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Analytical techniques for the determination of
tryptamines and b-carbolines in plant matrices
and in psychoactive beverages consumed
during religious ceremonies and neo-shamanic
urban practices
Alain Gaujac,
a,e,f
Sandro Navickiene,
c
Mark I. Collins,
d
Simon D. Brandt
e
and
Jailson Bittencourt de Andrade
a,b
*
The consumption of ayahuasca, a hallucinogenic beverage used by indigenous communities in the Amazon, is increasing
worldwide due to the expansion of syncretic religions founded in the north of Brazil in the first half of the twentieth century,
such as Santo Daime and União do Vegetal. Another example is the jurema wine, a drink that originated from indigenous
cultures of the northeast of Brazil. It is currently used for several religious practices throughout Brazil involving urban
neo-shamanic rituals and syncretic Brazilian religions, such as Catimbó and Umbanda. Both plant products contain
N,N-dimethyltryptamine which requires co-administration of naturally occurring monoamine oxidase inhibitors, for example
b-carboline derivatives, in order to induce its psychoactive effects in humans. This review explores the cultural use of
tryptamines and b-carbolines and focuses on the analytical techniques that have been recently applied to the determination
of these compounds in ayahuasca, its analogues, and the plants used during the preparation of these beverages. Copyright ©
2012 John Wiley & Sons, Ltd.
Keywords: ayahuasca; jurema wine; plants; tryptamines; b-carbolines; detection; hallucinogens
Introduction
Since the emergence of civilizations, the consumption of psycho-
active plants has been used to induce altered states of cons-
ciousness. In pre-Columbian societies, the use of these plants
was normally associated with mystical-religious rituals and
preparation for war. Colonization of the Americas resulted in
European explorers coming into contact with a variety of
psychoactive plants, including tobacco (Nicotiana spp.), maracujá
or passion fruit (Passiflora spp.), guaraná (Paulinia cupana) and
yopo (Anadenanthera peregrina).
[1–4]
Four centuries after the
global spread of tobacco, consumption of the plant-derived
beverage ayahuasca, which originated in indigenous Amazon
cultures, is attracting devotees throughout the world as a result
of the creation of syncretic religious groups in Brazil during the
twentieth century.
[5]
Two of these religions, Santo Daime and
União do Vegetal (UDV), are represented in various countries
around the world including Australia, the United States, and
Europe. In some countries, a number of legal disputes have
been described concerning the legalization of ayahuasca and
consumption during religious rituals.
[6–9]
In addition, ‘ayahuasca
tourism’is becoming increasingly common in those equatorial
South American countries that share areas of the Amazon
rainforest.
[6,10]
Moreover, the Internet also offers a great variety
of opportunities to purchase psychoactive plant materials.
[11–13]
Among the many compounds found in some of these
plants, the tryptamine and b-carboline derivatives (Figure 1)
represent simple indole alkaloids that are commonly present
in the biota.
Ayahuasca is most commonly produced as a decoction using
leaves of chacrona (Psychotria viridis) and sections of the stem
of the yage vine (Banisteriopsis caapi). Important key components
of the vine are b-carboline derivatives that act as inhibitors of
monoamine oxidase (MAO). The leaves of P. viridis contain the
psychoactive/hallucinogenic N,N-dimethyltryptamine (DMT) and
* Correspondence to: Jailson Bittencourt de Andrade, Universidade Federal
da Bahia (UFBA). Rua Barão de Jeremoabo, s/n. Ondina. CEP 40170–115.
Salvador-Ba, Brazil. E-mail: jailsong@ufba.br
aUniversidade Federal da Bahia (UFBA), Ondina, Salvador-Ba, Brazil
bInstituto Nacional de Ciência e Tecnologia, Centro Interdisciplinar de Energia e
Ambiente, Campus Universitário de Ondina, Salvador-Ba, Brazil
cUniversidade Federal de Sergipe (UFS), Campus São Cristóvão, São Cristóvão-
Se, Brazil
dUniversidade Estadual do Ceará (UECE), Campus do Itaperi, Fortaleza-
Ce, Brazil
eLiverpool John Moores University, School of Pharmacy and Biomolecular
Sciences, Liverpool UK
fInstituto Federal de Educação, Ciência e Tecnologia de Sergipe (IFS), Campus
São Cristóvão, São Cristóvão-Se, Brazil
Drug Test. Analysis (2012) Copyright © 2012 John Wiley & Sons, Ltd.
Review
Drug Testin
g
and Anal
y
sis
Received: 18 November 2011 Revised: 23 February 2012 Accepted: 23 February 2012 Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/dta.1343
the combination with reversible MAO inhibitors (MAOIs) renders
the DMT orally active.
[14–17]
In an analogous fashion, the jurema wine, originally consumed
only by pre-colonial indigenous tribes in the northeast of Brazil, has
become a part of the liturgy of the Catimbó and Afro-Brazilian
religious groups since colonization. The wine is predominantly
produced using the root bark of the jurema tree (Mimosa spp.), which
also contains DMT.
[18–21]
In large urban centres, it is common to
obtain the bark of M. tenuiflora (black jurema) from online
sources
[11,13]
and to use the seeds of Peganum harmala as a source
of MAOIs. P. harmala is a Mediterranean shrub that contains a
number of b-carbolines also present in B. caapi.
[22]
Studies involving the chemical characterization of these plants,
together with the development of analytical techniques for the
measurement of tryptamines and b-carbolines in plant matrices,
as well as in ritual beverages, are essential given the current
expansion in their use for religious, recreational, and clinical
research purposes. The need for an in-depth approach towards
analytical characterization becomes obvious in cases of untoward
effects or even fatal intoxications which can, for example, arise
from ill-informed combinations of plant products with other
psychoactive substances.
[23–26]
At the same time, consideration
needs to be given to the promising therapeutic potential that was
reported for constituents present in these plant materials.
[27–33]
In
addition, a wide variation of concentration levels of ayahuasca
components that differ not only from church to church, but also
between different batches of the same church, were also reported.
