- Email: [email protected]
Brephedrone: A new psychoactive substance seized in Brazil
Accepted Manuscript Title: Brephedrone: a new psychoactive substance seized in Brazil Authors: Yuri Machado, Jos´e Coelho Neto, Paulo Eduardo Nunes Barbosa, Rog´erio Ara´ujo Lordeiro, Rosemeire Brondi Alves PII: DOI: Reference:
S0379-0738(17)30144-5 http://dx.doi.org/doi:10.1016/j.forsciint.2017.04.007 FSI 8820
To appear in:
FSI
Received date: Revised date: Accepted date:
11-1-2017 27-3-2017 8-4-2017
Please cite this article as: Yuri Machado, Jos´e Coelho Neto, Paulo Eduardo Nunes Barbosa, Rog´erio Ara´ujo Lordeiro, Rosemeire Brondi Alves, Brephedrone: a new psychoactive substance seized in Brazil, Forensic Science Internationalhttp://dx.doi.org/10.1016/j.forsciint.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Brephedrone: a new psychoactive substance seized in Brazil Yuri Machadoa,c,*, José Coelho Netoa,b, Paulo Eduardo Nunes Barbosaa, Rogério Araújo Lordeiroa, Rosemeire Brondi Alvesc a
Divisão de Laboratório, Instituto de Criminalística da Polícia Civil de Minas Gerais, Rua Juiz de Fora, 400, CEP 30180-060 Belo Horizonte, MG, Brazil b Departamento de Física e Química, Pontifícia Universidade Católica de Minas Gerais, Avenida Dom José Gaspar, 500, CEP 30535-901 Belo Horizonte, MG, Brazil c Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901 Belo Horizonte, MG, Brazil
* Corresponding author at: Divisão de Laboratório, Instituto de Criminalística da Polícia Civil de Minas Gerais, Rua Juiz de Fora, 400, CEP 30180-060 Belo Horizonte, MG, Brazil. Tel.: 055 31 3330 1773; fax: 055 31 3330 1768. Email address: [email protected].
HIGHLIGHTS Brephedrone, a synthetic cathinone, was identified in street seized samples. Sample characterization was made using ATR-FTIR, GC—MS, LC—MS/MS and NMR. Brephedrone had never been reported in Brazil before this seizure.
ABSTRACT At the beginning of 2015, sixty-two capsules containing red-brown crystals seized in a historical city in Brazil were sent to this forensic laboratory for drug testing analysis. The material was identified as being Brephedrone, a new psychoactive substance and a bromine synthetic cathinone that it is related to serotonin transportation. This substance was analyzed by ATR-FTIR, GC-MS, LC-MS, 1H,
13
C and 2D NMR. Brephedrone apprehensions have been previously reported in
Finland, France and Spain. It was the first detection of this substance in the State of Minas Gerais. No reports or information regarding any other apprehension nor identification of Brephedrone in Brazil were known prior to the present case.
KEYWORDS: brephedrone, 4-bromomethcathinone, 4-BMC, synthetic cathinone, new psychoactive substance
1. Introduction Abusive use of controlled and proscribed substances has been, for many decades now, both a public health and security problem all over the world. In Brazil, the listing of those substances is done by ‘Agência Nacional de Vigilância Sanitária’ (ANVISA), a federal agency responsible for regulating the pharmaceutical market, among other attributions. The roll of controlled and proscribed substances is published in ‘Portaria 344/98’, a document issued and updated regularly by ANVISA [1,2]. The growing number of New Psychoactive Substances (NPS), designation used as a reference to chemical compounds sold on the streets or available online and which are able to induce effects like those of caused by ecstasy, methamphetamine and other illicit drugs, presents a challenge do agencies such as ANVISA, as new unidentified substances appear on the market almost on a daily basis, seeking to a breach though legal control mechanisms [3–6]. One of the most common types of NPS is the synthetic cathinones class, derived from the structure of the cathinone molecule (FIGURE 1), a phenethylamine found on the Catha edulis plant [7–9]. These substances are known on the illicit drug market by names like Khat, Meow Meow, bath salts, MCAT and many others [10]. According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), 103 new cathinones have been identified from 2004 to 2016 in Europe [11]. According to the Drug Enforcement Administration (DEA), from 2013 to 2015, the 20 most frequent synthetic cathinones generated 51,284 reports and 35 new ones were reported for the first time in 2015 [12]. Like other NPSs, the knowledge about effects and risks associated to the use of such substances are very limited and usually restricted to cells and animals. The common side effects, reported by users, include cardiovascular, psychiatric and neurological signs and symptoms [13].
