- Email: [email protected]
Rapid detection of NBOME's and other NPS on blotter papers by direct ATR-FTIR spectrometry
Accepted Manuscript Title: Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry Author: Jos´e Coelho Neto PII: DOI: Reference:
S0379-0738(15)00171-1 http://dx.doi.org/doi:10.1016/j.forsciint.2015.04.025 FSI 7981
To appear in:
FSI
Received date: Revised date: Accepted date:
22-1-2015 6-4-2015 20-4-2015
Please cite this article as: Jos´e Coelho Neto, Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.04.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acknowledgments
\section*{Acknowledgements}
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We would like to thank all our laboratory colleagues, who contributed blotter samples from their cases, including Rog\'egio Araujo Lordeiro and Paulo Eduardo Nunes Barbosa, also responsible for running {LC-MS} and {GC-MS} tests and providing helpful discussions. Special thanks to Marco Ant\^onio Fonseca Paiva, director of the Instituto de Criminal\'istica da Pol\'icia Civil de Minas Gerais, who encouraged and supported research efforts within the Institute.
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Title Page (with authors and addresses)
Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry
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Jos´e Coelho Neto Divis˜ao de Laborat´orio, Instituto de Criminal´ıstica da Pol´ıcia Civil de Minas Gerais Rua Juiz de Fora, 400, CEP 30180-060, Belo Horizonte, MG, Brasil
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Departamento de F´ısica e Qu´ımica, Pontif´ıcia Universidade Cat´olica de Minas Gerais Avenida Dom Jos´e Gaspar, 500, CEP 30535-901, Belo Horizonte, MG, Brasil
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Corresponding author Email address: [email protected] (Jos´e Coelho Neto)
Preprint submitted to Forensic Science International
April 3, 2015
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Highlights (for review)
Highlights
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Quick direct detection method for NPS on blotter papers via ATRFTIR. Sample integrity preserved (no extraction required), allowing further testing. Inhomogeneity (possibly dosage-related) of blotter papers on the market evidenced. 25B-, 25C- and 25I-NBOME’s detected on most blotters tested. Methallylescaline (MAL) detected on some blotters tested.
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Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry
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Abstract
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Blotter paper is among the most common forms of consumption of new psychotropic substances (NPS), formerly referred as designer drugs. In many cases, users are misled
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to believe they are taking LSD when, in fact, they are taking newer and less known drugs like the NBOMEs or other substituted phenethylamines. We report our findings in quick testing of blotter papers for illicit substances like NBOMEs and other NPS by taking ATR-
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FTIR spectra directly from blotters seized on the streets, without any sample preparation. Both sides (front and back) of each blotter were tested. Collected data were analysed by
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single- and multi-component spectral matching and submitted to chemometric discrimi-
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nant analysis. Our results showed that, on 66.7% of the cases analysed, seized blotters contained one or more types of NBOMEs, confirming the growing presence of this novel
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substances on the market. Matching IR signals were detected on both or just one side of the blotters and showed variable strength. Although no quantitative analysis was made, detection of these substances by the proposed approach serves as indication of variable and possibly higher dosages per blotter when compared to LSD, which showed to be below the detection limit of the applied method. Blotters containing a mescaline-like compound, later confirmed by GC-MS and LC-MS to be MAL (methallylescaline), a substance very similar to mescaline, were detected among the samples tested. Validity of direct ATRFTIR testing was confirmed by checking the obtained results against independent GC-MS or LC-MS results for the same cases/samples.
Preprint submitted to Forensic Science International
April 3, 2015
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Keywords: ATR-FTIR, Blotter papers, NPS (new psychotropic substances), NBOME,
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MAL (methallylescaline), Designer drugs, Discriminant analysis.
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1. Introduction In recent years, spreading of new, unregulated substances of abuse, referred generally
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as NPS, also known as novel designer drugs or legal highs (an allusion to the deceptive legal status of such substances on most countries), has become an issue regarding public
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health in many parts of the world, demanding constant improvements both on the available knowledge pertaining to the chemistry, pharmacology and toxicology of such substances
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and on their legal regulation as well[1, 2]. Among those substances, which include a growing number of substituted phenethylamines, the NBOME series, a generic denomination
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for phenethylamines presenting a 2 − methoxybenzyl group replacing a hydrogen on the amine, have been attracting attention from medical and legal authorities, due to its association with several intoxication cases and even deaths[3–6], causing it to be outlawed in
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various countries, including Brazil[7, 8].
Like LSD, NBOMEs produce hallucinogenic effects acting as partial ou full agonists
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to serotonin 5 − HT 2A receptors[9], but presenting increased affinity and selectivity[10].
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It’s undesirable effects, however, include tachycardia, hypertension, agitation, confusion, pupil dilation, aggressiveness, seizures and other effects[11–14], making it much more
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dangerous and potentially fatal than LSD. Lower cost, higher availability and the fact that NBOMEs are still considered legal in many countries seem to be causing increased use of such substances and even encouraging commerce of NBOMEs as counterfeit LSD. Recent reports showed that blotters containing NBOMEs were sold in Spain, Austria and possibly other countries as if they contained LSD[15–17], exposing LSD-accustomed users to unexpected dangers. Forensic analysis of blotters is normally conducted by GC-MS or LC-MS and require sample preparation which include extraction of the analytes by soaking blotters on organic solvents, followed by filtration and/or dilution prior to injection, as described by
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Zuba et al. [18], procedures which are both time consuming and sample-destructive. In this work we report our attempts to rapidly detect NBOMEs and other NPS by taking ATR-FTIR spectra directly from the blotters, with no sample preparation. Analysis of the
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spectra obtained from both faces of each blotter was carried out considering three different levels: single spectrum database comparison, multi-component database comparison
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(deconvolution) and discriminant analysis (chemometrics). Aside from a faint indentation mark on each side, produced by the ATR vise accessory, samples were undamaged and
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preserved for further testing.
