Another chemically masked drug: p-tosyl methylamphetamine. Michael Collins*, Ananta Bhattarai and Helen Salouros. 1National Measurement Institute, Australian Forensic Drug Laboratory, Riverside Corporate Park, North Ryde, Sydney, NSW 2113, Australia. *Corresponding Author: Michael Collins Australian Forensic Drug Laboratory, National Measurement Institute, Sydney, New South Wales, Australia This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1002/dta.2363 This article is protected by copyright. All rights reserved. Abstract In July/August 2017, postal consignments from China each containing 1 kilogram bags described as “Nourishing Whitening Powder” were seized by the Australian Border Force (ABF). The total weight of the consignments exceeded 24 kilograms and initial testing revealed nothing of an illicit nature. However, the nature of the consignments including the labels were suspicious and samples were transferred to the National Measurement Institute Australia (NMIA). Using NMR, MS and IR spectroscopy the substance was identified as the p-tosyl derivative of methylamphetamine. This was confirmed by preparing the p-tosyl derivative using an authentic methylamphetamine sample. The resulting product was analysed by GC-MS and NMR and the spectra matched those of the seized material. Keywords: Methylamphetamine, precursor chemicals, chemical derivatives. This article is protected by copyright. All rights reserved. Introduction Methylamphetamine is one of the most heavily manufactured illicit drugs in the world today.[1] It is produced on a massive scale, usually from one of two precursors – ephedrine/pseudoephedrine or phenyl-2-propanone – and usually using one of only a handful of synthetic routes.[2] Areas of mass production are Southeast Asia, Southwest Asia, Eastern Europe and Mexico. It is trafficked in large quantities from production areas to target countries such as the United States and Australia and great efforts are made by the drug traffickers to conceal the drug from detection by Customs and Border Force officials. This has usually involved physical concealment methods such as disguising the drug as a shipment of some other commodity such as a foodstuff or an industrial product. In recent years attempts have been made to chemically mask illicit drugs by small changes to their molecular structure in order to evade detection devices. An example was a recent large seizure of MDMA disguised as its tertiary butoxycarbonyl (t-BOC) derivative [3, 4] quickly followed by an even larger seizure of a t-BOC-methylamphetamine derivative. The t-BOC derivatives are easy to make, equally easy to convert back into their parent drug and represent a very effective concealment method. In July/August 2017, several postal consignments from China containing 1 kilogram bags labelled “Nourishing Whitening Powder” were seized by the Australian Border Force (ABF). The total weight of the consignments exceeded 24 kilograms. The seized samples tested negative for the usual drugs and custody of the material was transferred to the Australian Forensic Drug Laboratory (AFDL) at the National Measurement Institute Australia (NMIA) for further analysis. This case report describes the identification of the seized material as the para-tosyl (p-tosyl) derivative of methylamphetamine (Figure 1) and its characterization using proton and carbon nuclear magnetic resonance spectroscopy (NMR), ATR-infrared This article is protected by copyright. All rights reserved. analysis (IR) and gas chromatography mass spectrometry (GC-MS). The identification was confirmed by preparing the p-tosyl derivative of methylamphetamine and comparing mass and NMR spectra of the two samples. Experimental Section Reagents and Standards Toluene-p-sulphonyl chloride and hydrochloric acid (36%) were purchased from Merck (Kilsyth, Vic, Australia). Sodium hydroxide pellets were purchased from UNIVAR Ajax Finechem (Seven Hills, NSW, Australia). Deuterochloroform (Product #DLM-7-100S, Lot #12L-417) and dimethylsulfoxide-d (Product #DLM-10-25, Lot #13B-030) were purchased 6 from Cambridge Isotope Laboratories Inc (Andover, MA, USA). Preparation of the p-tosyl derivative of methylamphetamine. Due to the sensitive nature of the seized material a full description of the synthesis of the p- tosyl derivative of methylamphetamine is not provided. However it was prepared using a general procedure for the preparation of p-tosyl derivatives as outlined in Vogel‟s Textbook of Practical Organic Chemistry. [5] Nuclear magnetic resonance spectroscopy 1H NMR spectra of the seized material were initially acquired as solutions in CDCl on a 3 Bruker Avance 500 MHz NMR Spectrometer (11.76 T) equipped with a BBFO 5 mm Probe at a temperature of 295 K. A 90° pulse (13.2 s) with a 5 second relaxation delay and an acquisition time of 4.4 seconds was used. Eight scans were acquired and data was collected over a spectral width of 15 ppm into 64 K data points. Bruker TopSpin software was used to This article is protected by copyright. All rights reserved. operate the NMR Spectrometer and process raw data. Following the initial spectrum acquisition in CDCl subsequent spectra were recorded in d -DMSO. 13C NMR spectra were 3 6 obtained at 125 MHz and 512 scans were acquired over a spectral width of 260 ppm into 64K data points using a 30° pulse (3.3 s), a 2 