Subacute Toxicity and Pharmacokinetic Evaluation of the Synthetic Cannabinoid 4F-MDMB-BUTINACA in Rats: A Forensic and Toxicological Perspective

Abstract

Background: 4-MDMB-BUTINACA, a next-generation synthetic cannabinoid, presents significant public health and forensic challenges due to its evolving nature and potential toxicity. Methods: This study evaluates the subacute toxic effects and pharmacokinetics of 4−Fluoro MDMB−BUTINACA (4F-MDMB-BUTINACA) in adult male albino rats, administered orally for seven days at doses of 1 mg/kg, 5 mg/kg, and 15 mg/kg. The hematological, biochemical, and histopathological parameters were assessed and compared to controls. Results: The pharmacokinetics were determined using GC–MS/MS with a positive chemical ionization and granisetron as an internal standard. A histological analysis revealed inflammatory cell aggregation, congestion, hemorrhage, edema, and fibrosis in various tissues, with renal examinations showing tubule degradation, glomerular atrophy, Bowman’s space expansion, edema, and hemorrhage. The liver exhibited cellular infiltration, while cardiac muscle fibers showed myocardial fiber degradation and inflammatory cell aggregation. Biochemical assays indicated significant alterations (p < 0.05) in the serum levels of AST, ALT, ALP, GGT, total protein, albumin, triglycerides, urea, MCHC, MCV, RDW, platelets, neutrophils, eosinophils, and basophils compared to the controls. Conclusions: The validated bioanalytical method revealed rapid absorption of 4F-MDMB-BUTINACA, with a plasma half-life of 2.371 h, a volume of distribution of 2272.85 L, and a plasma clearance rate of 664.241 L/h. In conclusion, 4F-MDMB-BUTINACA is a highly toxic synthetic cannabinoid, particularly affecting the liver, kidneys, and heart.

1. Introduction

Synthetic cannabinoids (SCs) encompass a broad range of novel psychoactive substances (NPS) commonly used recreationally under the term “legal highs” [1]. These substances are frequently consumed through smoking in e-cigarettes or inhaled as vaporized scents [2,3]. The risk associated with synthetic cannabinoids has increased, particularly with the rise in acute and fatal toxicity cases over the past two decades. Additionally, the diversity of reported toxic synthetic cannabinoids has expanded, with new indole- and indazole-3-carboxamide derivatives emerging in acute fatal intoxications. Notably, the indazole-3-carboxamide synthetic cannabinoid known as 4F-BINACA (4-fluoro-MDMB-BUTINACA), depicted in Figure 1, has recently been linked to fatal intoxication cases [4,5]. First identified in the USA and Europe in 2018 [6], this novel synthetic cannabinoid was further characterized for its psychoactive effects in early 2020 [6]. Its toxicity, particularly in conjunction with alcohol, has been reported in acute intoxication cases [7,8] and occasionally in fatal poisoning incidents [4,8,9,10]. This substance warrants further investigation to elucidate its metabolism, toxic effects, and pharmacokinetic properties, which will enhance our understanding and improve methods for identifying and quantifying its toxicity in animal models.

The development and application of advanced analytical techniques for detecting and characterizing synthetic cannabinoids have been the subject of extensive research. Krotulski et al. (2021) emphasized the necessity for continued research, enhanced detection methods, and a multidisciplinary approach to address the associated public health and legal challenges [11]. Lin-Na et al. (2021) investigated the metabolic pathways of 4F-MDMB-BUTINACA and identified key metabolites in zebrafish, suggesting potential biomarkers for forensic analysis [12]. Wang et al. (2021) provided valuable insights for forensic toxicology by analyzing seized samples of 4F-MDMB-BUTINACA and MDMB-4en-PINACA using various spectroscopic techniques [7]. Kevin et al. (2022) highlighted the complex pharmacological profiles and unforeseen adverse reactions of AMB-FUBINACA, underscoring the need for comprehensive profiling [13]. Sparkes et al., 2022 demonstrated significant CB2 receptor affinity and distinct off-target effects of 5F-MDMB-BUTINACA and its analogs, essential for regulatory and therapeutic strategies [14]. Abbott et al., 2023 stressed the importance of updating detection libraries to combat the introduction of synthetic cannabinoids in the English prison system [15]. Patel et al. (2023) contributed to the understanding of synthetic cannabinoid receptor agonists at the CB2 receptor by examining their signaling mechanisms and toxicity [16]. Finally, Simon et al. (2023) highlighted the lethal potential and unpredictability of 4F-MDMB-BUTINACA through a fatal case of synthetic cannabinoid overdose, emphasizing the need for continuous forensic research to mitigate public health risks [5].

Despite their increasing association with severe intoxications and fatalities, the pharmacological mechanisms and toxicological effects of synthetic cannabinoids remain poorly understood, marking them as a significant global public health concern [17]. Case reports indicate that synthetic cannabinoids can be absorbed orally and through inhalation, but their bioavailability is not fully characterized, with no reported cases involving parenteral or rectal administration [18]. Due to their high lipophilicity, synthetic cannabinoids often exhibit a strong binding affinity to plasma proteins, potentially leading to expanded distribution volumes [17]. Brandon et al. (2021) demonstrated varying log D7.4 values among indole- and indazole-3-carboxamide synthetic cannabinoids, ranging from 2.81 for AB-FUBINACA (the least lipophilic) to 4.95 for MDMB-4en-PINACA (the most lipophilic) [19].

