In Vitro Metabolite Profiling of ADB-FUBINACA, A New Synthetic Cannabinoid

Abstract

Metabolite profiling of novel psychoactive substances (NPS) is critical for documenting drug consumption. N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (ADB-FUBINACA) is an emerging synthetic cannabinoid whose toxicological and metabolic data are currently unavailable. We aimed to determine optimal markers for identifying ADB-FUBINACA intake. Metabolic stability was evaluated with human liver microsome incubations. Metabolites were identified after 1 and 3 h incubation with pooled human hepatocytes, liquid chromatography- high resolution mass spectrometry in positive-ion mode (5600+ TripleTOF®, Sciex) and several data mining approaches (MetabolitePilot™, Sciex). Metabolite separation was achieved on an Ultra Biphenyl column (Restek®); full-scan TOF-MS and information-dependent acquisition MS/MS data were acquired. ADB-FUBINACA microsomal half-life was 39.7 min, with a predicted hepatic clearance of 9.0 mL/min/kg and a 0.5 extraction ratio (intermediate-clearance drug). Twenty-three metabolites were identified. Major metabolic pathways were alkyl and indazole hydroxylation, terminal amide hydrolysis, subsequent glucuronide conjugations, and dehydrogenation. We recommend ADB-FUBINACA hydroxyalkyl, hydroxydehydroalkyl and hydroxylindazole metabolites as ADB-FUBINACA intake markers. N-dealkylated metabolites are not specific ADB-FUBINACA metabolites and should not be used as definitive markers of consumption. This is the first ADB-FUBINACA in vitro metabolism study; in vivo experiments enabling pharmacokinetic and pharmacodynamics studies or urine from authentic clinical/forensic cases are needed to confirm our results.

1. INTRODUCTION

Synthetic cannabinoids (SCs) are novel psychoactive substances (NPS) eliciting cannabimimetic psychoactive effects [1]. SCs were originally produced for biomedical research but now are abused, leading many countries to schedule these drugs as controlled substances [2-5]. Clandestine laboratories continue producing new compounds that mimic effects of scheduled NPS to circumvent legislation. More than 130 SCs were identified to date [6]. Little pharmacological or toxicological data are available when substances first appear. Analytical methods require constant updating, and pharmacodynamic and pharmacokinetic studies must be conducted for the continual emergence of NPS. With its patent filed only in 2009, ADB-FUBINACA, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (Fig. 1) is one of the newest SC [7]. In 2013, the drug was identified for the first time in illegal herbal blends in Japan, in association with two other SCs ADBICA and XLR-11 [8]. The drug was reported for the first time in Europe in the same year in Hungary [6] as Facebook logo shaped tablets, and in biological samples from patients who swallowed or crushed and insufflated the product [9, 10].

Fig. (1).

Structure of ADB-FUBINACA and Schedule I controlled analogs.

There are currently no in vivo human or animal ADB-FUBINACA pharmacodynamic data. In vitro experiments demonstrated potent CB₁ cannabinoid receptor binding (K_i = 0.36 nM, EC₅₀ = 0.98 – 1.2 nM) [7, 11] and CB₂ (EC₅₀ = 3.5 nM) [11]. CB₁ binding affinity that correlates with cannabinoid psychoactive effects [12] is 140 times stronger than for THC, comparable to other indole and indazole SCs with an aminomethylbutanamide or aminodimethylbutanamide structure [11]. Affinity for CB₁ receptors is slightly lower for the desmethyl and indole analogs, AB-FUBINACA (scheduled) [13] and ADB-FUBICA, and higher with the N-pentyl and N-fluoropentyl analogs, ADB-PINACA (scheduled) [13] and 5F-ADB-PINACA [11]. ADB-FUBINACA reportedly produces drowsiness, respiratory deprivation, CO₂ retention, hypotonia, hypothermia, vomiting, sinus tachycardia, mydriasis, extrapyramidal movement disorder, hyperkinesis, acoustic and visual hallucinations, agitation and mixed symptoms [2, 10]. Structural analogs were involved in several intoxication and fatalities [14-19], with the first ADB-FUBINACA death reported in 2016 [20].