Brephedrone (FIGURE 1), also known as 4-BMC, 4-bromomethcathinone or 1-(4bromophenyl)-2-(methylamino)propan-1-one, is a synthetic cathinone reported to have been sold on the illicit drug markets of Spain, France and Finland [14–16].
2
Available preliminary studies suggested that Brephedrone is able to inhibit serotonin transport, acting as an antidepressant [17,18]. Other recent works seek to elucidate its effect on the dopamine and serotonin transport [19,20]. In this work, we report our findings about the characterization of a sample seized on the streets of the historical city of Ouro Preto, Brazil, found to contain Brephedrone. At the time of the seizure (August 2015), Brephedrone has not been included in ‘Portaria 344/98’ yet, therefore, it was not under legal control in Brazil. As of August 31, 2016, ‘Portaria 344/98’ was updated by ANVISA, including Brephedrone as a proscribed substance [21].
2. Material and methods 2.1. Case history On February 8, 2015, a bottle containing gelatinous capsules (FIGURE 2) was received by our forensic laboratory for drug testing. The bottle contained 62 capsules, with a mass of 10 g total, with red-brown crystals inside. Such material was seized by Minas Gerais state police at historical city of Ouro Preto, Brazil.
2.2. Chemicals
Ammonium hydroxide, acetonitrile (ACN), formic acid, methanol and acetone were purchased from Synth (Diadema, SP, Brazil). Methanol-d4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrapure water was obtained by Milipore Direct Q3.
2.3. Purification
Routine tests (infrared analysis and gas chromatography-mass spectrometry) made on the crystals indicated the presence of an unknown substance, never detected before in our laboratory. In order to allow the unique identification and characterization of the compound present on the
3
original crystals, the material was purified by recrystallization, following the procedure described below. The crystals were removed from the capsules, resulting in net mass of 3.3g, which was transferred to a beaker and solubilized in warm methanol. Enough methanol was used to ensure complete solubilization of the crystals. The solution was filtered on qualitative filter paper and 50 mL of acetone was added. The resulting suspension was kept under refrigeration at -4 ° C for 24 hours and then was again filtered. The retained solid was recrystallized using the same procedures. At the end of the purification process, 2.2g of a white crystalline solid was obtained. 2.4. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis ATR-FTIR spectra was taken using a NicoletTM iZ10 Spectrometer equipped with EverGlo IR source, DLaTGS room temperature IR detector and single-bounce Smart OrbitTM accessory module with diamond ATR crystal. All hardware from Thermo Fischer Scientific Inc. (USA). Each spectrum was averaged over 16 scans, taken at 4 cm-1 resolution, with maximum detector window aperture and minimum interferometer mirror speed, in the range of 400 - 4000 cm-1.
2.5. Gas chromatography coupled with mass spectrometry (GC-MS) analysis
The sample was prepared for analysis by transferring 1 mg of the white solid to a microcentrifuge tube and adding 1 mL of HPLC grade methanol. GC-MS analysis was performed on an Agilent GC 7890A coupled with an Agilent XL MSD 5975C quadrupole mass spectrometer. The chromatographic separation was performed with a 433 HP-5MS capillary column (30 m x 250 µm x 0.25 µm). A volume of 1 µL of the sample solution was injected with a split ratio of 75:1. Helium was used as carrier gas with a constant flow of 1.0 mL/min. The temperature of the GC-MS interface and the injector was set at 280°C and 250°C, respectively, and the oven temperature was programmed as follows: 50°C for 1 min, increased to 270°C at 25°C/min and holding at 270ºC for 2 min (total run time: 11.8 min). The m/z scan range wat set to 35-550.
