Identification of New Synthetic Cannabinoid ADB-CHMINACA (MAB-CHMINACA) Metabolites in Human Hepatocytes
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ADB-CHMINACA (MAB-CHMINACA) is a new synthetic cannabinoid with high potency and many reported adverse events and fatalities. The drug is currently scheduled in several countries in Europe and the USA. Analytical methods need to be developed to confirm ADB-CHMINACA intake for clinical and forensic programs. For many synthetic cannabinoids, parent compound is not detectable in biological samples after intake, making the detection of metabolites the only way to prove consumption. Therefore, detection of ADB-CHMINACA metabolites in biological specimens is critical. Since there are currently no published data on ADB-CHMINACA metabolism, we aimed to identify its major metabolites. Cryopreserved human hepatocytes were incubated with 10 μmol/L ADB-CHMINACA for 3 h. Incubations were analyzed with liquid chromatography on a biphenyl column, high resolution tandem mass spectrometry (orbitrap), and metabolite identification software. A reference standard of six commercially available potential metabolites was simultaneously analyzed under the same conditions to allow correct assignment of isomers. We detected ten major metabolites. Biotransformations mainly occurred at the cyclohexylmethyl tail of the compound, as also observed with structural analogs’ metabolism. Minor reactions also occurred at the tert-butyl chain. Only two analytical standards of potential metabolites matched an actual metabolite detected in hepatocyte incubations. We recommend A9 (ADB-CHMINACA hydroxycyclohexylmethyl), A4 (ADB-CHMINACA 4″-hydroxycyclohexyl), and A6 (ADB-CHMINACA hydroxycyclohexylmethyl) as metabolite targets to document ADB-CHMINACA intake in clinical and forensic cases. Additionally, these results will guide analytical standard manufacturers to better provide suitable references for further studies on ADB-CHMINACA metabolism.
KEY WORDSADB-CHMINACA hepatocyte metabolism high resolution mass spectrometry MAB-CHMINACA synthetic cannabinoid
Synthetic cannabinoids (SC) are novel psychoactive substances (NPS) producing their effects via the endocannabinoid system, as central (CB1) and peripheral (CB2) cannabinoid receptor agonists (1). SC potency and binding affinity for CB1 and CB2 are often much higher than classic cannabinoids, such as Δ9-tetrahydrocannabinol (THC), the main psychoactive constituent of cannabis (1, 2, 3, 4). Consequently, SC are actively abused since 2008 as “legal highs” (5). Many countries scheduled SC as illicit substances, but clandestine laboratories continue to produce new molecules to circumvent the law. In Japan, where the scheduling process is the fastest, 858 SC were controlled by April 2015 (6). As the occurrence of consumption and the diversity of SC increased, SC intake is associated with considerable morbidity and mortality (7, 8, 9, 10).
Adverse effects associated with ADB-CHMINACA intake were observed in four patients who smoked a white powder labeled “AM-2201.” Observed effects included vomiting, seizures, limb twisting, muscle tremors, aggression, slurred speech, pressure spikes, wheezing, respiratory failure, and losses of consciousness. Blood concentrations ranged from 1.3 to 14.6 μg/L between 1 and 2 h after intake (13). In 2014, ADB-CHMINACA was involved in a fatal case of SC intoxication. Herbal blends that were ingested contained 133 ± 4.5 mg/g ADB-CHMINACA and 49.2 ± 2.5 mg/g 5F-ADBICA, another SC (12,14). Postmortem femoral vein blood, right heart blood, and left heart blood concentrations of ADB-CHMINACA were 6.1 ± 0.2, 10.6 ± 0.7, and 9.3 ± 0.3 μg/L, respectively (15). Multiple outbreaks of severe intoxications associated with ADB-CHMINACA intake were also reported in the USA (Kansas, Louisiana, Mississippi, Maryland, Texas, and Virginia), including hundreds of hospitalizations and at least three fatalities (7,16). Because of increasing prevalence and safety concerns, ADB-CHMINCA was controlled in Japan in 2014 (17), in Singapore in 2015 (18), and in several European countries. The drug was placed into schedule I in the USA in late 2015 (16).
Clinical evaluation of ADB-CHMINACA intake is not possible, due to lack of toxicology data, making analytical toxicology the most efficient way to prove consumption. Urine is the most common matrix for drug testing because of the longer window of detection compared to blood and oral fluid, a higher concentration of metabolites for several days after intake, and a generally larger sample volume. However, SC are highly metabolized and detection of parent SC in urine is rare (19, 20, 21, 22, 23). This is the case for AB-CHMINACA, desmethyl analog of ADB-CHMINACA (Fig. 1), for which 15 different metabolites were detected in a urine specimen from an authentic case of abuse, with no detectable parent compound (24). This is also the case of MDMB-CHMICA, another ADB-CHMINACA analog with an indole core and a terminal methyl ester instead of a carboxamide (Fig. 1) (25). In a fatal case of intoxication involving ADB-CHMINACA, the drug was quantified in 14 different postmortem body fluids and tissues (6.1 to 156 μg/L or g) but was not detectable in urine (15). Under these conditions, detection of ADB-CHMINACA urinary metabolites may be critical to document intake. To date, nothing is known about ADB-CHMINACA metabolism, although several theoretical metabolite standards are commercially available (most likely based on in silico predictions or extrapolation from analog metabolism). Therefore, we aimed to investigate human ADB-CHMINACA metabolism to identify specific urinary markers of intake.