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2. Materials and Methods 2.1. Samples
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A total of 77 blotter papers decorated with various artwork patterns were taken randomly as samples from 27 street apprehensions conducted by local police forces on the
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state of Minas Gerais, Brazil, during 2014. Multiple blotters, presenting the same artwork
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or not, were taken from the same apprehension when possible and tested individually. Spectra of each single blotter were collected once at the back side, which normally pre-
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sented no artwork, and once at the front side, defined as where the main artwork was located.
2.2. Instrumentation
ATR-FTIR spectra of blotters were taken using a NicoletT M iZ10 spectrometer equipped with EverGlo IR source, DLaTGS room temperature IR detector and single-bounce Smart OrbitT M accessory module with diamond ATR crystal. All hardware from Thermo Fischer Scientific Inc. (USA). Each spectrum were averaged over 16 scans, taken at 4cm−1 resolution, maximum detector window aperture and minimum interferometer mirror speed, in the range of 400 − 4
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4000cm−1 . Background signal was averaged over 8 scans prior to each measurement. Collection of each spectrum, including background measurement, usually took around
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2 minutes. 2.3. Data analysis
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Analysis of collected spectra was carried out using the software suite accompanying the spectrometer, which included the OMNICT M 9.1.27, used for data acquisition
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and single spectrum database comparison, the OMNICT M SpectaT M 2.0, used for multicomponent deconvolution and database comparison, and the TQ AnalystT M 9.1.17, used
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for chemometric discriminant analysis. All software from Thermo Fischer Scientific Inc. (USA).
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Spectral IR libraries had to be used for single- and multi-component sample spectrum analysis, as standards of target substances were not available for direct comparison. Libraries used included HR Comprehensive Forensic FT-IR Collection and HR Georgia
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State Forensic Drugs Library, available commercially from Thermo Fischer Scientific Inc.
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(USA), and the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) Infrared Library[19], freely available on the internet. NBOMEs spectra were available
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only on the SWGDRUG library.
Single spectrum database comparison consisted in performing an automated search on all available spectra on the selected libraries to find the best single matches. Each single search took around 5 seconds. For multi-component deconvolution and database comparison, up to 4 of the available spectra on the selected libraries were automatically combined into one by the OMNICT M SpectaT M to produce the best matches possible, revealing the presence of other compounds other than the best single matches previously found. Each multi-component search took around 1 minute. Finally, for discriminant analysis, a chemometrical method was created using the tools provided by the TQ AnalystT M 5
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software and used to classify the samples based on the collected spectra. The method contained a NBOME class constructed based upon spectra taken from blotters previously tested by ATR-FTIR and confirmed, by GC-MS or LC-MS, to contain at least one type
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of the NBOMEs previously detected (25B-, 25C- and 25I-NBOME, as showed on the results), which were used as class standards. Other classes, including a blank paper class,
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were created using the same procedure and the best available samples and added to the method. As target substances in pure form were not available to be used as class stan-
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dards, sets of samples reproducing the observed behavior during the first two rounds of analysis (described on the results) were considered as best available samples and used to
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construct each class. Therefore, all classes for target substances were built using standards (samples) sharing the same type of matrix (paper). After calibration, performed automat-
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ically by the software, the resulting method were used to classify other samples not used as class standards. Though construction and calibration of the method took a couple of hours, including selection of best standard candidates for each class, after completion,
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classification of a new sample spectrum was virtually instantaneous.
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3. Results and Discussion
3.1. Single spectrum database comparison As could be expected a priori, taking ATR-FTIR spectra directly from a blotter produced results matching primarily paper/cellulose on most samples analysed, specially when the back side was tested. On the front side of some blotters, the presence of plastic polymers was detected, which was attributed to a protective film found covering the artwork, possibly created during painting/printing process. On a certain number of blotters, however, the collected spectra deviated considerably from the paper/cellulose and the plastic film profiles, indicating a strong presence of other substances. From all samples tested,
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both on back and front sides, blotters showing signals matching primarily 25B-NBOME, 25C-NBOME, 25I-NBOME and a mescaline-like compound were found. Comparative examples between spectra obtained from blotters and references are showed in Fig. 1 for
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25I-NBOME and Fig. 2 for the mescaline-like compound. Detection of such substances dominating the spectral profile while still impregnated on the paper suggests a very high
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dosage for these blotters.
At this primary level of analysis, NBOMEs were detected on 9 blotters, taken from 7
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different cases, corresponding to 11.7% of the total number of blotters tested and 25.9% of apprehension cases. The mescaline-like compound was detected on 5 blotters, from
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2 different cases, corresponding to 6.5% of blotters and 7.4% of cases. Negative results, matching paper/cellulose or plastic film were obtained on 63 blotters, from the 18 remain-
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ing cases, corresponding to 81.8% of total blotters and 66.7% of cases. Results obtained for each side of tested blotters are summarized on Table 1.
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Table 1: Results from the single spectrum database comparison for tested blotters.