In our study, three experimental phases were conducted to investigate the toxicity and pharmacokinetic behavior of 4F-MDMB-BUTINACA in Wistar albino rats. First, the GC–MS/MS method was validated to ensure precise and accurate quantification of 4F-MDMB-BUTINACA levels in rat plasma. Following successful validation, six Wistar albino rats were orally administered a dose of 50 mg/kg to characterize the pharmacokinetic profile of 4F-MDMB-BUTINACA, including parameters such as the half-life (t_1/2), volume of distribution (Vd), and clearance rate (CL). In a separate set of experiments designed to assess toxicity, three groups of Wistar albino rats (each consisting of six rats) were administered varying doses of 4F-MDMB-BUTINACA (1, 5, and 15 mg/kg/day) orally on a daily basis for a duration of seven days. A fourth group of six rats served as a control and received no treatment. The hematological and biochemical parameters were assessed, and histopathological examinations of vital organs were performed. This comprehensive approach aims to enhance our understanding of the pharmacological and toxicological properties of synthetic cannabinoids, with 4F-MDMB-BUTINACA serving as a model.

2. Materials and Methods

2.1. Drug Preparation and Animals

The drug 4−Fluoro MDMB−BUTINACA was initially dissolved in dimethyl sulfoxide (DMSO). This solution was then diluted with olive oil to achieve the desired drug concentration, ensuring that the final DMSO concentration did not exceed 5%. A 5% olive oil–DMSO mixture served as the vehicle control. The animal experimental procedures, authorized by the research committee under Decision No. NAUSS-Rec-24-02, utilized thirty Wistar albino rats, each weighing between 160 and 200 g. The rats were randomly allocated into two principal experiments. The first experiment, aimed at evaluating the pharmacokinetic profile, involved six rats receiving an oral dose of 50 mg/kg of 4F-MDMB-BUTINACA, as detailed in Section 2.6. The second experiment assessed toxicity and, as described in Section 2.7, comprised four groups of six rats each. Group 1 served as the control, while Groups 2, 3, and 4 were administered oral doses of 1, 5, and 15 mg/kg/day of 4−Fluoro MDMB−BUTINACA, respectively, for a duration of seven days. All the rats had ad libitum access to water and a commercial pelleted diet (Saudi Grains Organization, Riyadh, Saudi Arabia). They were housed under standardized conditions, with the temperature maintained at 23 ± 2 °C, relative humidity at 50–60%, and a 12 h light/dark cycle.

2.2. Chemicals and Reagents

The analytical reference standard 4−Fluoro MDMB−BUTINACA (C₁₉H₂₆FN₃O₃) utilized in this study was procured from Cayman Chemicals, Estonia. Granisetron (C₁₈H₂₄N₄O), obtained from MedChemExpress, USA, was used as the internal standard (IS) to ensure accurate quantification through the analytical techniques employed. Dimethyl sulfoxide (DMSO), used for dissolving the drug, was sourced from Biotraxx, Cyprus. Olive oil was purchased from a local market in Saudi Arabia (KSA). The chemicals employed in liquid–liquid extraction (LLE) and gas chromatography–mass spectrometry (GC–MS), including methyl-tert-butyl ether (MTBE) and ethyl acetate, were of analytical grade, with ammonium hydroxide (NH₄OH) also acquired from Sigma-Aldrich, Germany. The plasma samples were analyzed using biochemistry kits from Sysmex, Japan. The biochemical assay kits for measuring hepatic enzymes, including aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), and alkaline phosphatase (ALP, EC 3.1.3.1), as well as renal parameters, such as urea, creatinine, albumin, total protein, and cholesterol, were obtained from United Diagnostics Industry, Dammam, KSA.

2.3. Preparation of the Calibrators and QC Samples for GC-MS Validation

To prepare the calibrator solutions, 10 mg of 4−Fluoro MDMB−BUTINACA were dissolved in 10 mL of methanol to obtain a stock solution with a concentration of 1 mg/mL. A standard solution with a concentration of 100 µg/mL was prepared by diluting 1 mL of this stock solution with 9 mL of methanol. Subsequent dilutions resulted in standard solutions with concentrations of 10 µg/mL, 1 µg/mL, and 0.1 µg/mL. Calibrators were prepared in rat plasma, with concentrations ranging from 0.5 to 1000 ng/mL. This was achieved by adding the analyte reference standard and a fixed amount of internal standard (Granisetron, 10 µg/mL) to the plasma matrix. For quality control (QC) purposes, 500 µL of the plasma samples were spiked with the internal standard and analyzed after adding the analyte reference standards to reach low, medium, and high concentrations (10, 100, and 500 ng/mL, respectively).

2.4. Procedures of Liquid–Liquid Extraction of the Drug from the Plasma

The liquid–liquid extraction procedure began by spiking a 500 µL plasma sample containing the drug with 50 µL of the internal standard. Subsequently, 1 mL of acetonitrile was added to the mixture in a polypropylene tube. The mixture was vortexed for 3 min and then centrifuged for 5 min at 3000 rpm. The supernatant was evaporated to approximately 0.5 mL under a nitrogen stream at 35 °C. Following evaporation, the sample was reconstituted with 250 µL of 1% NH₄OH and extracted with 3 mL of a mixture of MTBE and ethyl acetate (80:20, v/v). The organic layer was separated, evaporated to dryness, and reconstituted with 50 µL of ethyl acetate. Finally, the reconstituted sample was transferred to GC–MS/MS vials for analysis.

2.5. Validation of GC–MS/MS Analysis

This study employed various validation parameters to ensure the high quality of the techniques and to establish the basis for the proposed analytical procedure. The sensitivity, selectivity, linearity, precision, accuracy, and recovery were assessed according to the FDA M10 Bioanalytical Method Validation guidelines [20].

Analyses were performed using an Agilent GC–MS/MS system equipped with an Agilent 7693 autosampler. The GC column utilized was an HP-5MS (5% Phenyl Methyl Silox) polysiloxane capillary column, with dimensions of 30 m in length, 250 µm in diameter, and a film thickness of 0.25 µm. Helium served as the carrier gas, flowing at a rate of 2.25 mL/min, while ammonia was used as the reagent gas in the positive chemical ionization source, at a flow rate of 1.5 mL/min. A temperature gradient was applied over time, as detailed in

Table 1. Temperature gradient details.

