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Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers
Forensic Science International 309 (2020) 110184
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Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers Damila Rodrigues de Moraisa , Kelly Francisco da Cunhab , Taís Betoni Rodriguesb , Rafael Lanarob,c , Lilian de Melo Barbosad , Jorge Jardim Zaccae , Marcos Nogueira Eberlina , Jose Luiz Costac,f,* a
ThoMSon Mass Spectrometry Laboratory, University of Campinas, Institute of Chemistry, Campinas, São Paulo, 13083-970, Brazil Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, São Paulo, 13083-887, Brazil c Campinas Poison Control Center, Faculty of Medical Sciences, University of Campinas, Campinas, São Paulo, 13083-970, Brazil d Technical-Scientific Police Superintendence, Criminalistics Institute, Campinas, São Paulo, 13018-170, Brazil e Brazilian Federal Police, Scientific and Technical Department - PF, Brasília, Distrito Federal, 70037-900, Brazil f Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-871, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2019 Received in revised form 16 January 2020 Accepted 3 February 2020 Available online 6 February 2020
NBOMe and NBOH are new psychoactive substances with potent activity on serotonin 5-HT2a receptors causing serious toxic effects, including serotonin toxidrome and death. The aim of this work was to develop a comprehensive MS/MS protocol, using triple quadrupole mass spectrometers coupled to LC and GC, for rapid screening and quantitation of NBOMes and NBOHs in seized blotter papers. Different scan methods (neutral loss, precursor ion or multiple reaction monitoring) were used to obtain structural information of phenylethylamine class. The developed protocol was validated for qualitative and quantitative analysis, showing a satisfactory limit of detection (1 ng/mL), with excellent selectivity, imprecision (intra and interday imprecision lower than 1.2 % RSD) and accuracy (between -7.1 and +5.6 %, n = 15), as well as bias values. The analysis of real samples shown that NBOH compounds were the most frequently detected, with concentrations ranging from 0.1 to 1,929 mg per blotter sample. Triple quadrupole mass spectrometers can be a useful tool for identification of new psychoactive substances. A comprehensive protocol using both LC–MS/MS and GC–MS/MS, with different scanning modes, have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers. © 2020 Elsevier B.V. All rights reserved.
Keywords: New psychoactive substances Phenethylamine derivatives NBOMe NBOH LC–MS/MS GC–MS/MS
1. Introduction New psychoactive substances (NPS) are synthetic drugs, which contain chemical structures and biological activity similar of illegal or controlled substances. NPS are synthesized in other to be a legal alternative to circumventing existing drug laws [1–3]. NBOMe and NBOH are NPS with hallucinogenic effects (similar to LSD). These compounds are synthetized as derivatives from the 2C-X of psychoactive phenethylamines [4–6]. The NBOMe compounds began to be marketed by the internet in the year 2010 in the form of blotter papers, similar to those of LSD (consumed sublingually), or powder (consumed intranasally) [7]. The growing consumption of NBOMes is attributed to their low cost, high availability and the legal access to many compounds of the class
* Corresponding author at: Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-871, Brazil. E-mail address: [email protected] (J.L. Costa). http://dx.doi.org/10.1016/j.forsciint.2020.110184 0379-0738/© 2020 Elsevier B.V. All rights reserved.
in several countries [8]. According to Coelho Neto et al. [9] NBOH series are compounds recently reported, appeared around 2017, to have been found on blotter papers as a legal alternative for the NBOMe series which also act as a potent agonist on serotonin (5HT2a, 5-hydroxytryptamine) receptors [10], however the physiological and toxicological properties of NBOH are unknown [4]. The growing number of NPS, as NBOMe and NBOH series, and the increase in trafficking in synthetic drugs are also major concerns for the control of the illicit drug market [11]. Fast changing in the drugs structure also result in a much larger number of different drug substances to control [12]. Therefore, it is of great importance to develop and effective (rapid, comprehensive, and selective) protocol for NBOMe and NBOH screening and quantitation. Several analytical methods have been developed to identify and quantify NBOMe and NBOH compounds in blotter paper by LC and GC chromatography coupled to tandem with mass spectrometry but they require standards or high resolution and high accuracy mass measurements [7,13–15]. MS/MS methods via multiple reaction monitoring (MRM) have been mostly implemented in
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triple quadrupole (QqQ) instruments, but MRM method development requires reference materials, hence new NBOMe and NBOH drugs would not be detected. There are, however, different modes of acquisition in QqQ instruments which have been poorly explored. For instance, the precursor ion and neutral loss scan modes have been used to selectively screen for several drugs with sharing moieties [16–18] or classes of compounds with a characteristic neutral loss. In this work we developed, validated and applied to real samples a protocol to screen for NBOMe and NBOH by either LC– MS/MS or GC–MS/MS, or both. The protocol is shown to be quite comprehensive since it monitors either a characteristic neutral loss for NBOMe, a product ion for NBOMe and NBOH, or a common fragment ion for NBOMe. The use of three screening features – a common neutral loss, a common fragment or a common moiety – seems to provide a way to detect new designer drugs. 2. Material and methods
ratio 1:10. For quantitative LC–MS/MS analysis, the samples were diluted with extraction solvent (methanol containing the internal standard) in different ratios (1:10, 1:100 or 1:1000) in order to achieve the linearity range. In GC–MS/MS method, samples were derivatizated before instrumental analysis. 2.4. Standard and sample derivatization for GC–MS/MS method analysis Standard mix solutions and samples were derivatizated according to Zuba, Sekuła and Buczek [15]. Standard mix solutions and samples (100 mL) were evaporated under a stream of N2 and room temperature (25 C). Then, derivatives were prepared by dissolving dry extracts separately in 100 mL of derivatizing agent (TFAA:chloroform, 1:1, v/v), and, after vortex mixing, the reaction mixture was incubated in a capped tube at 70 C for 40 min. After cooling to room temperature, the samples were evaporated to dryness under a stream of N2 and reconstituted with 100 mL of ethyl acetate in a vial (1 mL of injection).
2.1. Chemicals and reagents 2.5. Precursor and product ion scan method by LC–MS/MS Solvents employed in the sample extraction and chromatographic analyses were: trifluoroacetic anhydride (TFAA) 99 %, HPLC grade ethyl acetate, chloroform, and formic acid 95 % from Sigma–Aldrich (Saint Louis, MO, USA), HPLC grade acetonitrile and methanol from Merck (Darmstadt, Germany), and water was purified by a Milli-Q gradient system (Millipore, Milford, MA, USA). Reference materials of 25C-NBOH, 25B-NBOH, 25I-NBOH, 25HNBOMe, 25C-NBOMe, 25I-NBOMe, 25B-NBOMe, 25G-NBOMe, 25D-NBOMe, 25E-NBOMe, 25N-NBOMe, 25T2-NBOMe, acrylfentanyl, fentanyl, thiofentanyl, valeryl fentanyl, were purchased from Cayman Chemical Company (Ann Arbor, MI, USA); 2C-I, LSD and LSD-d3 (internal standard) were purchased from Cerilliant (Round Rock, TX, USA). The full name and structure of all analytes are shown in the Supplementary Fig. 1. 2.2. Standard solutions preparation Individual stock solutions were prepared by dilution of standard materials as follow: 1000 mg/mL of 25C-NBOH, 25BNBOH, 25I-NBOH, 25H-NBOMe, 25C-NBOMe, 25I-NBOMe, 25BNBOMe, 25G-NBOMe, 25D-NBOMe, 25E-NBOMe in methanol; 100 mg/mL of LSD in acetonitrile; 100 mg/mL of 25N-NBOMe, 25T2NBOMe and 2C-I in methanol. For GC–MS/MS and LC–MS/MS screening methods, individual working solutions and mix working solutions with all analytes were prepared in concentrations ranging from 1 to 10 mg/mL, using methanol as solvent. LSD-d3 was used as internal standard at 200 ng/mL in methanol (extraction solvent). All solutions (stock, work and calibrator) were stored at 20 C. 2.3. Samples preparation Different blotter paper samples which were suspected to contain illicit compounds, were provided by the TechnicalScientific Police Superintendence from São Paulo State, Brazil. Individual blotter paper sample (c.a. 1.0 cm 1.0 cm) was placed in 2 mL polypropylene tube. Then 1,000 mL of methanol containing the internal standard (LSD-d3, at 200 ng/mL) was added, the tubes were capped, vortexed for 5 min and centrifuged at 14,000 rpm for 5 min at ambient temperature. The supernatant (c.a. 500 mL) was transferred to other polypropylene tube and diluted according to each analysis performed. For LC–MS/MS qualitative analysis (precursor ion method), samples were diluted in the ratio 1:20, and for GC–MS/MS qualitative analysis samples were diluted in the
The solution, 1 mL, was injected into the Nexera UFLC chromatographic system coupled to a LCMS8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The analytes were separated using a RaptorTM Biphenyl (100 mm 2.1 mm ID, 2.7 mm, Restek) column with pre column RaptorTM Biphenyl (5 mm 2.1 mm DI, 2.7 mm, Restek) maintained at 40 C. The mobile phase, consisting 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) (75:25, isocratic elution). The flow rate was kept constant at 0.5 mL/min. The separated analytes were identified with a triple quadrupole mass spectrometer with positive electrospray ionization (ESI). The applied ESI conditions were: capillary voltage 4.50 kV, desolvation temperature 250 C, heat temperature 400 C, drying gas (N2) flow 15 L/min, nebulizing gas (N2) flow 3 L/min and collision gas (Ar) 230 kPa. The mass spectrometer was set to operate of the precursor ion scan mode for the fragment ion of m/z 121 (characteristic of NBOMe series) with a collision energy (CE) of 20 V, and of m/z 107 (characteristic of NBOH series) with CE of 27 V, event time of 0.340 s, scan velocity of 454 Da/s, mass range from m/z 300 to m/z 450 with a dependent event product ion scan experiment activated for compounds with intensity greater than 25,000 cps, for the both events. In the product ion scan mode, the conditions were: CE of 15 V (for the monitoring of the fragment ion of m/z 121), and CE of 20 V (for the monitoring of the fragment ion of m/z 107), event time of 0.500 s, scan velocity of 769 Da/s and a range from m/z 80 to m/z 450, for the both scans. The compounds were identified by comparison of spectrum obtained by product ion scan with an inhouse MS/MS spectrum library, built by the injection of standards at the same ionization conditions described in this item. Data was acquired and analyzed (including library search) using the software Labsolution (version 5.53 SP2, Shimadzu). 2.6. Neutral loss scan and single ion monitoring method by GC–MS/MS The solution, 1 mL, was injected in splitless mode into the GCMS TQ8040, a gas chromatographic system coupled to triple quadrupole mass analyzer with electron ionization (EI) source (Shimadzu, Kyoto Japan). The chromatographic conditions were developed and optimized using derivatizated reference material working solutions, with mass spectrometer set to fullscan mode. The analytes were separated using a HP-5MS (30 m 0.25 mm 0.25 mm, Agilent, USA) and helium at a constant flow rate of 1.0 mL/min was used as the carrier gas.
