Oct-14 Clinician Pathology Yes Yes Synthetic cannabinoid 23-Sep-14 Researcher metabolism Emergency 21-Sep-14 Clinician Yes Yes Department 19-Sep-14 Clinician Mental Health Yes Yes (5) 16-Sep-14 Clinician Intern Yes No 09-Sep-14 Clinician General Practice Yes No 09-Sep-14 Clinician Internal Medicine Yes No 09-Sep-14 Clinician General Practice Yes No Drug market epidemiology emerging 08-Sep-14 Researcher psychoactive substances Emergency 08-Sep-14 Clinician Yes Yes Department

Chapter 2:- A National Approach to Synthetic drugs

2.3 Discussion Respondents represent both clinical and research interests. As anticipated, the clinical respondents are in the front line fields of emergency medicine and mental health. Notably respondent three reported an increase of 15 cases in twelve months relating to synthetic cannabinoids.

All respondents indicated they would be interested in sharing data and case studies that they had access to. The student respondent contributed the following statement .

“This stuff opens doors within the mind previously unknown... The clarity and understanding of 'how things work' when inebriated is astounding ... I reckon I'm close to finding something significant!”

2.4 Conclusion At the time of publication there was very little known about synthetic cannabinoids and NPS.

Publication of this manuscript showed that there was significant interest from a widely read medical journal regarding the clinical need for this concern to be published. This survey demonstrates the interest and willingness of clinical and research groups to collaborate in a nationally orientated approach to information sharing.

Chapter 3: Pill Testing at Music Festivals

3 Chapter 3 Introduction

One of the issues distracting to the core focus of developing a consistent drug testing program is pill testing at music and dance festivals. NPS have influenced this area due to several factors.

The drugs are undetectable by traditional methods. Therefore, attendees in possession are unlikely to attract attention of law enforcement conducting swab or canine screening tests. If a personal or property search is warranted, the substance may be discovered, however, in the absence of robust legislation for NPS conviction is unlikely.

The potency of NPS is often much greater than traditional drugs leading to a commercial gain for sellers. Using fentanyl as an example. Fentanyl is a synthetic opioid with a potency around 50 times that of heroin. If a drug distributor has a very dilute supply of heroin a small amount of fentanyl can be added to give the end user a similar effect. Overall more ‘hits’ can be sold from the same gram weight of fentanyl versus heroin leading to greater profits. In the same manner as synthetic cannabinoids or bath salts, manufacturers slightly modify the chemical structure of the drug leading to analogues. This has also occurred with fentanyl. Fentanyl analogues such as acetyl fentanyl, butyrfentanyl, furanylfentanil and carfentanil, their effects and toxicities and have been discussed elsewhere (120-122). Carfentanil is the most potent fentanyl analogue at 10,000 times that of morphine (123). With this increased potency even more doses can be sold from a very small amount however the inaccuracy of dosing, poor heterogeneity and inexperience of illicit drug manufacturers has led to a number of overdoses (124).

In a bid to minimise the harms associated with drug use at music and dance events, despite lack of evidence and modelling,some groups have initiated mobile pill testing. Pill testing is aimed at sampling a drug to inform the user of its content and purity. Our research group had significant concerns around the lack of understanding of the testing shortfalls, and therefore aimed to highlight the limitations of pill testing unknowns and discuss the challenges associated with this process in more depth.

Chapter 3: Pill Testing at Music Festivals

3.1 Statement of Contribution

This is a co-author statement attesting to the candidate’s contribution to the publication listed below.

I attest that Research Higher Degree candidate Michelle Williams contributed to the publication listed below by providing the concept and correcting the manuscript.

Schneider, J.; Galettis, P.; Williams, M.; Lucas, C.; Martin, J. (2016) Pill testing at music festivals: can we do more harm? Internal medicine journal, 46, 1249-1251.

Chapter 3: Pill Testing at Music Festivals

This statement explains the contribution of all authors in the article listed above.

Author contribution percentage and description of contribution to the article listed above.

Author Contribution (%) Description of Signature Date contribution to article

Jenny Schneider 40 Provided concept 03/08/18 Wrote the manuscript

Peter Galettis 20 Provided 03/08/18 analytical information

Wrote the manuscript

Michelle Williams 30 Provided concept 03/08/18

Corrected the manuscript

Catherine Lucas 5 Provided clinical 03/08/18 information

Corrected the manuscript

Jenny Martin 5 Provided clinical 03/08/18 information Corrected the manuscript

Michelle Williams

03/08/18

56 Chapter 3: Pill Testing at Music Festivals

ADRT Signature: Date: 9/8/18

ADRT Name: Derek Laver

57 Chapter 3: Pill Testing at Music Festivals

58 Chapter 3: Pill Testing at Music Festivals

Chapter 3: Pill Testing at Music Festivals

Chapter 4 Methods for the detection of Cathinones in Oral Fluid

4 Chapter 4 Introduction Cathinones or bath salts are named due to the crystalline nature of the packaged product. The term bath salts, plant food or research chemicals were used as marketing devices to avoid detection.

Designed to mimic amphetamines, cocaine or hallucinogens these drugs vary in potency but are generally more potent than the drug which they mimic (125).

Very little is known about the concentrations in oral fluid or the usage patterns as there are few methods for the detection of this class of NPS available.

The aim of this part of the research was to develop a method for the detection of cathinones in neat oral fluid.

Chapter 4 Methods for the detection of Cathinones in Oral Fluid

4.1 Statement of Contribution

This is a co-author statement attesting to the candidate’s contribution to the publication listed below.

I attest that Research Higher Degree candidate Michelle Williams contributed to the publication listed below by providing the experimental design, executed the experiment, performed analysis, writing the manuscript.

Williams, M., Martin, J., & Galettis, P. (2017). A Validated Method for the Detection of 32 Bath Salts in Oral Fluid. J Anal Toxicol, 41(8), 659-669.

Chapter 4 Methods for the detection of Cathinones in Oral Fluid

This statement explains the contribution of all authors in the article listed above.

Author contribution percentage and description of contribution to the article listed above

Author Contribution (%) Description of Signature Date contribution to article

Michelle Williams 80 Experimental 07/08/18 design, executed the experiment, performed analysis, wrote the manuscript

Peter Galettis 10 Experimental 07/08/18 design, correct the manuscript

Jennifer Martin 10 Corrected the 07/08/18 manuscript

Michelle Williams 07/08/18

ADRT Signature: Date: 9/8/18

ADRT Name: Derek Laver

64 Chapter 4 Methods for the detection of Cathinones in Oral Fluid

Chapter 4 Methods for the detection of Cathinones in Oral Fluid

Chapter 5 Methods for the detection of Synthetic Cannabinoids in Oral Fluid

5 Chapter 5 Introduction

Synthetic cannabinoids are the second main group of NPS. These drugs originate as chemicals sold as a crystalline substance. This is dissolved in a solvent ranging from methanol or ethanol to petrol and lighter fluid. This liquid is then added to a combustible plant material. The addition may occur via spray or by combination in a cement mixer or similar device. The poor controls in the mixing mean that distribution is unequal giving rise to hot spots within the final package. The uncontrolled nature of the preparation also means that toxicities can arise from the solvent, pesticide residues on the plant or, as these products are designed to be smoked, the pyrolysis products thereof.

To begin to inform usage patterns, detection windows and provide workplaces with a means of detection and deterrent, a method for the detection of synthetic cannabinoids in neat oral fluid was developed.

This section discusses the specific drugs included, the preparation and collection of samples and validation parameters for the method.

5.1 Statement of Contribution

This is a co-author statement attesting to the candidate’s contribution to the publication listed below.

Williams, M., Martin, J., & Galettis, P. (2018). A Validated Method for the Detection of Synthetic Cannabinoids in Oral Fluid. J Anal Toxicol, bky043-bky043. doi:10.1093/jat/bky043

This statement explains the contribution of all authors in the article listed above.

Author contribution percentage and description of contribution to the article listed above.

Author Contribution (%) Description of Signature Date contribution to article

Michelle Williams 80 Experimental 07/08/18 design, executed the experiment, performed analysis, wrote the manuscript

Peter Galettis 10 Experimental 07/08/18 design, correct the manuscript

Jennifer Martin 10 Corrected the 07/08/18 manuscript

Michelle Williams 07/08/18

ADRT Signature: Date: 9/8/18

ADRT Name: Derek Laver

79 80

Chapter 6 Quantisal Validation data

6 Quantisal validation Devices for the collection of oral fluid vary considerably with two main variants, buffered and non- buffered. Non-buffered systems collect neat oral fluid with or without the use of a collection pad for the donor to manipulate within the oral cavity thus aiding collection. Buffered systems typically also have a collection pad and one of the most widely known is the Quantisal collection device. This device consists of two main components. The collection pad is a flat segment of filter paper like material attached to a plastic stick. Within the handle of the stick the collection pad extends and contains an indicator that turns blue when sufficient volume of oral fluid has been collected. The other main component to the system is the collection tube which contains the buffer. This buffer is designed to stabilise any drugs within the sample and also provides a greater volume, than the 1mL collected on the test pad, for analysis.

The aims of this section are to apply previously validated and published methods for the analysis of neat oral fluid to use with the Quantisal buffer.

6.1 Materials and methods The method developed and validated for the detection of cathinones in neat oral fluid is presented in

Chapter Four. The method developed and validated for the detection of synthetic cannabinoids in neat oral fluid is presented in Chapter Five.

6.2 Modifications to published methods The Quantisal system collects 1mL±10% of oral fluid and adds this to 3mL±10% collection buffer.

Samples were prepared by adding 1ml of previously spiked neat oral fluid to 3mL of Quantisal buffer.