Temp. Gradient °C/min	Temperature °C	Hold Time min	Run Time min
-	150	2	0.5
30	320	5.3333	13

. A 2 µL sample volume was introduced into the system via splitless injection. Both the injector and interface were maintained at a constant temperature. Ionization was achieved using positive chemical ionization (PCI) with an energy level of 70 electron volts. The multiple reaction monitoring (MRM) mode was employed to target specific precursor-to-product ion transitions, providing both qualitative and quantitative data. Monitoring unique fragmentation patterns enabled precise detection and quantification of the target analytes. Plastic test tubes (Kartell SP A, Milan, Italy) and syringes (HLB Therapeutics, Seongnam, South Korea) were utilized for sample storage and handling.

2.5.1. Evaluation of the GC–MS/MS Selectivity

In accordance with the FDA M10 guidelines for bioanalytical method validation, evaluating the selectivity of GC–MS/MS for detecting 4−Fluoro MDMB−BUTINACA and the internal standard (granisetron) in blood samples requires demonstrating that the method can accurately differentiate between the drug and the internal standard from endogenous compounds and potential interferences. This involves analyzing three blank blood samples, which are spiked with the drug and internal standard at low concentrations, to ensure that no significant signals are detected from matrix substances in the absence of the analytes. Additionally, the method’s selectivity is confirmed by verifying that the MRM (multiple reaction monitoring) transitions specific to the drug and internal standard are clearly resolved and do not overlap with other peaks in the chromatogram.

2.5.2. Evaluation of the GC–MS/MS Sensitivity

The sensitivity evaluation was conducted by determining the limit of quantification (LOQ) and the limit of detection (LOD) for each analyte. The LOD is defined as the lowest concentration of an analyte in a sample that can be consistently detected and identified, characterized by a signal-to-noise ratio of ≥3, a well-defined peak shape, acceptable chromatography peak shape, satisfactory retention time, and good resolution. The LOQ is determined as the concentration at which the signal-to-noise ratio is ≥10.

2.5.3. Evaluation of the GC–MS/MS Linearity

The linearity was assessed by calculating the correlation coefficient (r²) and constructing a calibration curve using standard solutions of 4−Fluoro MDMB−BUTINACA. These solutions were prepared in drug-free blood samples (blanks) across nine concentration levels: 0.5, 2, 5, 10, 20, 50, 100, 500, and 1000 ng/mL.

2.5.4. Evaluation of the GC–MS/MS Accuracy and Precision

The accuracy and precision of SC 4−Fluoro MDMB−BUTINACA were evaluated at three quality control concentrations: low (LQC, 10 ng/mL), medium (MQC, 100 ng/mL), and high (HQC, 500 ng/mL). Quality control samples were analyzed over three separate days, with each sample tested in five replicates. The precision was assessed by calculating the relative standard deviation (RSD) for both inter-assay and intra-assay variations, which should not exceed 15% RSD. The accuracy was determined by evaluating the bias of the results.

2.5.5. Evaluation of the Method’s Recovery and Stability

In our routine laboratory work, we assess the recovery and stability of any newly purchased substance according to FDA M10 guidelines. For assessing the recovery, QC samples at concentrations of 10 ng/mL, 100 ng/mL, and 500 ng/mL are prepared and analyzed to compare the peak of the analyte extracted from biological matrices with that of the analyte in ethanol, providing insights into the efficiency of the extraction process. High and consistent recovery values indicate a robust method. For assessing the stability, QC samples are subjected to freeze–thaw cycles, where they are frozen at −20 °C and thawed at 25 °C, with concentrations analyzed immediately after the final thaw and compared to those of freshly prepared samples. Additionally, the autosampler stability is tested by storing QC samples on the GC–MS/MS autosampler at 25 °C for 24 h, with a subsequent analysis to compare their concentrations to those of freshly prepared samples. The stability is evaluated by calculating the percentage change in the concentration, ensuring adherence to method validation criteria from the following equation:

$$\mathrm{Percentage} \mathrm{Change}={\displaystyle \frac{\mathrm{Initial} \mathrm{Concentration}- \mathrm{Measured} \mathrm{concentration}}{\mathrm{Initial} \mathrm{Concentration}}} \times 100$$

2.6. Animal Experiments for Evaluating the Pharmacokinetic Profile

For the initial assessment of the pharmacokinetic profile, a cohort of six Wistar albino rats was administered an oral dose of 50 mg/kg body weight of 4F-MDMB-BUTINACA prepared in 5% DMSO as the vehicle. Blood samples were collected at 0.5, 1, 2, 4, 6, and 8 h post-administration via retro-orbital puncture using a heparinized capillary tube to minimize distress. Approximately 0.5 mL of blood was collected at each time point, transferred to EDTA-coated microcentrifuge tubes, and centrifuged at 3000 rpm for 10 min to separate the plasma. The plasma samples were subjected to liquid–liquid extraction (LLE), with 50 µL of an internal standard (granisetron, 10 µg/mL) added to each 500 µL plasma sample, as detailed in Section 2.4. The concentration of 4F-MDMB-BUTINACA in the plasma was quantified using an optimized LLE–GC–MS/MS method. The pharmacokinetic parameters, including half-life, volume of distribution, and clearance rate, were determined from the concentration-time data using noncompartmental analysis, as described by Yu and Cao (2017) [21]. Briefly, to determine the pharmacokinetic profile of 4F-MDMB-BUTINACA following oral administration (50 mg/kg) in rats, a non-compartmental analysis (NCA) was conducted using WinNonLin software (version 6.4). The plasma concentration data, quantified by GC–MS/MS, were collected at predetermined time points for each rat. In WinNonLin, the dataset was imported, and the extravascular model was selected to account for oral dosing. Key pharmacokinetic parameters were calculated using the trapezoidal rule, including AUC (area under the curve), C_max (maximum concentration), T_max (time to reach C_max), λz (terminal elimination rate constant), t_1/2 (half-life), CL/F (clearance), and Vd/F (volume of distribution). The terminal phase was fitted using log-linear regression, and the concentration-time profiles were graphically represented for each subject. Visual inspection ensured appropriate model fitting. The calculated parameters and concentration-time curves were compiled, and the results were interpreted to assess the absorption, distribution, and elimination of 4F-MDMB-BUTINACA. The analysis was reported with a focus on the key pharmacokinetic insights, providing a foundation for further research.

