Characterization of URB Series Synthetic Cannabinoids by HRMS and UHPLC–MS/MS

A large number of synthetic cannabinoids are included in new psychoactive substances (NPS) and constitute an open research area in analytical pharmaceutical and toxicology when methods are needed to unambiguously identify these substances and their metabolites in biological fluids. A full molecular characterization of five synthetic molecules of the URB series that is able to interact with the endocannabinoid system was achieved with a high-resolution mass spectrometry (HRMS) in positive ion electrospray ionization and collisional experiments on the protonated parent ions, obtaining characteristic fragmentation patterns. Ultra-high-performance liquid chromatography coupled with a triple quadrupole (UHPLC-MS/MS) has also been used, which can help develop methods for screening and confirming synthetic cannabinoids in biological fluids.


Introduction
Cannabis contains more than 500 compounds, of which at least 100 have pharmacological properties [1] and are therefore classified as phytocannabinoids [2,3]. It is currently the most widely used illicit drug in Europe, with around 22.2 million European adults reporting its use in 2021 [4]. In recent decades, studies on the endocannabinoid system (ECS) have commanded great interest, above all due to the implications deriving from cannabinoid (CB) receptors, which were discovered in the 1990s and are involved in many physiological and pharmacological events [5][6][7][8][9][10]. CB 1 receptors are mainly present in some parts of the central nervous system (CNS), (predominantly the cerebellum, basal ganglia, hippocampus, and cerebral cortex), while CB 2 are mainly found in the immune system (primarily B cells and natural killers). Despite this, the CB 1 s are present, albeit scarcely, in districts other than the CNS [8] and the CB 2 s centrally [11], respectively. For more detail on the CB 1 and CB 2 receptors, see [9,12]. When CB receptors are activated, there is a decrease in the concentrations of cyclic adenosine monophosphate (cAMP), a messenger that is essential for regulating many cellular functions. In fact, following the binding with suitable ligands, they determine an agonist or antagonist activity, depending on whether they are stimulated or blocked. On the other hand, they can also determine a modulating action through the inhibition of the enzymatic proteins responsible for the metabolism of the endogenous molecules that physiologically bind to them [10]. Indeed, the latter, among which the most studied are N-arachidonoylethanolamine (anandamide, AEA) [13] and 2-arachidonoyl-sn-glycerol (2-AG) [14,15], plays a very important role in the regulation of many physiological processes after being released on demand from phospholipid membranes [16,17]. The basic concept is that endocannabinoids are produced (and consequently act) when and where it is needed. On the contrary, the administration of non-endogenous molecules that bind to the CB receptors [18,19], in particular when the action is aimed at the CNS, can cause effects of various kinds, such as those exerted by the main psychoactive drug of the cannabis, that is ∆ 9 -tetrahydrocannabinol (THC) [20], or those carried out by non-psychotropic cannabidiol (CBD) [21], which have antipodal pharmacological effects despite their similar structural characteristics. ECS is therefore to be considered as a reference system for the discovery of new drugs.
Evidence of the importance of substances capable of interfering with CB receptors is represented by the presence on the market of medicines containing active pharmaceutical ingredients that are able to bind to CB receptors, such as Cesamet ® (nabilone), prescribed for the improvement of chemotherapy-induced nausea and vomiting (CINV) states in patients who are not responding to conventional antiemetic therapies. Marinol ® (dronabinol), administered for CINV and appetite stimulation in patients with AIDS (acquired immune deficiency syndrome), and Sativex ® (THC and cannabidiol), used for the symptomatic relief of the pain and/or the management of the neuropathic pain and spasticity in adults with multiple sclerosis and not responsive to other antispasticity therapies or other marketed drugs. More recently, a new drug containing cannabidiol (Epidiolex ® ) has been approved by the Food and Drug Administration for the treatment of seizures associated with two rare and severe forms of epilepsy, that is, Lennox-Gastaut syndrome and Dravet syndrome, in patients who are two years of age and older [2,[21][22][23].
On the other hand, direct and indiscriminate activation of the ECs receptors, particularly of the central CB 1 , can cause several side effects, such as dry mouth, muscle weakness, myalgia, palpitations, blurred vision, memory impairment, disorientation, hallucinations, and paranoia [24][25][26][27]. This problem is accentuated in all situations in which there is an uncontrolled intake that leads to abuse and involves substances of both natural and synthetic origin.
