LC–HR-MS(/MS) and GC–MS analyses of compounds 1–5

Firstly, exact molecular mass numbers of compounds 1–5 were confirmed by measurements in the full scan MS mode using LC–HR-MS (Fig. 2). In addition, the accurate masses of the predominant product ions, obtained by the LC–HR-MS/MS analysis (Fig. 3), provided further structural information. Based on the obtained exact molecular masses, chemical formulae for protonated molecular ions and product ions of compounds 1–5 are summarized in Table 1 together with calculated mass numbers. The errors between the observed and theoretical mass numbers of all protonated molecular ions and fragment ions were smaller than 5 ppm. The LC–MS fragmentation routes of these five protonated compounds were proposed (Fig. 4).

Fig. 2 Mass spectra of compounds 1–5 obtained by liquid chromatography–high resolution-mass spectrometry (LC–HR-MS) in the single stage mode Full size image

Fig. 3 Spectra of product ion spectra of compounds 1–5 obtained by LC–HR-MS in the tandem (MS/MS) mode Full size image

Table 1 Accurate mass numbers of the protonated molecular and predominant product ions, and their proposed chemical formulae obtained for compounds 1–5 measured by liquid chromatography–high-resolution mass spectrometry (/mass spectrometry) Full size table

Fig. 4 Proposed fragmentation routes of protonated compounds 1–5 Full size image

For GC–MS analysis, total ion current chromatogram (TIC) and electron ionization mass spectra of compounds 1–5 are shown in Figs. 5 and 6, respectively. In each of all TIC chromatograms, only a single peak appeared (Fig. 5), showing that each sample contained a target compound with very high purity (probably more than 95 %). The GC–MS fragmentation routes of these five compounds were also proposed (Fig. 6).

Fig. 5 Total ion current chromatogram for samples containing compounds 1–5 obtained by gas chromatography–electron ionization-mass spectrometry (GC–EI-MS) Full size image

Fig. 6 Mass spectra of compounds 1–5 obtained by GC–EI-MS together with their probable fragmentation modes Full size image

Confirmation of compound 5 and its similarity with compounds 1–4

Compound 5 is a known compound; its structure was confirmed by the comparing its GC–MS and NMR data with the data of compound AB-FUBINACA reported by Uchiyama [9] and that in the GC–MS databases from Cayman Chemical [11]. However, high-resolution mass spectrum and MS/MS analysis of compound 5 have not been reported and are shown in Table 1, Figs. 2e and 3e; ions of [M + Na]+ and [M + H]+ were observed in the ESI mass spectrum (Fig. 2e). In the product ion mass spectrum (Fig. 3e), fragment ions at m/z 352.1465 (B 5 ), 324.1511 (C 5 ), and 253.0775 (D 5 ) were formed by the sequential loss of NH 3 , CO and amino-methylpropyl from the protonated molecule (Fig. 4). The product ion mass spectrum at m/z 109.0453 (E 5 ) was a rearrangement product of 1-fluoro-4-methylbenzene. The mass difference value between product ions m/z 253.0775 (D 5 ) and 109.0453 (E 5 ) was 144.0324, which indicated the indazole-carbaldehyde moiety.

Great similarities were found for the MS and NMR spectra of compounds 1–5, which revealed the structure similarity among these five compounds. For example, the same neutral loss of ammonia [M + H–NH 3 ] + were observed in the LC–MS spectra (Figs. 2, 3), which corresponds to the loss of amino side chain. In addition, the same unsaturation degrees of 10 were observed. Therefore, the structure of four unknown compounds 1–4 were elucidated by comparing the MS and NMR spectral data with the known compound 5.

Identification of compound 1

The LC–MS and GC–MS spectra of compound 1 are shown in Figs. 2a, 3a, and 6a, respectively. The chemical structure of compound 1 was predicted by comparing its LC–MS and GC–MS spectra with those of compound 5 (AB-FUBINACA) (Figs. 2e, 3e, 6e). The accurate mass spectrum of compound 1 was measured by LC–HR-MS/MS in the positive mode. The ion peak observed at m/z 365.1979 (A 1 ) (Table 1) suggested that the protonated molecular formula of the compound was C 21 H 25 N 4 O 2 + (calcd. 365.1972). Product ions at m/z 91.0546 (E 1 ), 235.0870 (D 1 ), and 320.1764 (C 1 ) were found in the LC–HR-MS/MS spectrum (Fig. 3a). The product ion at m/z 91.0546 (E 1 ) (predicted chemical formula: C 7 H 7 +) indicated the presence of an un-substituted benzyl moiety. The mass difference value between product ions at m/z 235.0870 (D 1 ) and 91.0546 (E 1 ) was 144.0324, which was identical with that between 253.0775 (D 5 ) and 109.0453 (E 5 ); compound 1 was deduced to have the same indazole-carbaldehyde moiety as compound 5. The difference value between product ions C 1 (C 20 H 22 N 3 O+) and D 1 (C 15 H 11 N 2 O+) of compound 1 was higher than that between product ions C 5 (C 19 H 19 FN 3 O+) and D 5 (C 15 H 10 FN 2 O+) of compound 5 by 14 amu, which indicated that compound 1 had an additional methylene moiety than that group of compound 5. According to the LC–MS spectral data, compound 1 was presumed to be N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-benzyl-1H-indazole-3-carboxamide. The fragment ions at m/z 91, 235 and 320 of compound 1 in the GC–MS spectrum supported the presumed structure of compound 1 (Fig. 6a).

