Chemical characterisation of synthesised reference standards
UPLC–QToF-MS, GC–MS, ATR-FTIR spectroscopy, NMR (1H, 13C and 19F) spectroscopy and Raman spectroscopy data and experimentally derived pKa’s, log P (octanol/water) and log D7.4 (octanol/water) values for the characterisation of the synthesised reference standards were provided (see supplementary material, section C). Log P is the partition coefficient of the non-ionised form of the analyte, whilst log D is the pH-dependent coefficient of distribution for ionisable compounds such as the MT-45s at the physiological pH (pH 7.4).
The MT-45 and fluorinated analogue reference standards were tested using a range of commonly used presumptive tests (see supplementary material). MT-45 and its fluorinated analogues gave similar, positive results for Scott’s (cobalt (II) thiocyanate) test, normally used as a presumptive test for cocaine, to those previously reported for the structurally similar diphenidine and 2-, 3- and 4-methoxphenidine (MXP), most likely due to the presence of tertiary amines in all . However, the MT-45s did not give a positive result in the modified Scott’s test, which allows them to be differentiated from diphenidine and the MXPs [McKenzie et al., unpublished data]. The MT-45s all gave a yellow result in the Mecke’s test as did diphenidine and 2-, 3- and 4-MXP [McKenzie et al., unpublished data]. Diphenidine and the MT-45s produced similar responses to the Marquis test (immediate yellow) but were differentiated from the MXPs which produce a transient pink or red/brown reaction .
Identification of seized material
The seized tablet was analysed by GC–MS, ATR-FTIR spectroscopy and NMR spectroscopy. Presumptive testing provided the same results for the seized tablet as observed for MT-45 and its fluorinated analogue reference standards. Preliminary GC–MS analysis indicated that it contained a previously unreported fluorinated MT-45 derivative rather than 2FPPP. The mass spectrum of the seized sample revealed a molecular ion at m/z 366, and a comparison to the mass spectra of the MT-45 and 2F-MT-45 reference standards is provided in the supplementary material. MT-45 could be chromatographically separated from the fluorinated MT-45s; however, the regioisomers could not be separated by retention time or mass using GC–MS. Comparison of the seized tablet with the synthesised reference standards using ATR-FTIR spectroscopy indicated that 2F-MT-45 was present with two discriminatory areas in the fingerprint region of the spectra, 800–650 nm and 1550–1400 nm (see supplementary material).
NMR spectroscopy data for the seized sample unequivocally confirmed the presence of 2F-MT-45 in the seized tablet along with unidentified excipients (Table 1). The data indicated the presence of a mono-fluorinated diphenylethyl group, a piperazine group (broadened by exchange processes) and a cyclohexane group. The connections between these groups and the position of the fluorine were determined using HMBC and NOESY/HOESY data (see supplementary material). The HMBC/NOESY/HOESY correlations to/from the 1′ proton confirmed that the molecule was fluorinated at the 1′-2 position. This was further confirmed when the 1H NMR and 19F NMR data from the F-MT-45 reference standards were compared with the seized sample (Figs. 3 and 4). The position of the fluorine singlet in the decoupled spectra showed the identical signals for the 2F-MT-45 and the seized sample (both δ: −119.6 ppm), and the proton-coupled data provides greater localised structural information and further evidence that the seized sample contains 2F-MT-45. To the author’s knowledge, this is the first identification of a fluorinated MT-45 analogue in a seized sample.
Metabolite identification studies
Metabolite identification studies were carried out using the synthesised MT-45 and 2F-MT-45 reference standards. No previous metabolite identification studies have been carried out on MT-45 using human liver microsomes or hepatocytes, although metabolites have previously been identified using rat hepatocytes and detected in mouse urine . MT-45 has been reported in biological samples from fatal and nonfatal intoxications of drug users; however, no metabolites have been identified in human samples. 2F-MT-45 has not previously been reported in any intoxication cases and thus this work gives information on urinary metabolites should such a case arise in the future or to provide metabolite data to be added to screening methods. Such studies are required to provide analytical targets for urinary metabolites for toxicological screening methods in clinical and forensic toxicology studies. At the physiological pH (7.4) at which all metabolomics studies were carried out, MT-45 and 2F-MT-45 are protonated and have log D7.4 values of 3.17 and 3.56 (see supplementary material), respectively, making them both relatively lipophilic substances.
In a previous in vitro study using rat hepatocytes , the identification of 14 MT-45 metabolites (10 phase I and 4 phase II) was reported. In this study, 15 unique (plus 1, tentatively identified) MT-45 metabolites have been identified following MT-45 incubation with human liver microsomes, human hepatocytes, mouse hepatocytes and using mouse in vivo studies (see Fig. 5). An additional metabolite (M17), previously identified by , was identified in human urine only in this study. The chromatographic and mass spectral data for the metabolites identified (M1–M17) is provided in Table 2. Chromatograms and time-course data for MT-45 metabolite identification studies using human liver microsomes and human hepatocytes are provided in Fig. 6. Data for mouse hepatocyte and mouse in vivo experiments are provided in the supplementary information (section E). Twelve unique 2F-MT-45 metabolites were identified following 2F-MT-45 incubation with human liver microsomes, human hepatocytes, mouse hepatocytes and in vivo mouse studies, and these are summarised in Fig. 7 and Table 3 (2F-M1–2F-M12).