4
2.6. Liquid Chromatograph accoupled with mass spectrometry (LC-MS)
LCMS analyses were performed on a Shimadzu LCMS 8030 triple quadrupole mass spectrometer with ESI source (Shimadzu Corporation, Kyoto, Japan) coupled with UPLC Shimadzu UPLC CLASS 8030 binary Liquid Chromatography System. LabSolutionsTM was used for data acquisition and processing. Chromatographic separations were performed by Shimadzu Shim-pack C18 column (30x100 mm, 2.2 μm in particle size) with isocratic elution at a total flow rate of 0.4 mL/min composed of 0.1% formic acid and 0.2% ammonium hydroxide in water (Mobile Phase A) at a flow rate of 0.2 mL/min and 0.1% formic acid, 0.2% ammonium hydroxide and 5% water in acetonitrile (Mobile Phase B) at a flow rate of 0.2 mL/min. The oven temperature was set at 40 ºC. The conditions for mass spectrometry were optimized as follow: positive mode electrospray ionization (ESI). The conditions of the interface were: DL temperature, 200 °C; Nebulizing Gas flow, 3 L/min; Heat Block temperature, 400 °C; Drying Gas flow, 15 L/min; Collision Induced Dissociation (CID) gas pressure, 230 kPa; Dwell time, 100 ms; and Interface Voltage, 4.5 kV. A Q3 full scan was used over the range of 110 to 400 amu to seek out the ions and a multiple reaction monitoring (MRM) was used after parent ion selection.
2.7. Nuclear magnetic resonance spectroscopy (NMR) analysis The NMR analysis was performed at Universidade Federal de Minas Gerais. A 10 mg of solid was dissolved in 1 mL methanol-d4 with tetramethylsilane (TMS) as internal standard. 1H,
13
C,
DEPT 135 (Distorsionless Enhancement by Polarization Transfer) and HMBC (Heteronuclear Multiple-Bond Correlation) experiments were recorded using Bruker Avance DRX 400 MHz Spectrometer. 3. Results and discussion
3.1. ATR-FTIR analysis The attenuated total reflection infrared (ATR-IR) spectrum of Brephedrone is shown in FIGURE 3. Bands observed at 2915 cm-1 and 2747 cm-1 can be assigned to aryl C-H and alkyl C-
5
H stretch vibration respectively. A strong band observed at 1583 cm-1 can indicate stretch aromatic ring vibrations (C=C). One important feature of cathinones is the conjugation of the carbonyl group with the phenyl group [22]. A strong band observed at 1678 cm-1 can be assigned to C=O stretch vibration. Two bands at 3432 cm-1 and 3362 cm-1 are indicated N-H stretch. The out of plane C–H bending bands in the region of 675–900 cm-1 are frequently used to differentiate between di-substituted aromatic compounds[23]. A strong band at 833 cm-1 indicates C-H out-of-plane bending vibration relating to para-substituted aromatic compound. This bending can be observed in other para-substituted substances such as 4-methylmethcathinone and 4fluoromethcathinone with the values at 827 cm-1 [23] and 847 cm-1 [24] respectively.
3.2. GC-MS analysis
The EI (electron ionization) mass spectrum of the Brephedrone is shown in Fig. 4 and 5. The EI mass spectrum had a main fragment at m/z 58. Additionally, m/z 75, m/z 155 and m/z 183 were found. These four peaks are in agreement with Paillet-Loilier (2014) [25]. The 155 m/z and 183 m/z fragments demonstrate a characteristic isotope pattern indicating one bromine atom present attached to aromatic ring.
