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

© Kristian Björnstad, 2009

ISBN 978-91-7409-420-6

“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

1

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 16th 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).

2

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 19th 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).

3

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.

4

OMe

OMe

MeO

OMe

OMe

MeO

α-asarone β-asarone

NH

N

Dimethyltryptamine (DMT)

O O HO

O

C

C

N

Scopolamine

O HO

O

C

C

N

Atropine

NH

OH

Ephedrine

NH

NH

H

H3CO

Ibogaine

O NH

N

Harmine

O NH

N

Harmaline

H3CO

N

NH2

O

Lysergic acid amide (LSA)

NH2

OMe

OMe

MeO

Mescaline

NH

N

OH

Psilocin

NHN

HH

H

OHO

O

Yohimbine

OMe

OMe

MeO

OMe

OMe

MeO

OMe

OMe

MeO

OMe

OMe

MeO

α-asarone β-asarone

NH

N

NH

N

Dimethyltryptamine (DMT)

O O HO

O

C

C

NO O HO

O

C

C

N

Scopolamine

O HO

O

C

C

N O HO

O

C

C

N

Atropine

NH

OHNH

OH

Ephedrine

NH

NH

H

H3CO

NH

NH

H

H3CO

Ibogaine

O NH

NO N

H

N

Harmine

O NH

NO N

H

N

Harmaline

H3CO

N

NH2

O

H3CO

N

NH2

O

Lysergic acid amide (LSA)

NH2

OMe

OMe

MeO

NH2

OMe

OMe

MeO

Mescaline

NH

N

OH

NH

N

OH

Psilocin

NHN

HH

H

OHO

O

NHN

HH

H

OHO

O

Yohimbine

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).

5

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

6

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).

7

Root of A. calamus - α-and β-asarone

Oil of A. calamus - α- and β-asarone

Dried P. semilanceata –psilocybin and psilocin

Seeds of A. nervosa -LSA

L. williamsii -mescaline

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

Root of A. calamus - α-and β-asarone

Oil of A. calamus - α- and β-asarone

Dried P. semilanceata –psilocybin and psilocin

Seeds of A. nervosa -LSA

L. williamsii -mescaline

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

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.

8

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.

Vacuum

LCIon

sourceMass

analyzer Detector

LC MS

Vacuum

LCIon

sourceMass

analyzer Detector

Vacuum

LCIon

sourceMass

analyzer Detector

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

9

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).

Figure 4. A schematic of the electrospray ionization (ESI) interface.

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

LC flow

Metal capillary

Droplets containing mobile phase and ions

N2 gas flow

MS-inlet

LC flow

Metal capillary

Droplets containing mobile phase and ions

N2 gas flow

MS-inlet

Heat

Heat

MS-inlet

Corona discharge needle

Auxillary gas – N2

Nebulizer gas – N2

LC flow e e

Quartz tube

Heat

Heat

MS-inlet

Corona discharge needle

Auxillary gas – N2

Nebulizer gas – N2

LC flow e e

Quartz tube

10

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.

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).

Figure 7. Schematic of triple quadrupole mass spectrometer.

Ions

To detector

+

+

--

Ions

To detector

+

+

--

Ions

To detector

+

+

--

Ion

sourceDetector

Quadrupole 1

Mass filter

Quadrupole 2

Fragmentation zone

Quadrupole 3

Mass filter

Ion

sourceDetector

Quadrupole 1

Mass filter

Quadrupole 2

Fragmentation zone

Quadrupole 3

Mass filter

11

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

12

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

13

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

14

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. C18 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 C18 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.

15

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)):

16

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).

17

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.

0

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nsit

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(cp

s)

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1500

2000

2500

3000

3500

4000

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nsit

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)

Mescaline 250 µg/L

Mescaline-D9Mescaline

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2500

3000

3500

4000

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nsit

yfo

r m

esca

line

(cps

)

Mescaline 250 µg/L

Mescaline-D9MescalineMescaline-D9Mescaline

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 concentration

(µg/L)

Observed mean concentration

(µg/L) Bias (%)

Repeatability (CV%)

Total CV (CV%)

Number of observations

(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

18

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.

