Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers

Journal Pre-proof Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers Damila Rodrigues de Morais (Conceptualization) (Methodology) (Validation) (Investigation)Writing - original draft, Visualization), Kelly Francisco da Cunha (Conceptualization) (Methodology) (Validation) (Investigation) (Writing - original draft) (Visualization), Ta´ıs Betoni Rodrigues (Investigation) (Writing review and editing) (Visualization), Rafael Lanaro (Validation) (Investigation) (Writing - review and editing) (Visualization), Lilian de Melo Barbosa (Investigation) (Resources), Jorge Jardim Zacca (Investigation) (Resources), Marcos Nogueira Eberlin (Resources) (Writing - review and editing) (Funding acquisition), Jose Luiz Costa (Conceptualization) (Writing - review and editing) (Funding acquisition) (Project administration)

PII:

S0379-0738(20)30046-3

DOI:

https://doi.org/10.1016/j.forsciint.2020.110184

Reference:

FSI 110184

To appear in:

Forensic Science International

Received Date:

25 June 2019

Revised Date:

16 January 2020

Accepted Date:

3 February 2020

Please cite this article as: de Morais DR, da Cunha KF, Betoni Rodrigues T, Lanaro R, de Melo Barbosa L, Jardim Zacca J, Nogueira Eberlin M, Costa JL, Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers, Forensic Science International (2020), doi: https://doi.org/10.1016/j.forsciint.2020.110184

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

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Full Title: Triple quadrupole–mass spectrometry protocols for the analysis of NBOMes and NBOHs in blotter papers

Short Title: Mass spectrometry protocols for NBOMes and NBOHs analysis

Damila Rodrigues de Moraisa, Kelly Francisco da Cunhab, Taís Betoni Rodrigues b,

Rafael Lanarob,c, Lilian de Melo Barbosad, Jorge Jardim Zaccae, Marcos

aThoMSon

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Nogueira Eberlina, Jose Luiz Costac,f Mass Spectrometry Laboratory, University of Campinas, Institute of Chemistry,

Campinas, São Paulo, 13083-970, Brazil bDepartment

of Pharmacology, Faculty of Medical Sciences, University of Campinas, Campinas,

São Paulo, 13083-887, Brazil

Poison Control Center, Faculty of Medical Sciences, University of Campinas,

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cCampinas

Campinas, São Paulo, 13083-970, Brazil

Police Superintendence, Criminalistics Institute, Campinas, São Paulo,

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dTechnical-Scientific

13018-170, Brazil eBrazilian

Federal Police, Scientific and Technical Department - PF, Brasília, Distrito Federal,

fFaculty

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70037-900, Brazil

of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-

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871, Brazil

Correspondence to: Jose Luiz Costa, Faculty of Pharmaceutical Sciences, University of Campinas, Campinas, São Paulo, 13083-871, Brazil. E-mail: [email protected], Phone:

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+55 19 3521 7232 and Fax: +55 19 3521 7592

Graphical abstract

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Highlights

Triple quadrupole mass spectrometers can be a useful tool for identification of NPS

New strategies to the analysis NBOMes and NBOHs by LC-MS/MS and GC-MS/MS

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Neutral loss and precursor ion scan modes were useful for drug

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identification

NBOH concentrations ranges from 0.1 to 1,929 μg/blotter sample

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Abstract

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

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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 μg 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

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developed and showed to be useful to screening NBOMe and NBOH in blotter papers.

Keywords: new psychoactive substances, phenethylamine derivatives,

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1. Introduction

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NBOMe, NBOH, LC-MS/MS, GC-MS/MS.

New psychoactive substances (NPS) are synthetic drugs, which contain

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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

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hallucinogenic effects (similar to LSD). These compounds are synthetized as derivatives from the 2C-X of psychoactive phenethylamines [4–6].

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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 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 (5-HT2a, 5-

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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

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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 triple quadrupole (QqQ) instruments, but MRM method development requires

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reference materials, hence new NBOMe and NBOH drugs would not be detected. There are, however, different modes of acquisition in QqQ

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instruments which have been poorly explored. For instance, the precursor ion and neutral loss scan modes have been used to selectively screen for several

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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

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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

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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

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provide a way to detect new designer drugs. 2. Material and methods 2.1. Chemicals and reagents 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),

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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, 25H-NBOMe, 25C-NBOMe, 25I-NBOMe, 25B-NBOMe, 25G-NBOMe, 25D-NBOMe, 25ENBOMe, 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 Figure 1.

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2.2. Standard solutions preparation

Individual stock solutions were prepared by dilution of standard materials as follow: 1,000 µg/mL of 25C-NBOH, 25B-NBOH, 25I-NBOH, 25H-NBOMe, 25CNBOMe, 25I-NBOMe, 25B-NBOMe, 25G-NBOMe, 25D-NBOMe, 25E-NBOMe

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in methanol; 100 µg/mL of LSD in acetonitrile; 100 µg/mL of 25N-NBOMe, 25T2-NBOMe and 2C-I in methanol.