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The initial column temperature 80 C was maintained for 1.00 min, then increased linearly at a rate of 20 C/min to 320 C and maintained for 5.0 min. The GC injector was maintained at 280 C, the ion source and interface temperature were 300 C, ionization energy at 70 eV and positive ions were analyzed. Acquisition was carried out in the neutral loss scan mode, for the neutral loss of 233 Da (characteristic of NBOMe series, see Fig. 1), CE of 10 V, event time of 0.350 s, scan velocity of 416 Da/s, and mass range from m/z 390 to m/z 525; and concomitant single monitoring of the ions of m/z 91 and m/z 121 (characteristics of NBOMe series). Argon was used as collision gas at 200 kPa. Chromatograms and spectra were registered from 9.00–18.00 min. Data was acquired and analyzed using the software GCMSsolution (version 4.45, Shimadzu). 2.7. Neutral loss and single ion monitoring scan method by GC–MS/ MS, and precursor and product ion scan method by LC–MS/MS validation Both methods, neutral loss scan by GC–MS/MS and precursor and product ion scan by LC–MS/MS, were validated over three consecutive days, with parameters such as selectivity, limit of detection (LOD) and imprecision (under repeatability and reproducibility) according to the “Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens” manual, outlined by the UNODC (United Nations Office On Drugs And Crime) [19]. The analyte identification criteria were a peak eluting within 2 % of the average of the calibrator retention time, a signal-to-noise ratio of at least 3, and the mass spectrum should have a good visual match to that of the standard or should achieve a fit factor of 85 % or more in the library search (compared to our with in-house MS/ MS spectrum library), on a scale in which a perfect fit achieves a fit factor of 100 % [19]. The LOD is usually defined as the lowest concentration that reached the identification criteria, but in this method validation the administrative LOD value was defined based on the laboratory’s administratively decision point for reporting these analytes, which consider also the concentrations frequently observed on seized material [14]. The selectivity is concerned with the extent to which other substances (compounds usually found to be present in seized drugs) interfere with the identification and, where appropriate, quantification, of the analytes of interest [19]. Selectivity was tested with a mixture of substances commonly present in seized blotter papers, such as 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25E-NBOMe, 25G-NBOMe, 25H-NBOMe, 25I-NBOMe, 25T2NBOMe, 25B-NBOH, 25C-NBOH, 25I-NBOH, LSD, and other psychoactive substances, at concentrations between 1.0–2.5 mg/mL for GC–MS/MS and 0.3 - 0.5 mg/mL for LC–MS/MS method. The analyzed substances investigated in selectivity studies are described at Supplementary Table 1. Both methods were
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considered as selective if there were no peaks present at the same NBOMes and NBOHs retention time. Additionally, the LC–MS/ MS method was considered as selective if no other compounds were identified by comparison with an in-house MS/MS spectrum library as NBOMes and NBOHs. Intraday imprecision was determined by analyzing five replicates in the same day and interday imprecision was determined by analyzing five replicate samples, in three consecutive days conditions, of 0.45 mg/mL of mix of NBOMes and NBOHs standard for LC–MS/MS method and of 4.0 mg/mL of mix of NBOMes derivatizated for GC–MS/MS method. The method retention time should be within 2 % of those obtained in reference material analysis. No more than one sample in five (20 %) should give a false negative result. 2.8. Multiple reactions monitoring quantitation method by LC–MS/MS The solution, 2 mL, was injected into the Nexera UFLC chromatographic system coupled to a LCMS8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The analytes were separated using a ForceTM Biphenyl (50 2.1 mm ID, 1.8 mm, Restek, USA) column and the oven was operated in a temperature of 40 C. The mobile phase, consisting 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). Chromatography separation was carried out using a gradient elution programmed as follows: 5 % B for 0.20 min, followed by a linear change to 35 % B over 1.30 min, held at 35 % B for 2.10 min and returned to initial conditions over 0.10 min (total run time of 5.00 min). The flow rate was kept constant at 0.5 mL/min. Identification and quantitation of the separated analytes were done with a triple quadrupole mass spectrometer operating in the precursor ion scan mode with dependent event of product ion scan via positive electrospray ionization (ESI). The applied ESI conditions were: capillary voltage 4.50 kV, desolvation temperature 250 C, heat temperature 400 C, drying gas (N2) flow 15 L/ min, nebulizing gas (N2) flow 3 L/min and collision gas (Ar) 230 kPa. The analyses were performed in multiple reactions monitoring mode (MRM). The MRM conditions used are presented in Supplementary Table 2, where first transition was used for quantitation and second transition used for identification. Data was acquired and treated using the software Labsolution (version 5.53 SP2, Shimadzu). 2.9. Multiple reactions monitoring method by LC–MS/MS validation The method was validated over three consecutive days, with parameters such as selectivity, limit of detection (LOD) and quantification (LOQ), linearity, accuracy bias, imprecision (under repeatability and reproducibility) and recovery rates based on the “Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized
Fig. 1. Proposed fragmentation of NBOMe and NBOH derivatives in GC–MS/MS, after derivatization of NBOMes with TFAA.