In 2008, a synthetic cannabinoid, namely JWH-018 [28], was first identified in Europe as a constituent of the herbal products available on the web, and was advertised as incense, air fragrances, and herbal blends not intended for human consumption [29]. These products were sold under fanciful trade names (Spice, K2), and quickly spread to several countries as they could be consumed alternatively to cannabis because they were not detectable by the classic drug tests in urine. This situation has allowed the marketing of products able to induce similar or more powerful effects than cannabis without incurring penalties because they are not known and are not listed among the prohibited substances [30]. Since then, the number of "spice" products and new synthetic cannabinoids has steadily increased, so much so that, very recently, 280 individual synthetic cannabinoid receptor agonists (SCRAs) have been reported to the United Nations Office on Drugs and Crime (UNODC) Early Warnings Advisory from 90 different countries up to 2019 [31], and others have been identified in recent years. SCRAs are part of the large family of the so-called new psychoactive substances (NPS), to which, according to UNODC, all substances of abuse belong, either in a pure form or as a preparation, which are not controlled by the 1961 Single Convention on Narcotic Drugs or the 1971 Convention on Psychotropic Substances, but which may pose a public health threat [32]. Among the main classes of NPS, we can include, in addition to SCRAs, synthetic opioids, stimulants, psychedelics, hallucinogens, and so on [33]. Moreover, NPS from 133 countries and territories have been reported [34]. Consequently, the increase in NPS [33][34][35][36] and smart drugs has led to restrictive and monitoring actions by the institutions involved. The continuous emergence of NPS on the market, however, makes legislative restrictions inadequate and the monitoring task of prevention agencies more difficult.
The recent appearance and widespread diffusion of legal hemp-based products (resin, leaves, and inflorescences) with a low THC content but adulterated with highly active synthetic cannabinoids has made the scenario even more complex. In fact, industrial hemp with a low THC concentration is cheaper and the addition of a small amounts of synthetic cannabinoids is enough to mimic the indiscriminate activity aimed at CB targets. This dangerous way of spreading synthetic cannabinoids puts people's health at risk, not only of those who knowingly buy the drug but also of those who are convinced that they are buying a harmless natural product [4].
At present, most of the cannabimimetic substances identified in seizures of illegal materials in Europe and in other countries derive from commercial products already known, with various abbreviations (e.g., AM, AB, CP, JWH, HU, RCS, UR, URB, and WIN), and belonging to different chemical classes (e.g., adamantoylindoles, aminoalkylindoles, benzoylindoles, cyclohexylphenols, dibenzopyrans, naphthoylindoles, naphthylmethylindoles, naphthylmethylindenes, phenylacylindoles, tetramethylcyclopropylketoindoles, indazole carboxamides, and naphthoylpyrroles) [37]. Recently, an interesting study aiming to explore the steric requirements at CB 1 and CB 2 receptors and the role of the related amino acid side chain has been developed that uses some SCRAs [38].
The continuous introduction of new analogues of existing CB drugs is certainly a legal problem, but it is also a major cause of analytical and toxicological uncertainty [39]. In fact, the lack of certified reference standards and the inadequacy of knowledge relating to biological activity and metabolism make it difficult to determine and evaluate new compounds, especially biological matrices. Therefore, the increase in knowledge and the characterization of new CB compounds would be important for their identification.
In this work, a full molecular characterization by high-resolution mass spectrometry (HRMS) and ultra-high-performance liquid chromatography (UHPLC)-MS/MS of five synthetic molecules of the URB series designed to interact indirectly or directly with ECS ( Figure 1) is presented for the first time. of synthetic cannabinoids is enough to mimic the indiscriminate activity aimed at CB targets. This dangerous way of spreading synthetic cannabinoids puts people's health at risk, not only of those who knowingly buy the drug but also of those who are convinced that they are buying a harmless natural product [4]. At present, most of the cannabimimetic substances identified in seizures of illegal materials in Europe and in other countries derive from commercial products already known, with various abbreviations (e.g., AM, AB, CP, JWH, HU, RCS, UR, URB, and WIN), and belonging to different chemical classes (e.g., adamantoylindoles, aminoalkylindoles, benzoylindoles, cyclohexylphenols, dibenzopyrans, naphthoylindoles, naphthylmethylindoles, naphthylmethylindenes, phenylacylindoles, tetramethylcyclopropylketoindoles, indazole carboxamides, and naphthoylpyrroles) [37]. Recently, an interesting study aiming to explore the steric requirements at CB1 and CB2 receptors and the role of the related amino acid side chain has been developed that uses some SCRAs [38].
The continuous introduction of new analogues of existing CB drugs is certainly a legal problem, but it is also a major cause of analytical and toxicological uncertainty [39]. In fact, the lack of certified reference standards and the inadequacy of knowledge relating to biological activity and metabolism make it difficult to determine and evaluate new compounds, especially biological matrices. Therefore, the increase in knowledge and the characterization of new CB compounds would be important for their identification.
