Introduction

New psychoactive substances (NPS) have become a global and continuously growing phenomenon over the last decade, posing a risk to public health while remaining a challenge to law enforcement agencies [1, 2]. From 2009 to 2017, over 800 NPS have been reported in 111 countries and territories, most of them being classified as phenethylamines, synthetic cathinones or synthetic cannabinoids [3].

Phenethylamines are agonists of the serotonin (5-HT, 5-hydroxytryptamine) receptors [4]. The N-benzyl substitution in phenethylamines (usually from the 2C-X family, such as 2C-I and 2C-B) improve the binding and functional activity in 5-HT receptors, with N-(2-hydroxybenzyl) substituted substances usually presenting the highest affinities to the \(\hbox {5-HT}_{\mathrm{2A}}\) receptor [4,5,6].

Since 2015, a new NPS group based on N-benzyl-2,5-dimethoxyphenethylamines (Fig. 1), commonly described as NBOHs, emerged as recreational drugs. NBOHs, substances structurally closely related to NBOMes, are usually found on blotter papers and have become an option for LSD (lysergic acid diethylamide) users. Self-reported use of NBOHs has been reported in United States [7], Australia [8], and Germany [9]. These substances were already identified for street samples seized in Brazil [10], Finland, Germany, and Slovenia [11].

Fig. 1
figure 1

Structures of N-benzylphenethylamines: 2Cs, 2C-I, 2C-B, and 2C-C; NBOMes 25I-NBOMe, 25B-NBOMe, and 25C-NBOMe; NBOHs 25I-NBOH, 25B-NBOH, 25C-NBOH, 25H-NBOH, and 25E-NBOH

Full size image

Like most NPS, knowledge about the toxicology of NBOHs is very limited or virtually nonexistent. According to information gathered from user forums (not to be considered as canon), the effects include tachycardia, and auditory and visual hallucinations [12, 13]. While both NBOHs and NBOMes apparently taste bitter, thus providing a simple way for users to distinguish them from LSD [14], NBOHs are reported to induce weaker effects and to last less than NBOMes, being unaccompanied with “body load”, an unpleasant physical sensation reported by drug users [14].

Together with usual difficulties associated with the identification of NPS (unknown substance, never before reported, not present in reference spectral libraries, no certified standards available, etc), detection of NBOHs present yet another important trait: thermal degradation under standard gas chromatography, which seriously increases the risk of misidentification [15, 16] and demands the development of other detection methods [17,18,19,20,21].

Herein, we report our efforts for the identification and structural elucidation of three new NBOHs detected in seized samples submitted to our forensic laboratory: 25B-NBOH, 25C-NBOH and 25E-NBOH.

Materials and methods

Chemicals and reagents

Acetone, chloroform, diethyl ether, methanol, sodium hydroxide, and hydrochloric acid were purchased from Synth (Diadema, SP, Brazil); methanol LC grade from Merck (Darmstadt, Germany); ammonium formate and methanol-\(\textit{d}_{4}\) with 99.8% isotopic purity, containing 0.03% (v/v) tetramethylsilane (TMS), from Sigma-Aldrich (St. Louis, MO, USA); the ultrapure water from Milipore Direct Q3 (Billerica, MA, USA); silica gel 60 (70–230 mesh) from Merck.

Samples and extraction

Blotter papers suspected to contain illicit substances were seized between 2017 and 2018 by police forces from Minas Gerais. These blotters usually represented a colorful side including drawings of cartoon and game characters, scientists, deities, etc. and a white side (Fig. 2). The samples were grouped according to previous routine analysis, such as gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), as follows: one hundred blotter papers for samples named as 01 and 02, and twenty-five blotter papers for sample named as 03.

Fig. 2
figure 2

Appearances of blotter papers containing illicit substances

Full size image

Each sample group (01, 02, and 03) was submitted to three successive extractions: 30 mL of chloroform (2 \(\times\)) and 30 mL of methanol in beakers by 20 min in magnetic stirrer. After mixing the 90 mL of solvent extracted, 1 mL of NaOH solution (1%, w/v) was added. Each solution was evaporated to dryness getting a solid reddish residue. Each residue was submitted to column chromatography using silica gel as stationary phase. The elution was performed with chloroform, acetone, and methanol, in increasing polarity order, providing the corresponding fractions. The acetone fraction was evaporated to dryness and 20 mL of saturated solution of hydrochloric acid in diethyl ether was added to the residue, providing, after new evaporation to dryness, a beige solid labeled as compound 01 (15.1 mg), compound 02 (13.3 mg), and compound 03 (4.5 mg).