0

20000

40000

60000

80000

100000

120000

140000

0 1 2 3 4 5 6 7 8 9

Time (min)

Inte

nsi

ty(c

ps)

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20000

40000

60000

80000

100000

120000

140000

0 1 2 3 4 5 6 7 8 9

Time (min)

Inte

nsi

ty(c

ps)

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,

19

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.

Clinical sample 9300 µg/L

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160000

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Mescaline-D9Mescaline

Clinical sample 9300 µg/L

0

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100000

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140000

160000

0 1 2 3 4 5 6 7 8 9

Time (min)

Inte

nsit

y(c

ps)

Mescaline-D9MescalineMescaline-D9Mescaline

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

20

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

time (mean; min)

Target concentration

(µg/L)

Observed concentration (mean; µg/L)

Bias (%)

Repeatability CV (%)

Total CV (%)

Concentration range for

peak area ratio Psilocin-D4 (IS)1

5.39

Psilocin 5.43 1000 500 100 50

1017 508 102 53

1.7 1.6 2.0 6.0

4.8 2.2 1.9 5.5

4.9 6.1 4.1 8.3

5-1000 µg/L r2 = 0.9990

Ephedrine 5.61 1000 500 100 50

1078 564 104 56

7.8 12.8 4.0 12.0

5.9 4.8 3.9 9.5

6.1 10.7 8.4 11.7

5-5000 µg/L r2 = 0.9994

LSA 5.86 1000 500 100 50

1068 518 106 61

6.8 3.6 6.0 22.0

7.6 5.6 8.7 6.8

7.7 6.5 10.0 13.5

10-5000 µg/L r2 = 0.9998

Scopolamine 6.15 1000 500 100 50

1010 510 102 52

1.0 2.0 2.0 4.0

10.2 6.7 9.0

14.6

13.8 8.3 11.9 25.4

10-5000 µg/L r2 = 0.9998

DMT-D4 (IS)1

6.37

DMT 6.44 1000 500 100 50

1051 527 102 55

5.1 5.4 2.0 10.0

7.5 5.1 4.6 8.5

7.8 6.2 6.3 13.9

5-5000 µg/L r2 = 0.9994

Atropine 6.80 1000 500 100 50

1022 538 103 51

2.2 7.6 3.0 2.0

5.5 5.2 6.6 7.2

6.0 6.9 10.1 26.4

5-5000 µg/L r2 = 0.9998

Harmane-D2 (IS)1

6.87

Harmaline 7.38 1000 500 100 50

977 498 102 51

-2.3 -0.4 2.0 2.0

2.7 4.4 4.0 6.0

9.9 7.5 7.3 13.7

5-5000 µg/L r2 = 1.0000

Harmine 7.42 1000 500 100 50

991 503 101 49

-0.9 0.6 1.0 -2.0

3 5.2 6.5 7

6.1 5.2 9.3 15.3

5-5000 µg/L r2 = 0.9994

Yohimbine 7.67 1000 500

1098 564

9.8 12.8

6.9 5.1

10.8 7.6

5-5000 µg/L

1 The respective internal standards used for quantification of the plant-derived substances.

21

0

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Intensity (cps) peak 1,6,9

1

3

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2000

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Inte

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Intensity (cps) peak 1,6,9

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6 9

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45

6

7

8

9

10

11

12 13

Control sample solution Sample positive for DMT, harmaline and harmine

Sample positive for ephedrine Sample positive for LSA

Sample positive for atropine an scopolamine Sample E after hydrolysis

Sample positive for psilocin Sample G after hydrolysis

A B

C D

E F

G H

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12 13

Control sample solution Sample positive for DMT, harmaline and harmine

Sample positive for ephedrine Sample positive for LSA

Sample positive for atropine an scopolamine Sample E after hydrolysis

Sample positive for psilocin Sample G after hydrolysis

A B

C D

E F

G H

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.

22

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.

