Forensic Chemistry 1 (2016) 13–21

Contents lists available at ScienceDirect

Forensic Chemistry
journal homepage: www.elsevier.com/locate/forc

Full length articles

Evaluating the selectivity of colorimetric test (Fast Blue BB salt) for the
cannabinoids identification in marijuana street samples by UV–Vis, TLC,
ESI(+)FT-ICR MS and ESI(+)MS/MS
Nayara A. dos Santos a,b, Lindamara M. Souza b, Eloilson Domingos b, Hildegardo S. França a,
Valdemar Lacerda Jr. b, Adilson Beatriz c, Boniek G. Vaz d, Rayza R.T. Rodrigues b, Verônica V. Carvalho d,
Bianca B. Merlo e, Ricardo M. Kuster b, Wanderson Romão a,b,⇑
a
Instituto Federal do Espírito Santo, 29106-010 Vila Velha – ES, Brazil
b
Laboratório de Petroleômica e Química Forense, Departamento de Química, Universidade Federal do Espírito Santo, 29075-910 Vitória – ES, Brazil
c
Instituto de Química, Universidade Federal de Mato Grosso do Sul – UFMS, Campo Grande, MS, Brazil
d
Instituto de Química, Universidade Federal de Goiás, 74001-970 Goiânia, GO, Brazil
e
Superintendência de Polícia Técnico-Científica do Estado do Espírito Santo, 29045-402 Vitória – ES, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Marijuana is one of the most marketed illicit drugs in the world. Originated from Cannabis sativa L,
Available online 14 July 2016 D9-tetrahydrocannabinol (or D9-THC) is proscribed in Brazil by the law number 11.343 from August
23rd of 2006 as well as its isomers, salts, ethers and esters. However, D9-THC is the main responsible
Keywords: for the hallucinogenic effects of the plant. The principal screening test used by the Brazilian Forensic
Marijuana Police is a colorimetric test called Fast Blue B or BB salt, in which the development of a reddish color
D9-THC in a basic medium indicates the presence of cannabinoids such as D9-THC, cannabinol (CBN) and/or
Fast Blue BB salt
cannabidiol (CBD). However, a better understanding of this chemical reaction is required. In this work,
ESI-FT-ICR MS
the products of the colorimetric test were identified by positive-ion mode electrospray ionization
Fourier transform ion cyclotron resonance mass spectrometry (ESI(+)FT-ICR MS), collision-induced
dissociation (CID) experiments or ESI(+)MS/MS, ultraviolet–visible (UV–Vis) spectroscopy and thin layer
chromatography (TLC). A chemical structure was proposed for the product of the reaction (m/z 626)
formed by Fast Blue BB salt (4-amino-2,5-diethoxybenzanilide diazotated zinc double salt) and D9-THC
molecule. Besides, the selectivity of the colorimetric test was evaluated in presence of interferences from
other plants such as Jacaranda decurrens Cham and Paullinia cupana Kunth.
Ó 2016 Elsevier B.V. All rights reserved.

1. Introduction More than 535 different natural compounds identified in the

According to the 2014 World Drug Report (WDR) from the Uni-
ted Nations Office on Drugs and Crime (UNODC), marijuana is one
of the most consumed drugs by the world population. This drug is
obtained from the Cannabis sativa L. plant, which is cultivated in
almost every country of the world [1,2]. According to the appre-
hension report from the Brazilian Federal Police, a growing number
of seizures has been observed as a function of time during the last
20 years, Fig. 1 [3].
⇑ Corresponding author at: Laboratório de Petroleômica e Química Forense,
Departamento de Química, Universidade Federal do Espírito Santo, 29075-910
Vitória – ES, Brazil.
E-mail address: wandersonromao@gmail.com (W. Romão).
C. sativa L. plant, which 70 are cannabinoids responsible for its
psychoactive effects [4,5]. The term cannabinoid is attributed to
the group of molecules composed 21 carbon atoms present in
C. sativa L., and their products of transformation.
Other species, previously reported to the genus Cannabis, such
as Cannabis indica Lam. and Cannabis ruderalis Janisch, are currently
recognized as varieties of C. sativa L., [5] and the methodologies
employed to their production are diverse, varying from environ-
mental conditions, geographic origin, plant maturity, and male or
female species, to even indoor or outdoor production process [5].
The study and the improvement of these preparation methodolo-
gies has provided a wide variety of products with different levels
of D9-THC (6a,10a-trans-6a,7,8,10a-tetrahydro-6,6,9-trimethyl-3-
pentyl-6H-dibenzo[b,d]pyran-1-ol) that is one of the main
psychotropic substances found in marijuana. D9-THC has the

http://dx.doi.org/10.1016/j.forc.2016.07.001
2468-1709/Ó 2016 Elsevier B.V. All rights reserved.
14 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21