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Negative
Positive
plastic film
NBOMEs
Mescaline-like
back side
69 (89.6%)
1 (1.3%)
4 (5.2%)
3 (3.9%)
front side
55 (71.4%)
13 (16.9%)
6 (7.8%)
3 (3.9%)
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paper/cellulose
3.2. Multi-component deconvolution database comparison After searching for the best single match on the available IR libraries, every blotter spectrum was submitted to deconvolution trough the multi-component database search engine available from the spectrometer software suite. On this second round of analysis, new matching spectra were constructed by mathematically combining up to four reference spectra available on the libraries. As a result, a number of spectra previously matched primarily as paper/cellulose or plastic film (negative results) now included IR signals of one 7
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Figure 1: Spectral comparison showing IR profiles obtained from the backside of a sample blotter and reference spectra. (a) Spectral profile from the back side of a blotter containing 25I-NBOME. (b) IR spectrum
standard).
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1.1: Complete spectrum
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of 25I-NBOME (from SWGDRUG IR library[19]). (c) IR spectrum from blank absorbent paper (local
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1.2: Fingerprint region
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Figure 2: Spectral comparison showing IR profiles obtained from the backside of a sample blotter and reference spectra. (a) Spectral profile from the back side of a blotter containing a mescaline-like compound. (b) IR spectrum of Mescaline (from HR Georgia State Forensic Drugs Library). (c) IR spectrum from blank
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2.1: Complete spectrum
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absorbent paper (local standard).
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2.2: Fingerprint region
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or more NBOMEs or the mescaline-like compound, accounting for small IR peaks deviating from the previous negative match, located at the same regions as showed in Fig. 1.2 and Fig. 2.2, increasing the number of positive matches. Even fainter signal traces, found
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on blotters from other 2 cases previously negative, were assigned as DOB1 . Detection of weaker IR signals matching target substances hidden under the paper/cellulose and/or
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plastic film matrix, made possible through deconvolution, suggests much smaller dosages per blotter than those tested positive on the first round of testing. Results obtained by this
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approach are summarized on Table 2.
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Table 2: Results from the multi-component spectrum database comparison for tested blotters. Negative
Positive
NBOMEs
Mescaline-like
DOB
back side
21 (27.3%)
36 (46.7%)
14 (18.2%)
6 (7.8%)
front side
23 (29.9%)
34 (44.1%)
14 (18.2%)
6 (7.8%)
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(paper and/or film)
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After completion of the second round of analysis, NBOMEs detection increased to 39
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blotters, taken from 18 cases, corresponding to 50.6% of total blotters tested and 66.7% of apprehension cases. The mescaline-like compound detection increased to 14 blotters,
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taken from the same 2 cases as before, corresponding to 18.2% of blotters tested and 7.4% of cases. DOB was faintly detected on 6 blotters, taken from 2 cases, corresponding to 7.8% of blotters and 7.4% of cases. Negative results, matching paper/cellulose and/or protective film combinations were reduced to 18 blotters, taken from 5 cases, corresponding to 23.4% of blotters and 18.5% of cases. Up to this point, even before confirming the identities of the substances detected, the overall available results, which were based only on the use of direct ATR-FTIR spectrom1
2, 5 − Dimethoxy − 4 − bromoamphetamine.
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etry alone, aside from being able to detect target substances, also made evident the large inhomogeneity among tested blotters, characterized by the changes in signal strength from blotter to blotter, observed even within blotters presenting the same artwork, taken from
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the same blotter sheet, and the observation that IR signatures of target substances appeared mostly on one side (front or back) of each blotter. Such observations represent qualitative
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evidence of different dosages and different production processes per sample.
To confirm the presence of the detected substances, results obtained so far were con-
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fronted with those obtained by GC-MS or LC-MS for samples taken from the same cases, using procedures and methods similar to those described by Zuba et al. [18]. The compar-
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ison showed that, although all NBOME positive matches obtained from direct ATR-FTIR spectrometry were confirmed, the exact type of NBOME not always agreed, with 25B-
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NBOME, 25C-NBOME and 25I-NBOME sometimes being identified as one another and vice-versa. Such occasional divergence may be attributed to the use of spectral libraries instead of spectra taken from local standards, which were not available, combined with
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possible matrix (paper or plastic film) interference and the strong similarity between the
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chemical structures and IR spectral profiles of the NBOMEs detected. The same type of effect was observed on the blotters identified as containing DOB, which were confirmed as
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containing DOC2 by LC-MS. For the blotters initially marked as containing the mescalinelike compound, both GS-MS and LC-MS confirmed that, in fact, they contained MAL (Methallylescaline), a much less known psychedelic drug whose structure is very similar to that of mescaline, differing only by a 2 − propene group attached to the methyl of the methoxy on the 4−position. As no IR spectrum for MAL were available on any of the libraries used for comparison, the closest match found was mescaline. Having a much lower threshold dosage (somewhere around 20 − 40mg) than mescaline (threshold around 2
2, 5 − Dimethoxy − 4 − chloroamphetamine.