[34]
Occasionally, an extremely concentrated form called ‘ayahuasca
honey’can also be encountered which derives its name from high
viscosity similar to honey syrup. Detailed studies on the identity
and levels of psychoactive substances found in these preparations
and appropriately defined criteria for their determination are
required. This might be of particular interest in cases where there is
the concomitant use of other additives such as Cannabis,
[35,36]
P. harmala,tobacco,andjuremawine,whereprecautionsorquality
control might be lacking. The objective of this review is to present
some cultural and chemical features of DMT-containing plant
products. An account is provided of recent developments in
analytical approaches towards the determination of tryptamines,
b-carbolines and tetrahydro-b-carbolines detected in tissues of
M. tenuiflora, P. viridis, P. aquatica, B. caapi and P. harmala, as well
as in ayahuasca samples.
Psychoactive beverages used for ritual purposes:
ayahuasca and jurema wine
Ayahuasca
Ayahuasca (aya = soul, spirit; huasca = vine), a word belonging
to the Quechua dialect still spoken in some regions of South
America, is a drink that is mostly prepared using a decoction
of two plants: the leaves of the DMT-containing chacrona
(Psychotria viridis) and sections of the stem of the jagube vine
(Banisteriopsis caapi) that provides three major MAOI compo-
nents such as harmine, harmaline and tetrahydroharmine (THH)
(Figure 1). The chemical composition of ayahuasca can differ
between indigenous tribes due to the use of different plant
species
[1,14,15,37]
although the same psychoactive constituents
are present in all preparations.
[38–41]
Ayahuasca is known by
various indigenous names, including yajé,natema and caapi,
and was first described by Villavicencio in 1858. Seven years
earlier, the English explorer Richard Spruce made contact with
the Tukanoan Indians, in Rio Uaupés (Brazilian Amazonia), but
his findings concerning the use of a liana called caapi were
not published before 1908 when the plant was identified as
Banisteria caapi.
[37]
Clinical research on the physiological and psy-
chological effects of ayahuasca in humans re-emerged in the
early 1990s which offered important insights into psychopharma-
cological, biochemical and pharmacokinetic properties of this
hallucinogenic plant mixture. More importantly, these investiga-
tions set the stage for a range of clinical studies that followed
across several disciplines until the present day.
Brazilian legislation, based on a constitutional right to freedom of
religion, permits the consumption of ayahuasca within a religious
context, including children and pregnant women which, in this
case, requires parental consent.
[42,43]
Norms concerning the use of
ayahuasca in Brazil for religious purposes were published by the
N
H
N
R1
R2
N
H
N
O
R
N
H
N
O
R
N
H
NH
O
R
R
Harmol
CH3
R4
R3
Tryptamine
R1,R2R3,R4
,= H
5-Hydroxytryptamine (serotonin)
R1,R2R4R3
,= OH= H;
N-Methyltryptamine (NMT)
R1R2,= H
R3,R43
= H;= CH
N,N-Dimethyltryptamine (DMT)
= H
R3,R43
= CH
R,R
12 ;
5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT)
= H
R3R4
3
= OCH ;
Tryptamine Derivatives
β-Carboline Derivatives
R = H
Harmine
3
R = CH
Harmalol
R = H
Harmaline
3
R = CH
Tetrhydronorharmine
R = H
Tetrahydroharmine
3
R = CH
CH3
3
= CH
R,R
12 ;
Figure 1. General chemical structure of the tryptamine and b-carboline
derivatives.
A. Gaujac et al.
Drug Testin
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wileyonlinelibrary.com/journal/dta Copyright © 2012 John Wiley & Sons, Ltd. Drug Test. Analysis (2012)
Brazilian National Council on Drug Policies (CONAD) in January
2010
[44]
which prohibits the marketing of ayahuasca, its therapeutic
use, ayahuasca tourism, and its use with illicit drugs. Under this
Resolution, consumption is permitted in a religious context and
the same document also emphasizes the need for more multidisci-
plinary areas of research on ayahuasca.
[9,44]
The government of the
State of Acre in Brazil has published a Resolution concerning the
authorization of extraction and transport of Banisteriopsis spp. vines,
as well as the leaves of the Psychotria viridis shrub carried out by
religious organizations in the State of Acre for the purposes of
ayahuasca preparation.
[45]
It should be noted that while the
Brazilian government only legitimized the production and
consumption of ayahuasca derived from the B. caapi vine
[44]
the
Acre Resolution covers every species of the Banisteriopsis genus.
Jurema wine
Species of the Mimosaceae botanical subfamily, locally known
in northeast Brazil as jurema (from the Tupi yurema, meaning
succulent thorn bush), are considered to be amongst the most
potent plant sources of DMT. These medium-sized trees are used
by various indigenous groups, such as the Kariri-Xocó whose
communities are located on the left border of the São Francisco
River, the boundary between the Brazilian States of Sergipe
and Alagoas. The inner barks of stems and roots are used to
prepare a beverage called vinho da jurema (jurema wine), or
ajucá (by the Pancarú Indians) and cotcha-lhâ, by the Fulniô
Indians.
[46]
During the Toré, a ritual dance designed to demon-
strate the power of resistance and express the depth of Brazil’s
northeastern indigenous culture, the Indians drink vinho da
jurema including a number of additives such as tobacco and
Passiflora juice or a tea made from its leaves.
[47]
This beverage
can be produced using several species such as M. tenuiflora
(black jurema), M. ophthalmocentra (red jurema) and M. verrucosa
(white jurema or sweet jurema according to the Kariri-Xocó
Indians) and other plants of the Mimosaceae subfamily.
[19,20,47]
A mixture of plants is essential to potentiate the psychoactive
activity of the DMT, since the Mimosa spp. does not appear to
contain any appreciable quantities of MAO inhibitors. Although
there have not been any studies that reported oral psychoactivity
of jurema wine, it may be relevant to observe that indigenous
groups and members of Brazilian syncretic religions use large
quantities of tobacco.
[19]
It is known that tobacco smoke contains
a number of constituents that possess MAOI activity
[48–50]
which
indicates that orally administered DMT might become psychoactive
under these conditions.
The concomitant use of plants belonging to the Passiflora
species is common in these indigenous communities, while in
the syncretic Brazilian groups, besides the smoked tobacco used
during the rituals, sugarcane alcohol (cachaça) is also widely
used together with other additives during the preparation of
the psychoactive beverage.
[47,51]
A number of studies have
identified the presence of MAOI constituents in Passiflora species,
especially in P. incarnata.
[52–55]
Seeds of Peganum harmala, which
have been shown to be highly effective in inhibiting monoamine
oxidase, have also been described to potentiate the oral psy-
choactivity of jurema wine.