second relaxation delay and an acquisition time of 4.4 seconds. Gas chromatography mass spectrometry GC-MS analyses were performed on an Agilent 6890N GC interfaced with an Agilent 5973 MSD using a HP-5MS column (30 m x 0.25 mm x 0.25 m). Helium was used as the carrier gas in the constant flow rate mode. Injection port temperature was 240 °C and the MS interface temperature was 300 °C. The oven temperature program was 55 °C (3 minutes), ramped at 30 °C/min to 300 °C (no hold), and ramped at 20 °C/min to 325 °C (3 minutes). Injections (1 L) were made in pulsed splitless mode and a mass/charge range of m/z 40 to 500 was scanned. Determination of Accurate Mass using Liquid Chromatography-Quadrupole Time of Flight/Mass Spectrometry (LC-QTOF/MS) A 1ppm methanolic solution of the unknown and synthesized material were in turn introduced into the electrospray source of the LC/QTOF and a calibrated mass spectrum obtained with resolution better than 10,000 (full width at half maximum). A short guard column (C18) was used for infusion. The test sample solution was injected into the mobile phase flow at 0.2 mL/min (50:50) methanol water, followed by 0.1% formic acid in 50:50 methanol/water for a run time of 2 minutes). Full scan spectra (100 -1700 m/z) were collected every 0.5 seconds. For the seized material the calculated mass was 303.1293 amu and the This article is protected by copyright. All rights reserved. experimentally determined mass was 303.1294. For the synthesised material the experimentally determined mass was 303.1290 amu. Chiral Analysis Methylamphetamine was liberated from a portion of the seized p-tosyl methylamphetamine sample and its enantiomeric composition determined using a Mosher Acid derivatisation method. The liberated methylamphetamine (1 mg) was dissolved in hexane saturated with concentrated ammonia (2 mL). To this was added 2 drops of 2% (s)(+)-a-methoxy-a- (triflouromethyl)-phenylacetayl chloride (Mosher‟s Acid) in hexane. The solution was heated at 65 oC for 15 minutes and the hexane solution was analysed by GC/MS and compared to the GC/MS chromatographic trace of the Mosher acid derivative of both d- and l- methylamphetamine certified reference standards. Infra-red spectroscopy FTIR spectra were obtained on a Bruker ALPHA ATR Platinum Diamond instrument. Thirty-two background scans were acquired and the scan truncation range was 400–4000 cm- 1. Thirty-two scans of the sample were acquired in transmittance mode. A small amount of sample (~2 mg) was placed on the diamond ATR and the clamp engaged. Results and Discussion A sample of the seized material was delivered by Australian Border Force to the NMIA for analysis. The sample presented as a fine white powder and was analysed by GC-MS and the TIC exhibited a single chromatographic peak. The mass spectrum (Figure 2) obtained from this peak did not provide a good match to any spectral library entry but did return some poor This article is protected by copyright. All rights reserved. matches for various benzenesulfonamide compounds. An infrared spectrum of the compound (Figure 3) showed strong absorbances at 1288 cm-1 and 1150 cm-1, characteristic for sulphonamides. The 1H NMR spectrum in CDCl (Figure 4) looked similar to a 3 phenethylamine with an -methyl group such as methylamphetamine. The aliphatic portion of the spectrum contained two benzylic protons, one methine proton, a singlet integrating for three protons at 2.75 ppm, a slightly broader singlet integrating for three protons at 2.36 ppm and a methyl doublet at 0.98 ppm. This is consistent with methylamphetamine with the exception of the singlet at 2.36 ppm, i.e. an aromatic methyl group. So, looking at the mass, infrared and NMR spectra it seemed possible that this apparent unknown was just the p-toluenesulfonyl derivative of methylamphetamine, i.e. p-tosyl methylamphetamine. In Figure 2 possible mass fragmentations for p-tosyl methylamphetamine are shown that explain the major ions seen in the mass spectrum. Integration of the aromatic region of the 1H NMR spectrum, obtained in CDCl revealed nine 3, protons, consistent with the five protons due to methylamphetamine and an additional four protons which would be expected from the p-tosyl moiety. Because there was overlap of proton resonances in the aromatic region another spectrum was acquired in d -DMSO and 6 Figure 5 shows the aromatic portion of this spectrum (see Figure S1 for complete spectrum). It is clear from the spectrum that a p-di-substituted aromatic ring is present, again consistent with the p-tosyl moiety. Subsequent proton and carbon spectra were acquired using d - 6 DMSO as solvent. The 13C NMR spectrum exhibited four resonances in the aliphatic region as expected for methylamphetamine and an additional resonance at 21.54 ppm which is consistent with an aromatic methyl group of the p-tosyl moiety (Figure 6). There were 4 resonances in the aromatic section of the 13C spectrum additional to what would be expected This article is protected by copyright. All rights reserved. for methylamphetamine alone and consistent with a p-tosyl group. A series of two- dimensional (2D) NMR experiments including heteronuclear single quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC) and distortionless enhancement by polarization transfer (DEPT) were performed to assign all carbon chemical shifts and provide further confirmation of identity. Proton and carbon chemical shifts listing long range correlations are presented in Table 1. The 2D NMR images are provided in supplementary material (Figures S2 to S4). As a final proof of structure a sample of methylamphetamine was derivatized with p-tosyl chloride using the method described in Vogel‟s Practical Organic Chemistry.