The LC–QTOF-MS spectrum of the target compound showed the protonated molecular ion ([M + H]+) at m/z 327.0801 (C 17 H 13 ClFN 4 +) and an isotopic ion [M + 2 + H]+ at m/z 329.0776 due to the presence of chlorine atom (Fig. 2a). The difference in the chemical formula between the target compound (C 17 H 12 ClFN 4 ) and alprazolam (C 17 H 13 ClN 4 ) was observed to be one fluorine. The difference in the chemical formula between flubromazolam (C 17 H 12 BrFN 4 ) and the target compound (C 17 H 12 ClFN 4 ) was observed to replace bromine with chlorine. Moreover, the differences in the chemical formulae between the product ions C 16 H 11 ClFN 3 +· = m/z 299.0625, C 17 H 13 FN 4 +· = m/z 292.1124, C 15 H 11 ClFN 2 + = m/z 273.0592, C 15 H 7 ClFN 2 + = m/z 269.0281, C 14 H 9 ClFN 2 + = m/z 259.0437, C 14 H 6 ClFN+ = m/z 242.0169, C 14 H 8 FN 2 + = m/z 223.0662, and C 15 H 9 FN+ = m/z 222.0714 of the target compound and the product ions C 16 H 12 ClN 3 +· = m/z 281.0719, C 17 H 14 N 4 +·= m/z 274.1212, C 15 H 12 ClN 2 + = m/z 255.0682, C 15 H 8 ClN 2 + = m/z 251.0369, C 14 H 10 ClN 2 + = m/z 241.0526, C 14 H 7 ClN+ = m/z 224.0260, C 14 H 9 N 2 + = m/z 205.0764, and C 15 H 10 N+ = m/z 204.0805 of alprazolam were consistent with the presence of fluorine substituent. The chemical formulae were the same between the product ions C 10 H 9 ClN 3 + = m/z 206.0478, C 8 H 6 ClN 2 + = m/z 165.0212, and C 7 H 5 ClN+ = m/z 138.0107 of the target compound and the product ions C 10 H 9 ClN 3 + = m/z 206.0476, C 8 H 6 ClN 2 + = m/z 165.0216, and C 7 H 5 ClN+ = m/z 138.0106 of alprazolam. The similar phenomenon was observed between the target compound and flubromazolam. The differences in the chemical formulae between the product ions C 16 H 11 ClFN 3 +· = m/z 299.0625, C 14 H 9 ClFN 2 + = m/z 259.0437, and C 7 H 5 ClN+ = m/z 138.0107 of the target compound and the product ions C 16 H 11 BrFN 3 +· = m/z 343.0114, C 14 H 9 BrFN 2 + = m/z 302.9928, and C 7 H 5 BrN+ = m/z 181.9600 of flubromazolam were consistent with replacing bromine with chlorine. The chemical formulae were the same between the product ions C 17 H 13 FN 4 +· = m/z 292.1124, C 17 H 12 FN 2 + = m/z 263.0980, C 16 H 12 FN+· = m/z 237.0949, C 14 H 8 FN 2 + = m/z 223.0662, and C 15 H 9 FN+ = m/z 222.0714 of the target compound and the product ions C 17 H 13 FN 4 +· = m/z 292.1118, C 17 H 12 FN 2 + = m/z 263.0982, C 16 H 12 FN+· = m/z 237.0951, C 14 H 8 FN 2 + = m/z 223.0670, and C 15 H 9 FN+ = m/z 222.0715 of flubromazolam (Table 1; Fig. 2b–d). These findings indicated that the target compound was an alprazolam derivative with one fluorine atom substituted on the phenyl ring without chlorine atom and a flubromazolam derivative with replacing bromine with chlorine, i.e., flualprazolam (Fig. 1).

Fig. 2 Precursor ion spectrum of the target compound, and product ion spectra of the target compound, alprazolam and flubromazolam obtained by liquid chromatography–electrospray ionization-quadrupole time-of-flight-mass spectrometry Full size image

Table 1 Accurate masses of the protonated molecules and product ions and their proposed chemical formulae obtained for the target compound, alprazolam, and flubromazolam measured by liquid chromatography–quadrupole time-of-flight-mass spectrometry Full size table

The electron ionization mass spectrum of the target compound (Fig. 3a) was also compared with those of alprazolam (Fig. 3b) and flubromazolam (Fig. 3c). The differences between the fragment ions at m/z 326, 297, 291, 257, and 222 in the target compound and the fragment ions at m/z 308, 279, 273, 239, and 204 in alprazolam were 18, respectively, all due to the presence of the fluorine atom. The differences between the fragment ions at m/z 326, 297, and 137 in the target compound and the fragment ions at m/z 370, 341, and 181 in flubromazolam were 44, respectively, all due to replacement of bromine by chlorine. The fragment ions at m/z 291, 222, 111, 102, and 75 in the target compound are also observed in flubromazolam. These observations support the assumption that the target compound was substituted by one fluorine atom on the phenyl ring as compared with alprazolam and by replacing bromine with chlorine as compared with flubromazolam.

Fig. 3 Mass spectra of the target compound, alprazolam, and flubromazolam obtained by gas chromatography–electron ionization-mass spectrometry Full size image

The specific substitution positions of the fluorine atom were further elucidated by NMR analysis. We compared the chemical shifts of the corresponding carbons of the target compound with those observed in the alprazolam. The chemical shifts of C-6a to C-10a in the target compound were similar to the phenyl ring with chlorine atom present in the alprazolam (Table 2), but different from the aromatic protons on another phenyl ring. The presence of fluorine atom resulted in the splitting of the peaks in the 13C spectrum. For the target compound, doublet splitting of the carbon signal in the aromatic carbon region and spin-spin coupling constants (J C–F = 249.7 Hz; J C–F = 11.5 and 21.3 Hz) were characteristic for 19F–13C interactions [12]. In Fig. 4, one quaternary carbon that directly connected with the fluorine substituents was seen at δ C 161.7 ppm. The comparison of the DEPT and 13C spectra (Fig. 4) proved the presence of another aromatic quaternary carbon (127.2 ppm) with C–F spin-spin coupling constants of 11.5 Hz, which were characteristic for J C-F spin-spin coupling [12]. Therefore, this indicated that fluorine atom was substituted in the ortho position of the phenyl moieties (Fig. 5). Using both the 1D and 2D NMR spectra, the final assignments of the observed carbon and hydrogen chemical shifts for the target compound are shown in Table 2.

Fig. 4 13C nuclear magnetic resonance (NMR) spectrum (100 MHz, CD 3 OD) of the target compound Full size image

Fig. 5 1H NMR spectrum (400 MHz, CD 3 OD) of the target compound Full size image

Table 2 1H and 13C nuclear magnetic resonance chemical shifts and diagnostic correlations in two-dimensional spectra of the target compound Full size table

On the basis of the above instrumental data, the target compound was identified to be flualprazolam.