23

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

substances covered in

this study

Suspected substances

according to clinical data

N (% of all samples)

Samples positive

for illicit drugs1

(N)

Bioanalytically confirmed

positive samples

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

+ ephedrine

1 (1.0 %) 1 1 (2.4 %)

Ephedrine 2 (1.9%) 1 4 (9.8%)2

Harmaline + harmine +

DMT

1 (1.0%) 1 1 (2.4%)

LSA 8 (7.8%)3 2 (4.9%)

Psilocin 27 (26.2%) 3 22 (53.7%)

Total 53 (51.4%) 10 41 (100%)

Other substances4

Myristicin (nutmeg) 6 (5.8%)

Ibotenic acid

(red fly agaric)

2 (1.9%)

Mitragynine (kratom) 1 (1.0%) 1

Kavain (kava) 1 (1.0%) 1

Thevetia peruviana

(yellow oleander)

1 (1.0%)

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.

24

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.

25

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

symptoms reported

(in order of frequency)

Number

of cases

(N)

Age range

(mean, median)

of patients

(years)

Symptom

duration

(h)

Poisoning

Severity Score

(PSS)1

Asarone Nausea, prolonged

vomiting, tachycardia

5 15-26

(19.6, 19)

7-15 2

Atropine Mydriasis, tachycardia,

hallucinations

2 15-48 >13 2

Atropine +

scopolamine

Mydriasis, tachycardia,

hallucinations

4 17-18

(17.2, 17)

7 2

Atropine +

scopolamine +

ephedrine

Mydriasis, tachycardia,

hallucinations

1 15 - 1

Ephedrine Dizziness, tachycardia,

nausea

4 20-31

(26.7, 28)

7 2

Harmaline +

harmine + DMT

Hallucinations,

vomiting, mydriasis

1 17 7 2

LSA Vomiting, mydriasis,

leukocytosis

2 16-17 >10 2

Psilocin Mydriasis,

hallucinations, agitation

22 17-40

(22.5, 19)

2-10 1 or 2

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.

26

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.

mexicana, Copelandia cyanescens

Fresh or dried mushrooms

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).

27

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.

28

0

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120 130 140 150 159 174 185 205 220 235 254

m/z

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0200000400000600000800000

100000012000001400000160000018000002000000

120 130 140 150 160 174 185 197 210 225 245

m/z

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ce

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193165

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193165β-asarone

α-asarone

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100000012000001400000160000018000002000000

120 130 140 150 160 174 185 197 210 225 245

m/z

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208

β-asarone

α-asarone

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.

0

1000

2000

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Clinical sample

cis-TMC?

trans-TMC

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.

29

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.

A

224.1

181.1

151.1

OO

O

CH3

CH3

H3COH

181.

115

1.1O

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H3COH

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m/z

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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.

30

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

31

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.

32

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.

33

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.

34

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.

35

REFERENCES

1. Oxford University Press. Oxford English dictionary. 2000 [cited; Available

from: http://dictionary.oed.com/entrance.dtl. 2. Rätsch, C., The Encyclopedia of Psychoactive Plants. 2005, Rochester: Park

Street Press. 3. Beck, O., et al., Presence of phenylethylamine in hallucinogenic Psilocybe

mushroom: possible role in adverse reactions. J Anal Toxicol, 1998. 22(1): p. 45-9.

4. Halpern, J.H. and H.G. Pope, Jr., Hallucinogens on the Internet: a vast new

source of underground drug information. Am J Psychiatry, 2001. 158(3): p. 481-3.

5. Ott, J., Pharmacotheon - Entheogenic drugs, their plant sources and history. 1st

ed. 1993, Kennewick, WA: Natural Products Co. 6. Stafford, P.G. and J. Bigwood, Psychedelics encyclopedia. 3rd expanded ed ed.

1992, Berkeley, CA: Ronin Pub. 91, 420 p. 7. Evans Schultes, R. and A. Hofmann, Plants of the gods : origins of

hallucinogenic use. 1979, New York: McGraw-Hill. 192 s. 8. Schultes, R.E., Hallucinogens of plant origin. Science, 1969. 163(864): p. 245-

54. 9. Nichols, D.E., Hallucinogens. Pharmacol Ther, 2004. 101(2): p. 131-81. 10. Bruhn, J.G., et al., Mescaline use for 5700 years. Lancet, 2002. 359(9320): p.