Instrumentation

Attenuated total reflectance Fourier transform infrared spectroscopy analysis

Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) analyses were performed directly from the solids, except for compound 03 due to the small amount available. They were obtained using a NicoletTM iZ10 spectrometer equipped with EverGlo infrared (IR) source, DLaTGS room temperature IR detector under \(\hbox {N}_2\) purge and single-bounce Smart \(\hbox {Orbit}^{\mathrm{TM}}\) accessory module with diamond ATR crystal (Thermo Fischer Scientific, Madison, WI, 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\(^{-1}\).

Gas chromatography–mass spectrometry analysis

Samples were prepared by transferring 1 mg of solid to a microcentrifuge tube and adding 1 mL of LC grade methanol. Each solution was transferred to a vial for analysis. Analysis were performed on an Agilent GC 7890A (Agilent Technologies, Santa Clara, CA, USA) coupled with an Agilent XL MSD 5975C quadrupole mass spectrometer. The chromatographic separation was performed with a 433 HP-1MS capillary column (30 m \(\times\) 0.25 mm, i.d., film thickness 0.25 \(\upmu\)m; Agilent Technologies). A volume of 1 \(\upmu\)L of the sample solution was injected with a split ratio of 20:1. Helium was used as carrier gas with a constant flow of 1.0 mL min\(^{-1}\). The temperature of injector and the GC–MS interface was set at 280 and \(300\,^{\circ }\hbox {C}\), respectively. The oven temperature was initially maintained at \(150\,^{\circ }\hbox {C}\) for 1.5 min; increased to \(250\,^{\circ }\hbox {C}\) at \(30\,^{\circ }\hbox {C min}^{-1}\) and maintained for 1 min; increased to \(300\,^{\circ }\hbox {C}\) at \(50\,^{\circ }\hbox {C min}^{-1}\) and maintained for 3 min (total run time: 11.8 min). The solvent delay was set to 1.5 min. The mass scan range was set to m/z 35–550.

Liquid chromatography coupled to tandem mass spectrometry analysis

LC–MS/MS analyses were performed on a Prominence UFLC system (Shimadzu Corporation, Kyoto, Japan) coupled with a Shimadzu LCMS 8030 triple quadrupole mass spectrometer with electrospray ionization source (ESI) set in the positive ionization mode. LabSolutionsTM (Shimadzu Corporation) was used for data acquisition and processing.

Direct flow injections were performed with a total flow rate of 0.4 mL min\(^{-1}\). The mobile phase consisted of 50% of ultra-pure water (A) and 50% of acetonitrile (B), both containing ammonium formate (0.024%, w/v). The injection volume was 1 \(\upmu\)L and the total run time was 1 min.

The MS conditions were as follows: desolvation line temperature, \(250\,^{\circ }\hbox {C}\); heat block temperature, \(400\,^{\circ }\hbox {C}\); nebulizing gas flow, 3 L \(\hbox {min}^{-1}\); drying gas flow, \(\hbox {15 L min}^{-1}\); collision-induced dissociation (CID) gas pressure, 230 kPa; dwell time, 100 ms; and interface voltage, 4.5 kV. A Q3 full scan were set from m/z 110 to 500 to seek out the protonated molecule ([M+H]\(^{+}\)), and product ion scan (PIS) were performed with collision energies of − 10, − 20, − 30, and − 40 eV for the purpose of analyzing the fragmentation pattern.

Nuclear magnetic ressonance spectroscopy analysis

The nuclear magnetic resonance (NMR) experiment was conducted using 12 mg of compound 01, 12 mg of compound 02 and 3.5 mg of compound 03. Each solid was dissolved in 0.6 mL methanol-\(\textit{d}_{4}\) with tetramethylsilane (TMS) as internal standard. \(^{1}\)H, \(^{13}\)C, DEPT 135 (Distortionless Enhancement by Polarization Transfer), HSQC (Heteronuclear Single Quantum Coherence) and HMBC (Heteronuclear Multiple-Bond Correlation) experiments were recorded using a Bruker Avance DRX 600 MHz spectrometer (Bruker, Ettlingen, Germany). Acquisition was conducted by TopSpin software 4.0.6 version (Bruker) and processing was conduct by MestReNova 12.0 (MestreLab Research, Santiago de Compostela, Spain).

Results

ATR-FTIR analysis

ATR-FTIR spectra of compounds 01 and 02 indicated the presence of small impurities that were not eliminated during the purification processes. The single infrared spectral profile analysis using available forensic libraries (SWGDRUG and Europe Response infrared library) indicated presence of signals matching with NBOH drugs [22, 23]. Interpretation of the ATR-FTIR spectra indicated the same findings from our previous work [15]: the higher degree of similarity with 25R-NBOMe when compared with 2C-R and a systematic decrease of the spectral band centered around 1250 cm\(^{-1}\), characteristic of asymmetric C–O–C vibrations of NBOMe compounds, indicating the substitution of \(\hbox {OCH}_3\) by OH (Fig. 3).