Fig. 1. Number of seizures of marijuana samples as function of time. Data reported by Brazilian Federal Police [7].

molecular formula (M) of C21H30O2 and a molar mass (Mw) of
314 Da, being insoluble in water and soluble in ethanol and
petroleum ether [6,7]. D9–THC can be found in the different parts
of the plant with various percentages: (i) 10–12 wt% in flowers,
(ii) 1–2 wt% in leaves, (iii) 0.1–0.3 wt% in stalk, and (iv) <0.03 wt%
in root [3].
In Brazil, the C. sativa L. plant, botanically classified in 1753 by
Carl Von Linné, is popularly known in portuguese as ‘‘maconha” (as
marijuana). It is mainly cultivated in temperate and tropical zones
and the concentration of its cannabinoids depends upon the age
and storage conditions of the plant [4,8].
Most chemical screening assays use the Fast Blue B or Fast Blue
BB salts (FBBS and FBBBS, respectively), and their structures are
described in Fig. 2d–e [9,10]. These colorimetric assays allied to
other analytical techniques, such as the thin layer chromatography
(TLC), are employed to identify the main cannabinoids present in
the seized marijuana samples. According to the literature, the main
cannabinoids identified are D9-THC, CBN, and cannabidiol (CBD)
[7] (Figs. 2a–c, respectively). The presence of cannabinoids is con-
firmed by means of the development of a reddish color after the
reaction. Because it is only a screening assay, the final confirmation
of the presence of the D9-THC must be made by other analytical
techniques with higher selectivity power such as GC-MS, NMR or
LC-MS [7,10].
Costa et al. [10] reported false positive results for the marijuana
colorimetric test using the FBBS (Fig. 2d) when some plants culti-
vated in Brazil and Venezuela are analyzed. Among them stand
out: Jacaranda decurrens Cham, popularly known as ‘‘Carobinha”
(rural bush with few branches) [11]; and Paullinia cupana Kunth,
known as Guaraná (plant which has been used in the treatment
of various diseases) [10,12]. Besides, little information is described
in the literature about the study of the chemical reaction that
occurs between the cannabinoids (D9-THC, CBN, and CBD) and
the FBBS and FBBBS reagents , Fig. 2c–d. Therefore, in this work,

Fig. 2. Main cannabinoids presents in Marijuana samples and the reagents employed in the colorimetric assays.
 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21 15

the products of the colorimetric assay between the marijuana sam-

ples and FBBBS, Fig. 2d, were studied with the following tech-
niques: positive-ion mode electrospray ionization Fourier
transform ion cyclotron resonance mass spectrometry (ESI(+)FT-
ICR MS) [13], collision-induced dissociation (CID) experiments or
ESI(+)MS/MS, ultraviolet–visible (UV–Vis) spectroscopy and TLC.
A mechanism for the reaction between FBBBS and D9-THC is pro-
posed. In addition, the selectivity of the colorimetric assay is eval-
uated in presence of extracts rich in polyphenols, being obtained
from Carobinha and Guaraná plants.

