Pyrantel pamoate (purity 99.7%) standard product was purchased from National Institutes for Food and Drug Control (Beijing, China). Standard products of PRA (purity 99.6%), FBT (purity 99.7%) and FEN (purity 100%) were purchased from China Institute of Veterinary Drug Control (Beijing, China). OXF (purity 96.75%) standard product was purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany). FEN–D3 standard product, as an internal standard (IS), was purchased from Shanghai Zhenzhun Biomedical Technology Co., Ltd. (Shanghai, China). HPLC grade acetonitrile and methanol were obtained from Merck (Darmstadt, Germany). LC–MS grade formic acid was obtained from Anaqua Chemicals Supply (Wilmington, DE, United States). N, N–dimethyl–formamide (DMF) was procured from Macklin (Shanghai, China). LC–MS grade ammonia solution was procured from Aladdin (Shanghai, China). Analytical grade ethyl acetate was procured from Guangdong guanghua Sci–Tech Co., Ltd. (Guangdong, China). Deionized water used during the entire analysis was purified using a Direct–Q5 UV system from Millipore (Boston, MA, United States). Blank dog plasma used in the experiment was collected from different beagle dogs and stored at −80°C until use (The Experimental Animal Center of Nanjing Agricultural University, Nanjing, China).

Quantitative analysis was performed on an ACQUITY™ UPLC system (Waters Corp., Milford, MA, United States), coupled with a Waters Xevo TQD triple quadrupole mass spectrometer (Waters Corp., Milford, MA, United States) equipped with electrospray ionization (ESI) and a Nitrogen generator (Peak Corp., Glasgow, United Kingdom). The analytes were separated on Waters Acquity UPLC BEH C18 (2.1 × 50 mm, 1.7 μm) with a thermostated column oven maintained at 35°C. The mobile phase consisted of solvent A (acetonitrile) and solvent B (0.1% formic acid) at a flow rate of 0.4 mL/min according to the following linear gradient: 0–6.0 min, 10%–100% A; 6.0–6.5 min, 100% A; 6.5–7.0 min, 100%–10% A; 7.0–9.0 min 10% A. The autosampler temperature was set at 6°C, and the injection volume was 5 μL.

Mass spectrum analysis was performed in the positive ionization mode under multiple reaction monitoring by monitoring ion transitions of m/z 207.1 > 150 for PYR, m/z 313.2 > 203.1 for PRA, m/z 447.1 > 415.1 for FBT, m/z 300 > 268.1 for FEN, m/z 316 > 159 for OXF and m/z 303 > 268.1 for IS, respectively. The source parameters for mass spectrum analysis were as follows: capillary voltage, 1.0 kV; source temperature, 150°C; desolvation temperature, 500°C; desolvation gas flow, 800 L/h; cone gas flow, 50 L/h; dwell time, 80 ms. The cone voltages were 40 V for PYR, FEN, OXF and IS, 35 V for PRA and 30 V for FBT, and collision energies were 27 eV for PYR, 15 eV for PRA, 13 eV for FBT, 20 eV for FEN, 36 eV for OXF and 20 eV for IS, respectively. Mass Lynx software version 4.1 was used to controll all UPLC and MS parameters.

Individual stock solutions of PYR, PRA, FBT, FEN and OXF were prepared for standards and quality control samples in DMF at 1 mg/mL. IS stock solution was also prepared in DMF at 400 μg/mL. The mixed working solutions were obtained by mixing the stock solution in different volumes and then performing appropriate dilution in methanol: water (50:50, V/V). The IS working solution of 200 ng/mL was also prepared with DMF from the primary stock solution. All the primary stock solutions were stored at −20°C for more than 2 months. The working solutions for the analytes and IS were stored at 4°C and brought to room temperature before use.

Calibration standards (CSs) and quality control (QC) samples were prepared by spiking 10 μL mixed woking solutions to 90 μL of blank dog plasma. CSs of PYR and OXF were made at 4, 8, 16, 40, 80, 160 and 240 ng/mL concentrations, while QC samples were prepared at 180 ng/mL (high quality control, HQC), 120 ng/mL (middle quality control, MQC), 12 ng/mL (low quality control, LQC) and 4 ng/mL (LLOQ). For PRA, CSs were made at 15, 30, 60, 150, 300, 600 and 900 ng/mL concentrations, while QC samples were prepared at 675 ng/mL (HQC), 450 ng/mL (MQC), 45 ng/mL (LQC) and 15 ng/mL (LLOQ). For FBT, CSs were made at 2, 4, 8, 20, 40, 80 and 120 ng/mL concentrations, while QC samples were prepared at 90 ng/mL (HQC), 60 ng/mL (MQC), 6 ng/mL (LQC) and 2 ng/mL (LLOQ). For FEN, CSs were made at 10, 20, 40, 100, 200, 400 and 600 ng/mL concentrations, while QC samples were prepared at 450 ng/mL (HQC), 300 ng/mL (MQC), 30 ng/mL (LQC) and 10 ng/mL (LLOQ).