The structure of compound 1 was further elucidated by NMR analysis. The NMR spectra of this compound suggested the presence of two amide carbonyl groups [δ C 160.9, δ H 7.60 (1-CONH) and δ C 171.7, δ H 7.72 and 7.26 (1‴-CONH 2 )] as shown in Table 2. The analyses by 1H and 13C NMR, DEPT, HH COSY, HSQC and HMBC spectra of compound 1 revealed the presence of a 1-benzyl-1H-indazole moiety (positions 3′ to 7′a and positions 1″ to 7″) and an N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-carboxamide moiety (positions 1, 1-CONH, and 1‴-CONH 2 , and positions 1‴–6‴). Unfortunately, no HMBC correlation between the two moieties was observed. However, the chemical shifts of corresponding carbons of compound 1 [δ C 160.9 (C-1), 136.9 (C-3′), 122.2 (C-3′a), 121.7 (C-4′), 122.7 (C-5′), 127.0 (C-6′), 110.7 (C-7′), 140.7 (C-7′a), 52.4 (C-1″), and 171.7 (C-1‴)] were similar to those of AB-FUBINACA [δ C 161.2 (C-1), 137.1 (C-3′), 122.3 (C-3′a), 121.8 (C-4′), 122.8 (C-5′), 127.0 (C-6′), 110.6 (C-7′), 140.6 (C-7′a), 51.6 (C-1″), and 172.6 (C-1‴)] (Table 2). This result suggested that carboxamide carbon (C-1, δ C 160.9) in the N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-carboxamide moiety was attached to the carbon at the 3′-position (δ C 136.9) of the 1-benzyl-1H-indazole moiety as AB-FUBINACA [9].

Table 2 Nuclear magnetic resonance (NMR) data for compound 1 Full size table

Finally, on the basis of mass spectra and NMR data as shown above, the structure of compound 1 was determined as an AB-FUBINACA analog [IUPAC: N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-benzyl-1H-indazole-3-carboxamide] and named ADB-BINACA (Fig. 1). Compound 1 has been reported to have an affinity for CB 1 receptor [10], but this is the first report in which compound 1 has been detected in an illegal product.

Identification of compounds 2 and 3

The accurate mass spectra of compounds 2 and 3 were measured by LC–HR-MS(/MS) in the positive mode. The ion peak observed at m/z 368.1773 (A 2 ) and 382.1931 (A 3 ) (Table 1) suggested that the protonated molecular formulae of these two compounds were C 21 H 23 FN 3 O 2 + (calcd. 368.1769) and C 22 H 25 FN 3 O 2 + (calcd. 382.1925), respectively.

Compound 2 was presumed to be N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide based on the product ion/fragment patterns of the LC–HR-MS(/MS) analysis (Figs. 2b, 3b) and GC–MS analysis (Fig. 6b). The observed product ions at m/z 109.0452 (E 2 ), 252.0825 (D 2 ) and 351.1505 (B 2 ) of compound 2 (Fig. 3b) were similar to those of compound 5 (AB-FUBINACA) (Fig. 3e) obtained by LC–HR-MS/MS analysis. The product ion at m/z 109.0452 (E 2 ) (predicted chemical formula: C 7 H 6 F+) indicated the presence of a fluorobenzyl moiety. By comparing the product ion at m/z 252.0825 (D 2 ) (predicted chemical formula: C 16 H 11 FNO+) in compound 2 (Fig. 3b) with the product ion at m/z 253.0775 (D 5 ) (predicted chemical formula: C 15 H 10 FN 2 O+) in compound 5, one can predicted compound 2 has an 1-pentyl-1H-indole-3-carbonyl moiety. The mass difference value between product ions at m/z 252.0825 (D 2 ) and 109.0452 (E 2 ) also indicated the indole-carbonyl moiety by comparing of the product ion pattern of a known synthetic cannabinoid that has the same moiety, such as ADBICA [7].

