Mass Spectrometric Investigation of Intoxications with Plant- Derived Psychoactive Substances

From DEPARTMENT OF CLINICAL NEUROSCIENCE Karolinska Institutet, Stockholm, Sweden

MASS SPECTROMETRIC INVESTIGATION OF INTOXICATIONS WITH PLANT- DERIVED PSYCHOACTIVE SUBSTANCES

Kristian Björnstad

Stockholm 2009

All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet. Printed by E-Print AB, Stockholm 2009

“It’s an insane world, but I’m proud to be part of it.” - Bill Hicks

To my parents

ABSTRACT

The flora of the world contains many plants and fungi with stimulant, hallucinogenic and narcotic effects. For centuries, many of these have been used in initiation rites, physical and spiritual healing and rites of divination. Many of the plants are not placed under any restrictions regarding their use and sale and, with use of the Internet, they are easily obtained.

LC-MS/MS and GC-MS methods were developed and used for the detection of 12 plant-derived psychoactive substances, in urine samples, in cases of intoxication. The investigated substances were: asarones, atropine, DMT, ephedrine, harmaline, harmine, ibogaine, LSA, mescaline, psilocin, scopolamine and yohimbine.

Urine samples (n=462) from patients admitted to the Maria youth clinic were analyzed for the presence of mescaline. No samples were positive for mescaline, but the method was validated using a clinical sample from a German intoxication case.

Urine samples (n=103) from patients admitted to emergency departments all over Sweden were investigated for all 12 substances included in this study. All patients either admitted intake of a psychoactive plant substance or were suspected thereof. In 41 of the 103 samples at least one of the investigated substances was present. The most common substance was psilocin, found in 22 urine samples. Mydriasis, tachycardia, visual hallucinations, nausea and vomiting were the symptoms most often reported. These symptoms can be regarded as minor or moderate in terms of severity. The results suggest a low occurrence of psychoactive plant use in Sweden.

Studies were done in attempt to elucidate the metabolic pattern of α- and β-asarone in humans. Cis(β)-2,4,5-trimethoxycinnamic acid was regarded to be the most abundant metabolite, evidence of a hydroxylated metabolite, thought to be hydroxylated β- asarone, was also found.

Today these substances are often marketed on the Internet as “safe” and “legal highs”, which may lead to an increased use and calls for continuous investigation into psychoactive plant intoxications.

LIST OF PUBLICATIONS

I. Björnstad K , Helander A, Beck O. Development and Clinical Application of an LC-MS-MS Method for Mescaline in Urine. J Anal Toxicol . 2008;32(3):227-31.

II. Björnstad K , Beck O, Helander A. A multi-component LC-MS/MS method for detection of ten plant-derived psychoactive substances in urine. J Chromatogr B Analyt Technol Biomed Life Sci , 2009; 877(11-12):1162-1168

III. Björnstad K , Hultén P, Beck O, Helander A. Bioanalytical and clinical evaluation of 103 suspected cases of intoxications with psychoactive plant materials. (Submitted)

IV. Björnstad K , Helander A, Hultén P, Beck O. Bioanalytical Investigation of Asarone in Connection with Acorus calamus Oil Intoxications. (Manuscript)

The original articles (I and II) have been printed with permission from the publishers.

CONTENTS

Introduction ...... 1 Plant derived psychoactive substances ...... 1 Historical perspective...... 1 Associated risk...... 2 Investigated substances ...... 3 Analysis ...... 7 Gas chromatography-mass spectrometry (GC-MS)...... 7 Liquid chromatography-mass spectrometry (LC-MS)...... 8 Criteria for identification...... 11 Aims of the thesis ...... 12 Materials and methods ...... 13 Ethical approval...... 13 Patient samples...... 13 Paper I ...... 13 Paper II-IV...... 13 Analytical Procedure...... 13 Immunological screening...... 13 Solid phase extraction (Paper I)...... 14 Hydrolysis (Paper II-IV) ...... 14 LC-MS/MS (paper I-IV) ...... 14 GC-MS (paper IV)...... 15 Incubations with Cunninghamella elegans (paper IV) ...... 15 Method validation...... 15 Poison severity scoring ...... 15 Results and discussion ...... 17 Paper I...... 17 Method validation of the screening system ...... 17 Method validation of the confirmation procedure...... 18 Clinical samples...... 18 Paper II ...... 19 Method validation...... 19 Clinical application...... 22 Paper III ...... 22 Bioanalytical and clinical data ...... 22 Drug acquisition, preparations and dose...... 25 Samples not investigated bioanalytically...... 26 Paper IV...... 26 Clinical results ...... 26 Analysis of asarones, TMA-2 and TMC ...... 26 Search for hydroxylated and demethylated asarone metabolites.... 29 General discussion ...... 30 Conclusions ...... 32 Acknowledgements ...... 33 References ...... 35

LIST OF ABBREVIATIONS

APCI Atmospheric Pressure Chemical Ionization CEDIA Cloned Enzyme Donor Immunoassay CV Coefficient of Variation DMT Dimethyltryptamine EI Electron Impact ESI Electrospray Ionization GC Gas chromatography GC-MS Gas chromatography-mass spectrometry HPLC High Performance Liquid Chromatography HPPD Hallucinogen Persisting Perception Disorder IS Internal Standard LC Liquid Chromatography LC-MS Liquid Chromatography-Mass Spectrometry LOD Limit of Detection LSA Lysergic Acid Amide LSD Lysergic Acid Diethylamide MAO Mono Amine Oxidase MS Mass spectrometry m/z Mass over Charge PSS Poison Severity Score SPE Solid Phase Extraction spp. Species SIM Selected Ion Monitoring SRM Selected Reaction Monitoring TMA-2 2,4,5-trimethoxyamphetamine TMC 2,4,5-trimethoxycinnamic acid WHO World Health Organization

INTRODUCTION

PLANT DERIVED PSYCHOACTIVE SUBSTANCES According to the Oxford English Dictionary, the definition of a psychoactive substance is a substance “that possesses the ability to affect the mind, emotions, or behaviour” (1). There are numerous plants and fungi containing psychoactive substances. Some of the better known plants are: Papaver somniferum (opium poppy), Erythroxylum coca (coca bush) and Cannabis sativa (marijuana) (2). Today, most countries have a restrictive legislation against the use and sales of these plants and substances. However, the flora of the world contains many other plants with stimulant, hallucinogenic and narcotic effects that are not placed under any restrictions regarding their use and sale. Although specific species of plants and fungi are restricted to certain areas, in general, plants and fungi with psychoactive properties can be found all over the world, for example Trichocereus pachanoi (San Pedro) in Ecuador, Psilocybe semilanceata (liberty cap) found in Sweden, Tabernanthe iboga used in Gabon, Argyreia nervosa (Hawaiian baby wood rose) growing in Australia and Brugmansia suaveolens (angel’s trumpet) found in Nepal (2, 3 ). A few psychoactive plants and fungi are indigenous to Sweden, but the vast majority of these plants are not and therefore hard to come by. However, with the emerging of the Internet psychoactive plants and fungi from all over the world can easily be obtained from online shops (4).

Historical perspective The use of psychoactive plants for initiation rites, physical and spiritual healing and rites of divination is well described and shown to occur in many parts of the world (2, 5-9). There is a wide variety of plants and fungi used, ranging from small cacti to large trees (5, 6 ).For example Lophophora williamsii (peyote), a small cactus found in parts of the USA and Mexico, has a history of use for divination rites and medical healing (6) that can be traced back to pre-Columbian times and there are even findings dating back as far as 5700 years (10 ). In the 16 th century the Spaniards came in contact with peyote, they related it with heathen sacrificial rites of the Aztecs and in 1620 its use was prohibited (6). Today, in the USA, members of the Native American Church use Peyote legally, a use that is protected by law on account of religious freedom (6).