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100mg), MAL would make a much better candidate for putting on paper, although at least 4 to 5 blotters would be needed to achieve the threshold, as the maximal dosage a single blotter could hold is considered to be around 5mg[20, 21]. All blotters still reported as
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negative by direct ATR-FTIR spectrometry after the multi-component analysis turned up
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contain LSD when examined by GC-MS or LC-MS. 3.3. Discriminant analysis
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To complete the analysis of the IR spectra taken from the blotters and avoid relaying solely on single- or multiple-component profile matching, a multiclass discriminant anal-
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ysis method, available from the chemometric tools provided by the TQ AnalystT M was implemented. The method contained 4 classes, NBOME, MAL, LSD and BLANK, con-
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structed upon spectra taken from a selected part of the samples tested previously and some common blank paper samples. With no pure standards available, sets of samples reproducing the observed behaviors, i.e. stronger and weaker NPS signals, detection on opposing
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sides and the presence of protective films, were selected to characterize each class. By
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selecting samples in this way, all classes shared a similar matrix (paper) imbued within them. The NBOME class was created using 28 selected spectra taken from 17 blotters
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spawning 6 cases, previously matched and confirmed as containing one or more types of the NBOMEs detected in previous rounds of analysis (25B-, 25C- and 25I-NBOME), as class standards. No attempt was made to use discriminant analysis to distinguish between the three types of NBOMEs detected, due to the cross-identification between these species reported during multi-component analysis and the fact that many blotters contained more than one of them. MAL class contained 12 spectral standards taken from 6 blotters spawning 1 case. The LSD class was based on 18 spectra taken from 9 blotters spawning 2 cases, whose selection was guided by the LC-MS results during confirmation of our previous tests. For construction of the BLANK class, 9 spectra, taken from 3 different types 12
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of absorbent paper were used. A DOC class was not created as only 6 blotters from 2 cases were available. Method calibration parameters were set to use a single variance distribution for all classes, without any data normalization. The resulting method was able to
fied by the pairwise distance plot showed in Fig. 3.
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separate all standards used for its construction without any misclassification, as exempli-
Figure 3: Pairwise distance plot for the Mahalanobis distance between MAL and BLANK classes after
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calibration of the discriminant analysis method created to classify blotters.
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After calibration, remaining blotter samples not used for method creation (except
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those previously identified as containing DOC, whose class was not implemented on the method), along with 12 spectra from 4 other types of absorbent paper, were submitted to classification. A total of 39 blotters (22 NBOMEs from 12 cases, 8 MAL from 2 cases and 9 LSD from 3 cases) were tested. As each single blotter generated 2 spectral profiles (front and back), true positive hits were required to have both sides placed on the same class, otherwise resulting in misclassification. The resulting confusion matrix thus obtained is showed in Table 3.
Although LSD-containing blotters could not be identified by previous approaches, possibly due to extremely low dosages, usually on the microgram range[20], the constructed method was able to classify most samples correctly, which is promising. The obtained 13
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Table 3: Confusion matrix for the classification of blotters and blank papers obtained from the constructed discriminant analysis method. Predicted MAL
LSD
BLANK
NBOMEs
18
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1
3
MAL
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8
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0
LSD
1
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7
BLANK
1
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Actual
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NBOMEs
method for blotter classification presented accuracy of 91%, sensitivity of 82% and speci-
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ficity of 94%, averaged over all classes. Though we deem such results satisfactory, specially when we consider that most misclassifications occurred for samples presenting low
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intensity NPS IR signals (possibly related to lower dosages), it is possible that better classification results, including separation between the NBOMEs detected, could be achieved
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4. Conclusions
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if pure standards were available to be used in the building of classes.
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We have show that rapid detection of NBOMEs and other NPS on blotters is possible by direct ATR-FTIR spectrometry. Three types of NBOMEs (25B-NBOME, 25CNBOME and 25I-NBOME) were detected on 50.6% of blotters tested, corresponding to 66.7% of apprehension cases, evidencing the strong presence of these substances on the blotter market. Other NPS were also detected, including a mescaline-like compound, later identified as MAL (methallylescaline), a much lesser known mescaline-like compound, apparently never reported to have been found on blotter. Evidence of great inhomogeneity in drug dosages present on tested samples was found. Further quantitative studies, which demand the use of NPS standards in pure form, have to be conducted in order to evaluate drug dosage per blotter and establish the detection limits of the proposed analyses. 14
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No reference dealing with ATR-FTIR applied to the analysis of blotters were found on the literature, making the current study a totally new approach. As advantages, the proposed approach offers quick results, sample preservation and easy implementation. Af-
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ter collecting a spectrum directly from the surface of a blotter, the proposed procedures (single- and multi-component database comparison and discriminant analysis) can be ap-
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plied hierarchically, as in the present study, or independently from each other. However, as each blotter is basically treated as a mixture of paper and one or more NPS, IR spectrom-
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etry discriminating power is reduced and the technique can no longer be considered Category A according to SWGDRUG recommendations[22], therefore requiring confirmation
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from other analytical techniques, like GC-MS or LC-MS. Nevertheless, even if considered only as a quick preliminary test, the proposal presented constitutes one more useful tool to
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aid forensic experts dealing with a growing number of NPS, specially if we consider that, when combined with one of the mentioned hyphenated techniques, the obtained results are in good agreement with SWGDRUG recommendations for proper identification of drugs,
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References
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[4] D. G. E. Caldicott, S. J. Bright, M. J. Barratt, NBOME - a very different kettle of fish..., Med. J. Aust. 199 (5) (2013) 322–323, (Letter to the editor).
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[8] Agˆencia Nacional de Vigilˆancia Sanit´aria - Minist´erio da Sa´ude, Resoluc¸a˜ o da Diretoria Colegiada - RDC N o¯ 06, http://bvsms.saude.gov.br/bvs/saudelegis/ anvisa/2014/rdc0006_18_02_2014.pdf, (Published on 19-02-2014), 2014. [9] D. E. Nichols, Hallucinogens, Pharmacol. Ther. 101 (2) (2004) 131–181.