In this work, a full molecular characterization by high-resolution mass spectrometry (HRMS) and ultra-high-performance liquid chromatography (UHPLC)-MS/MS of five synthetic molecules of the URB series designed to interact indirectly or directly with ECS ( Figure 1) is presented for the first time. The molecules presented in Figure 1 are defined as synthetic cannabinoids under the broader definition of molecules interfering with the endocannabinoid system without exerting a direct action on the CB receptors (a more classical definition). On the other hand, the EMCDDA (European Monitoring Center for Drugs and Drug Addiction) also includes synthetic cannabinoids molecules that do not bind the receptor.
The compounds of the URB series studied here are representative of molecules able to interfere indirectly (enzyme inhibitors) or directly (ligands) with the biological targets of the endocannabinoid system. These include the following molecules: (A) Two carbamates that inhibit fatty acid amide hydrolase (FAAH), which catalyze the intracellular hydrolysis of the endocannabinoid AEA, and are able to exert anxiolytic and analgesic properties (cyclohexylcarbamic acid 3′-carbamoylbiphenyl-3-yl ester, URB597) [40] or restricted peripheral analgesic effects (cyclohexylcarbamic acid 3′-carbamoyl-6-hydroxybiphenyl-3-yl ester, URB937) [41]. They play a very important role in the panorama of FAAH inhibitors because they were the first ever with a good overall profile. Still today, they constitute reference molecules in the specific research oriented toward the study of molecules that are able to interfere with the endocannabinoid system. So much of this is true that URB597 is the most widely used pharmacological tool in the field, the use of The molecules presented in Figure 1 are defined as synthetic cannabinoids under the broader definition of molecules interfering with the endocannabinoid system without exerting a direct action on the CB receptors (a more classical definition). On the other hand, the EMCDDA (European Monitoring Center for Drugs and Drug Addiction) also includes synthetic cannabinoids molecules that do not bind the receptor.
The compounds of the URB series studied here are representative of molecules able to interfere indirectly (enzyme inhibitors) or directly (ligands) with the biological targets of the endocannabinoid system. These include the following molecules: (A) Two carbamates that inhibit fatty acid amide hydrolase (FAAH), which catalyze the intracellular hydrolysis of the endocannabinoid AEA, and are able to exert anxiolytic and analgesic properties (cyclohexylcarbamic acid 3 -carbamoylbiphenyl-3-yl ester, URB597) [40] or restricted peripheral analgesic effects (cyclohexylcarbamic acid 3 -carbamoyl-6-hydroxybiphenyl-3-yl ester, URB937) [41]. They play a very important role in the panorama of FAAH inhibitors because they were the first ever with a good overall profile. Still today, they constitute reference molecules in the specific research oriented toward the study of molecules that are able to interfere with the endocannabinoid system. So much of this is true that URB597 is the most widely used pharmacological tool in the field, the use of which serves to further understand pharmacological applications. Both are covalent and irreversible inhibitors. (B) A reverse carbamic analogue of FAAH inhibitors that inactivates the enzyme degrading 2-AG, which is a monoglyceride lipase (MGL), and is able to relieve the stress-induced pain and induce neuroprotection (biphenyl-3-yl carbamic acid cyclohexyl ester, URB602 [42]). It is a covalent and partially reversible inhibitor. (C) A benzoxazinone derivative initially studied as an MGL inhibitor (6-methyl-2-p-tolylaminobenzo[d]oxazin-4-one, URB754) [43,44]. (D) Finally, a peripherally restricted mixed CB 1 antagonist/CB 2 agonist pyrrolic drug endowed with anti-obesity, neuroprotective, and antitumor properties [45,46], and an interesting anti-inflammatory profile (data not available) {[4-amino-1-(4-chlorobenzyl)-2methyl-5-phenyl-1H-pyrrol-3-yl] (phenyl)methanone, URB447. These compounds were included among the monitored commercial substances. Notably, URB597 was found in herbal products in both Japan [47] and the United States of America [48], which was notified by the European Union Early Warning System in 2013 (found in Poland) [49], while URB754 has been reported in the literature as a new designer drug [50].
Although, at the moment, the phenomena of abuse and fatality have not been correlated with the considered drugs, the molecular characterization and MS fragmentation behavior of the protonated molecules reported here are important for the unambiguous identification of the substances. They also play a key role in the future development of LC-MS/MS or LC-HRMS screening methods that are useful for their research on seized samples.
In fact, the substances studied in this work are potentially dangerous because they are active at very low concentrations (lower than most direct cannabinoids). Furthermore, they are emerging drugs and sufficient epidemiological data to describe their consumption have not yet been acquired (on the other hand, if no one includes them in the screening of real intoxication cases, no one can estimate their prevalence).