Another example is the brew ayahuasca, made from various plants found in the Amazon region. It has a history of use in magic rituals, divination and healing, also here the use dates back to pre-Columbian times (11 ). The first scientific studies of these rituals were done in the middle of the 19 th century, by the English botanist Richard Spruce (6). The decoction was, and is still, used by shamans to let them see the spirits of plants and animals. During the inebriation the shaman is able to identify the cause of illnesses and speak to the spirits in order to get to know and understand their purpose of being (2). Both the use of ayahuasca and Peyote was abolished by western civilizations when they first came in contact with it. Great effort was put in to eradicate their use, it was not uncommon to flog or even kill users (11 ) (6). In Gabon the plant Tabernanthe iboga is used by the Bwiti cult for medical, initiation and religious purposes. This use is fairly new, comparing to ayahuasca and peyote, with the earliest recordings being from 1864(6). When initiated into the cult, a person takes a huge dose of iboga that can be as high as hundred times of what is normally used in religious ceremonies. This massive dose induces a coma, in which the person’s soul travels into the “other world”, occasionally ingestion of doses this high have resulted in death (2).

Associated risk Most reports on psychoactive plant use concern case reports and these often reflect the worst case scenario. However, some papers on general risk and certain afflictions have been published. A risk assessment of ritual use of oral dimethyltryptamine (DMT) and harmala alkaloids has been done and the conclusion was that there is a minimal potential for dependence and psychological disturbances (12 ). In comparison to other commonly abused psychoactive substances, e.g. heroin, DMT, psilocybin and psilocin show a high safety ratio (13 ). Several of the plant derived psychoactive substances are hallucinogens (2), a term often given different explanations in the literature. In this thesis, hallucinogens will mean substances capable of causing hallucinations, if not stated otherwise. D. E. Nichols has concluded that most hallucinogens do not cause life-threatening physiological changes (9). According to Nichols, hallucinogens are considered to be those “substances with psychopharmacology resembling that of the natural products mescaline and psilocybin and the semi-synthetic substance known as lysergic acid diethylamide (LSD-25)” (9).

Use of hallucinogens can result in hallucinogen persisting perception disorder (HPPD) (14 ). It is diagnosed based on 3 criteria (adapted from Halpern, 2003(14 )):

1) Reoccurrence of perceptual symptoms that occurred under intoxication with a hallucinogen 2) The symptoms in criterion 1 effect social, occupational or other areas of functioning. 3) The symptoms cannot be related to another medical or mental condition (14 ).

When using hallucinogenic drugs there is always a risk for a so called “bad trip”. These can result in dysphoria, anxiety, fear, panic, frightening hallucinations and paranoia. In an uncontrolled setting these effects might lead to a dangerous behaviour (15 ). In the literature, the most common psychoactive plants associated with severe intoxications and traumas, from intentional ingestion, are those containing atropine and scopolamine e.g. angel’s trumpet and Jimson weed (16-20 ). Hallucinogens are not considered to produce dependence in the way that they do not produce a craving for the drug. However, there might be a pattern of use that start to effect e.g. work, school and relationships (15 ).In regards to withdrawal effects, the World health Organization has concluded that there is no evidence for such effects being associated with hallucinogens (ecstasy not included)(21 ).

Investigated substances In this thesis, a total of 12 plant derived substances were investigated. In paper I mescaline (fig.1) was examined. In Paper II atropine, DMT, ephedrine, harmaline, harmine, ibogaine, LSA, psilocin, scopolamine and yohimbine (fig.1) were investigated. In paper IV α- and β-asarone (trans- and cis-asarone) (fig.1) were examined and in Paper III all 12 substances were investigated. An overall reason for undertaking the study of these substances was the interest put forth by the National Institute of Public Health regarding psychoactive plant use. These substances were selected after checking lists of top sellers on various Internet shops, as well as considering the effects of the plants. For mescaline, recent confiscation of peyote cacti by the authorities was also a reason.

α-asarone β-asarone Atropine Dimethyltryptamine (DMT) Ephedrine

OMe OMe O OH C N N H N O OH NH C MeO MeO OMe OMe

Harmaline Harmine Ibogaine Lysergic acid amide (LSA) H3CO NH2 H N N N O N O N O N NH H H H

H3CO

Mescaline Psilocin Scopolamine Yohimbine

NH2 OH O C N O N O OH NH N H H C MeO OMe NH OMe H O O OH Figure 1. Structural formulas of investigated substances.

α- and β-asarone α- and β-asarone (fig.1) are present in the roots of several plants, among them Acorus calamus (sweet flag) (fig. 2) (5). In the wild A. calamus is found in North America, Europe and Central Asia (2) and has been used in ayurvedic medicine for thousands of years for insomnia, neurosis and fevers (6, 22 ). The first scientific reports of the psychoactive properties of A. calamus were published by Hoffer and Osmond in 1967(23 ). The psychoactive properties have been suggested to be caused by 2,4,5- trimethoxyamphetamine (TMA-2), because α- and β-asarone are natural precursors to this drug (6).

Atropine and scopolamine Several plants containing atropine and scopolamine (fig.1) have been used as inebriants in many cultures in different parts of the world (2, 16 ). The substances can be found in plants such as Brugmansia spp . (e.g. Angel’s trumpet) and Datura spp . (e.g. Jimson weed) etc (2, 18, 24 ). Today different species of the plants grow naturally in Africa, the Americas, Asia, Australia and Europe (2). Consumption of the plants often lead to hallucinations but also severe adverse effects like respiratory depression and even self- mutilation have been reported (18, 19 ).

Dimethyltryptamine (DMT) Dimethyltryptamine (fig.1) containing plants, such as Psychotria viridis (fig. 2) and Diplopterys cabrerana , are used when making the psychoactive South American brew ayahuasca (25 ). Many DMT containing plants are only found in Central and South America, but e.g. Phalaris spp . can be found in Africa, Australia, Eurasia, and North America(2). DMT is orally inactive but is made active with help of mono amine oxidase (MAO) inhibitors, also part of the ayahuasca brew (see harmaline, harmine) (25, 26 ). The psychoactive effects of DMT are powerful hallucinations and alterations of perception (25 ).

Ephedrine Ephedrine (fig.1) can be found in several Ephedra species, e.g. E. sinica and E. equisetina . In China E. sinica has been used as medication for the common cold, for thousands of years (24 ). Ephedra spp . are present in Eurasia and the Americas (2). The action of ephedrine is similar to that of amphetamine but it is not as potent (24 ). Ephedrine is also widely used for promoting weight loss (27 ).

Harmaline and harmine Peganum harmala (Syrian rue) native to Asia and Southern Europe (2) and the South American liana Banisteriopsis caapi (fig. 2) are two plants that contain harmaline and harmine (28 ). Banisteriopsis caapi is used when making ayahuasca and its role is to make DMT orally active, by way of inhibiting MAO (25, 26, 28 ).

Ibogaine Ibogaine can be found in high quantities in the African shrub Tabernanthe iboga (29 ). When taken in small amounts, T. iboga can reduce hunger and fatigue, but in higher doses it is hallucinogenic (30 ). It has also been reported that ibogaine can be helpful in treatment of substance abuse, especially opioid withdrawal (31, 32 ).