1866. 11. Grob, C.S., et al., Human psychopharmacology of hoasca, a plant hallucinogen

used in ritual context in Brazil. J Nerv Ment Dis, 1996. 184(2): p. 86-94. 12. Gable, R.S., Risk assessment of ritual use of oral dimethyltryptamine (DMT)

and harmala alkaloids. Addiction, 2007. 102(1): p. 24-34. 13. Gable, R.S., Comparison of acute lethal toxicity of commonly abused

psychoactive substances. Addiction, 2004. 99(6): p. 686-96. 14. Halpern, J.H. and H.G. Pope, Jr., Hallucinogen persisting perception disorder:

what do we know after 50 years? Drug Alcohol Depend, 2003. 69(2): p. 109-19. 15. Johnson, M., W. Richards, and R. Griffiths, Human hallucinogen research:

guidelines for safety. J Psychopharmacol, 2008. 22(6): p. 603-20. 16. Gopel, C., C. Laufer, and A. Marcus, Three cases of angel's trumpet tea-induced

psychosis in adolescent substance abusers. Nord J Psychiatry, 2002. 56(1): p. 49-52.

17. Balikova, M., Collective poisoning with hallucinogenous herbal tea. Forensic

Sci Int, 2002. 128(1-2): p. 50-2. 18. Forrester, M.B., Jimsonweed (Datura stramonium) exposures in Texas, 1998-

2004. J Toxicol Environ Health A, 2006. 69(19): p. 1757-62.

36

19. Marneros, A., P. Gutmann, and F. Uhlmann, Self-amputation of penis and tongue after use of Angel's Trumpet. Eur Arch Psychiatry Clin Neurosci, 2006. 256(7): p. 458-9.

20. Diker, D., et al., Coma as a presenting sign of Datura stramonium seed tea

poisoning. Eur J Intern Med, 2007. 18(4): p. 336-8. 21. World Health Organization. Neuroscience of psychoactive substance use and

dependence. 2004 [cited; xx, 264 p.]. Available from: https://www04.sub.su.se/login?url=http://site.ebrary.com/lib/sthlmub/Doc?id=10062376 Fulltext.

22. Vohora, S.B., S.A. Shah, and P.C. Dandiya, Central nervous system studies on

an ethanol extract of Acorus calamus rhizomes. J Ethnopharmacol, 1990. 28(1): p. 53-62.

23. Hoffer, A. and H. Osmond, The hallucinogens. 1967, New York: Academic P.

626 s. 24. Samuelsson, G. and Apotekarsocieteten, Drugs of natural origin : a textbook of

pharmacognosy. 5., rev. ed. 2004, Stockholm: Apotekarsocieteten. 620 s. 25. Riba, J., et al., Human pharmacology of ayahuasca: subjective and

cardiovascular effects, monoamine metabolite excretion, and pharmacokinetics. J Pharmacol Exp Ther, 2003. 306(1): p. 73-83.

26. Callaway, J.C., G.S. Brito, and E.S. Neves, Phytochemical analyses of

Banisteriopsis caapi and Psychotria viridis. J Psychoactive Drugs, 2005. 37(2): p. 145-50.

27. Magkos, F. and S.A. Kavouras, Caffeine and ephedrine: physiological,

metabolic and performance-enhancing effects. Sports Med, 2004. 34(13): p. 871-89.

28. Frison, G., et al., A case of beta-carboline alkaloid intoxication following

ingestion of Peganum harmala seed extract. Forensic Sci Int, 2008. 179(2-3): p. e37-43.

29. Alper, K.R., Ibogaine: a review. Alkaloids Chem Biol, 2001. 56: p. 1-38. 30. Cheze, M., et al., Determination of ibogaine and noribogaine in biological fluids

and hair by LC-MS/MS after Tabernanthe iboga abuse Iboga alkaloids distribution in a drowning death case. Forensic Sci Int, 2008. 176(1): p. 58-66.