ADB-FUBINACA pharmacodynamic and pharmaco-kinetic studies are still lacking. Many SCs are active at low doses and extensively metabolized with little to no parent compound found in urine, making urinary metabolite detection critical for documenting consumption in forensic and clinical cases [21-25]. One ADB-FUBINACA human liver microsome (HLM) in vitro metabolism study identified a single hydroxyalkyl metabolite [26]. Identifying the SC responsible for resultant toxicities also is important for educating the public on the drug’s dangers.

Our aim was to determine ADB-FUBINACA human metabolism to improve analytical identification of this new SC in biological samples. In vitro HLM [27-29] and human hepatocytes incubations [30-33] with high resolution mass spectrometry (HRMS) analysis proved useful for predicting human SC metabolism. Human hepatocytes contain all required hepatic metabolic enzymes and endogenous cofactors and provide the natural orientation of membrane enzymes, better predicting metabolite production in in vivo conditions than enzymes alone or HLM [34].

2. EXPERIMENTAL

2.1. Chemicals and Reagents

ADB-FUBINACA and diclofenac standards were purchased from Cayman Chemical (Ann Arbor, MI, USA) and Toronto Research Chemicals (Toronto, Canada) respectively. LC-MS grade water, methanol, and formic acid (Optima™ LC/MS) were acquired from Fisher Scientific (Fair Lawn, NJ, USA), and trypan blue and LC-MS grade acetonitrile from Sigma-Aldrich^® (St. Louis, MT, USA). Distilled water was produced by an ELGA PURELAB^® Ultra Analytic purifier (Siemens Water Technologies, Lowell, MA, USA). Fifty-donor pooled HLM, ten-donor-pooled cryopreserved human hepatocytes, InVitroGRO™ hepatocyte thawing (HT) medium, and InVitroGRO™ Krebs-Henseleit buffer (KHB) were obtained from BioreclamationIVT (Westbury, NY, USA). Solutions A (NADPH regeneration system) and B (glucose-6-phosphate dehydrogenase) were purchased from BD Biosciences (San Jose, CA, USA).

2.2. Metabolic Stability Assessment with Human Liver Microsomes (HLM)

2.2.1. Incubation

The reaction mixture included 780 µL distilled water, 100 µL 0.5 M potassium phosphate buffer pH 7.4, 10 µL solution B, and 10 µL 100 µmol/L ADB-FUBINACA in methanol. After vortexing, HLM suspensions (20 mg/mL) were thawed at 37ºC, 50 µL added to the reaction mixture and gently mixed. The suspension was pre-incubated at 37ºC for 3 min, and the reaction initiated by adding 50 µL solution A. Samples (100 µL) were collected after 0, 3, 8, 13, 20, 45 and 60 min incubation and the reaction quenched with an equal volume of ice-cold acetonitrile. Samples were stored at – 80ºC until analysis.

HLM samples were thawed and diluted 100-fold with mobile phase A before injection onto the LC-HRMS. Liquid chromatography (LC)-HRMS parameters for HLM incubations are described below.

2.2.2. LC-MS Parameters

HLM LC-MS analysis was performed on a 3200 QTRAP^® mass spectrometer (triple quadrupole-linear ion trap) equipped with an TurboV ion source operated in positive electrospray mode from Sciex (Foster City, CA, USA) and coupled with an LC-20ADXR HPLC system from Shimadzu (Columbia, MD, USA). Data were acquired with analyst TF software version 1.5 (Sciex).

Separation was performed using a Kinetex^® C₁₈ column (100 x 2.1 mm, 2.6 µm) combined with a 50 x 2.1 mm guard column of identical mobile phase from Phenomenex (Torrance, CA, USA). Elution was achieved within 15 min with a gradient mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.3 mL/min. Injection volume was 5 µL. Autosampler and column oven temperatures were 4 and 40ºC, respectively. Gradient conditions started at 5% B for 0.5 min, increased to 95% B in 9.5 min, were held for 2.5 min, returned to initial conditions in 0.1 min and re-equilibrated for 2.4 min.

The mass spectrometer was operated in multiple reaction monitoring (MRM) mode. Three transitions were monitored between 6.5 and 8.5 min (accumulation time, 0.5 s): transition m/z 383.2 > 253.3 with a 33 V collision energy (CE) and a 4 V collision cell exit potential (CXP), transition m/z 383.2 > 109.0 with a 57 V CE and a 4 V CXP, and transition m/z 383.2 > 366.3 with a 17 V CE and a 6 V CXP. The first transition was for quantification and the last two for identification confirmation. Source parameters were: gas 1, 45 psi; gas 2, 70 psi; curtain gas, 30 psi; temperature, 500ºC; ion spray voltage, 4 kV; declustering potential, 31 V.