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Materials and Biological Specimens” manual, outlined by the UNODC [19]. The method selectivity was determined evaluating other compounds usually found to be present in blotter paper samples. Potential interferences encountered other drugs of abuse and pharmaceuticals (see Supplementary Table 1) were evaluated by fortifying them at 500 ng/mL into low QC and negative samples. No interference was noted if all analytes in the low QC quantified within 20 % of target with acceptable qualifier/quantifier MRM ratios and no peak in the negative sample satisfied LOD criteria. The limits of detection (LOD) and quantification (LOQ) represent the lowest concentration of the substance under evaluation that can be detected and quantified, respectively. Regarding LOD and LOQ, these parameters were calculate taking into account the instrumental signal-to-noise ratio (S/N > 3 for LOD, S/N > 10 for LOQ), but in this validation the administrative LOD and LOQ values were defined based on the laboratory’s administratively decision point for reporting this analyte, which consider the concentrations frequently observed on seized material [14]. The linearity was determined with calibration range from 1 to 100 ng/mL in methanol. A high linear correlation coefficient (r) value (r 0.99) was used as criterion of good linearity. The calibration curves were constructed by plotting the peak area versus analyte standard concentration using the leastsquares linear regression method (1/x2 weighting) and correlation. For concentration determination of each analyte in blotter paper samples the analyte peak area was used and the concentrations were calculated using the calibration curve. Accuracy bias and imprecision were determined using quality control (QC) samples prepared by spiking 3, 30 and 75 ng/mL (QC1, QC2 and QC3, respectively) of each standard in blotter paper extract methanol solution. Three replicates of QC samples were run in the same batch together with calibration standards, and the calibration curve was then obtained and the QC concentrations determined. The accuracy bias was determined dividing the mean concentration for analytical runs (intraday n = 3; interday n = 9) by the expected concentration, and expressed as percentage. Accuracy bias values in the range of 80–120 % were considered acceptable. The intra- and interday imprecision was obtained by calculating the relative standard deviation (%RSD) for the mean concentration (n = 5 and n = 15, respectively) and one-way analysis of variance (ANOVA) was performed on each QC concentration to assess potentially significant interday variability at p < 0.05. Imprecision values with %RSD less than 20 % were considered acceptable. Once was not possible to have a blank matrix of samples (blotter paper), the accuracy of the method was determined as recovery experiments. Two different concentrations (30 and 75 ng/mL) of analyte working solutions were added to blotter paper samples solution and analyzed by the proposed method. Results were calculated as experimental values compared to theoretical values (initial analyte sample concentration+added working solution) and were expressed as percent recovery. The recovery rates were considered acceptable above 80 %.
corresponding to a NBOMe moiety- are screened, most new illegal substances of both series are expected to be detected. Fig. 2 presents a chromatogram obtained by the analysis of working solution of three NBOH and nine NBOMe at 0.3 mg/mL – this concentration is equivalent to 6 mg/blotter, considering the sample preparation procedure, the dilution factor and high recovery rates (around 100 %. This LOD was therefore adequate, since because NBOMe compounds are normally present in blotter papers in the range of 50 mg to 2000 mg per blotter sample [14]. The similarity of in house library against NBOMe and NBOH standard spectra compounds for LOD solutions were high, varying between 87 % and 98 % (Supplementary Table 3). The S/N ratios actually indicated that the developed method could detect NBOMe and NBOH compounds in concentrations even lower than 0.3 mg/mL. Supplementary Table 3 shows results obtained for imprecisions, calculated as relative standard deviation (RSD%), with results between 0.2 and 1.2 %. No interferences were observed at negative and/or LOD samples in presence of potential interferences encountered, others drugs of abuse and pharmaceuticals at 0.5 mg/mL. No carryover was observed in blank samples after analysis of a 3.8 mg/mL of NBOMe and NBOH standards. 3.2. Neutral loss scan and single ion monitoring method by GC–MS/MS Neutral loss scan monitoring method was developed based on a characteristic neutral loss of 233 Da [15] which is common for the TFAA derivatizated NBOMe series. For single ion monitoring, the two fragments m/z 121 and m/z 91 were selected. As Fig. 3 shows, the developed GC–MS/MS method was shown to provide also an adequate screening tool to identify NBOMes in blotter paper samples. Again, as common fragments were monitored, new design NBOMes could also be detected. TFAA derivatization for NBOH series failed since no peaks were detected in the corresponding chromatograms. The administrative LOD value obtained via the GC–MS/MS neutral loss scan and single ion monitoring scan were 2.5 mg/mL (equivalent to 25 mg/blotter paper) for all compounds, excepted for 25E-NBOMe (LOD of 4.0 mg/mL, equivalent to 40 mg/blotter paper). Again, these LOD values are therefore adequate because NBOMe compounds are found in concentration higher than 50 mg per blotter sample [14]. Supplementary Table 4 summarizes the intraday and interday imprecisions calculated by relative standard deviation (RSD%). The imprecision was low with values between 0.01 and 0.27 %, therefore in the range of 2 % recommended by the UNODC guide (UNODC, 2017), for all analytes in the concentration of 0.45 mg/mL, without false negatives. No interferences were observed at negative and/or LOD samples in the presence of the potential interferences encountered, other
3. Results and discussions 3.1. Precursor and product ion scan method by LC–MS/MS The precursor ion method was developed based on two characteristic fragment ions common for NBOMe and NBOH series compounds, that is the ion of m/z 121 and m/z 107, respectively. In order to enhance the selectivity of the method and compounds identification, a product ion scan dependent event and in-house library were created. The method showed to be a simple and efficient screening tool for NBOMes and NBOHs in blotter paper samples. Since the precursors of a characteristic fragment ion –
Fig. 2. Representative chromatogram of NBOMes working solution mixture (0.3 m g/mL) obtained by LC–MS/MS precursor and product ion scans modes. Legend: (1) 25C-, (3) 25B-, (5) 25I-NBOH, and, (2) 25H-, (4)25N-, (6) 25D-, (7) 25C-, (8) 25T2-, (9) 25B-, (10) 25G-, (11) 25E- and (12) 25I-NBOMe.
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Fig. 3. Representative chromatogram of NBOMes working solution mixture (2.5 mg/mL), derivatized with TFAA and analyzed by GC–MS/MS in neutral loss scan and single ion monitoring modes. Legend: (1) 25H-, (2) 25D-, (3) 25E-, (4) 25G-, (5) 25C-, (6) 25B-, (7) 25I- and 25N-NBOMe-TFA.
drugs of abuse at 2.5 mg/mL as well as pharmaceuticals at 1.0 mg/ mL. No carryover was observed in blank samples after analysis of a 10 mg/mL of NBOMe standards. 3.3. Multiple reactions monitoring quantitation method by LC–MS/MS Fig. 4 presents the LC–MS/MS chromatogram obtained by the fast MRM quantitation method developed to quantify thirteen compounds, that is, LSD and twelve phenethylamines (from NBOMe, NBOH and 2C series) which are usually present in blotter papers. Supplementary Table 5 shows the validation results by the MRM quantitation LC–MS/MS method for LSD, 2C-I, NBOH (25C-, 25B- and 25I-NBOH) and NBOMe (25H-, 25C-, 25I-, 25B-, 25G-, 25D- and 25E-NBOMe). A linear curve with six calibration points
from 1 to 100 ng/mL was performed using a 1/x2 weight linear regression, r > 0.996, for the twelve compounds, which presented a satisfactory linearity. Angular coefficients imprecisions for three days of analysis ranged from 1.4 for 25B-NBOH to 8.8 % for 25I-NBOMe. LOD and LOQ were 1 and 3 ng/mL, respectively, for all compounds. Intraday and interday imprecisions were calculated by one-way analysis of variance (ANOVA, p < 0.05) and showed low values of %RSD for all analytes in three concentrations of QC (3, 50 and 75 ng/mL). The accuracy bias were within -7.1 and +5.6 % (n = 15), as well as bias values. No carryover was observed in blank samples after analysis of a 100 ng/mL sample (higher calibrator) and no interferences were observed at negative and/or LOQ samples in presence of potential interferences encountered, others drugs of abuse and pharmaceuticals at 500 ng/mL.
Fig. 4. Representative LC–MS/MS chromatogram of MRM quantitation method obtained from a working solution mixture of (1) LSD; (2) 2C-I; (4) 25C-, (5) 25B- and (6) 25INBOH; (3) 25H-, (7) 25D-, (8) 25C-, (9) 25B-, (10) 25G-, (11) 25E-, (12) 25I-NBOMe at 10 ng/mL.
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Table 1 Qualitative and quantitative results obtained by proposed triple quadrupole analytical protocol (LC–MS/MS and GC–MS/MS) to the analysis of phenylethylamines in seized blotter papers. Sample #
MRM LC-MS/MS method
Blotter paper concentrataion (mg/blotter)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Precursor and product ion scan LC-MS/MS method
Neutral loss and SIMb GC-MS/MS method
MS similarity in comparison to in house library (%)
Identification
LSD
2C-I 25CNBOH
25INBOH
25HNBOMe
25CNBOMe
25BNBOMe
25INBOMe
25CNBOH
25INBOH
25HNBOMe
25CNBOMe
25INBOMe
25CNBOMe
25INBOMe
ND ND ND ND ND 86.1 ND ND ND ND ND ND ND ND ND ND
12.0 0.3 ND 0.2 ND ND ND ND ND 6.3 ND ND 4.4 ND ND ND
715 5.2 61.2 0.07 1.4 10.5 802 1374 433 254 1829 509 1929 301 1498 ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.6
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 70.6
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1.9
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 14.9
79 96 94 84 92 99 96 94 97 91 91 93 92 97 84 ND
95 96 97 98 98 97 98 94 97 97 98 92 96 97 98 ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 88
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 97
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 91
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Detected
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Detected
340 1.0 5.0 1.7 0.04 ND 47.0 47.0 28.0 33.9 12.6 24.0 43.2 19.0 65.0 0.1
ND: not detected.