[56,57]
The use of jurema wine has a long history, stretching from its
indigenous origins in the sertão, i.e. the northeastern region of
Brazil, to current days where it is consumed throughout the
country by the members of Catimbó-Jurema and followers from
other religions. This is a typical example of syncretic evolution
of the original indigenous tradition. The jurema use was adopted
by the Afro-Brazilian religions which incorporated the Jurema cult
in their own traditions when fugitive African slaves were
harboured by the northeastern indigenous tribes during their
escape to the quilombos (communities of escaped slaves).
Nowadays, we can see the incorporation of jurema use into neo-
shamanic practices and its popularization via the Internet. In contrast
to ayahuasca, the additives used in the preparation of jurema wine,
by the Indians and members of these religions, remain a closely
guarded secret. In Brazil, there is historical documentation describing
the indictment and imprisonment of indigenous Indians who
consumed the drink.
[46,51]
Jurema rituals were almost extinguished
by the devastating impact of Portuguese Christian colonization;
however, since the end of the twentieth century the movement
has witnessed a substantial resurgence.
[47]
Plants used for Ayahuasca
Psychotria spp
Psychotria spp. belongs to the Rubiaceae family, which also
includes coffee. Some species of the genus Psychotria are
used by Amazonian Indians as additives in the preparation of
ayahuasca, namely, P. viridis, P. carthaginensis,P. psychotriaefolia
and P. poeppigiana. In the Amazon, P. viridis (Figure 2b), is a shrub
that reaches a maximum height of 2–3m
[1]
and which is popu-
larly known as ‘chacrona’,‘chacruna’,or‘rainha’. Native to the
Amazon rainforest, where the plant is becoming increasingly rare,
it has become commercially cultivated due to the demand for its
leaves, which are used to prepare ayahuasca, although this
practice is frowned upon by the Brazilian authorities. In Brazil,
the churches tend to be located in the countryside nearby urban
centres where there is always a possibility of maintaining their
own plantations called reinados das rainhas (kingdoms of the
Queens). Plantations have also been reported in Hawaii and
California.
[58]
The leaves of P. viridis are collected in the early
morning or the late afternoon for the production of ayahuasca.
The first description of the presence of DMT in these leaves was
published in 1970, as was the first report of the presence of
the chemical in a member of the Rubiaceae family.
[59]
The
leaves have been reported to contain between 0.10 and 0.61%
DMT, together with traces of N-methyltryptamine (NMT) and
2-methyl-tetrahydro-b-carboline (MTHC).
[58]
Banisteriopsis spp
Some species of the Banisteriopsis genus, including B. argentea,
B. inebrians, B. caapi and B. muricata, are used to prepare ayahuasca
and other similar psychoactive beverages, since they contain the
MAOI needed to ensure oral psychoactivity of DMT.
[60,61]
None-
theless, B. caapi (Figure 2d), is the plant most commonly used
for this purpose.
[14,37,58]
The entire plant contains b-carboline
and tetrahydro-b-carboline alkaloids (in concentrations varying
from 0.11 to 1.95%), although the stem of the vine is the
part normally used. The main alkaloids present are harmine,
harmaline, and THH. The levels of harmine, which exerts a
reversible MAOI effect, are equivalent to between 40 and 96%
of the total alkaloid content of the plant. On the other hand,
it has also been reported that these constituents were absent
in B. caapi samples.
[58]
Similarly the species P. viridis,B. caapi is
also cultivated in Brazil by some religious groups and plantations
have also been reported in Hawaii.
[41]
Determination of psychoactive plant constituents
Drug Testin
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Drug Test. Analysis (2012) Copyright © 2012 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/dta
Plants used for Jurema wine
Mimosa spp
Several members of the Mimosa genus (Leguminosae family)
are known as jurema by rural communities in Brazil.
[1]
Some
species such as M. tenuiflora,M. Ophthalmocentra,M. verrucosa
and M. scabrella may contain considerable amounts of psychoactive
tryptamines, especially in their barks.
[19,47,58,62,63]
In Brazil, M. tenuiflora (Willd.) Poir. [syn. M. hostilis (Mart.) Benth.]
(Figure 2a), known as ‘jurema-preta’(black jurema), is used as the
main ingredient in jurema wine since the inner bark of the stem
and roots is rich in DMT. Native to low rainfall regions that
experience periodical drought, this plant is abundantly found in
northeast Brazil, in dry valleys in southern Mexico, in the north
of Venezuela and Colombia, as well as in Honduras and El
Salvador. In its native habitat it reaches a height of 2.5–5 m and
readily colonizes degraded terrain, grows rapidly, and is able to
generate new shoots after cutting.
[64]
In Mexico, where it is
known as tepescohuite,
[21]
it appears that there are no reports of
its usage as a psychotropic product, although the dried and
ground bark is used for wound healing and treatment of skin
burns.
[65]
Jurema wine made from the inner bark of M. tenuiflora
in addition to Peganum harmala seeds which provide the same
active principles found in ayahuasca, namely DMT and MAOIs.
[56,57]
The illicit use of M. tenuiflora has become a concern that is currently
being addressed by the Brazilian Federal Police.
[13]
Plants used for ayahuasca and jurema
wine analogues
Phalaris spp
The presence of tryptamines in Phalaris species was first
described in phytochemical studies for agricultural purposes.
P. arundinacea (reed canary grass), P. canariensis and P. aquatica
are found worlwide. P. aquatica (Figure 2c) is a grass native to
the Mediterranean region, and is common in wetlands and that
is considered to be toxic to ruminant livestock. Instances of
animal poisoning involving Phalaris species, sometimes fatal,
have been reported in Australia, South Africa, Argentina, Brazil,
and the USA.
[66–70]
Within its genus, P. aquatica contains the
highest levels of DMT in addition to other tryptamines, such as
5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) and NMT.
[71]
It
has also been increasingly used for the preparation of ayahuasca
analogues.
[56,57]
Peganum harmala L
Syrian rue, or P. harmala (Figure 2e), is a shrub native to the dry
regions of the Mediterranean, North Africa, the Middle East, India,
and Mongolia.
[1,22]
In North Africa, its seeds are used to the
present day as ritual incense. It is an ancient ritual plant, and in
folk medicine it is still used for gynecological purposes and as a
vermifuge. This plant is increasingly used in North America and
Figure 2. (a) Morphology of M. tenuiflora (illustration from J.B. Clark);
[37]
(b) Morphology of P. viridis (illustration from I. Brady);
[37]
(c) Phalaris aquatica,
eighteenth-century illustration;
[58]
(d) Morphology of B. caapi (Illustration from E.W. Smith);
[37]
(e) Peganum harmala, seventeenth-century illustration.