Lysergic acid amide (LSA) The similarities in effects between LSA and lysergic acid diethylamide (LSD) have resulted in plants containing LSA being used as narcotics, although it is less potent than LSD (24 ). LSA is present in the seeds of e.g. Ipomea violacea (morning glory) and Argyreia nervosa (Hawaiian baby wood rose) (fig.2) (24, 33 ). I. violacea is used as an

5 ornamental plant in many parts of the world. A. nervosa occur in Africa, Australia and India, but is now planted as an ornamental in all tropical regions (2).

Mescaline There has been a documented use of mescaline containing cacti dating back as far as 5700 years (10 ). Different cacti have been used throughout the Americas, e.g. Lophophora williamsii (Peyote) (fig. 2) in Mexico and Trichocereus pachanoi (San Pedro) in Ecuador (6). Mescaline effects include visual and auditory hallucinations (5).

Psilocin Most psychoactive fungi contain psilocybin and to a lesser extent also psilocin (2). The substances can be found in numerous species, of which many belong to the Psilocybe genus e.g. Psilocybe semilanceata (fig. 2) (5). Both the traditional use and modern day recreational use of these fungi is well known (34 ). Upon ingestion, psilocybin is converted, through dephosphorylation, into psilocin which is the active principal and acts as a hallucinogen (35 ). Mushrooms belonging to the Psilocybe genus can be found all over the world (2).

Yohimbine Yohimbine is present in the bark of e.g. Pausinystalia yohimbe, native to Nigeria, Cameroon and Congo (2). It has been used against erectile dysfunction and for promoting weight loss (36 ). But it is also known to produce psychoactive effects at higher doses (2). Yohimbine containing plants are native to Africa, the Americas and Southeast Asia (2).

Leaves of P. viridis - DMT Bark of B. caapi – harmaline and harmine

Dried P. semilanceata – Seeds of A. nervosa - L. williamsii - psilocybin and psilocin LSA mescaline

Root of A. calamus - α- Oil of A. calamus - α- and β- β and -asarone asarone Figure 2. Plant material sold for use as psychoactives. From top to bottom and left to right: P. viridis – DMT; B. caapi – harmaline and harmine; P. semilanceata – psilocybin and psilocin; A. nervosa – LSA; L. williamsii (Peyote) – mescaline; A. calamus - α- and β-asarone; A. calamus oil - α- and β-asarone. (Photographs ©A. Helander)

ANALYSIS Gas chromatography-mass spectrometry (GC-MS) In the analytical setting, GC-MS is a well established and important technique (37 ). Separation of analytes in GC is done by vaporizing the sample which is then carried by the flowing gas phase through a capillary tube coated with stationary phase. Analytes are separated according to their volatility and affinity to the stationary phase (37 ). For GC-MS, the most widely used method of ionization is electron impact (EI) (37 ), which is achieved through impact with electrons of high energy (70eV), emitted from a filament (37 ). Often a quadrupole mass analyzer is used for ion selection.

GC-MS is suitable for lipophilic and thermostable substances (38 ). It offers great separation power and specificity, but has the drawback that sample preparation, e.g. derivatization and sample clean-up, is most often needed (39 ). An advantage of GC-MS, over LC-MS, is the amount of spectral information collected, especially when using full scan acquisition of data (39 ).

Liquid chromatography-mass spectrometry (LC-MS) Reversed phase liquid chromatography is a common and widely used method of separation coupled to mass spectrometry (40 ). Separation is achieved on a column packed with stationary phase made of a nonpolar solid material and as mobile phase a polar solvent with buffer is used (41 ). The use of LC-MS in the field of analytical toxicology started in the beginning of the 1990s (38 ). Before this, GC-MS had been the preferred method of choice because of its great specificity and high ability of separation (39 ). For GC-MS there is a greater need for an appropriate sample clean up compared to LC-MS (39 ). However, when using LC-MS, thorough investigations of matrix effects have to be conducted (38 ). LC-MS has proven to be suitable for more polar and unstable analytes (39 ). A schematic of the LC-MS system is shown in fig 3.

Ion Mass LC source analyzer Detector

Vacuum

LC MS Figure 3 . The setup of an LC-MS system, comprising of an LC, ion source, mass analyzer and detector.

Interfaces for ionization Before analytes can enter the mass analyzer they have to be transferred from liquid phase to gas phase, which can be achieved by different ionization interfaces. The two most common interfaces used in LC-MS under atmospheric pressure are the electrospray ionization interface (ESI) and the atmospheric pressure chemical ionization interface (APCI) (42 ). ESI was first described by the group of John B. Fenn

8 in 1985(43 ) and was awarded the Nobel Prize in Chemistry 2002. The ESI has the ability to ionize a great range of molecules with regard to both molecular weight and polarity (42 ). While APCI can be used at greater flow rates (42) and is also less susceptible to matrix effects (44 ). In ESI ions are formed by applying a high voltage to the metal capillary which affects the liquid surface and produces a spray of charged droplets. The droplets, containing ions and solvent, are subjected to a warm dry gas that let the ions evaporate from the surface and then enter the mass analyzer (fig. 4) (42, 45 ). In APCI, ions are formed through gas phase reactions between vaporized analytes and vaporized mobile phase. A corona discharge needle, emitting electrons, facilitate the formation of ions (fig. 5) (42 ).

Droplets containing mobile phase and ions

LC flow

N2 gas flow Metal capillary MS-inlet Figure 4. A schematic of the electrospray ionization (ESI) interface .

MS-inlet

Heat Nebulizer gas

–N2

LC flow e e

Heat Auxillary gas

–N2 Quartz tube

Corona discharge needle

Figure 5. A schematic of the atmospheric pressure chemical ionization (APCI) interface.

Quadrupole Mass Analyzer In mass spectrometry ions are selected according to their mass over charge ratio ( m/z ). A basic schematic of the quadrupole mass analyzer is shown in fig 6. Mass filtering is achieved by the ability to change polarity of the DC voltage and radio frequency voltage applied to the metal rods that make up the quadrupole (42 ). Only ions with the selected m/z are allowed to pass through to the detector, all others are deflected.

tor tec de To

- -

s Ion +

Figure 6. A schematic of the quadrupole mass analyzer.

Tandem mass spectrometry (MS/MS) In a quadrupole tandem mass analyzer 3 quadrupoles are aligned (fig. 7). Quadrupole 1 and 3 are used as mass analyzers and quadrupole 2 is used as a fragmentation zone. Fragmentation is done by introducing a collision gas as well as applying collision energy (42 ). The first quadrupole is used to select a precursor ion. In the collision cell the precursor ion is fragmented to produce product ions and neutral fragments. The third quadrupole is then used to select product ions of interest for detection (46 ). This procedure is called selected reaction monitoring (SRM). Nordgren et al has showed that dilution followed by direct injection of the sample can be sufficient when using LC-MS/MS as a screening procedure (47 ).

Quadrupole 1 Quadrupole 2 Quadrupole 3 Mass filter Fragmentation zone Mass filter

Ion Detector source

Figure 7. Schematic of triple quadrupole mass spectrometer.

Criteria for identification Although the instrumentations described above are very selective, the risk of false positives always need to be considered. In order to minimize these there are guidelines on criteria for identification published in the Official Journal of the European Communities. There is a criterion on a minimum of 4 identification points (table 1) for positive identification as well as criteria for ranges of relative intensities between product ions etc.

