Differentiation of AB-FUBINACA and its five positional isomers using liquid chromatography–electrospray ionization-linear ion trap mass spectrometry and triple quadrupole mass spectrometry
Positional isomer differentiation is crucial for forensic analysis. The aim of this study was to differentiate AB-FUBINACA positional isomers using liquid chromatography (LC)–electrospray ionization (ESI)-linear ion trap mass spectrometry (LIT-MS) and LC–ESI-triple quadrupole mass spectrometry (QqQ-MS).
AB-FUBINACA, its two fluorine positional isomers on the phenyl ring, and three methyl positional isomers in the carboxamide side chain were analyzed by LC–ESI-LIT-MS and LC–ESI-QqQ-MS.
Four of the positional isomers, excluding AB-FUBINACA and its 3-fluorobenzyl isomer, were chromatographically separated on an ODS column in isocratic mode. ESI-LIT-MS could discriminate only three isomers, i.e., the 2-fluorobenzyl isomer, the N-(1-amino-2-methyl-1-oxobutan-2-yl) isomer, and the N-(1-amino-1-oxobutan-2-yl)-N-methyl isomer, based on their characteristic product ions observed at the MS3 stage in negative mode. ESI-QqQ-MS differentiated all six isomers in terms of the relative abundances of the product ions that contained the isomeric moieties involved in collision-induced dissociation reactions. The six isomers were more clearly and significantly differentiated upon comparison of the logarithmic values of the product ion abundance ratios as a function of collision energy.
The present LC–MS methodologies were useful for the differentiation of a series of AB-FUBINACA positional isomers.
KeywordsAB-FUBINACA Positional isomer differentiation Liquid chromatography Electrospray ionization Linear ion trap mass spectrometry Triple quadrupole mass spectrometry
Over the past 50 years, pharmaceutical companies and academic laboratories have developed synthetic cannabinoids (SCs) as potential pharmaceutical agents for the treatment of pain. These compounds are CB1 and/or CB2 agonists and elicit similar effects to that of Δ9-tetrahydrocannabinol (THC), the active component in cannabis [1, 2, 3]. In late 2008, the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) detected several SCs, including JWH-018, JWH-073, and CP-47,497, in herbal smoking mixtures marketed as incense or room odorizers often known as ‘K2’ or ‘Spice’. Subsequently, various herbal products containing SCs have been distributed worldwide , resulting in numerous serious incidents involving individuals under their influence. To immediately deter the epidemic of drug abuse, many countries have increased the number of individually named controlled substances. However, structurally distinct designer drugs aimed at circumventing such legal measures have continued to be synthesized. In an attempting to address worldwide social concerns, the UK government has implemented a blanket scheduling of SCs through the introduction of generic legislation based on modifications to core structures, as recommended by the UK home office from the Advisory Council on the Misuse of Drugs (ACMD) in 2009 . However, this regulation did not deter the prevalence of SCs. Indeed, according to reports from the Forensic Early Warning System (FEWS) in 2012, noncontrolled SCs such as AM2201, RCS-4, and UR-144, so-called ‘second-generation’ synthetic cannabinoids, were frequently detected in forensic seizures [6, 7, 8].
Materials and methods
N-(1-Amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(2-fluorobenzyl)-1H-indazole-3-carboxamide (isomer-1), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(3-fluorobenzyl)-1H-indazole-3-carboxamide (isomer-2), N-(1-amino-1-oxopentan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (isomer-3), N-(1-amino-2-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (isomer-4), and N-(1-amino-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-N-methyl-1H-indazole-3-carboxamide (isomer-5) (Fig. 1) were purchased from Cayman Chemical (Ann Arbor, MI, USA). Their standard stock solutions (200 μg/mL) were prepared in methanol and stored at − 20 °C. The working standard solutions (20 μg/mL) to be injected into the mass spectrometer were prepared by diluting the stock solutions.
LC–ESI-LIT-MS was performed on a Prominence Ultrafast Liquid Chromatograph (Shimadzu, Kyoto, Japan) linked to an LXQ LIT mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) equipped with an ESI source. Instrumental control, data acquisition, and analysis were performed using Xcalibur software ver. 2.0 (Thermo Fisher Scientific). The analytes were separated using two ODS columns: (1) L-column 2 ODS column (150 × 1.5 mm i.d., particle size 5 μm; Chemicals Evaluation and Research Institute, Tokyo, Japan) and (2) YMC-Ultra HT Pro C18 column (75 × 2.0 mm i.d., particle size 2 μm; YMC, Kyoto, Japan) at a column oven temperature of 40 °C. The injection volume was 1 μL. Other LC parameters using the (1) L-column 2 ODS column and (2) YMC-Ultra HT Pro C18 column were as follows, respectively: (1) flow rate, 0.1 mL/min; elution mode, gradient with 10 mM ammonium acetate/5% methanol in distilled water (A) and 10 mM ammonium acetate/5% distilled water in methanol (B) from 100% A to 100% B over 15 min, and by isocratic elution with the final solvent composition for 10 min; and (2) flow rate, 0.25 mL/min; elution mode, isocratic with 50% A/50% B for 30 min. The MS parameters were as follows: polarity, positive and negative; scan mode, product ion scan; activation type, collision-induced dissociation (CID); isolation width, m/z 2.00; normalized CE, 35.0%; activation Q, 0.250; activation time, 30 ms; collision gas, helium.
