Pharmacokinetic and toxicological evaluation of AB-CHMINACA in rodents: A comprehensive study

Abstract

Objective

This study aimed to develop and validate a sensitive analytical method for detecting and quantifying AB-CHMINACA, and to comprehensively evaluate its pharmacokinetics, metabolic pathways, and acute toxicity in rodent models.

Methodology

Male Wistar rats and Swiss albino mice were used for pharmacokinetics, metabolism, and acute toxicity studies. AB-CHMINACA was dissolved in Dimethyl sulfoxide (DMSO; 2%–5%) and diluted in corn oil for oral gavage. Blood and urine samples were collected from rats for pharmacokinetic and metabolite analyses using Gas Chromatography–Tandem Mass Spectrometry (GC-MS/MS) and liquid chromatography–quadrupole time-of-flight mass spectrometry, respectively. Mice were monitored for behavioral toxicity and necropsied for histopathological analysis. Pharmacokinetic parameters were calculated using WinNonlin Phoenix 6.0, and metabolites were identified through mass spectrometry.

Results

Method validation showed excellent linearity (0.5–1000 ng/mL, r² > .99), with precision (variability 2.3%–7.2%) and accuracy (95%–105%), adhering to FDA guidelines. The method’s limit of detection (LOD) and limit of quantification (LOQ) were 0.5 and 1.0 ng/mL, respectively. Pharmacokinetic analysis revealed a biphasic elimination (t½ 13.8 h), Vd of 228.75 L, and Cl of 11.4 L/h. Four major Phase I metabolites were identified, with Met 4 (Di-Hydroxy-AB-CHMINACA) being the most abundant in urine at 8–24 h. Acute toxicity in mice showed dose-dependent symptoms, including lethargy, seizures, and mortality at 50 mg/kg.

Conclusion

These results enhance the understanding of AB-CHMINACA's pharmacokinetic behavior and metabolic profile, contributing valuable insights to forensic toxicology and highlighting the dose-dependent nature of its acute toxicity.

Keywords

AB-CHMINACA pharmacokinetics metabolites LC-Q/TOF-MS acute toxicity forensic toxicology

Introduction

The increasing popularity of synthetic cannabinoids (SCs) globally presents significant public health challenges due to their rapidly evolving chemical structures.¹ SCs form the largest category of novel psychoactive substances (NPS) to emerge in the past decade and are often marketed as “legal” alternatives to cannabis.² These compounds interact with cannabinoid receptors CB1 and/or CB2 and were initially developed to explore the endogenous cannabinoid system or as potential pharmacological treatments.³

SC synthesis began in the mid-1960s, but recreational use did not gain traction until the early 2000s.⁴ Most of the abused SCs act as CB1 receptor agonists, exhibiting significantly higher binding affinity than delta-9-tetrahydrocannabinol (THC). As a result, they produce more intense cannabimimetic effects, including pronounced cognitive impairment, altered sensory perception, and transient hallucinations. Additionally, SC use has been linked to adverse physiological effects not commonly associated with cannabis, such as seizures, vomiting, hyperglycemia, hypokalemia, stroke, myocardial infarction, and acute kidney injury.⁵ While controlled SC administration studies would be valuable, conducting such research in humans is currently impractical due to the absence of sufficient preclinical toxicological data.^5–7

Reports from the American Association of Poison Control Centers⁸ show a rise in SC-related exposures, from 2096 in 2010 to 6968 in 2011, followed by a decline to 5230 in 2012 and further down to 2668 in 2013. The decrease in 2013 was likely due to the classification of SCs as Schedule I substances, increased public awareness, and better management by physicians. The cases rose again to 3677 in 2014. SCs are now regulated under the U.S. Controlled Substances Act,⁹ banned by WADA,¹⁰ and controlled in many countries.²

AB-CHMINACA, one of the potent SCs, has demonstrated efficacy far exceeding that of known full agonists of the CB1 receptor.¹¹ Initially developed by Pfizer for potential therapeutic application, its development was discontinued in 2013 due to adverse effects.¹² Studies indicate that AB-CHMINACA's potency is 11‒58 times greater than THC across various tests,¹ raising concerns over its potential for abuse and toxicity.

