Metabolism of N-ethylhexedrone and buphedrone: An in vivo study in mice using HPLC-MS/MS
Introduction
N-ethylhexedrone (NEH) and buphedrone (BUPH) are synthetic drugs derived from cathinone, a natural psychoactive alkaloid isolated from khat plant (Catha edulis), that is structurally similar to amphetamine [1], [2], [3]. Often labelled as “legal highs”, “bath salts”, “plant food” or “research chemicals”, synthetic cathinones (cathinone derivatives, SC) are readily accessible at low cost via internet, in head shops or through drug dealers, owing their popularity to psychoactive properties similar to amphetamine and to other common illicit drugs, such as cocaine and 3,4-methylenedioxymethamphetamine (MDMA) [4], [5], [6]. Together with synthetic cannabinoids, opioids, benzodiazepines and other stimulants, SC are included in a group of compounds denominated as “new psychoactive substances” (NPS) [4], [7]. More than 670 NPS have been identified in Europe over the past decade, including a total of 130 SC [4]. The constant entrance of new, or newly synthesized cathinones into the recreational drug market, is enabled by the multitude of possible substitutions to the core skeleton of cathinones. Therefore, addition of substituents to the α-carbon, to the N-terminus and/or to the aromatic ring of cathinones may result in new molecules that are not detected in routine drug screening [8], [9], [10].
It has been proposed that SC can be grouped into four families according to the N-alkyl and aromatic ring substituents [11]: N-alkylated cathinones with or without aromatic ring substituents, such as NEH and BUPH; 3′,4′-methylenedioxy-N-alkylated cathinones; N-pyrrolidine cathinones with or without aromatic ring substituents and 3′,4′-methylenedioxy-N-pyrrolidine cathinones. Hence, depending on their chemical structure, SC may undergo different preferred metabolic pathways [12], [13]. Nonetheless, independent or combined N-dealkylation of the primary amine and β-keto reduction to the corresponding alcohol appears to be common phase I metabolic pathways among the four families of SC [12], [14], [15], [16]. Additionally, hydroxylation of the benzene ring, a major pathway for amphetamines, has also been observed in rat urine following SC administration [13], [17]. Phase I hydroxylated SC metabolites may also undergo phase II metabolism, being excreted in urine as glucuronides or sulfates [17], [18], [19] and also conjugated with dicarboxylic acids [20], [21]. Even though it has been suggested that SC are usually consumed in such doses that allow for the detection of their unchanged form in urine [3], [5], [13], [22], additional information on drug metabolism and on metabolite/drug ratios could be useful to provide estimates of time of drug consumption in addition to the confirmation of drug intake. In a study comprising the analysis of human urine to uncover the metabolic profiles of a range of SC, parent drugs corresponding to N-alkylated cathinones with or without aromatic ring substituents, were either absent or less abundant than metabolites [12]. This could be explained by the amount consumed and time of consumption, which were unknown to the authors. In fact, urinary recovery of mephedrone, one of the cathinones considered in that study [12], after 24–48 h post drug administration was practically null, while some metabolites were still being excreted [23]. Furthermore, since the use of SC has been associated with several cases of acute and fatal intoxications, it is imperative that these new substances be subject to controlled pharmacological studies, including evaluation of metabolic and excretion profiles, not only to enable intake confirmation through target biomarkers, but also to link adverse effects to the responsible compound, which can be the drug itself or a metabolite [8], [24], [25].
Cheap and fast screening methods of drugs of abuse, such as immunoassays, have been used in clinical and forensic studies [26]. However, immunoassays usually only cover a certain number of drugs or drug classes, implying limitations in the identification of new compounds, and are prone to generating false positive or false negative results [27], [28]. Thus, in recently reviewed literature regarding analytical methods for the identification and quantification of synthetic cathinones in biological matrices, gas or liquid chromatography coupled to mass spectrometry (GC–MS or HPLC-MS, respectively) have been the most commonly mentioned methodologies and urine the most analyzed matrix [29]. Achievement of high specificity and sensitivity is possible, in tandem MS methods (e.g. HPLC-MS/MS), when 2 or 3 ion transitions per compound are recorded in multiple reaction monitoring (MRM) mode, allowing for more than 40 synthetic cathinones, as well as other drugs, and some metabolites to be correctly identified in urine at the same time [28], [30], [31], [32].
This study contributes to uncover the metabolism of NEH and BUPH through the identification and quantification of excreted metabolites in urine following in vivo studies in mice. These two cathinones were synthesized in-house, as well as metabolites, selected among those expected from literature [12], [13], [20] and predicted in silico. The parent drugs were used for the in vivo studies. Following a single dose administration of NEH or BUPH to two groups of mice, 24 h urine was collected and analyzed by HPLC-MS/MS, using optimized and validated methods. HRMS was also used to screen for other phase I and phase II metabolites.
Section snippets
Chemicals and materials
N-ethylhexedrone (NEH) 2-(ethylamino)-1-phenyl-1-hexanone hydrochloride, buphedrone (BUPH) 2-(methylamino)-1-phenylbutan-1-one hydrochloride, and corresponding metabolites 2-(ethylamino)-1-phenylhexan-1-ol hydrochloride (H1), 2-amino-1-phenylhexan-1-one (H2), 2-amino-1-phenylhexan-1-ol hydrochloride (H3), 4-(2-(ethylamino)-1-hydroxyhexyl)phenol hydrochloride (H4), 4-(2-amino-1-hydroxyhexyl)phenol hydrochloride (H5), 2-(methylamino)-1-phenylbutan-1-ol hydrochloride (B1),
Results and discussion
To the best of our knowledge, this is the first controlled in vivo study in mice concerning the metabolism of NEH and BUPH. The lack of commercially available standards of SC or metabolites may at times constitute an impairment to the development of such studies [5], [29]. Thus, for this work, NEH, BUPH and some selected metabolites (Fig. 1, Fig. 2) were synthesized in-house. Considering the metabolic pathways known to occur for cathinones [12], [13], [20] and metabolite prediction in silico,
Conclusions
In this work, NEH, BUPH and corresponding metabolites were quantified for the first time in 24 h urine samples from mice exposed to both drugs, using a HPLC-MS/MS method. Identification was confirmed by comparison with standards synthetized in-house. Metabolites quantified in this study suggest that both drugs follow the same metabolic pathways as other N-alkylated cathinones, with or without aromatic ring substituents. The most excreted metabolites were the β-keto-N-dealkylated compounds,
CRediT authorship contribution statement
Joana Carrola: Methodology, Investigation, Validation, Writing - original draft. Noélia Duarte: Supervision, HRMS data analysis, Writing - review & editing. Pedro Florindo: Investigation. Sara Henriques: Investigation. Gustavo da Silva: Investigation. Lubertus Bijlsma: Investigation. Rui Moreira: Supervision. Catarina Correia: Investigation (HRMS assays). Maria de Jesus Perry: Investigation. Álvaro Lopes: Conceptualization, Project administration, Funding acquisition, in silico metabolites
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was financially supported by Fundação para a Ciência e a Tecnologia (FCT), through PTDC-SAU-TOX/32515/2017 and, in part, UID/DTP/04138/2013 (iMed.ULisboa) and by the European Commission (HOME/2014/JDRU/AG/DRUG/7086). The authors acknowledge the financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa – RNEM; LISBOA-01-0145-FEDER-402-022125). Joana Carrola also acknowledges RNEM for her post-doctoral fellowship.
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