During extraction, 400µL of oral fluid buffer mix was added to 200µL ACN containing internal standards. This was then vortexed, centrifuged and transferred to autosampler vials as described.

Chapter 6 Quantisal Validation data

6.2.1 Ion Suppression During LC-MS-MS validation the LOD of 1ng/mL obtained in neat oral fluid samples could not be achieved for synthetic cannabinoids. To improve sensitivity the injection volume was increased from

1µL to 5µL. This modification provided the sensitivity at the lower end of the calibration curve and did not cause saturation or any adverse effects at the higher end. Figure 6.1 demonstrates the increase in signal attained by increasing the injection volume. The 1µL injection produced maximum counts per second of 1.4e4 whereas the 5µL injection produced 3.5e4. Figure 6.2 compares the signal from

1µL injection in the cathinone method (7.5e4) to 5µL injection in the cannabinoid method (2.2e5) for the lowest quality control of 3ng/mL. This demonstrates the increase in the injection volume produces enough signal for accurate quantitation.

Chapter 6 Quantisal Validation data

Figure 6.1 Comparison of 1µL and 5µL injection at LOD (1ng/mL) synthetic cannabinoids

Chapter 6 Quantisal Validation data

Figure 6.2 Comparison between cathinone signal and cannabinoid signal at Low QC (3ng/mL)

Chapter 6 Quantisal Validation data

6.3 Validation data Intraday, interday imprecision and accuracy values for QCs are presented in Tables 1 and 2 for

cathinones and synthetic cannabinoids respectively.

Validation criteria was that intraday and interday imprecision must not exceed 15%CV. This method

demonstrated a CV range of 3.2%-14.9% for intraday and 6.3% - 14.7% for interday imprecision.

Accuracy ranged between 86.6% - 109.2% where a variation between 85%-115% was acceptable for

validation.

Table 6.1 Accuracy and imprecision parameters for cathinones.

Low (3ng/mL) Mid (30ng/mL) and High (300ng/mL)

Intraday (n=7) Interday (n=13) Accuracy (n=13)

L M H L M H L M H PMA 10.4 9.6 7.1 9.9 12.3 10.6 95.5 102.7 99.0 MDAI 10.7 10.9 9.1 9.4 10.4 12.0 91.0 97.7 100.2 MDA 6.6 8.0 8.4 8.0 12.8 11.5 93.0 104.2 98.2 MDMA 8.1 8.0 6.2 9.1 10.6 8.4 90.2 97.6 95.9 TMA 12.1 10.7 9.7 13.1 14.7 12.8 91.3 105.3 101.3 Methedrone 8.2 12.3 5.6 10.1 11.3 10.8 90.4 95.1 91.9 Cathinone 10.0 11.1 10.1 8.4 10.2 12.6 96.0 90.2 95.4 2-FMA 12.4 8.1 6.8 12.0 7.2 9.0 86.6 98.3 96.1 Methylone 5.3 11.7 12.4 10.4 11.4 11.1 87.2 96.8 100.7 Ephedrone 3.2 8.6 12.3 11.6 7.7 12.0 95.9 88.4 90.7 Flephedrone 13.9 10.2 10.7 14.1 8.5 11.5 94.3 89.2 91.0 4-MTA 8.4 13.2 10.4 7.9 13.5 14.3 96.7 95.1 94.4 4-MEC 12.7 5.9 4.4 12.3 10.6 7.5 91.9 90.8 92.1 Pentedrone 7.4 9.7 5.5 10.1 12.1 8.4 87.6 89.9 92.2 2-DMA 8.1 11.5 5.7 8.4 8.7 9.8 94.2 99.3 96.3 MBDB 6.3 6.4 3.9 8.7 8.3 7.0 92.3 95.3 95.6 Mephedrone 13.9 7.3 3.2 12.9 9.4 8.0 89.7 91.2 92.2 MDEA 10.1 7.9 3.2 13.0 10.6 7.1 89.2 95.3 94.4 Butylone 12.0 6.1 3.6 11.7 6.9 7.1 99.4 92.2 92.6 MPBP 7.1 8.4 5.7 9.7 10.7 9.3 87.7 99.5 98.3 2C-B 9.9 13.7 12.7 11.7 11.8 12.8 97.4 102.8 101.9 DOET 7.8 13.0 11.6 14.1 12.4 11.5 91.7 101.3 100.3 DOB 11.7 11.5 10.4 11.6 9.5 11.9 94.0 103.6 98.5 2C-T-2 9.0 12.8 11.5 8.4 12.9 13.8 102.2 101.9 100.5 2C-T-7 10.2 7.5 10.8 13.8 12.1 13.2 100.0 101.4 101.6

Chapter 6 Quantisal Validation data

TFMPP 10.6 11.7 9.9 12.8 13.4 12.3 92.1 98.6 101.7 PVP 12.1 8.9 7.1 11.5 9.0 9.9 89.9 101.3 99.3 MDPV 10.1 7.7 5.9 10.1 8.9 8.8 91.1 99.3 97.7 25C-NBOMe 8.9 12.6 12.5 12.4 12.1 11.8 107.4 99.5 108.0 25B-NBOMe 4.6 8.1 14.4 6.3 13.3 12.0 108.3 109.2 107.6 Naphyrone 9.9 10.9 9.8 11.8 9.6 12.6 91.7 99.6 94.8 25T4-NBOMe 13.4 9.3 14.9 10.9 11.3 12.1 102.9 98.9 108.1

The synthetic cannabinoid method demonstrated intraday and interday impresision of 4.2% - 13.7%

and 6.1% - 14.1% respectively with an accuracy of 86.4% - 109.1%

Table 6.2 Accuracy and imprecision parameters for synthetic cannabinoids.

Low (3ng/mL) Mid (30ng/mL) and High (300ng/mL)

Intraday (n=7) Interday (n=13) Accuracy (n=13) L M H L M H L M H STS-135 7.6 11.0 8.8 8.3 9.8 7.5 89.6 86.5 87.6 JWH-250 10.2 11.5 9.8 12.0 8.9 10.8 90.4 90.7 90.1 JWH-251 8.6 9.9 8.0 9.3 11.3 10.0 88.3 92.4 98.4 UR-144 13.7 12.7 4.2 13.9 13.4 6.1 90.9 91.7 89.1 RCS-4 9.3 10.3 9.4 9.9 11.1 8.3 89.5 91.1 86.4 JWH-73 12.5 12.7 6.4 12.3 11.4 8.1 89.9 96.5 91.7 XLR-11 9.2 6.8 6.2 10.0 9.9 8.1 91.3 91.5 90.6 JWH-18 10.6 10.1 6.5 11.9 10.9 9.7 93.3 90.9 92.5 AM2201 9.0 13.4 4.4 10.5 12.8 6.1 88.2 95.1 90.2 AKB-48 4.4 13.4 6.8 8.3 13.6 7.4 102.1 93.1 89.5 JWH-19 13.1 11.5 4.9 12.0 9.9 6.6 90.5 90.2 89.3 JWH-200 12.1 8.5 10.2 11.0 10.8 12.8 100.6 97.8 97.1 AB- 9.8 5.4 7.2 11.6 11.5 8.9 102.1 101.3 104.2 CHMINACA AB- 6.4 9.1 11.6 7.9 9.1 12.0 88.8 88.1 97.0 FUBINACA AB-PINACA 13.5 9.1 8.6 14.1 10.8 10.9 93.2 95.1 100.5 AB-005 9.8 6.5 7.5 10.3 12.7 9.1 109.1 105.7 105.6 AM2233 9.3 8.6 6.6 11.2 12.9 10.3 99.3 103.6 106.1 JWH-122 13.4 12.8 7.9 13.1 11.4 8.3 88.3 91.9 88.6 JWH-20 6.1 8.6 6.6 7.9 8.8 8.3 95.6 100.1 93.5

Repeatability values of mean and standard deviation of the QCs for cathinones and synthetic

cannabinoids are presented in tables 6.3 and 6.4 respectively.

Chapter 6 Quantisal Validation data

Table 6.3 Repeatability values of cathinones.

Low SD Mid SD High SD (3ng/mL) (30ng/mL) (300ng/mL) PMA 2.9 0.3 30.8 3.8 297.1 31.5 MDAI 2.7 0.3 29.3 3.0 300.7 36.2 MDA 2.8 0.2 31.3 4.0 294.7 33.8 MDMA 2.7 0.2 29.3 3.1 287.8 24.1 TMA 2.7 0.4 31.6 4.6 304.0 39.0 Methedrone 2.7 0.3 28.5 3.2 275.8 29.8 Cathinone 2.9 0.2 27.1 2.8 286.2 36.2 2-FMA 2.6 0.3 29.5 2.1 288.4 26.0 Methylone 2.6 0.3 29.0 3.3 302.1 33.7 Ephedrone 2.9 0.3 26.5 2.0 272.2 32.6 Flephedrone 2.8 0.4 26.8 2.3 273.1 31.3 4-MTA 2.9 0.2 28.5 3.8 283.3 40.5 4-MEC 2.8 0.3 27.2 2.9 276.2 20.8 Pentedrone 2.6 0.3 27.0 3.3 276.6 23.3 2-DMA 2.8 0.2 29.8 2.6 288.9 28.3 MBDB 2.8 0.2 28.6 2.4 286.7 20.0 Mephedrone 2.7 0.3 27.4 2.6 276.5 22.0 MDEA 2.7 0.3 28.6 3.0 283.1 20.0 Butylone 3.0 0.3 27.6 1.9 277.7 19.8 MPBP 2.6 0.3 29.9 3.2 295.0 27.5 2C-B 2.9 0.3 30.8 3.6 305.7 39.0 DOET 2.8 0.4 30.4 3.8 301.0 34.5 DOB 2.8 0.3 31.1 3.0 295.6 35.2 2C-T-2 3.1 0.3 30.6 3.9 301.4 41.5 2C-T-7 3.0 0.4 30.4 3.7 304.9 40.3 TFMPP 2.8 0.4 29.6 4.0 305.1 37.4 PVP 2.7 0.3 30.4 2.7 298.0 29.4 MDPV 2.7 0.3 29.8 2.6 293.2 25.8 25C-NBOMe 3.2 0.4 29.8 3.6 323.9 38.1 25B-NBOMe 3.2 0.2 32.8 4.3 322.7 38.7 Naphyrone 2.8 0.3 29.9 2.9 284.4 35.9 25T4-NBOMe 3.1 0.3 29.7 3.4 324.4 39.3

