Pharmacokinetics of matrine and amoxicillin in pigs after gavage administration of each drug alone and in combination

Pharmacokinetics of matrine and amoxicillin in pigs after gavage administration of each drug alone and in combination | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Pharmacokinetics of matrine and amoxicillin in pigs after gavage administration of each drug alone and in combination RuoNan Li, DanNa Zhou, HuiYu Hu, FuHao Wang, XiaoLing Lv, Lei Sun, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4566584/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Matrine (MT) exhibits antibiotic resistance reversal and antiviral activities in vitro . However, the in vivo efficacies of MT and amoxicillin (AMO)-MT combination cannot be accurately evaluated due to the lack of pharmacokinetics (PK) data. The aim of this study was to investigate the PKs of MT and AMO in pigs after gavage administration of each drug alone and in combination. Results Twenty-four pigs were randomly distributed to three treatments, namely group A (MT, 50 mg/kg), group B (AMO, 50 mg/kg), and group C (MT, 50 mg/kg + AMO, 50 mg/kg). The maximum concentration (C max ), area under the curve from time 0 to 36 h (AUC 0 → 36 h ), apparent volume of distribution (V d ) and total body clearance (Cl B ) for group A were 1389.73 ± 320.05 µg/L, 4138.16 ± 1245.23 h*µg/L, 63.08 ± 22.24 L/kg and 12.98 ± 3.71 L/h/kg, respectively, versus 21153.92 ± 16967.88 µg/L ( P < 0.01), 40239.83 ± 22044.18 h*µg/L ( P < 0.01), 9.93 ± 3.86 L/kg ( P < 0.01), and 1.61 ± 0.81 L/h/kg ( P < 0.01) for group B. Significantly greater T max , Cl B , V d ( P < 0.05) and lower C max , AUC 0 → 36 h ( P < 0.05) were observed in group A compared to those in group C. Conclusions Compared with AMO, MT was poorly absorbed, extensively distributed and rapid eliminated in pigs. The PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PK drug-drug interaction between MT and AMO. Pharmacokinetics Matrine Amoxicillin Pig Drug-drug interaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Bacterial and viral infectious diseases have caused huge economic losses to global pig industry. Some phytochemicals have been found to have unique antibacterial and/or antiviral activities, and their use alone or in combination with existing antibiotics may become a promising anti-infective treatment strategy [ 1 ]. Matrine (MT, Fig. 1 A) is one of the major quinolizidine alkaloids isolated from Sophora flavescens Ait and Sophora alopecuroides [ 2 ]. Previous in vitro experiments showed that MT could reverse the resistance of Haemophilus parasuis to cefaclor [ 3 ], and restore the susceptibility of Escherichia coli ( E. coli ) to a variety of antibiotics [ 4 , 5 ]. Another study revealed the inhibitory effect of MT on biofilm formation of antibiotic-resistant E. coli [ 6 ], which helped to increase the susceptibility of these resistant strains to antibiotics and decrease their pathogenicity to hosts. In addition, MT also exhibited activity against porcine circovirus type 2 [ 7 – 10 ] and porcine reproductive and respiratory syndrome virus [ 7 , 8 , 11 ]. Such antibiotic resistance reversal and antiviral activity make MT a potential therapeutic or prophylactic drug for porcine colibacillosis, porcine circovirus associated disease and porcine reproductive and respiratory syndrome. Amoxicillin (AMO, Fig. 1 B) is a β-lactam antibiotic that was once widely used to treat porcine colibacillosis. Unfortunately, it is becoming ineffective due to increasing resistance to E. coli [ 12 ], which poses a serious challenge for the treatment of porcine colibacillosis. Using AMO in combination with MT may be an effective strategy for addressing this challenge. Nevertheless, it remains unclear whether MT can also exert antibiotic resistance reversal and antiviral activity in pigs. The lack of pharmacokinetics (PK) data is one of the important reasons for the inability to accurately evaluate the in vivo efficacy of MT. To our knowledge, the PK