Fig. 3
figure 3

Comparison of infrared (IR) spectrograms for 2C-E (from SWGDRUG IR library), 25E-NBOMe (from SWGDRUG IR Library version 2.0), and compound 01, identified as 25E-NBOH. The arrows show the decrease of spectral band indicating the substitution of \(\hbox {OCH}_3\) by OH in the 25E-NBOH

Full size image

GC–MS analysis

The GC–MS analysis of compounds 01, 02 and 03 (Fig. 4) indicated the presence of, respectively, 2C-E (RT: retention time = 4.58 min), 2C-C (RT = 4.85 min) and 2C-B (RT = 5.15 min) as most intense peaks according to mass spectra library available (Table 1). The mass spectra for the secondary peaks (RTs = 2.33, 2.34, and 2.31 min, respectively; Fig. 5) were all similar but they had no reasonable match on MS libraries currently. These results have already been predicted for other NBOHs corroborating the finding that this group of substances are thermolabile under the GC temperature conditions [16]. Considering the results of mass spectrum libraries and the presence of secondary peaks, there was an indication that substances 01, 02 and 03 were 25E-NBOH, 25C-NBOH, and 25B-NBOH, respectively. The thermal fragmentation reactions and proposed mass fragmentation pathways for these substances are presented in Fig. 6 according to the previous works [16, 24].

Fig. 4
figure 4

Total ion chromatograms by gas chromatography–mass spectrometry (GC–MS) for samples 01 (25E-NBOH), 02 (25C-NBOH) and 03 (25B-NBOH), and respective mass spectra of the main peaks

Full size image
Table 1 Gas chromatography–mass spectrometry ion data for analyzed samples
Full size table
Fig. 5
figure 5

GC–MS spectra of secondary peaks

Full size image
Fig. 6
figure 6

Thermal fragmentation reaction produced by GC–MS, and proposed mass fragmentation pathways for 25R-NBOH/2C-R

Full size image

LC–MS/MS analysis

The positive full scan and PIS mass spectra of compounds 01, 02, and 03 are presented in Fig. 7. For compound 02, the relative intensity of the ion at m/z 322 was 3:1 of signal at m/z 324. For compound 03, the relative intensity of the ion at m/z 368 was 1:1 of signal at m/z 370. These isotopologue patterns indicate the presence of chlorine and bromine atoms in chemical structures of 02 and 03, respectively. The precursor ion and product ions of the analyzed substances (Fig. 8) are compatible with mass fragmentation pathway proposed in the previous works [15, 25] and can be summarized as shown in Table 2.

Fig. 7
figure 7

Full scan mass spectra (a) and product ion spectra of 01 (25I-NBOH), 02 (25C-NBOH), and 03 (25B-NBOH) at different collision energies of 10 (b), 20 (c), 30 (d), and 40 eV (e) recorded by liquid chromatography–tandem mass spectrometry (LC–MS/MS)

Full size image
Fig. 8
figure 8

Proposed mass fragmentation pathways for 25R-NBOH produced by LC–MS/MS

Full size image
Table 2 Precursor and product ion data by liquid chromatography–tandem mass spectrometry for analyzed samples
Full size table

NMR analysis

NMR spectroscopic results of 01, 02, and 03 are presented in Tables 3, 4, and 5, respectively. Minor impurities were detected in the three spectra without prejudice to the data interpretation. All experiments (\(^{1}\)H, \(^{13}\)C, DEPT 135, HSQC and HMBC) were used for characterizing 01 and 02. However, due to the limited amount of 03, \(^{13}\)C signals were assigned only by HSQC and HMBC experiments.

Table 3 \(^{1}\)H and \(^{13}\)C nuclear magnetic resonance (NMR) spectral data for compound 01 (25E-NBOH) obtained at 600 MHz (methanol-\(\textit{d}_{4}\))
figure a
Full size table
Table 4 \(^{1}\)H and \(^{13}\)C NMR spectral data for compound 02 (25C-NBOH) obtained at 600 MHz (methanol-\(\textit{d}_{4}\))
figure b
Full size table
Table 5 \(^{1}\)H and \(^{13}\)C NMR spectral data for compound 03 (25B-NBOH) obtained at 600 MHz (methanol-d4 )
figure c
Full size table