FEN–D3 was selected as an internal standard. FEN–D3 is highly similar in chemical structure to FEN. When there are complex matrix components in the sample, FEN-D3 can undergo similar physicochemical processes together with FEN, reducing the errors caused by the matrix effect. For PYR, PRA, FBT and OXF, although their chemical structures are not exactly the same as that of FEN, the stability and behavioral characteristics of FEN-D3 in the entire detection system can serve as a reference standard. During the simultaneous detection of multiple components, the stability of its chemical properties helps to provide a relatively stable signal in the complex instrumental analysis environment, which can be used to compare and calibrate the detection signals of other drugs.

For analysis, 100 μL of plasma was mixed with 10 μL of IS woking solution (200 ng/mL), followed by vortexing for 10 s. Then, 50 μL of 1 M ammonia solution and 50 μL of DMF were added and vortexed for 30 s. After vortexing, 500 μL of acetonitrile and 800 μL of ethyl acetate were added, then vortexed for 5 min, and centrifuged at 12,000 r/min for 7 min at 4°C. The supernatant was transferred to a new 5 mL centrifuge tube. In the remaining precipitation, 200 μL of acetonitrile and 800 μL of ethyl acetate were added, then vortexed for 5 min and centrifuged at 12,000 r/min for 10 min at 4°C. The two supernatants were mixed in the 5 mL centrifuge tube and then evaporated to dryness under nitrogen stream at 40°C. The residue was reconstituted with two dilution solvents containing 500 μL of acetonitrile: water (1:1, V/V) and 500 μL of acetonitrile: water (2:8, V/V). After samples were filtered by 0.22 um filter membrane, a 5 μL aliquot was injected into the UPLC–MS/MS system.

The specificity and selectivity of this method was documented by analyzing six blank plasma samples from different dogs and spiked plasma samples at the LLOQ concentrations and the upper limit of quantification (ULOQ) concentrations for each analyte. The peak area of the interference components should be less than 20% of the peak area of the LLOQ standard and less than 5% of the peak area of the IS.

Calibration curves were acquired by plotting the peak area ratios of the transition pair of analytes to that of IS versus the nominal concentration using a linearly weighed (1/x) least squares regression method in duplicate. Calibration curves were considered acceptable when the correlation coefficient (r2) was greater than 0.99 and the acceptance criterion for each back–calculated standard concentration was ±15% deviation from the nominal value except at LLOQ, which was set at ± 20%.

Intra–batch and inter–batch precision and accuracy were determined by analyzing five replicates at LLOQ in addition to three different QC levels as described above on three consecutive days. The mean accuracy should be within ±15%, except for the LLOQ where it can be within ±20% of the nominal concentration. Similarly, the precision (coefficient of variation, CV) should not exceed 15%, except for the LLOQ where it can be less than 20%.

The extraction recoveries of PYR, PRA, FBT, FEN, OXF and IS from dog plasma were evaluated in six replicates by comparing the mean peak area responses of pre–extraction fortified samples at LQC, MQC and HQC levels with those of post–extraction fortified samples at the same concentrations.

The matrix factors of each analyte and IS were determined by comparing the mean peak area responses of post–extraction fortified samples at three QC levels with those of solutions prepared in moblile phase solutions (neat samples) at equivalent concentrations. The IS–normalized matrix factors were calculated by dividing the matrix factor of the analytes by the matrix factor of the IS. Since it is impossible to completely eliminate the effect of the matrix, the consistency in matrix effect is examined in different plasma sources. The precision should be ≤15% for the IS–normalized matrix factors at each level.

Dilution reliability was investigated to ensure that samples with concentrations above ULOQ could be diluted with blank matrix without affecting accuracy and precision. PYR and OXF were spiked at a concentration of 720 ng/mL in dog plasma, while PRA, FBT and FEN were spiked at concentrations of 2,700, 360 and 1,800 ng/mL. All of the analytes were diluted with pooled dog plasma five folds in five replicates and analyzed. Acceptance criteria were defined as precision (CV%) not exceeding 15%, and accuracy being within ±15% of the nominal concentration.