Despite the classification of SCs as Schedule I substances and growing public awareness of their risks, the toxic dose of SCs, including AB-CHMINACA, remains undetermined in humans. Animal studies have not yet provided sufficient data regarding their toxicity. With AB-CHMINACA's pharmacological profile largely unexplored, there is a pressing need for studies that evaluate its pharmacokinetics, metabolism, and toxicological effects.

This study seeks to address this knowledge gap by examining the pharmacokinetic behavior, half-life, volume of distribution, and clearance of AB-CHMINACA in rat blood samples using GC-MS/MS. The data generated will help elucidate the compound's pharmacological and toxicological properties, contributing valuable information for forensic toxicology and public health authorities.

Materials and methods

Chemicals and reagents

AB-CHMINACA (≥98% purity) was obtained from Cerilliant Corporation and Cayman Chemical (USA). HPLC-grade solvents—methanol, water, ammonium formate, and formic acid were purchased from Sigma-Aldrich. Dimethyl sulfoxide (DMSO) was sourced from Biotraxx (Cyprus), and corn oil was purchased from a local supplier (Saudi Arabia). Ultrapure water was prepared using a Milli-Q purification system (Millipore, France).

Instrumentation

GC-CI-MS/MS

Gas chromatography–chemical ionization tandem mass spectrometry (GC-CI-MS/MS) was performed on an Agilent 7890B GC system coupled to a 7000C triple quadrupole mass spectrometer, equipped with a 7693 autosampler and an HP-5MS capillary column (30 m × 0.25 mm, 0.25 µm film). A 2 µL injection was introduced in splitless mode. The oven temperature program was: initial 200 °C (2 min), ramped at 15 °C/min to a final temperature of 300 °C, held to complete a total run time of 12 min. Helium served as the carrier gas at 1 mL/min. The injector and transfer line were maintained at 280 °C. The system operated in multiple reaction monitoring (MRM) mode with ammonia as the reagent gas and argon at 1.5 mL/min as the collision gas.

LC-ESI-QTOF/MS

Liquid chromatography–electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS) was conducted using an Agilent 6530B QTOF system (Agilent Technologies, Santa Clara, CA, USA) with a TurboIonSpray source in positive mode (ESI+, 2500 V). Parameters were: curtain gas at 30 psi; ion source gases 1 and 2 at 60 psi; source temperature at 600 °C. The TOF scan range was 100–650 Da (acquisition time 0.07 s), and the MS/MS scan range was 25–650 Da (0.05 s). The declustering potential was 80 V; collision energy was set at 10 V.

Chromatographic separation was achieved using an Agilent ZORBAX Eclipse Plus C18 reverse-phase column (2.1 × 100 mm, 1.8 µm particle size), maintained at 30 °C with a flow rate of 0.7 mL/min. The autosampler was maintained at 5 °C. Mobile phase A was 10 mM ammonium formate in water, and mobile phase B was 0.1% formic acid in methanol. The gradient program: 10% B (0.30 min) → linear increase to 98% B (7.30 min), held until 8.80 min, then re-equilibrated to 10% B at 8.81 min; total runtime: 11.0 min.

Standard and quality control (QC) sample preparation

Stock solutions

A primary stock solution of AB-CHMINACA (1 mg/mL) was prepared in methanol. Serial dilutions yielded secondary working solutions of 100, 10, and 1 µg/mL, and 100 and 10 ng/mL. Granisetron was used as the internal standard (IS) at 10 µg/mL in methanol.

Calibration standards and QC samples

Blank human plasma was fortified with AB-CHMINACA and 50 µL of IS to prepare calibration standards at 0.5, 2, 10, 25, 50, 100, 500, and 1000 ng/mL. A blank with only IS was included. QC samples were also prepared in plasma at the following concentrations:

Low QC (LQC): 50 ng/mL

Medium QC (MQC): 250 ng/mL

High QC (HQC): 500 ng/mL

Each QC sample included 50  µL of IS. Details of calibration and QC preparations are provided in Table 1.

Table 1.

Preparation of calibration standards for drug quantification in plasma.