[5] The mass spectrum, IR spectrum, proton and carbon NMR spectra of the synthesised p-tosyl methylamphetamine matched those of the seized material (Figures 7, 8, 9 and 10 respectively). The calculated mass for the synthesised p-tosyl derivative of methylamphetamine is 303.1293 amu and the mass determined for the unknown white powder was 303.1294 amu. The mass determined for the synthesised p-tosyl methylamphetamine was 303.1290 amu. The p-tosyl derivative of methylamphetamine has been reported in the literature before [6] although only as a minor by-product. During the Birch reduction of ephedrine, both the aromatic ring and the hydroxyl group were reduced to give 1-(1, 4-cyclohexadien-1-yl)-N- methyl-2-propanamine and the p-tosyl derivative of this compound was prepared by Zvilichovsky et al.[6] This cyclohexadienyl compound is often used as a route specific marker compound by forensic chemists engaged in illicit drug profiling. Cyclohexadienyl compounds can undergo a degree of re-aromatization and Zvilichovsky et al. reported that This article is protected by copyright. All rights reserved. some of the p-tosyl derivative of 1-(1, 4-cyclohexadien-1-yl)-N-methyl-2-propanamine re- aromatized resulting in the p-tosyl derivative of methylamphetamine (Figure 11). Unlike the t-BOC derivatives of MDMA and methylamphetamine which gave very high yields of the parent amines following dilute acid reflux the p-tosyl group is not so easily cleaved. But it seems unlikely that a clandestine manufacturer would prepare large quantities of the p-tosyl methylamphetamine and ship it to Australia without first having determined an efficient method of deprotection. The authors attempted to remove the p-tosyl group using a standard procedure outlined in Vogel‟s Textbook of practical organic chemistry [5] which employs refluxing 50% sulphuric acid. However, the best yield obtained after three attempts was only 30% and it seems unlikely that this would be acceptable to a drug trafficking cartel. The literature cites other methods for cleavage of the p-tosyl group from various sulphonamides with refluxing hydrobromic acid in acetic acid being one of the most common and with an average yield of between 40 and 50%. Others include the sonication in methanol for 20-40 minutes in the presence of magnesium metal [7] and stirring at room temperature in methanol/benzene in the presence of magnesium metal.[7] While much is known about the pharmacology of methylamphetamine virtually nothing has been written about its p-tosyl derivative. But the p-tosyl methylamphetamine may have some physiological activity by virtue of its potential hydrolysis to the parent methylamphetamine in the presence of stomach acid and stomach enzymes. However the toxicity of the p-tosyl derivative may be so high as to make this an unattractive administration route for the recreational drug user. This article is protected by copyright. All rights reserved. The range of amine protecting reagents employed in legitimate organic synthesis is large and quite apart from the t-BOC and p-tosyl groups includes the following: fluorenylmethyloxycarbonyl chloride (FMOC-Cl), benzyl chloroformate (Cbz), 2,2,2- trichloroethyl chloroformate (Troc), benzoyl chloride (Bz), p-nitrobenzenesulfonyl chloride (nosylates) and p-bromobenzenesulfonyl chloride (brosylates).[8] These groups are easily attached, fulfil their role as protecting groups, and then easily removed by comparatively simple chemistry which is why they are so popular amongst synthetic chemists. Clandestine drug manufacturers and traffickers are nothing if not adaptable. It would be naïve to assume that attempts at chemical masking will cease and it is probable that clandestine manufacturers will simply use different protecting groups as current ones are identified. In the last two years we have seen both MDMA and methylamphetamine trafficked into Australia in large quantities „masked‟ as their t-BOC derivatives. At the time this represented a clever way of circumventing presumptive testing protocols at the Australian Border. It would seem though that once the clandestine producers realised that the „masked‟ MDMA and methylamphetamine had been identified they simply switched to another protecting group. However using the same analytical techniques the p-tosyl methylamphetamine was quickly identified too. In part this is probably because whenever a seized substance is not one of the „usual suspects‟, e.g. heroin, cocaine, etc, it attracts the attention of forensic chemists and is subjected to rigorous analytical chemistry procedures. Conclusion Mass, NMR and infrared spectral techniques were used to identify samples of methylamphetamine that had been „disguised‟ as the p-tosyl derivative of methylamphetamine. Identity was confirmed by the laboratory preparation of the p-tosyl derivative from methylamphetamine and p-tosyl chloride and comparison of its spectra with This article is protected by copyright. All rights reserved.