2. Procedure

2.1. Reagents and materials

Eight marijuana samples were used for the colorimetric assays

as well as parts of the C. sativa L. plant, such as stalk, flower, leaf
and root. All samples were seized in Espírito Santo (ES) state (Bra-
zil), being provided by the Civil Police of ES (the cooperation agree-
ment, process number 23068.011398/2012-72). The samples of the
Carobinha and Guarana plants were purchased from local market.
The solvents and reagents used for the colorimetric assays, TLC
and UV–Vis analyses were: acetonitrile (ACN), petroleum ether, Fig. 3. TLC sprayed with FBBBS for the cannabinoids detection using (a) chloroform
and (b) cyclohexane:toluene:diethylamine (75:15:10 v%) as eluents.
chloroform, cyclohexane, toluene, diethylamine, sodium and
ammonium hydroxide (grade of analytical purity higher than
99.5%, from VetecÒ Química Fina Ltda, Brazil) and the revealing Table 1
Fast blue BB salt (4-amino-2,5-diethoxybenzanilide diazotated zinc Rf values for D9-THC and eight samples (S1–S8) of marijuana analyzed by TLC sprayed
double salt, 4-benzoylamino-2,5-diethoxybenzenediazonium chlo- with FBBBS using two systems of elution: (1) chloroform; and (2) cyclohexane:-
toluene:diethylamine (75:15:10 v/v %).
ride hemi(zinc chloride) salt), from Sigma-Aldrich Chemicals, USA.
The reagents formic acid (HCOOH), ammonium hydroxide Samples Chloroform Cyclohexane:toluene:diethylamina
(NH4OH), and sodium trifluoroacetate (NaTFA) used in the ESI- (75:15:10 v/v %)
FT-ICR MS analysis were acquired from Sigma-Aldrich Chemicals, D9-THC CBN CBD
USA. D9-THC 0.69 0.65 – –
S1 0.69 0.63 0.58 0.51
2.2. Colorimetric assay using Fast Blue BB Salt (FBBBS) S2 0.69 0.63 0.58 0.51
S3 0.69 0.61 – –
S4 0.69 0.60 0.54 0.49
The crude extracts of marijuana, carobinha and guarana (which S5 0.69 0.60 0.54 0.49
correspond to all parts of the plant mixed) and parts of the C. sativa S6 0.69 0.60 0.54 0.49
L. (flower, leaf, stalk and root) were subjected to the colorimetric S7 0.69 0.62 0.53 0.49
S8 0.69 0.60 – –
test. Around 2 mg of each sample was dissolved in 1 mL of ACN,
x ± sd 0.69 ± 0.00 0.61 ± 0.01 0.55 ± 0.02 0.50 ± 0.01
and afterwards, 100 lL of an aliquot of the extract was spotted
on a filter paper. Then, over the same region were added 2 mg of
FBBBS and 100 lL of NaOH 0.1 M. The tiles were visualized under
resolution of 4 cm-1. Initially, UV–Vis spectra were acquired for
natural daylight. The development of a reddish color indicates a
the marijuana, carobinha and guarana extracts in ACN. After, the
positive result for the cannabinoids.
reagent solution FBBBS and the colorimetric test solution (contain-
ing 100 lL of the extract of each system, 2 mg of FBBBS and 100 lL
2.3. Thin layer chromatography (TLC) of NH4OH) were analyzed. This procedure was performed in quartz
cuvettes.
Eight samples of pressed marijuana (named samples S1-S8)
were subjected to the simple extraction process of cannabinoids
in microtubes. A standard D9-THC solution (2 mg mL-1) was used 2.5. ESI(+)-FT-ICR MS and ESI(+)MS/MS
as reference material. A mass of around 2 mg of sample dissolved
in petroleum ether was used for the TLC analysis. Solutions were The mass spectrometer (model 9.4 T Solarix, Bruker Daltonics,
applied in the chromatographic plate, with a stationary phase of Bremen, Germany) was set over a mass range of m/z 200–2000.
silica gel, by using a capillary tube. The chromatographic chamber The ESI(+)-FT-ICR MS analyzes were performed with samples con-
was prepared 45 min before performing analyses. Two elution sys- taining the marijuana crude extract, the revealing FBBBS and the
tems were evaluated: (i) chloroform (100 mL) and (ii) cyclohexane: extract containing a mixture of both. The samples were prepared
toluene:diethylamine 75:15:10%v/v. The plates were sprayed with in ACN in a concentration of 2 mg mL-1, and for the acquisition in
FBBBS solution to stain cannabinoids. the ESI(+) mode, the solution was prepared in 0.1 % of formic acid.
For each analysis, the injection flow rate was 5 lL min-1. The
2.4. UV–Vis spectroscopy remaining parameters of the ESI(+) source are: (i) capillary voltage
(cone): 3500–4100 V; (ii) end plate offset = 500 V; (iii) drying gas
The UV–Vis analyses were performed in a Perkin ElmerÒ spec- temperature and flow rate: 250 °C and 2 L min-1; (iv) nebulizer
trophotometer, model Lambda 45, USA, in a dual beam mode, in gas pressure: 1 bar; (v) skimmer = 15 V; and (vi) collision volt-
the region from 200 to 700 nm, with a total of 64 scans and a age = (±)1 V. In the ion transmission, ion accumulation times in
16 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21