2.6.1. Calculations of the Pharmacokinetic Parameters

Plasma samples from six albino rats administered 4F-MDMB-BUTINACA at a dosage of 50 mg/kg were analyzed using the earlier described liquid–liquid extraction, with granisetron serving as the internal standard, and quantified by LLE–GC–MS/MS. The concentration-time data were imported into WinNonlin Phoenix 6.4 (Pharsight, Mountain View, CA, USA), where the pharmacokinetic parameters were computed using noncompartmental analysis. The key parameters that were determined included the initial concentration (C0), apparent volume of distribution (Vd = D/Cp), elimination rate constant (K), terminal phase half-life (t_1/2 = 0.693/K), and apparent total clearance (CL = K × Vd), following the methodology outlined by Yu and Cao [21].

2.6.2. Statistical Analysis

A non-compartmental pharmacokinetic data analysis was performed in WinNonlin Phoenix 6.4. The results were presented as mean ± standard error of the mean (SEM). The statistical significance was determined using Student’s t-test, with a significance level set at p < 0.05 using SPSS version 22 (SPSS Inc., Chicago, IL, USA) (Carrillo et al., 1996) [22].

2.7. Animal Experiments for Evaluating the Sub-Acute Toxicity

In accordance with OECD 407 guidelines for subacute toxicity evaluation [23], a total of 24 rats were randomly assigned to four groups. Group 1 served as a control, while Groups 2, 3, and 4 received daily oral doses of 1 mg/kg, 5 mg/kg, and 15 mg/kg of 4F-MDMB-BUTINACA, respectively. During the seven-day treatment period, the rats were closely monitored twice daily for signs of morbidity and mortality. Clinical symptoms and body weights were recorded regularly, and blood samples were collected at the end of the study for subsequent hematological and biochemical analyses.

2.7.1. Hematological Parameters

The hematological parameters—including hemoglobin (Hb) concentration, red blood cell count (RBC), packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell count (WBC)—were measured in heparinized blood using a Sysmex XE-2100 Analyzer (Sysmex, Kobe, Japan).

2.7.2. Biochemical Parameters

To assess the functionality of various organs and systems within the body, and to evaluate the potential toxicity of 4F-MDMDB-BUTINACA, several blood test parameters were measured at the end of the treatment period. These tests are instrumental in detecting toxic effects and other conditions affecting the liver, kidneys, heart, and metabolic system. Consequently, the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatine kinase (CK), total protein, albumin, cholesterol, high-density lipoprotein (HDL), triglycerides, urea, creatinine, total bilirubin, and direct bilirubin were measured using a Beckman Coulter DxC 700 AU (Beckman Coulter, Brea, CA, USA).

2.7.3. Histopathology

At the end of the treatment period of the toxicity experiment, the rats were euthanized using diethyl ether. A necropsy was performed, which included a thorough examination for gross lesions. Samples of the liver, kidney, and heart were then fixed in 10% neutral buffered formalin. A histopathological analysis was conducted using paraffin embedding techniques as described by Carleton et al. (1980) [24]. In summary, the tissues were preserved in formalin, subsequently washed, dehydrated through graded ethanol, cleared in xylene, embedded in paraffin, sectioned at a thickness of 5 μm, mounted on slides, stained with hematoxylin and eosin (H&E), and examined microscopically.

3. Results and Discussions

3.1. GC–MS/MS Method Suitability for Routine Analysis

The objective of employing GC–MS in this research was to assess the pharmacokinetic profile of 4−Fluoro MDMB−BUTINACA in rat plasma using GC–CI–MS/MS. Optimizations of the gas chromatography (GC) settings, chromatographic parameters, MS/MS conditions, and extraction techniques enhanced the sensitivity and reduced interferences. The refined chromatographic conditions resulted in short retention times for 4−Fluoro MDMB−BUTINACA and the internal standard. For MS/MS optimization, a full scan identified the precursor ions at m/z 364 and 304 for 4−Fluoro MDMB−BUTINACA and at m/z 313 and 176 for the internal standard granisetron, as detailed in

Table 2. Retention times, quantifier and qualifier transition ions, and corresponding collision energies for 4−Fluoro MDMB−BUTINACA and granisetron (internal standard) in GC–CI–MS/MS analysis.

Compound	Retention Time (min)	Quantifier Transition Ion		Collision Energy (eV)	Qualifier Transition Ions		Collision Energy (eV)
4−Fluoro MDMB−BUTINACA	8.149	364	304	20	364	345	20
					364	219
Granisetron (IS)	9.550	313	176	12	313	126	12
					313	96

.

Product ion scans with collision energies ranging from 12 to 25 eV were employed to fragment these ions, which were subsequently detected. The resulting chromatograms and spectra validated the method’s suitability for routine analysis. The results regarding selectivity, sensitivity, and linearity of the GC–MS/MS method illustrate its ability to accurately identify and quantify 4F-MDMB-BUTINACA without interference from matrix components, as depicted in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6 and Figure 7.

The analyte remained quantifiable at the specified retention times (RT) and mass-to-charge ratio (m/z) values of the monitored ions across a concentration range of 0.5–1000 ng/mL, as illustrated in Figure 7.

The intra-assay (within-run) precision and accuracy data for 4−Fluoro MDMB−BUTINACA across different concentrations (10 ng/mL, 100 ng/mL, and 500 ng/mL) in the rat plasma samples demonstrate the consistent performance of the analytical method, as illustrated in

Table 3. Intra-assay (within-run) precision (% RSD) and accuracy (% Bias) data for 4−Fluoro MDMB−BUTINACA in rat plasma samples.