Chapter 6 Quantisal Validation data

Table 6.4 Repeatability values for synthetic cannabinoids. Low Mid High SD SD SD (3ng/mL) (30ng/mL) (300ng/mL) STS-135 2.7 0.2 26.0 2.6 262.9 19.6 JWH-250 2.7 0.3 27.2 2.4 270.2 29.3 JWH-251 2.6 0.2 27.7 3.1 295.3 29.6 UR-144 2.7 0.4 27.5 3.7 267.4 16.4 RCS-4 2.7 0.3 27.3 3.0 259.1 21.6 JWH-73 2.7 0.3 29.0 3.3 275.1 22.4 XLR-11 2.7 0.3 27.5 2.7 271.7 22.0 JWH-18 2.8 0.3 27.3 3.0 277.5 26.8 AM2201 2.6 0.3 28.5 3.6 270.6 16.6 AKB-48 3.1 0.3 27.9 3.8 268.5 19.9 JWH-19 2.7 0.3 27.1 2.7 267.9 17.7 JWH-200 3.0 0.3 29.3 3.2 291.2 37.3 AB- 3.1 0.4 30.4 3.5 312.7 27.7 CHMINACA AB- 2.7 0.2 26.4 2.4 291.1 34.8 FUBINACA AB-PINACA 2.8 0.4 28.5 3.1 301.4 33.0 AB-005 3.3 0.3 31.7 4.0 316.9 28.8 AM2233 3.0 0.3 31.1 4.0 318.4 32.8 JWH-122 2.6 0.3 27.6 3.1 265.9 22.0 JWH-20 2.9 0.2 30.0 2.6 280.5 23.2

6.4 Conclusion

This method was adapted from the previously validated and published methods for the detection of cathinones and synthetic cannabinoids in neat oral fluid. With a modification to sample preparation excluding the addition of water in place of a direct sampling of oral fluid Quantisal Buffer mixture, the same dilution concentrations are achieved. The presence of ion suppression when detecting synthetic cannabinoids required the increase of the injection volume to 5µL for that method. These methods are accurate, precise, repeatable and have the same benefits of the original methods.

These advantages are that a very small volume of original oral fluid sample is used, the sample preparation step is simple and rapid, and linearity, LOQ, LOD, separation of isobaric compounds are retained.

Chapter 6 Quantisal Validation data

The methods outlined above combined with the original methods now allow for the analysis of cathinones and synthetic cannabinoids in either neat oral fluid or Quantisal buffer. This permits analysis and comparison of samples collected via both means which will be addressed in the following section.

Chapter 7 Stability and Recovery

7 Chapter 7 Introduction

The selection of collection device is vital to ensure the integrity of the sample during transport to the laboratory. Chapter one discussed examples where the collection device added interfering substances to the sample (44). Also the adsorption of THC to the inside of the collection device is well documented (42). However, while some data may be applicable to NPS very few investigations have been executed.

This section aims to provide analyses on the recovery of NPS in neat and buffered oral fluid collection systems at three temperature conditions.

Furthermore, the effect of a collection pad is investigated independently to the presence of the buffer within the collection system.

Chapter 7 Stability and Recovery

7.1 Statement of Contribution

This is a co-author statement attesting to the candidate’s contribution to the publication listed below.

Stability and Recovery of NPS in oral fluid from neat and buffered collection devices

Submitted to Journal of Analytical Toxicology 27/08/2018

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Chapter 7 Stability and Recovery

This statement explains the contribution of all authors in the article listed above.

Author contribution percentage and description of contribution to the article listed above

Author Contribution (%) Description of Signature Date contribution to article

Michelle Williams 80 Experimental 07/08/18 design, executed the experiment, performed analysis, wrote the manuscript

Peter Galettis 10 Experimental 07/08/18 design, correct the manuscript

Jennifer Martin 10 Corrected the 07/08/18 manuscript

Michelle Williams

07/08/18

ADRT Signature: Date: 9/8/18

ADRT Name: Derek Laver

101 Chapter 7 Stability and Recovery

7.2 Abstract Stability and recovery of commonly used drugs has been widely acknowledged as an issue. In the new version of AS 4760:2006 to be released later this year the required recovery of drug from collection devices is >70%. AS 4760:2006 is the current standard in Australia governing the collection, detection and quantitation of drugs in oral fluid. Here we investigate the stability and recovery of 51 Novel Psychoactive Compounds (NPS) across two classes, cathinones and synthetic cannabinoids. The investigation looks at three concentrations (3ng/mL, 30ng/mL and 300ng/mL) across three storage conditions (room temperature, refrigerated and frozen) and compared a non- buffered glass collections device, the Biophor tube, with the buffered Quantisal system, used both with and without the collection pad. Recovery of high concentration samples ranged from 0% to

121% for non-buffered at room temperature and 0%-111% when buffered. Refrigeration improved recovery to 35%-101% and 66%-104% for non-buffered and buffered respectively. Freezing improved recovery for high (300ng/mL) and mid (30ng/mL) concentration samples. In samples where the oral fluid was applied to the collection pad, simulating real world use the results were erratic due to drug retention by and disintegration of the collection pad. Overall, samples should be stored refrigerated and buffer should be used with caution. The use of systems which involve a collection pad and buffer pose an additional set of variables that need further investigation, particularly in scenarios where a defined cut-off concentration or action point exist.

7.3 Introduction Oral fluid as a matrix for drug testing is gaining popularity and is becoming used for roadside, workplace and pre-employment testing (126). In Australia, workplace oral fluid testing is governed by the standard AS 4760:2006. This standard describes the drug/drug metabolites to be tested, the screening and confirmatory cut-off concentrations, and procedures to be followed regarding donor identification, specimen labelling and chain of custody. The drugs specified in this standard are

Tetrahydrocannabinol (THC), Amphetamine (Amp), Methylamphetamine (Met)

Methylenedioxymethamphetamine (MDMA), Methylenedioxyamphetamine (MDA), Cocaine,

Benzoylecgonine, Codeine, Morphine and 6-Acetylmorphine. At the time of development these drugs were reflective of the drugs in society and aligned with the standard for urine testing, except for benzodiazepines. However, in recent times with the increase in prescription of synthetic opioids such

102 Chapter 7 Stability and Recovery

as oxycodone and the rise in Novel Psychoactive Substances (NPS) in combination with improved device technology this standard required updating. The anticipated AS/NZS 4760:2018 standard has not yet been finalised but has lowered the cut-off concentration for THC for both screening and confirmatory testing (127). Oxycodone has been included as a mandatory drug with benzodiazepines and NPS being optional though this may change prior to final publication. Further updates recognise the extensive role of on-site or instant screening, as well as the need for high quality transport devices and sets out a minimum recovery of 70% drug/drug metabolite from these devices.

The stability and recovery of drugs of abuse in urine is an area that has been researched extensively

(128). However less is known regarding oral fluid other than the loss of THC due to adsorption to the wall of plastic containers (42). One study has investigated this for NPS by comparing the recovery from polypropylene and borosilicate tubes determining that lipophilic synthetic cannabinoids are subject to loss when stored at room temperature in polypropylene tubes (82).

Oral fluid, as a matrix can have some challenges with small sample volume possibly the greatest.

Because of this, some devices utilise a buffer solution which theoretically stabilises the drug in solution and increases the sample available for analysis. Some devices use a collection pad prior to insertion into the buffer however this can introduce another two sources of error (volume collected and drug recovery from test pad) in addition to any error in dilution volumes. Other challenges outlined by Wille et al. (2018) in a recent review are the stability of the drug in the oral fluid matrix and the use of immunological versus targeted and non-targeted screening methods (39).

Some manuscripts investigate stability of NPS in buffered solutions however they apply spiked oral fluid directly to the buffer at the ratio described by the manufacturer with the total volumes not always reflective of real world use (41). Others utilise the collection pad however dip the pad into spiked oral fluid allowing it to absorb until the indicator changes colour (69).

The storage of samples prior to, or during transport to the laboratory is another critical pre-analytical step particularly in a country as geographically dispersed as Australia, where testing, often in a remote location, may be a number of days transport away from the testing laboratory in a capital city.

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Currently the literature contains one manuscript discussing the recovery of 11 synthetic cannabinoids from polypropylene and borosilicate tubes (82) and another discussing the recovery of 9 cathinones from Quantisal Buffer versus neat oral fluid in plastic (41). Both manuscripts demonstrate the poor recovery of drug at room temperature indicating that both collection device and storage conditions are important.

Stability is defined by the Scientific Working Group for Forensic Toxicology (SWGTOX) as the analytes resistance to chemical change in a matrix under specific conditions for given time intervals

(129). Appendix C of the proposed AS/NZS 4760:2018 specifies that collection devices shall achieve a minimum recovery of 70% drug (drug metabolite) however a specific definition of recovery is not provided (127). Stability of a drug will directly influence the possible recovery thus both stability and recovery are directly linked. For the purposes of this manuscript percentage recovery is calculated and is used as a measure of the drug stability in the specific conditions in addition to any losses encountered due to adsorption to the container walls, adherence to the collection pad or buffer-matrix interactions.