It was observed thirteen types of H-signal (two of these signals were overlapped) and nineteen types of C-signal for compound 01; eleven types of H-signal and seventeen types of C-signal for compound 02 and 03. In general, these signals, supported by HSQC and HMBC experiments, were attributed to one 1,2,4,5-tetra-substituted aromatic ring containing two methoxy, one ethylmethylamine group and an ethyl (01), chlorine (02) or bromine group (03). It was observed that the amine group was attached to one (2-hydroxyphenyl)methyl group. Based on structural elucidation using NMR, compound 01 was identified as 2-(((4-ethyl-2,5-dimethoxyphenethyl)amino)methyl)phenol (25E-NBOH, 2C-E-NBOH), compound 02 as 2-(((4-chloro-2,5-dimethoxyphenethyl)amino)methyl)phenol (25C-NBOH, 2C-C-NBOH), and compound 03 as 2-(((4-bromo-2,5-dimethoxyphenethyl)amino)methyl)phenol (25B-NBOH, 2C-B-NBOH). These data were in agreement with those observed by ATR-FTIR, GC–MS and LC–MS/MS.

NBOH outlook

The number of identifications of N-benzylphenethylamines (NBOMes and NBOHs) and LSD by our laboratory in seized samples between 2014 and the first semester of 2019 is shown in Fig. 9a, updating our previous work [10]. NBOMes dominated the blotter paper drug market up to the beginning of 2016. Starting at this point, NBOH identification has increased, overcoming NBOMes at the second semester of 2016. In contrast, the number of NBOMes identifications has decreased and are, nowadays, less significant than LSD (305 identifications of NBOHs, 86 of LSD and 20 of NBOMes, between July 2018 and June 2019).

Fig. 9
figure 9

Changes of N-benzylphenethylamine identification between first trimester (T) of 2014 and second trimester of 2019 (a) and NBOH identification between third trimester of 2015 and second trimester of 2019 (b). Asterisk: local ban of several NBOMe substances; double asterisk: local ban of 25I-NBOH; triple asterisk: local ban of other NBOH substances

Full size image

From 2015 to 2017, the only NBOH detected was 25I-NBOH. The other NBOHs (Fig. 9b) started appearing in the last two years, with 25B-NBOH first identified in 2017, and 25C-NBOH and 25E-NBOH in 2018, totalizing 376 NBOHs identifications in blotter paper (Jan 2017 to Jun 2019). Although 25H-NBOH has been also identified in 2017, it was considered primarily as a minor component, always associated with other major component NBOHs, and was not included in the number of identifications considered until January 2018.

Discussion

To our knowledge, the present study is the first academical work to identify and to characterize 25B-NBOH, 25C-NBOH, and 25E-NBOH in authentic street seized samples. With the addition of 25I-NBOH, the first substance of these group reported, NBOHs has become a consolidated option in the recreational drug market. Regarding the legal aspect, none of NBOH substances are controlled by any United Nations Conventions. Due to the efforts of local forensic laboratories, some NBOHs are scheduled in Brazil as prohibited substances: 25I-NBOH (October 2016), 25B-NBOH, 25C-NBOH, 25E-NBOH and 25H-NBOH (December 2018) [26]. Scheduling of the N-benzylphenethylamines seems to have impact in local drug market, namely decreasing the availability of scheduled drugs. The dynamics of such process, specially its rate, however, is not clearly enough; while an NBOH was first identified in a seized sample about 1.5 years after the ban of the corresponding NBOMe, newer NBOHs emerged only seven months after the ban of 25I-NBOH. Although these numbers help us to illustrate the dynamics of the drug market, they should not be considered accurately because they are significantly influenced by the agility of police investigation, seizures of drug materials and the ability of forensic laboratories to establish the identity of new substances.

The knowledge about pharmacology and toxicology of these NBOHs, as well others NPS, are limited. 25I-NBOH showed a higher selectivity for the \(\hbox {5-HT}_{\mathrm{2A}}\) receptor when compared to \(\hbox {5-HT}_{\mathrm{2C}}\) (449.8-fold higher) followed by 25C-NBOH (43.7-fold higher), 25E-NBOH (31.0-fold higher), and 25B-NBOH (8.7-fold higher) [5]. Many studies on toxicokinetics and toxicodynamics of NBOHs remain to be explored.

Conclusions

The permanent emergence of NPS is, undoubtedly, a result of the efforts of drug dealers in circumventing drug controls. In this context, here we have presented data on the identification and structural characterization of three NPS, members of the NBOH class: 25B-NBOH, 25C-NBOH and 25E-NBOH. To our knowledge, this is the first report to identify and to characterize these three substances in authentic street seized samples which have emerged in the Brazilian market after 25I-NBOH was scheduled. As predicted in a previously published work, we have confirmed that these three substances are thermolabile, degrading into the corresponding 2C-X molecule during its injection in standard gas chromatography, easily leading them to be misidentified as such, a grave error from the forensic point of view.