Stability experiments were conducted in working solutions of the analytes and IS for short term stability at 4°C and stock solutions for long term stability at −20°C, respectively. Peak area ratio responses (Analytes/IS) of stored solutions were compared with those of freshly prepared solutions. The acceptance criterion was ±10.0% deviation when comparing the values of fresh solutions. The stabilities of the analytes in beagle dog plasma were evaluated by analyzing five replicates at LQC and HQC levels. Types of stabilities were following as: bench top stability (room temperature), refrigerator stability (4°C), autosampler stability for processed samples (6°C), freeze–thaw stability (−80°C), short term stability (−20°C) and long term (–80°C) storage stability. The samples were considered stable if the accuracy was within ±15% of the nominal concentration and the precision (CV%) were ≤15%.

Carry–over effect was estimated by injecting blank samples after injecting calibration standards at the ULOQ concentrations. The measured peak area should not exceed 20% of the LLOQ and 5% of the IS.

The study design was an open label, balanced, randomized, crossover, two–treatment, two–period, two–sequence and single–dose study to investigate the bioequivalence between a single oral dose of Drontal® Plus Tasty containing 150 mg FBT, 144 mg pyrantel pamoate and 50 mg PRA (BAYER, Germany) and self–developed compound febantel tablets of the same specification in 40 beagle dogs under fasting conditions. Adaptive feeding and observation were carried out for 7 days before the bioequivalence study. Physical examination, blood routine and blood biochemical tests were performed the day before the trial to ensure the health of the dogs. The study protocol was conducted in accordance with the Ethical Guidelines for Investigations in Laboratory Animals and approved by the Ethics Committee of Nanjing Agricultural University (Nanjing, China). Prior to the study, the dogs were fast and maintained with water for 12 h. The subjects were orally administered a single dose of test or reference formuations with 100 mL of water with a washout period of 2 weeks. Blood samples were collected into heparinized tubes at 0.00 (pre–dose), 0.17, 0.33, 0.67, 1.00, 1.50, 2.00, 2.50, 3.00, 4.00, 5.00, 6.00, 7.00, 8.00, 9.00, 10.00, 11.00, 12.00, 14.00, 24.00 and 30.00 h after oral administration. However, two dogs vomited out pills during blood sample collection in this study. Therefore, only 38 dogs completed the crossover process. The number of blood collections for drug analysis was 21 samples in each study period. The collected blood samples were centrifuged in a pre–cooled centrifuge at 2,150 g for 10 min, and the separated plasma was transferred to a clean centrifuge tube and frozen in a −80°C refrigerator until analysis. During the entire study, subjects had a standard diet while water intake was unmonitored.

The pharmacokinetic parameters for the analytes were evaluated by non–compartmental analysis using Phoenix WinNonlin software version 8.1 (Certara Corporation, Radnor, PA, United States). The maximum plasma concentration (Cmax), the area under the plasma concentration-time curve from time zero to the last sampling time (AUC0–30), the area under the plasma concentration-time curve from time zero to infinity (AUC0–inf), as the primary target, and their geometric mean ratios (test/reference) using log transformed data were assessed to determine whether the formulations were pharmacokinetically equivalent. The drug formulations were considered pharmacokinetically equivalent if the geometric mean ratios and 90% confidence intervals for these parameters were within 80%–125%.

The aim of this study was to provide a bio–sample detection method for the bioequivalence study of compound febantel tablets in dogs. FEN–D3, as an isotope–labeled internal standard, can maximize the elimination of errors in the analysis process. Considering the following points: (1) FBT is metabolized rapidly and the absorption of PYR is limited, resulting in relatively low blood drug concentrations of both. (2) Given that a substantial quantity of samples is involved in the bioequivalence study, it is essential to minimize the time required for sample pretreatment and elution analysis to the greatest extent possible. (3) As five target analytes need to be determined concurrently, a high level of separation is demanded. Therefore, in order to develop and validate a rapid, sensitive, selective and simple analytical method for the extraction and quantification of PYR, PRA, FBT and its active metabolites, FEN and OXF in dog plasma, the extraction procedure, mass spectrum paramaters and chromatographic separation condition were suitably optimized.

Chromatographic conditions were optimized to obtain sharp peak shape, high response and short run time for the analytes and IS, with the improvement on base–line noise and reduction on solvent consumption. Different mobile phase composition and mobile phase additives were tested for optimal resolution, speed and sensitivity on Waters Acquity UPLC BEH C18 (50 mm × 2.1 mm, 1.7 μm) column. Different conbinations of acetonitrile or methanol and additives such as formic acid and ammonium formate in different volume ratios were tried. It was observed that acetonitrile was selected as the organic modifier since it provided shorter run time and sharper peak shape for the anlytes and IS compared to methanol. What’s more, formic acid provided better peak shape and reponse than ammonium formate in the positive mode. Finally, the best chromatographic conditions were acquired using acetonitrile with 0.1% formic acid (10:90, V/V) as initial mobile phase under a gradient elution mode. The retention time for PYR, PRA, FBT, FEN, OXF and FEN–D3 was 1.41 min, 3.55 min, 4.28 min, 3.29 min, 2.17 min and 3.29 min, respectively. The reproducibility of retention times for the analytes, expressed as CV, was ≤0.67% for 100 injections on the same column.