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[13] M. H. Y. Tang, C. K. Ching, M. S. H. Tsui, F. K. C. Chu, T. W. L. Mak, Two cases of severe intoxication associated with analytically confirmed use of the novel psychoactive substances 25B-NBOMe and 25C-NBOMe, Clin. Toxicol. 52 (5) (2014)
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S0379-0738(15)00171-1 http://dx.doi.org/doi:10.1016/j.forsciint.2015.04.025 FSI 7981
To appear in:
FSI
Received date: Revised date: Accepted date:
22-1-2015 6-4-2015 20-4-2015
Please cite this article as: Jos´e Coelho Neto, Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry, Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.04.025 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Acknowledgments
\section*{Acknowledgements}
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We would like to thank all our laboratory colleagues, who contributed blotter samples from their cases, including Rog\'egio Araujo Lordeiro and Paulo Eduardo Nunes Barbosa, also responsible for running {LC-MS} and {GC-MS} tests and providing helpful discussions. Special thanks to Marco Ant\^onio Fonseca Paiva, director of the Instituto de Criminal\'istica da Pol\'icia Civil de Minas Gerais, who encouraged and supported research efforts within the Institute.
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Title Page (with authors and addresses)
Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry
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Jos´e Coelho Neto Divis˜ao de Laborat´orio, Instituto de Criminal´ıstica da Pol´ıcia Civil de Minas Gerais Rua Juiz de Fora, 400, CEP 30180-060, Belo Horizonte, MG, Brasil
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Departamento de F´ısica e Qu´ımica, Pontif´ıcia Universidade Cat´olica de Minas Gerais Avenida Dom Jos´e Gaspar, 500, CEP 30535-901, Belo Horizonte, MG, Brasil
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Corresponding author Email address: [email protected] (Jos´e Coelho Neto)
Preprint submitted to Forensic Science International
April 3, 2015
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Highlights (for review)
Highlights
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Quick direct detection method for NPS on blotter papers via ATRFTIR. Sample integrity preserved (no extraction required), allowing further testing. Inhomogeneity (possibly dosage-related) of blotter papers on the market evidenced. 25B-, 25C- and 25I-NBOME’s detected on most blotters tested. Methallylescaline (MAL) detected on some blotters tested.
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Rapid detection of NBOME’s and other NPS on blotter papers by direct ATR-FTIR spectrometry
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Abstract
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Blotter paper is among the most common forms of consumption of new psychotropic substances (NPS), formerly referred as designer drugs. In many cases, users are misled
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to believe they are taking LSD when, in fact, they are taking newer and less known drugs like the NBOMEs or other substituted phenethylamines. We report our findings in quick testing of blotter papers for illicit substances like NBOMEs and other NPS by taking ATR-
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FTIR spectra directly from blotters seized on the streets, without any sample preparation. Both sides (front and back) of each blotter were tested. Collected data were analysed by
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single- and multi-component spectral matching and submitted to chemometric discrimi-
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nant analysis. Our results showed that, on 66.7% of the cases analysed, seized blotters contained one or more types of NBOMEs, confirming the growing presence of this novel
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substances on the market. Matching IR signals were detected on both or just one side of the blotters and showed variable strength. Although no quantitative analysis was made, detection of these substances by the proposed approach serves as indication of variable and possibly higher dosages per blotter when compared to LSD, which showed to be below the detection limit of the applied method. Blotters containing a mescaline-like compound, later confirmed by GC-MS and LC-MS to be MAL (methallylescaline), a substance very similar to mescaline, were detected among the samples tested. Validity of direct ATRFTIR testing was confirmed by checking the obtained results against independent GC-MS or LC-MS results for the same cases/samples.
Preprint submitted to Forensic Science International
April 3, 2015
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Keywords: ATR-FTIR, Blotter papers, NPS (new psychotropic substances), NBOME,
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MAL (methallylescaline), Designer drugs, Discriminant analysis.
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1. Introduction In recent years, spreading of new, unregulated substances of abuse, referred generally
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as NPS, also known as novel designer drugs or legal highs (an allusion to the deceptive legal status of such substances on most countries), has become an issue regarding public
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health in many parts of the world, demanding constant improvements both on the available knowledge pertaining to the chemistry, pharmacology and toxicology of such substances
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and on their legal regulation as well[1, 2]. Among those substances, which include a growing number of substituted phenethylamines, the NBOME series, a generic denomination
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for phenethylamines presenting a 2 − methoxybenzyl group replacing a hydrogen on the amine, have been attracting attention from medical and legal authorities, due to its association with several intoxication cases and even deaths[3–6], causing it to be outlawed in
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various countries, including Brazil[7, 8].
Like LSD, NBOMEs produce hallucinogenic effects acting as partial ou full agonists
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to serotonin 5 − HT 2A receptors[9], but presenting increased affinity and selectivity[10].
