Disposition of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and its metabolite 4-bromo-2-hydroxy-5-methoxyphenethylamine in rats after subcutaneous administration

Article abstract of DOI:10.1016/j.toxlet.2008.01.017

The psychedelic compound 4-bromo-2,5-dimethoxyphenethylamine (2C-B) has appeared as an agent in drug abuse or overdose cases in humans. The human pharmacokinetics of this drug is unknown and only partial information is available on its metabolites. Our experimental study was focused on the disposition and kinetic profile of 2C-B in rats after subcutaneous administration using a GC-MS validated method. One of the major metabolites 4-bromo-2-hydroxy-5-methoxyphenethylamine (2H5M-BPEA) was confirmed in rat tissues of lung, brain, liver and was quantitatively evaluated as well. The disposition of 2C-B was characterized by its estimated half-life 1.1 h and estimated volume of distribution 16 L/kg. The lung susceptibility for drug retention and gradual temporal release parallel to the brain were ascertained. The drug penetrating the blood/brain barrier was without significant delay. 2C-B brain to serum ratio attained a maximum value of 13.9 and remained over the value of 6.5 to the end of our observation (6 h after the dose). The distribution of the hydroxylated metabolite 2H5M-BPEA into the lipophilic brain tissue was less efficient in relation to the parent compound. The kinetics of the drug partitioning between blood to brain may be important for the subsequent assessment of its psychotropic or toxic effects.

Full text of DOI:10.1016/j.toxlet.2008.01.017

Available online at www.sciencedirect.com
Toxicology Letters 178 (2008) 29–36
Disposition of 4-bromo-2,5-dimethoxyphenethylamine (2C-B) and
its metabolite 4-bromo-2-hydroxy-5-methoxyphenethylamine
in rats after subcutaneous administration
a
b
a,∗
Miroslava Rohanov a´ , Tom a´ sˇ P a´ len ´ı cˇ ek , Marie Bal ´ı kov a´
a
Institute of Forensic Medicine and Toxicology, 1st Faculty of Medicine, Charles University in Prague, 121 08 Prague 2, Czech Republic
b
´
Prague Psychiatric Center, Ustavn ´ı 91, 181 03 Prague 8-Bohnice, Czech Republic
Received 1 November 2007; received in revised form 27 January 2008; accepted 28 January 2008
Available online 7 February 2008
Abstract
The psychedelic compound 4-bromo-2,5-dimethoxyphenethylamine (2C-B) has appeared as an agent in drug abuse or overdose cases in humans.
The human pharmacokinetics of this drug is unknown and only partial information is available on its metabolites. Our experimental study was
focused on the disposition and kinetic profile of 2C-B in rats after subcutaneous administration using a GC–MS validated method. One of the major
metabolites 4-bromo-2-hydroxy-5-methoxyphenethylamine (2H5M-BPEA) was confirmed in rat tissues of lung, brain, liver and was quantitatively
evaluated as well. The disposition of 2C-B was characterized by its estimated half-life 1.1 h and estimated volume of distribution 16 L/kg. The lung
susceptibility for drug retention and gradual temporal release parallel to the brain were ascertained. The drug penetrating the blood/brain barrier
was without significant delay. 2C-B brain to serum ratio attained a maximum value of 13.9 and remained over the value of 6.5 to the end of our
observation (6 h after the dose). The distribution of the hydroxylated metabolite 2H5M-BPEA into the lipophilic brain tissue was less efficient in
relation to the parent compound. The kinetics of the drug partitioning between blood to brain may be important for the subsequent assessment of
its psychotropic or toxic effects.
©
2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: 2C-B; 4-bromo-2,5-dimethoxyphenethylamine; Pharmacokinetics; Disposition; Metabolites; Rats
1
. Introduction
C-B or BDMPEA (4-bromo-2,5-dimethoxyphenethyl-
known under a variety of street names such as “Nexus, Venus,
Bromo, Erox, or Synergy”.