Calibration Level	Concentration (ng/mL)	Volume of drug solution	Volume of plasma	Volume of IS (10 µg/mL)
1	0.5	25 µL (10 ng/mL)	475 µL	50 µL
2	2	10 µL (100 ng/mL)	490 µL	50 µL
3	10	50 µL (100 ng/mL)	450 µL	50 µL
4	25	125 µL (100 ng/mL)	375 µL	50 µL
5	50	25 µL (1 µg/mL)	475 µL	50 µL
6	100	5 µL (10 µg/mL)	495 µL	50 µL
7	500	25 µL (10 µg/mL)	475 µL	50 µL
8	1000	50 µL (10 µg/mL)	450 µL	50 µL
9	Blank	—	500 µL	50 µL

Human plasma was selected as the blank matrix for method validation owing to its comparable protein composition, viscosity, and pH to rodent plasma, ensuring similar extraction efficiency and analyte recovery. This matrix is also widely standardized and readily available, facilitating reliable method reproducibility. The validated method was subsequently applied to rodent plasma samples to confirm matrix compatibility.

Animals and dosing

Subjects

Male Wistar rats (12 weeks, 150–160 g) and Swiss albino mice (6 weeks, 20–25 g) were used, with n = 6 per group. Animals were housed under controlled conditions (23 ± 2 °C, 50–60% relative humidity, 12 h light/dark cycle) with free access to food and water and acclimatized for 7 days.

Drug administration

AB-CHMINACA was initially dissolved in 2%–5% DMSO and further diluted in corn oil for oral gavage. Control animals received vehicle only (DMSO in corn oil).

Experimental design

The study evaluated the pharmacokinetics, metabolism, urinary excretion, and acute toxicity of AB-CHMINACA following oral administration in rodents. Seven experimental groups were established, each comprising six animals (n = 6) (Table 2).

Male Wistar rats were assigned to vehicle control and treatment groups receiving AB-CHMINACA at 50 or 100 mg/kg for pharmacokinetic and metabolic profiling. A separate rat cohort received the same doses and was housed in metabolic cages for urine collection at 8 and 24 h post-dose. Rats were monitored for clinical signs throughout the study.

Swiss albino mice were used for acute toxicity assessment and were divided into vehicle control and treatment groups (50 and 100 mg/kg). Clinical and behavioral observations were recorded at 1, 2, 3, 5, and 24 h after dosing. At study termination, animals underwent necropsy, and liver and kidney tissues were collected for histopathological analysis.

Table 2.

Experimental design for pharmacokinetic, metabolism, urinary excretion, and acute toxicity evaluation of AB-CHMINACA in rodents.

Group	Species	Treatment	Purpose
1	Rats	Vehicle	PK/metabolism control
2	Rats	50 mg/kg	PK/metabolism
3	Rats	100 mg/kg	PK/metabolism
4	Rats	50 and 100 mg/kg	Urine collection (8 and 24 h)
5	Mice	Vehicle	Toxicity control
6	Mice	50 mg/kg	Acute toxicity
7	Mice	100 mg/kg	Acute toxicity

Note: Each treatment group consisted of six animals (n = 6).

Sample collection and preparation

Blood and urine

Blood samples were collected from rats at 0.5, 1, 2, 4, 6, 8, and 24 h post-dose. Plasma was separated and stored at −20 °C. Urine was collected pre-dose and over 8–24 h in metabolic cages.

Extraction procedures

Plasma: Liquid-liquid extraction with 1-chlorobutane, drying under nitrogen, reconstitution for GC-MS/MS analysis.

Urine: 100 µL urine mixed with 900 µL methanol:acetonitrile (1:1), vortexed 1 min, sonicated 3 min, centrifuged at 8000 r/min for 5 min. Supernatant dried under nitrogen, reconstituted in 500 µL water:methanol (80:20), vortexed, centrifuged, and 10 µL injected for analysis.

Method Validation

The analytical method was validated according to standard bioanalytical guidelines to ensure reliability and reproducibility.

Selectivity: Six different sources of drug-free human plasma were analyzed to confirm the absence of interfering peaks at retention times and m/z values corresponding to AB-CHMINACA and the internal standard (IS).

Sensitivity: The limit of detection (LOD) and lower limit of quantification (LLOQ) were determined based on signal-to-noise ratios of ≥3 and ≥10, respectively, by analyzing low-concentration samples in triplicate.

Linearity: Calibration curves were constructed using spiked plasma samples across a concentration range of 0.5‒1000 ng/mL. Each concentration was analyzed in duplicate, with linear regression (1/x weighting) applied. The correlation coefficient (R²) was required to be ≥ .99.

