Chemicals and reagents

Cerilliant (Round Rock, TX) standard solutions of fentanyl, para-fluorofentanyl (pFF) and fentanyl-D5 were purchased from Sigma-Aldrich in methanol for preparation of calibration curves (St. Louis, MO). Fentanyl-D5 was used as an internal standard for both compounds. Lithium heparin Sprague–Dawley rat plasma that was used as the matrix for plasma samples was obtained from Innovative Research (Novi, MI). Fentanyl hydrochloride and para-fluorofentanyl hydrochloride for animal experiments were purchased from Cayman Chemical Company (Ann Arbor, MI). Drug solutions for animal treatments were prepared fresh each day and were made in saline (Moltox, Boone, NC) using hydrochloride salt forms of fentanyl and pFF at a concentration of 300 μg/mL. Mobile phase A consisted of 0.1% acetic acid (100% LC–MS grade) purchased from Millipore Sigma (Darmstadt, Germany) and water (LC–MS grade) purchased from Fisher Chemical (Waltham, MA). Mobile phase B consisted of pure methanol (LC–MS grade) from Fisher Chemical (Waltham, MA). Formic acid (LC–MS grade) and acetonitrile (LC–MS grade) were purchased from Fisher Chemical (Waltham, MA). The compressed 5.0 ultra-high purity (UHP) grade argon gas tank used for the collision gas was purchased from Linde (Danbury, CT). The nitrogen gas tank that was used for the evaporation step was a nitrogen compressed gas tank obtained from Linde (Danbury, CT). A nitrogen generator was used to supply the heating, drying, and nebulizing gases from SouthTek (Wilmington, NC).

Standard preparation

A stock solution was prepared by combining and then diluting the two individual methanol standards to a concentration of 1,000 ng/mL in methanol. This solution was then further diluted to 100 ng/mL and 10 ng/mL in methanol to create stock solutions for all calibrators. The calibrators, when spiked into plasma (100 μL), were at final concentrations of 100, 75, 50, 25, 10, 5, 1, and 0.5 ng/mL. Quality control methanol stocks were created by combining and then diluting individual standards to a concentration of 800 ng/mL (high QC), 40 ng/mL (medium QC) and 1,500 ng/mL (low QC) in methanol. The low QC stock was then further diluted to a final concentration of 15 ng/mL in methanol. When spiked into plasma (100 μL), the final concentrations of each quality control were as follows: 80 ng/mL (high QC), 40 ng/mL (medium QC), and 1.5 ng/mL (low QC). The internal standard solution was prepared by diluting fentanyl-D5 to a final concentration of 100 ng/mL in methanol. All solutions were stored in the freezer at −20 ℃ in amber vials until use.

Plasma preparation methods

Plasma was prepared using protein precipitation with chilled crash solution (1% formic acid in acetonitrile). Briefly, 300 μL of crash solution, 100 μL of spiked, blank, or animal experiment plasma and 30 μL of internal standard solution were aliquoted to a 1.5 mL microcentrifuge tube and placed in an Eppendorf F1.5 ThermoMixer (Eppendorf, Enfield, CT) for 10 min at a speed of 1500 rpm. The crashed plasma sample was then centrifuged for 5 min at 7,900 rpm. The supernatant was drawn off, transferred to HybridSPE phospholipid filters (Supelco, Bellefonte, PA) and extracted by vacuum. The remaining acetonitrile in the extractant was evaporated by a direct flow of nitrogen gas. The extractant was then reconstituted in 100 μL of water + 0.1% acetic acid and injected at an injection volume of 3 μL onto the liquid chromatography tandem mass spectrometer (LC–MS/MS).

Brain region preparation methods

Individual brain regions were weighed and then processed by tissue homogenization. Briefly, 500 μL of mobile phase A was aliquoted to a 1.5 mL microcentrifuge tube. Then, the brain region sample was added and homogenized using a Benchmark D1000 Homogenizer (Benchmark Scientific, Sayreville, NJ) for 10 s on a setting of 2. The homogenized sample was then centrifuged for 5 min at 7,900 rpm. 100 μL of supernatant was drawn off, transferred to a microvial insert and 30 μL of internal standard solution was added. A corresponding calibration curve containing both fentanyl and pFF was prepared in mobile phase A and all samples and calibrators were then injected at an injection volume of 3 μL onto the liquid chromatography tandem mass spectrometer (LC–MS/MS). The method used was modified from our previously validated plasma method for fentanyl (Canfield and Sprague 2023) to include pFF. Mobile phase A was used as the matrix for all calibrators to correspond with the solution used for brain homogenization. Concentrations of both compounds in each brain region were then quantified using LabSolutions Insight software (version 5.93) and then the concentration per tissue was calculated in ng/g. In order to evaluate for any potential matrix effects, five (5) samples of pre-frontal cortex were used from rats that were not treated with fentanyl or pFF. We utilized the post-extraction addition method for assessing matrix effects according to the Academy Standards Board: Standard Practice for Method Validation in Forensic Toxicology (American Academy of Forensic Sciences, 2019). The area under the curve of five post-extraction addition high concentration quality control (HQC) brain samples were compared to the area under the curve of five neat HQC samples. Matrix effects were deemed acceptable within ± 25%.

