**3. Discussion**

#### *3.1. GC–MS and HR-MS*

From the GC–MS spectrum (Figure 1), the 1B-LSD molecular ion (*m*/*z* 393) could be identified, as well as some characteristic clusters of these types of substances. More precisely, the clusters at *m*/*z* 293, *m*/*z* 292 and *m*/*z* 291, were consistent with the additional CH2 substituent of 1B-LSD compared to that reported by Brandt et al. [9], when compared with 1P-LSD, which presents a fragment cluster at *m*/*z* 277, *m*/*z* 278 and *m*/*z* 279, as also previously reported [5]. In the case of LSD, with no substituent at the indole nitrogen, this fragment cluster of ions is shifted towards the cluster at *m*/*z* 221, *m*/*z* 222 and *m*/*z* 223 [5,9]. With the exception of *m*/*z* 322, which represents the loss of the acyl radical from 1B-LSD, the remaining three species might have represented the butyl-substituted counterparts of those species, previously described as LSD, detected at *m*/*z* 280 (retro-Diels Alder), *m*/*z* 223 and *m*/*z* 196 (loss of *N*,*N*-diethylacrylamide). A neutral loss of *N*,*N*-diethylformamide from the M•<sup>+</sup> might have led to the formation of the *m*/*z* 291 ion [5]. The EI+ characterization of 1B-LSD, 1P-LSD and LSD, presented by Brandt and other authors, was perfectly consistent with the results of the present study, and allows the unequivocal identification of 1B-LSD [5–9].

In the case of UHPLC–qTOF-MS analysis, the extraction of the ion chromatogram (XIC) with the [M + H]<sup>+</sup> of 1B-LSD (*m*/*z* 394.249) revealed a clear and abundant peak. From the respective MS<sup>2</sup> fragmentation pattern, five abundant ions were identified which related to the suspected molecular structure, namely the *m*/*z* 293.165 (loss of [(diethylamino)methylidyne]oxidanium ion), 223.124 (loss of but-1-en-1-one), 208.075 (loss of a methyl), 192.091 (loss of methylamine) and 180.081 (internal rearrangement of tetrahydropyridine with loss of a methyl). The obtained results were in accordance with previously reported works on the ESI+ analysis of 1B-LSD itself, and also 1P-LSD [5,9,11–14].

Regarding the Orbitrap-MS, the MS2 fragmentation pattern was in full accordance with the qTOF-MS2 results, with the four most abundant ions being *m*/*z* 293.16454 (loss of [(diethylamino)methylidyne]oxidanium ion), 223.12265 (loss of but-1-en-1-one), 208.07528 (loss of a methyl) and fragments 180.08114 and 181.08117 (internal rearrangement of tetrahydropyridine with loss of a methyl). Once again, these fragments are typical of 1B-LSD and 1-P-LSD [5,9,11–14].

For both the qTOF and Orbitrap-MS analysis, some of the fragments identified with the Orbitrap-MS analysis were also consistent with those of the GC–MS analysis, even though different ionization modes were used (ESI+ vs. EI+).

#### *3.2. NMR*

Despite the fact that the sample aliquot was so small, the 1H-NMR spectrum still revealed the identity of a complex mixture (Figure 4) in which many signals could be identified (like protons 14 to 16 of the aromatic ring) as typical of LSD-like substances which share the same backbone structure. However, the signals that are most noteworthy are those which allow the identification of 1B-LSD in this mixture, i.e., those assigned to the butyl (CH3 and CH2) part of the molecule with δH (MeOD-d4): 1.09 ppm [4] (3H, t, J = 7.4 Hz), 1.86 ppm [3] (2H, m, J = 7.4 Hz) and 2.98 ppm [2] (2H, t, J = 7.4 Hz). These results are consistent with a recent study by Brandt et al. on this substance [9].

On the other hand, the direct 1D 13C NMR measurement was not possible due to the reduced quantity of the sample and the presence of relatively large quantities of other substances extracted from the original matrix. However, with 2D HSQC, it was possible to confirm the presence of the methyl and methylene groups. The bond connections between the protons in this butyl group were confirmed by the 2D DQF-COSY. Furthermore, the long-range 1H-13C HMBC showed the correlation of these two methylene groups with a C=O at 171.6 ppm. These results are consistent with a recent work of Brandt et al. on this substance [9].