Accuracy and Precision: Quality control samples at low (LQC), medium (MQC), and high (HQC) concentrations were analyzed in triplicate over multiple days. Accuracy was assessed as percent relative error, and precision by relative standard deviation (RSD), both required to be within ±15% for intra- and inter-day analyses.

Toxicity assessment

Behavioral toxicity assessment

Mice were monitored for clinical signs of toxicity including hyperactivity, sedation, convulsions, tremors, and respiratory distress. Observations were conducted at 1, 2-, 3-, 5, and 24-h post-administration.

Clinical monitoring and necropsy

Body weight and clinical status were recorded. Necropsies were conducted on all treated animals to assess gross pathological changes.

Data analysis

Pharmacokinetics

Pharmacokinetic parameters (C_max, T_max, t½, AUC, clearance) were calculated using non-compartmental analysis with Phoenix WinNonlin version 6.0.

Metabolite identification and quantification

Metabolites of AB-CHMINACA were characterized based on accurate mass shifts, retention times, and MS/MS fragmentation patterns using Mass Hunter software. Diagnostic ions and neutral losses characteristic of hydroxylation (+16 Da), ketone formation (–2 Da), and multiple hydroxylations were used to elucidate structural modifications. For instance, Met 1 (m/z 387.239) displayed a fragment ion at m/z 342 (loss of −45 Da), consistent with the elimination of a –CH₃CO group. Each proposed metabolite structure was further supported by theoretical isotope distribution and matched empirical fragmentation spectra.

Authentic reference standards for individual AB-CHMINACA metabolites were not commercially available at the time of analysis. Consequently, metabolite quantification was performed using a semi-quantitative approach. A validated calibration curve for the parent compound (AB-CHMINACA) was established in human plasma over the concentration range of 0.5–1000 ng/mL (r² > .99). This calibration was subsequently applied as a reference for relative quantification of the detected metabolites.

For each metabolite, peak-area ratios (metabolite/internal standard) were compared to the corresponding AB-CHMINACA calibration response, and results were expressed as relative abundance (%) with respect to the parent analyte. This semi-quantitative strategy, commonly adopted when authentic metabolite standards are unavailable, enabled reliable inter-sample comparison and interpretation of metabolic profiles.

Results

Method validation results

The method validation for AB-CHMINACA analysis was carried out to ensure its robustness and reliability. The calibration curve for AB-CHMINACA was established over a linear range of 0.5–1000 ng/mL, with an r² value greater than .99, demonstrating excellent linearity. The precision of the method was evaluated both within-day and between-day. Within-day precision ranged from 2.3% to 6.5%, while between-day precision was between 3.1% and 7.2%. The accuracy of the method was found to be 95%‒105% across the tested concentration range, adhering to FDA acceptance criteria. The detection limits were also evaluated, with a LOD of 0.5 ng/mL and a LOQ of 1.0 ng/mL, indicating the method's sensitivity for detecting low concentrations of AB-CHMINACA.

Pharmacokinetics of AB-CHMINACA

The pharmacokinetic profile of AB-CHMINACA was assessed in plasma using GC-CI-MS/MS. The plasma concentration–time curve showed a biphasic elimination pattern, with a rapid distribution phase followed by a slower elimination phase. Key pharmacokinetic parameters included a half-life (t½) of 13.8 h, indicating a relatively prolonged elimination. The volume of distribution (Vd) was 228.75 L, and plasma clearance (Cl) was 11.4 L/h, suggesting moderate distribution and clearance. AB-CHMINACA had a retention time of 11.1 min, while the internal standard eluted at 10.1 min, allowing for clear differentiation between the two compounds. The precursor ion for AB-CHMINACA was observed at m/z 340, and for the internal standard at m/z 313, both providing reliable identification during analysis.

Metabolite identification and profiling

AB-CHMINACA metabolites were identified using LC-Q/TOF-MS, confirming four major Phase I metabolites primarily formed through hydroxylation and ketone formation (Table 3). These included:

Met 1 (Hydroxy-AB-CHMINACA): Precursor ion at m/z 373.48; fragment ions at m/z 95, 239, 257, and 342.

Met 2 (Ketone-AB-CHMINACA): Precursor ion at m/z 370.47; fragment ions at m/z 95, 145, 239, 255, 326, and 340.