This manuscript aims to further the literature by evaluating the effect of temperature on the stability and recovery of 51 Novel Psychoactive Substances, 32 cathinone or ‘bath salt’ drugs and 19 synthetic cannabinoids. Analysis was conducted using a borosilicate glass tube and two variations of Quantisal device usage. The Quantisal device was assessed with and without the sample being applied to the collection pad to independently evaluate the effect of the collection pad independent to the buffer.

The volumes used are reflective of those in real world application of the devices and evaluate low

(3ng/mL) mid (30ng/mL) and high (300ng/mL) concentrations across three temperature conditions

(room temperature, refrigerated and frozen).

7.4 Materials and Methods

7.4.1 Chemicals and reagents One mg/mL solutions of each Cathinone, Ephedrone, Methylone, Flephedrone, 3,4-

Methylenedioxyamphetamine (MDA), para-Methoxyamphetamine (PMA), Methedrone, 3,4,5-

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Chapter 7 Stability and Recovery

Trimethoxyamphetamine (TMA), Methylenedioxyamphetamine (MDMA), Butylone, Mephedrone, 3,4-

Methylenedioxyethylamphetamine (MDEA), 4-Methylethcathinone (MEC), Pentedrone, N-methyl-1,3- benzodioxolylbutanamine (MBDB), 4-Methylthioamphetamine (MTA), α-pyrrolidinovalerophenone

(Alpha-PVP), 1-(4-methylphenyl)-2-(1-pyrrolidinyl)-1-butanone (MPBP), 4-Bromo-2,5- dimethoxyphenethylamine (2C-B), 3,4-methylenedioxy-pyrovalerone (MDPV),

Dimethoxybromoamphetamine (DOB), 4-(ethylthio)-2,5-dimethoxy-benzeneethanamine (2C-T-2), 1-

[3-(trifluoromethyl)phenyl]-piperazine, dihydrochloride (TFMPP), 4-ethyl-2,5-dimethoxy-α-methyl- benzeneethanamine (DOET), 2,5-dimethoxy-4-(propylthio)-benzeneethanamine (2C-T-7), naphyrone), 2-(4-chloro-2,5-dimethoxyphenyl)-N-(2-methoxybenzyl)ethanamine (25C-NBOMe), 4- bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe) and 2,5- dimethoxy-N-[(2-methoxyphenyl)methyl]-4-[(1-methylethyl)thio]-benzeneethanamine (25T4-

NBOMe),STS-135, JWH-250, JWH-251, UR-144, RCS-4, JWH-73, XLR-11, JWH-18, AM-2201, AKB-

48, JWH-19, JWH-122, JWH-20, JWH-200, AB-CHMINACA, AB-FUBINACA, AB-PINACA, AM-2233 and AB-005 were purchased from Lipomed (Arlesheim, Switzerland). 5,6-Methylenedioxy-2- aminoindane (MDAI), 2-fluoromethamphetamine (FMA), 2,5 dimethoxyamphetamine (DMA 1mg/ml solutions were purchased from Chiron AS (Trondheim, Norway). Amphetamine-d5, methamphetamine-d9 and MDMA-d5 were purchased from Cerilliant (Round Rock, TX, USA). JWH-

250 d5, JWH-73 d7, JWH-18 d11 and JWH122-d9 were purchased from Cayman Chemicals (Ann

Arbour MI, USA). Water was purified using a Merk Millipore, Milli-Q Advantage A10 system

(Darmstadt, Germany). Reagent grade ≥95% Formic acid and LCMS grade Chromasolv ®

Acetonitrile were from Sigma Aldrich (St Louis, MO, USA)

Oral fluid samples were collected from a number of laboratory staff by direct expectoration into a sterile tube. These samples were tipped into a tube when pooled, leaving any sediment in the primary container for discard. The oral fluid samples were not centrifuged prior to spiking.

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Chapter 7 Stability and Recovery

7.4.2 Preparation of internal standards, calibration solutions and quality controls A stock solution at 10µg/mL in ACN of all analytes was prepared from the individual primary standards of 1mg/mL. The calibration curve was generated by adding 50µL, 20µL, 10µL or 5µL stock solution to 1mL blank oral fluid producing the highest points on the calibration curve (500ng/mL,

200ng/mL, 100ng/mL and 50ng/mL respectively). These were diluted further in blank oral fluid to produce the lower end of the calibration curve at 20ng/mL 10ng/mL, 7.5ng/mL, 5ng/mL, 2.5ng/mL and

1ng/mL.

Biophor and Quantisal devices were kindly donated by Clonal Technologies (Brisbane Australia).

7.4.3 Preparation of samples Two collection devices were investigated with the Quantisal device evaluated with and without the collection pad. This investigation was performed to evaluate the effect of the Quantisal Buffer independent to the recovery efficiency of the entire system. Three temperature conditions investigated were, ambient laboratory ~22°C, refrigerated 4°C and frozen -18°C. The samples were not protected from light however those stored at ambient temperature were placed on a lower shelf to shield from direct exposure to fluorescent lights. Samples were prepared on study day 0 by adding

3000µL, 300µL or 30µL of stock solution to 100mL of pooled oral fluid giving final concentrations of high 300ng/mL, medium 30ng/mL and low 3ng/mL. Samples were extracted on study days 1, 2, 4, 7,

14, 21 and 30 with the adjustments outlined below and in Table 7.1.

To minimise variation in the volume of Quantisal Buffer present in the tubes, the buffer was poured into a glass bottle. All tubes were then washed with purified water and allowed to dry. Three millilitres was pipetted into each tube using a positive displacement pipette prior to the addition of spiked oral fluid.

Samples in the Biophor tube were pipetted directly into the tube which was then capped and placed in the relevant storage condition. Samples where the Quantisal test pad was not used were pipetted directly into the tube, capped, inverted to mix thoroughly and stored. Samples where the Quantisal test pad was used had 0.5 mL slowly pipetted along the length on one side of the test pad then repeated on the other side. Pads where oral fluid dripped off were discarded and application

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Chapter 7 Stability and Recovery

repeated. The test pad and stick were placed into the tube which was capped and inverted to mix prior to storage.

Due to limited numbers of tubes, samples for days 4, 7 and 14 were taken from the same Biophor or

Quantisal-without pad tubes at room temp and in refrigerated only. The frozen samples were excluded from multiple sampling to minimise any free-thaw stability concerns and samples using the test pad were excluded to ensure the volume of buffer in contact with the test pad was consistent with the other samples. These time points were chosen as they were deemed the least important in a real world simulation. The earlier time points are reflective of samples being transported to the laboratory for analysis immediately following a non-negative on-site screen and the later time points reflective of long term storage. The 30 day samples at room temperature and refrigerated with the Quantisal test pad showed considerable fungal growth and were excluded from analysis. There was no sample for day seven-Biophor-frozen and day 14 Quantisal with pad for both room temp and refrigerated. Table

7.1 outlines the samples collected at each time point. The black bold outline indicates the samples were shared for included time points and shaded cells indicate a sample that was not prepared or excluded from analysis.

Table 7.1 Time points and samples collected for stability and recovery. Day 1 Day 2 Day 4 Day 7 Day 14 Day 21 Day 30 Biophor Room temp x x x x x x x Fridge x x x x x x x Freezer x x x x x x Quantisal without Room temp x x x x x x x pad Fridge x x x x x x x Freezer x x x x x x x Quantisal with Room temp x x x x x Excluded pad Fridge x x x x x Excluded Freezer x x x x x x x

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Chapter 7 Stability and Recovery

On the day of extraction, relevant samples were removed from storage, thawed completely at room temperature and inverted to mix thoroughly. Duplicate samples were prepared by adding 100µL oral fluid to 300µL water and 200µL ACN containing internal standards. These tubes were centrifuged at

5000 r.p.m. (2,300g) for 5 minutes. A 250µL aliquot was transferred to another tube for storage at -

80°C until analysis. Prior to analysis, samples for each batch were thawed completely, mixed and

100µL aliquot transferred to autosampler vials prior to injection into the LC-MS/MS system.

The UHPLC system was a Shimadzu, Nexera X2 LC-30AD pumps, SIL-30AC autosampler with a

DGU-20A5 degassing unit and CTO-20A column oven (Kyoto, Japan). Chromatographic separation was performed on a Kinetex (Biphenyl-Cannabinoids, F5-Cathinones) column (50mmx3mmx2.6µm) purchased from Phenomenex (Torrence, CA, USA) held at 40°C. Formic acid 0.1% (A) and

Acetonitrile (B) were used as the mobile phases at a flow rate of 0.5mL/min.

The mass spectrometer(MS) was a 6500QTRAP (SCIEX, Framingham, MA, USA). The MS operated in electrospray positive mode with the following settings: Curtain gas-20, Collision Gas medium, Ion

Spray Voltage-5500, Temperature-450°C Ion Source Gas 1-15 and Ion Source Gas 2-20. Scheduled

MRM mode was used for compound detection with a detection window set to 20sec around the expected retention time. Data acquisition was controlled by Analyst 1.6.3 and processed with

MultiQuant 3.0. (SCIEX, Framingham, MA, USA).

7.4.4 Validation of methods All methods used for analysis were validated to National Association of Testing Authorities (NATA) guidelines (130) where limit of detection (LOD), limit of quantitation (LOQ), selectivity, linearity of calibration, imprecision, repeatability, ion suppression and measurement of uncertainty (MOU) were evaluated. The validation procedure is regularly used in our laboratory and has been fully described elsewhere (131, 132). The method used to analyse samples in Quantisal Buffer differs from that described by the addition of 400µL buffer oral fluid mixture to 200µL ACN containing internal standard. No water is added to these samples and the injection volume into the LC-MS/MS is 5µL for synthetic cannabinoids as opposed to 1µL for cathinones.