Results and Discussion
The tuning parameters optimized in UHPLC-MS/MS to obtain fragment ions of high abundance for use in the multiple reaction monitoring (MRM) method are reported in Table 1 and Figure 2. tion have not yet been acquired (on the other hand, if no one includes them in the sc ing of real intoxication cases, no one can estimate their prevalence).

Results and Discussion
The tuning parameters optimized in UHPLC-MS/MS to obtain fragment ions of abundance for use in the multiple reaction monitoring (MRM) method are reporte Table 1 and Figure 2.  The identity of the observed fragment ions was confirmed by HRMS and collisioninduced dissociation (CID) measurements in an Orbitrap comparing the theoretical to the experimental accurate masses, as reported in Table 2 (fragments of data are partially shown). The characteristic fragmentation patterns of URB compounds were subsequently interpreted as follows.  (Figure 3).       The main fragmentation pathways are driven by α-cleavages to the carbonyl moieties (Table 2, Figure 6). The main fragmentation pathways are driven by α-cleavages to the carbonyl moieties (Table 2, Figure 6).  The main fragmentation pathways are driven by α-cleavages to the carbonyl moieties (Table 2, Figure 6).     Other main characteristic fragments are observed at m/z 213 due to the loss of cyclohexyl isocyanate and ammonia, and m/z 338 due to the loss of ammonia only (Figures 8  and 9).  Other main characteristic fragments are observed at m/z 213 due to the loss of cyclohexyl isocyanate and ammonia, and m/z 338 due to the loss of ammonia only (Figures 8  and 9). Other main characteristic fragments are observed at m/z 213 due to the loss of cyclohexyl isocyanate and ammonia, and m/z 338 due to the loss of ammonia only (Figures 8 and 9). Other main characteristic fragments are observed at m/z 213 due to the loss of cyclohexyl isocyanate and ammonia, and m/z 338 due to the loss of ammonia only (Figures 8  and 9).