[58]
A. Gaujac et al.
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Europe to produce drinks containing DMT and b-carbolines
that are analogous to ayahuasca.
[22,57]
Three to four grams
of the seeds is considered to be sufficient to inhibit the action
of monoamine oxidase. The alkaloid content of P.harmala seeds
is around 2–6 %, and consists principally of harmine and
harmaline.
[58]
Analytical methods
Earlier work
The first descriptions of methods used for the determination
of tryptamines and b-carbolines in these plant species and
their beverages date from the mid-twentieth century. Most
of them were based on liquid-liquid extraction (LLE) and
purification was usually performed by column chromatography
and crystallization techniques.
[46,72–77]
Since the late 1960s, the
use of high performance liquid chromatography (HPLC) and
gas chromatography (GC) coupled with mass spectrometry (MS)
became more prominent.
[14,59–61]
The Brazilian chemist Oswaldo Gonçalves de Lima was the first
scientist to study the chemical composition of the jurema wine,
as well as its preparation, and also the first to isolate DMT from
the bark of black jurema. This study also provided a detailed
account of the ceremony and preparation of the vinho da jurema
by Indians of the Pancarú tribe (Pernambuco, Brazil). This study
also described the isolation of the alkaloid fraction of the root
bark of M. tenuiflora which led to the identification of ‘Nigerina’,
i.e. ‘nigerine’(0.31% dry weight, DW).
[46]
Years later, this was
confirmed to be DMT following the analysis of M. tenuiflora bark
by Pachter and co-workers. This sample was provided by de Lima
and was found to contain up to 0.57% of DMT in the dried
plant.
[77]
Meckes-Lozoya et al.
[21]
identified serotonin and DMT
in samples of M. tenuiflora root bark using GC-MS and Batista
et al.
[20]
isolated the alkaloid fraction of M. ophthalmocentra and
reported the presence of DMT (1.6%, DW) and NMT (0.0012%
DW), respectively.
Regarding ayahuasca, and the plants employed in its prepara-
tion, descriptions of analytical quantitative methods date back
to the early 1970s. The first description of P. viridis analysis was
provided in 1969
[78]
and Rivier and Lindgren carried out a major
investigation into the analytical chemistry of ayahuasca and
reported the findings in a landmark paper in 1972.
[14]
The authors
reported the results of their work carried out on the upper Rio
Purus region near the border between Peru and Brazil, in which
they reported the use of ayahuasca by the Sharanahua and
Culina Indians. The procedure for chemical analysis of the parts
of the plants used in its preparation has also been described.
Implementation of liquid-liquid extraction (LLE) was followed by
an analysis by GC-MS. The leaf samples of P. viridis showed a
DMT content (DW) of approximately 0.34%. The same substance
was also found at higher concentration levels (0.66% DW) in the
leaves of P. carthaginensis. The presence of NMT and MTHC was
also detected at trace levels. However, one of the leaf samples
was reported to contain 85% NMT and 12% MTHC (total alkaloid
content 0.11%, DW). Dry matter samples of stems, branches,
leaves, and roots of B. caapi revealed the presence of b-carbolines
ranging from 0.05 to 1.90% with the majority being represented
by harmine, followed by THH, harmaline, and harmol, respec-
tively.
[14]
Also in this work, some samples of ayahuasca were an-
alyzed, stating for each 100 mL of the beverage the presence of
6.6–19 mg for harmine, 1.5–9.8 mg for tetrahydroharmine, 0.3–
1.6 mg for harmaline and 5.4–16.0 mg for DMT.
In 1984, and with the use of appropriate standard solutions,
samples of ayahuasca from Peru were analyzed qualitatively
and quantitatively using two-dimensional thin-layer chromatog-
raphy (TLC), HPLC, and GC-MS.
[15]
The majority of alkaloids
obtained from five Peruvian ayahuasca samples were quantified
by HPLC-UV (260 nm) which led to the detection of DMT
(0.6 mg/mL), harmine (4.67 mg/mL), THH (1.60 mg/mL) and
harmaline (0.41 mg/mL), respectively. The same samples were
also freeze dried and subjected to analysis by HPLC. The reported
values were DMT (6.4 mg/g or 0.64%), harmine (23.8 mg/g or
2.38%), THH (11.1 mg/g or 1.11%), and harmaline (5.1 mg/g or
0.51%). Six samples of B. caapi were also evaluated quantitatively
by HPLC leading to harmine (0.57–6.35 mg/g or 0.057–0.635% ),
THH (0.25–3.8 mg/g or 0.025–0.38%), harmaline (0.5–3.8 mg/g
or 0.05–0.38%), harmol (0.01–1.2 mg/g or 0.001–0.12%) and har-
malol (trace–0.35 mg/g or trace –0.035%). Analyses conducted
by GC-MS also confirmed the presence of DMT (1–1.6 mg/g or
0.1–0.16%) in leaves of P. viridis.
[15]
Recent analyses
Sample preparation techniques
Most of the methods described for the determination of trypta-
mines and b-carbolines present in plant matrices and ayahuasca
employ sample preparation techniques that require large quanti-
ties of toxic organic solvents and that are time-consuming. For
herbal samples (Tables 1 and 2), maceration in a suitable solvent,
LLE and use of a continuous-flow Soxhlet extraction, are the most
commonly used procedures.
[22,38,79–83]
An effective alternative
technique, especially useful when combined with GC, is matrix
solid-phase dispersion (MSPD).
[84,85]
This approach was described
first in 1989
[86]
and was recently employed by Gaujac et al. for the
quantitative determination of DMT in the bark of M. tenuiflora.
[87]
This procedure offered the advantage of low solvent consump-
tion while providing excellent indices of selectivity, precision,
and recovery. Following optimization using a multivariate proce-
dure, recoveries were reported in the range of 81.7–116.2%.
[87]
Callaway et al. reported the quantification of b-carbolines and
DMT in P. viridis leaves and B. caapi stems obtained by sonication
of a 100 mg sample for 10 min using a minimal volume of meth-
anol (2 mL). The mixture was allowed to stand for 24 h and then
centrifuged before dilution a small aliquot of the supernatant in
the mobile phase. Validation data for the proposed technique
were not reported.