Table 1 . Example of the number of identification points earned by GC-MS (EI), LC-MS and LC- MS/MS. Technique Number of ions (n = an integer) Identification points GC-MS (EI) N n LC-MS N n LC-MS/MS 1 precursor, 2 product ions 4

AIMS OF THE THESIS

• To develop sensitive and selective methods using LC-MS/MS for the analysis of psychoactive plant derived substances in urine.

• Apply developed methods in clinical studies of intoxications.

• Review intoxications with plant-derived psychoactive substances, presented at emergency departments.

• Evaluate the value of analytical investigation in cases of intoxication

MATERIALS AND METHODS

In this section a summary of the instrumentation and methodology is presented. For a more complete presentation please see original papers, provided in the back, or references within this section.

Ethical approval For all projects included in this thesis, ethical approval was granted by the local ethical committee at the Karolinska University Hospital (Stockholm, Sweden) (Dnr 00-236 and Dnr 2008/1087-32).

PATIENT SAMPLES Paper I The patient samples analyzed in Paper I were collected through the Maria Youth clinic, Stockholm, Sweden. Patients were selected on two criteria 1) it was their first visit to the clinic, 2) they were admitted at the emergency room at the clinic. A total of 462 samples were collected and analyzed. 96% of the patients were between the ages of 14- 20.

Paper II-IV For Paper II-IV, samples were collected from emergency rooms at hospitals all over Sweden. This was done through collaboration with the Swedish Poisons Information Centre along with information about the project sent to the emergency rooms in the country. Samples were taken when patients were suspected of, or admitted, intake of a psychoactive plant material. A total of 103 samples were collected and analyzed. About 80% of the patients were between the ages of 13-27.

ANALYTICAL PROCEDURE Immunological screening All urine samples investigated were initially screened for amphetamines (cutoff: 500 µg/L), opiates (cutoff: 300 µg/L), buprenorphine (cutoff: 5 µg/L), methadone (cutoff: 300 µg/L), propoxyphene (cutoff: 300 µg/L), cannabinoids (cutoff: 25 µg/L), benzodiazepines (cutoff: 200 µg/L), and cocaine (cutoff: 150 µg/L), using an

13 immunological drug screening with CEDIA reagents (Microgenics, Passau, Germany) on an Olympus AU640 (Tokyo, Japan) according to routine methods.

Solid phase extraction (Paper I) One milliliter of the urine sample was mixed with sodium carbonate buffer and mescaline-D9 and subjected to SPE. C 18 SPE cartridges were conditioned with methanol, distilled water and sodium carbonate buffer at a flow rate of 1 mL/min. After application of sample, the cartridges were washed with sodium carbonate buffer and acetic acid and dried under vacuum. The analyte was eluted with methylenchloride:isopropanol containing 2% ammonium hydroxide followed by methanol. Both fractions were collected into the same tube and evaporated to dryness under nitrogen gas. The final content was dissolved in 5% methanol containing ammonium acetate.

Hydrolysis (Paper II-IV) Of the investigated substances, psilocin and scopolamine are known to undergo conjugation with glucuronic acid (34, 48 ). In order to liberate glucuronide conjugates, samples where intoxication with these substances was suspected were treated with β- glucuronidase (E. coli K12, 140 U/mL).

LC-MS/MS (paper I-IV) The LC-MS/MS system comprised of a Perkin Elmer (Norwalk, CT, USA) series 200 autosampler and two micropumps, coupled to a PESciex 2000 tandem mass spectrometer (Applied Biosystems, Streetsville, ON, Canada). Two analytical columns were used for analysis: for mescaline a HyPURITY

C18 column (Thermo Hypersil-Keystone,Waltham, MA) was used and for all other substances a Hypersil GOLD column equipped with a UNIGUARD C 18 column (ThermoFisher, Waltham, MA, USA) was used. Separation was achieved using gradient elution with acetonitrile and formic acid for all substances except mescaline. For mescaline, gradient elution with methanol and ammonium acetate was used. For all analysis the mass spectrometer was operating in SRM mode. For Paper I an APCI interface and positive mode was used. For all substances in Paper II, and TMA-2 in Paper IV, an ESI interface operating in positive mode was used. In Paper IV, TMC was analyzed with ESI in negative mode.

The software used was Analyst 1.4.2 from Applied Biosystems (Streetsville, ON, Canada).

GC-MS (paper IV) For GC-MS analysis, an Agilent Technologies (Santa Clara, CA, USA) 6890N Network GC system coupled to a 7683 series injector, autosampler and a 5973 Network mass selective detector was used. Analysis was carried out both in full scan mode and selected ion monitoring (SIM) mode. Separation was achieved on a HP-Ultra (Agilent) 17 m × 0.2 mm fused silica capillary column and helium was used as carrier gas.

Incubations with Cunninghamella elegans (paper IV) Fungus culture, C. echinulata var. elegans , was added to an agar plate and incubated for 5 days at 27 °C. The fungus was peeled off and suspended in sterile saline solution. Part of the suspension was added to a flask with Sabouraud dextrose broth and left to incubate at 27 °C. After 3 days, the broth was replaced with new broth and oil from A. calamus was added. The mixture was left to incubate for 7 days at 27 °C. The experiment was terminated by the addition acetonitrile (49 ).

Method validation For investigation of performance of the developed methods, triplicates of each tested substance concentration was injected over 5 consecutive days. This approach was based on the guidelines found in EP 15-A2 set by the Clinical and Laboratory Standards Institute (CLSI.). EP 15-A2 contains a spread sheet for easy determination of repeatability, reproducibility and total CV.

POISON SEVERITY SCORING Poison severity scoring was done according to the scheme published by Persson et al (50 ). Scoring is done on a scale from 0-4. In the paper a description of specific symptoms, resulting in the respective score, for a number of organs and body systems is presented. However, the general definition of scoring is (adapted from Persson et al. 1998(50 )):

0 = “no symptoms or signs related to poisoning”. 1 = mild, transient or spontaneously dissipating symptoms. 2 = “pronounced or prolonged symptoms”. 3 = “severe or life-threatening symptoms”. 4 = “death” (50 ).

RESULTS AND DISCUSSION

PAPER I Method validation of the screening system Since 55-60% of ingested mescaline is excreted in unchanged form, it was used as the analyte (51, 52 ). For mescaline and the internal standard (IS), in spiked controls and clinical sample, the retention times were about 4.5 min (Fig. 8). When analyzing negative urines and clinical samples positive for other illicit drugs, no interfering peaks were observed.

35000 4000 Mescaline 250 µg/L Mescaline 30000 3500 Mescaline-D9 3000 25000

2500 20000 2000 15000 1500 10000 1000 Intensity formescaline (cps) 5000

Intensity for (cps) mescaline-D9 500

0 0 0 1 2 3 4 5 6 7 8 9 Time (min) Figure 8 . Chromatograms from the SRM analysis showing the peaks for mescaline ( m/z 212.3 180.3) and the IS mescaline-D9 ( m/z 221.3 186.3) for a spiked human urine sample used as calibrator (mescaline concentration 250 µg/L).

When urine was used as matrix, the limit of detection (LOD) of the method was 3 to 5 µg/L (signal-to-noise ratio of 3), and a good linearity (peak area ratio = 0.00071 x mescaline concentration - 0.033; r2 = 0.9998) was established for urines spiked with mescaline in the concentration range 5 to 10000 µg/L. The intra- and inter-assay variations of the method were determined and the total coefficient of variation (CV) for spiked samples containing 10 to 1000 µg/L mescaline was <8.5% (Table 2).