Fig. 4. Colorimetric test using FBBBS reagent for the cannabinoids detection in (a–h) C. sativa L., (i) J. decurrens Cham. and (j) P. cupana Kunth plants.

Fig. 5. UV–Vis spectra for solutions of (a) J. decurrens Cham., (d) P. cupana Kunth, (g) marijuana and (b,e,h) FBBBS, and (c,f,i) of the mixture of FBBBS reagent with the three
plants. Note that a band at 471 nm is only observed for marijuana sample with FBBBS, corroborating with the selectivity of the FBBBS reagent.
 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21 17

Fig. 6. ESI(+)-FT ICR mass spectra for samples of (a) marijuana, (b) FBBBS and (c) mixture of FBBBS with marijuana.

I H
O C5 H11 O C5 H11
NaOH O C5 H11

O O + H2 O
O

II OEt
OEt

N N NH N N NH

EtO O EtO O

III
O C5 H11 OEt
O C5 H11
OEt
N N NH H
O N N NH
EtO O O
EtO O
1
IV
O C5 H11
OEt O C5 H11
H OEt
N N NH H
O N N NH
O
EtO O
EtO O
2
C 38H 47 N3 O5
V
O C5 H11 HO C5 H11
OEt OEt
H
N N NH N N NH
O O
EtO O EtO O
2 3
C 38H 47 N3 O5