Plasma Concentration (ng/mL)	Mean Recovery Concentration (ng/mL)	SD	RSD%	Bias
			<15%	±<15%
10	9.372	0.236	2.52	−6.28
100	95.586	2.466	2.58	−4.41
500	498.248	8.207	1.647	−0.35

.

The relative standard deviations (RSD%) for all three concentrations were below the acceptable threshold of 15%, with values of 2.52%, 2.58%, and 1.647%, respectively. This indicates the high precision of the method across different runs. The accuracy, expressed as %Bias, was within ±15%, with values of −6.28% at 10 ng/mL, −4.41% at 100 ng/mL, and −0.35% at 500 ng/mL, reflecting the method’s reliability in quantifying the analyte close to its true value. The low RSD% and %Bias at all the concentration levels underscore the method’s robustness and reproducibility, essential for consistent and reliable detection of 4−Fluoro MDMB−BUTINACA in the plasma samples.

On the other hand, the inter-assay (between-run) precision and accuracy data over three days for the quality control samples (QC1 at 10 ng/mL, QC2 at 100 ng/mL, and QC3 at 500 ng/mL) also indicate a strong performance of the analytical method, as illustrated in

Table 4. Inter-assay (between-run) precision (% RSD) and accuracy (% Bias) data for 4−Fluoro MDMB−BUTINACA in rat plasma samples.

Expected	QC1-10 ng/mL	QC2-100 ng/mL	QC3-500 ng/mL
Day 1	9.372	95.586	498.248
Day 2	9.35	97.05	487.804
Day 3	9.62	97.742	489.66
Mean	9.447	96.793	491.904
S.D.	0.150	1.101	5.572
RSD%	1.587	1.137	1.133
Bias%	−5.527%	−3.207%	−1.619%

.

The RSD% values were 1.587% for QC1, 1.137% for QC2, and 1.133% for QC3, all well below the 15% threshold, confirming the method’s precision within a single analytical run. The %Bias values for QC1, QC2, and QC3 were −5.527%, −3.207%, and −1.619%, respectively, all within the acceptable range of ±15%, as illustrated in Table 4. The consistency in the mean recoveries (9.447 ng/mL, 96.793 ng/mL, and 491.904 ng/mL) close to the expected concentrations further supports the method’s accuracy. The standard deviations (S.D.) were low (0.150, 1.101, and 5.572), reinforcing the method’s precision.

Both the inter-assay and intra-assay precision and accuracy data demonstrate the validated GC–MS/MS method’s reliability for quantifying 4−Fluoro MDMB−BUTINACA in rat plasma samples. The low %RSD values in both Table 3 and Table 4 indicate that the method yields reproducible results across multiple runs and within single runs. The %Bias values, all within ±15%, suggest that the method accurately measures the analyte concentrations, with minimal deviation from the expected values. These findings confirm that the method is both precise and accurate, making it suitable for the pharmacokinetic and toxicological studies of 4−Fluoro MDMB−BUTINACA. The high precision and accuracy at different concentration levels ensure that the method can reliably detect and quantify the analyte across a range of concentrations typically encountered in biological samples.

3.2. Pharmacokinetic Profile of 4F-MDMB-BUTINACA Following Oral Administration at a Dose of 50 mg/kg

Following the oral administration of a 50 mg/kg dose of 4F-MDMB-BUTINACA to a cohort of six rats, the plasma concentration of the drug was quantified at various time points using a validated GC–MS/MS method. The concentration of 4F-MDMB-BUTINACA increased up to 6 h post-administration. Unfortunately, due to the death of 5 out of the 6 rats after 6.5 h, data beyond this time point could not be collected. The logarithm of the drug concentrations was plotted against the time post-administration, as illustrated in

Table 5. Concentrations of 4-FLUORO-MDMB-BUTINACA in rat plasma at various time points post-administration. Data are presented as mean ± standard deviation (n = 6), except for the 4- and 6-hour time points, where n reflects the number of surviving rats.

Post-administration time (hours)	0.5 (n = 6)	1 (n = 6)	2 (n = 6)	4 (n = 4)	6 (n = 1)
Mean plasma concentration in (ng/kg) for oral administeration of 50 mg/kg of the substance	23.557	28.64	41.135	87.18	110.371
Log10 for dose 50 mg/kg	1.372	1.457	1.614	1.940	2.043

and Figure 8.

From

Table 6. Pharmacokinetic parameters of 4-FLUORO-MDMB-BUTINACA measured in rat plasma following oral administration of 50 mg/kg. Data are presented as mean ± standard deviation (n = 6).

Intercept	1.3424
Slope	0.1269
C0	21.99885109 ng/kg
K	0.2922507 h−1
Dose	50,000,000 ng/kg
Vd	2272.85 L
t1/2	2.371 h.
Cl	664.241 L/h
AUC	75.2739 ng·h/mL

and Figure 8, the calculated pharmacokinetic parameters further elucidate the drug’s behavior: an intercept of 1.342 and a slope of 0.1269 denote the linearity in the elimination phase; an initial concentration (C0) of 21.998 ng/kg and an elimination rate constant (K) of 0.2923 h⁻¹ reflect rapid absorption and moderate elimination.

The extensive volume of distribution (Vd) of 2272.85 L suggests significant tissue distribution, while the half-life (t_1/2) of 2.371 h indicates a moderate duration of systemic presence. The clearance rate (Cl) of 664.241 L/h signifies efficient drug elimination, and an area under the curve (AUC) of 75.2739 ng·h/mL represents the overall systemic exposure. Collectively, these findings demonstrate the comprehensive pharmacokinetic profile of 4−Fluoro MDMB−BUTINACA, highlighting its absorption, extensive distribution, and efficient clearance in the rat model after oral administration of the drug.