3.3. LC-MS analysis The positive full scan mass spectrum is shown in Fig 6 and the MS/MS fragmentation spectrum of Brephedrone is shown in Fig 7. In the TIC at retention time 2.5 min was observed a single peak at m/z 242 and m/z 244 corresponding to the [M+H]+ ions. This characteristic isotope pattern indicating one bromine atom present in the molecule is observed. Five MRM transitions found for Brephedrone are shown in TABLE 1. Fragments 145 m/z and 144 m/z correspond to the found in the work of Adamowicz [26].
6
3.4. NMR analysis The proton and carbon NMR resonance assignments are shown in TABLE 2. The results for the observed hydrogen shifts are similar to those found in the work of Foley [17].
4. Conclusions In this work, a clear identification and characterization of the new psychoactive substance, Brephedrone, by ATR-FT-IR, GC-MS, LC-MS and NMR is presented. This bromine synthetic cathinone was found in the form of crystal solid inside gelatinous capsules that were seized by local police forces. No other substance was identified in the material with the techniques used. To the best of our knowledge, this was the first time Brephedrone was identified in street samples seized in Brazil and Latin America. The presence of this substance in the streets of Brazil, as well as in other parts of the world, is an example illustrating the growing presence of synthetic cathinones as a major class of NPS in the global illicit drugs market
ACKNOWLEDGEMENTS We are very grateful to the criminal expert Frederico Nunes Valadão and graduation student Marina Jurisch for useful discussions. José Coelho Neto is supported by FIPPUC-MG. Rosemeire Brondi Alves is supported by CNPq research fellowship. Special thanks to Sergio Bellas Romariz, director of the Instituto de Criminalística da Polícia Civil de Minas Gerais, who encouraged and supported research efforts within the Institute.
7
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[21]
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[22]
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[23]
J.D. Power, P. McGlynn, K. Clarke, S.D. McDermott, P. Kavanagh, J. O’Brien, The analysis of substituted cathinones. Part 1: Chemical analysis of 2-, 3- and 4-methylmethcathinone, Forensic Sci. Int. 212 (2011) 6–12. doi:10.1016/j.forsciint.2011.04.020.
[24]
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[25]
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[26]
P. Adamowicz, B. Tokarczyk, Simple and rapid screening procedure for 143 new psychoactive substances by liquid chromatography-tandem mass spectrometry, Drug Test. Anal. (2015) 31–33. doi:10.1002/dta.1815.
9
FIGURE 1 - Chemical structures of synthetic cathinones. (two column fitting image
FIGURE 2 – Bottle containing the capsules and one specimen of the capsule. (one column fitting image)
10
FIGURE 3. ATR-IR spectra of 4-BMC. (two column fitting image)
11
FIGURE 4 – GC-MS Total Ion Chromatogram (TIC) of Brephedrone. (two column fitting image)
FIGURE 5 – EI mass spectra of Brephedrone and fragmentation proposal. (two column fitting image)
12
FIGURE 6 –MS spectrum of Brephedrone in the positive ion mode. (one or two column fitting image)
FIGURE 7 – MS/MS spectra of Brephedrone (one or two column fitting image)
13
FIGURE 8 - Brephedrone structure. (two column fitting image)
14
TABLE 1: MRM parameters employed in LC–MS/MS method. Q1, Q1 Pre Bias; V, volt; CE, Collision Energy; Q3, Q3 Pre Bias.