URB602
The main fragment ions of URB602 ([M + H] + m/z 296) are those at m/z 214, due to the loss of cyclohexyl, at m/z 196 and m/z 170, depending on the losses of water and carbon anhydride, respectively (Figures 11 and 12, CE 20 V).

URB602
The main fragment ions of URB602 ([M + H] + m/z 296) are those at m/z 214, due to the loss of cyclohexyl, at m/z 196 and m/z 170, depending on the losses of water and carbon anhydride, respectively (Figures 11 and 12, CE 20 V).

URB602
The main fragment ions of URB602 ([M + H] + m/z 296) are those at m/z 214, due to the loss of cyclohexyl, at m/z 196 and m/z 170, depending on the losses of water and carbon anhydride, respectively (Figures 11 and 12, CE 20 V).

URB602
The main fragment ions of URB602 ([M + H] + m/z 296) are those at m/z 214, due to the loss of cyclohexyl, at m/z 196 and m/z 170, depending on the losses of water and carbon anhydride, respectively (Figures 11 and 12, CE 20 V).

URB754
Characteristic MS/MS fragmentation patterns are shown in Figures 14 and 15 (C V).

N
H N 160 106 Figure 13. Scan of URB602 compound obtained by HRMS.

URB754
Characteristic MS/MS fragmentation patterns are shown in Figures 14 and 15 (CE 40 V).

URB754
Characteristic MS/MS fragmentation patterns are shown in Figures 14 and 15 (CE 40 V).

URB754
Characteristic MS/MS fragmentation patterns are shown in Figures 14 and 15 (CE 40 V).  The main fragment ions of URB754 ([M + H] + at m/z 267) are 6-methyl-4-oxo-4H-3,1benzoxazin-2-ylium at m/z 160, due to the loss of 4-methylaniline, and the ion at m/z 249, due to the loss of water caused by the opening of the 1,3-oxazin-6-one ring. A fragment ion at m/z 106 was also observed, caused by the formation of the toluidine derivative ion. The description of the accurate mass is shown in Figure 16. The main fragment ions of URB754 ([M + H] + at m/z 267) are 6-methyl-4-oxo-4H-3,1benzoxazin-2-ylium at m/z 160, due to the loss of 4-methylaniline, and the ion at m/z 249, due to the loss of water caused by the opening of the 1,3-oxazin-6-one ring. A fragment ion at m/z 106 was also observed, caused by the formation of the toluidine derivative ion. The description of the accurate mass is shown in Figure 16.

URB447
URB447 shows a quasi-molecular ion [M + H] + at m/z 401 and 403, due to the presence of the characteristic isotopic pattern of chlorine 35 Cl and 37 Cl (Figure 17).

URB447
URB447 shows a quasi-molecular ion [M + H] + at m/z 401 and 403, due to the presence of the characteristic isotopic pattern of chlorine 35 Cl and 37 Cl (Figure 17).

URB447
URB447 shows a quasi-molecular ion [M + H] + at m/z 401 and 403, due to the presence of the characteristic isotopic pattern of chlorine 35 Cl and 37 Cl (Figure 17).   A direct cleavage of the C-N bond between the 4-chlorobenzyl group and the Npyrrolyl group results in a 4-chlorobenzylium ion at m/z 125 or 4-amino-3-benzoyl-2-methyl-5-phenyl-1H-pyrrol-1-ylium ion at m/z 276 (C18H16N2O) (Figures 18 and 19). Analogue fragment ions at m/z 127 and at m/z 298 were observed starting from a similar precursor ion at m/z 403 (figure not shown). The description of the accurate mass is shown in Figure  20.  A direct cleavage of the C-N bond between the 4-chlorobenzyl group and the Npyrrolyl group results in a 4-chlorobenzylium ion at m/z 125 or 4-amino-3-benzoyl-2-methyl-5-phenyl-1H-pyrrol-1-ylium ion at m/z 276 (C18H16N2O) (Figures 18 and 19). Analogue fragment ions at m/z 127 and at m/z 298 were observed starting from a similar precursor ion at m/z 403 (figure not shown). The description of the accurate mass is shown in Figure  20.  (Figures 18 and 19). Analogue fragment ions at m/z 127 and at m/z 298 were observed starting from a similar precursor ion at m/z 403 (figure not shown). The description of the accurate mass is shown in Figure 20. A direct cleavage of the C-N bond between the 4-chlorobenzyl group and the Npyrrolyl group results in a 4-chlorobenzylium ion at m/z 125 or 4-amino-3-benzoyl-2-methyl-5-phenyl-1H-pyrrol-1-ylium ion at m/z 276 (C18H16N2O) (Figures 18 and 19). Analogue fragment ions at m/z 127 and at m/z 298 were observed starting from a similar precursor ion at m/z 403 (figure not shown). The description of the accurate mass is shown in Figure  20.