3.3. Monitoring the Sub-Acute Toxicity of the 4F-MDMB BUTINACA on the Studied Animals

The effects of various doses of 4−Fluoro MDMB−BUTINACA administered orally to Wistar rats were evaluated. Both the control group and Group 1, which received a daily dose of 1 mg/kg, showed no significant adverse effects over the course of a week. In contrast, Group 3, which was treated daily with 5 mg/kg, exhibited signs of depression, fatigue, and tachypnea, with a mortality rate of 50% by the third day. All the rats in Group 4, receiving 15 mg/kg daily, died within 24 h. Measurements of body weight taken before and after the trial indicated no significant changes in the average body weight across all the groups.

3.4. Hematological Changes with 4F-MDMB BUTINACA Oral Administration

Hematological values, which include a wide range of blood parameters, such as red blood cell (RBC) count, white blood cell (WBC) count, hemoglobin levels, and platelet count, and differentials like neutrophils, lymphocytes, and monocytes, can be indicative of both acute and chronic toxicity in rats exposed to toxic substances. These parameters can show changes due to the body’s response to the toxic agent, reflecting damage to organs, bone marrow, or systemic effects that influence blood cell production and lifespan.

The administration of 4F-MDMB-BUTINACA in rats for one week resulted in significant hematological changes. As illustrated in

Table 7. Hematological changes in rats given various levels of 4F-MDMB-BUTINACA orally for one week. Data are presented as mean ± standard deviation (n = 6).

Parameters	Control (n = 6)	4F-MDMB-BUTINACA (1 mg/kg/Day) (n = 6)	4F-MDMB-BUTINACA (5 mg/kg/Day) (n = 6: 3 Survived, 3 Died)
WBC	3.8 ± 0.47	2.67 ± 0.26	2.00 ± 0.32
RBC	6.82 ± 0.33	7.30 ± 0.28	7.18 ± 0.38
HGB	13.17 ± 0.34	13.40 ± 0.56	13.60 ± 0.32
HCT	42.93 ± 2.8	47.43 ± 2.5	43.87 ± 0.58
MCV	63.07 ± 1.7	65.43 ± 6 *	61.40 ± 2.9 *
MCH	19.33 ± 0.58	18.37 ± 0.9	19 ± 0.6
MCHC	30.70 ± 0.41	28.50 ± 2.5 *	31 ± 0.76 *
RDW	18.10 ± 0.5	22.17 ± 5.1 *	19.57 ± 0.9 *
PLT	748.33 ± 49	824.67 ± 19 *	789.67 ± 7.4 *
NE	0.13 ± 0.13	0.05 ± 0.001 *	0.33 ± 0.06 *
LY	2.33 ± 0.3	2.30 ± 0.2	1.60 ± 0.32
ES	0.33 ± 0.033	0.001 ± 0.011 *	0.001 ± 0.01 *
BA	0.33 ± 0.033	0.001 ± 0.012 *	0.067 ± 0.66 *

Values are expressed as the mean ± standard error. Statistical significance is indicated by * (p < 0.05). (n = 6) for each group unless otherwise specified. The 5 mg/kg/day dose group included 6 rats, with 3 survivors and 3 fatalities. The abbreviations used in the table are as follows: BA—basophils, ES—eosinophils, HCT—hematocrit, HGB—hemoglobin, MCH—mean corpuscular hemoglobin, MCHC—mean corpuscular hemoglobin concentration, MCV—mean corpuscular volume, LY—lymphocytes, NE—neutrophils, PLT—platelets, RDW—red cell distribution width, RBC—red blood cell count, WBC—white blood cell count.

, a notable decrease in the white blood cell (WBC) counts was observed in both the 1 mg (2.67 ± 0.26) and 5 mg (2.00 ± 0.32) groups compared to the control (3.8 ± 0.47), suggesting potential immunosuppressive effects. The red blood cell (RBC) counts increased slightly in the 1 mg group (7.30 ± 0.28) and 5 mg group (7.18 ± 0.38) relative to the control (6.82 ± 0.33), accompanied by slight elevations in the hemoglobin (HGB) and hematocrit (HCT) levels, indicating a possible compensatory response. The mean corpuscular volume (MCV) showed a dose-dependent variation, with a significant increase in the 1 mg group (65.43 ± 6) and a decrease in the 5 mg group (61.40 ± 2.9). The mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) displayed mixed trends, with the MCHC significantly lower in the 1 mg group (28.50 ± 2.5) and higher in the 5 mg group (31 ± 0.76). The elevated red cell distribution width (RDW) in the 1 mg group (22.17 ± 5.1) indicated increased red cell size variability. The platelet (PLT) counts were higher in the treated groups, suggesting potential inflammatory or bone marrow activation. The differential leukocyte counts revealed significant changes in neutrophils (NE), eosinophils (ES), and basophils (BA), indicating immune modulation.

3.5. Serobiochemical Changes with 4F-MDMB BUTINACA Oral Administration

The serobiochemical analysis of rats administered 4F-MDMB-BUTINACA for one week demonstrated significant changes indicative of potential organ toxicity. As detailed in

Table 8. Serobiochemical changes in rats given various levels of (4F-MDMB-BUTINACA) orally for a week.