Table 2 . Reproducibility of the LC-MS/MS screening system for urinary mescaline

Target mescaline Observed mean Number of concentration concentration Bias Repeatability Total CV observations (µg/L) (µg/L) (%) (CV%) (CV%) (n) 1 10.0 10.5 5.0 6.3 8.5 15 100.0 95.9 -4.1 6.3 7.3 15 1000.0 1025.5 2.6 8.0 8.0 15

Investigations of mescaline stability during storage was done by comparing spiked urines kept refrigerated and spiked urines kept at ambient temperature (21 °C) for 7 days. The observed differences were <7%, which is within the limits for the intra- and inter-assay CV. The influence of ion suppression was investigated by infusing mescaline at a constant rate and at the same time injecting drug free urines. The influence of 10 patient urines was investigated (representative sample shown in Fig. 9). No distinct ion suppression was observed at the retention time of mescaline and the IS.

140000

120000

100000

80000

60000 Intensity (cps)

40000

20000

0 0 1 2 3 4 5 6 7 8 9 Time (min) Figure 9 . Influence of ion suppression studied by infusing mescaline at a constant rate, while drug free urines (n = 10) were injected via the autosampler. The result for one representative sample is shown.

Method validation of the confirmation procedure To assess the recovery of mescaline in the SPE clean-up procedure, comparison of peak areas ratios of mescaline and the IS for urine samples analyzed with and without the SPE step was done. This was done in triplicates at a mescaline concentration of 100 µg/L and the mean recovery of urinary mescaline in the SPE method was 99% (range 92-111%).

Clinical samples The reason for choosing a youth clinic for collection of samples was the general belief that adolescents constitute a high risk group in terms of hallucinogen use. The result of the immunologic drug screening by CEDIA (Microgenics, Passau, Germany) showed that 148 of 462 (32%) urine samples were positive for illicit drugs. The most common illicit drug was cannabis. However, none of the 462 urine samples were positive for mescaline using the LC-MS/MS screening method and no false negatives were detected by the confirmation system. Since no positive samples were detected, a clinical sample from a German intoxication case was supplied by Professor Hans Maurer (Saarland University, Homburg,

Germany) in order to validate the method. The mescaline concentration of this sample was 9300 µg/L (Fig. 10). This concentration is well above the LOD for the screening method, but lower than concentrations previously reported for mescaline in urine (53, 54 ). This suggests that the screening method is sensitive enough to detect mescaline in clinical urine samples. However, for correct quantification of mescaline, samples should be diluted. The results of this study suggests no or a low occurrence of mescaline use in the targeted age group.

160000 Clinical sample 9300 µg/L 140000 Mescaline Mescaline-D9 120000

100000

80000

Intensity (cps) 60000

40000

20000

0 0 1 2 3 4 5 6 7 8 9 Time (min) Figure 10 . Chromatograms from the SRM analysis showing the peaks for mescaline ( m/z 212.3 180.3) and the IS mescaline-D9 ( m/z 221.3 186.3) for a human urine sample collected after intake of mescaline (concentration 9300 µg/L)

PAPER II Method validation The use of multi-component methods has previously been demonstrated and was successfully implemented in this study (47, 55 ). The LC-MS/MS method was able to separate 10 plant-derived substances, within 8 min, total analysis time 14 min. Psilocin, which, eluted first did so after ~5 min and was well separated from the front (k’ value 4.5) (Fig. 11A). Retention times and validation data, such as analytical recovery, imprecision and linearity ranges, for each analyte and internal standard are given in Table 3. After comparing previously reported urine levels of the various substances and the findings of this study, the LC-MS/MS method is considered suitable for investigation of these substances (28, 30, 34, 35, 56-60 ). However, dilution might be necessary in some cases, for correct quantification. The maximum influence of ion suppression, due to matrix effects, occurred at the time of the column void volume and had almost recovered before the first analyte had

19 eluted. Due to the use of an acetonitrile gradient, the presence of ion enhancement was observed (61 ). The increase in baseline, as a result of ion enhancement, was 50-80% for the late eluting substances (harmaline, harmine, yohimbine and ibogaine). With the use of matrix standards and deuterated internal standards these effects were compensated for. By comparing the standard samples used as controls over a 9 month period, the standards were indicated to be stable when kept in freezer (-20°C). The deviation from target values was typically <10%. Psilocin, which is known to be light sensitive (62, 63 ), showed a 40% decrease in signal after 2 days, if exposed to light.

Table 3 . Reproducibility of the LC-MS/MS multi-component method for 10 plant-derived psychoactive substances in urine.

Substance Retention Target Observed Bias Repeatability Total Concentration time concentration concentration (%) CV CV range for (mean; min) (µg/L) (mean; µg/L) (%) (%) peak area ratio

Psilocin-D4 (IS) 1 5.39

Psilocin 5.43 1000 1017 1.7 4.8 4.9 500 508 1.6 2.2 6.1 5-1000 µg/L 100 102 2.0 1.9 4.1 r2 = 0.9990 50 53 6.0 5.5 8.3

Ephedrine 5.61 1000 1078 7.8 5.9 6.1 500 564 12.8 4.8 10.7 5-5000 µg/L 100 104 4.0 3.9 8.4 r2 = 0.9994 50 56 12.0 9.5 11.7

LSA 5.86 1000 1068 6.8 7.6 7.7 500 518 3.6 5.6 6.5 10-5000 µg/L 100 106 6.0 8.7 10.0 r2 = 0.9998 50 61 22.0 6.8 13.5

Scopolamine 6.15 1000 1010 1.0 10.2 13.8 500 510 2.0 6.7 8.3 10-5000 µg/L 100 102 2.0 9.0 11.9 r2 = 0.9998 50 52 4.0 14.6 25.4

DMT-D4 (IS) 1 6.37

DMT 6.44 1000 1051 5.1 7.5 7.8 500 527 5.4 5.1 6.2 5-5000 µg/L 100 102 2.0 4.6 6.3 r2 = 0.9994 50 55 10.0 8.5 13.9

Atropine 6.80 1000 1022 2.2 5.5 6.0 500 538 7.6 5.2 6.9 5-5000 µg/L 100 103 3.0 6.6 10.1 r2 = 0.9998 50 51 2.0 7.2 26.4

Harmane-D2 (IS) 1 6.87

Harmaline 7.38 1000 977 -2.3 2.7 9.9 5-5000 µg/L 500 498 -0.4 4.4 7.5 r2 = 1.0000 100 102 2.0 4.0 7.3 50 51 2.0 6.0 13.7

Harmine 7.42 1000 991 -0.9 3 6.1 500 503 0.6 5.2 5.2 5-5000 µg/L 100 101 1.0 6.5 9.3 r2 = 0.9994 50 49 -2.0 7 15.3

Yohimbine 7.67 1000 1098 9.8 6.9 10.8 500 564 12.8 5.1 7.6 5-5000 µg/L 1 The respective internal standards used for quantification of the plant-derived substances.