2.2.3. Calculations

In vitro microsomal half-life (T_1/2) and clearance (CL_{int, micr}), predicted intrinsic clearance (CL_int), predicted human hepatic clearance (CL_hep), and extraction ratio (ER) were calculated based on Baranczewski [35] and McNaney’s [36] models, without consideration of plasma protein binding. T_1/2 was calculated based on the percentage of ADB-FUBINACA MS signal remaining at each time point.

2.3. Metabolite Profiling with Human Hepatocytes

2.3.1. Incubation

Hepatocytes were thawed at 37ºC, washed twice with InVitroGRO™ HT medium and KHB, and centrifuged at 100 g for 5 min. Supernatant was removed and the pellet re-suspended in 2 mL KHB, yielding a 2 x 10⁶ cells/min concentration. Cell viability, assessed with trypan blue exclusion dye (0.4%, v/v), was ≥80%. ADB-FUBINACA was dissolved in methanol and diluted in KHB to a final 20 µmol/L concentration. The reaction mixture was prepared with 250 µL hepatocyte suspension and 250 µL drug solution and incubated in a Forma™ Steri-Cycle™ CO₂ incubator at 37ºC (Thermo Scientific). Samples (500 µL) were incubated for 0, 1 and 3 h and the reaction quenched with an equal volume of ice-cold acetonitrile. Diclofenac was incubated in a separate mixture under the same conditions and 4’-hydroxydiclofenac and acyl-β-D-glucuronide diclofenac monitored to ensure hepatocyte metabolic activity. In addition, a negative control with ADB-FUBINACA in buffer without hepatocytes was incubated to rule out non-enzymatic reactions. Samples were stored at – 80ºC until analysis.

Hepatocyte samples were thawed and centrifuged at 4ºC, 15,000 g for 10 min, to remove cell debris. Supernatants were diluted 5-fold with 0.1% formic acid in water (mobile phase A) before injection.

2.3.2. LC-HRMS Parameters

LC-HRMS analysis of hepatocyte incubations was performed on a 5600⁺ TripleTOF^® quadrupole-time of flight mass spectrometer equipped with a DuoSpray source (Sciex) operated in positive electrospray mode, coupled with an LC-20ADXR HPLC system from Shimadzu. Data were acquired with Analyst TF software version 1.6 (Sciex).

Separation was performed on an Ultra Biphenyl column (100 x 2.1 mm, 3 µm) combined with a 10 x 2.1 mm guard column of identical phase from Restek^® (Bellefonte, PA, USA). Elution was achieved within 15 min with a gradient mobile phase composed of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a flow rate of 0.5 mL/min. Injection volume was 15 µL. Autosampler and column oven temperatures were set at 4 and 30ºC respectively. Gradient conditions started with 20% B for 0.5 min, increased to 95% B in 10.5 min and then held for 2 min, returned to initial conditions in 0.1 min and re-equilibrated for 1.9 min. Efflux was diverted to waste before 2 and after 13 min.

The mass spectrometer operated in full-scan TOF-MS and information-dependent acquisition (IDA) MS/MS modes. One MS cycle was composed of one full-scan TOF-MS survey scan followed by up to eight IDA MS/MS scans (accumulation time, 0.5 s). The eight ions detected with the most intense signal in the survey scan (intensity threshold, 100 cps), triggered an MS/MS event with fragmentation (isolation window, m/z 1.5). IDA mode included a list of expected metabolites (Supplementary material, Table S1 ^{(5.3MB, pdf)} ) that were fragmented in priority, regardless of their intensity (mass tolerance, 50 mDa). Full MS scans were acquired from m/z 100 – 1000 with high resolution and a 0.1 s accumulation time. IDA MS/MS scans were acquired from m/z 30 – 1000 with high resolution; collision energy was 35 ± 15 V and accumulation time 0.05 s. Source parameters were: gas 1, 60 psi; gas 2, 75 psi; curtain gas, 45 psi; temperature, 650ºC; ion spray voltage, 4 kV; declustering potential, 80 V.

