HR-MAS NMR for Rapid Identification of Illicit Substances in Tablets and Blotter Papers Seized by Police Department

Illicit substances found in blotter papers and tablets seized by police are traditionally identified and characterized from extracts of these materials. However, the procedures involved in extraction stages can result in artifacts and even contamination of the samples to be analyzed. On the other hand, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) is a technique that requires no pretreatment steps, enabling direct analysis of the material, including the analysis of new illegal synthetic psychoactive substances. This study presents and discusses applications of the HR-MAS NMR in the analysis of tablets and blotter papers seized. Additional analysis in solution of the extracts of these materials was performed to compare the obtained spectral resolution signals. The results demonstrated that the HR-MAS NMR allowed the rapid identification of 3,4-methylenedioxy-N-methylcathinone (methylone), 4-methylmethcathinone (mephedrone), 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2methoxyphenyl)methyl]ethanamine (25B-NBOMe) in samples of tablets and blotter papers seized in Goiás State, Brazil.

The increasing proliferation of the designer drugs has hindered their identification based only on the traditional comparison of results obtained from the analysis with available spectral libraries. 11Added to this, the difficulties associated with the unequivocal assignment of the substitution pattern in the aromatic rings present in new amphetamine derivatives and other molecules with similar chemical structure, absent in equipment databases, makes detection techniques traditionally used in forensic science fairly limited. 12,13n this context, nuclear magnetic resonance (NMR) spectroscopy, one of the most used tools for identification and structural characterization of organic compounds, demonstrates great potential to solve problems in forensic chemistry, especially in identification of new compounds without analytical standards.This technique also belongs to the list of analytical techniques recommended by the Scientific Working Group for the Analysis of Seized Drugs (SWGDRUG) aiming at the elaboration of expert reports of a definitive character. 14mong NMR techniques, high-resolution (HR) NMR for liquid and in solution samples is the most frequently used.Its main disadvantage is the need for sample extraction, which leads to loss of information about the natural state of the sample.Another drawback is the amount of sample required to obtain a good signal/noise ratio without increased cost and analysis time.On the other hand, the investigation of samples without any pretreatment and with high-resolution can be carried out by high-resolution magic angle spinning (HR-MAS) NMR spectroscopy.][17] In this regard, the present study describes the use of 1 H HR-MAS NMR analyses for unprocessed heterogeneous materials (without any preliminary treatment), aiming to identify illicit substances in samples of blotter papers and tablets seized by police department in Goiás State, Brazil.
In order to compare spectral resolutions, in solution NMR analyses for extracted compounds were also evaluated.

Heterogeneous samples
Samples of blotter paper of approximately 0.8 cm 2 were fragmented into pieces.Four pieces were inserted into a zirconium rotor with internal volume of 50 µL (Figure 3).The samples of tablets were powdered and 15 mg were transferred to an identical rotor.In both experiments 40 µL of CD 3 OD were added to the rotors.

In solution samples
Samples of the blotter papers were extracted with 510 µL of CD 3 OD during 1 h.The solution was transferred to a 5 mm diameter NMR tube.Samples of tablets were powdered, 10 mg transferred to an Eppendorf flask, 1 mL of   MeOH was added, and the mixture was stirred in vortex for 15 s.After centrifugation for 30 s, the solution, in another flask, was evaporated using nitrogen gas.A volume of 500 µL of CD 3 OD were transferred to a 5 mm NMR tube.

NMR conditions
All NMR data were acquired at 25 °C using a Bruker Avance III 500 NMR spectrometer (Karlsruhe, Germany) operated at 11.75 T ( 1 H resonance frequency 500.13MHz), equipped with a 5 mm triple resonance broad band (TBI) probe (in solution analyses) and a 4 mm HR-MAS probe spinning at 7 kHz (unprocessed samples).The spectra were recorded using a composite pulses presaturation (CPPR) sequence for water signal suppression.Sixty four (64) free induction decays (FIDs) were collected into 122 k data points using a 12.4 µs pulse width (90 pulse angle), spectral width of 12500 Hz, relaxation delay of 1.0 s and an acquisition time of 4.99 s.An exponential weighing factor corresponding to line broadening of 0.3 Hz was applied to the FIDs.The following abbreviations are used to designate NMR absorption patterns: s (singlet), brd (broad doublet), brt (broad triplet), d (doublet), dt (double triplet), t (triplet), q (quartet), dd (double doublet), ddd (double double doublet) and m (multiplet).