4-BMC
Precursor ion (m/z)
Product ion (m/z)
Q1 (V)
CE (V)
Q3 (V)
242
145
-17
-25
-15
242
144
-17
-35
-27
242
132
-17
-25
-27
242
104
-17
-45
-19
242
103
-17
-50
-19
TABLE 2: 1H and
13
C chemical shift values (δ, ppm) of Brephedrone in CD3OD at 25 ºC with
TMS as internal standard. Only the most important correlations in the HMBC experiment for assignment were indicated (s = singlet; d = doublet; q = quartet; δ = chemical shift in ppm). δC
DEPT 135
δH
HMBC
1
196.5
C
•
H2´/C1
2
60,7
CH
5.18 (q, J = 7.2 Hz, 1H)
H2/C3; H2/C1´
3
16,2
CH3
1.60 (d, J = 7.2 Hz, 3H)
H3/C2; H3/C1
1’
131,1
C
•
•
2’
131,8
CH
8,00 (d, J = 8.5 Hz, 2H)
H2´/C1
3’
133,8
CH
7,78 (d, J = 8.5 Hz, 2H)
•
4’
133,2
C
•
•
1’’
31,9
CH3
2,81 (s, 3H)
•
Number of atom
15
16
S0379-0738(17)30144-5 http://dx.doi.org/doi:10.1016/j.forsciint.2017.04.007 FSI 8820
To appear in:
FSI
Received date: Revised date: Accepted date:
11-1-2017 27-3-2017 8-4-2017
Please cite this article as: Yuri Machado, Jos´e Coelho Neto, Paulo Eduardo Nunes Barbosa, Rog´erio Ara´ujo Lordeiro, Rosemeire Brondi Alves, Brephedrone: a new psychoactive substance seized in Brazil, Forensic Science Internationalhttp://dx.doi.org/10.1016/j.forsciint.2017.04.007 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Brephedrone: a new psychoactive substance seized in Brazil Yuri Machadoa,c,*, José Coelho Netoa,b, Paulo Eduardo Nunes Barbosaa, Rogério Araújo Lordeiroa, Rosemeire Brondi Alvesc a
Divisão de Laboratório, Instituto de Criminalística da Polícia Civil de Minas Gerais, Rua Juiz de Fora, 400, CEP 30180-060 Belo Horizonte, MG, Brazil b Departamento de Física e Química, Pontifícia Universidade Católica de Minas Gerais, Avenida Dom José Gaspar, 500, CEP 30535-901 Belo Horizonte, MG, Brazil c Departamento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, CEP 31270-901 Belo Horizonte, MG, Brazil
* Corresponding author at: Divisão de Laboratório, Instituto de Criminalística da Polícia Civil de Minas Gerais, Rua Juiz de Fora, 400, CEP 30180-060 Belo Horizonte, MG, Brazil. Tel.: 055 31 3330 1773; fax: 055 31 3330 1768. Email address: [email protected].
HIGHLIGHTS Brephedrone, a synthetic cathinone, was identified in street seized samples. Sample characterization was made using ATR-FTIR, GC—MS, LC—MS/MS and NMR. Brephedrone had never been reported in Brazil before this seizure.
ABSTRACT At the beginning of 2015, sixty-two capsules containing red-brown crystals seized in a historical city in Brazil were sent to this forensic laboratory for drug testing analysis. The material was identified as being Brephedrone, a new psychoactive substance and a bromine synthetic cathinone that it is related to serotonin transportation. This substance was analyzed by ATR-FTIR, GC-MS, LC-MS, 1H,
13
C and 2D NMR. Brephedrone apprehensions have been previously reported in
Finland, France and Spain. It was the first detection of this substance in the State of Minas Gerais. No reports or information regarding any other apprehension nor identification of Brephedrone in Brazil were known prior to the present case.
KEYWORDS: brephedrone, 4-bromomethcathinone, 4-BMC, synthetic cathinone, new psychoactive substance
1. Introduction Abusive use of controlled and proscribed substances has been, for many decades now, both a public health and security problem all over the world. In Brazil, the listing of those substances is done by ‘Agência Nacional de Vigilância Sanitária’ (ANVISA), a federal agency responsible for regulating the pharmaceutical market, among other attributions. The roll of controlled and proscribed substances is published in ‘Portaria 344/98’, a document issued and updated regularly by ANVISA [1,2]. The growing number of New Psychoactive Substances (NPS), designation used as a reference to chemical compounds sold on the streets or available online and which are able to induce effects like those of caused by ecstasy, methamphetamine and other illicit drugs, presents a challenge do agencies such as ANVISA, as new unidentified substances appear on the market almost on a daily basis, seeking to a breach though legal control mechanisms [3–6]. One of the most common types of NPS is the synthetic cathinones class, derived from the structure of the cathinone molecule (FIGURE 1), a phenethylamine found on the Catha edulis plant [7–9]. These substances are known on the illicit drug market by names like Khat, Meow Meow, bath salts, MCAT and many others [10]. According to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), 103 new cathinones have been identified from 2004 to 2016 in Europe [11]. According to the Drug Enforcement Administration (DEA), from 2013 to 2015, the 20 most frequent synthetic cathinones generated 51,284 reports and 35 new ones were reported for the first time in 2015 [12]. Like other NPSs, the knowledge about effects and risks associated to the use of such substances are very limited and usually restricted to cells and animals. The common side effects, reported by users, include cardiovascular, psychiatric and neurological signs and symptoms [13].