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7.4.5 Data analysis The drug present was analysed at the end of the study and compared to the drug present at day zero.

Where the recovery was less than 70% the drug was deemed to be unstable under those conditions.

The recovery of samples that were applied to the test pad prior to analysis were also compared to the initial spiked concentration In 2015 when NATA withdrew accreditation for section 3 of AS 4760:2006 interested parties began the process of updating this standard. During this update, the inclusion of on-site screening and, collection and transport devices was warranted. The preliminary guidelines required devices to achieve a minimum recovery of 70%. This value may seem low however, the balance between quality and achievability must be obtained. 70% was accepted in the final version of the standard as the minimum recovery acceptable for a collection device used by a service seeking accreditation to AS/NZS 4760:2019 (7).

7.5 Results and Discussion The results from each section will be presented and discussed separately by drug class and storage condition. Sections 7.5.1 and 7.5.2 discuss the results of the recovery of synthetic cannabinoids and cathinones respectively when stored in the Biophor or Quantisal with buffer alone. All results pertaining to the use of the test pad with the Quantisal system are presented in section 7.6.

7.5.1 Synthetic Cannabinoids Most synthetic cannabinoids showed acceptable recovery when stored in the Biophor device compared to Quantisal buffer (Table 7.2) where poor stability (red), particularly among the JWH analytes was noted. UR-144 and XLR-11 showed the poorest recovery with 27% and 46% respectively, at the highest concentration. The XX-INACA (AB-FUBINACA, AB-CHMINACA, AB-

PINACA) analytes displayed opposite properties to the JWH group with poorer stability in the Biophor compared to Quantisal buffer alone. AB-005 was not stable in either device with adequate stability only observed at the highest concentration in Biophor.

Table 7.2 Recovery of synthetic cannabinoids when stored at room temperature. Biophor Quantisal no pad

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High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30 ng/mL) (3ng/mL) STS-135 112 83 96 74 73 72 AB-005 83 56 50 61 64 55 AB- 74 64 52 101 114 82 CHMINACA AB- 71 44 40 94 119 50 FUBINACA AB-PINACA 83 69 47 110 94 85 AKB-48 112 107 96 35 34 63 AM2201 105 97 79 81 64 74 AM 2233 99 84 76 87 83 69 JWH-122 116 101 103 51 61 72 JWH-18 116 91 100 47 58 60 JWH-19 104 86 95 61 55 64 JWH-20 103 102 104 56 63 64 JWH-200 110 79 68 72 101 55 JWH-250 117 89 82 62 51 61 JWH-251 114 82 98 45 32 60 JWH-73 117 90 99 47 61 65 RCS-4 101 80 81 51 57 67 UR-144 90 99 91 27 32 61 XLR-11 110 85 73 46 51 63

When stored refrigerated synthetic cannabinoids had acceptable (>70%) recoveries (Table 7.3) at high concentrations with some moderate losses shown by recoveries of 57%, 39%, 63% 69% 59% and 65% for AB-005, AB-FUBINACA, JWH-19, JWH-200, JWH-251 and UR-144 respectively at medium and low concentrations.

Table 7.3 Recovery of synthetic cannabinoids at 4°C.

Biophor Quantisal no pad High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) STS-135 86 93 78 84 98 84 AB-005 77 80 57 89 89 76 AB- 91 83 88 81 117 77 CHMINACA AB- 96 81 89 94 78 39 FUBINACA AB-PINACA 102 87 91 83 100 88 AKB-48 99 94 89 82 103 91 AM2201 94 82 82 79 102 92

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AM 2233 88 86 84 85 74 88 JWH-122 84 99 90 84 91 85 JWH-18 98 104 78 105 94 88 JWH-19 94 110 76 87 63 89 JWH-20 92 95 79 85 101 84 JWH-200 93 78 86 90 106 69 JWH-250 107 90 85 75 98 88 JWH-251 96 89 59 85 95 90 JWH-73 86 90 96 80 82 80 RCS-4 83 81 79 71 86 90 UR-144 91 83 84 87 65 77 XLR-11 97 89 74 96 79 85

All synthetic cannabinoids showed acceptable recovery at high and medium concentrations when stored frozen (Table 7.4). Low concentrations when stored in the Biophor device showed some degradation with recovery between 39%-69%. Analytes, AB-005 and JWH-20 showed the greatest losses with recovery of 39% and 41% respectively.

Table 7.4 Recovery of cannabinoids when at -20°C.

Biophor Quantisal no pad High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) STS-135 80 74 66 102 80 90 AB-005 83 75 39 97 96 85 AB- 96 82 73 100 89 89 CHMINACA AB- 83 84 72 114 66 95 FUBINACA AB-PINACA 92 86 84 109 88 94 AKB-48 99 76 56 101 84 94 AM2201 85 83 71 104 93 99 AM 2233 87 86 85 113 108 83 JWH-122 101 84 59 100 98 90 JWH-18 103 83 52 111 82 88 JWH-19 99 78 59 116 103 95 JWH-20 113 87 41 90 99 89 JWH-200 92 77 54 100 89 82 JWH-250 84 76 60 103 78 90 JWH-251 99 87 56 105 84 90 JWH-73 83 79 74 95 95 96

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RCS-4 90 87 63 105 93 92 UR-144 108 74 69 100 91 94 XLR-11 85 84 81 97 94 92

Overall, recovery is best when the sample is stored refrigerated in either device. Only the Biophor had acceptable recovery for the majority of compounds when stored at room temperature. This is consistent with published literature, where better recovery of synthetic cannabinoids are observed in glass collection devices than polypropylene at room temp (82). Data published by Kneisel et al.

(2013) demonstrated this poor recovery of most (10/11) synthetic cannabinoids when stored at room temperature in polypropylene containers at 72 hours ranging from 9.1% to 54% (82). The device used here is different to that published but is still polypropylene thus the presence of Quantisal Buffer improves recovery of synthetic cannabinoids when stored at room temperature compared to a polypropylene tube without Quantisal Buffer. However, the Biophor demonstrated improved recovery over either of the polypropylene tubes when stored at room temperature.

7.5.2 Cathinones Recovery of cathinones is acceptable for 21 of the 32 compounds in the Biophor device at room temperature (Table 7.5) with some inconsistencies between each concentration level noted for

Butlyone, 2C-T-2, 2C-T-7, 25B-NBOMe and 25T4-NBOMe. The recovery greater than 100% of PMA,

MDAI, MDA, MDMA, TMA and MDPV particularly at high concentrations may be due to these drugs being produced by the breakdown of others within the mixture and may warrant further investigation.

Methedrone, Cathinone, Methylone, Ephedrone, Flephedrone, 4-MTA, 4-MEC, Pentedrone and

Mephedrone showed very poor recovery in Biophor which was ameliorated by the Quantisal buffer but not to an acceptable degree.

Table 7.5 Recovery of cathinones when stored at room temperature.

Biophor Quantisal no pad High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) PMA 110 106 107 111 93 44 MDAI 103 104 75 107 101 101

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MDA 102 98 88 97 97 84 MDMA 102 93 85 100 107 91 TMA 121 114 92 102 99 79 Methedrone 34 54 39 85 84 63 Cathinone 0 0 0 0 0 0 FMA 105 101 88 100 103 84 Methylone 23 34 10 68 69 48 Ephedrone 0 1 0 15 18 0 Flephedrone 0 3 0 18 21 6 4-MTA 82 92 87 88 87 47 4-MEC 14 35 34 60 73 46 Pentedrone 12 35 43 69 72 43 2-DMA 103 116 92 108 108 78 MBDB 93 99 91 100 101 84 Mephedrone 7 19 12 52 53 37 MDEA 92 106 87 99 111 82 Butylone 69 82 67 93 98 80 MPBP 89 101 104 92 93 73 2C-B 95 96 86 101 111 86 DOET 94 110 94 100 99 84 DOB 97 108 107 102 107 75 2C-T-2 89 95 61 93 86 39 2C-T-7 79 78 54 90 82 33 TFMPP 91 88 83 90 100 61 PVP 84 102 98 92 91 64 MDPV 105 124 123 92 93 84 25C-NBOMe 77 92 97 94 93 78 25B-NBOMe 69 91 99 99 98 73 Naphyrone 70 75 74 76 77 61 25T4- 58 65 72 77 63 9 NBOMe

Refrigeration of cathinones in either device improves recovery for many drugs with Quantisal Buffer alone producing more acceptable recovery than Biophor (Table 7.6). Recovery for Methedrone, and,

4-MTA are similar between each device and it is likely that the improved recovery is a function of being refrigerated. Recovery of Cathinone, Methylone, Ephedrone, Flephedrone, 4-MEC, Pentedrone and Mephedrone are increased in the Quantisal Buffer making the difference between acceptable and unacceptable recovery for Cathinone, Ephedrone and Flephedrone. Of note is the poor recovery of the three NBOMe drugs in the Biophor device at high and low concentrations.