Parameters	Control (n = 6)	4F-MDMB-BUTINACA (1 mg/kg/Day) (n = 6)	4F-MDMB-BUTINACA (5 mg/kg/Day) (n = 6: 3 Survived, 3 Died)
AST (U/L)	115.65 ± 2.1	145.18 ± 3.3 *	146.1 ± 1.2 *
ALT (U/L)	64.28 ± 0.43	75.8 ± 9.1 *	78.06 ± 6.7 *
GGT (U/L)	45.6 ± 3.7	56.43 ± 2.2 *	46.18 ± 1.4 *
ALP (U/L)	562.03 ± 16.4	623.32 ± 14	591.5 ± 17.5 *
HDL (mmol/L)	1.47 ± 0.1	1.14 ± 0.04	1.21 ± 0.07
LDH (U/L)	238.4 ± 5.4	392.9 ± 57.2	204.28 ± 9.1
CHOL (mmol/L)	2.26 ± 0.2	1.85 ± 0.2	1.88 ± 0.05
TG (mmol/L)	0.81 ± 0.07	0.93 ± 0.31 *	0.92 ± 0.2
TBIL (umol/L)	1.523 ± 0.44	2.5 ± 0.02 *	2.32 ± 0.1 *
DBIL (umol/L)	0.157 ± 0.1	0.27 ± 0.03	0.5 ± 0.1
TP (g/L)	61.1 ± 0.31	55.21 ± 1.4 *	60.7 ± 0.5 *
ALB (g/L)	32.3 ± 0.14	29.51 ± 1.2 *	32.75 ± 0.92 *
UREA (mmol/L)	5.84 ± 0.5	5.36 ± 0.6	8.95 ± 0.26 *
Na (mmol/L)	140.9 ± 0.2	139.88 ± 0.52	142.4 ± 0.75
K (mmol/L)	5.36 ± 0.14	5.12 ± 0.5	4.76 ± 0.17
CL (mmol/L)	99.03 ± 0.9	97.24 ± 1.8	101.33 ± 1
PHOS (mmol/L)	3.24 ± 0.8	3.147 ± 0.1	2.5 ± 0.3
IRON (umol/L)	43.5 ± 6.5	33.38 ± 5.8	58.15 ± 18
Ca (mmol/L)	3.057 ± 0.05	3.1 ± 0.05	2.73 ± 0.6

Values are expressed as mean ± standard error. Statistical significance is indicated by * (p < 0.05). The table abbreviations are defined as follows: AST (aspartate aminotransferase, U/L), ALT (alanine aminotransferase, U/L), GGT (gamma-glutamyl transferase, U/L), ALP (alkaline phosphatase, U/L), HDL (high-density lipoprotein, mmol/L), LDH (lactate dehydrogenase, U/L), CHOL (cholesterol, mmol/L), TG (triglycerides, mmol/L), TBIL (total bilirubin, µmol/L), DBIL (direct bilirubin, µmol/L), TP (total protein, g/L), ALB (albumin, g/L), UREA (urea, mmol/L), Na (sodium, mmol/L), K (potassium, mmol/L), CL (chloride, mmol/L), PHOS (phosphate, mmol/L), IRON (iron, µmol/L), and Ca (calcium, mmol/L).

, elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were observed in both the 1 mg/kg (AST: 145.18 ± 3.3; ALT: 75.8 ± 9.1) and 5 mg/kg (AST: 146.1 ± 1.2; ALT: 78.06 ± 6.7) dose groups compared to the control group (AST: 115.65 ± 2.1; ALT: 64.28 ± 0.43), suggesting hepatocellular damage. Additionally, the gamma-glutamyl transferase (GGT) and alkaline phosphatase (ALP) levels were elevated, particularly in the 1 mg/kg dose group, further supporting potential liver dysfunction. Although the high-density lipoprotein (HDL) levels were reduced and the total cholesterol (CHOL) levels were lower in the treated groups, the triglyceride (TG) levels showed a slight increase, indicating disturbances in lipid metabolism. The total bilirubin (TBIL) and direct bilirubin (DBIL) levels were significantly elevated in the treated groups, which also suggests hepatic stress. The total protein (TP) and albumin (ALB) levels were diminished in the 1 mg/kg group, indicating impaired protein synthesis. Moreover, a significant increase in the urea levels in the 5 mg/kg dose group (8.95 ± 0.26) points to potential nephrotoxicity. Although the electrolyte and mineral levels exhibited only minor variations, notable changes in the phosphate (PHOS) and calcium (Ca) levels were observed, highlighting possible effects on mineral balance and renal function. Collectively, these findings indicate that administration of 4F-MDMB-BUTINACA at both doses produces considerable toxic effects on the liver, kidney, and metabolic functions in rats.

3.6. Diagnosis of the Subacute Toxicity of 4-MDMB-Butinaca and Histopathological Changes

For a better diagnosis and monitoring of the toxicity baseline, hematological values are compared with periodic tests to identify significant changes over time. Besides hematological tests, a comprehensive approach including biochemical assays (liver and kidney function tests), histopathological examinations (tissue biopsies), and clinical observations were essential for a full assessment of acute and chronic toxicity, as illustrated in Figure 9, Figure 10 and Figure 11.

As illustrated in Table 8, the control rats showed no microscopic alterations after one week (group 1). Acute histopathological alterations include liver dilated portal vein and sinusoids, edema, congestion, and inflammatory cell aggregations around and between cells, as well as perivascular fibrosis and vascular wall thickening. In the kidneys, alterations include congestion and edema of renal blood vessels and within the Bowman’s space, as well as leakage of hemorrhage in between the tubules, glomeruli atrophy, increased Bowman’s space, necrosis, and tubular degeneration. In the heart, there is congestion, hemorrhage, myocardial fiber degeneration, and accumulated inflammatory cells.

The photomicrographs of the liver samples revealed significant histopathological changes following treatment with 4F-MDMB-BUTINACA. Figure 9A depicts the control liver with a normal structure, including a central vein (CV), binucleated hepatocytes (HC), blood sinusoids (s), and Kupffer cells (k). In contrast, Figure 9B shows the liver of a rat administered 1 mg/kg of 4F-MDMB-BUTINACA, exhibiting inflammation (blue arrows), perivascular fibrosis (orange arrow), cellular and vascular edema, and congestion (red arrows). Further exacerbation is observed in Figure 9C, where the liver of a rat treated with 5 mg/kg of 4F-MDMB-BUTINACA presents intravascular hemorrhage (green arrow), a thickened vein wall and congestion (white arrow), perivascular fibrosis (orange arrow), cellular and vascular edema, and congestion (red arrows), along with inflammation (blue arrow). These images underscore the dose-dependent hepatic damage induced by 4F-MDMB-BUTINACA.