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It’s undesirable effects, however, include tachycardia, hypertension, agitation, confusion, pupil dilation, aggressiveness, seizures and other effects[11–14], making it much more
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dangerous and potentially fatal than LSD. Lower cost, higher availability and the fact that NBOMEs are still considered legal in many countries seem to be causing increased use of such substances and even encouraging commerce of NBOMEs as counterfeit LSD. Recent reports showed that blotters containing NBOMEs were sold in Spain, Austria and possibly other countries as if they contained LSD[15–17], exposing LSD-accustomed users to unexpected dangers. Forensic analysis of blotters is normally conducted by GC-MS or LC-MS and require sample preparation which include extraction of the analytes by soaking blotters on organic solvents, followed by filtration and/or dilution prior to injection, as described by
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Zuba et al. [18], procedures which are both time consuming and sample-destructive. In this work we report our attempts to rapidly detect NBOMEs and other NPS by taking ATR-FTIR spectra directly from the blotters, with no sample preparation. Analysis of the
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spectra obtained from both faces of each blotter was carried out considering three different levels: single spectrum database comparison, multi-component database comparison
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(deconvolution) and discriminant analysis (chemometrics). Aside from a faint indentation mark on each side, produced by the ATR vise accessory, samples were undamaged and
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preserved for further testing.
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2. Materials and Methods 2.1. Samples
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A total of 77 blotter papers decorated with various artwork patterns were taken randomly as samples from 27 street apprehensions conducted by local police forces on the
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state of Minas Gerais, Brazil, during 2014. Multiple blotters, presenting the same artwork
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or not, were taken from the same apprehension when possible and tested individually. Spectra of each single blotter were collected once at the back side, which normally pre-
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sented no artwork, and once at the front side, defined as where the main artwork was located.
2.2. Instrumentation
ATR-FTIR spectra of blotters were taken using a NicoletT M iZ10 spectrometer equipped with EverGlo IR source, DLaTGS room temperature IR detector and single-bounce Smart OrbitT M accessory module with diamond ATR crystal. All hardware from Thermo Fischer Scientific Inc. (USA). Each spectrum were averaged over 16 scans, taken at 4cm−1 resolution, maximum detector window aperture and minimum interferometer mirror speed, in the range of 400 − 4
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4000cm−1 . Background signal was averaged over 8 scans prior to each measurement. Collection of each spectrum, including background measurement, usually took around
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2 minutes. 2.3. Data analysis
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Analysis of collected spectra was carried out using the software suite accompanying the spectrometer, which included the OMNICT M 9.1.27, used for data acquisition
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and single spectrum database comparison, the OMNICT M SpectaT M 2.0, used for multicomponent deconvolution and database comparison, and the TQ AnalystT M 9.1.17, used
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for chemometric discriminant analysis. All software from Thermo Fischer Scientific Inc. (USA).
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Spectral IR libraries had to be used for single- and multi-component sample spectrum analysis, as standards of target substances were not available for direct comparison. Libraries used included HR Comprehensive Forensic FT-IR Collection and HR Georgia
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State Forensic Drugs Library, available commercially from Thermo Fischer Scientific Inc.
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(USA), and the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) Infrared Library[19], freely available on the internet. NBOMEs spectra were available
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only on the SWGDRUG library.
Single spectrum database comparison consisted in performing an automated search on all available spectra on the selected libraries to find the best single matches. Each single search took around 5 seconds. For multi-component deconvolution and database comparison, up to 4 of the available spectra on the selected libraries were automatically combined into one by the OMNICT M SpectaT M to produce the best matches possible, revealing the presence of other compounds other than the best single matches previously found. Each multi-component search took around 1 minute. Finally, for discriminant analysis, a chemometrical method was created using the tools provided by the TQ AnalystT M 5
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software and used to classify the samples based on the collected spectra. The method contained a NBOME class constructed based upon spectra taken from blotters previously tested by ATR-FTIR and confirmed, by GC-MS or LC-MS, to contain at least one type
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of the NBOMEs previously detected (25B-, 25C- and 25I-NBOME, as showed on the results), which were used as class standards. Other classes, including a blank paper class,
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were created using the same procedure and the best available samples and added to the method. As target substances in pure form were not available to be used as class stan-
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dards, sets of samples reproducing the observed behavior during the first two rounds of analysis (described on the results) were considered as best available samples and used to
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construct each class. Therefore, all classes for target substances were built using standards (samples) sharing the same type of matrix (paper). After calibration, performed automat-
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ically by the software, the resulting method were used to classify other samples not used as class standards. Though construction and calibration of the method took a couple of hours, including selection of best standard candidates for each class, after completion,
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classification of a new sample spectrum was virtually instantaneous.
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3. Results and Discussion
3.1. Single spectrum database comparison As could be expected a priori, taking ATR-FTIR spectra directly from a blotter produced results matching primarily paper/cellulose on most samples analysed, specially when the back side was tested. On the front side of some blotters, the presence of plastic polymers was detected, which was attributed to a protective film found covering the artwork, possibly created during painting/printing process. On a certain number of blotters, however, the collected spectra deviated considerably from the paper/cellulose and the plastic film profiles, indicating a strong presence of other substances. From all samples tested,
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both on back and front sides, blotters showing signals matching primarily 25B-NBOME, 25C-NBOME, 25I-NBOME and a mescaline-like compound were found. Comparative examples between spectra obtained from blotters and references are showed in Fig. 1 for
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25I-NBOME and Fig. 2 for the mescaline-like compound. Detection of such substances dominating the spectral profile while still impregnated on the paper suggests a very high
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dosage for these blotters.
At this primary level of analysis, NBOMEs were detected on 9 blotters, taken from 7
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different cases, corresponding to 11.7% of the total number of blotters tested and 25.9% of apprehension cases. The mescaline-like compound was detected on 5 blotters, from
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2 different cases, corresponding to 6.5% of blotters and 7.4% of cases. Negative results, matching paper/cellulose or plastic film were obtained on 63 blotters, from the 18 remain-
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ing cases, corresponding to 81.8% of total blotters and 66.7% of cases. Results obtained for each side of tested blotters are summarized on Table 1.
