Rapid detection of tert-butoxycarbonyl-methamphetamine by direct analysis in real time time-of-flight mass spectrometry
Phenethylamines constitute the majority of drug-related arrests in Japan. Recently, the smuggling of tert-butoxycarbonyl (t-Boc)-protected phenethylamines has become of increasing concern, because of the difficult identification of these masked substances.
In this study, a rapid and accurate method for the detection of t-Boc-methamphetamine (t-Boc-MP) by direct analysis in real time–time-of-flight-mass spectrometry (DART–TOF-MS) was developed. The efficiency of the method was evaluated by comparison with conventional gas chromatography–MS (GC–MS) and liquid chromatography–TOF-MS (LC–TOF-MS) techniques.
During GC–MS analysis of t-Boc-MP, MP was generated in the injection port, which can lead to an analytical error. In the LC–TOF-MS spectrum, fragment ions were detected, which were generated by McLafferty rearrangement in the ion source. On the other hand, in the DART–TOF-MS analysis of t-Boc-MP, pyrolysis could be suppressed by using a micro-syringe injection method, and the fragment ions generated by McLafferty rearrangement were still observed. Moreover, protonated t-Boc-MP could be detected.
Hence, DART–TOF-MS provides a rapid and accurate method for the detection of t-Boc-MP, allowing suppression of the pyrolysis reaction and identification of both fragment ions and protonated t-Boc-MP. To our knowledge, this is the first report for detecting t-Boc-MP by MS techniques.
Keywordstert-Butoxycarbonyl-methamphetamine (t-Boc-MP) “Masked” methamphetamine Rapid detection by DART–TOF-MS McLafferty rearrangement Pyrolysis
In Japan, methamphetamine (MP), amphetamine, and their salts are strictly regulated by the Stimulants Control Law, and represent the majority of drug-related arrests. MP is seized in various forms such as crystal, tablet, and powder forms, and some MP seizures have been found to contain adulterants such as sodium benzoate and sodium thiosulfate . In particular, N-isopropylbenzylamine, which is a structural isomer of MP , is one of the most common adulterants. In addition, the use of masked illegal substances as a method of drug smuggling is of increasing concern. In 2004, the Australian Custom Police seized methyl-3-[3′,4′-(methylene dioxy)phenyl]-2-methyl glycidate, which upon hydrolysis and decarboxylation efficiently produced 3,4-methylenedioxyphenyl-2-propanone, a precursor of 3,4-methylenedioxymethamphetamine (MDMA) .
Recently, phenethylamines bearing a tert-butoxycarbonyl (t-Boc) group on the amine were seized [4, 5, 6]. The t-Boc group is one of the most commonly used amino-protecting groups in organic synthesis, such as peptide synthesis, because of the simple protection-deprotection procedures . In general, the t-Boc group can be removed by treatment with a strong acid, such as trifluoroacetic acid, to give the original compound in high yield . Deprotection can also be efficiently achieved by dissolution in distilled water at 100 °C within several tens of minutes, without using a strong acid . In particular, seizures of t-Boc-protected MDMA and MP, phenethylamines containing secondary amino groups, have been reported. Namely, t-Boc-MDMA was found in a viscous and bright red liquid hair product by the Australian Custom Police. The substance was initially believed to be the MDMA precursor safrole, but further detailed analysis identified it as t-Boc-MDMA . Moreover, t-Boc-MP mixed with a liquid detergent was seized in New Zealand . In view of the simple protection-deprotection procedures, a variety of phenethylamine drugs could be easily masked by t-Boc protection of the amino group. However, no analytical data for t-Boc-protected phenethylamines is available to date, making drug detection difficult and inaccurate. Moreover, t-Boc-MP and t-Boc-MDMA are unregulated. Clearly, the objective of the amine-masking strategy would be to reduce the risk of smuggling illicit drugs by converting them into unregulated substances. Hence, in order to prevent their distribution worldwide, a rapid and accurate analytical method for the detection of t-Boc-masked drugs is needed to quickly provide the administrative authorities with the drug data.
Liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–MS (GC–MS) are the most widely used techniques for illegal drug analysis. LC–time-of-flight-MS (LC–TOF-MS) is particularly useful for identifying unknown compounds, because it can easily detect protonated molecules and accurately measure the molecular mass [10, 11]. However, analytical techniques based on chromatographic separations, such as LC–TOF-MS and GC–MS, usually require relatively long measurement times. On the other hand, MS using a direct analysis in real time (DART) ion source developed by Cody et al.  is a rapid analytical method as compared to GC–MS and LC–TOF-MS. Moreover, high-resolution TOF-MS allows the estimation of chemical formulas from accurate mass data. Thus, DART–TOF-MS has been widely used as a screening technique in the field of food hygiene and forensic science [13, 14]. On the basis of these considerations, we envisioned that DART–TOF-MS would provide an efficient screening method for t-Boc-MP.
In this study, a rapid and accurate DART–TOF-MS screening method for t-Boc-MP was developed. The efficiency of the method was evaluated by comparison with GC–MS and LC–TOF-MS analyses.
Materials and methods
MP hydrochloride, which was provided by the Ministry of Health, Labor and Welfare (Tokyo, Japan), was used in this study. Di-tert-butyl dicarbonate was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). All other reagents were of special grade from Kanto Kagaku Co., Ltd. (Tokyo, Japan). t-Boc-MP was synthesized according to the procedure described by Davis et al.  for the preparation of t-Boc-amine. The purity of synthesized t-Boc-MP was confirmed using GC–flame ionization detection (≧ 98%). The microsyringe used (capacity 10 μL, 23–26-gauge needle) was purchased from Hamilton Inc. (Reno, NV, USA).
Agilent 7890A GC/5975C MSD system (Agilent Technologies, Santa Clara, CA, USA); column: DB-5MS (30 × 0.25-mm i.d., film thickness 0.25 μm; Agilent Technologies); inlet temperature: 250 °C; oven temperature: 2 min at 60 °C, 10 °C/min to 300 °C, and 5 min at 300 °C; transfer line temperature: 280 °C; injection volume: 1 μL; injection mode: split (20:1) or splitless; carrier gas: He (1.2 mL/min); ionization conditions: electron ionization, 70 eV, 150 °C; mass range: m/z 40–500.
LC–TOF-MS was used in MSE mode, which allowed alternating low and high collision energies in one injection, thus providing precursor and fragment/product ion information, respectively. The following operational conditions were used.
ACQUITY UPLC instrument (Waters, Milford, MA, USA); column: ACQUITY UPLC HSS C18 column (150 × 2.1-mm i.d., particle size 1.8 μm; Waters); solvent A: 5 mM ammonium formate in water, pH 3; solvent B: 0.1% formic acid in acetonitrile; flow rate: 0.4 mL/min; elution program: 80% A/20% B (2-min hold) to 20% A/80% B (2–15 min, 8-min hold); injection volume: 1 μL; column temperature: 50 °C.
Xevo G2 QToF mass spectrometer (Waters); ion source: electrospray ionization in positive mode; ion source temperature: 150 °C; capillary and cone voltages: 830 and 40 V, respectively; collision energy function 1: 6 V; collision energy function 2: 10–40 V; mass range: m/z 50–1000; scan time: 0.2 s; lock mass: leucine enkephalin (m/z 556.2771).
DART ion source instrument
DART-SVP™ (Ionsense, Saugus, MA, USA); ionization mode: positive mode; helium gas flow: 3.5 L/min; ion source temperature: 200 °C; discharge electrode needle voltage: 3200 V.
JMS-100LP AccuTOF LC-Plus (JEOL, Tokyo, Japan); orifice 1 voltage: 10 V; orifice 2 voltage: 5 V; orifice 1 temperature: 180 °C; ring lens voltage: 5 V; ion guide peak voltage: 300 V; reflectron voltage: 980 V; mass range: m/z 10–1000; internal mass number calibration: polyethylene glycol 200 and 400; sample injection method: microsyringe.