[38]
The extraction of tryptamines was described by Zhou et al.
who used 0.2 g of dry plant matrix (P. aquatica) and macerated
the sample in 10 mL of 1% HCl, with periodic agitation.
[71]
After
3–4 days the mixture was centrifuged and the supernatant
passed through a solid-phase extraction (SPE) column. Analytes
retained on the column were eluted with 2 mL of an alkaline
alcoholic mixture containing NH
4
OH. No recovery tests were
reported.
[71]
Wang et al. used various parts of B. caapi, including
leaves, stems, large branches, and bark, and employed an
extraction into hot water followed by HPLC analysis although
method validation data were not provided.
[32]
Maceration in
methanol and extraction of b-carbolines by LLE with chloroform
was reported for the analysis of P. harmala seeds
[79,82,83]
and
Pulpati et al. offered a methanol extraction of P. harmala seeds
(1 g) with methanol (3 x 50 mL) under reflux conditions (1 h).
[88]
Determination of psychoactive plant constituents
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Table 1. Methods for the determination of b-carbolines in B. caapi and P. harmala
B. caapi P. harmala
Reference Callaway et al.
[38]
Wang et al.
[32]
Kartal et al.
[79]
Hemmateenejad
et al.
[82]
Monsef-Esfahani et al.
[83]
Pulpati et al.
[88]
Herraiz et al.
[22]
Material analyzed Stem Leaves, stems, large
branches and bark
Seeds Seeds Seeds Seeds Leaves, sections of stem,
flowers, roots, fruits
and seeds
Analytes of interest Harmine, harmaline
and THH
THNH, harmol, THH,
harmaline, harmine and
compounds from other
chemical classes
Harmol, harmalol,
harmine and
harmaline
Harmine, harmane,
harmalol and
harmaline
Harmol, harmalol,
harmine and
harmaline
Harmine and harmaline
and compounds from
other chemical classes
Harmol, harmalol,
harmine, harmaline
and THH
Sample preparation
method
Sonification in
methanol and
resuspension of
residue in mobile
phase
Maceration in hot water Maceration in methanol
and extraction by LLE
with chloroform
Maceration in methanol
and extraction by LLE
with chloroform
Maceration in methanol
and extraction by LLE
with chloroform
Reflux in methanol (1 h) Maceration in an acid
solution of HClO
4
and
methanol (1:1)
Separation / detection
technique
HPLC and detection
by fluorescence
HPLC-DAD HPLC-UV at 330 nm HPLC-UV at 330 nm HPLC-UV at 330 nm HPTLC-UV at 366 nm
(densitometer-TLC
scanner)
HPLC-DAD
Figures of merit –
Linear range:
1.0–500.0 mg/mL
(for THNH and THH)
0.2–100 mg/mL, (for
harmol, harmaline
& harmine)
r² >0.999; LOD <10.25
mg/mL; LOQ <31.0
mg/mL; RSD <4.609%
Linear range:
1.0–10.0 mg/mL;
94 <R**<107%
RSD <5%;
Linear range:
0.5–20 mg/mL
r
2
>0.998 LOD
<0.1 mg/mL
LOQ = 0.5 mg/mL
0.6 <RSD <10.2%
Linear range:
4–24ng/spot (for
harmine) 8–24ng/
spot (for harmaline);
r
2
>0.993; LOD=2 ng;
LOQ = 4 ng;
97.7 <R<98.4%
Instrumental
precision:
RSD <1.53%;
Repeatability:
RSD <1.62%;
–
Concentration levels* Harmine: 0.31 –
8.43 mg/g
(0.031 –0.843%);
Harmaline: 0.03 –
0.83 mg/g
(0.003 –0.083%);
Harmine: 10
-3
–0.672%;
Harmaline: 10
-4
–
0.058%; THH: 0.004–
0.34%; THNH: 0 –
0.014%; Harmol:
4.10
-4
–0.019%
Harmol: 1.094%;
Harmine: 0.476%;
Harmaline: 0.611%;
Harmine: 1.84%;
Harmane: 0.18%;
Harmaline: 3.90%;
Harmalol: 0.25%
(in dried seeds)
Harmine: 0.465
g/100 g (0.465%);
Harmaline: 0.355
g/100 g (0.355%)
Harmine: 0,44% (w/w);
Harmaline: 0.096%
(w/w);
(in seeds) Harmalol:
6 mg/g (0.6%);
Harmol: 0.03 mg/g
(0.003%); Harmaline:
56 mg/g (5.6%);
Harmine: 43 mg/g
(4.3%); THH: 1.1 mg/g
(0.11%)
THH: 0.05 –2.94 mg/g
(0.005 –0.294%)
* Concentration levels based on dry weight (DW) of vegetable matter.
** Recovery (R).
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Table 2. Methods for the determination of tryptamines in M. tenuiflora,P. viridis and P. aquatica
M. tenuiflora P. viridis P. aquatica
Reference Nicasio et al.
[81]
Vepsäläinen et al.
[80]
Gaujac et al.
[87]
Callaway et al.
[38]
Zhou et al.
[71]
Material analyzed Inner bark, seeds, leaves and
flowers. Cultures in vitro
(plantules and callus)
Root inner bark Seeds and flowers. Inner barks
of stems and roots.
Leaves Entire plant
Analytes of interest DMT, tryptamine, serotonin DMT and yuremamine DMT DMT DMT, 5-MeO- DMT, gramine,
hordenine, bufotenine and
tryptamine
Sample preparation method Soxhlet reflux extraction with
chloroform and NH
3
solution
(27%) (49:1)
Extracts obtained by
maceration in methanol
and resuspension in mobile
phase
Matrix solid-phase dispersion
(MSPD)
Maceration in methanol
(67%) + acetonitrile
(11%) + 0.1 mol/L
ammonium acetate (22%)
Maceration in HCl (1%),
centrifugation and solid-
phase extraction (SPE)
Separation / detection
technique
HPLC-UV at 280 nm HPLC-DAD NMR (
13
C- and
1
H NMR)
GC-MS HPLC and fluorescence
detection
HPTLC and HPLC- MS
Figures of merit Linear range: 2 –40 mg/mL –
Linear range: 0.62 –20 mg/g;
r
2
= 0.9962; LOD = 0.12 mg/g;
LOQ = 1.25 mg/g; 81.7<R**
<116.2%; Precision inter-day:
RSD <16.8%; Precision intra-day:
RSD <7.4%
–
Linear range: 120 –3840 ng/spot;
r
2
>0.991; Precision
intra-day: RSD <5%
Concentration levels*
(in barks) DMT: 0.11 –0.35%;
Tryptamine: 0.0022 –0.0071%;
(in flowers) DMT: 0.03%;
Tryptamine: 0.0075%; (in leaves)
DMT: 0.01 –0.09%; Serotonin:
0.009%; (in cultures) For all
analytes: <0.08%
–
(in inner barks)
DMT: 1.26 –9.35 mg/g (0.126 –
0.935%) (in seeds and flowers) DMT:
Below the LOQ
DMT: 0 –17.75 mg/g
(0 –1.775%)
DMT: 66.3 –177 mg/kg
(0.00663 –0.0177%);
5-MeO-DMT: 176 mg/kg
(0.0176%)
* Concentration levels based on dry weight (DW) of vegetable matter.