Control sample solution Sample positive for DMT, harmaline and harmine 150000 10 7 60000 3 50000 A B 8 11 100000 40000 7 9 6 30000 6 10 13 50000 1 2 12 (cps) Intensity 9 11 20000 1 Intensity (cps) 4 5 10000 0 0 4 5 6 7 8 9 10 4 5 6 7 8 9 10 Time (min) Time (min)

Sample positive for ephedrine Sample positive for LSA

6 9 30000 2000000 9 2000 25000 3 Intensity(cps) peak 1,6,9 Intensity(cps) peak 1,6,9 6 CD25000 1500000 1500 20000 20000 1 15000 1000000 15000 1000 4 10000 10000 1 500000 500 Intensity (cps) peak 3 peak (cps) Intensity 5000 4 peak (cps) Intensity 5000

0 0 0 0 4 5 6 7 8 9 10 4 5 6 7 8 9 10 Time (min) Time (min)

Sample positive for atropine an scopolamine Sample E after hydrolysis

30000 30000 6 6 9 25000 25000 8 F 9 E 20000 20000 5 8 1 15000 15000 1

Intensity (cps) 10000 (cps) Intensity 10000

5000 5 5000

0 0 4 5 6 7 8 9 10 4 5 6 7 8 9 10 Time (min) Time (min)

Sample positive for psilocin Sample G after hydrolysis

25000 9 250000 2 6 20000 GH200000 2 15000 150000 1 10000 100000 6 9 Intensity (cps) Intensity (cps) 5000 50000 1

0 0 4 5 6 7 8 9 10 4 5 6 7 8 9 10 Time (min) Time (min) Figure 11 . Ion chromatograms obtained with the LC-MS/MS multi-component method for measurement of 1) Psilocin-D4 (deuterated internal standard, IS), 2) psilocin, 3) ephedrine, 4) LSA, 5) scopolamine, 6) DMT-D4 (IS), 7) DMT, 8) atropine, 9) harmane-D2 (IS), 10) harmaline, 11) harmine, 12) yohimbine, and 13) ibogaine. A) A spiked urine sample used as control containing 500 µg/L of each substance. B) Clinical urine sample from a case of intoxication with Syrian rue (Peganum harmala), being positive for DMT, harmaline and harmine. C) Clinical urine sample positive for ephedrine. D) Urine sample from intoxication with Hawaiian baby woodrose seeds (Argyreia nervosa), being positive for LSA (a double peak for LSA was also obtained with the standard in A). E) Urine sample from intoxication with Angel’s trumpet (Brugmansia arborea), being positive for scopolamine and atropine. F) The same urine sample as in E after enzymatic hydrolysis with ß-glucuronidase. G) Urine sample from intoxication with Psilocybe cubensis mushroom, being positive for psilocin. H) The same urine sample as in G after enzymatic hydrolysis with ß-glucuronidase.

Clinical application The LC-MS/MS method was able to detect intake of 8 of the 10 substances investigated, in clinical urine samples. The 8 substances were DMT, harmine and harmaline (Fig. 11B), ephedrine (Fig. 11C), LSA (Fig. 11D), atropine and scopolamine (Fig. 11E), and psilocin (Fig. 11G). The result of enzymatic hydrolysis, with ß-glucuronidase, of samples containing scopolamine and psilocin is shown in fig. 11E-F (scopolamine) and 11G-H (psilocin). Although psilocin and scopolamine, in most cases, were detected without enzymatic hydrolysis, there was a marked increase of detected substance after enzymatic hydrolysis. This was expected according to previous findings (34, 35, 48, 56 ). Approximately one third of investigated samples were positive for illicit drugs in the immunochemical screening. Their presence did not show any analytical interference.

PAPER III Bioanalytical and clinical data A total of 103 urine samples were collected during 2005-2008. Samples were collected from all over Sweden. The patients were aged 13-52 years (mean 22, median 19) with ~80% being aged 13-27 years and ~60% being 20 years or younger. The majority of the patients (63%) were males. Of the 103 samples, 19 (~18%) were positive for illicit drugs using immunological screening. Often it was the patients themselves that contacted the emergency wards, after experiencing unwanted or unanticipated symptoms. This is not surprising, considering how these substances are marketed (64 ). In 41 urine samples, one or more of the 11 plant-derived substances investigated were detected (Table 4) and only 2 of the 11 substances were not found: ibogaine and yohimbine. Psilocin was the most common substance found (54%) in the bioanalytically confirmed cases. In most of the bioanalytically confirmed cases (93%), the results and clinical record were in agreement. This figure is high, compared to the frequency of self-reports seen in illicit drug intoxications (65, 66 ). Table 4 shows 1) the number of suspected and confirmed cases for substances investigated, 2) suspected substances not possible to analyze for and 3) in which cases other illicit drugs could be identified. Concentration ranges, of the various substances, found in the bioanalytically confirmed cases are seen in table 5.

Table 4 . Intake of psychoactive plant materials and illicit drugs according to clinical suspicion and the bioanalytically confirmed plant-derived substances in urine samples. Plant-derived Suspected substances Samples positive Bioanalytically confirmed substances covered in according to clinical data for illicit drugs 1 positive samples this study N (% of all samples) (N) N (% of confirmed cases) Asarone 8 (7.8%) 1 5 (12.2%) Atropine 1 (1.0%) 1 2 (4.9%) Atropine + scopolamine 5 (4.9%) 2 4 (9.8%) Atropine + scopolamine 1 (1.0 %) 1 1 (2.4 %) + ephedrine Ephedrine 2 (1.9%) 1 4 (9.8%) 2 Harmaline + harmine + 1 (1.0%) 1 1 (2.4%) DMT LSA 8 (7.8%) 3 2 (4.9%) Psilocin 27 (26.2%) 3 22 (53.7%) Total 53 (51.4%) 10 41 (100%)

Other substances 4 Myristicin (nutmeg) 6 (5.8%) Ibotenic acid 2 (1.9%) (red fly agaric) Mitragynine (kratom) 1 (1.0%) 1 Kavain (kava) 1 (1.0%) 1 Thevetia peruviana 1 (1.0%) (yellow oleander) Tribulus terrestris 1 (1.0%) 1 Hoodia gordonii 1 (1.0%) “Spice” 2 (1.9 %) 3 “Psychedelic snuff” 1 (1.0%) Benzodiazepines 2 (1.9%) 1 Synthetic drugs 14 (13.6%) 2 “Unknown”2 18 (17.5%) 3 Total 103 (100%) 19

1 The illicit drugs detected were amphetamines, opiates, buprenorphine, cannabinoids, and propoxyphene. Many

samples were also positive for benzodiazepines but this had often been administered at the emergency department

and was therefore not included.

2 In 2 intoxications with an “unknown” or unspecified plant drug, ephedrine was detected in the urine samples.

3 One clinical case of LSA poisoning also involved intake of “Spice”.

4 These substances were not detectable by the analytical methods used in this study.

Table 5 . Concentration ranges of investigated substances found in the cases reviewed in this study. Substance Observed levels in urine (µg/L)

α- and β-Asarone 1-70 Atropine 29-1000 DMT 3000 Ephedrine 100-90000 Harmaline 2300 Harmine 1200 LSA 31-49 Psilocin Without enzymatic hydrolysis: 0-5600 With enzymatic hydrolysis: 101-15000 Scopolamine Without enzymatic hydrolysis: 102-403 With enzymatic hydrolysis: 1130-4950

In the bioanalytically confirmed cases, the symptoms reported, their duration, and the PSS grading was investigated (Table 6). Mydriasis, tachycardia, visual hallucinations, nausea and vomiting were the most reported symptoms. Hallucination was the most common symptom resulting in a PSS of 2. All intoxications involving plant-derived materials resolved after rest and symptomatic treatment. Although all cases investigated came from emergency departments, no PSS over 2 was noted, that reflects only minor or moderate signs. Symptoms observed for intoxications of α-, β-asarone, atropine, scopolamine, ephedrine and psilocin were in accordance with previously reported symptoms (18, 36, 67, 68 ). For the LSA intoxications, the symptoms reported in this study were partly in agreement with earlier findings (33, 69, 70 ). The main symptom lacking was hallucinations, which were not seen in any of the confirmed cases in this study. The patient positive for DMT, harmaline and harmine admitted intake of P. harmala seeds. The symptoms observed have previously been described in P. harmala intoxications (28, 71 ). However, the presence of DMT can not be attributed to P. harmala. A possible explanation is that the patient was aware of the illegality of DMT and made the choice not to tell the physician about any intake of this.