LC–ESI-QqQ-MS was performed on an Agilent 1260 Infinity LC system linked to a 6470A triple quadrupole LC/MS tandem mass spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an ESI source. Instrumental control, data acquisition, and analysis were performed using Mass Hunter software ver. B.07.00 (Agilent Technologies). An L-column 2 ODS column (150 × 1.5 mm i.d., particle size 5 μm; Chemicals Evaluation and Research Institute) was used at a column oven temperature of 40 °C. The injection volume was 5 μL. The flow rate was 0.1 mL/min. The elution mode was isocratic with 10 mM ammonium acetate/60% methanol in distilled water for 25 min. The MS parameters were as follows: polarity, positive; scan mode, product ion scan; fragmentor voltage, 80 eV; cell accelerator voltage, 5 eV; collision gas, nitrogen; CE, 0–90 eV.
Statistical analyses were performed using BellCurve for Excel ver. 2.02 (Social Survey Research Information Co., Ltd., Tokyo, Japan), which is an add-in software to Microsoft Excel 2010. The homogeneity of variances was calculated by Bartlett’s test to determine if the obtained data were normally distributed (parametric, p > 0.05) or not (nonparametric, p < 0.05). If the data were parametric, one-way analysis of variance (ANOVA) followed by multiple pairwise comparisons as post hoc analysis using Tukey’s test was performed. For nonparametric data, Kruskal–Wallis test as nonparametric ANOVA, followed by the Steel–Dwass test as a nonparametric multiple pairwise comparison, was performed.
Results and discussion
Linear ion trap multiple-stage mass spectrometry
ESI-LIT mass spectra of the six isomers were recorded in positive mode. The precursor ions at the MS2–MS6 stages were set at m/z 369, 352, 324, 253, and 225, respectively. The obtained mass spectra in the MS1–MS6 stages are shown in Fig. S2. Spectra in stages greater than MS7 were not obtained because of the lack of product ions. In the mass spectra of each isomer, identical fragment/product ions were observed at m/z 369 ([M+H]+) and 391 ([M+Na]+) in the MS1 stage, at m/z 352 ([M–NH2]+) in MS2, at m/z 324 ([M–CONH2]+) in MS3, at m/z 253 ([M–(C4H9N)CONH2]+) in MS4, at m/z 109 (fluorobenzyl cation), 225 ([M–CO(C4H9N)CONH2]+) and 235 in MS5, and at m/z 198 and 205 in MS6. Although the abundances of the ions at m/z 198 in MS6 appeared to slightly differ in isomer-2, the other mass spectra were very similar. Therefore, ESI-LIT-MS operated in positive mode could not be used to effectively differentiate the six isomers.
Triple quadrupole energy-resolved mass spectrometry
We have investigated the differences between AB-FUBINACA and its five positional isomers (two fluorine positional isomers on the phenyl ring and three methyl positional isomers in the carboxamide side chain) by LC–ESI-LIT-MS and LC–ESI-QqQ-MS. Excluding AB-FUBINACA and its 3-fluorobenzyl isomer (isomer-2), four of the isomers were separated on an ODS column operated in isocratic mode. Multiple-stage mass spectrometry using LIT in negative mode allowed three isomers, namely, the 2-fluorobenzyl isomer (isomer-1), the N-(1-amino-2-methyl-1-oxobutan-2-yl) isomer (isomer-4), and the N-(1-amino-1-oxobutan-2-yl)-N-methyl isomer (isomer-5), to be differentiated based on their characteristic product ions observed in the MS3 stage. Energy-resolved mass spectrometry, which employs QqQ-MS as a function of CE, revealed that the relative abundances of the product ions containing the isomeric moieties, produced by CID reactions, were different for the six isomers. Furthermore, all six isomers were clearly differentiated by plotting the logarithmic values of the characteristic product ion abundance ratios against the CE. Based on the above results, the combination of LC with ESI-QqQ-MS is effective for the differentiation of a series of AB-FUBINACA positional isomers.
This work was supported by JSPS KAKENHI (No. 15H00542).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
This article does not contain any studies involving human participants or animals performed by any of the authors.
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