** Recovery (R).
Determination of psychoactive plant constituents
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Herraiz et al., on the other hand, described the maceration of
0.2–0.5 g of P. harmala components (leaves, sections of stem,
flowers, roots, fruits, or seeds) in 20 mL of a 1:1 mixture containing
0.6 mol/L HClO
4
and methanol (1:1). Following centrifugation HPLC
analysis was employed after dilution of the supernatant.
[22]
The procedures required to prepare ayahuasca samples
(Table 3) for GC analysis can be more time-consuming than those
required for HPLC analyses, largely due to incompatibility of GC
capillary columns with water. However, a successful implementa-
tion of LLE has been reported using n-butyl chloride as the
organic solvent.
[89]
A variation of the theme was offered by
Gambelunghe et al. who reported a GC-MS analysis of an
ayahuasca sample seized in Italy.
[40]
In this particular case,
sodium hydroxide and an internal standard (diphenylhydramine)
were added to 5 mL ayahuasca followed by extraction into ethyl
ether and centrifugation. Method validation data were not
reported.
[40]
On the other hand, a C
18
cartridge has also been
employed for the determination of ayahuasca alkaloids by GC
nitrogen phosphorus detection (NPD) which showed that SPE
procedures can be equally applied. Minimal sample manipulation
and small amounts of organic solvents were required and
recoveries exceeded 68% for measurements in triplicate at
concentrations of 0.3, 1.5, and 3.0 mg/mL.
[39]
McIlhenny et al. pre-
pared ayahuasca samples from parts of specimens of P. viridis and
B. caapi collected from cultivations in the district of South Kona,
Hawaii (established using clones originating from Peru).
[41]
The
B. caapi vines were macerated and boiled slowly, together with
P. viridis leaves, for 10 h in 11 litres of double-distilled water. Ali-
quots (100 mL) of each ayahuasca preparation were diluted and
analyzed by HPLC tandem mass spectrometry (MS/MS). The
samples of ayahuasca derived from two extracts prepared
simultaneously, in which the biomass of B. caapi was maintained
constant and the quantity of P. viridis was varied (either 150 or
300 leaves).
[41]
Moura et al. prepared extracts of P. viridis for
the quantification of DMT by LLE with hexane. An average recov-
ery of 70% was obtained in experiments using three different
concentration levels.
[90]
Separation and quantification methods
Kartal and colleagues carried out a full validation exercise for
the determination of harmol, harmalol, harmine and harmaline
in P. harmala seeds using HPLC-UV. Several chromatographic
parameters were also measured, including capacity factor and
resolution. Harmol, harmine and harmaline were determined in
samples at concentrations of 1.0, 0.4 and 0.6%, respectively.
[79]
The method described by Gaujac et al. included a full evaluation
of figures of merit throughout all stages of the process of GC-MS
analysis of the M. tenuiflora bark. The levels of DMT varied from
1.26 to 9.35 mg/g in samples of stem and root bark that had been
collected in regions characterized by different pluviometric
regimes.
[87]
Vepsäläinen et al., using HPLC-UV and nuclear
magnetic resonance (NMR) spectroscopy, discovered the pres-
ence of a new phytoindole in the bark of M. tenuiflora and it
was also observed that heat or pH fluctuations impact on stability
of this molecule. The phytoindole was termed yuremamine and
further studies would be required in order to determine any
potential MAOI activity.
[80]
Nicasio et al. used reversed phase
HPLC, with UV detection at 280 nm, to measure tryptophan,
tryptamine, serotonin and DMT in the bark, flowers, and
leaves of M. tenuiflora, as well as in the callus and plantules
using micropropagation techniques. The authors conducted
two measurements in different times of the year to assess the
variation of concentration levels of these analytes between
winter and summer seasons.
[81]
An HPLC method with a non-polar column and fluorescence
detection was reported by Callaway et al.,
[38]
who employed
a method that was based on their earlier work,
[89]
to measure
b-carbolines and DMT in parts of B. caapi and in the leaves of
P. viridis, respectively. In dry B. caapi material the concentrations
obtained were 0.31–8.43 mg/g (harmine), 0.03–0.83 mg/g
(harmaline) and 0.05–2.94 mg/g (THH), respectively. In dry leaves
of P. viridis the maximum concentration of DMT measured was
17.75 mg/g. Diurnal fluctuations were also reported where higher
concentrations were detected during daytime (with peaks at
06:00 am and 06:00 pm). Since DMT levels tended to reduce
at dusk it was suggested that DMT might be produced in
the leaves to aid absorption of solar radiation.
[38]
A proof-of-
principle study using capillary electrophoresis laser-induced
fluorescence electrospray ionization mass spectrometry (CE-LIF-
ESI-MS) method was presented by Huhn et al.
[91]
The com-
bination of both detection systems was particularly helpful as it
allowed for the ability to obtain favorable peak shapes (50 Hz
sampling rate) and structural information based on ESI-MS/MS
detection. In case of co-elution or incomplete resolution of peaks,
quantitative determinations would be possible only with the use
of extracted ion electropherograms. Both detection methods
could conveniently detect a set of six b-carboline standards
around 770 amol levels. A diluted ayahuasca sample revealed
the presence of DMT, harmaline, harmine and THH (no quantita-
tion) and an ethanolic extract obtained from P. viridis leaves
(ultrasonication at 45 C) showed DMT and an unidentified
species with a protonated molecule at m/z 189 and product ions
at m/z 165, 147, 119, 104, and 87, respectively.