2.3.3. Metabolite Identification

Data from hepatocyte incubations were analyzed with MetabolitePilot™ software (v. 1.6; Sciex) for ADB-FUBINACA metabolites identification. Processing settings are described in Table 1.

Table 1.

MetabolitePilot™ processing settings. MS(/MS), (tandem) mass spectrometry.

Parent Compound	ADB-FUBINACA
Formula	C21H23N4O2F
Theoretical m/z (positive mode)	383.1878
Biotransformations
C7H5F loss, C6H11NO loss, C6H12N2O loss, NH loss, amide hydrolysis, carboxylation, defluorination, defluorobenzylation, desaturation, glucuronidation, glutathione conjugation, hydrogenation, amide hydrolysis to carboxylic acid, internal hydrolysis, ketone formation, hydroxylation, sulfation, and combinations
Chromatographic parameters
Min peak width	2.5 s
Min chromatographic intensity	500 cps
Retention time window	1 to 13 min
MS parameters
m/z tolerance	50 ppm
Min MS peak intensity	100 cps
Mass range window	150 to 800 m/z
Isotopic pattern intensity tolerance	20%
Isotopic pattern m/z tolerance	25 ppm
MS/MS parameters
m/z tolerance	50 ppm
Min MS/MS peak intensity	50 cps
Characteristic product ions	Find at least 1
Neutral loss	Find at least 1

3. RESULTS

3.1. Metabolic Stability Assessment

ADB-FUBINACA had a 39.7 ± 0.1 min half-life (T_1/2) and an in vitro microsomal intrinsic clearance (CL_{int micr}) of 17.5 ± 0.1 µL/min/mg in HLM incubations. Intrinsic (CL_int) and hepatic (CL_hep) clearances were estimated at 16.5 and 9.0 mL/min/kg, respectively, with a 0.5 extraction ratio (ER).

3.2. Metabolite Profiling

ADB-FUBINACA peak area was 7.8 x 10⁶, 4.8 x 10⁶, and 2.3 x 10⁶ respectively after 0, 1, and 3 h incubation with human hepatocytes. Parent MS/MS spectrum (m/z 383.1878, retention time: 6.58 min) presented major characteristic fragments produced by aminodimethylbutanamide loss (m/z 253.0772), carboxamide loss (m/z 338.1663) or fluorobenzylium ion formation (m/z 109.0448) (Fig. 2). Two minor fragments also were formed by loss of dimethylbutanamide (m/z 270.1037) and amine (m/z 366.1612). ADB-FUBINACA fragments m/z 109, 253, and 338 were previously observed by Uchiyama et al. and Takayama et al. by LC-ESI-MS/MS (QTOF and triple quadrupole, respectively) [8, 26].

Fig. (2).

ADB-FUBINACA and major metabolites’ MS/MS spectrum and assigned fragmentation patterns. Metabolites are presented in ascending retention time order.

Twenty-one and twenty-two ADB-FUBINACA metabolites were identified in hepatocyte samples after 1 and 3 h incubation, respectively, totaling twenty-three different metabolites (labeled M1 to M23 in ascending retention time (RT) order)

Accurate mass for diagnostic product ions are given in Fig. (2) and S1 ^{(5.3MB, pdf)} (Supplementary material). Peak area for ADB-FUBINACA at 0 h was 7.8 x 10⁶. Metabolites are listed by ascending RT and ranked depending on signal intensity after 1 and 3 h incubation. MS, mass spectrometry; ND, not detected; RT: retention time; *, no MS/MS.

(Fig. 3). Biotransformations, retention times, accurate mass molecular ion, nominal mass product ions, and MS peak areas for parent and metabolites are listed in Table 2. MS/MS spectra and fragmentation patterns are reported in Fig. (2) (major metabolites) and S1 ^{(5.3MB, pdf)} (Supplementary material). Accurate mass errors were within 3.6 ppm. The main biotransformations included hydroxylation at the dimethylpropane chain (M1, M11, M15 to M17, M19, and M21) or the indazole ring (M1, M4, M11, M13, and M14), amide hydrolysis (M9, M18, M19, and M21 to M23), subsequent corresponding glucuronide conjugations (M1, M2, M4 to M6, M10, M12, and M18), and dehydrogenation (M15, M21, and M23). Other reactions included epoxidation followed by epoxide hydrolysis (M7), methylene-fluorophenyl hydroxylation then glucuronidation (M8), and N-dealkylations (M2, M3, M9, and M13). All metabolites were absent from the control 0 h incubation. No metabolite was the result of a non-enzymatic transformation as none was detected after 3 h incubation without hepatocytes. Structure elucidation of the top ten metabolites is described below.