Liquid chromatography tandem mass spectrometry methods

A triple-quadrupole LCMS-8050 CL from Shimadzu U.S.A. manufacturing (Canby, OR) was utilized for sample analysis with a gradient separation method. The method utilized water + 0.1 vol% acetic acid and 100% methanol for the mobile phases and the flow rate was a constant 0.75 mL/min at 70 ℃ over the 6.75 min gradient between 1 and 99% MeOH with a total method run time of 10 min. The gradient began with 1% mobile phase B until 5 min and then the concentration increased to 55% mobile phase B. The gradient then held until 6.76 min where the concentration again increased to 99% mobile phase B. This was held until 8 min where equilibration occurred before the next sample by decreasing mobile phase B back to 1% until the end of the 10-min run. The stationary phase consisted of a Raptor 50 mm X 2.1 mm, 2.7 µm, biphenyl column (Restek, Bellefonte, PA) for the separation of the analytes and a Raptor guard column (Restek, Bellefonte, PA). MRM transitions, Q1 and Q3 pre-bias voltages, collision energy, and retention time for each compound are displayed in Table S1. Concentrations were quantified using LabSolutions Insight software (version 5.93). Representative chromatograms for a plasma calibrator and plasma sample for fentanyl and pFF are displayed in Figure S1. Representative chromatograms for a brain region calibrator and brain region sample for fentanyl and pFF are displayed in Figure S2.

Method validation

The method used for drug quantification in plasma was previously validated for fentanyl (Canfield and Sprague 2023) according to the Academy Standards Board: Standard Practice for Method Validation in Forensic Toxicology (American Academy of Forensic Sciences, 2019). Therefore, the plasma methods for pFF only were validated prior to sample testing. Parameters assessed were calibration model, bias, precision, limit of detection (LOD), lower limit of quantification (LLOQ), ionization suppression/enhancement, carryover and interference studies.

Calibration models/linearity were determined using the 8 non-zero calibrators indicated above over 5 days in rat plasma matrix samples. Models were then evaluated using residual plots and R2 values.

LLOQ and LOD were both defined as the lowest non-zero calibrator (0.5 ng/mL) and were assessed in triplicate over 3 days. Precision (within-and between-run) and bias were assessed for LLOQ and LOD, and % CV and % bias were calculated and determined to be acceptable within ± 20%. Precision (within- and between-run) and bias were assessed using three QC (quality control) concentrations (high, medium, and low) in triplicate over 5 runs in rat plasma. % CV and % bias were then calculated and determined to be acceptable within ± 20%. % CV values for within-run and between-run precision for all LLOQ, LOD and QC samples were calculated using the ANOVA approach as defined in the Academy Standards Board: Standards Practice for Method Validation in Forensic Toxicology Sect. 8.2.2.3.4 (American Academy of Forensic Sciences, 2019).

Ionization suppression/enhancement was determined using the post-extraction addition (American Academy of Forensic Sciences, 2019). Ten different lots of Sprague–Dawley rat plasma were fortified with low and high QCs and ISTD before and after extraction. Ten neat samples were also prepared at the low and high QC concentrations in mobile phase A. Matrix effects were determined by comparing area ratios of post-extraction samples to neat samples and extraction recovery was also determined by comparing the area ratios of pre- and post-extraction samples. Matrix effects were deemed acceptable within ± 25%.

Carryover was assessed by reinjection of an extracted blank matrix after the highest calibrator over 5 days. Carryover was determined to be negligible if analyte response in the blank was < 10% of the analyte response in the lowest calibrator.

Matrix interferences were assessed by the analysis of ten different lots of Sprague–Dawley rat plasma that were not fortified. This was to ensure the matrix itself was not interfering with pFF. Internal standard interference was assessed by analyzing a blank sample with internal standard to assess whether the internal standard was interfering with the detection of pFF. Analyte interference was assessed by analyzing a spiked sample without the addition of internal standard. Exogenous interferences were not assessed for this method due to the studies involving controlled drug-administration to drug-free rats.

Animal experiments

For PK/PD experiments, animals were housed one per cage (cage size: 21.0 × 41.9 × 20.3 cm) at a room temperature of 24–25 ℃, maintained on a 12:12-light/dark schedule. Food and water were provided ad libitum. The same was true for the brain concentration study except animals were housed two per cage and were given a 1-week acclimation period. Animal maintenance and research were conducted in accordance with the eighth edition of the Guide for Care and Use of Laboratory Animals as set forth by the National Institutes of Health, and all protocols were approved by the Bowling Green State University Animal Care and Use Committee (Protocol Number: 2109741–2). All methods were carried out in compliance with relevant institutional, Federal and ARRIVE guidelines and regulations. At the completion of the study, animals were euthanized by carbon dioxide exposure.