Results and discussion
Structural confirmation of the synthesized t-Boc-MP
Pyrolysis of t-Boc-MP in the GC–MS injection port
Methamphetamine (MP)-to-tert-butoxycarbonyl-methamphetamine (t-Boc-MP) peak area ratios and t-Boc-MP peak areas measured by gas chromatography–mass spectrometry (GC–MS) at different inlet temperatures with split injection at 20:1
Inlet temperature (°C)
Peak area ratio (MP-to-t-Boc-MP; ± SD, n = 3)
(× 107, ± SD, n = 3)
3.7 ± 0.2
3.0 ± 0.1
0.0022 ± 0.000049
2.6 ± 0.3
0.0145 ± 0.00061
2.3 ± 0.7
MP-to-t-Boc-MP peak area ratios and t-Boc-MP peak areas measured by GC–MS for each injection mode at inlet temperature of 200 °C
Peak area ratio (MP-to-t-Boc-MP; ±SD, n = 3)
(× 107, ± SD, n = 3)
0.012 ± 0.00034
80.2 ± 0.06
0.0024 ± 0.00017
5.27 ± 0.01
3.70 ± 0.21
0.67 ± 0.02
Comparison of sample injection methods
Effect of solvent
In addition, all spectra showed a peak at m/z 499.352, which was attributed to the dimer of t-Boc-MP, whereas the peaks at m/z 399.300 and 443.003, corresponding to the dimers of t-Boc-MP with MP and with isobutene-desorbed t-Boc-MP, respectively, were detected in all spectra except for the ethyl acetate sample. However, in the case of the ethyl acetate sample, the most intense peak was found at m/z 177.122, which corresponded to the dimer form of ethyl acetate. Moreover, the impurity peaks related to the solvent and water molecules were detected in the mass range below m/z 150.128 in all solvent samples. The impurity peaks related to methanol had lower ionic strengths as compared to those for the other solvents. Hence, in this respect, methanol proved to be the best solvent for DART–TOF-MS analysis of t-Boc-MP.
Effect of ion source temperature
Effect of sample concentration
In this study, a DART–TOF-MS method was developed for the rapid and accurate detection of t-Boc-MP by using a microsyringe injection technique, methanol as a solvent, an ion source temperature of 200 °C, and a sample concentration of 10 μg/mL. This method, as a screening test, proved to be superior to GC–MS and LC–TOF-MS analyses. During GC–MS analysis, t-Boc-MP underwent pyrolysis in the injection port under certain operational conditions, which can lead to an analytical error. In the LC–TOF-MS spectrum, ion fragments generated by McLafferty rearrangement were detected, whereas the protonated t-Boc-MP parent ion was not found. On the other hand, in the DART–TOF-MS analysis, pyrolysis was not observed with any of the solvents. Moreover, similarly to LC–TOF-MS, in the DART–TOF-MS spectrum, the fragment ions formed by McLafferty rearrangement were detected, as well as the protonated t-Boc-MP peak. Hence, DART–TOF-MS provides a rapid and accurate method for the detection of t-Boc-MP with significant reduction of the analytical error by suppression of the pyrolysis reaction, and for the identification not only with fragment ions but also with the protonated t-Boc-MP. To our knowledge, this is the first report dealing with detection of t-Boc-MP by MS techniques.
A portion of this work was supported by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare, Japan.
Compliance with ethical standards
Conflict of interest
There are no financial or other relationships that could lead to a conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
- 2.Sanderson RM (2008) Identification of N-methylbenzylamine hydrochloride, N-ethylbenzylamine hydrochloride, and N-isopropylbenzylamine hydrochloride. Microgram J 6:36–45Google Scholar
- 5.Westphal F, Grreser U, Holz K, Erkens M (2016) Strukturaufkärung und analytische Daten eines ungewöhnlichen MDMA-Derivates (in German). Toxichem Krimtech 83:92–102Google Scholar
- 6.Herald NZ (2017) Masked meth: first time method used, as four men charged with importing $100 m of the substance. http://www.Nzherald.co.nz/nz/news/article.cfm?c_id=1&objectid=11818866. Accessed 15 Mar 2017
- 11.Hernandez F, Ibenez M, Botero-Coy A-M, Bade R, Bustos-Lopez MC, Rincon J, Moncavo A, Bijlsma L (2015) LC-QTOF-MS screening of more than 1,000 licit and illicit drugs and their metabolites in wastewater and surface waters from the area of Bogotá, Colombia. Anal Bioanal Chem 21:6405–6416CrossRefGoogle Scholar
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