Fig. (3).

Combined extracted ion chromatogram of ADB-FUBINACA and metabolites obtained from hepatocyte incubation after 3 h. ADB-FUBINACA metabolites are numbered M1 to M23 in ascending order of retention time.

Table 2.

Elemental composition, accurate mass molecular ion, nominal mass for diagnostic product ions, MS peak areas, and RT in hepatocyte incubations.

ID	Biotransformation	[M+H]+	RT (min)	Elemental Composition	Mass Error (ppm)	Diagnostic Product Ions (m/z)	Incubation (1 h)		Incubation (3 h)
							Peak Area	Rank	Peak Area	Rank
ADB-FUBINACA		383.1890	6.58	C21H23N4O2F	3.3	109, 253, 270, 338, 366	4.8 x 106		2.3 x 106
M1	Aliphatic hydroxylation + indazole dihydroxylidation + glucuronidation	607.2061	2.91	C27H31N4O11F	2.5	109, 285, 461, 590	N.D.		7.9 x 103	21
M2	N-Dealkylation + indazole hydroxylidation + glucuronidation	462.1314	3.53	C21H20N3O8F	1.5	109, 269, 286, 445	2.3 x 104	12	4.9 x 104	16
M3	Methylenefluorophenyl loss	275.1509	3.65	C14H18N4O2	2.5	145, 162, 230, 258	8.4 x 104	8	1.3 x 105	13
M4	Indazole dihydroxylation + glucuronidation	591.2107	3.72	C27H31N4O10F	1.8	109, 267, 285, 370, 461, 546, 574	5.7 x 104	10	2.3 x 105	8
M5	Indazole hydroxylation + glucuronidation	575.2160	3.86	C27H31N4O9F	2.1	109, 269, 354, 445, 530, 558	1.6 x 104	15	1.3 x 105	12
M6	Indazole hydroxylation + glucuronidation	575.2159	3.93	C27H31N4O9F	2.0	109, 269, 354, 445, 530, 558	1.2 x 105	6	2.3 x 105	9
M7	Dihydrodiol formation	417.1946	3.95	C21H25N4O4F	3.2	109, 241, 287, 372, 400	9.3 x 104	7	2.6 x 105	6
M8	Methylbenzene hydroxylation + glucuronidation	575.2160	4.17	C27H31N4O9F	2.1	125, 145, 269, 354, 530, 558	8.4 x 104	9	1.7 x 105	11
M9	Methylenefluorophenyl loss + amide hydrolysis	276.1350	4.42	C14H17N3O3	2.5	145, 162, 230	N.D.		7.9 x 103	22
M10	Indazole hydroxylation + glucuronidation	575.2156	4.44	C27H31N4O9F	1.4	109, 269, 354, 445, 530, 558	2.0 x 104	14	5.2 x 104	15
M11	Aliphatic hydroxylation + indazole hydroxylation	415.1791	4.48	C21H23N4O4F	3.6	109, 269, 340, 398	7.4 x 103	19	2.0 x 104	20
M12	N-hydroxylation + glucuronidation	575.2154	4.69	C27H31N4O9F	1.0	109, 253, 324, 338, 382, 558	2.1 x 104	13	8.4 x 104	14
M13	N-Dealkylation + indazole hydroxylation	286.0986	4.76	C15H12N3O2F	0.0	109, 269	6.1 x 103	20	N.D.
M14	Indazole hydroxylation	399.1836	5.45	C21H23N4O3F	2.2	109, 269, 286, 354	1.7 x 105	5	3.4 x 105	5
M15	Aliphatic hydroxylation + dehydrogenation	397.1678	5.60	C21H21N4O3F	1.9	109, 253, 270, 324, 361	4.5 x 105	2	8.5 x 105	2
M16	Aliphatic hydroxylation	399.1838	5.63	C21H23N4O3F	2.9	109, 253, 324, 382	1.6 x 106	1	1.7 x 106	1
M17	Aliphatic carboxylation	413.1635	5.65	C21H21N4O4F	3.7	109, 253, 324, 368, 396	1.1 x 104	18	4.2 x 104	17
M18	Amide hydrolysis + glucuronidation	560.2041	5.92	C27H30N3O9F	0.3	109, 253, 270, 338, 366, 384	1.5 x 104	16	3.2 x 104	19
M19	Amide hydrolysis + aliphatic hydroxylation	400.1677	6.06	C21H22N3O4F	2.5	109, 253, 324	1.5 x 104	17	2.6 x 105	7
M20	Not identified (+O, -4H)	395.1525	6.48	C21H19N4O3F	2.9	109, 253	5.4 x 104	11	2.0 x 105	10
M21	Amide hydrolysis + aliphatic hydroxylation + dehydrogenation	398.1513	6.75	C21H20N3O4F	0.7	109, 253, 324, 352	3.1 x 103*	21	3.3 x 104	18
M22	Amide hydrolysis	384.1726	7.17	C21H22N3O3F	2.1	109, 253, 338	1.8 x 105	4	3.7 x 105	4
M23	Amide hydrolysis + dehydrogenation	382.1572	7.43	C21H20N3O3F	2.7	109, 253, 324	1.8 x 105	3	6.6 x 105	3