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Table 1: Results from the single spectrum database comparison for tested blotters.
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Negative
Positive
plastic film
NBOMEs
Mescaline-like
back side
69 (89.6%)
1 (1.3%)
4 (5.2%)
3 (3.9%)
front side
55 (71.4%)
13 (16.9%)
6 (7.8%)
3 (3.9%)
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paper/cellulose
3.2. Multi-component deconvolution database comparison After searching for the best single match on the available IR libraries, every blotter spectrum was submitted to deconvolution trough the multi-component database search engine available from the spectrometer software suite. On this second round of analysis, new matching spectra were constructed by mathematically combining up to four reference spectra available on the libraries. As a result, a number of spectra previously matched primarily as paper/cellulose or plastic film (negative results) now included IR signals of one 7
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Figure 1: Spectral comparison showing IR profiles obtained from the backside of a sample blotter and reference spectra. (a) Spectral profile from the back side of a blotter containing 25I-NBOME. (b) IR spectrum
standard).
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1.1: Complete spectrum
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of 25I-NBOME (from SWGDRUG IR library[19]). (c) IR spectrum from blank absorbent paper (local
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1.2: Fingerprint region
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Figure 2: Spectral comparison showing IR profiles obtained from the backside of a sample blotter and reference spectra. (a) Spectral profile from the back side of a blotter containing a mescaline-like compound. (b) IR spectrum of Mescaline (from HR Georgia State Forensic Drugs Library). (c) IR spectrum from blank
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2.1: Complete spectrum
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absorbent paper (local standard).
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2.2: Fingerprint region
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or more NBOMEs or the mescaline-like compound, accounting for small IR peaks deviating from the previous negative match, located at the same regions as showed in Fig. 1.2 and Fig. 2.2, increasing the number of positive matches. Even fainter signal traces, found
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on blotters from other 2 cases previously negative, were assigned as DOB1 . Detection of weaker IR signals matching target substances hidden under the paper/cellulose and/or
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plastic film matrix, made possible through deconvolution, suggests much smaller dosages per blotter than those tested positive on the first round of testing. Results obtained by this
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approach are summarized on Table 2.
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Table 2: Results from the multi-component spectrum database comparison for tested blotters. Negative
Positive
NBOMEs
Mescaline-like
DOB
back side
21 (27.3%)
36 (46.7%)
14 (18.2%)
6 (7.8%)
front side
23 (29.9%)
34 (44.1%)
14 (18.2%)
6 (7.8%)
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(paper and/or film)
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After completion of the second round of analysis, NBOMEs detection increased to 39
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blotters, taken from 18 cases, corresponding to 50.6% of total blotters tested and 66.7% of apprehension cases. The mescaline-like compound detection increased to 14 blotters,
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taken from the same 2 cases as before, corresponding to 18.2% of blotters tested and 7.4% of cases. DOB was faintly detected on 6 blotters, taken from 2 cases, corresponding to 7.8% of blotters and 7.4% of cases. Negative results, matching paper/cellulose and/or protective film combinations were reduced to 18 blotters, taken from 5 cases, corresponding to 23.4% of blotters and 18.5% of cases. Up to this point, even before confirming the identities of the substances detected, the overall available results, which were based only on the use of direct ATR-FTIR spectrom1
2, 5 − Dimethoxy − 4 − bromoamphetamine.
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etry alone, aside from being able to detect target substances, also made evident the large inhomogeneity among tested blotters, characterized by the changes in signal strength from blotter to blotter, observed even within blotters presenting the same artwork, taken from
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the same blotter sheet, and the observation that IR signatures of target substances appeared mostly on one side (front or back) of each blotter. Such observations represent qualitative
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evidence of different dosages and different production processes per sample.
To confirm the presence of the detected substances, results obtained so far were con-
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fronted with those obtained by GC-MS or LC-MS for samples taken from the same cases, using procedures and methods similar to those described by Zuba et al. [18]. The compar-
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ison showed that, although all NBOME positive matches obtained from direct ATR-FTIR spectrometry were confirmed, the exact type of NBOME not always agreed, with 25B-
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NBOME, 25C-NBOME and 25I-NBOME sometimes being identified as one another and vice-versa. Such occasional divergence may be attributed to the use of spectral libraries instead of spectra taken from local standards, which were not available, combined with
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possible matrix (paper or plastic film) interference and the strong similarity between the
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chemical structures and IR spectral profiles of the NBOMEs detected. The same type of effect was observed on the blotters identified as containing DOB, which were confirmed as
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containing DOC2 by LC-MS. For the blotters initially marked as containing the mescalinelike compound, both GS-MS and LC-MS confirmed that, in fact, they contained MAL (Methallylescaline), a much less known psychedelic drug whose structure is very similar to that of mescaline, differing only by a 2 − propene group attached to the methyl of the methoxy on the 4−position. As no IR spectrum for MAL were available on any of the libraries used for comparison, the closest match found was mescaline. Having a much lower threshold dosage (somewhere around 20 − 40mg) than mescaline (threshold around 2
2, 5 − Dimethoxy − 4 − chloroamphetamine.