Met 3 (Di-Hydroxy-AB-CHMINACA): Precursor ion at m/z 387.47;fragment ions at m/z 95, 239, 255, 271, and 356 (Figure 1).

Met 4 (Hydroxy-Ketone-AB-CHMINACA): Precursor ion at m/z 390.47; fragment ions at m/z 93, 237, 257, 273, 291, 340, and 358 (Figure 2).

Figure 1.

Metabolite product Met 4 Hydroxylation + Ketone formation (cyclohexylmethyl) AB-CHMINACA, proposed fragmentation mechanism and mass spectrum upon ionization (ESI) at 25 MeV.

Figure 2.

Metabolite Met 3 Di-Hydroxy AB-CHMINACA, proposed fragmentation mechanism, and mass spectrum upon ionization (ESI) at 25 MeV.

Table 3.

Assumed main metabolites of the study compound, molecular structure, molecular weight of the compounds and their recorded ions, and percentage mass error.

	Formula	RT min	[M + H]+m/z	Molecular mass m/z	Mass Er. ppm
MAB-CHMINACA	C₂₀H₂₈N₄O₂	7.39	357.5	356.47	0.9
Met 1	C₂₀H₂₉N₄O₃	6.26	374.5	373.48	0.1
Met 2	C₂₀H₂₆N₄O₃	6.33	371.5	370.47	0.4
Met 3	C₂₀H₂₇N₄O₄	5.55	388.5	387.47	0.1
Met 4	C₂₀H₂₀N₄O₄	5.70	391.5	390.47	0.4

All four metabolites (Met 1–Met 4) were detected in urine samples collected at 8 and 24 h post-administration in both low- and high-dose groups (Table 4). The data revealed a dose-dependent increase in metabolite formation across both blood and urine matrices, with Met 3 consistently exhibiting the highest abundance, followed by Met 4, Met 1, and Met 2. The relative abundance (%) of each metabolite at both time points further confirmed enhanced metabolic activity at higher doses. The proposed metabolic pathways and sequence of metabolite formation are illustrated in Figure 3, highlighting the biotransformation profile of AB-CHMINACA.

Figure 3.

Proposed metabolic reactions to the drug AB-CHMINACA in urine samples from rats, and the possible conformation sequence of the metabolites.

Table 4.

Relative abundance (%) of AB-CHMINACA metabolites in rat plasma and urine after 8 h and 24 h of administration.

Metabolite	Matrix	8 h (50 mg/kg)	24 h (50 mg/kg)	8 h (100 mg/kg)	24 h (100 mg/kg)	Trend
Met 1 (Hydroxy-AB-CHMINACA)	Plasma	8.4 ± 1.1	6.2 ± 0.8	12.6 ± 1.4	10.1 ± 0.9	↑ dose-dependent
Met 2 (Ketone-AB-CHMINACA)	Plasma	5.9 ± 0.7	4.8 ± 0.5	9.2 ± 1.2	7.6 ± 0.9	↑ dose-dependent
Met 3 (Di-Hydroxy-AB-CHMINACA)	Plasma	21.3 ± 2.6	18.4 ± 2.1	33.5 ± 3.2	29.8 ± 2.7	↑ dose-dependent
Met 4 (Hydroxy-Ketone-AB-CHMINACA)	Plasma	10.5 ± 1.3	8.7 ± 1.0	15.4 ± 1.8	13.1 ± 1.2	↑ dose-dependent
Met 1	Urine	9.8 ± 1.0	7.9 ± 0.9	14.7 ± 1.6	12.8 ± 1.3	↑ dose-dependent
Met 2	Urine	7.1 ± 0.8	5.4 ± 0.6	10.5 ± 1.0	8.7 ± 0.9	↑ dose-dependent
Met 3	Urine	26.9 ± 2.9	24.5 ± 2.6	41.3 ± 3.7	38.8 ± 3.1	↑ dose-dependent (Major)
Met 4	Urine	12.1 ± 1.3	10.4 ± 1.1	18.5 ± 1.8	15.9 ± 1.4	↑ dose-dependent

Table 5.

Effects of AB-CHMINACA at Low, Medium, and High Doses on Experimental Mice Over 24 H Compared With the Control Group.