Brephedrone (FIGURE 1), also known as 4-BMC, 4-bromomethcathinone or 1-(4bromophenyl)-2-(methylamino)propan-1-one, is a synthetic cathinone reported to have been sold on the illicit drug markets of Spain, France and Finland [14–16].
2
Available preliminary studies suggested that Brephedrone is able to inhibit serotonin transport, acting as an antidepressant [17,18]. Other recent works seek to elucidate its effect on the dopamine and serotonin transport [19,20]. In this work, we report our findings about the characterization of a sample seized on the streets of the historical city of Ouro Preto, Brazil, found to contain Brephedrone. At the time of the seizure (August 2015), Brephedrone has not been included in ‘Portaria 344/98’ yet, therefore, it was not under legal control in Brazil. As of August 31, 2016, ‘Portaria 344/98’ was updated by ANVISA, including Brephedrone as a proscribed substance [21].
2. Material and methods 2.1. Case history On February 8, 2015, a bottle containing gelatinous capsules (FIGURE 2) was received by our forensic laboratory for drug testing. The bottle contained 62 capsules, with a mass of 10 g total, with red-brown crystals inside. Such material was seized by Minas Gerais state police at historical city of Ouro Preto, Brazil.
2.2. Chemicals
Ammonium hydroxide, acetonitrile (ACN), formic acid, methanol and acetone were purchased from Synth (Diadema, SP, Brazil). Methanol-d4 was purchased from Sigma-Aldrich (St. Louis, MO, USA). The ultrapure water was obtained by Milipore Direct Q3.
2.3. Purification
Routine tests (infrared analysis and gas chromatography-mass spectrometry) made on the crystals indicated the presence of an unknown substance, never detected before in our laboratory. In order to allow the unique identification and characterization of the compound present on the
3
original crystals, the material was purified by recrystallization, following the procedure described below. The crystals were removed from the capsules, resulting in net mass of 3.3g, which was transferred to a beaker and solubilized in warm methanol. Enough methanol was used to ensure complete solubilization of the crystals. The solution was filtered on qualitative filter paper and 50 mL of acetone was added. The resulting suspension was kept under refrigeration at -4 ° C for 24 hours and then was again filtered. The retained solid was recrystallized using the same procedures. At the end of the purification process, 2.2g of a white crystalline solid was obtained. 2.4. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analysis ATR-FTIR spectra was taken using a NicoletTM iZ10 Spectrometer equipped with EverGlo IR source, DLaTGS room temperature IR detector and single-bounce Smart OrbitTM accessory module with diamond ATR crystal. All hardware from Thermo Fischer Scientific Inc. (USA). Each spectrum was averaged over 16 scans, taken at 4 cm-1 resolution, with maximum detector window aperture and minimum interferometer mirror speed, in the range of 400 - 4000 cm-1.