3.4. Authentic forensic cases
4. Conclusions
Blotter papers suspected to contain psychoactive phenethylamines were seized by law enforcement and evaluated by the developed protocol of analysis: precursor and product ion scan by LC–MS/MS, neutral loss scan and single ion monitoring by GC–MS/ MS and MRM quantitation by LC–MS/MS (Table 1). The most common phenethylamines derivatives in blotter paper samples were 25I- and 25C-NBOH. The 2C-I, LSD, 25HNBOMe, 25C-NBOMe, 25I-NBOMe and 25B-NBOMe were also found. The 2C-I was detected in five samples, but in concentrations lower than 12 mg per blotter sample. At such low concentration, 2C-I was probably present as minor contaminant (unreacted starting material) from the synthesis of 25I-NBOH. Precursor and product ion scan LC–MS/MS method and neutral loss and SIM GC– MS/MS method presented satisfactory results comparing to MRM LC–MS/MS for NBOMes. In previous study, Edmunds et al. [20] developed a method to evaluate of illicit 25X-NBOMe seizures by UPLC-PDA, and applied it to analysis of samples from Western Australia, seized between January/2014 and February/2016. The authors detected 25CNBOMe, 25I-NBOMe and 25B-NBOMe in samples, with concentrations ranging from 114 to 1,480 mg per blotter sample. Our blotter papers were seized between January/2017 and June/2017, in Sao Paulo state (Southeast Brazil). The phenethylamines profiles observed in our samples were different, where NBOH compounds were the most frequently detected, with concentrations from 0.1 to 1,929 mg per blotter sample. The greater prevalence of NBOH compounds as hallucinogenic drugs was also observed by Machado et al. [21], whom recently published a qualitative chemical characterization of 25B-, 25C- and 25E-NBOH detected in seized blotter papers. Just in one sample we detected NBOMe compounds (sample 16). According to Poklis et al. [14], NBOMe compounds found per blotter ranged from 50 mg to 2000 mg and sample quantity lower than 30 mg per blotter sample was considered impurities, thus 25H-NBOMe, 25I-NBOMe and 25B-NBOMe could be considered interferents in sample 16.
Frequently described as powerful quantitative instruments, triple quadrupole mass spectrometers can be also useful tool for identification of new psychoactive substances. In the present work, a comprehensive protocol using both LC–MS/MS and GC–MS/MS, with different scanning modes (MRM, SIM, neutral loss, precursor ion and product ion), have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers. When applied to authentic forensic samples, the approach was shown to be a powerful, selective and sensitive tool for untargeted screening. As general structural moieties were monitored, the protocol is expected to work also to detect new phenylethylamines analogues with high efficiency. CRediT authorship contribution statement Damila Rodrigues de Morais: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Visualization. Kelly Francisco da Cunha : Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Visualization. Taís Betoni Rodrigues: Investigation, Writing - review & editing, Visualization. Rafael Lanaro: Validation, Investigation, Writing review & editing, Visualization. Lilian de Melo Barbosa: Investigation, Resources. Jorge Jardim Zacca: Investigation, Resources. Marcos Nogueira Eberlin: Resources, Writing - review & editing, Funding acquisition. Jose Luiz Costa: Conceptualization, Writing review & editing, Funding acquisition, Project administration. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgements The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, process number 23038.006844/2014-
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46), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process numbers 2018/00432-1, 2018/11849-0 and 2016/ 23157-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process number 131780/2017-4 and 425814/ 2018-1) by the fellowships and the financial support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.forsciint.2020.110184. References [1] J. Lobo Vicente, H. Chassaigne, M.V. Holland, F. Reniero, K. Kolár, S. Tirendi, I. Vandecasteele, I. Vinckier, C. Guillou, Systematic analytical characterization of new psychoactive substances: a case study, Forensic Sci. Int. 265 (2016) 107– 115, doi:http://dx.doi.org/10.1016/j.forsciint.2016.01.024. [2] J. Suzuki, M.A. Dekker, E.S. Valenti, F.A. Arbelo Cruz, A.M. Correa, J.L. Poklis, A. Poklis, Toxicities associated with NBOMe ingestion— a novel class of potent hallucinogens: a review of the literature, Psychosomatics 56 (2015) 129–139, doi:http://dx.doi.org/10.1016/j.psym.2014.11.002. [3] M. Ibáñez, J.V. Sancho, L. Bijlsma, A.L.N. van Nuijs, A. Covaci, F. Hernández, Comprehensive analytical strategies based on high-resolution time-of-flight mass spectrometry to identify new psychoactive substances, TrAC Trends Anal. Chem. 57 (2014) 107–117, doi:http://dx.doi.org/10.1016/j.trac.2014.02.009. [4] L.C. Arantes, E.F. Júnior, L.F. de Souza, A.C. Cardoso, T.L.F. Alcântara, L.M. Lião, Y. Machado, R.A. Lordeiro, J.C. Neto, A.F.B. Andrade, 25I-NBOH: a new potent serotonin 5-HT2A receptor agonist identified in blotter paper seizures in Brazil, Forensic Toxicol. 35 (2017) 408–414, doi:http://dx.doi.org/10.1007/ s11419-017-0357-x. [5] V.B. Kueppers, C.T. Cooke, 25I-NBOMe related death in Australia: a case report, Forensic Sci. Int. 249 (2015) e15–e18, doi:http://dx.doi.org/10.1016/j.forsciint.2015.02.010. [6] L.M. Nielsen, N.B. Holm, S. Leth-Petersen, J.L. Kristensen, L. Olsen, K. Linnet, Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH, Drug Test. Anal. 9 (2017) 671–679, doi:http://dx.doi.org/10.1002/dta.2031. [7] D. Zuba, K. Sekuła, Analytical characterization of three hallucinogenic N -(2methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs, Drug Test. Anal. 5 (2013) 634–645, doi:http://dx.doi.org/10.1002/dta.1397.
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[8] J. Coelho Neto, Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry, Forensic Sci. Int. 252 (2015) 87–92, doi:http:// dx.doi.org/10.1016/j.forsciint.2015.04.025. [9] J. Coelho Neto, A.F.B. Andrade, R.A. Lordeiro, Y. Machado, M. Elie, E. Ferrari Júnior, L.C. Arantes, Preventing misidentification of 25I-NBOH as 2C-I on routine GC–MS analyses, Forensic Toxicol. 35 (2017) 415–420, doi:http://dx. doi.org/10.1007/s11419-017-0362-0. [10] D.R. de Morais, I.L. Barbosa, K.F. Cunha, G.L. Tripodi, C.F.F. Angolini, M.F. Franco, E.M. de Aquino, M.N. Eberlin, J.L. Costa, EASI-IMS an expedite and secure technique to screen for 25I-NBOH in blotter papers, J. Mass Spectrom. 52 (2017) 701–706, doi:http://dx.doi.org/10.1002/jms.3977. [11] UNODC, World Drug Report 2017, (2017) . [12] D. Pasin, A. Cawley, S. Bidny, S. Fu, Characterization of hallucinogenic phenethylamines using high-resolution mass spectrometry for non-targeted screening purposes, Drug Test. Anal. 9 (2017) 1620–1629, doi:http://dx.doi. org/10.1002/dta.2171. [13] S. Botch-Jones, J. Foss, D. Barajas, F. Kero, C. Young, J. Weisenseel, The detection of NBOMe designer drugs on blotter paper by high resolution time-of-flight mass spectrometry (TOFMS) with and without chromatography, Forensic Sci. Int. 267 (2016) 89–95, doi:http://dx.doi.org/10.1016/j.forsciint.2016.08.008. [14] J.L. Poklis, S.A. Raso, K.N. Alford, A. Poklis, M.R. Peace, Analysis of 25I-NBOMe, 25B-NBOMe, 25C-NBOMe and other dimethoxyphenyl- N -[(2-methoxyphenyl) methyl]ethanamine derivatives on blotter paper, J. Anal. Toxicol. 39 (2015) 617–623, doi:http://dx.doi.org/10.1093/jat/bkv073. [15] D. Zuba, K. Sekuła, A. Buczek, 25C-NBOMe—new potent hallucinogenic substance identified on the drug market, Forensic Sci. Int. 227 (2013) 7–14, doi: http://dx.doi.org/10.1016/j.forsciint.2012.08.027. [16] J. Marcos, O.J. Pozo, Current LC–MS methods and procedures applied to the identification of new steroid metabolites, J. Steroid Biochem. Mol. Biol. 162 (2016) 41–56, doi:http://dx.doi.org/10.1016/j.jsbmb.2015.12.012. [17] H. Schulte, H. Miyagawa, Y. Sakamoto, T. Kamata, S. Matsuta, H. Nishioka, M. Katagi, K. Zaitsu, A. Ishii, K. Tsuboi, H. Tsuchihashi, Comprehensive Analysis of Naphthoylindole-type Synthetic Cannabinoids by GC-MS/MS, (2014) . [18] J. Dahl, A. Rigdon, Designer Cannabinoids, (2011) . [19] UNODC, Guidance for the Validation of Analytical Methodology and Calibration of Equipment Used for Testing of Illicit Drugs in Seized Materials and Biological Specimens, (2009) . [20] R. Edmunds, R. Donovan, D. Reynolds, The analysis of illicit 25X-NBOMe seizures in Western Australia, Drug Test. Anal. 10 (2018) 786–790, doi:http:// dx.doi.org/10.1002/dta.2260. [21] Y. Machado, J. Coelho Neto, R.A. Lordeiro, R.A. Alves, E. Piccin, Identification of new NBOH drugs in seized blotter papers: 25B-NBOH, 25C-NBOH, and 25ENBOH, Forensic Toxicol. 38 (2020) 203–215, doi:http://dx.doi.org/10.1007/ s11419-019-00509-7.