UHPLC-MS/MS
The characterization of compounds by UHPLC-MS/MS was carried out with a UH-PLC system (Nexera X2, Shimazdu, Kyoto, Japan) coupled with a triple quadrupole mass spectrometer (Hybrid System 4000 Qtrap, AB Sciex, Framingham, MA, USA). Chromatographic separation was performed on a 50 × 2.1 mm KINETEX EVO 1.9 µm C18 column (Phenomenex, Bologna, Italy). The mobile phase was composed of solvent "A" (0.1% formic acid) and "B" (0.1% formic acid in acetonitrile with 5% water). The initial chromatographic conditions were 20% "B", and the gradient was programmed as follows: 0-0.1 min hold 20% "B", 0.1-6.0 min to 80% "B", and 6.0-6.1 min to 100% "B", and it was kept for 3 min. Finally, the initial condition was restored and kept for 2 min to equilibrate the system. Total run time was 11 min at the constant flow rate (0.5 mL/min); the column temperature was set at 40 • C; the autosampler was maintained at 10 • C. The mass spectrometer operated in positive ion mode using ESI TurboIonSpray ® under the following conditions: curtain gas (CUR) 30.0 psi, ion spray voltage (IS) 4000 V, temperature (TEM) 500 • C, ion source gas 1 (GS1) 40.0 psi, and ion GS2 55.0 psi. Identification was performed using MRM mode. MRM transitions were experimentally established. At least three MRM transitions were monitored for each compound. A total of 19 transition were monitored with an MRM pause time of 5 ms and a dwell time of 20 ms for each transition; the total scan time was 0.47 s.

HRMS Compound Characterization
The methanolic solutions of the individual standards (URB597, URB937, URB602, URB754, and URB447) were prepared separately in 10 mL volumetric flasks at an approximate concentration of 100 or 1000 µg/mL. Each drug was diluted to a concentration from 0.1 to 1 ppm in methanol. The determination of the exact mass of ionic species in the URB compounds was performed on HRMS experiments using a Linear Trap Quadrupole (LTQ) Orbitrap XL hybrid mass spectrometry (Thermo Scientific TM , ThermoFisher Scientific, Waltham, MA, USA) equipped with an electrospray ion source operated in positive ion mode. Direct infusion of compounds into the MS ion source was performed using a syringe pump at a flow rate of 10 µL/min. The protonated molecules of each compound were isolated in the linear ion trap and fragmented at varying collision energies in order to identify the most abundant and characteristic product ions.

Conclusions
The present study integrates and extends the mass spectrometry characterization of synthetic drugs that are able to interact with the cannabinoid system. It provides the first description of URB937, URB602, URB754, and URB447 compounds by CID in tandem with the quadrupole mass spectrometry and hybrid ion trap-orbitrap mass spectrometry. It should also be considered that, so far, the analyses and characterizations have been carried out in MS (EI) [44,45,51,52] or MS (ESI) [41] only. The methods used here have made it possible to unequivocally characterize the molecules under examination with the aim to determine the operating conditions for their accurate identification and, therefore, laying the foundations for the future development of analytical methods aimed at the early screening of NPS in seizures or biological samples. However, these substances have long been monitored as potential alerts [48,53]. In this respect, studies on the metabolism of the URB compounds are necessary, and the work is in progress.