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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
5000
10000
15000
20000
25000
30000
35000
0 1 2 3 4 5 6 7 8 9
Time (min)
Inte
nsit
yfo
r m
esca
line-
D9
(cp
s)
0
500
1000
1500
2000
2500
3000
3500
4000
Inte
nsit
yfo
r m
esca
line
(cps
)
Mescaline 250 µg/L
Mescaline-D9Mescaline
0
5000
10000
15000
20000
25000
30000
35000
0 1 2 3 4 5 6 7 8 9
Time (min)
Inte
nsit
yfo
r m
esca
line-
D9
(cp
s)
0
500
1000
1500
2000
2500
3000
3500
4000
Inte
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)
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)
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
0
20000
40000
60000
80000
100000
120000
140000
160000
0 1 2 3 4 5 6 7 8 9
Time (min)
Inte
nsit
y(c
ps)
Mescaline-D9Mescaline
Clinical sample 9300 µg/L
0
20000
40000
60000
80000
100000
120000
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
500000
1000000
1500000
2000000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s) p
eak
3
0
5000
10000
15000
20000
25000
30000
Intensity (cps) peak 1,6,9
1
3
6
9
0
500
1000
1500
2000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s) p
eak
4
0
5000
10000
15000
20000
25000
Intensity (cps) peak 1,6,9
14
6 9
0
50000
100000
150000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
6
7
9
10
11
0
5000
10000
15000
20000
25000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
2
6
9
0
50000
100000
150000
200000
250000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
2
6 9
0
5000
10000
15000
20000
25000
30000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
5
6
8
9
0
5000
10000
15000
20000
25000
30000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)1
5
6
89
0
10000
20000
30000
40000
50000
60000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1 2
3
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
0
500000
1000000
1500000
2000000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s) p
eak
3
0
5000
10000
15000
20000
25000
30000
Intensity (cps) peak 1,6,9
1
3
6
9
0
500
1000
1500
2000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s) p
eak
4
0
5000
10000
15000
20000
25000
Intensity (cps) peak 1,6,9
14
6 9
0
50000
100000
150000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
6
7
9
10
11
0
5000
10000
15000
20000
25000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
2
6
9
0
50000
100000
150000
200000
250000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
2
6 9
0
5000
10000
15000
20000
25000
30000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1
5
6
8
9
0
5000
10000
15000
20000
25000
30000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)1
5
6
89
0
10000
20000
30000
40000
50000
60000
4 5 6 7 8 9 10
Time (min)
Inte
nsity
(cp
s)
1 2
3
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
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
500000
1000000
1500000
2000000
2500000
3000000
3500000
0 5 10 15
Time (min)
Ab
un
dan
ce
A
0
200000
400000
600000
800000
1000000
1200000
1400000
120 130 140 150 159 174 185 205 220 235 254
m/z
Ab
un
dan
ce
0200000400000600000800000
100000012000001400000160000018000002000000
120 130 140 150 160 174 185 197 210 225 245
m/z
Ab
un
dan
ce
208
193165
208
193165β-asarone
α-asarone
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
0 5 10 15
Time (min)
Ab
un
dan
ce
A
0
200000
400000
600000
800000
1000000
1200000
1400000
120 130 140 150 159 174 185 205 220 235 254
m/z
Ab
un
dan
ce
0200000400000600000800000
100000012000001400000160000018000002000000
120 130 140 150 160 174 185 197 210 225 245
m/z
Ab
un
dan
ce
208
193165
208
193165β-asarone
α-asarone
0
100000
200000
300000
400000
500000
600000
700000
800000
0 5 10 15
Time (min)
Ab
un
dan
ce
B
0
50000
100000
150000
200000
120 131 141 152 163 174 183 197 211 227 241 259
m/z
Ab
un
dan
ce
0
100000
200000300000
400000
120 130 140 150 159 169 180 193 211 225
m/z
Ab
un
dan
ce
165
165 193
193
208
208
β-asarone
α-asarone
0
100000
200000
300000
400000
500000
600000
700000
800000
0 5 10 15
Time (min)
Ab
un
dan
ce
B
0
50000
100000
150000
200000
120 131 141 152 163 174 183 197 211 227 241 259
m/z
Ab
un
dan
ce
0
100000
200000300000
400000
120 130 140 150 159 169 180 193 211 225
m/z
Ab
un
dan
ce
165
165 193
193
208
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
3000
4000
5000
6000
7000
8000
0 2 4 6 8 10 12 14
Time (min)
Inte
nsi
ty (
cps)
TMC standard
C. elegans
Clinical sample
cis-TMC?
trans-TMC
0
1000
2000
3000
4000
5000
6000
7000
8000
0 2 4 6 8 10 12 14
Time (min)
Inte
nsi
ty (
cps)
TMC standard
C. elegans
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
O
O
CH3
CH3
H3COH
181.
115
1.1
0
200000
400000
600000
800000
1000000
1200000
1400000
120 130 140 151 160 170 179 189 199 208 218 231 242 260
m/z
Ab
un
dan
ce
A
224.1
181.1
151.1
OO
O
CH3
CH3
H3COH
181.
115
1.1O
O
O
CH3
CH3
H3COH
181.
115
1.1
0
200000
400000
600000
800000
1000000
1200000
1400000
120 130 140 151 160 170 179 189 199 208 218 231 242 260
m/z
Ab
un
dan
ce
B
151.1
181.1
224.1
0
500000
1000000
1500000
2000000
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3500000
4000000
4500000
5000000
120 130 140 151 161 171 182 193 202 213 224 236 250
m/z
Ab
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151.1
181.1
224.1
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
120 130 140 151 161 171 182 193 202 213 224 236 250
m/z
Ab
un
dan
ce
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
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