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Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers Damila Rodrigues de Moraisa , Kelly Francisco da Cunhab , Taís Betoni Rodriguesb , Rafael Lanarob,c , Lilian de Melo Barbosad , Jorge Jardim Zaccae , Marcos Nogueira Eberlina , Jose Luiz Costac,f,* a
ThoMSon Mass Spectrometry Laboratory, University of Campinas, Institute of Chemistry, Campinas, São Paulo, 13083-970, Brazil Department of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas, São Paulo, 13083-887, Brazil c Campinas Poison Control Center, Faculty of Medical Sciences, University of Campinas, Campinas, São Paulo, 13083-970, Brazil d Technical-Scientific Police Superintendence, Criminalistics Institute, Campinas, São Paulo, 13018-170, Brazil e Brazilian Federal Police, Scientific and Technical Department - PF, Brasília, Distrito Federal, 70037-900, Brazil f Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-871, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 June 2019 Received in revised form 16 January 2020 Accepted 3 February 2020 Available online 6 February 2020
NBOMe and NBOH are new psychoactive substances with potent activity on serotonin 5-HT2a receptors causing serious toxic effects, including serotonin toxidrome and death. The aim of this work was to develop a comprehensive MS/MS protocol, using triple quadrupole mass spectrometers coupled to LC and GC, for rapid screening and quantitation of NBOMes and NBOHs in seized blotter papers. Different scan methods (neutral loss, precursor ion or multiple reaction monitoring) were used to obtain structural information of phenylethylamine class. The developed protocol was validated for qualitative and quantitative analysis, showing a satisfactory limit of detection (1 ng/mL), with excellent selectivity, imprecision (intra and interday imprecision lower than 1.2 % RSD) and accuracy (between -7.1 and +5.6 %, n = 15), as well as bias values. The analysis of real samples shown that NBOH compounds were the most frequently detected, with concentrations ranging from 0.1 to 1,929 mg per blotter sample. Triple quadrupole mass spectrometers can be a useful tool for identification of new psychoactive substances. A comprehensive protocol using both LC–MS/MS and GC–MS/MS, with different scanning modes, have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers. © 2020 Elsevier B.V. All rights reserved.
Keywords: New psychoactive substances Phenethylamine derivatives NBOMe NBOH LC–MS/MS GC–MS/MS
1. Introduction New psychoactive substances (NPS) are synthetic drugs, which contain chemical structures and biological activity similar of illegal or controlled substances. NPS are synthesized in other to be a legal alternative to circumventing existing drug laws [1–3]. NBOMe and NBOH are NPS with hallucinogenic effects (similar to LSD). These compounds are synthetized as derivatives from the 2C-X of psychoactive phenethylamines [4–6]. The NBOMe compounds began to be marketed by the internet in the year 2010 in the form of blotter papers, similar to those of LSD (consumed sublingually), or powder (consumed intranasally) [7]. The growing consumption of NBOMes is attributed to their low cost, high availability and the legal access to many compounds of the class
* Corresponding author at: Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-871, Brazil. E-mail address: [email protected] (J.L. Costa). http://dx.doi.org/10.1016/j.forsciint.2020.110184 0379-0738/© 2020 Elsevier B.V. All rights reserved.
in several countries [8]. According to Coelho Neto et al. [9] NBOH series are compounds recently reported, appeared around 2017, to have been found on blotter papers as a legal alternative for the NBOMe series which also act as a potent agonist on serotonin (5HT2a, 5-hydroxytryptamine) receptors [10], however the physiological and toxicological properties of NBOH are unknown [4]. The growing number of NPS, as NBOMe and NBOH series, and the increase in trafficking in synthetic drugs are also major concerns for the control of the illicit drug market [11]. Fast changing in the drugs structure also result in a much larger number of different drug substances to control [12]. Therefore, it is of great importance to develop and effective (rapid, comprehensive, and selective) protocol for NBOMe and NBOH screening and quantitation. Several analytical methods have been developed to identify and quantify NBOMe and NBOH compounds in blotter paper by LC and GC chromatography coupled to tandem with mass spectrometry but they require standards or high resolution and high accuracy mass measurements [7,13–15]. MS/MS methods via multiple reaction monitoring (MRM) have been mostly implemented in
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triple quadrupole (QqQ) instruments, but MRM method development requires reference materials, hence new NBOMe and NBOH drugs would not be detected. There are, however, different modes of acquisition in QqQ instruments which have been poorly explored. For instance, the precursor ion and neutral loss scan modes have been used to selectively screen for several drugs with sharing moieties [16–18] or classes of compounds with a characteristic neutral loss. In this work we developed, validated and applied to real samples a protocol to screen for NBOMe and NBOH by either LC– MS/MS or GC–MS/MS, or both. The protocol is shown to be quite comprehensive since it monitors either a characteristic neutral loss for NBOMe, a product ion for NBOMe and NBOH, or a common fragment ion for NBOMe. The use of three screening features – a common neutral loss, a common fragment or a common moiety – seems to provide a way to detect new designer drugs. 2. Material and methods
ratio 1:10. For quantitative LC–MS/MS analysis, the samples were diluted with extraction solvent (methanol containing the internal standard) in different ratios (1:10, 1:100 or 1:1000) in order to achieve the linearity range. In GC–MS/MS method, samples were derivatizated before instrumental analysis. 2.4. Standard and sample derivatization for GC–MS/MS method analysis Standard mix solutions and samples were derivatizated according to Zuba, Sekuła and Buczek [15]. Standard mix solutions and samples (100 mL) were evaporated under a stream of N2 and room temperature (25 C). Then, derivatives were prepared by dissolving dry extracts separately in 100 mL of derivatizing agent (TFAA:chloroform, 1:1, v/v), and, after vortex mixing, the reaction mixture was incubated in a capped tube at 70 C for 40 min. After cooling to room temperature, the samples were evaporated to dryness under a stream of N2 and reconstituted with 100 mL of ethyl acetate in a vial (1 mL of injection).