3.2.1. Hydroxylated Metabolites

M14 and M16 (m/z 399.1827, 5.45 and 5.63 min respectively) were hydroxyl metabolites of ADB-FUBINACA as suggested by the 15.9949 Da increase from parent mass (+O).

M14 MS/MS spectrum included the fragments m/z 269.0721, 286.0986, 354.1612, and 382.1561 that were also 15.9949 Da larger and also included the fluorobenzylium ion (m/z 109.0448), indicating that the hydroxylation occurred on the benzene part of the indazole ring. It is noteworthy that no water loss was detected during M14 fragmentation as the delocalized electrons of the benzene ring strengthened the hydroxyl bond. M6 (m/z 575.2148, 3.93 min) likely is the corresponding glucuronide of M14. M4 (m/z 591.2097, 3.72 min) was formed by further hydroxylation of ADB-FUBINA hydroxyindazole-glucuronide on the benzene ring.

M16 MS/MS spectrum included fragments m/z 354.1612 and 382.1561 that were 15.9949 Da larger than those of the parent. Fragments m/z 109.0448 and 253.0772, also present in the parent MS/MS spectrum, further suggested that M16 hydroxylation occurred on the dimethylpropane chain. Fragment m/z 324.1507 was produced by a carboxamide and CHOH loss, perhaps the result of a hydroxylation on a methyl group of the dimethylpropane chain. Further hydroxylation of M16 generated M17 (m/z 413.1619, 5.65 min).

3.2.2. Metabolites Generated by Amide Hydrolysis

Metabolites generated by amide hydrolysis eluted later than non-hydrolyzed metabolites, as observed with other SCs with the same terminal carboxamide group [37].

M22 (m/z 384.1718, 7.17 min) was the result of amide hydrolysis as suggested by a 0.9840 Da increase (-NH₂, +OH). The MS/MS spectrum showed fragments m/z 109.0448, 253.0772, 338.1663, and 366.1612, also present in parent spectrum, demonstrating that ADB-FUBINACA was not transformed except amide hydrolysis on the terminal amine function. Similarly, M19 (m/z 400.1667, 6.06 min) presented a 0.9840 Da increase from M16 mass (ADB-FUBINACA hydroxydimethylpropyl) and the same fragmentation pattern, indicating that both amide hydrolysis and dimethylpropane hydroxylation occurred.

3.2.3. Dehydrogenated Metabolites

M23 (m/z 382.1561, 5.92 min) was formed by amide hydrolysis and dehydrogenation as suggested by a 1.0317 Da decrease (-NH₂, +OH, -H₂). Given the late RT, M23 could not possibly be an artifact derived from a water loss of a hydroxylated metabolite. MS/MS spectrum presented fragments m/z 109.0448 and 253.0772, indicating that the dehydrogenation occurred on the aminodimethylbutanamide portion of ABD-FUBINACA, leading to an imine formation. Fragment m/z 324.1507 was produced by the loss of the carboxylic acid and one methyl group (– 58.0054 Da).