Incubation with human liver microsomes (HLM) is the most common in vitro model for metabolite profiling due to low cost, availability, and simplicity of use. However, they may not predict in vivo metabolites or the relative abundance of metabolites, as observed with 5F-AKB48 (26) and AM-2201 (27,28). Human hepatocyte profiles generally match better with authentic urine SC metabolites because they are functioning complete cells and contain comprehensive phase I and II metabolic enzymes and cofactors, uptake and efflux transporters, and drug binding proteins (29, 30, 31, 32). Therefore, we incubated ADB-CHMINACA with human hepatocytes and identified metabolites with liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS), following our previously recommended workflow (33). Additionally, we compared our results to commercially available reference standards of theoretical ADB-CHMINACA metabolites.
Chemicals and Reagents
Incubation with Hepatocytes
Hepatocytes were thawed at 37°C and gently rinsed twice with CP Medium and once with KHB, then centrifuged at room temperature, 100×g, for 5 min. The pellet was solubilized in 2 mL KHB. Cell viability was measured with trypan blue exclusion dye to allow adjusting the buffer volume to a 2 × 106 viable cells/mL concentration. Two hundred fifty microliters suspension was gently mixed with 20 μmol/L ADB-CHMINACA in KHB with 0.7% methanol (10 μmol/L final concentration) and incubated for 0 and 3 h at 37°C in a Forma™ Steri-Cycle™ CO2 incubator (Thermo Scientific; Fremont, CA, USA). Metabolic reactions were quenched with 500 μL acetonitrile. Samples were centrifuged at 4°C, 15,000×g, for 5 min, and stored at −80°C until analysis. A control sample with diclofenac was incubated under the same conditions and 4′-hydroxydiclofenac and acyl-β-D-glucuronide diclofenac were monitored to ensure hepatocyte metabolic activity. Simultaneously, a negative control with ADB-CHMINACA standard without hepatocytes was incubated for 3 h to control for interferences and non-enzymatic reactions.
Samples were thawed at room temperature, vortex mixed, and centrifuged at 4°C, 15,000×g, for 5 min. One hundred microliter supernatants was transferred to disposable plastic microtubes with 100 μL acetonitrile, vortex mixed, and centrifuged at 4°C, 15,000×g, for 5 min. Supernatants were transferred to conical glass tubes and evaporated to dryness under nitrogen at 40°C in a TurboVap® LV evaporator (Zymark; Hopkinton, MA, USA). Residues were reconstituted with 150 μL mobile phase A/B 80:20 (v/v), centrifuged at 4°C, 15,000×g for 5 min, and supernatants transferred into autosampler vials with glass inserts. Fifteen microliters was injected onto the chromatographic system.
Ten microliters ADB-CHMINACA M1, M2, M3, M7, M10, and M11 standards was vortex mixed with 990 μL mobile phase A/B 80:20 (v/v) in individual glass autosampler vials. Fifteen microliters was injected onto the chromatographic system.
LC-MS analysis was performed on a Q Exactive™ Plus mass spectrometer equipped with a heated electrospray in positive-ion mode (Thermo Scientific) and coupled with an Ultimate™ 3000 LC system (Dionex; Sunnyvale, CA, USA). Data were acquired with Xcalibur software (v. 3.0.63, Thermo Scientific).
Separation was achieved on a 100 × 2.1 mm, 3 μm, Ultra Biphenyl column (Restek®; Bellefonte, PA, USA) and identically packed guard cartridge (10 × 2.1 mm). Elution was performed at 30°C with 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) at a 0.5 mL/min flow rate. Initial conditions (20% B) were maintained for 0.5 min, then %B was increased to 95% within 10.5 min and held for 2 min; the column was re-equilibrated within 2 min (15 min run time).