2.1. Chemicals and reagents 2.5. Precursor and product ion scan method by LC–MS/MS Solvents employed in the sample extraction and chromatographic analyses were: trifluoroacetic anhydride (TFAA) 99 %, HPLC grade ethyl acetate, chloroform, and formic acid 95 % from Sigma–Aldrich (Saint Louis, MO, USA), HPLC grade acetonitrile and methanol from Merck (Darmstadt, Germany), and water was purified by a Milli-Q gradient system (Millipore, Milford, MA, USA). Reference materials of 25C-NBOH, 25B-NBOH, 25I-NBOH, 25HNBOMe, 25C-NBOMe, 25I-NBOMe, 25B-NBOMe, 25G-NBOMe, 25D-NBOMe, 25E-NBOMe, 25N-NBOMe, 25T2-NBOMe, acrylfentanyl, fentanyl, thiofentanyl, valeryl fentanyl, were purchased from Cayman Chemical Company (Ann Arbor, MI, USA); 2C-I, LSD and LSD-d3 (internal standard) were purchased from Cerilliant (Round Rock, TX, USA). The full name and structure of all analytes are shown in the Supplementary Fig. 1. 2.2. Standard solutions preparation Individual stock solutions were prepared by dilution of standard materials as follow: 1000 mg/mL of 25C-NBOH, 25BNBOH, 25I-NBOH, 25H-NBOMe, 25C-NBOMe, 25I-NBOMe, 25BNBOMe, 25G-NBOMe, 25D-NBOMe, 25E-NBOMe in methanol; 100 mg/mL of LSD in acetonitrile; 100 mg/mL of 25N-NBOMe, 25T2NBOMe and 2C-I in methanol. For GC–MS/MS and LC–MS/MS screening methods, individual working solutions and mix working solutions with all analytes were prepared in concentrations ranging from 1 to 10 mg/mL, using methanol as solvent. LSD-d3 was used as internal standard at 200 ng/mL in methanol (extraction solvent). All solutions (stock, work and calibrator) were stored at 20 C. 2.3. Samples preparation Different blotter paper samples which were suspected to contain illicit compounds, were provided by the TechnicalScientific Police Superintendence from São Paulo State, Brazil. Individual blotter paper sample (c.a. 1.0 cm 1.0 cm) was placed in 2 mL polypropylene tube. Then 1,000 mL of methanol containing the internal standard (LSD-d3, at 200 ng/mL) was added, the tubes were capped, vortexed for 5 min and centrifuged at 14,000 rpm for 5 min at ambient temperature. The supernatant (c.a. 500 mL) was transferred to other polypropylene tube and diluted according to each analysis performed. For LC–MS/MS qualitative analysis (precursor ion method), samples were diluted in the ratio 1:20, and for GC–MS/MS qualitative analysis samples were diluted in the
The solution, 1 mL, was injected into the Nexera UFLC chromatographic system coupled to a LCMS8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The analytes were separated using a RaptorTM Biphenyl (100 mm 2.1 mm ID, 2.7 mm, Restek) column with pre column RaptorTM Biphenyl (5 mm 2.1 mm DI, 2.7 mm, Restek) maintained at 40 C. The mobile phase, consisting 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B) (75:25, isocratic elution). The flow rate was kept constant at 0.5 mL/min. The separated analytes were identified with a triple quadrupole mass spectrometer with positive electrospray ionization (ESI). The applied ESI conditions were: capillary voltage 4.50 kV, desolvation temperature 250 C, heat temperature 400 C, drying gas (N2) flow 15 L/min, nebulizing gas (N2) flow 3 L/min and collision gas (Ar) 230 kPa. The mass spectrometer was set to operate of the precursor ion scan mode for the fragment ion of m/z 121 (characteristic of NBOMe series) with a collision energy (CE) of 20 V, and of m/z 107 (characteristic of NBOH series) with CE of 27 V, event time of 0.340 s, scan velocity of 454 Da/s, mass range from m/z 300 to m/z 450 with a dependent event product ion scan experiment activated for compounds with intensity greater than 25,000 cps, for the both events. In the product ion scan mode, the conditions were: CE of 15 V (for the monitoring of the fragment ion of m/z 121), and CE of 20 V (for the monitoring of the fragment ion of m/z 107), event time of 0.500 s, scan velocity of 769 Da/s and a range from m/z 80 to m/z 450, for the both scans. The compounds were identified by comparison of spectrum obtained by product ion scan with an inhouse MS/MS spectrum library, built by the injection of standards at the same ionization conditions described in this item. Data was acquired and analyzed (including library search) using the software Labsolution (version 5.53 SP2, Shimadzu). 2.6. Neutral loss scan and single ion monitoring method by GC–MS/MS The solution, 1 mL, was injected in splitless mode into the GCMS TQ8040, a gas chromatographic system coupled to triple quadrupole mass analyzer with electron ionization (EI) source (Shimadzu, Kyoto Japan). The chromatographic conditions were developed and optimized using derivatizated reference material working solutions, with mass spectrometer set to fullscan mode. The analytes were separated using a HP-5MS (30 m 0.25 mm 0.25 mm, Agilent, USA) and helium at a constant flow rate of 1.0 mL/min was used as the carrier gas.
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The initial column temperature 80 C was maintained for 1.00 min, then increased linearly at a rate of 20 C/min to 320 C and maintained for 5.0 min. The GC injector was maintained at 280 C, the ion source and interface temperature were 300 C, ionization energy at 70 eV and positive ions were analyzed. Acquisition was carried out in the neutral loss scan mode, for the neutral loss of 233 Da (characteristic of NBOMe series, see Fig. 1), CE of 10 V, event time of 0.350 s, scan velocity of 416 Da/s, and mass range from m/z 390 to m/z 525; and concomitant single monitoring of the ions of m/z 91 and m/z 121 (characteristics of NBOMe series). Argon was used as collision gas at 200 kPa. Chromatograms and spectra were registered from 9.00–18.00 min. Data was acquired and analyzed using the software GCMSsolution (version 4.45, Shimadzu). 2.7. Neutral loss and single ion monitoring scan method by GC–MS/ MS, and precursor and product ion scan method by LC–MS/MS validation Both methods, neutral loss scan by GC–MS/MS and precursor and product ion scan by LC–MS/MS, were validated over three consecutive days, with parameters such as selectivity, limit of detection (LOD) and imprecision (under repeatability and reproducibility) according to the “Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized Materials and Biological Specimens” manual, outlined by the UNODC (United Nations Office On Drugs And Crime) [19]. The analyte identification criteria were a peak eluting within 2 % of the average of the calibrator retention time, a signal-to-noise ratio of at least 3, and the mass spectrum should have a good visual match to that of the standard or should achieve a fit factor of 85 % or more in the library search (compared to our with in-house MS/ MS spectrum library), on a scale in which a perfect fit achieves a fit factor of 100 % [19]. The LOD is usually defined as the lowest concentration that reached the identification criteria, but in this method validation the administrative LOD value was defined based on the laboratory’s administratively decision point for reporting these analytes, which consider also the concentrations frequently observed on seized material [14]. The selectivity is concerned with the extent to which other substances (compounds usually found to be present in seized drugs) interfere with the identification and, where appropriate, quantification, of the analytes of interest [19]. Selectivity was tested with a mixture of substances commonly present in seized blotter papers, such as 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25E-NBOMe, 25G-NBOMe, 25H-NBOMe, 25I-NBOMe, 25T2NBOMe, 25B-NBOH, 25C-NBOH, 25I-NBOH, LSD, and other psychoactive substances, at concentrations between 1.0–2.5 mg/mL for GC–MS/MS and 0.3 - 0.5 mg/mL for LC–MS/MS method. The analyzed substances investigated in selectivity studies are described at Supplementary Table 1. Both methods were
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considered as selective if there were no peaks present at the same NBOMes and NBOHs retention time. Additionally, the LC–MS/ MS method was considered as selective if no other compounds were identified by comparison with an in-house MS/MS spectrum library as NBOMes and NBOHs. Intraday imprecision was determined by analyzing five replicates in the same day and interday imprecision was determined by analyzing five replicate samples, in three consecutive days conditions, of 0.45 mg/mL of mix of NBOMes and NBOHs standard for LC–MS/MS method and of 4.0 mg/mL of mix of NBOMes derivatizated for GC–MS/MS method. The method retention time should be within 2 % of those obtained in reference material analysis. No more than one sample in five (20 %) should give a false negative result. 2.8. Multiple reactions monitoring quantitation method by LC–MS/MS The solution, 2 mL, was injected into the Nexera UFLC chromatographic system coupled to a LCMS8040 triple quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The analytes were separated using a ForceTM Biphenyl (50 2.1 mm ID, 1.8 mm, Restek, USA) column and the oven was operated in a temperature of 40 C. The mobile phase, consisting 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). Chromatography separation was carried out using a gradient elution programmed as follows: 5 % B for 0.20 min, followed by a linear change to 35 % B over 1.30 min, held at 35 % B for 2.10 min and returned to initial conditions over 0.10 min (total run time of 5.00 min). The flow rate was kept constant at 0.5 mL/min. Identification and quantitation of the separated analytes were done with a triple quadrupole mass spectrometer operating in the precursor ion scan mode with dependent event of product ion scan via positive electrospray ionization (ESI). The applied ESI conditions were: capillary voltage 4.50 kV, desolvation temperature 250 C, heat temperature 400 C, drying gas (N2) flow 15 L/ min, nebulizing gas (N2) flow 3 L/min and collision gas (Ar) 230 kPa. The analyses were performed in multiple reactions monitoring mode (MRM). The MRM conditions used are presented in Supplementary Table 2, where first transition was used for quantitation and second transition used for identification. Data was acquired and treated using the software Labsolution (version 5.53 SP2, Shimadzu). 2.9. Multiple reactions monitoring method by LC–MS/MS validation The method was validated over three consecutive days, with parameters such as selectivity, limit of detection (LOD) and quantification (LOQ), linearity, accuracy bias, imprecision (under repeatability and reproducibility) and recovery rates based on the “Guidance for the Validation of Analytical Methodology and Calibration of Equipment used for Testing of Illicit Drugs in Seized
Fig. 1. Proposed fragmentation of NBOMe and NBOH derivatives in GC–MS/MS, after derivatization of NBOMes with TFAA.