[91]
Hemmateenejad et al. applied multivariate statistical proce-
dures to optimize an HPLC procedure (UV detection at 330 nm)
for the determination of harmine, harmane, harmalol and
harmaline in P. harmala seeds
[82]
and the chromatographic
conditions, including column and mobile phase, were similar to
those described earlier by Kartal et al.
[79]
In validation tests the
method gave a precision value of 4.6%, excellent linearity
(r
2
>0.999) and limits of detection and quantification in the
ranges of 3.1–10.3 mg/mL and 9.3–31.0 mg/mL, respectively. In
seeds collected from plants in Iran, concentrations of harmine,
harmane, harmaline and harmalol were 1.84, 0.16, 3.90, and
0.25%, respectively.
[79]
Other excellent validation results were
obtained by Monsefi-Esfarani et al. using an adaptation of the
same method with changes in mobile phase pH. Calibration
curves were linear (r
2
>0.998) for all analytes in the concentra-
tion range of 0.5–20 mg/mL and method RSD values ranged from
0.6–10.2% for all analytes. LODs were less than 0.1 mg/mL and
LOQs equal to 0.5 mg/mL.
[83]
Pulpati et al. reported a high-
performance thin-layer chromatography (HPTLC) method for
the quantification of harmine, harmaline, vasicine and vasici-
none from P. harmala seeds.
[88]
These compounds were
detected by a densitometric method and the seeds were
found to contain 0.44% of harmine and 0.096% of harmaline
(both DW) with suitable figures of merit. Herraiz et al.
measured harmol, harmalol, harmine, harmaline, and THH in
extracts prepared using different parts of P. harmala.Quantifi-
cation of the b-carbolines employed reversed phase HPLC
with UV-diode array detection (DAD).
[22]
Standardized aqueous extracts of B. caapi were obtained by
Wang et al.
[32]
These were prepared using different parts of the
A. Gaujac et al.
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Table 3. Methods for the determination of tryptamines and b-carbolines in ayahuasca
Ayahuasca
Reference Callaway
[34]
Huhn et al.
[91]
Pires et al.
[39]
Gambelunghe et al.
[40]
McIlhenny et al.
[41]
Moura et al.
[90]
Material analyzed Real ayahuasca samples Real ayahuasca sample Real ayahuasca samples Real ayahuasca sample Extracts prepared
in the laboratory from
P. viridis leaves and
the B. caapi vine
Extracts prepared
from P. viridis
leaves, and real
ayahuasca
samples
Analytes of interest DMT, harmine, harmaline
and THH
DMT, norharmane,
harmane, harmine,
harmaline, harmol
and THH
DMT, harmine, harmaline
and THH
DMT, harmine and
harmaline
DMT, NMT, harmol,
harmalol, harmine,
harmaline and THH
DMT
Sample preparation
technique
For DMT: LLE; For b-carbolines:
dilution of extracts in
mobile phase
Ayahuasca sample was
diluted with a buffer
Solid-phase extraction Extraction with ethyl ether
and centrifugation
Dilution of samples
in the mobile phase
LLE
Separation / detection
techniques
DMT: GC-NPD; b-carbolines:
HPLC-FL
CE -LIF-MS GC-NPD GC-MS HPLC-MS/MS qNMR
Figures of merit ––
Linear range: 0.02 –
4.0 mg/mL;
0.9941 <r² <0.9971;
LOD = 0.01 mg/mL;
LLOQ = 0.02 mg/mL;
1.3% <RSD <9.7%
–
Linear range: 5 –100 ng/mL
and 5 –100 mg/mL;
r
2
>0.9965; LOD <0.0079
ppm; LOQ <0.24 ppm
Linear range:
25 –1000 mg/L;
r² = 0.999;
LOD = LLOQ =
12.5 mg/L;
R*= 70%
RSD <5.1%;
Concentration levels DMT: 0 –14.15 mg/mL;
THH: 0.48 –23.80 mg/mL;
Harmaline: 0 –0.90 mg/mL;
Harmine: 0.45 –22.85 mg/mL
–DMT: 0.42 –0.73 mg/mL;
Harmine: 0.37 –
0.83 mg/mL;
Harmaline: 0.64 –
1.72 mg/mL; THH: 0.21 –
0.67 mg/mL
DMT: 24 mg/100 mL;
Harmaline 6 mg/100 mL
Harmine: 34 mg/100 mL
DMT: 0.12 –3.19 mg/mL;
NMT: 0.0052 –
0.0313 mg/mL;
Harmalol: 0.0026 –
0.0310 mg/mL; Harmol:
0.0009 –0.0633 mg/mL;
Harmine: 0.91 –
16.1 mg/mL; Harmaline:
0.054 –1.55 mg/mL;
THH: 1.22 –11.90 mg/mL
–
* Recovery (R).
Determination of psychoactive plant constituents
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plant, including leaves, stem bark, and entire branches, and
were collected from different geographical locations in the
Hawaiian Islands of Oahu and Hilo during different seasons.
Determinations of tetrahydronorharmine (THNH), harmol, THH,
harmaline and harmine were performed using HPLC-DAD. The
concentrations measured are listed in Table 1. Validation test
results were not reported. Zhou et al. developed a method
to quantify tryptamines and a b-carboline in 14 P. aquatica
populations using HPTLC.
[71]
Visualization of spots included the
use of an acidified anisaldehyde reagent spray that produced
intense colours which were amenable to quantitation using a
flatbed digital scanner. Good linearity was obtained in the
concentration range between 120–3840 ng per spot, with a cor-
relation coefficient above 0.991, for hordenine, methyltyramine,
gramine, and 5-MeO-DMT. The method provided good specificity
for the analytes of interest, as well as adequate repeatability with
a variation of less than 5%, on average, for analyses in duplicate.
Compound identification was confirmed by atmospheric pres-
sure chemical ionization (APCI) LC-MS.
[71]
Of particular note amongst the methods used to quantify
tryptamines and b-carbolines in ayahuasca (Table 3) is GC
coupled with either an NPD or a mass spectrometer.
[34,39,40]
NMR has also been used to quantify DMT.