profiles of MT have been described in rats [ 13 – 17 ], dogs [ 18 ], rabbits [ 19 ], and humans [ 20 ], and the PKs of AMO have also been investigated in pigs [ 21 – 24 ], but no data are available on the PKs of MT or the combination of MT and AMO in pigs. The present study was intended to ascertain the PKs of MT and AMO in pigs after gavage administration of each drug alone and in combination. The pharmacokinetic drug-drug interaction (PDDI) between MT and AMO was also investigated. The results would provide theoretical bases for evaluating the in vivo efficacies of MT and AMO-MT combination. Results Method validation As shown in Fig. 2 , the qualitative ions were m/z 249.1→176.0, m/z 249.1→148.0 for MT and m/z 366.0→208.1, m/z 366.0→114.3 for AMO, respectively; the quantification ions were m/z 249.1→148.0 for MT and m/z 366.0 → 114.2 for AMO, respectively. Representative multiple reaction monitoring (MRM) chromatograms obtained from blank plasma samples, blank plasma samples spiked with MT or AMO, and plasma samples after administration of MT or AMO were presented in Figs. 3 and 4 . The retention times of MT and AMO were approximately 1.558 and 2.630 min, respectively. There were no endogenous interfering peaks observed at the retention times of MT and AMO. The peak area ratios of the analytes at the concentration of 1, 25, 100 µg/L were: MT 13.85 ± 1.26%, 12.52 ± 2.14% and 12.82 ± 1.88%; AMO 40.21 ± 3.21%, 50.02 ± 1.70% and 54.10 ± 2.82%, respectively. There were significant matrix suppression effects in porcine plasma samples. The matrix-matched calibration curves showed good linearity (MT: y = 4216.10x – 8298.30, r = 0.9990; AMO: y = 62.817x + 129.90, r = 0.9949) over the concentration range from 1 to 100 µg/L. The accuracy and precision of MT and AMO were listed in Table 1 . The accuracy ranged from − 18.59 to 14.37% for intra-day determination and − 15.18 to 13.54% for inter-day determination, respectively. The corresponding precision ranged from 1.99 to 7.87% and 0.71 to 7.62%, respectively. The developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods showed high sensitivity in determining MT and AMO with a lower limit of quantification (LLOQ) of 5 µg/L. The results of stability evaluation were listed in Table 2 . Good stability was observed for MT and AMO under the conditions mentioned above. Table 1 Precision and accuracy of the LC-MS/MS methods for the determination of MT and AMO in pig plasma samples Analyte Nominal concentration (µg/mL) Intraday (n = 6) Interday (n = 18) Determined concentration (µg/l) a RSD (%) RE (%) Determined concentration (µg/l) a RSD (%) RE (%) MT 5 4.13 ± 0.19 4.60 -17.33 4.24 ± 0.22 5.16 -15.18 4.32 ± 0.28 6.46 -13.55 4.27 ± 0.16 3.77 -14.65 100 96.40 ± 3.59 3.73 -3.60 96.27 ± 3.23 3.35 -3.73 96.49 ± 3.45 3.58 -3.51 95.90 ± 3.21 3.35 -4.10 500 537.28 ± 12.42 2.31 7.46 542.94 ± 11.99 2.21 8.59 549.67 ± 11.11 2.02 9.94 541.85 ± 10.80 1.99 8.22 AMO 5 4.74 ± 0.23 4.82 –5.19 4.40 ± 0.34 7.62 –12.07 4.38 ± 0.34 7.87 –12.43 4.07 ± 0.25 6.07 –18.59 100 113.82 ± 4.33 3.80 13.83 112.67 ± 1.64 1.45 12.67 113.38 ± 2.61 2.30 13.38 110.80 ± 3.16 2.85 10.80 500 571.83 ± 22.29 3.90 14.37 567.68 ± 4.01 0.71 13.54 567.38 ± 28.79 5.07 13.48 563.83 ± 28.68 5.09 12.77 Table 2 Stability of MT and AMO in pig plasma samples under various conditions (n = 5) Condition Nominal concentration (µg/mL) MT (n = 5) AMO (n = 5) Determined concentration (µg/mL) a RSD (%) RE (%) Determined concentration (µg/mL) a RSD (%) RE (%) –20°C, 7 days 5 4.39 ± 0.40 9.21 –12.24 4.57 ± 0.27 5.93 –8.52 100 89.23 ± 4.07 4.56 –10.77 93.89 ± 6.84 7.28 –6.11 500 470.39 ± 30.46 6.48 –5.92 540.48 ± 25.61 4.74 8.10 Room temperature, 24 h 5 4.69 ± 0.40 8.45 –6.28 4.76 ± 0.37 7.83 –4.72 100 92.07 ± 6.16 6.69 –7.93 107.59 ± 7.90 7.34 7.59 500 479.25 ± 36.63 7.64 –4.15 553.42 ± 30.84 5.57 10.68 Three freeze/thaw cycles 5 4.65 ± 0.44 9.49 –7.08 4.63 ± 0.59 12.74 –7.40 100 89.11 ± 5.72 6.42 –10.89 93.44 ± 1.44 1.54 –6.56 500 458.97 ± 28.88 6.29 –8.21 487.27 ± 40.65 8.34 –2.55 