M15 (m/z 397.1670, 5.60 min) was formed by ADB-FUBINACA hydroxylation and dehydrogenation as suggested by the 13.9792 Da decrease (+O, -H₂). Fragments m/z 109.0448, 253.0772, 270.1037, and 324.1507 indicate imine formation and methyl hydroxylation. Fragment m/z 379.1565 confirmed hydroxylation (water loss).

M20 (m/z 395.1514, 6.48 min) was generated by hydroxylation and a di-dehydrogenation, as suggested by the 11.9636 Da increase (+O, -2H₂). Fragments m/z 109.0448 and 253.0772, also detected in ADB-FUBINACA’s mass spectrum, indicated that reactions occurred on the amino-dimethylbutanamide moiety. Fragment m/z 324.1507 indicated dehydrogenation to the imine function.

3.2.4. Dealkylated Metabolites

M13 (m/z 286.0986, 4.76 min) was formed by dimethylbutanamide cleavage and a hydroxylation, as suggested by the 97.0892 Da decrease (+O, -C₆H₁₁NO). Few fragments were produced as M13 signal intensity was low in the Full-MS scan after 1 h incubation and not detectable after 3 h incubation. Fragment m/z 109.0448 corresponds to unchanged methylenefluorophenyl and indicated that the reaction occurred on the indazole ring benzene group. Fragment m/z 269.0720 was formed by amine loss.

3.2.5. Epoxidized Metabolite

M7 (m/z 417.1933, 3.95 min) was a dihydrodiol, as suggested by a 34.0055 Da increase (+2O, +2H). Fragments m/z 287.0826, 304.1092, 372.1718, and 400.1667, were 34.0055 Da larger than the corresponding ADB-FUBINACA fragments while fragment m/z 109.0448 was still present, indicating that the reactions did only affect the indazole ring. We hypothesized that M7 was formed by epoxidation of the benzene moiety of ADB-FUBINACA’s indazole ring, followed by hydrolysis of the newly formed epoxide. This metabolic reaction was previously observed in several SCs containing indole or indazole ring metabolic pathways [33, 37].

4. DISCUSSION

4.1. Metabolic Stability Assessment

According to McNaney’s [36] and Lavé’s [38] classifications based on intrinsic clearance and extraction ratio estimations, respectively, ADB-FUBINCA is considered an intermediate-clearance drug (15<CL_int<40 and 0.3<ER<0.7). As a result, ADB-FUBINCA metabolites are expected to be found in urine several days after intake. First-pass hepatic elimination is expected to be significant, and hepatic clearance sensitive to changes in plasma protein binding, drug metabolism, and hepatic blood flow [39]. However, the extent of plasma protein binding can play an important role in a drug’s phar-macokinetics. If ADB-FUBINACA was a highly protein-bound drug, this would lower the hepatic clearance and extend the detection window.

4.2. Metabolic Profiling

4.2.1. Method

HRMS is a powerful tool in metabolite identification studies as it theoretically allows capturing every compound in a single injection, and facilitates molecular formula determination of metabolites and fragments. Nitrogen atoms of the indazole core and the two amide functions are easily charged in acidic conditions, making positive-ion mode ionization suitable for ADB-FUBINACA metabolites’ detection. A 50 ppm MS mass tolerance was chosen during preliminary automated metabolite identification as it is broad enough that no possible metabolite with a high mass error could be missed, but narrow enough to predict elemental composition. MS/MS IDA mode scan speed allowed monitoring eight compounds at each MS cycle with a ramped collision energy fragmentation to maximize the production of different fragments and facilitate identification.

Samples were prepared by simple dilution as opposed to extraction before injection to increase detection of potential metabolites. However, matrix suppression could impede detection of metabolites with low signal intensity. Similarly, relative metabolite rank based on MS peak intensity could be confounded by matrix effect and ionization efficiency.

4.2.2. Metabolite Identification

The suggested ADB-FUBINACA metabolic pathway is described in Fig. (4). M16 (ADB-FUBINACA hydroxy-alkyl), M15 (ADB-FUBINACA hydroxydehydroalkyl) and M14 (ADB-FUBINACA hydroxylindazole) are the three metabolites recommended as ADB-FUBINACA intake markers. Several glucuronidated metabolites (M1, M2, M4 to M6, M8, M10, M12, and M18) suggest that hydrolysis of biological samples (e.g. urine, blood) prior to extraction could increase non-glucuronidated metabolites’ concentrations and facilitate their detection.

Fig. (4).