Table 6. The three most common symptoms reported in 41 cases with bioanalytically confirmed intoxications by plant-derived substances, the range of symptom duration, and grading of the poisoning according to the Poisoning Severity Score. 1 PSS 1 = Minor poisoning (“mild, transient, and spontaneously resolving symptoms or signs”); PSS 2 = Moderate poisoning (“pronounced or prolonged symptoms or signs”) (50 ).

Substance Most common Number Age range Symptom Poisoning symptoms reported of cases (mean, median) duration Severity Score of patients (in order of frequency) (N) (years) (h) (PSS) 1 Asarone Nausea, prolonged 5 15-26 7-15 2 vomiting, tachycardia (19.6, 19) Atropine Mydriasis, tachycardia, 2 15-48 >13 2 hallucinations Atropine + Mydriasis, tachycardia, 4 17-18 7 2 scopolamine hallucinations (17.2, 17) Atropine + Mydriasis, tachycardia, 1 15 - 1 scopolamine + hallucinations ephedrine Ephedrine Dizziness, tachycardia, 4 20-31 7 2 nausea (26.7, 28) Harmaline + Hallucinations, 1 17 7 2 harmine + DMT vomiting, mydriasis LSA Vomiting, mydriasis, 2 16-17 >10 2 leukocytosis Psilocin Mydriasis, 22 17-40 2-10 1 or 2 hallucinations, agitation (22.5, 19)

Drug acquisition, preparations and dose In 50 cases information about how the drug was acquired was available. In 56% of these, the Internet was reported as the source for drug acquisition. Additional means of acquisition reported were: through friends, bought on the street, grown at home and collected in nature. Reported drug preparations are shown in Table 7. Mushrooms of Psilocybe spp . were most often used in the psilocin intoxications. Doses were reported in the range of 1-5g of dried mushrooms, which is consistent with previous reports (72, 73 ). Acorus calamus was used in form of the essential oil produced from the root. Doses of “a few drops” to 2.5 mL were given. Four of 8 patients, suspected of LSA intoxication, reported ingestion synthetic LSA, but this could not be confirmed analytically. To further investigate this, analysis was carried out on 3 preparations supposed to contain synthetic LSA (bought from an Internet shop). LSA could not be confirmed in any of these preparations.

Table 7. Plant species and preparations reported for the various substances . Substance Substance origin Preparations used α- and β-Asarone A. calamus Essential oil Atropine and scopolamine Datura spp. Brugmansia spp . Seeds, leaves and flowers Ephedrine - Tablets taken for weight loss Harmaline and harmine P. harmala Seeds LSA A. nervosa, I. violacea, synthetic Seeds and blotter Psilocin P. cubensis, P. semilanceata, P. Fresh or dried mushrooms mexicana, Copelandia cyanescens

Samples not investigated bioanalytically Of the 103 samples collected, 50 turned out to be related to either plant-derived substances not covered by the analytical methods or possible designer drugs. (Table 4).

PAPER IV Clinical results In all 7 cases investigated, the drug preparation reportedly ingested was calamus oil in either crude or capsule form. The Internet and “through friends” were claimed as means of acquisition. Doses stated were “a few drops” up to 2.5 mL for the oil and 850 mg for capsules. Prolonged vomiting was the most common symptom. This resulted in a PSS of 2 (out of 4) in most cases. The symptoms reported herein are in accordance with previous observations, including the lack of hallucinations (67 ). The first reference mentioning hallucinogenic effects of A. calamus is the “The Hallucinogens” (23 ), where the effects are attributed to α- and β-asarone. In more recent literature, the idea of α- and β-asarone converting into TMA-2 is suggested to be the reason for the hallucinogenic properties (5, 67, 74 ). However, no references could be found, besides “The Hallucinogens”, that mention specific cases where hallucinations actually have occurred. The fact that β-asarone have proven mutagenic, and hepatocarcinogenic, effects raise concerns about using A. calamus as an inebriant (75, 76 ).

Analysis of asarones, TMA-2 and TMC α- and β-asarone were identified by GC-MS analysis, in SIM mode, in 5 of 7 urine samples based on full scan data (Fig 12B).

No evidence, supporting the theory, of TMA-2 being formed could be found in the clinical samples. Studies done in rat hepatocytes and hypercholesterolemic rats have shown that trans- TMC is the major metabolite produced from α-asarone (77, 78 ). In this study, only trans-TMC was identified in the broth solution resulting from incubation of C. elegans with a calamus oil preparation (Fig. 13). In the clinical urine samples only a peak thought to correspond to cis-TMC was seen. This peak had similar relative abundances of product ions as trans-TMC. cis-TMC was indicated to be present in 6 of the 7 cases (Fig. 13). If this indeed is cis-TMC, it seems to be the most suitable analyte for investigating A. calamus intoxications. Since both α- and β-asarone were found in the urine samples, in similar amounts, the presence of trans-TMC would be expected, if it indeed is major metabolite in man as well. C. elegans was used as a model system because it has previously proven suitable for producing metabolites (49 ). However, C. elegans have been shown to possess a stereoselective metabolism differing from that found in mammalian enzyme systems (79 ). This could be a reason for the differences seen in analysis of TMC, in this study.

3500000 A α-asarone 3000000 2000000 208 1800000 1600000 1400000 2500000 1200000 1000000 193 800000

β-asarone Abundance 600000 165 2000000 400000 200000 0 1400000 208 120 130 140 150 160 174 185 197 210 225 245 1500000 1200000 m/z 1000000 800000 Abundance 193 1000000 600000 Abundance 400000 165 200000 0 500000 120 130 140 150 159 174 185 205 220 235 254 m/z 0 0 5 10 15 Time (min)

800000 B α-asarone 700000 400000 208 600000 300000 200000 193 β-asarone 100000 165

500000 Abundance 0 208 120 130 140 150 159 169 180 193 211 225 400000 200000 m/z 150000 300000 100000 165 193 Abundance 50000 200000 Abundance 0 120 131 141 152 163 174 183 197 211 227 241 259 100000 m/z 0 0 5 10 15 Time (min)

Figure 12 . GC-MS chromatograms in SIM ( m/z 208.1) and full spectra ( inset ) for ( A) standards of α- and β-asarone and ( B) a clinical urine sample.

8000 TMC standard cis -TMC? C. elegans 7000 Clinical sample

6000 trans -TMC

5000

4000

3000 Intensity (cps) Intensity

2000

1000

0 0 2 4 6 8 10 12 14 Time (min)

Figure 13 . Ion chromatograms of a TMC-standard, a broth solution originating from incubation of Acorus calamus oil with the fungus Cunninghamella elegans , and a representative clinical urine sample, as analyzed by LC-MS/MS. The ion transition monitored was m/z 237.2 →133.1.