PKs of MT and AMO in pigs Twenty-four pigs were randomly distributed to three treatments, namely group A (MT, 50 mg/kg), group B (AMO, 50 mg/kg), and group C (MT, 50 mg/kg + AMO, 50 mg/kg). The time course of MT and AMO in porcine plasma were shown in Fig. 5 , and the main PK parameters were summarized in Tables 3 and 4 . Compared with AMO, MT exhibited a poor absorption, extensive distribution and rapid elimination in pigs. The maximum concentration (C max ), area under the curve from time 0 to 36 h (AUC 0 → 36 h ), apparent volume of distribution (V d ), and total body clearance (Cl B ) for group A were 1389.73 ± 320.05 µg/L, 4138.16 ± 1245.23 h*µg/L, 63.08 ± 22.24 L/kg and 12.98 ± 3.71 L/h/kg, respectively, versus 21153.92 ± 16967.88 µg/L ( P < 0.01), 40239.83 ± 22044.18 h*µg/L ( P < 0.01), 9.93 ± 3.86 L/kg ( P < 0.01), and 1.61 ± 0.81 L/h/kg ( P < 0.01) for group B. MT was no longer detectable in porcine plasma at 16 h post-dosing, while three out of eight plasma samples still had enough AMO to be quantified using the LC-MS/MS method at the last time point (36 h post-dosing). The PK profile of MT in pigs after the combined administration of MT and AMO was significantly different from that after the administration of MT alone. Significantly greater time to maximum concentration (T max ), Cl B , V d ( P < 0.05) and lower C max , AUC 0 → 36 h ( P < 0.05) were observed in group A compared to those in group C. No significant differences in mean residence time (MRT), terminal elimination rate constant (λ z ), and elimination half-life (t 1/2 ) were found between the two groups ( P > 0.05). After the combined administration of MT and AMO, the T max of AMO was significantly increased ( P < 0.01) and the V d of AMO was significantly decreased ( P < 0.05) compared with those after the administration of AMO alone. No significant difference in C max , AUC 0 → 36 h , MRT, Cl B , λ z , and t 1/2 were found between the group B and group C. Table 3 the PK parameters of MT in pigs after gavage administration of MT alone (group A, n = 8) and in combination with AMO (group C, n = 8). Parameter Unit Group A Group C C max µg/L 1389.73 ± 320.05* 2009.96 ± 694.75* T max h 2.00 ± 0.00* 1.06 ± 0.42* AUC 0 → 36 h h*µg/L 4138.16 ± 1245.23** 8873.40 ± 3229.87** V d L/kg 63.08 ± 22.24* 30.31 ± 15.31* MRT h 4.14 ± 0.87 3.47 ± 0.46 Cl B L/h/kg 12.98 ± 3.71** 6.45 ± 2.88** λ z 1/h 0.21 ± 0.03 0.22 ± 0.02 t 1/2 h 3.31 ± 0.43 3.21 ± 0.25 C max maximum concentration, T max time to maximum concentration, AUC 0 → 36 h area under the curve from time 0 to 36 h, V d apparent volume of distribution, MRT mean residence time, Cl B total body clearance, λ z terminal elimination rate constant, t 1/2 elimination half-life. *Significant difference ( P < 0.05), ** Highly significant difference ( P < 0.01). Table 4 the PK parameters of AMO in pigs after gavage administration of AMO (group B, n = 8) and in combination with AMO (group C, n = 8). Parameter Unit Group B Group C C max µg/L 21153.92 ± 16967.88 16708.31 ± 10686.88 T max h 0.75 ± 0.58** 1.75 ± 0.71** AUC 0 → 36 h h*µg/L 40239.83 ± 22044.18 56259.82 ± 23364.90 V d L/kg 9.93 ± 3.86* 5.56 ± 2.07* MRT h 2.48 ± 0.73 3.37 ± 1.04 Cl B L/h/kg 1.61 ± 0.81 1.01 ± 0.37 λ z 1/h 0.18 ± 0.09 0.220 ± 0.08 t 1/2 h 5.26 ± 3.69 4.01 ± 1.94 C max maximum concentration, T max time to maximum concentration, AUC 0 → 36 h area under the curve from time 0 to 36 h, V d apparent volume of distribution, MRT mean residence time, Cl B total body clearance, λ z terminal elimination rate constant, t 1/2 elimination half-life. *Significant difference ( P < 0.05), ** Highly significant difference ( P < 0.01). Discussion Two reliable LC-MS/MS methods were developed and validated for the determination of MT and AMO in porcine plasma samples. Critical analytical performance parameters such as specificity, linearity, accuracy, precision, sensitivity, and stability were well demonstrated by validation data provided here. Unlike previous reports [ 14 , 16 , 20 , 25 , 26 ], significant