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Table 7.6 Recovery of cathinones when stored at 4°C

Biophor Quantisal no pad High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) PMA 84 89 123 97 93 87 MDAI 82 98 104 92 88 73 MDA 90 94 91 89 93 66 MDMA 96 93 101 95 92 85 TMA 93 100 98 96 95 75 Methedrone 83 94 91 98 81 75 Cathinone 35 37 0 66 70 75 FMA 81 90 92 97 85 90 Methylone 72 72 77 100 90 70 Ephedrone 41 32 41 89 84 71 Flephedrone 45 39 44 88 81 65 4-MTA 93 93 99 94 92 82 4-MEC 71 85 83 95 85 66 Pentedrone 69 76 88 91 98 108 2-DMA 101 97 111 103 97 61 MBDB 90 101 93 104 104 93 Mephedrone 72 65 81 90 84 85 MDEA 91 99 89 99 87 85 Butylone 88 92 90 90 88 96 MPBP 73 91 77 92 90 84 2C-B 79 79 85 99 91 87 DOET 79 91 81 95 89 77 DOB 73 93 71 97 85 75 2C-T-2 70 95 73 96 87 66 2C-T-7 67 84 61 96 86 71 TFMPP 72 79 75 93 95 80 PVP 74 92 71 95 90 77 MDPV 72 87 60 93 82 78 25C-NBOMe 62 84 54 90 89 75 25B-NBOMe 60 81 48 93 87 86 Naphyrone 86 86 56 94 90 76 25T4- 57 78 48 91 86 75 NBOMe

When stored frozen all drugs were recovered to an acceptable level in the Quantisal buffer (Table

7.7). Only MDAI and MDEA were recovered to an acceptable level when a low concentration was stored in the Biophor. The degradation of Cathinone to an undetectable level in Biophor may indicate

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very rapid degradation or possibly a concentration dependant adsorption to the container, both of which will require further investigation.

Table 7.7 Recovery of cathinones when stored at -20°C.

Biophor Quantisal no pad High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) PMA 95 79 64 107 94 104 MDAI 107 85 73 107 80 109 MDA 91 85 49 104 89 112 MDMA 91 85 62 100 88 89 TMA 91 85 45 103 92 93 Methedrone 97 80 69 96 88 89 Cathinone 98 46 0 105 73 88 FMA 95 84 66 102 99 106 Methylone 95 78 48 98 87 108 Ephedrone 91 55 40 106 74 104 Flephedrone 97 56 36 113 80 91 4-MTA 99 86 64 92 86 95 4-MEC 106 76 67 93 85 90 Pentedrone 102 74 63 92 85 86 2-DMA 95 88 65 91 87 98 MBDB 98 80 63 93 89 102 Mephedrone 92 71 54 96 87 90 MDEA 101 81 70 96 87 99 Butylone 99 82 58 92 93 73 MPBP 106 101 63 99 91 95 2C-B 101 74 46 93 98 89 DOET 101 74 57 96 91 100 DOB 102 76 56 102 91 99 2C-T-2 101 70 53 99 90 98 2C-T-7 105 69 51 95 93 86 TFMPP 99 67 51 99 86 99 PVP 104 75 56 100 93 91 MDPV 106 76 59 98 92 91 25C-NBOMe 106 67 48 93 94 95 25B-NBOMe 103 63 46 96 87 93 Naphyrone 109 71 54 97 93 103 25T4- 103 61 44 95 86 92 NBOMe

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The Quantisal device demonstrated acceptable recoveries across all analytes. The Biophor performed well at high concentrations however, decreased recovery was noted at low concentration.

Overall, when the sample is to be analysed for the presence of cathinones, it should spend minimal time at room temperature. Quantisal Buffer stabilised many of the very unstable cathinones when refrigerated or frozen with acceptable recovery of all analytes, at all concentrations when stored frozen.

A number of substituted cathinone analytes showed significant degradation when stored at room temperature in both devices. Cathinone and Flephedrone dropped to undetectable levels across all concentrations. The high losses were also observed in the literature though not to the same degree, particularly when Quantisal Buffer is used. The experimental design of Miller et al. (2017) stored the samples in a dark room and used smaller sample volumes of 100µL to 300µL oral fluid: buffer (41).

Our neat oral fluid samples were stored in borosilicate glass tubes rather than plastic thus direct comparisons should be made with caution. Figure 7.1 demonstrates the speed of the degradation observed for Cathinone (the drug) at the high concentration by graphing each sample analysed. This clearly demonstrates the rapid loss of Cathinone with a decrease from the 300ng/mL spiked into the sample to 215ng/mL at day 1 and a further decrease of two thirds by day four. Quantisal Buffer decreases this degradation in the short term however significant losses still occur at room temperature.

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Cathinone Recovery at High QC (300ng/mL) 350

300

250

200

150

Concentration ng/mL 100

0 0 5 10 15 20 25 30 Day

Biophor fridge Biophor room temp Quantisal fridge Quantisal room temp

Figure 7.1 Rapid degradation of cathinone, particularly when stored at room temperature.

7.6 Results where the Quantisal test pad was used In a real world scenario, many oral fluid samples are collected from the donor by chewing, sucking or otherwise holding the collection device with an absorbent sponge or pad in the mouth. This is held for a pre-determined amount of time or until an indicator alerts the collector that the desired volume has been collected. The Quantisal system uses a collection pad resembling filter paper attached to a plastic stick with an indicator window at the end that turns blue when 1mL±10% of oral fluid has been collected. The use of a collection pad introduces a further two sources of potential error in the collection process. The first is the volume of oral fluid collected. Whilst the Quantisal Buffer has been widely used and many manuscripts investigate this device they spike oral fluid, or synthetic substitute directly into the buffer at a ratio of 1oral fluid:3 buffer (41, 78, 133). Those that use the collection pad immerse it in spiked oral fluid making the assumption that it has drawn up 1mL (133).

Neither of these scenarios reflect the true application of the device. No collection agency will have a process where a set volume is collected into a container then accurately pipetted into the buffer nor where the collection pad is immersed in the collected sample. In situations where a donor is nervous, dehydrated or drug affected it can take in excess of 10 minutes for the indicator to turn blue. In these

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cases, the collection pad has usually been inserted and removed from the mouth multiple times to allow conversation and repeatedly moved around the oral cavity such that it is considerably disfigured.

It was noted during the sample preparation step of this study that the indicator in the stick turned blue with as little as 500µL oral fluid applied to one side of the device. Furthermore, when 1mL had been applied as indicated in the methods section many samples had to be repeated due to drips and those that did not drip were saturated to a level not observed in the device when used in the field, particularly where the collection interval is substantial.

The second source of potential error is the recovery of the drug from the test pad. The adhesion of drugs to the fibre of the test pad has not been investigated and it is possible for a sample result to be artificially lowered during the first days of storage which is when most analysis is conducted.

When the recovery of each analyte is investigated independently, the use of the Quantisal system appears to enhance the recovery of many drugs with JWH-73 reporting a recovery of 167% when stored in the fridge (Table 7.8). However due to the use of the Quantisal test pad this recovery is deceptive. To gain a more thorough understanding of the effect of the test pad, data for this group was normalised to the expected concentration. Due to the adherence of drug to the test pad, samples could not be compared to the analysed value on day 0 but must be compared to the value spiked onto the test pad. These values are 3ng/mL, 30ng/mL and 300ng/mL for the low, medium and high spiked concentrations respectively. In many cases such as AB-005, AB-FUBINACA and RCS-4 the calculated recovery is an over estimation due to artificially lowered Day 0 values caused by adherence of the drug to the test pad.

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Table 7.8 Recovery of cannabinoids in all storage conditions when sample is applied to the Quantisal test pad.

Quantisal with pad Quantisal with pad-normalised High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) Room STS-135 102 67 73 67 48 86 temp STS-135 Fridge 130 121 101 89 101 119 STS-135 Frozen 122 55 124 46 41 114 Room AB-005 85 64 62 64 51 63 temp AB-005 Fridge 125 125 91 100 117 111 AB-005 Frozen 92 81 141 54 63 120 AB- Room 107 122 104 89 114 124 CHMINACA temp AB- Fridge 88 98 95 67 92 125 CHMINACA AB- Frozen 91 76 123 53 63 120 CHMINACA AB- Room 117 98 103 117 118 87 FUBINACA temp AB- Fridge 111 137 64 124 154 227 FUBINACA AB- Frozen 132 72 73 47 33 96 FUBINACA Room AB-PINACA 116 102 101 90 86 99 temp AB-PINACA Fridge 111 116 106 102 110 123 AB-PINACA Frozen 105 74 114 59 60 105 Room AKB-48 57 47 80 29 24 87 temp AKB-48 Fridge 160 137 107 97 101 125 AKB-48 Frozen 154 151 113 49 41 127 Room AM2201 94 65 93 58 42 116 temp AM2201 Fridge 137 127 99 79 90 136 AM2201 Frozen 105 63 122 43 38 135 Room AM 2233 102 84 47 87 67 37 temp AM 2233 Fridge 102 111 96 95 110 100 AM 2233 Frozen 101 70 146 65 55 87 Room JWH-122 94 73 88 51 40 86 temp JWH-122 Fridge 143 151 112 79 93 111 JWH-122 Frozen 114 61 116 48 42 113 Room JWH-18 103 72 79 54 42 78 temp JWH-18 Fridge 160 129 113 86 102 117

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JWH-18 Frozen 102 55 131 47 43 110 Room JWH-19 86 71 80 46 35 72 temp JWH-19 Fridge 154 151 113 84 104 106 JWH-19 Frozen 115 52 130 49 35 100 Room JWH-20 86 67 77 41 32 67 temp JWH-20 Fridge 156 144 104 78 86 89 JWH-20 Frozen 108 59 128 47 39 98 Room JWH-200 88 80 43 83 72 28 temp JWH-200 Fridge 80 118 90 95 114 67 JWH-200 Frozen 69 87 165 49 67 66 Room JWH-250 81 52 67 51 39 77 temp JWH-250 Fridge 120 106 91 82 101 111 JWH-250 Frozen 99 52 129 44 41 117 Room JWH-251 66 53 68 42 32 77 temp JWH-251 Fridge 155 122 112 92 106 127 JWH-251 Frozen 111 60 134 50 42 117 Room JWH-73 91 62 77 52 38 95 temp JWH-73 Fridge 167 127 101 92 90 126 JWH-73 Frozen 95 61 133 44 40 134 Room RCS-4 80 55 72 55 40 91 temp RCS-4 Fridge 138 122 102 86 97 133 RCS-4 Frozen 93 55 131 46 38 136 Room UR-144 41 37 74 23 21 87 temp UR-144 Fridge 150 124 107 94 96 130 UR-144 Frozen 100 60 126 51 42 133 Room XLR-11 60 46 70 45 33 92 temp XLR-11 Fridge 125 100 97 90 85 132 XLR-11 Frozen 92 69 124 48 45 144

Overall, the recovery of synthetic cannabinoids are erratic and the only general conclusion that can be drawn is that recovery is best when samples are refrigerated.