2
2C-B possesses an affinity towards various central seroton-
ergic and adrenergic receptors (Glennon and Anderson, 1980;
Glennon et al., 1988, 1992; Villalobos et al., 2004; Lobos et
al., 1992; Monte et al., 1996; Neuvonen et al., 2006). Acti-
vation of the 5-HT2A (serotonin 2A) receptors by an agonist
ligand has been established to be bound with hallucinogenic
effect (e.g. Monte et al., 1996; Villalobos et al., 2004; Neuvonen
et al., 2006). In humans, 2C-B is usually consumed orally in
the range of 4–30 mg dosages, which can produce considerable
euphoria with increased receptiveness of the visual, auditory,
olfactory, and tactile sensations. Lower doses (4–10 mg) induce
entactogenic-stimulating effects while higher doses (10–20 mg)
induce psychedelic effects along with frightening hallucinations
and some sympathomimetic effects such as tachycardia, hyper-
tension and hyperthermia (Shulgin and Carter, 1975; Shulgin
andShulgin, 1991;Carmoetal., 2005). LaingandSiegel(2003b)
described its hallucinogenic potency as being 16 times stronger
amine) is a psychoactive agent related to both the mescaline-like
psychedelics and the amphetamine-like stimulants. The synthe-
sis of this compound has been described by Shulgin who also
tested its pharmacological effects on himself and a group of vol-
unteers (Shulgin and Carter, 1975; Shulgin and Shulgin, 1991).
2
C-B is a potential drug of abuse and it has appeared as an alter-
native or a complementary drug to Ecstasy on the illegal drug
market; earlier it was also sold via the Internet and nowadays
it appears especially on the dance scene in the form of vari-
ous tablets with questionable composition and potential risk of
intoxication (Giroud et al., 1998; de Boer et al., 1999a; de Boer
and Bosman, 2004; Laing and Siegel, 2003a). This drug is also
∗
Corresponding author. Tel.: +420 224 964 332; fax: +420 224 911 267.
E-mail address: mbali@lf1.cuni.cz (M. Bal ´ı kov a´ ).
0
378-4274/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2008.01.017

3
0
M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
analytical standard 4-bromo-2,5-dimethoxyphenylethylamine hydrochloride
than mescaline and Shulgin and Carter (1975) have described
that its potency is about one tenth of its three carbon counter-
part 4-bromo-2,5-dimethoxyamphetamine (DOB). The average
duration ofthe effectsin humans after2C-B ingested dose ranges
between 4 and 8 h depending on the dose and the susceptibility
of an individual (Shulgin and Shulgin, 1991; Karch, 2002).
At present, the pharmacokinetics of 2C-B based on controlled
study is unknown. To clarify the disposition of this drug in organ-
ism related to time after the dose may be important from the
perspective of successful detection and correct assessment of an
intoxication or drug abuse and from the perspective of under-
standing and explanation of its psychotropic effects. In the case
of 2C-B, the biotransformation scheme related to mescaline can
be assumed. It involves O-demethylation of methoxygroups of
the aromatic ring, beta-oxidation of the alkyl chain and oxida-
tive deamination involving different enzyme systems (Scheline,
(
2C-B) was provided by Sigma–Aldrich Ltd., Prague (purity > 99%).
Other reference standards, both O-monodesmethyl metabolites (2M5H-
BPEA and 2H5M-BPEA) were synthesized according to Kanamori et al. (2002)
1
13
with H and C NMR structural verification and provided as hydrochlorides by
Pharmaceutical Faculty of Charles University in Hradec Kr a´ lov e´ with estimated
purity of 81 and 80%.