matrix suppression effects were observed when analysing porcine plasma samples. Matrix-matched calibration curves were therefore used to compensate for matrix effects and to ensure accurate quantification. Satisfactory linearities ( r ≥ 0.999) were obtained within the concentration range from 1 to 100 µg/L. The plasma samples containing excessive concentrations of analytes (> 100 µg/L) were diluted with blank plasma extracts prior to analysis so that they fell within the linear range of the matrix matching standard curves. In this study, the PKs of MT and AMO in pigs were investigated after gavage administration of each drug alone and in combination. These results could be helpful to evaluate the in vivo efficacy of MT. Judged by the relatively low C max and AUC 0 → 36 h , MT appeared to be poorly absorbed through porcine intestine tract, which was consistent with previously observations in rats [ 14 ] and dogs [ 18 ]. Several in vitro experiments further confirmed that first pass metabolism, bile excretion, permeability, water solubility, and stability were not responsible for the relatively low C max and AUC 0 → 36 h [ 14 ]. One reason for this result might be that most of administered MT dose remained unabsorbed in porcine intestine tract. Since porcine intestine tract was considered to be the main site infected by E. coil and some other pathogens, the presence of high concentration of MT in porcine intestine tract provided a guarantee for its in vivo efficacy. Another possible explanation for the relatively low C max and AUC 0 → 36 h was that MT could be extensively distributed in the body and accumulated in some organs after administration. This speculation was supported by the fact that the V d of MT in pigs (63.08 ± 22.24 L/kg) was more than five times the volume of the whole body (10.66 ± 0.67 kg). Such distribution characteristics ensure a sufficient exposure of pathogens to MT, because most infections occurred in specific organs (such as trachea, lung, and heart) rather than in blood [ 27 , 28 ]. The PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PDDI between MT and AMO. As mentioned above, the co-administered AMO promoted the absorption of MT in the porcine intestine tract, while the absorption of AMO did not appear to be affected by the co-administered MT. The promotion of absorption of MT meant a decrease in the amount of MT in porcine intestine tract. Nevertheless, this did not change the fact that MT was still present in high concentrations in porcine intestine tract. Previous in vitro experiments showed that MT at concentrations of 0.3–0.5 mg/mL reversed the resistance of E. coli to ciprofloxacin [ 5 ]. We believed that the maximum concentration of MT in porcine intestine tract would far exceed this range after gavage administration at dose of 50 mg/kg. The PDDI between MT and AMO was observed during the distribution phase too. The co-administered AMO affected the distribution of MT, and vice versa. However, the V d of MT in pigs (30.31 ± 15.31 L/kg) was still approximately three times the volume of the whole body after the combined administration. This indicated that the characteristics of MT distribution in the body did not change. It should be noted that the accumulation of MT in porcine organs might pose a food safety risk to consumers. Further research on the tissue residue depletion of MT in pigs should be undertaken. Finally, administering MT in combination with AMO delayed the elimination of MT. As shown in Table 3 , the Cl B of MT in pigs was significantly decreased from 12.98 ± 3.71 L/h/kg to 6.45 ± 2.88 L/h/kg ( P < 0.01) after the combined administration. The significant decrease in Cl B could increase the concentration of MT in target organs, which might be helpful for MT to exert its resistance reversal activity and antiviral activity in vivo . Conclusion Compared with AMO, MT was poorly absorbed, extensively distributed and rapid eliminated in pigs. The PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PDDI