The calibration curve was linear over the range of 4–240 ng/mL for PYR and OXF, 15–900 ng/mL for PRA, 2–120 ng/mL for FBT, 10–600 ng/mL for FEN. A quadratic, 1/x, least–squares regression algorithm was used to plot the peak area ratio (analyte/IS) from MRM versus concentration. The calibration curve equation is y = ax + c, where y represents analyte/IS peak area ratio and x represents the plasma concentration of the analytes. The representative equations of the calibration curves were y = 0.0383196 x – 0.0142732, r2 = 0.999138 for PYR, y = 0.00980901 x – 0.00605049, r2 = 0.999027 for PRA, y = 0.0336798 x + 0.00546005, r2 = 0.998914 for FBT, y = 0.0433366 x– 0.0112174, r2 = 0.999105 for FEN and y = 0.0175517 x – 0.00655116, r2 = 0.999320 for OXF. The LLOQs were 4 ng/mL for PYR and OXF, 15 ng/mL for PRA, 2 ng/mL for FBT and 10 ng/mL for FEN and were adequate for PK studies following oral administration of compound febantel tablets.

Spiked dog plasma samples prepared at concentrations above ULOQ for all the analytes were diluted with five folds in five replicates and analyzed. The accuracy results for PYR were between 90.90% and 99.76%, for PRA were between 85.93% and 95.74%, for FBT were between 86.11% and 99.53%, for FEN were between 86.03% and 98.56%, for OXF were 86.90% and 97.50%, respectively, while the precisions (CV) for PYR were 4.44%, for PRA were 4.01%, for FBT were 5.65%, for FEN were 5.18% and for OXF were 4.44%, respectively. These data demonstrate that this dilution method has no impact on the accuracy and precision of sample detection.

No residues of the analytes and internal standard were detected in the extracted blank sample (without analytes and IS) after subsequent injection of the ULOQ.

The bioequivalence study based on plasma drug concentration represents an efficacious approach to determine the pharmaceutical equivalence or the pharmaceutical substitutability of two products containing the same active substance. As the Cmax of the parent compound is more sensitive to detecting absorption rate differences among dosage forms than that of the metabolite, the bioequivalence is evaluated based on the concentration of the parent compound. Nevertheless, to comprehensively understand the pharmacokinetic characteristics of the test formulation, the plasma drug concentrations of the metabolites were also determined and their corresponding pharmacokinetic parameters were analyzed. However, the bioequivalence of the metabolites was not calculated. Therefore, in the bioequivalence study of the compound febantel tablets, it is necessary to detect the plasma drug concentrations of five components, namely, PYR, PRA, FBT, FEN and OXF.

The UPLC–MS/MS analytical method was successfully applied to determine the analyte concentrations in dog plasma samples from the bioequivalence study. The study was conducted as an open label, balanced, randomized, crossover, two–treatment, two–period, two–sequence and single–dose design to compare the bioavailability of PYR, PRA, FBT, FEN and OXF between two products in 38 healthy beagle dogs. Each subject received a tablet from the test product (Self–developed preparation) and a tablet from reference product (Drontal® Plus Flavor) under fasting conditions with a washout period of 2 weeks.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.

The animal studies were approved by the Ethics Committee of Nanjing Agricultural University. The studies were conducted in accordance with the local legislation and institutional requirements. Written informed consent was obtained from the owners for the participation of their animals in this study.

FZ: Formal Analysis, Investigation, Methodology, Validation, Writing–original draft, Writing–review and editing. FG: Data curation, Formal Analysis, Methodology, Writing–review and editing. XZ: Investigation, Writing–review and editing. CL: Investigation, Writing–review and editing. JW: Investigation, Writing–review and editing. MW: Investigation, Writing–review and editing. RM: Investigation, Writing–review and editing. BC: Investigation, Writing–review and editing. QM: Investigation, Writing–review and editing. YW: Investigation, Writing–review and editing. ZW: Investigation, Writing–review and editing. DC: Investigation, Writing–review and editing. ZY: Conceptualization, Funding acquisition, Project administration, Resources, Writing–review and editing.

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by National Natural Science Foundation of China.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declare that no Generative AI was used in the creation of this manuscript.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.