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100mg), MAL would make a much better candidate for putting on paper, although at least 4 to 5 blotters would be needed to achieve the threshold, as the maximal dosage a single blotter could hold is considered to be around 5mg[20, 21]. All blotters still reported as
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negative by direct ATR-FTIR spectrometry after the multi-component analysis turned up
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contain LSD when examined by GC-MS or LC-MS. 3.3. Discriminant analysis
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To complete the analysis of the IR spectra taken from the blotters and avoid relaying solely on single- or multiple-component profile matching, a multiclass discriminant anal-
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ysis method, available from the chemometric tools provided by the TQ AnalystT M was implemented. The method contained 4 classes, NBOME, MAL, LSD and BLANK, con-
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structed upon spectra taken from a selected part of the samples tested previously and some common blank paper samples. With no pure standards available, sets of samples reproducing the observed behaviors, i.e. stronger and weaker NPS signals, detection on opposing
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sides and the presence of protective films, were selected to characterize each class. By
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selecting samples in this way, all classes shared a similar matrix (paper) imbued within them. The NBOME class was created using 28 selected spectra taken from 17 blotters
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spawning 6 cases, previously matched and confirmed as containing one or more types of the NBOMEs detected in previous rounds of analysis (25B-, 25C- and 25I-NBOME), as class standards. No attempt was made to use discriminant analysis to distinguish between the three types of NBOMEs detected, due to the cross-identification between these species reported during multi-component analysis and the fact that many blotters contained more than one of them. MAL class contained 12 spectral standards taken from 6 blotters spawning 1 case. The LSD class was based on 18 spectra taken from 9 blotters spawning 2 cases, whose selection was guided by the LC-MS results during confirmation of our previous tests. For construction of the BLANK class, 9 spectra, taken from 3 different types 12
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of absorbent paper were used. A DOC class was not created as only 6 blotters from 2 cases were available. Method calibration parameters were set to use a single variance distribution for all classes, without any data normalization. The resulting method was able to
fied by the pairwise distance plot showed in Fig. 3.
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separate all standards used for its construction without any misclassification, as exempli-
Figure 3: Pairwise distance plot for the Mahalanobis distance between MAL and BLANK classes after
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calibration of the discriminant analysis method created to classify blotters.
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After calibration, remaining blotter samples not used for method creation (except
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those previously identified as containing DOC, whose class was not implemented on the method), along with 12 spectra from 4 other types of absorbent paper, were submitted to classification. A total of 39 blotters (22 NBOMEs from 12 cases, 8 MAL from 2 cases and 9 LSD from 3 cases) were tested. As each single blotter generated 2 spectral profiles (front and back), true positive hits were required to have both sides placed on the same class, otherwise resulting in misclassification. The resulting confusion matrix thus obtained is showed in Table 3.
Although LSD-containing blotters could not be identified by previous approaches, possibly due to extremely low dosages, usually on the microgram range[20], the constructed method was able to classify most samples correctly, which is promising. The obtained 13
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Table 3: Confusion matrix for the classification of blotters and blank papers obtained from the constructed discriminant analysis method. Predicted MAL
LSD
BLANK
NBOMEs
18
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1
3
MAL
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8
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0
LSD
1
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7
BLANK
1
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Actual
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NBOMEs
method for blotter classification presented accuracy of 91%, sensitivity of 82% and speci-
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ficity of 94%, averaged over all classes. Though we deem such results satisfactory, specially when we consider that most misclassifications occurred for samples presenting low
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intensity NPS IR signals (possibly related to lower dosages), it is possible that better classification results, including separation between the NBOMEs detected, could be achieved
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4. Conclusions
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if pure standards were available to be used in the building of classes.
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We have show that rapid detection of NBOMEs and other NPS on blotters is possible by direct ATR-FTIR spectrometry. Three types of NBOMEs (25B-NBOME, 25CNBOME and 25I-NBOME) were detected on 50.6% of blotters tested, corresponding to 66.7% of apprehension cases, evidencing the strong presence of these substances on the blotter market. Other NPS were also detected, including a mescaline-like compound, later identified as MAL (methallylescaline), a much lesser known mescaline-like compound, apparently never reported to have been found on blotter. Evidence of great inhomogeneity in drug dosages present on tested samples was found. Further quantitative studies, which demand the use of NPS standards in pure form, have to be conducted in order to evaluate drug dosage per blotter and establish the detection limits of the proposed analyses. 14
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No reference dealing with ATR-FTIR applied to the analysis of blotters were found on the literature, making the current study a totally new approach. As advantages, the proposed approach offers quick results, sample preservation and easy implementation. Af-
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ter collecting a spectrum directly from the surface of a blotter, the proposed procedures (single- and multi-component database comparison and discriminant analysis) can be ap-
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plied hierarchically, as in the present study, or independently from each other. However, as each blotter is basically treated as a mixture of paper and one or more NPS, IR spectrom-
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etry discriminating power is reduced and the technique can no longer be considered Category A according to SWGDRUG recommendations[22], therefore requiring confirmation
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from other analytical techniques, like GC-MS or LC-MS. Nevertheless, even if considered only as a quick preliminary test, the proposal presented constitutes one more useful tool to
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aid forensic experts dealing with a growing number of NPS, specially if we consider that, when combined with one of the mentioned hyphenated techniques, the obtained results are in good agreement with SWGDRUG recommendations for proper identification of drugs,
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[13] M. H. Y. Tang, C. K. Ching, M. S. H. Tsui, F. K. C. Chu, T. W. L. Mak, Two cases of severe intoxication associated with analytically confirmed use of the novel psychoactive substances 25B-NBOMe and 25C-NBOMe, Clin. Toxicol. 52 (5) (2014)
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