Search for hydroxylated and demethylated asarone metabolites A possible hydroxylated metabolite was seen using GC-MS analysis of the fungus broth solution originating from incubation of calamus oil with C. elegans . It had a retention time of 8.43 min and contained the calculated molecular ion of hydroxylated asarone ( m/z 224.1). The mass spectrum showed presence of the expected tropylium ion at m/z 181.1 (Fig. 14B). After acetylation, a decrease in abundance of this peak was observed. In the analysis of the clinical urine samples, 4 of 7 cases showed the same peak at retention time 8.43 min and m/z 224.1 (Fig. 14A). Upon comparison of unacetylated and acetylated samples, this peak was thought to represent hydroxylated β-asarone. The presence of mono-demethylated metabolites could not be verified in the broth solution or any of the clinical samples by GC-MS analysis.

1400000 A 181.1 181.1 1200000 CH3 O 1000000

800000 O OH H3C 600000 O

Abundance CH3 151.1 151.1 400000 224.1

200000

0 120 130 140 151 160 170 179 189 199 208 218 231 242 260 m/z

5000000 181.1 B 4500000 4000000 3500000 3000000 2500000 151.1 2000000 Abundance 1500000 224.1 1000000 500000 0 120 130 140 151 161 171 182 193 202 213 224 236 250 m/z

Figure 14 . Spectrum for a possible hydroxylated asarone metabolite with the proposed fragmentation pattern indicated. Results are for the peak with a retention time of 8.43 min for ( A) an unacetylated clinical urine sample and ( B) an unacetylated broth solution originating from incubation of Acorus calamus oil with the fungus Cunninghamella elegans .

GENERAL DISCUSSION

LC-MS is becoming the standard technique for drugs of abuse analysis in forensic toxicology (38 ). Especially the potential of multi-component screening methods is of interest in the routine laboratory, being less time and resource consuming (38 ). The methods developed for this study were both a single target method and a multi- component method (80, 81 ). Both methods only needed an initial dilution prior to analysis, a concept previously described by Nordgren et al, that further saves both time and resources (47, 82 ). In Paper II a comparison between the developed methods and methods found in the literature, covering the related substances was done. It was found that the measuring range of the LC-MS/MS method covered the relative levels reported for the substances in urine

The Internet is a source of vast information on psychedelic drugs (83, 84 ) and it has been shown in other countries that such information has influenced drug use in adolescents (85 ). One study has also investigated how the Internet is used to spread information between users via instant messaging (86). Misinformation from the Internet has also led to at least one documented case of poisoning (87 ). Investigations of psychoactive plant use have often been limited to case reports (17, 19, 30, 69, 71, 88-90 ). Only for some, detailed investigations have been conducted (18, 91 ). The problem with legality of purchase and use of plant-derived psychoactive substances has been described by Richardson et al (64 ). From some countries there have been reports on an increased use in plant-derived psychoactive substances (92 ), and several countries, e.g. Sweden, Belgium, Germany and France, have past laws restricting use and sale of some of these plant materials. The fact that these plants often are marketed as “safe” and “legal highs” constitutes a problem (64 ). As seen in this study, most of the users ending up in the emergency departments due to intoxication with plant-derived psychoactive substances are young people. For many of them, the reason for contacting the emergency departments was likely that the symptoms that manifested where more overwhelming than they had anticipated. The results from this study suggest a low occurrence of psychoactive plant use in Sweden, not high enough to warrant calling it a big problem. However, the cases reviewed in this thesis are only a fraction of all the cases ending up at the emergency

30 departments and can not be said to reflect the total number of intoxications that occurred during the time frame of the thesis. Considering how these plants and substances are marketed, the ease with which they can be obtained and the many insincere vendors found on the Internet, it is warranted to call for surveillance of new tendencies and to act accordingly. The possibility that use of “legal highs” might trigger an interest in illicit drug use also needs to be considered. For some reason there seem to be a tendency, in western society, to regard something that is natural to also be safe and harmless. Although many of the psychoactive plants have been used for centuries, this is not a reason to regard them as harmless. In traditional settings they have always (with some exceptions) been handled with care and high degree of respect for the effects, e.g. often there would be a shaman guiding his people through the experience (2). Comparing this with how they sometimes are marketed and used today, I think that many of these societies would be appalled by the way their sacraments are treated.

CONCLUSIONS

• LC-MS/MS is a technique well suitable for detection of plant-derived psychoactive substances in clinical urine samples, following intoxications.

• The developed methods did not need a sample clean-up step prior to analysis and allowed for a rapid detection of the substances investigated. This makes them useful in a routine laboratory setting.

• When investigating A. calamus intoxications cis-TMC seems to be the most suitable analyte.

• Reports of the psychoactive properties of A. calamus are put in question and no evidence can be found supporting the idea of TMA-2 being an active component in intoxications.

• The number of intoxications with psychoactive plants, presented at emergency departments, in Sweden is low. However, the way in which such plants and substances are marketed and the ease with which they can be obtained warrants continuous investigations of such intoxications.

• The Internet was reported to be the most common means of acquisition of the different drug preparations.

• Physiological symptoms caused by the substances investigated in this thesis can be considered to be minor or moderate in terms of severity.

ACKNOWLEDGEMENTS

Many people have, in different ways, contributed to this thesis. From the bottom of my heart I thank all of you, who with your expertise or support have helped me over the years. I would especially like to thank:

Anders Helander , my main supervisor, for all your encouragement and always having time for questions and discussions. And for teaching me that a manuscript can always be polished one more time.

Olof Beck , my co-supervisor, for your expertise in mass spectrometry, support and showing me that Fantomen quotes are timeless “Då Fantomen provar en nyckel passar den.”

Jan Bruhn , my mentor, for your knowledge about these plants and substances and interesting discussions with a new take on problems.

My co-author, Peter Hultén at the Poisons Information Centre, for interesting cooperation and valuable discussions.

Jonas Bergström , for being a perfect role model for a fresh PhD student and showing me there is no such thing as to little time.

Helen Dahl , for all your help around the lab, the years of fun, discussions and always being full of good advice.

Naama Kenan , for friendship, discussions and always being annoyed at something, it made my situation feel so much better.

Yufang Zheng , for all the laughs and for not getting annoyed with me when my stress level was at the top.

Nikolai Stephanson , for all the help and for fruitful discussions whenever I got stuck.

All the staff at the Department of Clinical Pharmacology, for always helping me whenever I needed it.

Andreas Almlén , Marie Hegerstrand-Björkman, Bim Linderholm and Guido Stichtenoth at the Surfactant laboratory for the hours of cross-word puzzling taking my mind off work for a while.

The Center for Dependency Disorders in Stockholm , for granting me financial support to do this work.

Fredrik Lundberg , for making the life, of a skånepåg, in Stockholm so much easier. Thanks for always looking out for me.

Tobias Rudholm Feldreich , for all the fun we’ve had, being a great friend and for helping me mould this fine exterior.

Peter Börjesson , Rasmus Demse , Markus Jönsson , Martin Jönsson , Erik Rosell and Måns Rundqvist , my friends from the far south, thanks for always being able to take me back to the roots.

Gustav Björnstad , my brother, for being interested, inquisitive and always able to make me laugh.

Karl och Gunilla Björnstad , my parents, for you immense love and support.

Charlotte Björnstad , my fantastic wife, for always being there and believing in me. None of this would be possible without you. Ninakupenda sana!

This study was undertaken with financial support of the Swedish National Drug Policy Coordinator and the Swedish National Institute of Public Health.

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