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For GC-MS/MS and LC-MS/MS screening methods, individual working solutions and mix working solutions with all analytes were prepared in

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concentrations ranging from 1 to 10 µg/mL, using methanol as solvent. LSD-d3 was used as internal standard at 200 ng/mL in methanol (extraction solvent). All

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solutions (stock, work and calibrator) were stored at – 20 °C. 2.3. Samples preparation

Different blotter paper samples which were suspected to contain illicit

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compounds, were provided by the Technical-Scientific Police Superintendence from São Paulo State, Brazil. Individual blotter paper sample (c.a. 1.0 cm × 1.0

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cm) was placed in 2 mL polypropylene tube. Then 1,000 μL 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 μL) 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 ratio 1:10. For quantitative LC–MS/MS analysis, the samples were diluted with

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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 µL) were evaporated under a stream of N2 and room temperature (25 °C). Then, derivatives were prepared by dissolving dry extracts separately in 100 µL of derivatizing agent (TFAA:chloroform, 1:1, v/v), and, after vortex mixing, the

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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 µL of ethyl acetate in a vial (1 µL of injection).

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2.5. Precursor and product ion scan method by LC-MS/MS

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The solution, 1 µL, was injected into the Nexera UFLC chromatographic system coupled to a LCMS8040 triple quadrupole mass spectrometer

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(Shimadzu, Kyoto, Japan). The analytes were separated using a Raptor™ Biphenyl (100 mm x 2.1 mm ID, 2.7 μm, Restek) column with pre column Raptor™ Biphenyl (5 mm × 2.1 mm DI, 2.7 μm, Restek) maintained at 40ºC.

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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.

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The separated analytes were identified with a triple quadrupole mass spectrometer with positive electrospray ionization (ESI). The applied ESI

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conditions were: capillary voltage 4.50 kV, desolvation temperature 250 ºC, heat temperature 400 ºC, 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

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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 m/z/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 in-house 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).

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2.6. Neutral loss scan and single ion monitoring method by GC-MS/MS

The solution, 1 µL, 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

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conditions were developed and optimized using derivatizated reference material working solutions, with mass spectrometer set to fullscan mode. The analytes

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were separated using a HP-5MS (30 m × 0.25 mm × 0.25 µm, 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 ºC was maintained for 1.00 min, then increased linearly at a rate of 20 ºC/min to 320 ºC and maintained for 5.0 min. The GC injector was maintained at 280 ºC, the ion source and interface

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temperature were 300 ºC, 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 Figure 1), CE of 10

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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

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m/z 121 (characteristics of NBOMe series). Argon was used as collision gas at 200 kPa. Chromatograms and spectra were registered from 9.00 to 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

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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

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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

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validation the administrative LOD value was defined based on the laboratory’s

administratively decision point for reporting these analytes, which consider also

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the concentrations frequently observed on seized material [14]. The selectivity is concerned with the extent to which other substances

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(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

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seized blotter papers, such as 25B-NBOMe, 25C-NBOMe, 25D-NBOMe, 25ENBOMe, 25G-NBOMe, 25H-NBOMe, 25I-NBOMe, 25T2-NBOMe, 25B-NBOH, 25C-NBOH, 25I-NBOH, LSD, and other psychoactive substances, at

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concentrations between 1.0 - 2.5 μg/mL for GC-MS/MS and 0.3 - 0.5 μg/mL for LC-MS/MS method. The analyzed substances investigated in selectivity studies

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are described at Supplementary Table 1. Both methods were 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 µg/mL of mix of NBOMes

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and NBOHs standard for LC-MS/MS method and of 4.0 µg/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 µL, 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 Force™

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Biphenyl (50 x 2.1 mm ID, 1.8 µm, Restek, USA) column and the oven was operated in a temperature of 40 ºC. 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%

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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.

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Identification and quantitation of the separated analytes were done with a triple quadrupole mass spectrometer operating in the precursor ion scan mode

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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 ºC, heat temperature 400 ºC, drying gas (N2) flow 15 L/min,

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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

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was used for quantitation and second transition used for identification. Data was acquired and treated using the software Labsolution (version 5.53 SP2,

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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

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Calibration of Equipment used for Testing of Illicit Drugs in Seized 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.

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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

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values were defined based on the laboratory’s administratively decision point for reporting this analyte, which consider the concentrations frequently observed on

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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)

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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

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concentration determination of each analyte in blotter paper samples the analyte peak area was used and the concentrations were calculated using the calibration curve.