ADB-FUBINACA suggested metabolic pathway. M20 accurate mass and fragmentation pattern suggest the loss of four hydrogen atoms and the addition of an oxygen on ADB-FUBINACA dimethylbutanamide moiety but its structure was not fully elucidated. Bold arrow, major pathway; gluc, glucuronide.

Major metabolic pathways of AB-FUBINACA, ADB-FUBINACA’s analog (Fig. 1), in hepatocyte incubations included amide hydrolysis and alkyl and indazole hydroxylations [37]. Alkyl dehydrogenations, dihydrodiol formations, and glucuronidations also were observed to a lesser extent. ADB-FUBINACA metabolism in the present experiments was consistent with these results. ADB-FUBINACA N-dealkylated metabolites formed by dimethylbutanamide cleavage (M2 and M13) also were detected in AB-FUBINACA metabolism [37]. The same two metabolites could theoretically be formed after MDMB-FUBINACA metabolism as it presents the same indazole- methylenefluorophenyl structure. Similarly, metabolites formed by N-dealkylation (M3 and M9) could theoretically be formed after MAB-CHMINACA, ADB-CHMINACA, ADB-PINACA and 5-F-ADB-PINACA metabolism, as they present the same indazole-dimethylbutanamide structure. Metabolites generated by amide hydrolysis, M21, M22 and M23, could theoretically be formed after MDMB-FUBINACA O-demethylation. However, MDMB-FUBINACA, MAB-CHMINACA, ADB-CHMINACA, ADB-PINACA and 5-F-ADB-PINACA metabolism has yet to be elucidated. Given the constant emergence of new substances and depending on the legislation, being able to precisely distinguish one synthetic cannabinoid from another is critical. As M2, M3, M9, M13, M21, M22 and M23 may not be specific for ADB-FUBINACA intake, they are not recommended as ADB-FUBINACA consumption markers. Noteworthy, AB-FUBINACA carboxylic acid, the corresponding M22 ADB-FUBINACA metabolite, is specific for AB-FUBINACA metabolism, as there are no other compounds known that can produce it [37].

This in vitro hepatocyte marker profile should be confirmed with human urine samples after ADB-FUBINACA intake, as post-liver metabolism processes also can occur (e.g. re-absorption, extrahepatic metabolism, enterohepatic circulation). Unfortunately, authentic urine or blood specimens following ADB-FUBINACA consumption were not available despite our efforts to obtain them. It is important to verify in vitro hepatocyte metabolism results with human urine samples, although we have observed good correlation with authentic urine specimens and hepatocyte incubations for 10 µM FDU-PB-22, FUB-PB-22 [40], AB-PINACA [32] and AB-FUBINACA [37]. Castaneto et al. observed good agreement between metabolites identified during AB-FUBINACA hepatocyte incubations and authentic urine specimens following AB-FUBINACA intake, including the most intense signals for AB-FUBINACA carboxylic acid, AB-FUBINACA carboxylic acid-glucuronide, and AB-FUBINACA carboxylic acid-hydroxyalkyl metabolites [37].

CONCLUSION

The first ADB-FUBINACA metabolic profile is presented. ADB-FUBINACA pharmacokinetics were determined in human liver microsomes: ADB-FUBINACA was predicted as an intermediate-clearance compound with a 37.9 min microsomal half-life. ADB-FUBINACA metabolism was studied following human hepatocyte incubations: 23 meta-bolites were detected; M16 (ADB-FUBINACA hydroxyalkyl), M15 (ADB-FUBINACA hydroxydehydroalkyl) and M14 (ADB-FUBINACA hydroxylindazole) were detected with the most intense MS signals and are suggested as ADB-FUBINACA markers. Urine hydrolysis should be performed to improve detection capability.

Experiments were conducted to improve ADB-FUBINACA metabolite identification in human matrices for forensic or clinical cases. Identification of major metabolite markers is critical to guide synthesis efforts of manufacturers to provide suitable analytical standards and for further pharmacodynamic and pharmacokinetic studies. In the past, HLM and hepatocyte incubation approaches proved useful to predict human metabolism [27-33]. However, in vivo data are necessary to confirm results.

SUPPLEMENTARY MATERIAL

Supplementary material is available on the publisher’s web site along with the published article.

CONFLICT OF INTEREST

The authors confirm that they do not have any conflicts of interest with this article contents.