between MT and AMO. To our knowledge, this is the first to investigate the PK profiles of MT and the combination of MT and AMO in pigs. These results could be helpful to evaluate their in vivo efficacies. Methods Materials The standards of MT and AMO (greater than 98.0% purity) were purchased from Shanghai yuanye Biotechnology Co., Ltd. (Shanghai, China) and Dr. Ehrenstorfer GmbH (Augsburg, Germany), respectively. The standard stock solutions (1000 µg/mL) of MT and AMO were prepared separately in acetonitrile and stored at − 20°C. The working standard solutions (100 µg/mL) of each compound were prepared weekly by dilution of the stock standard solutions in acetonitrile and stored at 4°C. AMO soluble powder (30%) for animal study was purchased from Wuhan Hvsen Biotechnology Co., Ltd. (Wuhan, Hubei Province, China). LC-MS grade acetonitrile and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Deionized water (18.25 MΩ cm) produced by a Milli-Q system (Millipore Co., Bedford, MA, USA) was used in the whole study. Animals Twenty-four six weeks old crossbred (Landrace × Large White) healthy pigs weighing 10.66 ± 0.67 kg were purchased from Fujian Minlv Three Dimensional Agricultural Comprehensive Development Co., Ltd. (Ningde, Fujian Province, China). The pigs were randomly distributed to three treatments with eight replicates per treatment, namely group A (MT, 50 mg/kg); group B (AMO, 50 mg/kg); and group C (MT, 50 mg/kg + AMO, 50 mg/kg). The three groups were raised separately in three pig beds to acclimatize for seven days prior to the experiment. The environment temperature and humidity were maintained at 21 ± 6 ◦ C and 72–88%, respectively, and good ventilation was ensured. During the experiment, the pigs were fed with drug-free feed and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals [ 31 ] and were approved by the Research Ethics Committee of the College of Animal Science, Fujian Agriculture and Forestry University (No. PZCASFAFU21031). Analytical method The concentrations of MT and AMO in porcine plasma samples were determined using LC-MS/MS methods. Briefly, 500 µL of plasma sample was deproteinized with 500 µL of acetonitrile. After vortexing and centrifuging at 10000 rpm for 5 min, the supernatant was transferred to a clean centrifuge tube. For the determination of MT, the supernatant was filtered through 0.22-µm PTFE syringe filters (Lizhu Biological Technology Co., Guangzhou, Guangdong Province, China) prior to analysis. For the determination of AMO, the supernatant was diluted two times with deionized water, and then filtered through 0.22-µm PTFE syringe filters for LC-MS/MS analysis. The sample that contained MT was analyzed using an Agilent 6460 Triple Quadrupole LC-MS system (Agilent Technologies Inc., Santa Clara, CA, USA). Chromatographic separation was performed at 35 ◦ C using a ChromCore C 18 column (2.1 × 100 mm, 3 µm) from NanoChrom Technologies (Suzhou) Co., Ltd, (Suzhou, Jiangsu Province, China). The mobile phases were (A) 1% formic acid in water and (B) acetonitrile, and the corresponding gradient profile was 80% A for 0.2 min, then a linear gradient to 50% A at 2 min and return to 80% A in 0.5 min. The flow rate was 0.2 mL/min, and the injection volume was 2 µL. The mass spectrometer was operated in positive electrospray ionization (ESI+) mode with MRM resolution. Nitrogen was used as the desolvation gas at a flow rate of 720 L/h. Others parameters were listed in Table 5 . Table 5 The optimized MS/MS parameters for MT and AMO Compounds Qualitative ions (m/z) Quantification ions (m/z) Dwell time (s) Cone voltage (V) Collision energy (V) Capillary voltage (Kv) Source temperature (°C) Desolvation temperature (°C) MT 249.1 → 176.0 249.1 → 148.0 0.1 100 35 3.0 350 350 249.1 → 148.0 0.1 100 33 AMO 366.0 → 208.1 366.0 → 114.2 0.036 36 26 3.0 150 400 366.0 → 114.2 0.036 36 12 The sample that contained AMO was analyzed using a Waters UPLC-Xevo TQ-S Micro system (Waters, Milford, MA, USA). Chromatographic separation was performed at 