31. Alper, K.R., H.S. Lotsof, and C.D. Kaplan, The ibogaine medical subculture. J

Ethnopharmacol, 2008. 115(1): p. 9-24. 32. Mash, D.C., et al., Ibogaine in the treatment of heroin withdrawal. Alkaloids

Chem Biol, 2001. 56: p. 155-71. 33. Al-Assmar, S.E., The seeds of the Hawaiian baby woodrose are a powerful

hallucinogen. Arch Intern Med, 1999. 159(17): p. 2090. 34. Kamata, T., et al., Optimized glucuronide hydrolysis for the detection of

psilocin in human urine samples. J Chromatogr B Analyt Technol Biomed Life Sci, 2003. 796(2): p. 421-7.

35. Grieshaber, A.F., K.A. Moore, and B. Levine, The detection of psilocin in

human urine. J Forensic Sci, 2001. 46(3): p. 627-30.

37

36. Pittler, M.H., K. Schmidt, and E. Ernst, Adverse events of herbal food supplements for body weight reduction: systematic review. Obes Rev, 2005. 6(2): p. 93-111.

37. McNair, H.M. and J.M. Miller, Basic gas chromatography. Techniques in

analytical chemistry series. 1998, New York: Wiley. xii, 200 p. 38. Maurer, H.H., Current role of liquid chromatography-mass spectrometry in

clinical and forensic toxicology. Anal Bioanal Chem, 2007. 388(7): p. 1315-25. 39. Maurer, H.H., Position of chromatographic techniques in screening for

detection of drugs or poisons in clinical and forensic toxicology and/or doping control. Clin Chem Lab Med, 2004. 42(11): p. 1310-24.

40. Cole, R.B., Electrospray ionization mass spectrometry : fundamentals,

instrumentation, and applications. 1997, New York: Wiley. xix, 577 s. 41. Harris, D.C., Quantitative chemical analysis. 6. ed. 2002, New York: W.H.

Freeman. 744, [219] s., [12] pl.-s. i färg. 42. Johnstone, R.A.W. and M.E. Rose, Mass spectrometry for chemists and

biochemists. 2. ed. 1996, Cambridge: Cambridge University Press. xxii, 501 s. 43. Whitehouse, C.M., et al., Electrospray interface for liquid chromatographs and

mass spectrometers. Anal Chem, 1985. 57(3): p. 675-9. 44. Maurer, H.H., Hyphenated mass spectrometric techniques-indispensable tools

in clinical and forensic toxicology and in doping control. J Mass Spectrom, 2006. 41(11): p. 1399-413.

45. Nguyen, S. and J.B. Fenn, Gas-phase ions of solute species from charged

droplets of solutions. Proc Natl Acad Sci U S A, 2007. 104(4): p. 1111-7. 46. Hoffmann, E.d. and V. Stroobant, Mass spectrometry : principles and

applications. 2nd ed. 2001, Chichester ; New York: Wiley. xii, 407 p. 47. Nordgren, H.K. and O. Beck, Multicomponent screening for drugs of abuse:

direct analysis of urine by LC-MS-MS. Ther Drug Monit, 2004. 26(1): p. 90-7. 48. Renner, U.D., R. Oertel, and W. Kirch, Pharmacokinetics and

pharmacodynamics in clinical use of scopolamine. Ther Drug Monit, 2005. 27(5): p. 655-65.

49. Tevell, A., et al. Identification of drug metabolites produced by the fungus

Cunninghamella elegans using liquid chromatography/tandem mass spectrometry. in 16th International Conference of Racing Analysts and Veterinarians. 2006. Tokyo, Japan.

50. Persson, H.E., et al., Poisoning severity score. Grading of acute poisoning. J

Toxicol Clin Toxicol, 1998. 36(3): p. 205-13. 51. Charalampous, K.D., K.E. Walker, and J. Kinross-Wright, Metabolic fate of

mescaline in man. Psychopharmacologia, 1966. 9(1): p. 48-63. 52. Becker, H., [Constituents of the cactus Lophophora williamsii]. Pharm Unserer

Zeit, 1985. 14(5): p. 129-37. 53. Nolte, K.B. and R.E. Zumwalt, Fatal peyote ingestion associated with Mallory-

Weiss lacerations. West J Med, 1999. 170(6): p. 328.