Human in vitro studies
Five phase I MT-45 metabolites were detected following human in vitro studies (Fig. 6b). No phase II metabolites were detected in the human in vitro studies, suggesting that hepatic phase II metabolism was limited by the incubation conditions employed in this study. Three metabolites were identified following incubation of MT-45 with human hepatocytes: M1, formed as a result of N-dealkylation and M2 and M3, two mono-hydroxylated metabolites, (Figs. 5, 6a). Interestingly M1 (1,2-diphenylethylpiperazine) was not identified by Montesano et al.  in rat hepatocyte and mouse in vivo studies, who instead identified another N-dealkylated metabolite, 1-cyclohexyl-piperazine (labelled as M9 in that study, and M17 detected in human urine in this study).
M1 (1,2-diphenylethylpiperazine) was identified as one of three major metabolites, along with M2 and M3, using in vitro testing using human hepatocytes and human liver microsomes in this study and was also identified in verified human urine samples from users who had consumed MT-45 (Fig. 5). M1 is structurally similar to the dissociative substance, diphenidine (1,2-diphenylethylpiperadine; Fig. 1f). Two additional, minor (as estimated by peak area) metabolites were detected in the human liver microsome incubations only (Figs. 5 and 6b): the mono-hydroxylated metabolite M4 and the di-hydroxylated metabolite M6.
Four phase I 2F-MT-45 metabolites were detected following human in vitro studies (Fig. 7, Table 3); one metabolite resulted from N-dealkylation of the 2F-MT-45 parent structure and three due to mono-hydroxylations. As observed for MT-45, no phase II metabolites were detected in the human in vitro studies. Four metabolites were identified following incubation of 2F-MT-45 with human hepatocytes: 2F-M1 formed as a result of N-dealkylation and three mono-hydroxylated metabolites, 2F-M2, 2F-M3 and 2F-M4, were detected.
Mouse in vitro studies
Seven MT-45 metabolites were detected following incubation of MT-45 with mouse hepatocytes for 2 h (Fig. 5, Table 2; see supplementary material for chromatograms), three of which had not been observed previously in the human in vitro studies. M5 is hydroxylated on the phenethyl moiety similar to M2 detected in the human in vitro studies; M7 and M8 are phase II metabolites with glucuronidation occurring on the phenethyl moiety.
Seven 2F-MT-45 metabolites were detected following incubation of 2F-MT-45 with mouse hepatocytes (Fig. 7, Table 3), three of which (2F-M5, 2F-M6 and 2F-M7) had not been detected in the human in vitro studies. Metabolite 2F-M5, like 2F-M2, was hydroxylated on the phenethyl moiety; 2F-M6 is a minor metabolite which, like 2F-M3 and 2F-M4, was hydroxylated on the cyclohexyl moiety; 2F-M7 was dihydroxylated, with one hydroxylation on the phenethyl moiety and the other on the cyclohexyl moiety. The most prominent metabolites were the mono-hydroxylated 2F-M3 and 2F-M4 (m/z 383) metabolites.
Mouse in vivo studies
No MT-45 was detected in mouse urine collected after 24 h [see supplementary material, section E(ii)] and 10 metabolites were detected. Phase II (glucuronidated) metabolites were the most abundant as estimated by peak area, in agreement with previous findings in mouse urine . Metabolite M9 was the glucuronidated form of the N-dealkylated metabolite M1. Metabolites M10–M14 contained one hydroxyl group and one hydroxyl group with subsequent glucuronidation. M11 and M12 were hydroxylated on both the cyclohexyl moiety and phenethyl moiety with one hydroxyl group being subsequently glucuronidated. M10, M13 and M14 were di-hydroxylated on the phenethyl moiety, as indicated by the product ion at m/z 135 with one hydroxyl group being subsequently glucuronidated. Metabolite M15 was di-hydroxylated and subsequently glucuronidated on the diphenylethyl moiety.
No 2F-MT-45 was detected in mouse urine collected after 24 h. Five glucuronidated metabolites were identified (Fig. 7, Table 3). Metabolite 2F-M8 was hydroxylated and subsequently glucuronidated on the phenethyl moiety. Metabolite 2F-M9 was the glucuronidated form of 2F-M1. Three other metabolites containing one hydroxyl group and one hydroxyl group with subsequent glucuronidation on the 2-fluorophenyl phenethyl moiety were detected: 2F-M10, 2F-M11 and 2F-M12.
Confirmatory identification of MT-45 metabolites in human urine
All MT-45 metabolites identified in the human and mouse in vitro and mouse in vivo studies (M1–M16) were detected in the human urine samples obtained from two analytically confirmed cases of MT-45 intoxication (Fig. 5), confirming the applicability of the methodology to case samples. The relative concentrations of metabolites were similar in both cases with M1 and M3 being the major metabolites, followed by M2 and M4 (each about 50% response in UPLC–QToF-MS as compared with M1 and M3), M5 and M6 (about 25%), M7–M9, M12 and M17 (~ 5–10%), and finally M10, M11, M13, M15 and M16 (~ 1% or less). An additional metabolite, M17, was detected in the human urine samples with similar peak areas to M5 and M6 (25–30% of M1 and M3). This N-dealkylated metabolite had not been identified in the human in vitro, the mouse in vitro and mouse in vivo studies using female C57 mice in this work, but had been previously identified as a significant metabolite in a study using rat hepatocytes and male ICR (CD-1®) mice .