2.2. Animals and drug administration
All experiments were performed in compliance with international guidelines
and laws governing animal studies in the Czech Republic. Male rats Wis-
tar specific pathogen free (SPF; Velaz Ltd., Prague) weighing 0.230–0.250 kg
were housed individually in cages. They had free access to water and food
and were kept under a 12-h day and night cycle. They were administered a
5
0 mg/kg bolus dose of 2C-B hydrochloride dissolved in physiological solu-
tion in a volume of 2 ml/kg subcutaneously. The animals were sacrificed at
3
0, 60, 120, and 360 min after dosing (10 animals at each time interval) and
◦
serum, brain, liver and lung samples were collected and stored at −20 C until
1
978; Moffat et al., 2004; Charalampous et al., 1966). Today,
analyses.
results of some biotransformation studies of 2C-B on experi-
mentalanimalmodelsareavailable(Kanamorietal., 2002, 2003,
2.3. Sample preparation
2
005; Carmo et al., 2005, 2004; Theobald et al., 2007). De Boer
2
.3.1. Serum pretreatment
.5 ml aliquot of rat serum was transferred to a labeled tube. Internal standard
400 ng/ml MBDB) and 1 ml 0.1 M phosphate buffer (pH 6) were added and
mixed.
et al. also reported on the human metabolites found in the urine
specimenofasubjectabusing2C-B(deBoeretal., 1998;deBoer
et al., 1999b). In this single human urine sample, the presence
of the parent form and the 4-bromo-2,5-dimethoxyphenylacetic
acid was confirmed using GC–MS with reference standards. The
formation of some other metabolites was also indicated, how-
ever, they could not be identified unambiguously due to the lack
of reference standards. The systematic clinical study of this drug
with human volunteers remains restricted for ethical reasons and
therefore only various experimental animal studies have been
performed to date. In the experimental study with hepatocytes of
six species including humans (Carmo et al., 2005), the metabolic
pathways of 2C-B was constructed with the GC–MS identi-
fication of the metabolite 4-bromo-2,5-dimethoxyphenylacetic
acid in all species. The other metabolites formed by com-
bination of oxidative deamination and O-demethylation were
indicated too considering the mass spectrometry fragmentation
mechanisms.
Our study was focused on quantitative assessment of the
kinetic profiles of 2C-B and its prevailing O-demethylated
metabolite in serum, brain, liver and lung tissues of experimen-
tal rats after subcutaneous administration. The identification
of the monodemethylated metabolites and evaluation of their
abundance in serum and tissues was based on synthesized refer-
ence substances of 4-bromo-2-methoxy-5-hydroxyphenethyl-
amine (2M5H-BPEA) and 4-bromo-2-hydroxy-5-methoxy-
phenethylamine (2H5M-BPEA). The assessment of other poten-
tial metabolites of 2C-B in samples under our study cannot be
performed fully as the reference standards were not available.
0
(
2.3.2. Tissue pretreatment
1 g of tissue (brain, liver or lung) was homogenized with 4 ml methanol and
internal standard aliquot (400 ng/g MBDB for brain; 2000 ng/g MBDB for liver
and lung). The specimen was mixed and treated for 20 min in an ultrasonic bath
followed by water freezing (20 min) and centrifugation (5 min at 4000 rpm).
2
ml of supernatant were transferred to a clean glass test tube and evaporated.
The residue was reconstituted by adding 0.1 ml methanol and 1 ml of 0.1 M
phosphate buffer (pH 6).
2.3.3. Solid phase extraction procedure (SPE)
A pretreated specimen with buffer and internal standard was transferred
to the SPE disc preconditioned with 1 ml methanol and 1 ml phosphate buffer
(
pH 6). After sample application the disc was washed with 1 ml distilled
water, 1 ml 0.1 M HCl and 1 ml methanol and then finally dried with air for
min. Analytes were eluted three times with 1 ml of the fresh mixture of
5
dichlormethane/2-propanol/ammonium hydroxide (25%), 80/20/4, v/v/v. The
combined eluates were evaporated to dryness under an air stream at 40 C and
derivatized.