Fig. 7. Proposed mechanism for the product identified in the colorimetric test of marijuana using the reagent FBBBS.
18 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21
the hexapole and time-of-flight were 0.02 s and 0.9 ms, respec-
tively. Each spectrum was acquired by accumulation of 32 scans
of time-domain signals in 4 M (mega-point) [13–15].
All FT-ICR MS spectra were externally calibrated using a NaTFA
solution (m/z from 200 to 1200). The resolving power was of
approximately 500,000 at m/z 400 and a mass accuracy lower than
1 ppm provided the unambiguous molecular formula assignments
for singly charged molecular ions. The FT-ICR MS spectra were
acquired and processed using the software Data Analysis (Bruker
Daltonics, Bremen, Germany). The degree of unsaturation for each
molecule is determined from its DBE (double bond equivalent), Eq.
(1) [15–17]:
DBE ¼ c  h=2 þ n=2 þ 1
where c, h, and n are the numbers of carbon, hydrogen, and nitrogen
atoms, respectively, in the empirical formula determined from the
FT-ICR MS data.
The tandem mass spectrometry (MS2) experiments were per-
formed in a quadrupole analyzer coupled to the FT-ICR mass spec-
trometer. The ESI(+)-MS/MS spectra were acquired using an
infusion flow rate of 5 lL min-1, a capillary voltage of 4.0 kV, a neb-
ulizing temperature of 250 °C, argon as the collision gas, an ion
accumulation time of 1 s, an isolation window of 1.0 (m/z units),
and 15–30% of the collision energy. The collision-induced dissoci-
ation (CID) experiments were performed only for the ions of m/z
312 and 626, which correspond to FBBBS and the product of the
reaction between FBBBS and D9-THC molecule.
Fig. 8. ESI(+)MS/MS for ions of m/z (a) 312 and (b) 626.
3. Results and discussion
3.1. TLC
Fig. 3a–b show the TLC plates of the S1-S8 samples extracts
eluted in chloroform (system 1), and cyclohexane:toluene:diethyla
mine 75:15:10%v/v (system 2), both sprayed with the reagent
FBBBS, respectively. In Fig. 3a is observed a single spot with
Rf = 0.69 for all samples, which correspond to the D9-THC stan-
dard. For system 2, Fig. 3b, besides the spot corresponding to the
D9-THC, two new spots are also revealed for most of the samples
(S1, S2, S4–S7) with average Rf of 0.55 ± 0.02 and 0.49 ± 0.01,
Table 1, respectively. They correspond to the CBN and CBD (isomer
ð1Þ
of D9-THC) cannabinoids, respectively [14,18]. The presence of a
single spot for the samples S3 and S8 (that corresponds to D9-
THC) is consequence of the low concentration of the CBN and
CBD compounds or still due to the age of plant and its storage con-
dition, Fig. 3b.
Despite the absence of reference materials for the CBN and CBD
cannabinoids in the TLC analyses, the confirmation of these species
is supported by the specific color observed for each cannabinoid
after spraying with the FBBBS. For the CBD and CBN, orange and
purple colors are observed, respectively [19]. In addition, the dif-
ference of polarity also influences the distance traveled by the ana-
lytes. A smaller distance will be traveled by the cannabinoid that
interacts better with the stationary phase, i.e. the one of higher
polarity, that this case is the CBD. Thus, the polarity of the studied
 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21
compounds increases in the following order: D9-THC < CBN < CBD,
Fig. 3b. Note that CBD has two hydroxyl groups in its structure,
Fig. 2c, while CBN has higher hydrogen deficiency, or degree of aro-
maticity (DBE = 9), Fig. 2b–c. Both chemical properties contribute
for a higher interaction with the stationary phase.
3.2. Colorimetric test and UV–Vis analysis
Generally, the parts of the C. sativa L. plant are classified as a
function of D9-THC content. The literature described that the
D9-THC concentration within of plant decays from flower to root
[7,3,14]. Fig. 4e–h shows the results of the colorimetric test for
the parts of the C. sativa L. plant (leaf, 4e, flower, 4f, stalk, 4g,
and root, 4h), which positive results are observed only for leaf
and flower, Fig. 4e–f, that revealed a reddish spot. These results
are in accordance with Dias et. al. [14] that showed positive results
for flower samples, reaching a visual detection of cannabinoids at a
minimum concentration of 0.12 mg mL1.