Time	Effect (5 mg/kg, low)	Effect (50 mg/kg, medium)	Effect (100 mg/kg, high)
Immediately	Normal movement	Mild hyperactivity	Marked hyperactivity and tremors
After 15 min	Mild hyperactivity followed by relative lethargy after 10 min	Hyperactivity followed by piloerection and reduced movement	Intense hyperactivity, convulsions, and piloerection
After 1 h	Relative lethargy and light sensitivity	Noticeable lethargy and reduced responsiveness to external stimuli	Severe lethargy, light sensitivity, and loss of appetite
After 3 h	Lethargy and immobility but responds to stimuli	Pronounced lethargy and partial loss of response	Complete lethargy and reduced urination
After 6 h	Complete lethargy and loss of appetite	Extended lethargy with minimal activity	Deep coma observed in several mice; no response to external stimuli
After 24 h	All mice returned to normal behavior with increased appetite	Most mice recovered; mild fatigue persisted	4 of 6 mice recovered; 2 remained unresponsive (mortality 33%)
Overall mortality (%)	0%	0%	33%

Table 6.

Comparison between the current study's results and previous studies on the metabolism of some synthetic cannabinoids.

Compound	Species, route of administration	Matrix	Total Met	Biotransformation, (reaction sites)	Major phase I Met	Phase II Met	Other findings	Ref.
AB-CHMINACA	In vivo	Urine	4	Hydroxylation (cyclohexylmethyl), Keton Fornmation (cyclohexylmethyl)				Current Study
MAB-CHMINACA	In vitro, HLM	HLM	10	- hydroxylation of the cyclohexylmethyl tail - Cyclohexylmethyl ketolization - tert-butyl hydroxylation - dihydroxylation	ADB-CHMINACA hydroxycyclohexylmethyl	no phase II transformation was detected		Carlier, et al. (2017).
AMB-Fubinaca	In vivo	Urine	37	Oxidative Demethylation, Hydroxylation (Isobutyl group), Hydroxylation (Indazole Ring), subsequent carboxylation, Oxidative N-dealkylation (Fluoro-4-methyl-benzene), Hydroxylation (Methoxy group), Reduction (Isobutyl group), Oxidative deamination (2-Amino-3-methyl-butyric acid methyl), Reduction (Isobutyl group)	AMB-FUBINACA- OH	Glucuronides (n = 9)	19 main Metabolites	Al-Osaimi (2019)
AB-Fubinaca	In vitro, HLM	HLM	1	Monohydroxylation (aminooxobutane)	ADB-FUBINACA OH	ADB-FUBINACA OH (aminooxobutane moiety)	Only one metabolite detected	Takayama et al. (2014)
ADB-Fubinaca	In vitro, HLM	HLM	1	Monohydroxylation (aminooxobutane)	ADB-FUBINACA OH	ADB-FUBINACA OH (aminooxobutane moiety)	Only one metabolite detected	Takayama et al. (2014)
AB-PINACA	In vitro, HLM	HLM	3	Monohydroxylation (N-pentyl, aminooxobutane moiety)	AB-PINACA-N-OHpentyl	Not reported	Only two metabolites detected	Takayama et al. (2014)
AKB-48	In vitro, human hepatocytes	Human Hepat-ocytes	15	Mono or dihydroxylation (adamantane, Npentyl) Carbonylation (adamantane, N-pentyl)	Di-OH-AKB-48	Glucuronides	No pentanoic acid detected	Gandhi et al. (2013)
5F-AKB-48	In vitro, HLM	HLM	17	Defluorination Mono, di, or trihydroxylation (adamantane, indazole, N-fluoropentyl) Carboxylation (N-pentyl)	5F-AKB-48 OH or di-OH, 5F-AKB-48-NOH-fluoropentyl or 5F-AKB-48 OH-indazole	Glucuronides	Oxidative defluorination occurred without NADPH	Holm et al. (2015)
AM694	In vivo, human, oral and smoke (self-experiment) (N¼1)	Urine	6	Defluorination Monohydroxylation (N-fluoropentyl, N-pentyl) Carboxylation (N-pentyl)	AM694 N-OH-pentyl, AM694 N-pentanoic acid	Not specified	Dose: 167 mg/kg body mass and 16.7 mg/kg (smoke)	Grigoryev et al. (2013)
JWH-073	In vivo, rats, IP	Hair	3	Monohydroxylation (Nbutyl) Carboxylation (N-butyl)	JWH-073-N-3-OHbutyl	Not reported	JWH-073 parent concentration4 metabolites	Kim et al. (2013)
JWH-073	In vitro, HLM	HLM	5	Monohydroxylation (N-butyl, indole, naphthyl)	JWH-073-N-OH butyl or OH naphthyl	Not reported	JWH-073 parent concentration4 metabolites	Gambaro et al. (2014)
JWH-098	In vitro, rat liver slices	Rat liver slices	6	Monohydroxylation (N-pentyl, indole, naphthyl) O-demethylation (naphthyl), Carbonylation (N-pentyl), Demethylation (naphthyl), N-dealkylation	JWH-098 O-demethylated metabolite	Glucuronide OH-JWH-098	Prediction software	Strano-Rossi et al. (2014)
XLR-11	In vitro, human hepatocytes	Human hepatocytes	30	Defluorination, Carboxylation (N-pentyl, TMCP), Mono- or dihydroxylation, (TMCP, N-pentyl), carboxylation at N-pentyl, Aldehyde formation (TMCP), Hemi-acetal formation, (TMCP) carboxylation, at TMCP (1) Internal dehydration (TMCP) (1)	UR-144-N-5-OHpentyl and UR-144-N-pentanoic acid	Glucuronides (n = 16)		Wohlfarth et al. (2013)

