Proposal of 5-methoxy-N-methyl-N-isopropyltryptamine consumption 1 biomarkers through identification of in vivo metabolites from mice

New psychoactive substances (NPS) are a new breed of synthetically produced substances designed to mimic the effects of traditional illegal drugs. Synthetic cannabinoids and synthetic cathinones are the two most common groups, which try to mimic the effects of the natural compounds 9Δ-tetrahydrocannabinol and cathinone, respectively. Similarly, synthetic tryptamines are designer compounds which are based on the compounds psilocin, N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine found in some mushrooms. One of the most important tryptamine compounds found in seizures is 5-methoxy-N,N-diisopropyltryptamine, which has been placed as controlled substance in USA and some European countries. The control of this compound has promoted the rising of another tryptamine, the 5-methoxy-N-methyl-N-isopropyltryptamine, which at the time of writing this article has not been banned yet. So, it is undeniable that this new substance should be monitored. 5-methoxy-N-methyl-N-isopropyltryptamine has been reported by the Spanish Early Warning System and detected in our laboratory in two pill samples purchased in a local smart shop. This has promoted the need of stablishing consumption markers for this compound in consumers' urine. In the present work, the metabolism and pharmacokinetic of 5-methoxy-N-methyl-N-isopropyltryptamine has been studied by an in vivo approach, using adult male mice of the inbred strain C57BLJ/6. The use of ultra-high performance liquid chromatography coupled to high resolution mass spectrometry allowed the identification of four metabolites. After the pharmacokinetic study in serum and urine, the O-demethylated metabolite and the non-metabolised parent compound are proposed as consumption markers in hydrolysed urine. Data reported in this work will help hospitals and forensic laboratories to monitor the consumption and potential intoxication cases related to this tryptamine.


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New psychoactive substances (NPS) are a new breed of synthetically produced 17 substances designed to mimic the effects of traditional illegal drugs. Synthetic 18 cannabinoids and synthetic cathinones are the two most common groups, which try to 19 mimic the effects of the natural compounds 9 Δ-tetrahydrocannabinol and cathinone, 20 respectively. Similarly, synthetic tryptamines are designer compounds which are based 21 on the compounds psilocin, N,N-dimethyltryptamine and 5-methoxy-N,N-22 dimethyltryptamine found in some mushrooms. One of the most important tryptamine 23 compounds found in seizures is 5-methoxy-N,N-diisopropyltryptamine, which has been 24 placed as controlled substance in USA and some European countries. The control of this 25 compound has promoted the rising of another tryptamine, the 5-methoxy-N-methyl- N-26 isopropyltryptamine , which at the time of writing this article has not been banned yet. 27 So, it is undeniable that this new substance should be monitored. 28 5-methoxy-N-methyl-N-isopropyltryptamine has been reported by the Spanish Early 29 Warning System and detected in our laboratory in two pill samples purchased in a local 30 smart shop. This has promoted the need of stablishing consumption markers for this 31 compound in consumers' urine. In the present work, the metabolism and 32 pharmacokinetic of 5-methoxy-N-methyl-N-isopropyltryptamine has been studied by an 33 in vivo approach, using adult male mice of the inbred strain C57BLJ/6. The use of ultra-34 high performance liquid chromatography coupled to high resolution mass spectrometry 35 allowed the identification of four metabolites. After the pharmacokinetic study in serum 36 and urine, the O-demethylated metabolite and the non-metabolised parent compound are 37 proposed as consumption markers in hydrolysed urine.. Data reported in this work will 38 help hospitals and forensic laboratories to monitor the consumption and potential 39 intoxication cases related to this tryptamine. 40

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The term "new psychoactive substances" (NPS) includes synthetic compounds, but also 46 plant or fungal substances that are used as recreational drugs by elicitation of a 47 1 M H 2 PO 4 -/HPO 4 -2 buffer was prepared by dissolving the corresponding amount of 126 (NH 4 ) 2 HPO 4 in Milli-Q water and adjusting the pH to 7 with HCl. 127 128

Legal high samples containing 5-MeO-MiPT 129
Two different pills, Estrella (which means "Star" in Spanish) and Corazon ("Heart") 130 ( Figure 2) were bought in a local smart shop through its webpage. Both samples were 131 analysed in order to identify the NPS present in their composition. The tryptamine 5-132 MeO-MiPT was tentatively identified in both samples and unequivocally identified after 133 purchasing its reference standard. 134 135

Animal experiments 136
In vivo experiments were performed using C57BLJ6 adult male mice (Janvier, France). 137 The study protocol was approved by the ethical committee of Generalitat Valenciana 138 For obtaining blood samples, four groups of three animals were injected with the 5-147 MeO-MiPT solution. Ten, 20, 40 and 60 min after drug administration, animals were 148 quickly sacrificed by cervical dislocation and decapitated to obtain large volumes of 149 blood. For the control group (three animals), blood was collected in the same way, 60 150 min after vehicle injections. Blood of each animal was allowed to coagulate and 151 centrifuged. The serum from the animals of each group (either experimental or control) 152 was mixed, and aliquots were frozen for subsequent use. In conclusion, a total of five 153 serum samples (3 animals per sample) was obtained, one control and four obtained at 154 different times after 5-MeO-MiPT administration. 155 For urine samples, four groups of three animals were injected with 5-MeO-MiPT 156 solution, and four groups of three animals with the blank solution. Urine from control 157 and 5-MeO-MiPT groups were collected at 1, 2, 3, 4, 5, 6 and 24 h after the 158 administration of the drug or the vehicle solution. To do so, the three mice were put 159 together in a previously sterilized, regular home cage with a meshwire platform that 160 allowed safely collecting urine and avoiding those areas were excrements were present. 161 The sample obtained at a given time point after drug or vehicle injection, was 162 centrifuged, and the aliquots of the urine were frozen for its subsequent use. In

Identification of 5-MeO-MiPT in legal highs samples 228
The suspect screening of the legal highs samples (Estrella and Corazon) retrieved the 5-229 MeO-MiPT as a potential candidate to be present in both pills. After an accurate 230 fragment evaluation, the compound was tentatively identified in the two samples. 231