A similar impact of the drug administration was observed on the renal tissue structures demonstrating pathological changes induced by 4F-MDMB-BUTINACA. Figure 10A displays control renal tissue showing normal structures, including the glomerulus (G), Bowman’s space (BS), Bowman’s capsule (BC), distal tubule (DT), and proximal tubule (PT). In contrast, Figure 10B depicts the kidney of a rat treated with 1 mg/kg of 4F-MDMB-BUTINACA, revealing glomerular atrophy (blue arrow), increased Bowman’s space (green arrows), and mild hemorrhage within the glomeruli (red arrows) (H&E, 40×). Figure 10C illustrates the renal tissue of a rat treated with 5 mg/kg of 4F-MDMB-BUTINACA, showing glomerular edema (yellow arrow), mild tubular degeneration (blue arrows), necrosis (black arrow), and hemorrhage within the glomeruli (red arrows).

Moreover, the toxic effect of 4F-MDMB-BUTINACA was assessed through the heart histopathological examination. In Figure 11, photomicrographs of cardiac muscle tissues are presented. Figure 11A depicts control cardiac muscles, showing normal cardiac muscle fibers. In contrast, the cardiac muscles of rats administered 1 mg/kg of 4F-MDMB-BUTINACA exhibited vacuolar degeneration and intravascular congestion, as shown in Figure 11B. Furthermore, the cardiac muscles of rats administered 5 mg/kg of 4F-MDMB-BUTINACA displayed degeneration with swelling of the myocardial cell nuclei, increased eosinophilia, aggregation of inflammatory cells, vascular wall thickening, and congestion and dilatation of vascular channels, as illustrated in Figure 11C.

4. Conclusions

Due to the limited literature on the pharmacokinetics and toxicological profiles of newly emerging synthetic cannabinoids and the fact that only highly accurate instrumentation, such as chromatographic tools, has been successful in quantifying these substances and their metabolites in biological samples, our study aimed to provide new data on the pharmacokinetics and toxicity of synthetic cannabinoids. Specifically, we focused on substances associated with acute toxicity and fatal cases, such as 4−Fluoro MDMB−BUTINACA.

Our study first demonstrates that the GC–MS/MS method developed for analyzing 4−Fluoro MDMB−BUTINACA in rat plasma is both effective and reliable for routine pharmacokinetic and toxicological analyses. The optimized method showed high sensitivity, selectivity, and accuracy, as evidenced by precise and reproducible results across various concentration levels, with low relative standard deviations (RSD) and minimal bias. This confirms the method’s suitability for detecting and quantifying 4−Fluoro MDMB−BUTINACA in biological samples.

To investigate the pharmacokinetic profile of this synthetic cannabinoid, a group of six Wistar albino rats were orally administered a dose of 50 mg/kg of 4−Fluoro MDMB−BUTINACA. The pharmacokinetic analysis revealed rapid absorption, extensive tissue distribution, and moderate elimination, with a significant volume of distribution and a half-life of approximately 2.37 h. However, due to high mortality rates, data beyond 6.5 h could not be obtained, which limits the full assessment of the drug’s pharmacokinetic profile.

To enhance our understanding of the toxicity of 4−Fluoro MDMB−BUTINACA, four groups of rats were administered doses of 1, 5, and 15 mg/kg/day, while one group served as a control. This study reveals that lower doses (1 mg/kg) resulted in minimal adverse effects. In contrast, higher doses (5 mg/kg and 15 mg/kg) led to severe toxicity and high mortality rates. Notably, the group treated with the highest dose (15 mg/kg/day) exhibited 100% fatality, highlighting the concentration-dependent toxicity of 4F-MDMB-BUTINACA. These findings suggest a clear correlation between increasing doses and escalating toxic effects, with a marked threshold at the 15 mg/kg/day dosage. This finding is enhanced by significant hematological and biochemical changes, including reductions in white blood cell (WBC) counts associated with alterations in liver and kidney function markers. Histopathological analyses further support these results, demonstrating dose-dependent hepatic and renal damage.

Although the optimized liquid–liquid extraction (LLE) and gas chromatography–tandem mass spectrometry (GC–MS/MS) methods provided initial pharmacokinetic and toxicological profiles, this study is limited by the high mortality rate in the animal model, which restricted the ability to collect longitudinal data beyond 6.5 h. Additionally, the toxic concentrations associated with the fatalities may be specific to the route of administration and the animal species used. Thus, these factors should be considered in future studies. It is recommended that various animal models and routes of administration be investigated to explore the long-term effects of 4−Fluoro MDMB−BUTINACA and to determine its safety profile across different models, aiming for a better understanding of the concentration associated with severe toxic effects.

Finally, our study is the first that demonstrates that 4−Fluoro MDMB−BUTINACA produces dose-dependent toxicity in Wistar albino rats. Daily oral administration of a 1 mg/kg dose caused mild adverse effects, including slight hematological changes and minor hepatic alterations. In contrast, daily oral administration of a 5 mg/kg dose resulted in severe toxicity, characterized by high mortality, significant hematological and biochemical disturbances, and notable organ damage. A histopathological examination provided further evidence of dose-dependent toxicity to the liver, kidneys, and heart, correlating with elevated liver enzymes and altered blood parameters. A histopathological analysis of cardiac tissue after seven days of treatment revealed dose-dependent cardiotoxicity induced by 4F-MDMB-BUTINACA. In Group 2 (1 mg/kg), early signs of toxicity were evident, including vacuolar degeneration of myocardial fibers, inflammatory cell infiltration, and congestion, suggesting that even low doses can induce cardiac damage. Group 3 (5 mg/kg) showed more severe alterations, characterized by vacuolar degeneration, nuclear swelling of myocardial cells, increased eosinophilia, moderate interstitial inflammation, vascular wall thickening, and marked vascular congestion and dilatation. These findings highlight the escalating severity of cardiac injury with increasing doses, supporting the concentration-dependent toxic effects of 4F-MDMB-BUTINACA.

Limitations of this study include the short duration of exposure and the use of a single animal model, which may not fully represent long-term or species-specific impacts. These findings align with the existing literature on synthetic cannabinoids and underscore the need for cautious use and further investigation into their long-term impacts.