2.5. Gas chromatography coupled with mass spectrometry (GC-MS) analysis
The sample was prepared for analysis by transferring 1 mg of the white solid to a microcentrifuge tube and adding 1 mL of HPLC grade methanol. GC-MS analysis was performed on an Agilent GC 7890A coupled with an Agilent XL MSD 5975C quadrupole mass spectrometer. The chromatographic separation was performed with a 433 HP-5MS capillary column (30 m x 250 µm x 0.25 µm). A volume of 1 µL of the sample solution was injected with a split ratio of 75:1. Helium was used as carrier gas with a constant flow of 1.0 mL/min. The temperature of the GC-MS interface and the injector was set at 280°C and 250°C, respectively, and the oven temperature was programmed as follows: 50°C for 1 min, increased to 270°C at 25°C/min and holding at 270ºC for 2 min (total run time: 11.8 min). The m/z scan range wat set to 35-550.
4
2.6. Liquid Chromatograph accoupled with mass spectrometry (LC-MS)
LCMS analyses were performed on a Shimadzu LCMS 8030 triple quadrupole mass spectrometer with ESI source (Shimadzu Corporation, Kyoto, Japan) coupled with UPLC Shimadzu UPLC CLASS 8030 binary Liquid Chromatography System. LabSolutionsTM was used for data acquisition and processing. Chromatographic separations were performed by Shimadzu Shim-pack C18 column (30x100 mm, 2.2 μm in particle size) with isocratic elution at a total flow rate of 0.4 mL/min composed of 0.1% formic acid and 0.2% ammonium hydroxide in water (Mobile Phase A) at a flow rate of 0.2 mL/min and 0.1% formic acid, 0.2% ammonium hydroxide and 5% water in acetonitrile (Mobile Phase B) at a flow rate of 0.2 mL/min. The oven temperature was set at 40 ºC. The conditions for mass spectrometry were optimized as follow: positive mode electrospray ionization (ESI). The conditions of the interface were: DL temperature, 200 °C; Nebulizing Gas flow, 3 L/min; Heat Block temperature, 400 °C; Drying Gas flow, 15 L/min; Collision Induced Dissociation (CID) gas pressure, 230 kPa; Dwell time, 100 ms; and Interface Voltage, 4.5 kV. A Q3 full scan was used over the range of 110 to 400 amu to seek out the ions and a multiple reaction monitoring (MRM) was used after parent ion selection.
2.7. Nuclear magnetic resonance spectroscopy (NMR) analysis The NMR analysis was performed at Universidade Federal de Minas Gerais. A 10 mg of solid was dissolved in 1 mL methanol-d4 with tetramethylsilane (TMS) as internal standard. 1H,
13
C,
DEPT 135 (Distorsionless Enhancement by Polarization Transfer) and HMBC (Heteronuclear Multiple-Bond Correlation) experiments were recorded using Bruker Avance DRX 400 MHz Spectrometer. 3. Results and discussion
3.1. ATR-FTIR analysis The attenuated total reflection infrared (ATR-IR) spectrum of Brephedrone is shown in FIGURE 3. Bands observed at 2915 cm-1 and 2747 cm-1 can be assigned to aryl C-H and alkyl C-
5
H stretch vibration respectively. A strong band observed at 1583 cm-1 can indicate stretch aromatic ring vibrations (C=C). One important feature of cathinones is the conjugation of the carbonyl group with the phenyl group [22]. A strong band observed at 1678 cm-1 can be assigned to C=O stretch vibration. Two bands at 3432 cm-1 and 3362 cm-1 are indicated N-H stretch. The out of plane C–H bending bands in the region of 675–900 cm-1 are frequently used to differentiate between di-substituted aromatic compounds[23]. A strong band at 833 cm-1 indicates C-H out-of-plane bending vibration relating to para-substituted aromatic compound. This bending can be observed in other para-substituted substances such as 4-methylmethcathinone and 4fluoromethcathinone with the values at 827 cm-1 [23] and 847 cm-1 [24] respectively.
3.2. GC-MS analysis
The EI (electron ionization) mass spectrum of the Brephedrone is shown in Fig. 4 and 5. The EI mass spectrum had a main fragment at m/z 58. Additionally, m/z 75, m/z 155 and m/z 183 were found. These four peaks are in agreement with Paillet-Loilier (2014) [25]. The 155 m/z and 183 m/z fragments demonstrate a characteristic isotope pattern indicating one bromine atom present attached to aromatic ring.