Inclusion and Neutral Losses Lists for the Full-Scan Mass Spectrometry/Data-Dependent Tandem Mass Spectrometry (FullMS/ddMS2) and Full-Scan Mass Spectrometry/All-Ion Fragmentation/Data-Dependent Tandem Mass Spectrometry (FullMS/AIF/ddMS2) Acquisitions, Respectively, Used During High Resolution Mass Spectrometry Analysis of Hepatocytes Incubations. Bold Indicates Parent Compound
Neutral loss (m/z)
Neutral loss (m/z)
Neutral loss (m/z)
Compound Discoverer™ Processing Settings for Identifying ADB-CHMINACA Metabolites
Phase I expected transformations
Dihydrodiol formation (+2O +2H)
Oxidative deamination to alcohol (−N +O –H)
Oxidative deamination to ketone (−N +O –3H)
Phase II expected transformations
Acetylation (+2C +O +2H)
Glucuronidation (+6C +6O +10H)
Methylation (+C +2H)
Sulfation (+S +3O)
Maximum number of dealkylation steps on the same compound
Maximum number of phase II reactions on the same compound
Maximum number of reactions (phase I and II) on the same compound
RESULTS AND DISCUSSION
ADB-CHMINACA Metabolites in Human Hepatocytes
Accurate Mass Molecular Ion, Retention Time (RT), Elemental Composition, Nominal Mass for Diagnostic Product Ions, Mass Spectrometry Peak Areas (Extracted Ion Chromatogram), and ADB-CHMINACA Metabolites Rank Based on the Peak Area in Hepatocytes Incubations
Mass error (ppm)
Diagnostic product ions (m/z)a
Peak area at T3h
145, 241, 259, 326, 354
3.0 × 105
93, 255, 291, 358, 386
3.5 × 103
95, 145, 163, 257, 386
8.9 × 103
145, 273, 291, 358, 386
3.7 × 104
145, 257, 275, 342, 370
1.6 × 105
95, 239, 257, 342, 370
5.0 × 104
95, 145, 275, 342, 370
7.6 × 104
Ketone formation (cyclohexylmethyl)
145, 255, 340, 368
2.6 × 104
145, 257, 275, 342, 370
7.7 × 103
145, 257, 275, 342, 370
2.3 × 105
145, 163, 241, 259, 370
1.5 × 104
Cyclohexylmethyl Hydroxylation and Further Ketolization
Hydroxylation (+O) occurred in A4, A5, A6, A8, and A9, as indicated by the +15.9948 Da mass shift from parent. The five MS/MS spectra displayed the same fragments with characteristic neutral loss of amine (m/z 370.2119), carboxamide (m/z 342.2172), dimethylbutanamide (m/z 275.1386), and aminodimethylbutanamide (m/z 257.1282) groups indicating that neither the carboxamide linker nor the dimethylbutanamide side chain carried the transformation; the reaction did not occur either at the indazole core, as indicated by fragments m/z 145.0395 and 163.0500 also observed in parent, suggesting that hydroxylation occurred at the cyclohexylmethyl tail. Fragment m/z 95.0855 produced by the hydroxylated cyclohexylmethylium ion followed by water loss confirmed the cyclohexylmethyl group as the site of reaction. Interestingly, fragments m/z 239.1175 (water loss from m/z 257.1282), 257.1282, and 275.1386 presented with different intensity ratios in A4, A5, A6, A8, and A9: m/z 239.1175 signal was high in A5 but low or absent from other metabolites; m/z 257.1282 had the most intense signal in A4 and A9, while m/z 275.1386 was dominating in A6 and A8. As previously mentioned, cyclohexylmethyl hydroxylation was expected, as it is a major metabolic reaction of structural analogs (24,25,35). However, the MS/MS spectra do not allow accurate location of the hydroxyl group on the tail. The transformation may be generated by cytochrome P450 3A4 (CYP3) as it was identified as the main metabolic enzyme responsible for cyclohexylmethyl hydroxylation in AB-CHMINACA in vitro metabolism (24).
A7 was a minor metabolite produced by oxidation or hydroxylation followed by dehydrogenation, resulting in a ketone formation (+O –2H). Neutral loss of amine (m/z 368.1963), carboxamide (m/z 340.2023), and aminodimethylbutanamide (m/z 255.1125) groups and indazole fragments m/z 145.0396 and 163.0504 indicated a ketolization at the cyclohexylmethyl tail. Cyclohexylmethyl ketolization was previously reported in MDMB-CHMICA in vitro and in vivo metabolism with a low signal intensity (25). It was not detected in AB-CHMINACA metabolism (24).
A10 also was formed by hydroxylation (+O), as suggested by a +15.9952 Da mass shift from ADB-CHMINACA. However, A10 shared most of its fragments with parent such as m/z 97.1015 (cyclohexylmethyl tail), 145.0394 and 163.0498 (indazole core), and 241.1331 and 259.1443 (cyclohexylmethyl + indazole + carboxamide linker) designating the tert-butyl group of the side chain as the site of attack. It is noteworthy that the carboxamide loss was followed by subsequent hydroxymethyl loss (m/z 312.2064) and did not appear in A10 spectrum, as observed in the fragmentation of ADB-FUBINACA (36), ADB-PINACA, and 5F-ADB-PINACA (34) metabolites with the same transformation. tert-Butyl hydroxylation also occurred in MDMB-CHMICA (25,35) and AB-CHMINACA (24) metabolism, in which CYP3A3 was identified as the main enzyme responsible for the reaction (24).