Two plant drugs (J. decurrens Cham. and P. cupana Kunth) are
identified in the literature as possible interferers in the colorimet-
ric test performed with FBBS, Fig. 2d, providing false positive
results [10]. However, using FBBBS reagent instead of FBBS, nega-
tive results were obtained for both plant drugs, Fig. 4i–j, thus prov-
ing the best specificity of this reagent, Fig. 2e.
UV–Vis analysis was performed for the extracts containing the
plant drugs such as J. decurrens Cham. (Fig. 5a), P. cupana Kunth
(Fig. 5d) and C. sativa L. (Fig. 5g), the reagent FBBBS (Fig. 5b,e,h)
and reaction products, Fig. 5c, f and i.
Fig. 9. Proposed fragmentation mechanism of ion of m/z 312.
19
The UV–Vis spectrum for the marijuana extract shows absorp-
tion bands at k = 209 and 277 nm (p–p⁄ transitions) that gives a
transparent aspect to the solution. On the other hand, the P. cupana
Kunth extract, that has absorption bands with higher wave-lenghts
(k = 410 and 664 nm) gives a yellow–green color to the system. The
FBBBS reagent has bands at 228, 330 and 396 nm, and the last one
is responsible for its yellowish color. As a positive result was only
obtained for the C. sativa L. sample, a new band in the visible region
(k = 471 nm and A = 1.2  105 a.u.) is responsible for red color,
Fig. 5i, corresponding to n–p⁄ transitions of the electrons presents
in the diazo group (N2-R) bonded to an aromatic group. For the
extracts containing the reagent FBBBS with the J. decurrens Cham.
and P. cupana Kunth drugs (Fig. 5c and f, respectively), no band
was observed in the visible region, corroborating with the negative
results observed in the visual inspection of the colorimetric tests,
Fig. 4i and j.
4. ESI(+) FT-ICR MS and ESI(+)-MS/MS
Fig. 6a–c shows the ESI(+)FT-ICR MS spectra of extracts of a typ-
ical marijuana sample (6a), of reagent FBBBS (6b), and of the pro-
duct of the reaction between the marijuana sample and the
FBBBS (6c). In Fig. 6a, several signals are detected in the m/z
200–1000 region. Expanding the region around m/z 300, signals
corresponding to D9-THC and CBN molecules are identified as
[M + H]+ cations at m/z 315.23189 and 311.2006, and DBEs of 7
and 9, respectively. All detected signals have a mass accuracy
20 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21
Fig. 10. Proposed fragmentation mechanism of ion of m/z 626.
lower than 1 ppm. D9-THC and CBN have M = C21H30O2 and
C21H26O2, respectively. A DBE of 7 for D9-THC is related to the pres-
ence of an aromatic ring (DBE = 4), a cyclohexene (DBE = 2), and a
furan ring (DBE = 1), while for CBN, the DBE of 9 is due to the exis-
tence of two aromatic rings (DBE = 8) and also a cyclic ether group
(DBE = 1). CBD molecule is an isomer of D9-THC [20,21], and can
also be detected as an ion at m/z 315.23189. Despite both having
the same nominal mass, the concentration of D9-THC in marijuana
samples is always higher than of its isomers, as can be seen in the
TLC spot in Fig. 3b.
Fig. 6b shows the spectrum of the FBBBS reagent, also detected
as [M + H]+ cation, in which M = C17H18N3O3, at m/z 312.13409,
error of 0.56 ppm and DBE = 11. The value of DBE equals to 11 indi-
cates the presence of unsaturation related to two aromatic rings
(DBE = 8), a triple bond between two nitrogen atoms (DBE = 2),
and a carbonyl group (DBE = 1).
The product of the reaction between D9-THC (M = C21H30O2)
and FBBBS (M = C17H18N3O3) was identified with ESI(+)-FT-ICR
MS, Fig. 6c, presenting signals at m/z 626.35853 and 648.3405,
detected as protonated ion and sodium adduct ([M + H]+ and [M
+ Na]+, respectively) in which M = C38H47N3O5 and DBE = 17. The
product of the reaction between CBN and FBBBS is also
detected, in lower abundance, as the ion at m/z 622.3276 and
M = C38H42N3O5, corroborating with the TLC data, Fig. 3b.
The reaction mechanism of formation of the product identified
in Fig. 6c (M = C38H47N3O5 and DBE = 17) is elucidated in Fig. 7. The
chemical structure corroborates with the proposal by Kovar from
1989 [9]. The mechanism is described basically in four main steps.
The first one is an acid-base reaction, which the phenolic group
from D9-THC is converted to phenolate ion through the reaction
in basic medium producing a water molecule. The phenolate anion
has its negative charge stabilized by resonance effect (step I). Then,