The structure of compound 2 was further elucidated by GC–MS and NMR analyses. The fragment ions at m/z 109, 252 and 323 of compound 2 by GC–MS analysis (Fig. 6b) and the observed 1H and 13C NMR, DEPT, HH COSY, HSQC, and HMBC spectra for compound 2 suggested the presence of a 1-(4-fluorobenzyl)-1H-indole moiety (positions 2′ to 7′a and positions 1″–7″) and an N-(1-amino-3-methyl-1-oxobutan-2-yl)-carboxamide moiety (positions 1, 1-CONH, and 1‴-CONH 2 , and positions 1‴–5‴) as shown in Table 3. The key connections of the two moieties were revealed by the HMBC correlations. Namely, the HMBC correlations from the amide proton (1-CONH, δ H 7.57) and the indole proton (H-2′, δ H 8.35) to the carboxamide carbon atom (C-1, δ C 163.9) suggested that the carboxamide carbon (C-1) in the N-(1-amino-3-methyl-1-oxobutan-2-yl)-carboxamide moiety was attached to the carbon at the 3′-position of the 1-(4-fluorobenzyl)-1H-indole moiety. Therefore, the structure of compound 2 was determined to be N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide (AB-FUBICA).

Table 3 NMR data for compound 2 Full size table

The 13C NMR spectrum of compound 3 was very similar to that of compound 2 except for a dimethylpropyl moiety (position 2‴–6‴) as shown in Table 4. The difference between the molecular formulae of compound 3 (C 22 H 24 FN 3 O 2 ) and compound 2 (C 21 H 22 FN 3 O 2 ) is an additional CH 2 . The observed 1D and 2D NMR spectra of compound 3 suggested the presence of 1-(4-fluorobenzyl)-1H-indole and N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-carboxamide moieties. That is, compound 3 may have an additional methyl group at the 3‴-position in the structure of compound 2. Therefore, the structure of compound 3 was deduced to be N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indole-3-carboxamide (ADB-FUBICA). In addition, the product ions at m/z 109.0453 (E 3 ), 252.0824 (D 3 ), and 365.1668 (B 3 ) obtained by the LC–HR-MS/MS analysis (Fig. 3c), together with the fragment ions at m/z 109, 252 and 337 obtained by the GC–MS analysis (Fig. 6c) supported the presumed structure.

Table 4 NMR data for compound 3 Full size table

This is the first case in which compounds 2 and 3 has been detected in illegal products. Compounds 2 and 3 have been reported to have an affinity for CB 1 and CB 2 receptors [7].

Identification of compound 4

LC–HR-MS analysis of compound 4 gave an ion peak at m/z 350.1876, suggesting that the protonated molecular formula of compound was C 21 H 24 N 3 O 2 + (calcd. 350.1863). Compound 4 was presumed to be N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide based on the product ion/fragment patterns of the LC–HR-MS(/MS) analysis (Fig. 3d) and GC–MS analysis (Fig. 6d). The LC–HR-MS/MS profile of compound 4 was very similar to that of compound 2 (Fig. 3b). The product ion at m/z 91.0546 (E 4 ) (predicted chemical formula: C 7 H 7 +) indicated the presence of an un-substituted benzyl moiety. In addition, the fragment ions of compound 4 at m/z 349, 305, 234, 91 in GC–MS spectrum were all lower than those of compound 2 (367, 323, 252, 109) by 18 amu, which indicated the difference between compounds 4 and 2 was a single fluoro-substitution.

The structure of compound 4 was further elucidated by NMR analysis. The 13C NMR spectrum of compound 4 was very similar to that of compound 2 except for a benzyl moiety (positions 2″ to 7″) as shown in Table 5. The difference between the molecular formulae of compound 4 (C 21 H 23 N 3 O 2 ) and compound 2 (C 21 H 22 FN 3 O 2 ) is an additional hydrogen atom in the place of the absent fluorine atom. The observed 1H and 13C NMR, DEPT, HH COSY, HSQC, and HMBC spectra for compound 4 suggested the presence of 1-benzyl-1H-indole and N-(1-amino-3-methyl-1-oxobutan-2-yl)-carboxamide moieties. Compound 4 may have a benzyl group instead of the 4-fluorobenzyl group in the structure of compound 2.

Table 5 NMR data for compound 4 Full size table

Finally, on the basis of mass spectral and NMR data as shown above, the structure of compound 4 was finally determined as an AB-FUBICA analog [IUPAC: N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-benzyl-1H-indole-3-carboxamide], and named AB-BICA (Fig. 1).