◦
2
.3.4. Derivatization
The chemical derivatization by acetylation was performed by treatment
of dry residues with 200 ␮l of the acetylation mixture (acetic acid anhy-
dride/pyridine 10/1) at 60 C for 30 min. The derivatized samples were then
allowed to cool to ambient temperature prior to evaporation of excess acety-
lation agent. The residues were reconstituted in 100 ␮l ethylacetate and 1 ␮l
aliquot was analyzed using GC–MS.
◦
2
.4. GC–MS conditions
The Hewlett-Packard GC–MS system HP 6890-5973 (Agilent, Waldbronn
2
. Materials and methods
Germany) equipped with an autosampler, splitless injector and capillary HP5-
MS 30 m × 0.25 mm × 0.25 ␮m were used for the analyses. Helium was used
as the carrier gas at the flow rate of 1.0 ml/min. The oven temperature was
2
.1. Chemicals and reference standards
◦
◦
programmed starting from 85 C, held for 2 min, then with 30 C/min up to
◦
◦
◦
Chemicals used were of analytical grade. Extraction discs from SPEC
150 C, then with 15 C/min up to 250 C and held for 10 min. The injector
and the transfer line were operated at 250 C. Ionization was carried out in
◦
PLUS 3 ML DAU, Ansys Technologies were provided by Amedis Ltd. Prague.
-methylamino-1-(3,4-methylenedioxyphenyl)-butane hydrochloride (MBDB)
used as internal standard was kindly provided by UNDCP, Vienna. The reference
2
electron impact (EI) at 70 eV. The selected ion monitoring (SIM) mode was
used; quantifying and monitored ions were for the internal standard MBDB

M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
31
(
(
249, 176, 135, 114, 72), for 2C-B (301, 242, 229, 199, 148), for 2H5M-BPEA
329, 287, 228, 187, 148, 134, 77)—the underlined ions indicate the quantifying
ions.
2
2
.5. Method validation and determination of 2C-B and
H5M-BPEA concentrations
The GC–MS toxicological methods were developed and validated in com-
pliance to international standards (Peters and Maurer, 2002; Peters et al., 2007)
to determine the 2C-B and its main metabolite 2H5M-BPEA concentrations in
rat samples under the operating conditions described above. The calibration was
based on linear regression analysis using the ratio of peak areas of the ana-
lyte to the internal standard. Blank rat serum and tissue samples collected from
laboratory male Wistar rats were used for calibration and validation purposes.
Selectivity was checked with samples obtained from six different animals. 2C-
B and 2H5M-BPEA calibrators were obtained by adding appropriate amounts
of 2C-B, 2H5M-BPEA and internal standard to blank matrices to imitate the
concentration corresponding to values expected in real samples. Internal stan-
dard addition was 400 ng/ml for serum, 400 ng/g for brain and 2000 ng/g for
lung and liver. Six point calibrators with two replicates for each level were
used.
Fig. 1. Chromatogram corresponding to an authentic rat liver sample 60 min
after 2C-B subcutaneous administration: (1) MBDB.AC, internal standard; (2)
2C-B.AC, parent drug; (3) 2H5MBPEA.2AC, metabolite.
The limits of detection and quantification were determined separately using
spiked calibrators to lower concentrations in the range of 30–200 ng/ml (or ng/g)
of both analytes.
The control samples to evaluate precision and accuracy of both analytes
were prepared independently at three concentration levels in respect to the real
Fig. 2. Typical mass spectra of reference standards of acetylated 2C-B and its expected O-desmethyl metabolites: (a) 2C-B.AC, (b) 2H5M-BPEA.2AC, (c) 5H2M-
BPEA.2AC.