Acute toxicity symptoms

Administration of AB-CHMINACA at doses of 5, 50, and 100 mg/kg body weight produced a clear dose-dependent alteration in behavioral responses among mice (Table 5). At the lowest dose (5 mg/kg), animals exhibited mild hyperactivity shortly after administration, which gradually transitioned to relative lethargy and light sensitivity within 1 h. Complete lethargy was observed by 6 h, followed by full recovery and normal behavior within 24 h.

At 50 mg/kg, the onset of behavioral changes was faster and more pronounced. Mice showed hyperactivity, piloerection, and reduced movement within 15 min, progressing to marked lethargy and partial unresponsiveness between 3 and 6 h. Most mice recovered by 24 h, although mild fatigue persisted.

In contrast, the highest dose (100 mg/kg) elicited severe and sustained toxic effects. Mice demonstrated intense hyperactivity, tremors, and convulsions initially, followed by complete lethargy, loss of appetite, and deep coma in several cases within 6 h. Only four of six animals recovered movement by 24 h, indicating 33% mortality.

Overall, the findings indicate a dose-dependent increase in both the intensity and duration of toxic behavioral effects, with delayed recovery and higher mortality at elevated AB-CHMINACA doses.

Discussion

Method validation

The method validation was performed exclusively for AB-CHMINACA to ensure analytical robustness, reliability, and suitability for forensic and clinical applications. The calibration curve for AB-CHMINACA demonstrated excellent linearity across the range of 0.5–1000 ng/mL, with an r² > .99. The method exhibited strong precision, with within-day values ranging from 2.3% to 6.5% and between-day precision from 3.1% to 7.2%. Accuracy was maintained between 95% and 105% across all tested concentrations, meeting FDA acceptance criteria.

The method's LOD and LOQ were determined as 0.5 and 1.0 ng/mL, respectively, confirming its high sensitivity for detecting trace levels of AB-CHMINACA. As noted, no separate validation was conducted for metabolites; thus, metabolite data are presented qualitatively to support metabolic interpretation rather than quantitative assessment.

Pharmacokinetics of AB-CHMINACA in rats

This study aimed to characterize the pharmacokinetics of the SC, AB-CHMINACA, in rats. The plasma half-life (t^1/2) was found to be 13.8 h, differing from the 7–12 h range reported by Kevin et al.¹³ for intraperitoneally administered CUMYL-PICA and 5F-CUMYL-PICA. This discrepancy likely results from differences in administration routes. Since drug delivery methods influence pharmacokinetics, future studies should compare vaporized versus injected administration and their impact on plasma profiles.