the electrons of the phenolate anion of D9-THC attack the elec-
trophile, diazo group of FBBBS molecule (step II), in a slow step
of the reaction (rate-determining, step III), producing a non-
aromatic intermediate, structure 1, identified in Fig. 6c. The com-
pound 1 is converted in 2 and in 3 by tautomerization (steps IV
and V).
CID experiments were performed with the aim of confirming
the structure and connectivity of the reagent FBBBS (m/z 312)
and of the compound at m/z 626 (structures 1, 2 or 3) shown in
Fig. 6b–c, respectively. Fig. 8a shows the ESI(+)MS/MS spectra for
the [C17H18N3O3]+ ion at m/z 312, related to FBBBS molecule.
Fig. 9 represents the proposals of fragmentation mechanism for
this ion. The evidence of diazo group (N2-R) in the structure is con-
firmed from the transition described at step I, 312 ? 284 (related
to the loss of N2). The transitions 284 ? 256 and 266 ? 228 refer
to the loss of CH2 = CH2 through hydrogen rearrangement. The
transition 312 ? 266, reported in step II, indicates the elimination
of a CH3CH2OH group, whereas the loss of 57 Da stated in step III is
characterized by the simultaneous elimination of N2 and CH2CH3
groups.
Fig. 8b displays the ESI(+)MS/MS spectrum for the
[C38H47N3O5 + H]+ ion of m/z 626. Note that the CID experiment
revealed two main fragments at m/z 300 and 271 and others with
lower intensities at m/z 581, 552 and 524. Fig. 10 illustrates the
 N.A. dos Santos et al. / Forensic Chemistry 1 (2016) 13–21
fragmentation mechanism proposed for this ion of m/z 626 from
the MS/MS results. As described in step I, the fragment at m/z
300 was generated from the elimination of C21H28O2N group
(326 Da) through the homolytic cleavage between the two nitro-
gen atoms. The fragment at m/z 271 came from the loss of CH2CH3
group (29 Da) of fragment of m/z 300. The fragments at m/z 581,
552 and 524 are explained by the loss described in step II: loss of
 bridge, isolated from Cannabis sativa, Tetrahedron Lett. 53 (2012) 3560–3562.
OCH2CH3 (45 Da) radical, related to the transition 626 ? 581;
elimination of CH2CH3 (29 Da) radical, associated with the transi-
tion 581 ? 552; and elimination of CO (28 Da), related to the tran-
sition 552 ? 524, respectively.
5. Conclusion
The products of the colorimetric test, reaction between Fast Blue
BB salt (FBBBS) and D9-THC – present in marijuana samples – were
identified using ESI(+)-FT-ICR MS. The colorimetric test using the
reagent FBBBS shows a reddish color when in presence of cannabi-
noids in samples of C. sativa L., and the selectivity of this reagent is
higher than for the Fast Blue B salt (FBBS) in presence of interfer-
ences such as J. decurrens Cham and P. cupana Kunth. The positive
result for the colorimetric test using the reagent FBBBS was proved
with UV–Vis analyses due to the appearance of a band in the visible
region at approximately 471 nm.
The combination of TLC analyses (cyclohexane:toluene:diethyla
mine system 75:15:10 % v/v) with the reagent FBBBS allowed the
revelation and identification of the three main cannabinoids pre-
sent in marijuana: D9-THC, cannabinol (CBN) and cannabidiol
(CBD).
The products of the reaction between D9-THC and CBN
(C21H30O2) with the reagent FBBBS (C17H18N3O3), were identified
with the ESI(+)-FT-ICR MS technique, in which the signals at m/z
622.3276 and 626.35853 were detected in as protonated forms
([M + H]+) of the molecules with M = C38H43N3O5 and
C38H47N3O5. According to the ESI(+)-FT-ICR MS and ESI(+)MS/MS
results, a chemical mechanism was proposed for this reaction, thus
justifying the specificity and selectivity of this colorimetric assay.
Acknowledgments
The authors thank the Núcleo de Competências em Química do
Petróleo (UFES), coordinated by prof. Dr. Eustáquio V. R. Castro, for
the UV–VIS and ESI(+)-FT-ICR MS analyses, and FAPES
(65921380/2013), CAPES (23038.007083/2014-40), and CNPq
(445987/2014-6) for their financial support.
References
[1] United Nations Office On Drugs And Crime – UNODC, 2014 – Vienna. World
Drug Report. <http://www.unodc.org/wdr2014/> (accessed 20.01.2016).
[2] M.R. Boleda, M.T. Galceran, F. Ventura, Trace determination of cannabinoids
and opiates in wastewater and surface waters by ultra-performance liquid
21