3
2
M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
Table 1
2
Linearity of calibration of GC–MS assay in various rat matrices: regression coefficient (R ), limits of detection (LOD) and lower limit of quantification (LLOQ)
ANALYTE
SAMPLE
Calibration range (ng/ml; ng/g)
R2
LLOQ (S/N > 8)
LOD (S/N > 3)
2
C-B
Serum
Brain
Liver
Lung
100–3000
100–20,000
100–9000
100–40,000
0.999
0.998
0.999
0.996
100 ng/ml
70 ng/g
70 ng/g
50 ng/ml
30 ng/g
30 ng/g
50 ng/g
100 ng/g
2
H5M-BPEA
Serum
Brain
Liver
Lung
200–3000
130–6000
120–9000
120–9000
0.996
0.996
0.999
0.998
200 ng/ml
120 ng/g
120 ng/g
120 ng/g
120 ng/ml
80 ng/g
80 ng/g
80 ng/g
samples. The precision (repeatability) and accuracy values were evaluated by
analyzing six samples per day. Accuracy was calculated as the difference of
the mean of determined values (n = 6) from the theoretical one related to the
theoretical one.
both for parent 2C-B and its main metabolite 2H5M-BPEA.
The 2C-B limits of detection (LOD), S/N > 3, in serum and tis-
sues were at least 50 ng/ml (ng/g), lower limits of quantification
(
LLOQ), S/N > 8, at least 100 ng/ml (ng/g) or better. These LOD
and LLOQ values for 2H5M-BPEA were in selected tissues
0 ng/g (LOD) and 120 ng/g (LLOQ). These limits were rather
3
. Results
8
higher in the serum, LOD 120 ng/ml and LLOQ 200 ng/ml.
Repeatability of all assays was within 9%, except the value
near the LLOQ (11%). Accuracy was found to be within ± 11%
of the spiked value, except at the LLOQ where higher values
were found. The results are summarized in Table 2.
3
.1. GC–MS method validation parameters
Fig. 1 shows, as an example, the chromatogram related to
an authentic rat liver sample obtained 60 min after administra-
tion. The GC–MS spectra of acetylated standards of the parent
compound 2C-B and both potential O-desmethyl metabolites
2
H5M-BPEA and 2M5H-BPEA expected in rat samples in our
3.2. Structural identification of 2C-B and its
O-demethylated metabolites in rat samples
study are presented in Fig. 2.
The GC–MS method developed was found to be selective
both for serum and tissues and no interferences with analytes of
interest or internal standard were ascertained.
The method linearity was fulfilled in the concentration range
given in Table 1 with regression coefficients better than 0.996
Thedetectionandidentificationof2C-Binratsampleextracts
was based on direct comparison with the reference substance.
The identification and structural confirmation of 2H5M-BPEA
as the main metabolite determined in rat serum and tissues after
Table 2
Repeatability and accuracy of the GC–MS assay (n = 6)
Analyte
Sample
Spiked concentration
Repeatability
(%)
Accuracy
(%)
(
ng/ml; ng/g)
2
C-B
Serum
100
3.7
3.1
1.8
4.6
2.3
5.5
7.9
4.3
2.2
4.6
3.8
2.2
−1.8
+8.9
+0.4
−12.9
−10.1
+9.9
+19.8
+2.9
−3.0
−5.7
+3.9
−4.0
500
2
2
000
100
000
Brain
Liver
Lung
1
3
5,000
100
2
9
000
000
100
000
2
0,000
2
H5M-BPEA
Serum
Brain
Liver
Lung
200
2.4
2.2
4.1
6.2
7.3
3.3
10.6
8.3
6.7
10.8
4.6
−10.1
−0.7
+6.5
+12.3
−9.6
−2.2
+8.1
400
2
000
130
500
000
120
000
000
120
000
000
1
5
2
9
−1.7
+0.8
−1.4
−8.6
+4.9
2
9
5.3

M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
33
7
485 ± 1534 ng/g), nevertheless these were higher than those
in serum. After 6 h, 2C-B abundance in tissues still persisted
high above the detection limit (Fig. 3, Table 3).