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Materials and Biological Specimens” manual, outlined by the UNODC [19]. The method selectivity was determined evaluating other compounds usually found to be present in blotter paper samples. Potential interferences encountered other drugs of abuse and pharmaceuticals (see Supplementary Table 1) were evaluated by fortifying them at 500 ng/mL into low QC and negative samples. No interference was noted if all analytes in the low QC quantified within 20 % of target with acceptable qualifier/quantifier MRM ratios and no peak in the negative sample satisfied LOD criteria. The limits of detection (LOD) and quantification (LOQ) represent the lowest concentration of the substance under evaluation that can be detected and quantified, respectively. Regarding LOD and LOQ, these parameters were calculate taking into account the instrumental signal-to-noise ratio (S/N > 3 for LOD, S/N > 10 for LOQ), but in this validation the administrative LOD and LOQ values were defined based on the laboratory’s administratively decision point for reporting this analyte, which consider the concentrations frequently observed on seized material [14]. The linearity was determined with calibration range from 1 to 100 ng/mL in methanol. A high linear correlation coefficient (r) value (r 0.99) was used as criterion of good linearity. The calibration curves were constructed by plotting the peak area versus analyte standard concentration using the leastsquares linear regression method (1/x2 weighting) and correlation. For concentration determination of each analyte in blotter paper samples the analyte peak area was used and the concentrations were calculated using the calibration curve. Accuracy bias and imprecision were determined using quality control (QC) samples prepared by spiking 3, 30 and 75 ng/mL (QC1, QC2 and QC3, respectively) of each standard in blotter paper extract methanol solution. Three replicates of QC samples were run in the same batch together with calibration standards, and the calibration curve was then obtained and the QC concentrations determined. The accuracy bias was determined dividing the mean concentration for analytical runs (intraday n = 3; interday n = 9) by the expected concentration, and expressed as percentage. Accuracy bias values in the range of 80–120 % were considered acceptable. The intra- and interday imprecision was obtained by calculating the relative standard deviation (%RSD) for the mean concentration (n = 5 and n = 15, respectively) and one-way analysis of variance (ANOVA) was performed on each QC concentration to assess potentially significant interday variability at p < 0.05. Imprecision values with %RSD less than 20 % were considered acceptable. Once was not possible to have a blank matrix of samples (blotter paper), the accuracy of the method was determined as recovery experiments. Two different concentrations (30 and 75 ng/mL) of analyte working solutions were added to blotter paper samples solution and analyzed by the proposed method. Results were calculated as experimental values compared to theoretical values (initial analyte sample concentration+added working solution) and were expressed as percent recovery. The recovery rates were considered acceptable above 80 %.
corresponding to a NBOMe moiety- are screened, most new illegal substances of both series are expected to be detected. Fig. 2 presents a chromatogram obtained by the analysis of working solution of three NBOH and nine NBOMe at 0.3 mg/mL – this concentration is equivalent to 6 mg/blotter, considering the sample preparation procedure, the dilution factor and high recovery rates (around 100 %. This LOD was therefore adequate, since because NBOMe compounds are normally present in blotter papers in the range of 50 mg to 2000 mg per blotter sample [14]. The similarity of in house library against NBOMe and NBOH standard spectra compounds for LOD solutions were high, varying between 87 % and 98 % (Supplementary Table 3). The S/N ratios actually indicated that the developed method could detect NBOMe and NBOH compounds in concentrations even lower than 0.3 mg/mL. Supplementary Table 3 shows results obtained for imprecisions, calculated as relative standard deviation (RSD%), with results between 0.2 and 1.2 %. No interferences were observed at negative and/or LOD samples in presence of potential interferences encountered, others drugs of abuse and pharmaceuticals at 0.5 mg/mL. No carryover was observed in blank samples after analysis of a 3.8 mg/mL of NBOMe and NBOH standards. 3.2. Neutral loss scan and single ion monitoring method by GC–MS/MS Neutral loss scan monitoring method was developed based on a characteristic neutral loss of 233 Da [15] which is common for the TFAA derivatizated NBOMe series. For single ion monitoring, the two fragments m/z 121 and m/z 91 were selected. As Fig. 3 shows, the developed GC–MS/MS method was shown to provide also an adequate screening tool to identify NBOMes in blotter paper samples. Again, as common fragments were monitored, new design NBOMes could also be detected. TFAA derivatization for NBOH series failed since no peaks were detected in the corresponding chromatograms. The administrative LOD value obtained via the GC–MS/MS neutral loss scan and single ion monitoring scan were 2.5 mg/mL (equivalent to 25 mg/blotter paper) for all compounds, excepted for 25E-NBOMe (LOD of 4.0 mg/mL, equivalent to 40 mg/blotter paper). Again, these LOD values are therefore adequate because NBOMe compounds are found in concentration higher than 50 mg per blotter sample [14]. Supplementary Table 4 summarizes the intraday and interday imprecisions calculated by relative standard deviation (RSD%). The imprecision was low with values between 0.01 and 0.27 %, therefore in the range of 2 % recommended by the UNODC guide (UNODC, 2017), for all analytes in the concentration of 0.45 mg/mL, without false negatives. No interferences were observed at negative and/or LOD samples in the presence of the potential interferences encountered, other
3. Results and discussions 3.1. Precursor and product ion scan method by LC–MS/MS The precursor ion method was developed based on two characteristic fragment ions common for NBOMe and NBOH series compounds, that is the ion of m/z 121 and m/z 107, respectively. In order to enhance the selectivity of the method and compounds identification, a product ion scan dependent event and in-house library were created. The method showed to be a simple and efficient screening tool for NBOMes and NBOHs in blotter paper samples. Since the precursors of a characteristic fragment ion –
Fig. 2. Representative chromatogram of NBOMes working solution mixture (0.3 m g/mL) obtained by LC–MS/MS precursor and product ion scans modes. Legend: (1) 25C-, (3) 25B-, (5) 25I-NBOH, and, (2) 25H-, (4)25N-, (6) 25D-, (7) 25C-, (8) 25T2-, (9) 25B-, (10) 25G-, (11) 25E- and (12) 25I-NBOMe.
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Fig. 3. Representative chromatogram of NBOMes working solution mixture (2.5 mg/mL), derivatized with TFAA and analyzed by GC–MS/MS in neutral loss scan and single ion monitoring modes. Legend: (1) 25H-, (2) 25D-, (3) 25E-, (4) 25G-, (5) 25C-, (6) 25B-, (7) 25I- and 25N-NBOMe-TFA.
drugs of abuse at 2.5 mg/mL as well as pharmaceuticals at 1.0 mg/ mL. No carryover was observed in blank samples after analysis of a 10 mg/mL of NBOMe standards. 3.3. Multiple reactions monitoring quantitation method by LC–MS/MS Fig. 4 presents the LC–MS/MS chromatogram obtained by the fast MRM quantitation method developed to quantify thirteen compounds, that is, LSD and twelve phenethylamines (from NBOMe, NBOH and 2C series) which are usually present in blotter papers. Supplementary Table 5 shows the validation results by the MRM quantitation LC–MS/MS method for LSD, 2C-I, NBOH (25C-, 25B- and 25I-NBOH) and NBOMe (25H-, 25C-, 25I-, 25B-, 25G-, 25D- and 25E-NBOMe). A linear curve with six calibration points
from 1 to 100 ng/mL was performed using a 1/x2 weight linear regression, r > 0.996, for the twelve compounds, which presented a satisfactory linearity. Angular coefficients imprecisions for three days of analysis ranged from 1.4 for 25B-NBOH to 8.8 % for 25I-NBOMe. LOD and LOQ were 1 and 3 ng/mL, respectively, for all compounds. Intraday and interday imprecisions were calculated by one-way analysis of variance (ANOVA, p < 0.05) and showed low values of %RSD for all analytes in three concentrations of QC (3, 50 and 75 ng/mL). The accuracy bias were within -7.1 and +5.6 % (n = 15), as well as bias values. No carryover was observed in blank samples after analysis of a 100 ng/mL sample (higher calibrator) and no interferences were observed at negative and/or LOQ samples in presence of potential interferences encountered, others drugs of abuse and pharmaceuticals at 500 ng/mL.