The volume of distribution (Vd) was calculated at 228.75 L, reflecting the highly lipophilic nature of SCs. These compounds exhibit rapid distribution into lipid-rich tissues, particularly adipose tissue, leading to an early decline in blood concentrations and prolonged detection windows following chronic use.¹⁴ Previous interpretations attributing this pattern specifically to AB-CHMINACA sequestration have been updated to reflect current evidence. While AB-CHMINACA and related analogs display strong lipophilicity and extensive tissue distribution, direct quantitative data confirming adipose sequestration remain limited. Studies on structurally related SCs, including 5F-AMB, NNEI, and MN-18, support the hypothesis of preferential partitioning into fat due to their physicochemical properties and high logP values.¹⁵ This behavior likely contributes to delayed elimination and variable post-mortem redistribution patterns observed in this case. Additionally, blood protein binding may further influence elimination kinetics, although this parameter was not assessed in the present study.

Plasma clearance (CL) was 11.43 L/h, indicating relatively simple linear kinetics at the doses tested. This elimination rate appears faster than for other SCs. Studies examining various administration routes (intraperitoneal, intravenous, pulmonary) report faster peak concentrations and higher systemic exposure.^13,16,17

Drug elimination primarily depends on plasma volume passing through filtration organs, facilitating removal via urine, bile, dialysate, or exhaled gases. If significant drug amounts are sequestered in tissues or bound to plasma proteins, clearance from plasma slows. This relationship underscores the importance of volume of distribution in pharmacokinetics.

Metabolite profiling

Metabolite profiling using LC-Q/TOF-MS confirmed four major Phase I metabolites of AB-CHMINACA, mainly formed through hydroxylation and ketone formation: Met 1 (Hydroxy-AB-CHMINACA), Met 2 (Ketone-AB-CHMINACA), Met 4 (Di-Hydroxy-AB-CHMINACA), and Met 3 (Hydroxy-Ketone-AB-CHMINACA). All four were detected in urine at 8 and 24 h post-administration in both low- and high-dose groups. Met 3 was the most abundant, followed by Met 4, Met 1, and Met 2. Metabolite concentrations increased with dose, demonstrating dose-dependent metabolism. Similar metabolic interactions with comparable functional groups were reported in related SC compounds (Table 6).

Previous human hepatocyte incubation studies predicted these metabolites in vivo.¹⁸ Confirmation using authentic human urine samples would strengthen these findings but was unavailable.

Compared to Erratico et al.¹⁹ who identified M21 (a carboxylic acid metabolite formed via amidase hydrolysis) as the major Phase I metabolite, this study found hydroxylation and ketone formation as dominant pathways. Amidase enzymes selectively cleave the outer amide group, leading to M21 formation. CYP3A4 likely mediates hydroxylation at the methyl-cyclohexyl moiety, consistent with this study's findings, where Met 4 (Di-Hydroxy-AB-CHMINACA) was the major hydroxylated metabolite. These patterns align with metabolic profiles of similar SCs like MDMB-CHMICA.²⁰

Acute toxicity

AB-CHMINACA caused dose-dependent acute toxicity in rats, including lethargy, uncoordinated movements, excessive salivation, seizures, coma, hypothermia, and mortality at higher doses. These effects correspond with known toxic profiles of SCs, which include cardiovascular events (myocardial infarction, ischemic stroke), acute kidney injury, seizures, psychiatric symptoms (psychosis, paranoia, and suicidal ideation), hyperemesis, tachycardia, agitation, and nausea.²¹ The observed toxicity underscores the risks of SC use, especially at high doses, emphasizing the need for caution in clinical and forensic contexts. Understanding the pharmacodynamics and pharmacokinetics of SCs is crucial for managing overdoses and adverse effects.

Conclusion

In conclusion, the validated method presented in this study is robust for detecting and quantifying AB-CHMINACA and its metabolites, offering valuable insights into the pharmacokinetics and toxicity of this SC. The metabolite identification and profiling results are consistent with existing literature, and the pharmacokinetic parameters suggest that AB-CHMINACA behaves as a lipophilic compound with a long detection window in biological samples. The study also highlights the importance of acute toxicity data in understanding the potential risks associated with SCs and the need for continued research to develop more effective detection methods.

Footnotes

List of abbreviations

ORCID iD

Sachil Kumar

Ethics approval

Ethical approval was obtained from the Experimental Animals Ethics Committee of Naif Arab University for Security Sciences, in accordance with international standards for the care and use of experimental animals (Approval reference: NAUSS-REC-23-06).

Consent for publication

All authors give consent for publication.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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