Chemical synthesis, characterisation and in vitro and in vivo metabolism of the synthetic opioid MT-45 and its newly identified fluorinated analogue 2F-MT-45 with metabolite confirmation in urine samples from known drug users

Abstract

Purpose: The detection of a novel psychoactive substance, 2F-MT-45, a fluorinated analogue of the synthetic opioid MT-45, was reported in a single seized tablet. MT-45, 2F-, 3F- and 4F-MT-45 were synthesised and reference analytical data were reported. The in vitro and in vivo metabolisms of MT-45 and 2F-MT-45 were investigated.

Method: The reference standards and seized sample were characterised using nuclear magnetic resonance spectroscopy, ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry, gas chromatography-mass spectrometry, attenuated total reflectance-Fourier transform infrared spectroscopy and Raman spectroscopy. Presumptive tests were performed and physicochemical properties of the compounds determined. Metabolite identification studies using human liver microsomes, human hepatocytes, mouse hepatocytes and in vivo testing using mice were performed and identified MT-45 metabolites were confirmed in authentic human urine samples.

Results: Metabolic pathways identified for MT-45 and 2F-MT-45 were N-dealkylation, hydroxylation and subsequent glucuronidation. The major MT-45 metabolites identified in human in vitro studies and in authenticated human urine were phase I metabolites and should be incorporated as analytical targets to existing toxicological screening methods. Phase II glucuronidated metabolites were present in much lower proportions.

Conclusions: 2F-MT-45 has been detected in a seized tablet for the first time. The metabolite identification data provide useful urinary metabolite targets for forensic and clinical testing for MT-45 and allows screening of urine for 2F-MT-45 and its major metabolites to determine its prevalence in case work.

Keywords: 2F-MT-45; Clinical and forensic toxicology; MT-45; Metabolite identification; Novel psychoactive substances; Synthetic opioids.

Fig. 1

Chemical structures of a MT-45, including the structural notation used for nuclear magnetic resonance (NMR) spectroscopy data using the system reported by [16], b 2F-MT-45, c 3F-MT-45, d 4F-MT-45, e 2FPPP (fluorolintane), f diphenidine and g 2-methoxphenidine (2-MXP)

Fig. 2

Photograph of off-white/cream square tablet (in a clear snap-bag labelled with “2FPPP”), with 6-mm length, 6-mm width and 4-mm height/depth

Fig. 3

a ¹H NMR data for the seized sample, and b ¹H NMR data for the 2F-MT-45 reference standard. *Signals presumed to be originating from unknown excipient(s) present in the seized sample

Fig. 4

Comparison of a proton decoupled and b proton-coupled ¹⁹F NMR spectra for the seized tablet from bag labelled “2FPPP” and the synthesised fluorinated MT45 reference standards

Fig. 5

Overview of the metabolic profiling of MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH), mouse urine (MU) from in vivo testing and authenticated human urine (HU) samples. Product ion spectra and structural elucidation data are provided in the supplementary material, section F

Fig. 6

Chromatographic profiles and time-course data following incubation of MT-45 with a human hepatocytes after 120 min, and b human microsomes after 60 min obtained by ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry. Metabolite labelling information can be found in Fig. 5 and Table 2

Fig. 7

Overview of the metabolic profiling of 2F-MT-45 using human liver microsomes (HM), human hepatocytes (HH), mouse hepatocytes (MH) and mouse urine (MU) from in vivo testing. Product ion data and structural elucidation data are provided in supplementary material, section G