Fig. 4. Representative LC–MS/MS chromatogram of MRM quantitation method obtained from a working solution mixture of (1) LSD; (2) 2C-I; (4) 25C-, (5) 25B- and (6) 25INBOH; (3) 25H-, (7) 25D-, (8) 25C-, (9) 25B-, (10) 25G-, (11) 25E-, (12) 25I-NBOMe at 10 ng/mL.
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Table 1 Qualitative and quantitative results obtained by proposed triple quadrupole analytical protocol (LC–MS/MS and GC–MS/MS) to the analysis of phenylethylamines in seized blotter papers. Sample #
MRM LC-MS/MS method
Blotter paper concentrataion (mg/blotter)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
Precursor and product ion scan LC-MS/MS method
Neutral loss and SIMb GC-MS/MS method
MS similarity in comparison to in house library (%)
Identification
LSD
2C-I 25CNBOH
25INBOH
25HNBOMe
25CNBOMe
25BNBOMe
25INBOMe
25CNBOH
25INBOH
25HNBOMe
25CNBOMe
25INBOMe
25CNBOMe
25INBOMe
ND ND ND ND ND 86.1 ND ND ND ND ND ND ND ND ND ND
12.0 0.3 ND 0.2 ND ND ND ND ND 6.3 ND ND 4.4 ND ND ND
715 5.2 61.2 0.07 1.4 10.5 802 1374 433 254 1829 509 1929 301 1498 ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 0.6
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 70.6
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 1.9
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 14.9
79 96 94 84 92 99 96 94 97 91 91 93 92 97 84 ND
95 96 97 98 98 97 98 94 97 97 98 92 96 97 98 ND
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 88
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 97
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND 91
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Detected
ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND Detected
340 1.0 5.0 1.7 0.04 ND 47.0 47.0 28.0 33.9 12.6 24.0 43.2 19.0 65.0 0.1
ND: not detected.
3.4. Authentic forensic cases
4. Conclusions
Blotter papers suspected to contain psychoactive phenethylamines were seized by law enforcement and evaluated by the developed protocol of analysis: precursor and product ion scan by LC–MS/MS, neutral loss scan and single ion monitoring by GC–MS/ MS and MRM quantitation by LC–MS/MS (Table 1). The most common phenethylamines derivatives in blotter paper samples were 25I- and 25C-NBOH. The 2C-I, LSD, 25HNBOMe, 25C-NBOMe, 25I-NBOMe and 25B-NBOMe were also found. The 2C-I was detected in five samples, but in concentrations lower than 12 mg per blotter sample. At such low concentration, 2C-I was probably present as minor contaminant (unreacted starting material) from the synthesis of 25I-NBOH. Precursor and product ion scan LC–MS/MS method and neutral loss and SIM GC– MS/MS method presented satisfactory results comparing to MRM LC–MS/MS for NBOMes. In previous study, Edmunds et al. [20] developed a method to evaluate of illicit 25X-NBOMe seizures by UPLC-PDA, and applied it to analysis of samples from Western Australia, seized between January/2014 and February/2016. The authors detected 25CNBOMe, 25I-NBOMe and 25B-NBOMe in samples, with concentrations ranging from 114 to 1,480 mg per blotter sample. Our blotter papers were seized between January/2017 and June/2017, in Sao Paulo state (Southeast Brazil). The phenethylamines profiles observed in our samples were different, where NBOH compounds were the most frequently detected, with concentrations from 0.1 to 1,929 mg per blotter sample. The greater prevalence of NBOH compounds as hallucinogenic drugs was also observed by Machado et al. [21], whom recently published a qualitative chemical characterization of 25B-, 25C- and 25E-NBOH detected in seized blotter papers. Just in one sample we detected NBOMe compounds (sample 16). According to Poklis et al. [14], NBOMe compounds found per blotter ranged from 50 mg to 2000 mg and sample quantity lower than 30 mg per blotter sample was considered impurities, thus 25H-NBOMe, 25I-NBOMe and 25B-NBOMe could be considered interferents in sample 16.
Frequently described as powerful quantitative instruments, triple quadrupole mass spectrometers can be also useful tool for identification of new psychoactive substances. In the present work, a comprehensive protocol using both LC–MS/MS and GC–MS/MS, with different scanning modes (MRM, SIM, neutral loss, precursor ion and product ion), have been developed and showed to be useful to screening NBOMe and NBOH in blotter papers. When applied to authentic forensic samples, the approach was shown to be a powerful, selective and sensitive tool for untargeted screening. As general structural moieties were monitored, the protocol is expected to work also to detect new phenylethylamines analogues with high efficiency. CRediT authorship contribution statement Damila Rodrigues de Morais: Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Visualization. Kelly Francisco da Cunha : Conceptualization, Methodology, Validation, Investigation, Writing - original draft, Visualization. Taís Betoni Rodrigues: Investigation, Writing - review & editing, Visualization. Rafael Lanaro: Validation, Investigation, Writing review & editing, Visualization. Lilian de Melo Barbosa: Investigation, Resources. Jorge Jardim Zacca: Investigation, Resources. Marcos Nogueira Eberlin: Resources, Writing - review & editing, Funding acquisition. Jose Luiz Costa: Conceptualization, Writing review & editing, Funding acquisition, Project administration. Declaration of Competing Interest The authors declare that they have no conflicts of interest. Acknowledgements The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, process number 23038.006844/2014-
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46), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, process numbers 2018/00432-1, 2018/11849-0 and 2016/ 23157-0) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, process number 131780/2017-4 and 425814/ 2018-1) by the fellowships and the financial support. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.forsciint.2020.110184. References [1] J. Lobo Vicente, H. Chassaigne, M.V. Holland, F. Reniero, K. Kolár, S. Tirendi, I. Vandecasteele, I. Vinckier, C. Guillou, Systematic analytical characterization of new psychoactive substances: a case study, Forensic Sci. Int. 265 (2016) 107– 115, doi:http://dx.doi.org/10.1016/j.forsciint.2016.01.024. [2] J. Suzuki, M.A. Dekker, E.S. Valenti, F.A. Arbelo Cruz, A.M. Correa, J.L. Poklis, A. Poklis, Toxicities associated with NBOMe ingestion— a novel class of potent hallucinogens: a review of the literature, Psychosomatics 56 (2015) 129–139, doi:http://dx.doi.org/10.1016/j.psym.2014.11.002. [3] M. Ibáñez, J.V. Sancho, L. Bijlsma, A.L.N. van Nuijs, A. Covaci, F. Hernández, Comprehensive analytical strategies based on high-resolution time-of-flight mass spectrometry to identify new psychoactive substances, TrAC Trends Anal. Chem. 57 (2014) 107–117, doi:http://dx.doi.org/10.1016/j.trac.2014.02.009. [4] L.C. Arantes, E.F. Júnior, L.F. de Souza, A.C. Cardoso, T.L.F. Alcântara, L.M. Lião, Y. Machado, R.A. Lordeiro, J.C. Neto, A.F.B. Andrade, 25I-NBOH: a new potent serotonin 5-HT2A receptor agonist identified in blotter paper seizures in Brazil, Forensic Toxicol. 35 (2017) 408–414, doi:http://dx.doi.org/10.1007/ s11419-017-0357-x. [5] V.B. Kueppers, C.T. Cooke, 25I-NBOMe related death in Australia: a case report, Forensic Sci. Int. 249 (2015) e15–e18, doi:http://dx.doi.org/10.1016/j.forsciint.2015.02.010. [6] L.M. Nielsen, N.B. Holm, S. Leth-Petersen, J.L. Kristensen, L. Olsen, K. Linnet, Characterization of the hepatic cytochrome P450 enzymes involved in the metabolism of 25I-NBOMe and 25I-NBOH, Drug Test. Anal. 9 (2017) 671–679, doi:http://dx.doi.org/10.1002/dta.2031. [7] D. Zuba, K. Sekuła, Analytical characterization of three hallucinogenic N -(2methoxy)benzyl derivatives of the 2C-series of phenethylamine drugs, Drug Test. Anal. 5 (2013) 634–645, doi:http://dx.doi.org/10.1002/dta.1397.
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