Results and Discussion
The NMR spectra obtained for blotter papers using unprocessed or extracted samples are very similar, presenting the same signal patterns and chemical shifts (Figure 4).The multiplicities, coupling constants (J) and chemical shifts (d) are attributed to one 1,2,4,5-tetrasubstituted aromatic ring containing two methoxy and one isopropylamine attached groups.These informations, coupled with previous data coming from mass spectrometry (MS) (Figure S1), allowed the characterization of DOB.The full hydrogen signals assignment are presented in Table 1.
Similarly to DOB, the NMR spectra obtained for another blotter paper using unprocessed or extracted samples are very similar, presenting the same signal patterns and chemical shifts (Figure 5).The multiplicities, coupling constants (J) and chemical shifts are attributed to one 1,2,4,5-tetrasubstituted and one 1,2-disubstituted aromatic rings containing three methoxy and one ethylmethylamine attached groups.Bromine was also observed in MS spectra (Figure S2).This compound was characterized as 25B-NBOMe.The full hydrogen signals assignment are presented on Table 2.
Since the experiments with the HR-MAS probe were promising to identify illicit substances in blotter papers, without any extraction procedures, it was also used for identifying substances in tablets seized by police department, which have very distinct characteristics from blotter papers previously analyzed.For this, 15 mg of powdered tablet were analyzed, and mephedrone, the  major compounds on the mixture of substances, was identified (Figure 6).A p-disubstituted aromatic ring was characterized by two doublets at d 7.98 and 7.39 on 1 H NMR spectrum.One methine attached to methyl, methylamine and keto groups was identified on the basis of the quartet signal at d 5.21, with chemical shift typical   of keto and amine neighborhood.Three methyl groups were also identified by two singlets and one doublet signals.This was corroborated by gas chromatographymass spectrometry (GC-MS) (Figure S3).The complete assignment of the mephedrone NMR data is presented in Table 3.In this tablet, caffeine was also observed as a minor compound.
According to previous results, where spectra obtained from in solution or unprocessed samples were always similar, the next seized tablet sample was only analyzed by HR-MAS probe.A 1,4,5-trisubstituted aromatic ring was characterized by one double doublet at d 7.72 and two doublets at d 7.48 and 7.00 on 1 H NMR spectrum (Figure 7).One methylenedioxy group attached to aromatic ring was identified by the signal at d 6.12.Similarly to mephedrone, a keto-amine group was also observed.These NMR data were enough to characterize 3,4-methylenedioxy-N-methylcathinone, known as methylone.This was corroborated by GC-MS (Figure S4).The complete assignment of the methylone NMR data is presented in Table 4.  Thus, the results obtained by 1 H HR-MAS NMR technique exhibited an excellent discrimination power for the group of amphetamine derivatives and other molecules with similar chemical structure using minimal sample pretreatment (only powdering).

Conclusions
In this paper we demonstrated the efficiency of the 1 H HR-MAS NMR technique for unambiguous identification of new synthetic illicit substances, using minimal pretreatment of the tablets and blotter paper seized by police department in Goiás State.No significant difference on chemical profile was observed between unprocessed samples or extracted with solvent samples.
The great advantage observed using 1 H HR-MAS NMR technique is that no extraction procedure and purification of substances for further analysis was required, thus avoiding a possible contamination and the appearance of artifacts that could compromise the conclusions of the forensic examination.This technique also represents an important time saver, particularly considering the great number of drugs seized in Brazil.Therefore, despite the HR-MAS NMR technique not yet being diffused in forensics area, this work highlights its importance especially due to the limitations presented by traditional techniques compared with the growing emergence of new synthetic psychoactive illegal substances.

Figure 1 .
Figure 1.Chemical structures of some psychoactive substances.

Figure 3 .
Figure 3. Zirconium rotor (50 µL) and accessories, along with the sample of intact and fragmented blotter paper.

Figure 4 . 1 H
Figure 4. 1 H NMR spectra of blotter paper using (a) HR-MAS and (b) TBI probes showing the presence of DOB.The signal multiplicities are highlighted.

Figure 5 . 1 H
Figure 5. 1 H NMR spectra of blotter paper using (a) HR-MAS and (b) TBI probes showing the presence of 25B-NBOMe.The signal multiplicities are highlighted.

Figure 6 .
Figure 6. 1 H NMR spectra of tablets seized using (a) HR-MAS and (b) TBI probes showing the presence of mephedrone and other compounds (*: caffeine and impurity).The signal multiplicities are highlighted.

Figure 7 . 1 H
Figure 7. 1 H NMR spectrum of a new tablet sample using HR-MAS probe showing the presence of methylone.The signal multiplicities are highlighted.