The abundance of the metabolite 2H5M-BPEA in tissues
reached its maximum within 1 h (liver, lung) or 2 h (brain) after
the 2C-B dose (Fig. 4). The concentration levels of more polar
metabolite 2H5M-BPEA in the brain and lung were much lower
related to the parent compound reaching maximum values of
3
761 ± 1744 ng/g (lung) and 2726 ± 938 ng/g (brain) as demon-
strated in Fig. 4 and Table 3. In the liver, the important organ
for metabolism, the abundance of 2C-B and its metabolite were
relatively close. The kinetic curves of both compounds in the
liver demonstrate parallel temporal profiles, a gradual rise and
then decline, and both curves remained close to each other until
the end of our observation.
Fig. 3. Temporal distribution characteristic of 2C-B in rat serum, brain, liver
and lung after 50 mg/kg s.c. application. Mean values (n = 10).
our experimental 2C-B dose, was also based on the accordance
with mass spectra and retention of synthesized reference stan-
dard. The abundance of the other O-demethylated metabolite
The kinetic curves of lipophilic 2C-B in serum and all tissues
demonstrate significant drug retention in the lung and sufficient
influx into the brain. The brain/serum ratio was >6.5 starting
at 30 min and was kept above this value throughout the 6 h of
our experiment. The 2C-B penetration of the blood–brain bar-
rier was non problematic. The distribution of the hydroxylated
metabolite 2H5M-BPEA into the lipophilic brain tissue was less
efficient and lung retention less apparent related to the parent
drug.
2
M5H-BPEA (which could be distinguished from the previ-
ous one by GC–MS) was negligible in all studied samples and
therefore quantitative evaluation could not be performed.
3
.3. Temporal disposition profile of 2C-B and 2H5M-BPEA
metabolite
All animals tolerated the drug subcutaneous dose 50 mg/kg
during 6 h of our kinetic study. The determined kinetic profiles
of the parent 2C-B and its main metabolite 2H5M-BPEA found
in serum and various rat tissues are presented in the form of
mean values (n = 10) in Figs. 3 and 4. The values corresponding
to individual animals are summarized in Table 3.
4. Discussion
This study was performed to investigate the temporal dispo-
sition of the designer drug of abuse 2C-B, structurally related to
amphetamine and mescaline. Concern has been raised because
insufficient information is known about 2C-B pharmacokinet-
ics and metabolism which may be important from diagnostic
and therapeutic perspective of an intoxication. The experimen-
tal design was based on laboratory rats as the human clinical
study remains difficult from ethical reasons. The animal model
used in our study was rather different from 2C-B abuse common
mode in humans, even if unexpected events appear in toxicolog-
ical practice. The subcutaneous administration was applied to
rats to assure reproducible drug bioavailability between animals
which is important for toxicokinetic study and the high dose
was in favour of easier detection and identification of unknown
metabolites.
The results of the kinetic disposition study may be interest-
ing also in the connection with the subsequent assessment of
drug effects. The structural features of 2C-B, with halogen sub-
stitution at position 4 of the aromatic ring, correspond in an
optimal way to the requirements for hallucinogen-like activity
as expressed by others (e.g. Monte et al., 1996; Neuvonen et al.,
Absorption of the parent drug into the blood stream was rapid
after the subcutaneous dose; the maximum 2C-B serum con-
centration (mean cmax 2250 ± 253 ng/ml) was attained within
3
0 min (tmax = 30 min) as displayed in Fig. 3. The kinetic profiles
of 2H5M-BPEA metabolite in various tissues are demonstrated
in Fig. 4. The metabolite 2H5M-BPEA could not be detected in
serum samples during the whole time interval.