chromatography–tandem mass spectrometry, J. Chromatogr. A 1175 (2007)
38–48.
[3] Relatório anual Departamento de Polícia Federal. Relatório de atividades –
2008. <http://www.dpf.gov.br/institucional/relatorio-anual-pf/> (accessed
01.03.2016).
[4] M.A. Elsohly, D. Slade, Chemical constituents of marijuana: the complex
mixture of natural cannabinoids, Life Sci. 78 (2005) 539–548.
[5] F. Zulfiqar, S.A. Ross, D. Slade, S.A. Ahmed, M.M. Radwan, Z. Ali, I. Khan, M.A.
Elsohyl, Cannabisol, a novel D-9-THC dimer possessing a unique methylene
[6] M. Passagli, Toxicologia Forense: Teoria e Prática, second ed., Millennium,
Campinas, São Paulo, 2009, pp. 50–180.
[7] United Nations Office On Drugs And Crime – UNODC, 2009. Recommended
methods for the identification and analysis of cannabis and cannabis products.
Manual for use by national drug analysis laboratories. <http://www.unodc.org/
unodc/en/data-and-analysis/WDR-2009.html> (accessed 22.01.2016).
[8] R. Mechoulan, S. Bem-Shabat, Cannabidiol: an overview of some chemical and
pharmacological aspects. Part I: chemical aspects of CPL, Chem. Phys. Lipids
121 (2002) 35–43.
[9] K.A. Kovar, M. Laudszun, Chemistry and Reaction Mechanisms of Rapid Tests
for Drugs of Abuse and Precursors Chemicals. Pharmazeutisches Institut der
Universitat Tubingen Auf der Morgenstelle S, Tubingen Federal Republic of
Germany, 1989. <http://www.unodc.org/pdf/scientific/SCITEC6.pdf> (accessed
02.01.2016).
[10] D.C. Bordin, M. Messias, R. Lanaro, S.O.S. Cazenave, J.L. Costa, Análise forense:
pesquisa de drogas vegetais interferentes de testes colorimétricos para
identificação dos canabinoides da maconha (Cannabis sativa L.), Quím. Nova
35 (2010) 2040–2043.
[11] B.W. Bertoni, M.P.C. Telles, M.G. Malosso, S.C.Z. Torres, J.O. Pereira, Genetic
diversity in natural populations of Jacaranda decurrens Cham. determined
using RAPD and AFLP markers, Genet. Mol. Biol. 33 (2010) 532–538.
[12] E.M. Kuskoski, F. Roseane, A.A. Garcia, A.M. Troncoso, Chemical and
pharmacological properties of the fruit guaraná (Paullinia cupana), Vitae 12
(2005) 45–52.
[13] T.C. Carvalho, I.F. Oliveira, L.V. Tose, G. Vanini, J.B. Kill, A. Neto, L.F. Machado, J.
C. Laboissiere, V.L. Júnior, B.G. Vaz, W. Romão, Qualitative analysis of designer
drugs by paper spray ionisation mass spectrometry (PSI-MS), Anal. Methods 8
(2016) 614–620.
[14] I.R. Nascimento, H.B. Costa, L.M. Souza, L.C. Soprani, B.B. Merlo, W. Romão,
Chemical identification of cannabinoids in street marijuana samples using
electrospray ionization FT-ICR mass spectrometry, Anal. Methods 7 (2015)
1415–1424.
[15] T.M.C. Pereira, G. Vanini, E.C.S. Oliveira, F.M.R. Cardoso, F.P. Fleming, A.C. Neto,
V.L. Junior, E.V.R. de Castro, B.G. Vaz, W. Romão, Fuel 118 (2014) 348–354.
[16] C.A. Destefani, L.C. Motta, G. Vanini, L.M. Souza, J.F.A. Filho, C.J. Macrino, E.M.
Silva, S.J. Greco, D.C. Endringer, W. Romão, Europium-organic complex as
luminescent marker for the visual identification of gunshot residue and
characterization by electrospray ionization ft-icr mass spectrometry,
Microchemical J. 116 (2014) 216–220.
[17] H.B. Costa, L.M. Souza, L.C. Soprani, B.G. Oliveira, E.M. Ogawa, A.M.N. Korres, J.
A. Ventura, W. Romão, Monitoring the physicochemical degradation of
coconut water using ESI-FT-ICR MS, Food Chem. 174 (2014) 139–146.
[18] Rudolf Brenneisen Chemistry and Analysis of Phytocannabinoids and Other
Cannabis Constituents. In: Mahmoud A. ElSohly (Ed.), Marijuana and the
Cannabinoids, New Jersey, 2007.
[19] United Nations Office on Drugs and Crime – UNODC. Recommended methods
for the identification and analysis of cannabis and cannabis products. New
York. <http://www.unodc.org/documents/scientific/ST-NAR-40-Ebook.pdf>
(accessed 01.01.2016).
[20] S. Broecker, F. Pragst, Isomerization of cannabidiol and D9-
tetrahydrocannabinol during positive electrospray ionization. In-source
hydrogen/deuterium exchange experiments by flow injection hybrid
quadrupole-time-of-flight mass spectrometry, Rapid Commun. Mass
Spectrom. 216 (2012) 1407–1414.
[21] T.J. Kauppila, V. Arvola, M. Haapala, J. Pól, L. Aalberg, V. Saarela, S. Franssila, T.
Kotiaho, R. Kostiainen, Direct analysis of illicit drugs by desorption
atmospheric pressure photoionization, Rapid Commun. Mass Spectrom. 22
(2008) 979–985.