Second generation metabolites A1, A2, and A3 were produced by dihydroxylation (+2O), as indicated by a 31.9900-Da mass shift from parent. A1 and A3 followed a similar fragmentation pattern to A4, A5, A6, A8, and A9, yielding fragments by neutral loss of amine (m/z 386.2072), carboxamide (m/z 358.2115), dimethylbutanamide (m/z 291.1330), and aminodimethylbutanamide (m/z 273.1228) groups and characteristic fragments from the indazole core (m/z 145.0394 and 163.0500). This pattern suggested a dihydroxylation at the cyclohexylmethyl tail. Fragment m/z 93.0698 generated by dihydroxylation of the cyclohexylmethylium ion followed by two water losses further designated the tail as the site of attack. The signal of the aminodimethylbutanamide loss dominated in the A3 spectrum, as observed in major metabolites A4 and A9. However, this fragmentation was followed by two successive water losses (m/z 255.1122 and 237.1010) in A1.
A2 was dihydroxylated at the cyclohexylmethyl tail and at the tert-butyl group of the dimethylbutanamide side chain. As observed in A4, A5, A6, A8, and A9 MS/MS spectra, fragments m/z 95.0855, 145.0396, 163.0501, 239.1183, 257.1283, and 275.1385 indicated a hydroxycyclohexylmethyl metabolite. As shown for A10, amine (m/z 386.2069) and carboxamide (m/z 368.1961) neutral losses and subsequent hydroxymethyl loss (m/z 328.2008) indicated that the second hydroxylation occurred at the tert-butyl chain.
Comparison to Reference Standards
Optimal Targets for ADB-CHMINACA Intake
We recommend major metabolites A4 (ADB-CHMINACA 4″-hydroxycyclohexyl), A6 (ADB-CHMINACA hydroxycyclohexylmethyl), and A9 (ADB-CHMINACA hydroxycyclohexylmethyl) as biological markers for ADB-CHMINACA consumption. All three compounds are the result of a simple hydroxylation and carry ADB-CHMINACA indazole core substituted with a cyclohexylmethyl tail and a dimethylbutanamide side chain. Consequently, all three compounds specifically identify ADB-CHMINACA intake and none were reported as a metabolite from closely related analogs. MS/MS analysis does not allow precise location of A6 and A9 hydroxylation and requires comparison with reference standards that are not commercially available. These data inform manufacturers on their synthesis efforts to provide suitable standards. Currently, A4 is the most convenient target for ADB-CHMINACA intake, as its standard is available for purchase. Remarkably, although it is recommended for synthetic cannabinoids analysis (25,37, 38, 39, 40, 41), for ADB-CHMINACA identification, it is not necessary to hydrolyze urine samples as no phase II ADB-CHMINACA metabolite was detected.
In previous experiments involving human metabolism, hepatocyte incubations appeared successful to predict in vivo urinary metabolites (29, 30, 31, 32,42,43). Ideally, a confirmation of our results with urine specimens from authentic cases of ADB-CHMINACA intake would be advantageous (33); however, despite our efforts, we were unable to obtain such specimens. It is noteworthy that structural analogs AB-CHMINACA (24) and MDMB-CHMICA (25,35) in vivo metabolism corroborates our results: the most intense urinary metabolites are mono-hydroxylated at the cyclohexylmethyl tail, as observed in ADB-CHMINACA hepatocyte incubations.
For the first time, we determined ADB-CHMINACA (MAB-CHMINACA) human metabolism using hepatocyte incubations and LC-HRMS analysis with metabolite identification data-mining software. We detected 10 different phase I metabolites, most of the transformations occurring at the cyclohexylmethyl tail of the compound. We suggest A9 (ADB-CHMINACA hydroxycyclohexylmethyl), A4 (ADB-CHMINACA 4″-hydroxycyclohexyl), and A6 (ADB-CHMINACA hydroxycyclohexylmethyl) as targets for documenting ADB-CHMINACA intake in clinical and forensic specimens. These data also will provide support for analytical standard manufacturers to produce suitable metabolites for further pharmacodynamics and pharmacokinetic studies.
The authors would like to thank Tim Moeller from BioreclamationIVT for his assistance with the incubations, and Caroline Ding, Helen Sun, and Thermo Scientific for providing LC-HRMS instrumentation, Compound Discoverer® software and training via a National Institute on Drug Abuse Materials Transfer Agreement. This research was supported by the Intramural Research Program of the National Institute on Drug Abuse, National Institutes of Health.
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