The appearance of the parent 2C-B in all examined tis-
sues was rather delayed related to serum (tmax = 60 min) but
gradually accomplished significantly higher tissue concen-
trations as expected. Based on the data of this study, the
2
C-B abundance in tissues was in the order ranging from
lung > brain > liver > serum. The highest tissue concentrations of
the parent drug were found in the lung (cmax 27028 ± 7777 ng/g)
whereas the lowest ones were found in the liver (cmax
2006). There are many proofs that 2C-B links to hallucinogenic
drug activity with its affinity at 5-HT2A receptors that gives bet-
ter understanding to the effects of psychotropic drugs (Glennon
and Anderson, 1980; Glennon et al., 1988, 1992; Villalobos et
al., 2004; Lobos et al., 1992; Monte et al., 1996; Neuvonen et al.,
2006). The significant drug persistence in the brain ascertained
in our experimental rats is a prerequisite for 2C-B psychotropic
effects. As 2C-B is the lipophilic psychedelic substance, we
expected and found a high concentration in the brain, analo-
Fig. 4. Temporal distribution characteristic of 2H5M-BPEA in rat brain, liver
and lung after 2C-B 50 mg/kg s.c. application. Mean values (n = 10). In serum
this metabolite was not detected.

3
4
M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
Table 3
Temporal concentration profile of 2C-B and its metabolite 2H5M-BPEA in serum, brain, liver and lung of individual rats after 50 mg/kg s.c. 2C-B application
t (min)
2C-B
2H5M-BPEA
Brain (ng/g)
Animal n.
Serum (ng/ml)
Brain (ng/g)
Liver (ng/g)
Lung (ng/g)
Liver (ng/g)
Lung (ng/g)
3
3
3
3
3
3
3
3
3
3
0
0
0
0
0
0
0
0
0
0
1
2
3
4
5
6
7
8
9
2188
2583
2092
2625
2083
2208
1945
2638
1962
2178
10,412
21,370
15,273
14,706
15,318
16,911
14,005
16,115
12,801
9480
5631
6599
7572
6015
5553
4879
3152
4449
6500
3730
17,429
14,881
258,36
16,542
20,317
21,809
15,662
22,471
20,930
34,706
351
1150
966
326
853
754
380
410
770
766
3403
3311
5571
3200
3897
3275
2416
3962
2949
2343
132
513
536
62
640
500
433
274
178
432
10
Mean value (± S.D.)
2250 ± 253
14639 ± 3042
5408 ± 1237 21058 ± 5335
673 ± 273
3433 ± 872
370 ± 185
6
6
6
6
6
6
6
6
6
6
0
0
0
0
0
0
0
0
0
0
11
12
13
14
15
16
17
18
19
20
1468
2097
2498
2184
1706
895
2909
1397
2018
1751
21,712
24,515
24,994
11,696
15,213
13,568
16,035
11,820
17,404
14,067
7545
8150
8732
6083
7958
5747
5985
6658
11,360
6632
21,929
42,555
38,443
20,237
24,317
17,800
30,160
26,173
31,554
17,117
2174
2567
1426
912
1326
1913
3189
2064
4015
851
5642
4993
5392
3876
5048
5260
4516
4906
6572
4640
2055
7120
3631
1970
2658
6078
5329
3157
3750
1864
Mean value (± S.D.)
1892 ± 523
17102 ± 4493
7485 ± 1534 27028 ± 7777 2043 ± 954
5085 ± 683
3761 ± 1744
1
1
1
1
1
1
1
1
1
1
20
20
20
20
20
20
20
20
20
20
21
22
23
24
25
26
27
28
29
30
484
933
541
1142
770
963
1199
638
937
8538
13,746
10,979
9285
16,866
10,926
19,094
8882
3568
3192
7129
7255
8754
3516
4851
6346
5017
4570
14,274
15,688
22,680
19,947
25,469
15,425
8701
20,142
8739
29,893
3182
2605
1976
3190
2062
4007
1509
1335
3240
4154
2671
2157
5275
5484
5693
2453
3797
3878
3343
2732
592
662
2398
4289
2283
2260
3603
3381
2343
5073
8997
17,307
1390
Mean value (± S.D.)