Pharmacokinetic/pharmacodynamic experiments

Jugular vein cannula (JVC) male, Sprague–Dawley rats were obtained from Envigo (Indianapolis, IN). Seventeen animals were randomly allocated to one of three treatment groups: fentanyl (n = 6; 326.92 ± 3.99 g), para-fluorofentanyl (n = 6; 330.00 ± 4.49 g), and a saline control (n = 5; 325.90 ± 4.00 g). Time points for the study were 0 min, 30 min, 60 min, 120 min, 240 min, and 480 min post-dosing with 300 μg/kg sc. dose of each compound or 300 μL/kg saline. This dose was determined by a preliminary righting reflex study performed in our lab and based on our previous study (Canfield and Sprague 2023) and on the maximum dose of cyclopropylfentanyl used by Bergh et al. (2021). For pharmacokinetic evaluation, 400 μL of blood was drawn and then centrifuged at 7,900 rpm for 5 min and the plasma was pipetted into a microcentrifuge tube and placed in the freezer at −20 ℃ until analysis. On the day of LC–MS/MS testing, a fresh calibration curve was made, and the calibrators underwent the same extraction procedure as the samples. Concentrations of each compound were then quantified using LabSolutions Insight the same way as with the validation. Following the blood draw, pharmacodynamic parameters were assessed by performing a tail flick assay and measuring basal core body temperature at each time point. Core body temperature was recorded using a rectal probe thermometer. The rectal probe thermometer was a Thermalert Model TH-8 Temperature Monitor obtained from Physitemp (Clifton, NJ). The probe was lubricated with petroleum jelly and gently inserted into the rectum until a steady temperature was reached. Tail flick was assessed using a tail flick analgesia meter obtained from PanLab (Barcelona, Spain). The tail flick assay was performed on the middle third of the tail in triplicate using a focus of 70 and a 10 s cutoff to prevent tissue damage. After the baseline was established, if the tail flick assay timed out then subsequent replicates were not conducted.

Brain concentration experiments

To determine the brain concentrations of fentanyl and pFF, 12 male, Sprague–Dawley rats were obtained from Envigo (Indianapolis, IN). The 12 animals were randomly allocated to one of two treatment groups: fentanyl (n = 6; 355.00 ± 2.86 g) and para-fluorofentanyl (n = 6; 356.17 ± 1.25 g). Animals were treated with a 300 μg/kg sc. dose of either fentanyl or pFF. After 1 h, animals were killed by carbon dioxide exposure and the medulla, frontal cortex, hippocampus and striatum were dissected on ice. A blood sample was also obtained at this time point and processed as described above. Brain samples were immediately flash-frozen in liquid nitrogen and then transferred to a -80 ℃ freezer until analysis. Brain region concentrations of both drugs were determined using LabSolutions Insight (version 5.93) in ng/mL and then converted to ng/g of tissue using the weights of the whole tissue. A brain to plasma ratio was then calculated based on brain concentration of fentanyl or pFF in each region divided by the plasma concentration of the corresponding compound taken after 1 h in the same animal.

Pharmacokinetic and statistical analyses

All parent compound plasma concentration versus time rectangular plots were constructed and noncompartmental analysis was conducted utilizing Phoenix WinNonlin software (Version 8.3) to estimate PK parameters. The plasma Cmax was determined to be the highest observed plasma concentration and the corresponding timepoint was determined to be the Tmax. The terminal elimination rate constant (λz) was calculated by linear regression of the observed terminal natural log concentration versus time data and half-life (T½) was calculated as 0.693/λz. Pharmacokinetic data are presented as the mean ± S.E.M. for both study groups. The AUC0-∞ represents the total area under the plasma curve from time zero to infinity and was calculated using the linear trapezoidal rule with the terminal AUC being calculated as the last measured concentration divided by λz. VD/F is the ratio of the total dose present in the body to the plasma concentration when the distribution of the drug between the tissues and the plasma is at equilibrium following extravascular dosing. The Clp/F is the plasma clearance of the drug and is calculated as dose/AUC0-∞.

All statistical analyses of data were performed using R (version 4.2.2). Raw time course data for tail flick response and body temperature were normalized to percent maximum possible effect (%MPE, 10 s) or change from baseline for each rat (Δ temperature in °C), respectively. MPE was calculated using the following equation:

$$\frac baseline measure}}}} baseline measure }}}} \times $$

Normalized data are presented as mean ± S.E.M, where applicable. The normalized time course data were analyzed by an ANOVA followed by a Tukey’s post hoc test. To calculate the maximum change in temperature (maximum ∆ ℃), the maximum increase or decrease in core body temperature was compared to the animal’s baseline temperature. Maximum change in temperature was analyzed using a Student’s t test between fentanyl and pFF groups. Plasma concentrations and brain concentrations are presented as mean ± S.E.M, where applicable. All plasma pharmacokinetic parameters and brain concentrations were compared between fentanyl and pFF using a Student’s t test. All differences were considered significant with a p value < 0.05.