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Recovery of cathinones shows a similar pattern to the synthetic cannabinoids, in-that non-normalised values are artificially elevated, though not to the extent observed in the cannabinoids (Table 7.9). The three NBOMe drugs analysed showed recoveries of >90% when stored frozen in the Quantisal Buffer even at low concentrations however the use of the test pad decreased this to just over 50%. Unstable drugs such as Cathinone, Ephedrone and Flephedrone demonstrated the same instability at room temperature with modest recoveries when frozen.

Table 7.9 Recovery of cathinones in all storage conditions when sample is applied to the Quantisal test pad.

Quantisal with pad Quantisal with pad-normalised Condition High Medium Low High Medium Low (300ng/mL) (30ng/mL) (3ng/mL) (300ng/mL) (30ng/mL) (3ng/mL) PMA Room 136 125 91 103 99 65 temp PMA Fridge 135 121 149 98 92 88 PMA Frozen 89 71 117 52 47 62 MDAI Room 133 106 87 103 21 51 temp MDAI Fridge 123 125 82 97 96 59 MDAI Frozen 100 65 135 60 44 63 MDA Room 125 119 75 103 91 53 temp MDA Fridge 126 133 114 100 97 71 MDA Frozen 97 71 131 55 49 68 MDMA Room 129 104 118 100 81 83 temp MDMA Fridge 137 121 100 99 95 72 MDMA Frozen 86 68 114 49 50 72 TMA Room 121 129 95 100 100 68 temp TMA Fridge 116 122 82 95 94 54 TMA Frozen 91 68 157 52 45 61 Methedrone Room 105 81 91 77 56 72 temp Methedrone Fridge 132 128 99 90 85 79 Methedrone Frozen 103 67 113 53 40 62 Cathinone Room 5 1 66 3 1 37 temp Cathinone Fridge 110 106 166 86 78 74 Cathinone Frozen 90 67 76 52 42 55

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FMA Room 120 134 115 97 99 72 temp FMA Fridge 124 124 105 95 93 70 FMA Frozen 95 74 121 56 48 55 Methylone Room 96 46 105 76 35 57 temp Methylone Fridge 119 113 112 96 95 77 Methylone Frozen 90 67 135 57 49 63 Ephedrone Room 28 0 49 21 0 28 temp Ephedrone Fridge 109 116 102 88 91 69 Ephedrone Frozen 94 67 99 55 45 50 Flephedrone Room 31 2 55 23 1 32 temp Flephedrone Fridge 118 123 112 92 91 74 Flephedrone Frozen 97 72 96 54 46 43 4-MTA Room 110 65 91 82 47 75 temp 4-MTA Fridge 133 135 108 92 87 90 4-MTA Frozen 124 128 90 53 47 80 4-MEC Room 87 32 79 62 22 74 temp 4-MEC Fridge 124 128 90 86 85 85 4-MEC Frozen 99 71 96 49 43 64 Pentedrone Room 77 37 90 58 25 73 temp Pentedrone Fridge 123 117 82 74 63 78 Pentedrone Frozen 93 49 97 40 22 58 2-DMA Room 120 114 107 99 87 82 temp 2-DMA Fridge 126 124 107 93 103 85 2-DMA Frozen 100 68 132 56 47 80 MBDB Room 121 121 97 100 81 64 temp MBDB Fridge 107 128 85 81 93 68 MBDB Frozen 121 82 90 55 46 55 Mephedrone Room 71 23 87 51 16 64 temp Mephedrone Fridge 122 125 95 85 86 80 Mephedrone Frozen 99 70 103 49 43 69 MDEA Room 133 107 99 98 75 77 temp MDEA Fridge 120 127 98 87 90 80 MDEA Frozen 100 70 119 51 43 76 Butylone Room 122 90 105 91 62 81 temp Butylone Fridge 127 127 100 86 90 78

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Butylone Frozen 106 67 114 53 44 70 MPBP Room 140 104 104 100 75 77 temp MPBP Fridge 118 131 83 93 88 67 MPBP Frozen 88 74 91 49 46 51 2C-B Room 124 57 99 104 48 62 temp 2C-B Fridge 111 129 111 90 96 71 2C-B Frozen 88 80 154 53 51 62 DOET Room 123 112 108 107 89 71 temp DOET Fridge 121 132 97 95 101 69 DOET Frozen 92 69 132 53 48 65 DOB Room 122 124 123 107 90 76 temp DOB Fridge 107 129 94 86 99 71 DOB Frozen 96 69 170 55 45 77 2C-T-2 Room 106 70 101 87 54 61 temp 2C-T-2 Fridge 110 127 90 91 98 64 2C-T-2 Frozen 89 63 146 52 42 75 2C-T-7 Room 105 60 79 86 49 53 temp 2C-T-7 Fridge 109 117 83 89 96 60 2C-T-7 Frozen 92 74 139 54 49 67 TFMPP Room 116 46 109 95 37 76 temp TFMPP Fridge 119 131 115 90 98 77 TFMPP Frozen 96 76 102 54 48 51 PVP Room 116 93 99 96 70 67 temp PVP Fridge 119 122 101 93 91 74 PVP Frozen 90 68 85 51 46 45 MDPV Room 122 98 112 96 74 70 temp MDPV Fridge 117 126 98 89 93 66 MDPV Frozen 95 77 109 55 52 54 25C-NBOMe Room 113 107 100 95 87 58 temp 25C-NBOMe Fridge 111 125 89 91 101 54 25C-NBOMe Frozen 107 77 123 61 53 40 25B-NBOMe Room 112 109 115 89 90 69 temp 25B-NBOMe Fridge 103 117 104 82 93 62 25B-NBOMe Frozen 104 70 111 58 47 42 Naphyrone Room 99 83 69 81 69 58 temp

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Naphyrone Fridge 118 125 91 97 101 71 Naphyrone Frozen 100 78 97 59 54 52 25T4- Room 82 27 40 60 20 27 NBOMe temp 25T4- Fridge 100 109 73 76 87 54 NBOMe 25T4- Frozen 108 67 97 59 46 50 NBOMe

7.6.1 Adherence of drug to test pad While using a collection pad can provide the donor something to move around the mouth, facilitating saliva production. Doing so may pose an additional set of variables. The material of the test pad should not introduce any contaminants to the specimen. Likewise, it should not remove any analyte from it. Here, by comparing the effect of the Quantisal Buffer independent to the system (test pad and buffer) the effect of the test pad alone is clearly demonstrated. Figure 7.2 illustrates the adherence of the drug to the test pad for two representative drugs, JWH-73 and 4-MEC. In both cases, the samples stored at room temperature are analysed on day one with values considerably below the spiked value of 300ng/mL. As the test pad disintegrates, detaching from the stick around day 14, it liberates more drug and the analysed values increase before settling by day 21 to recovery values similar to those observed in the Quantisal Buffer without pad samples (47% for JWH-73 and 60% for

4-MEC)

When samples were refrigerated, slowing the disintegration of the test pad, the values continued to increase until the final analysis at day 21. Both drugs showed high recovery when spiked directly into

Quantisal Buffer (80% for JWH-73 and 95% for 4-MEC) thus the initial decrease is likely due to adherence and subsequent liberation from the test pad.

When the samples were frozen the initial values were all lower than the spiked value. The erratic nature of the recovery could be due to variations in the sample handling or in the individual test pad composition. As each sample was thawed only once there was no opportunity for the test pad to disintegrate fully thus liberating the drug from the fibres.

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Figure 7.2 Recovery of representative cannabinoid and cathinone from samples applied to the Quantisal collection pad.

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7.6.2 Dilution effect of buffer

AS 4760:2006 provides target concentrations in neat oral fluid and these are used by employers to action the results. When using a buffered system, a calculation based on the presumed dilution is necessary. Due to variability in buffer volume, in an attempt to provide maximum benefit-of-the-doubt to the donor some laboratories treat buffered samples as if they were neat oral fluid. This process discounts the dilution factor and reports the analysed value rather than a calculated one along with an interpretative comment stating the result will be underestimated. Subsequently, many samples testing non-negative (positive) at the work site are returned from the laboratory as confirmed negative, creating substantial expense and confusion for the workplace and the testing entity.

7.6.3 Limits of study In this study, breakdown products were not specifically assessed. Therefore, it is possible that the breakdown of one analyte could be artificially elevating the concentration of another for example XLR-

11 degrading to UR-144.

Recovery of drugs from neat oral fluid in a plastic collection or transport device was not conducted.

The implications are that the recovery of NPS, from polypropylene, neat oral fluid collection devices remains largely unknown.

7.7 Conclusion Overall, it can be seen that no device demonstrated appropriate recovery of >70% for all analytes across all conditions and concentrations. It is generally best to refrigerate samples rather than store at room temperature and whilst a buffer can aid in the recovery this is not true for all analytes. The use of a buffered system also adds complexities regarding dilution, which are further compounded by the use of a collection pad. Some of the issues relating to the use of a collection pad could be overcome during sample preparation. This process would require the test pad to be removed from the stick and deliberately broken down by vortexing within the buffer, followed by extraction or filtration. However, this labour-intensive process may still not recover all drugs from the fibres and may introduce further sources of error.