900 ± 279
12462 ± 3786
5420 ± 1774 17514 ± 6504 2726 ± 938
3748 ± 1253
3204 ± 999
3
3
3
3
3
3
3
3
3
3
60
60
60
60
60
60
60
60
60
60
31
32
33
34
35
36
37
38
39
40
61
102
105
104
105
78
82
63
51
76
883
1073
982
1125
1238
828
1276
646
840
215
263
324
172
160
204
320
211
346
217
1516
1859
2451
3637
1374
1658
2492
1955
1960
2667
411
801
458
474
402
692
519
436
516
500
294
379
152
168
172
300
486
264
180
136
1196
381
650
1034
N.D.
N.D.
467
283
262
714
928
Mean value (± S.D.)
83 ± 19
982 ± 187
243 ± 63
2157 ± 639
522 ± 122
253 ± 103
496 ± 359
gously as it was confirmed in our previous experimental study
with another structurally related hallucinogenic drug, DOB in
rats (Berankova et al., 2007). The lipophilicity of the parent drug
carries the responsibility for its transport and retention in all tis-
sues with impact on accumulation and elimination. However,
taking into account the electron withdrawing effects of bromine
substituent at the para position of the phenyl ring, having influ-
ence on the ionic form of the molecule, we cannot exclude that
several mechanisms contribute to the influx and following accu-
mulation of 2C-B in tissues, e.g. active transport in addition to
passive diffusion.
Our results demonstrate also the significant 2C-B drug reten-
tion in the lung. We have found very high concentration levels
of 2C-B in the lung and these experimental findings were in
accordance with previous reports on the more potent hallucino-
gen and structurally related substance DOB, differing only in
the alkyl counterpart of the molecule (Berankova et al., 2007).
The DOB accumulation in the lung was mentioned previously

M. Rohanov a´ et al. / Toxicology Letters 178 (2008) 29–36
35
by Sargent et al. (1975) and by Shulgin and Shulgin (1991) with
no mention about this phenomenon concerned 2C-B.
differences in susceptibility of hepatocytes from three human
donors.
2
C-B and some structurally related compounds, as mescaline
In conclusion, the temporal profile of the disposition of the
psychedelic 2C-B provides the first approximate estimation of
kinetic parameters of 2C-B based on controlled animal exper-
iment. The drug’s ability to accumulate in the lung and persist
in the brain after a higher dose can be important also from
the perspective of subsequent behavioral study and interpreta-
tion.
or DOB, have been studied on biotransformation with vari-
ous animal models (e.g. Scheline, 1978; Carmo et al., 2004,
005; Kanamori et al., 2002, 2003, 2005; de Boer et al., 1998,
999b; Theobald et al., 2007; Berankova and Balikova, 2005;
2
1
Berankova et al., 2007; Ewald et al., 2007). Summing up, two
metabolic pathways have been considered in the literature men-
tioned above, O-demethylation and oxidative deamination using
different enzyme systems. Nevertheless the temporal disposi-
tion of metabolites either their psychoactivities have not been
studied yet. In our experiments, the O-demethylated metabo-
lite 2H5M-BPEA was determined in rat serum and tissues as
the prevailing biodegradation product. The abundance of the
other O-demethylated metabolite 2M5H-BPEA was negligible
in all studied samples and its quantitative evaluation could not be
performed. Our experimental findings correspond to data pub-
lished by Kanamori et al. (2005) who found 2H5M-BPEA as
the major metabolite in isolated rat hepatocytes additionally to
the other metabolite 4-bromo-2,5-dimethoxyphenylacetic acid.
The abundance of both metabolites was dose and time depen-
dent; after higher dose the 2H5M-BPEA was prevailing above
the acidic metabolite. The presence of this latter metabolite, 4-
bromo-2,5-dimethoxyphenylacetic acid, was also indicated in
our rat samples analyzed by GC–MS considering fragmentation
mechanisms in mass spectra; however, it cannot be confirmed
fully and quantified as the appropriate reference standard was
not available.
Acknowledgements
The study has been supported by the grant of IGA MH No.
NR8332-3 and IGA MH CR No. NR8785-3. We thank Craig
Hampson BSc (Hons) for language correction.
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