[90]
Overall, an expan-
sion towards method validation seems indicated in order to
examine the reliability of measurements. Callaway
[34]
presented
a compilation of the results obtained for a large number of
ayahuasca samples with measurements of DMT, THH, harmine,
and harmaline. Decoctions of B. caapi were prepared in Brazil
by the three main religious groups involved in its use (Santo
Daime,União do Vegetal, and Barquinha), and preparations
of ayahuasca were also obtained from the Ecuadorian Shuar
Indian tribe. An earlier method
[89]
was used for the detection
of b-carbolines with separation by HPLC and fluorescence
detection. DMT was determined by GC-NPD and large differences
were found in the concentrations of the analytes in the samples,
with DMT levels varying between zero and 14.15 mg/mL.
Pires et al. appeared to be the first to report a validated
method for the simultaneous determination of both DMT
and the b-carbolines harmine, harmaline, and THH in real
samples of ayahuasca using GC-NPD.
[39]
For all of the analytes
the calibration curves showed excellent linearity in the concen-
tration range 0.02–4.0 mg/mL, with r² values varying between
0.9941 and 0.9971. The precision of the method was between
94.0 and 105.4%, and intra-day and inter-day coefficients of
variation were lower than 9.7%. LODs and LOQs were provided.
In stability tests using spiked water and ayahuasca, losses were
less than 10% after 24 h of storage at ambient temperature.
The ranges of concentrations measured in eight real ayahuasca
samples were 0.42–0.73 mg/mL (DMT), 0.37–0.83 mg/mL
(harmine), 0.64–1.72 mg/mL (harmaline) and 0.21–0.67 mg/mL
(THH). Despite originating from the same religious group
in Araçoiaba da Serra (Brazil), the concentrations in the beverages
varied between samples probably due to the use of different
quantities and proportions of the plants in each preparation,
as well as different alkaloid contents present in the plant
specimens.
[39]
McIlhenny et al. developed an HPLC electrospray ionization
(ESI) MS/MS method for the determination tryptamines and
b-carbolines present in ayahuasca samples prepared in the
laboratory.
[41]
This comprehensive MS/MS procedure was opti-
mized for the detection of 11 alkaloids and revealed that major
constituents of ayahuasca included THH, harmine, DMT, and
harmaline, followed by harmalol and NMT. In addition, 5-MeO-
DMT, 5-HO-DMT (bufotenin), and MTHC were also detected in
some but not all samples. Method validation included determina-
tion of precision, method bias, inter- and intra-day precisions,
limits of detection and quantification. The analytical curves
for the compounds were linear in the concentration ranges
employed (5–100 ng/mL and 5–100 mg/mL, depending on the
compound), with r
2
above 0.996.
[41]
Gambelunghe et al. measured concentrations of DMT and
harmine of 24.6 mg/100 mL and 34 mg/100 mL, respectively, in
a sample of ayahuasca that had been seized in Italy, but did
not provide any validation data.
[40]
An alternative approach was
offered by Moura et al.
[90]
who demonstrated that
1
H NMR could
be successfully employed for the detection of DMT in ayahuasca.
The optimized method was applied to water samples spiked
with DMT and 2,5-dimethoxybenzalde as the internal standard,
and excellent figures of merit were obtained in validation experi-
ments (Table 3). The authors analyzed extracts prepared from the
leaves of P. viridis, as well as eight samples of ayahuasca but
results were not reported.
[90]
Earlier work,
[34]
however, indicated
that typical levels of DMT in ayahuasca could well exceed the
linear range cited by Moura et al. (25–1000 mg/L).
[90]
The main
advantages of the
1
H NMR technique, compared to chromatogra-
phy, are that the analysis is fast (~30 s), non-destructive, and
that it can provide structural information as well. However, a
possible influence of matrix effects was not reported and the
method was developed in the absence of any b-carbolines
present in fortified aqueous samples. It is well-known that
ayahuasca preparations invariably contain material from one of
the Banisteriopsis spp., which provides the b-carbolines that are
vital for the oral psychoactivity of the DMT present in P. viridis
leaves. Further studies can shed more light on the question as to
whether such compounds could interfere with the determination
of DMT by NMR.
In summary, while the majority of analytical methods employed
in recent years involved the implementation of HPLC-UV
procedures, less expensive approaches towards quantitative
estimations of at least the major alkaloids found in these
psychoactive plant matrices, such as HPTLC, were found to
be suitable as well. The key alkaloids, i.e. DMT and the main
b-carbolines, are sufficiently volatile to undergo GC-based analy-
sis after extraction into a suitable organic solvent without the
need for derivatization. The complementary approach offered
by HPLC-based methods is increasingly supported by mass
spectrometric applications that offer improved sensitivity and
specificity when compared to UV/DAD detection. As described
above, the first comprehensive HPLC-MS/MS-based target
screening approach of ayahuasca and method validation was
reported by McIlhenny et al.
[41]
who also demonstrated that
matrix effects, particularly relevant where ESI is employed,
were not observed. The need for such a robust, sensitive, and
selective mass spectrometric analysis method is especially
helpful when considering bioanalytical research following
ayahuasca administration studies in humans.
[92,93]
In addition, it
is anticipated that future research on the characterization of these
diverse psychoactive brews will benefit from more comprehen-
sive unknown screening methodologies based on full-scan modes
in order to identify additional constituents that have not yet
been identified. Finally, a more thorough application of sensitive
and selective MS/MS-based methodologies is expected to shed
more light on the role of psychoactive tryptamines present in
mammalian tissues including humans.
[94]
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Conclusion
The use of psychoactive plant products known to contain
bioactive tryptamine and b-carboline derivatives is increasing
worldwide which reflects the expansion of syncretic religions
derived from South America and straightforward access of plant
products that contain these alkaloids. Recent research has
been reviewed concerning the implementation of analytical tech-
niques used for the detection of tryptamines and b-carbolines
present in plants and psychoactive beverages consumed
for religious and recreational purposes. For further studies, an
increased focus on method validation procedures is recom-
mended. Given the increasing interest in these plants and the
ritual beverages derived from them it is clear that suitable routine
analytical techniques will increasingly be required to accurately
measure the associated psychoactive compounds in a variety
of different matrices. This is especially the case within the clinical
and forensic context. An additional avenue for further explora-
tions includes a move towards minimal sample manipulation
and low to zero use of environmentally toxic solvents.
Acknowledgements
Helpful comments from Adjunct Professor J.C. Callaway, Ph.D. and
Prof. Mark Wainwright, Ph.D. are gratefully appreciated.
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