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Of the options investigated, the Quantisal Buffer, without the use of the collection pad provided the best overall results. A device that collects neat oral fluid which can be aliquoted into two separate fractions, one buffered the other neat would be desirable. This would eliminate the challenges associated with the use of a test pad and allow for the benefits of a buffer, however, may be cumbersome for collection staff to manage and therefore an unrealistic option.

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8 Discussion NPS were first noted in Australia in the resource sector where the work is very high risk such as underground mining, heavy transport and high voltage power cabling. Workers in these industries are regularly subject to routine drug testing under either of the Australian standards AS/NZS 4308:2008 for urine testing or AS 4760:2006 for oral fluid testing. These standards specify the analysed drugs of cannabis, amphetamine type substances, cocaine, opiates and benzodiazepines in urine only. The workers in these industries consumed NPS specifically because they mimicked the effects of traditional drugs yet remained undetectable on routine drug screening tests. Initially they were also legal and widely available, however, this changed frequently leading to a cycle of evolution and legislation where drug manufacturers would develop a new legal analogue in response to updated legislation. This evolution occurred rapidly, and many test manufacturers struggled to keep up. Other challenges faced in this continuously changing landscape were the lack of analytical standards leaving some research groups to synthesise their own, obtain from seized materials through links with law enforcement or purify from purchased herbal mixtures (80). Manufacturers of ELISA kits for immunoassay struggled to keep up as the time taken to raise antibodies and bring a product to market meant that the drugs had already evolved making the kit outdated. Cross reactivity of NPS to existing instant test kits were investigated with very poor results. Overall, there were no options for any workplace wishing to undertake drug testing for NPS. Furthermore, there was no information on usage patterns, toxicity, detection methods or appropriate collection devices.

Chapter One undertakes a review of the current literature on both NPS and traditional drug testing.

Very few manuscripts existed discussing NPS specifically with even fewer on analytical methods for their detection. Therefore, much of what was known was gleaned from the application of knowledge gained in the area of traditional drug detection to NPS.

Chapter Two addresses the ability to describe and quantify the problem of NPS. One aspect of this is the lack of shared data and information for clinicians on NPS. The Editorial requests the promotion of a national approach to data collection surrounding exposure and clinical effects.

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Chapter Three investigates a related issue of testing for NPS in the music and dance festival attending population. This type of testing typically uses an analytical technique with limited sensitivity such as infrared spectroscopy, immunoassay or colorimetric detection. These methods detect high concentrations of a drug, which even for traditional drugs such as Methamphetamine may have adverse effects (134). These detection methods are also typically targeted toward specific drugs or drug classes and NPS typically show poor cross reactivity to test kits designed for traditional drugs.

The increased potency of NPS leads to lower concentrations necessary in pills, along with poor cross reactivity mean that detection of NPS in pills via these methods is unlikely. In addition, these methods are not analysing for excipients, carrier compounds or bulking agents that may also be harmful.

Overall, the provision of pill testing services to patrons may provide some information about the composition of the pill however is unlikely to be comprehensive enough to enable safe consumption.

In response to the lack of available testing options, the core focus of this Thesis was the development of analytical methods for the detection of NPS in oral fluid. This provides an option for workplaces wishing to undertake this testing. The first method analysed 32 cathinone or ‘bath salt’ drugs and the other analysed 19 synthetic cannabinoids. Chapter Four describes the validation of the cathinone method and Chapter Five describes the synthetic cannabinoid method. Both methods were linear from 2.5ng/mL to 500ng/mL with cathinones having an accuracy of 85.3% - 108.4% of the target concentration and an imprecision of 1.9% – 14%CV. The synthetic cannabinoid analysis method demonstrated an accuracy of 90.5% – 112.5% and an imprecision of 4-14.7% CV.

These methods were further validated for use with the Quantisal Buffer, described in Chapter Six.

The Quantisal collection system contains a buffer and is a popular device used for the collection and transport of oral fluid samples requiring confirmatory analyses under AS 4760:2006. The advantages to the methods developed are that they analyse a range of drugs not previously available, consume a very small volume of original sample, use a rapid sample preparation step and short chromatographic separation time. Both methods employ a simple gradient to which additional analytes can be included as the standards become available.

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At the time of publication these methods were the first and largest available in Australia outside a forensic setting and were employed by a commercial pathology laboratory providing confirmatory testing services.

The final chapter employs these methods to evaluate the stability of NPS in oral fluid over 30 days in varying storage conditions. This work also aims to inform the choice of collection device by evaluating the recovery of a buffered and non-buffered system. At room temperature, recovery of synthetic cannabinoids is better in the Biophor, borosilicate glass tube (mean 88%, range 44%-117%) than Quantisal Buffer alone (mean 65% range 27% to 119%) Compared to almost equivalent recovery of cathinones (mean 73% Biophor and 76% Quantisal). When refrigerated, recovery improved in both devices across all concentrations. The very unstable cathinones, Methedrone,

Cathinone, Methylone, Ephedrone, Flephedrone, 4-MTA, 4-MEC, Pentedrone and Mephedrone showed improved recovery by refrigeration and best when in Quantisal Buffer. When frozen, recovery was best in the Quantisal Buffer with a mean recovery of 94% for both drug classes. The Biophor showed acceptable recovery at high and mid concentrations for most drugs. However, poorer mean recovery of 64% for synthetic cannabinoids and 54% for cathinones at low concentrations. When the

Quantisal system was used with the collection pad the recovery was poor and very erratic particularly in the first days following collection.

8.1 Challenges

NPS are a very closely related group of compounds. Isobaric pairs such as Pentedrone and 4-MEC are encountered frequently as are product fragments common to multiple precursor ions. The chromatographic separation of these isobaric compounds is critical in their identification. To achieve this, multiple columns were investigated throughout the method development process with baseline separation attained. The addition of more drugs may pose a challenge if they form an isobaric pair or triplet with drugs existing in the method.

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The methods were designed with the aim of being implemented into a routine pathology laboratory.

Therefore, the analytical run time had to be as short as possible. Initially all 51 drugs were included in a single method, Achieving separation of the isobaric compounds combined with those eluting at high organic content made the analytical run prohibitively long (~15min). Splitting the analysis into two methods allowed for different, more suitable columns to be used and shortened the run time to an acceptable length. The implications of this for the laboratory are that a 15 minute run will require over three hours prior to the first patient sample being analysed, assuming a ten point calibration curve and

3 QCs. Compared to less than one and two hours for the published synthetic cannabinoid and cathinone methods respectively. Most laboratories will decrease this further by performing a 4-6 point standard curve allowing for the first patient sample to be reported within an hour of analysis beginning.

8.2 Limitations of Thesis One of the major limitations encountered early in the Thesis was the lack of published data. Typically a literature review is one of the first tasks undertaken however 4-5 years ago when this work commenced there were less than fifteen manuscripts focusing on analytical methods to review. This limited the ability to build on published works e.g. refining methods around a known concentration range. This lead to the decision to develop methods with the following characteristics: -

 Simple sample preparation  As low LOQ as possible  As large calibration range as realistic  As many drugs as available  Fast as possible analytical run

The methods have a calibration range reaching unlikely high values along with QC values spread throughout the range rather than clustered around a cut-off concentration. Both these factors can be revised by any laboratory wishing to implement the methods and it is easier to decrease calibration range of known linearity than increase it. Overall, the lack of published data led to the development of more adaptable methods. Furthermore, the number of published works has increased sufficiently for a more thorough review specifically related to the detection of NPS in oral fluid as outlined in Chapter

One.

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A limitation of the analytical component is the lack of isotopic standards. This means that none of the cathinones and few of the cannabinoids have matched internal standards. Had the preparation involved an extraction technique such as SPE the impact of this may have been significant. The methods developed here are a simple protein precipitation and the lack of matched internal standards was of minimal impact.

8.3 Future directions The field of NPS is rapidly evolving with new or modified drugs discovered frequently. Two new class of NPS have become known within the market. Synthetic opioids such as fentanyl analogues, methoxetamine and U-47700 are one class, with synthetic benzodiazepines such as clonazolam and etizolam, the other. There is a significant amount of research yet to be undertaken with these two classes as there are few options for the detection and information on stability and recovery is scarce.

Investigations into the pharmacokinetics of NPS such as detection windows, the degree to which drugs are excreted into oral fluid and any relationship between oral fluid concentration and impairment are also necessary.

The matrices containing drug are also changing such as e-cigarette liquid. With this rapid change, methods developed must be quickly modified to remain current to the drugs on the market or risk becoming ineffective, either as a legislative or deterring instrument. Furthermore, failure to remain current in detection methods will decrease the ability to accurately quantify usage patterns and attribute harms associated with NPS use. Technology can assist in keeping up to a degree with the use of high resolution mass spectrometry. Accreditation bodies such as NATA are yet to embrace these technologies and have not yet permitted the use of high resolution mass spectrometry in accredited analysis.

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8.4 Conclusion

Overall, this Thesis has achieved the aims set out and shown:-

 There is interest from research and clinical groups in developing a cohesive national approach to the reporting and documentation of exposures to NPS.  Developed and validated analytical methods for the detection of cathinones and synthetic cannabinoids in neat oral fluid.  Modified the developed methods for use with the Quantisal buffer.  Performed stability analysis determining that refrigeration allows for the best recovery.  Demonstrated the best device for sample collection does not utilise a collection pad and Quantisal buffer may only stabilise some compounds.

Although oral fluid is an attractive matrix for laboratories and mobile testing facilities, predominantly used for its less invasive and observable collection process, there remains a constant need for improvement. The performance of instant test devices and the addition of NPS to these devices is an area for continued improvement and investigation.

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