38

54. Reynolds, P.C. and E.J. Jindrich, A mescaline associated fatality. J Anal Toxicol, 1985. 9(4): p. 183-4.

55. Kikura-Hanajiri, R., et al., Simultaneous determination of nineteen

hallucinogenic tryptamines/beta-calbolines and phenethylamines using gas chromatography-mass spectrometry and liquid chromatography-electrospray ionisation-mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci, 2005. 825(1): p. 29-37.

56. Sticht, G. and H. Kaferstein, Detection of psilocin in body fluids. Forensic Sci

Int, 2000. 113(1-3): p. 403-7. 57. Nakamura, Y., et al., [Retrospective study of two cases of acute datura

poisoning]. Chudoku Kenkyu, 2008. 21(2): p. 177-81. 58. Strano-Rossi, S., C. Colamonici, and F. Botre, Parallel analysis of stimulants in

saliva and urine by gas chromatography/mass spectrometry: perspectives for "in competition" anti-doping analysis. Anal Chim Acta, 2008. 606(2): p. 217-22.

59. del Mar Ramirez Fernandez, M., et al., Liquid chromatography-tandem mass

spectrometry method for the simultaneous analysis of multiple hallucinogens, chlorpheniramine, ketamine, ritalinic acid, and metabolites, in urine. J Anal Toxicol, 2007. 31(8): p. 497-504.

60. Konishi, T., K. Akaki, and K. Hatano, Determination of hyoscyamine and

scopolamine in serum and urine of humans by liquid chromatography with tandem mass spectrometry. Shokuhin Eiseigaku Zasshi, 2008. 49(4): p. 266-71.

61. Cech, N.B. and C.G. Enke, Practical implications of some recent studies in

electrospray ionization fundamentals. Mass Spectrom Rev, 2001. 20(6): p. 362-87.

62. Hasler, F., et al., Determination of psilocin and 4-hydroxyindole-3-acetic acid

in plasma by HPLC-ECD and pharmacokinetic profiles of oral and intravenous psilocybin in man. Pharm Acta Helv, 1997. 72(3): p. 175-84.

63. Tiscione, N.B. and M.I. Miller, Psilocin identified in a DUID investigation. J

Anal Toxicol, 2006. 30(5): p. 342-5. 64. Richardson, W.H., 3rd, C.M. Slone, and J.E. Michels, Herbal drugs of abuse: an

emerging problem. Emerg Med Clin North Am, 2007. 25(2): p. 435-57; abstract ix.

65. Heyerdahl, F., et al., Clinical assessment compared to laboratory screening in

acutely poisoned patients. Hum Exp Toxicol, 2008. 27(1): p. 73-9. 66. Lee, M.O., P.M. Vivier, and D.B. Diercks, Is the Self-report of Recent Cocaine

or Methamphetamine Use Reliable in Illicit Stimulant Drug Users Who Present to the Emergency Department with Chest Pain? J Emerg Med, 2008.

67. Vargas, C.P., et al., Getting to the root (Acorus calamus) of the problem. J

Toxicol Clin Toxicol, 1998. 36(3): p. 259-60. 68. Peden, N.R., et al., Clinical toxicology of "magic mushroom" ingestion.

Postgrad Med J, 1981. 57(671): p. 543-5. 69. Borsutzky, M., et al., [Hawaiian baby woodrose: (Psycho-) Pharmacological

effects of the seeds of Argyreia nervosa. A case-orientated demonstration]. Nervenarzt, 2002. 73(9): p. 892-6.

39

70. Cohen, S., Suicide Following Morning Glory Seed Ingestion. Am J Psychiatry, 1964. 120: p. 1024-5.

71. Mahmoudian, M., H. Jalipour, and P. Salehian, Toxicity of Peganum harmala :

review and a case report. Iran J Pharmcol Ther, 2002. 1(1): p. 1-4. 72. Halpern, J.H., Hallucinogens and dissociative agents naturally growing in the

United States. Pharmacol Ther, 2004. 102(2): p. 131-8. 73. Salzman, E. and B.H. Rumack, Mushroom poisoning : diagnosis and treatment.