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Accuracy bias and imprecision were determined using quality control (QC) samples prepared by spiking 3, 30 and 75 ng/mL (QC1, QC2 and QC3,

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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)

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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

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analyte sample concentration + added working solution) and were expressed as percent recovery. The recovery rates were considered acceptable above 80%.

3. Results and discussions

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3.1. Precursor and product ion scan method by LC-MS/MS

The precursor ion method was developed based on two characteristic

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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

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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

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the precursors of a characteristic fragment ion – corresponding to a NBOMe moiety- are screened, most new illegal substances of both series are expected to be detected. Figure 2 presents a chromatogram obtained by the analysis of

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working solution of three NBOH and nine NBOMe at 0.3 μg/mL – this concentration is equivalent to 6 μg/blotter, considering the sample preparation

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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 μg to 2,000 μg 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 μg/mL.

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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 μg/mL. No carryover was observed in blank samples after analysis of a 3.8 μg/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

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derivatizated NBOMe series. For single ion monitoring, the two fragments m/z 121 and m/z 91 were selected. As Figure 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

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monitored, new design NBOMes could also be detected. TFAA derivatization for NBOH series failed since no peaks were detected in the corresponding

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chromatograms.

The administrative LOD value obtained via the GC-MS/MS neutral loss

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scan and single ion monitoring scan were 2.5 μg/mL (equivalent to 25 μg/blotter paper) for all compounds, excepted for 25E-NBOMe (LOD of 4.0 μg/mL, equivalent to 40 μg/blotter paper). Again, these LOD values are therefore

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adequate because NBOMe compounds are found in concentration higher than 50 μg per blotter sample [14].

Supplementary Table 4 summarizes the intraday and interday

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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%

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recommended by the UNODC guide (UNODC, 2017), for all analytes in the concentration of 0.45 μg/mL, without false negatives. No interferences were observed at negative and/or LOD samples in the

presence of the potential interferences encountered, other drugs of abuse at 2.5 μg/mL as well as pharmaceuticals at 1.0 μg/mL. No carryover was observed in blank samples after analysis of a 10 μg/mL of NBOMe standards. 3.3. Multiple reactions monitoring quantitation method by LC-MS/MS

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Figure 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 25INBOH) 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

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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

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(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

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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

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potential interferences encountered, others drugs of abuse and pharmaceuticals at 500 ng/mL.

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3.4. Authentic forensic cases

Blotter papers suspected to contain psychoactive phenethylamines were

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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

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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, 25H-NBOMe, 25C-NBOMe, 25INBOMe and 25B-NBOMe were also found. The 2C-I was detected in five samples, but in concentrations lower than 12 μg 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

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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 25C-NBOMe, 25I-NBOMe and 25B-NBOMe in samples, with concentrations ranging from 114 to 1,480 μg 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

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different, where NBOH compounds were the most frequently detected, with concentrations from 0.1 to 1,929 μg 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

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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 μg to 2,000 μg and

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sample quantity lower than 30 μg per blotter sample was considered impurities,

interferents in sample 16.

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4. Conclusions

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thus 25H-NBOMe, 25I-NBOMe and 25B-NBOMe could be considered

Frequently described as powerful quantitative instruments, triple quadrupole mass spectrometers can be also useful tool for identification of new

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psychoactive substances. In the present work, a comprehensive protocol using both LC-MS/MS and GC-MS/MS, with different scanning modes (MRM, SIM,

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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.

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Credit Author Statement

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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.

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Conflict of interest

Acknowledgements

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The authors declare that they have no conflicts of interest.

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The authors thank Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, process number 23038.006844/2014-46), Fundação de

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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

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Tecnológico

(CNPq,

process

number

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131780/2017-4 and 425814/2018-1) by the fellowships and the financial support.

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Figure 1. Proposed fragmentation of NBOMe and NBOH derivatives in GC-

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MS/MS, after derivatization of NBOMes with TFAA.

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Figure 2. Representative chromatogram of NBOMes working solution mixture

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(0.3 μg/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)

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25C-, (8) 25T2-, (9) 25B-, (10) 25G-, (11) 25E- and (12) 25I-NBOMe.

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Figure 3. Representative chromatogram of NBOMes working solution mixture (2.5 μg/mL), derivatized with TFAA and analyzed by GC-MS/MS in neutral loss

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scan and single ion monitoring modes. Legend: (1) 25H-, (2) 25D-, (3) 25E-, (4)

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25G-, (5) 25C-, (6) 25B-, (7) 25I- and 25N-NBOMe-TFA.

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Figure 4. Representative LC-MS/MS chromatogram of MRM quantitation method obtained from a working solution mixture of (1) LSD; (2) 2C-I; (4) 25C-,

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(5) 25B- and (6) 25I-NBOH; (3) 25H-, (7) 25D-, (8) 25C-, (9) 25B-, (10) 25G-,

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(11) 25E-, (12) 25I-NBOMe at 10 ng/mL.