3.3. LC-MS analysis The positive full scan mass spectrum is shown in Fig 6 and the MS/MS fragmentation spectrum of Brephedrone is shown in Fig 7. In the TIC at retention time 2.5 min was observed a single peak at m/z 242 and m/z 244 corresponding to the [M+H]+ ions. This characteristic isotope pattern indicating one bromine atom present in the molecule is observed. Five MRM transitions found for Brephedrone are shown in TABLE 1. Fragments 145 m/z and 144 m/z correspond to the found in the work of Adamowicz [26].
6
3.4. NMR analysis The proton and carbon NMR resonance assignments are shown in TABLE 2. The results for the observed hydrogen shifts are similar to those found in the work of Foley [17].
4. Conclusions In this work, a clear identification and characterization of the new psychoactive substance, Brephedrone, by ATR-FT-IR, GC-MS, LC-MS and NMR is presented. This bromine synthetic cathinone was found in the form of crystal solid inside gelatinous capsules that were seized by local police forces. No other substance was identified in the material with the techniques used. To the best of our knowledge, this was the first time Brephedrone was identified in street samples seized in Brazil and Latin America. The presence of this substance in the streets of Brazil, as well as in other parts of the world, is an example illustrating the growing presence of synthetic cathinones as a major class of NPS in the global illicit drugs market
ACKNOWLEDGEMENTS We are very grateful to the criminal expert Frederico Nunes Valadão and graduation student Marina Jurisch for useful discussions. José Coelho Neto is supported by FIPPUC-MG. Rosemeire Brondi Alves is supported by CNPq research fellowship. Special thanks to Sergio Bellas Romariz, director of the Instituto de Criminalística da Polícia Civil de Minas Gerais, who encouraged and supported research efforts within the Institute.
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FIGURE 1 - Chemical structures of synthetic cathinones. (two column fitting image
FIGURE 2 – Bottle containing the capsules and one specimen of the capsule. (one column fitting image)
10
FIGURE 3. ATR-IR spectra of 4-BMC. (two column fitting image)
11
FIGURE 4 – GC-MS Total Ion Chromatogram (TIC) of Brephedrone. (two column fitting image)
FIGURE 5 – EI mass spectra of Brephedrone and fragmentation proposal. (two column fitting image)
12
FIGURE 6 –MS spectrum of Brephedrone in the positive ion mode. (one or two column fitting image)
FIGURE 7 – MS/MS spectra of Brephedrone (one or two column fitting image)
13
FIGURE 8 - Brephedrone structure. (two column fitting image)
14
TABLE 1: MRM parameters employed in LC–MS/MS method. Q1, Q1 Pre Bias; V, volt; CE, Collision Energy; Q3, Q3 Pre Bias.
4-BMC
Precursor ion (m/z)
Product ion (m/z)
Q1 (V)
CE (V)
Q3 (V)
242
145
-17
-25
-15
242
144
-17
-35
-27
242
132
-17
-25
-27
242
104
-17
-45
-19
242
103
-17
-50
-19
TABLE 2: 1H and
13
C chemical shift values (δ, ppm) of Brephedrone in CD3OD at 25 ºC with
TMS as internal standard. Only the most important correlations in the HMBC experiment for assignment were indicated (s = singlet; d = doublet; q = quartet; δ = chemical shift in ppm). δC
DEPT 135
δH
HMBC
1
196.5
C
•
H2´/C1
2
60,7
CH
5.18 (q, J = 7.2 Hz, 1H)
H2/C3; H2/C1´
3
16,2
CH3
1.60 (d, J = 7.2 Hz, 3H)
H3/C2; H3/C1
1’
131,1
C
•
•
2’
131,8
CH
8,00 (d, J = 8.5 Hz, 2H)
H2´/C1
3’
133,8
CH
7,78 (d, J = 8.5 Hz, 2H)
•
4’
133,2
C
•
•
1’’
31,9
CH3
2,81 (s, 3H)
•
Number of atom
15
16