1978, Cleveland: CRC Press. 263 p. 74. Dennehy, C.E., C. Tsourounis, and A.E. Miller, Evaluation of herbal dietary

supplements marketed on the internet for recreational use. Ann Pharmacother, 2005. 39(10): p. 1634-9.

75. Sivaswamy, S.N., et al., Mutagenic activity of south Indian food items. Indian J

Exp Biol, 1991. 29(8): p. 730-7. 76. Wiseman, R.W., et al., Structure-activity studies of the hepatocarcinogenicities

of alkenylbenzene derivatives related to estragole and safrole on administration to preweanling male C57BL/6J x C3H/HeJ F1 mice. Cancer Res, 1987. 47(9): p. 2275-83.

77. Hasheminejad, G. and J. Caldwell, Genotoxicity of the alkenylbenzenes alpha-

and beta-asarone, myristicin and elimicin as determined by the UDS assay in cultured rat hepatocytes. Food Chem Toxicol, 1994. 32(3): p. 223-31.

78. Antunez-Solis, J., et al., 2,4,5-trimethoxycinnamic acid: the major metabolite of

alpha-asarone, retains most of the pharmacological properties of alpha-asarone. J Enzyme Inhib Med Chem, 2008: p. 1.

79. McMillan, D.C., P.P. Fu, and C.E. Cerniglia, Stereoselective fungal metabolism

of 7,12-dimethylbenz[a]anthracene: identification and enantiomeric resolution of a K-region dihydrodiol. Appl Environ Microbiol, 1987. 53(10): p. 2560-6.

80. Björnstad, K., A. Helander, and O. Beck, Development and clinical application

of an LC-MS-MS method for mescaline in urine. J Anal Toxicol, 2008. 32(3): p. 227-31.

81. Bjornstad, K., O. Beck, and A. Helander, 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): p. 1162-8.

82. Nordgren, H.K., et al., Application of direct urine LC-MS-MS analysis for

screening of novel substances in drug abusers. J Anal Toxicol, 2005. 29(4): p. 234-9.

83. Gordon, S.M., R.F. Forman, and C. Siatkowski, Knowledge and use of the

Internet as a source of controlled substances. Journal of Substance Abuse Treatment, 2006. 30: p. 271-274.

84. Montagne, M., Drugs on the internet. I: introduction and web sites on

psychedelic drugs. Subst Use Misuse, 2008. 43(1): p. 17-25. 85. Boyer, E.W., M. Shannon, and P.L. Hibberd, The Internet and psychoactive

substance use among innovative drug users. Pediatrics, 2005. 115(2): p. 302-5. 86. Boyer, E.W., et al., Dissemination of psychoactive substance information by

innovative drug users. Cyberpsychol Behav, 2007. 10(1): p. 1-6.

40

87. Brush, D.E., S.B. Bird, and E.W. Boyer, Monoamine oxidase inhibitor poisoning resulting from Internet misinformation on illicit substances. J Toxicol Clin Toxicol, 2004. 42(2): p. 191-5.

88. Kontrimaviciute, V., et al., Distribution of ibogaine and noribogaine in a man

following a poisoning involving root bark of the Tabernanthe iboga shrub. J Anal Toxicol, 2006. 30(7): p. 434-40.

89. Borowiak, K.S., K. Ciechanowski, and P. Waloszczyk, Psilocybin mushroom

(Psilocybe semilanceata) intoxication with myocardial infarction. J Toxicol Clin Toxicol, 1998. 36(1-2): p. 47-9.

90. Satora, L., H. Goszcz, and K. Ciszowski, Poisonings resulting from the

ingestion of magic mushrooms in Krakow. Przegl Lek, 2005. 62(6): p. 394-6. 91. Forrester, M.B., Nutmeg intoxication in Texas, 1998-2004. Hum Exp Toxicol,

2005. 24(11): p. 563-6. 92. Kresanek, J., et al., Drug abuse in Slovak Republic. Przegl Lek, 2005. 62(6): p.

357-60.