Metabolism of N-ethylhexedrone and buphedrone: An in vivo study in mice using HPLC-MS/MS

Highlights

• N-ethylhexedrone and buphedrone were detected unaltered in mice urine.

• Similar metabolic pathways were observed for both cathinones.

• The most excreted metabolites, among those considered, derived from N-dealkylation.

• 4-aryl hydroxylation were firstly detected in non-ring substituted cathinones.

• N-acetyl, glucuronides and dicarboxylic acid conjugates were tentatively identified.

Abstract

N-ethylhexedrone (NEH) and buphedrone (BUPH) are synthetic drugs structurally related to natural cathinone. These synthetic cathinones (SC) are members of the heterogenous family of new psychoactive substances (NPS), which have caused major concern in scientific and forensic communities over the past years, due to their widespread consume. Thus, there is a constant need for monitoring the use of these new substances and gather knowledge on their metabolism and excretion profiles, in order to try to identify markers of NPS consumption.

This study aimed at the identification and quantification of NEH, BUPH and selected phase I metabolites using HPLC-MS/MS. NEH, BUPH and some related metabolites were synthesized in-house and quantified in 24 h mice urine, following single dose administration of each drug (64 mg kg⁻¹, i.p.). NEH and BUPH were quantified in mice urine at 58.3 ± 14.4 and 146.2 ± 14.9 µg mL⁻¹, respectively. Similar metabolic pathways were observed for both drugs. Among the metabolites studied, the most excreted ones derived from N-dealkylation of either NEH or BUPH (at around 80 µg mL⁻¹ of urine). Other metabolites resulting from ketone reduction and ketone reduction combined with N-dealkylation or 4-aryl hydroxylation (detected for the first time in non-ring substituted SC) were also identified and quantified. Urine samples were screened using liquid chromatography-high resolution mass spectrometry and various phase II metabolites, including N-acetylated, glucuronides and dicarboxylic acid conjugates were tentatively identified, some of them for the first time. This work is a contribution to the identification of metabolites from SC that can become potential markers to estimate drug consumption.

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.