35 ◦ C on a Waters ACQUITY UPLC BEH C 18 column (2.1 × 100 mm, 1.7 µm; Waters, Milford, MA, USA). The mobile phase composition was the same as that for the analysis of MT, and the corresponding gradient profile was 98% A for 0.2 min, then a linear gradient to 20% A at 3.5 min and return to 80% A in 1.5 min. The flow rate was 0.3 mL/min, and the injection volume was 3 µL. The MS was operated in ESI + with MRM resolution. The desolvation gas (N 2 ) flow was 800 L/h. Others parameters were listed in Table 5 . The analytical performances of the two LC-MS/MS methods were evaluated according to USFDA guidelines [ 30 ]. Five batches of blank porcine plasma samples from different sources and twenty-five blank porcine plasma samples spiked with MT (or AMO) at LLOQ were analyzed as mentioned above. The MRM chromatograms of blank samples and analyte-spiked blank samples were compared to evaluate the analytical specificity. Matrix effect was investigated as described in a previous study [ 31 ]. Three different concentrations of analytes (1, 25, 100 µg/L) were investigated by analyzing five replicates for each concentration. Matrix-matched calibration curves were established by spiking blank plasma extracts with known concentrations of MT or AMO. These curves were used to calculated the concentrations of MT and AMO in porcine plasma samples. The linearity of the matrix-matched calibration curves was represented by the correlation coefficient, and a correlation coefficient greater than 0.98 was acceptable. Three consecutive analytical batches including three concentrations of quality control (QC) samples (spiked at 5, 100 and 500 µg/L) with six replicates for each were performed to evaluate the analytical precision and accuracy. The relative standard deviation (RSD) and relative error (RE) were used as indicators for the evaluation of analytical precision and accuracy, respectively. The blank samples spiked with MT (or AMO) at 1, 5, 25, and 100 µg/L, with six replicates for each, were analyzed as mentioned above. The LLOQ, defined as the lowest concentration with RSD and RE ≤ 20% was used as the indicator for the evaluation of analytical sensitivity. The stabilities of MT and AMO in porcine plasma were evaluated under various conditions. The analyte-spiked blank samples at 5, 100, 500 µg/L were subjected to short-term condition (room temperature, 24 h), to long-term condition (–20°C, seven days), and to three cycles of freeze-thaw stability studies (freezing at − 20°C for 24 h then thawing at room temperature for 12 h). Then, these samples were analyzed as mentioned above. Experimental design The drug solutions for gavage administration included the MT solution (25 mg/mL, measured as MT), AMO solution (25 mg/mL, measured as AMO), and MT-AMO mixed solution (25 mg/mL, measured as MT, and 25 mg/mL, measured as AMO), which were prepared by dissolving appropriate amount of MT standard, AMO soluble power, and a mixture of both in sterile water, respectively. During the experiment, the pigs in group A received a single dose of MT at 50 mg/kg, the pigs in group B received a single dose of AMO at 50 mg/kg, and the pigs in group C received a single dose of MT at 50 mg/kg and AMO at 50 mg/kg, respectively. Blood (approximately 3 mL) were collected via the anterior vena cava at 0 (pre-dosing), 0.25, 0.5, 1, 2, 3, 4, 5, 8, 12, 16, 24, and 36 h post-dosing. Then, plasma was prepared and stored at − 20 ◦ C until analysis. Data analysis The PK parameters for each subject were determined with WinNonlin version 5.2.1 (Pharsight Co., Mountain View, CA, USA) by non-compartmental analysis. These parameters were as follows: C max , T max , AUC 0 → 36 h , V d , MRT, Cl B , λ z , and t 1/2 . The plasma drug concentrations, PK parameters, and other data involve in this study were presented as means ± standard deviation (SD). The differences in PK parameters between the treatment groups were determined by a two-tailed t-test using SPSS version 21 (IBM Co., Armonk, NY, USA), and a P -value of < 0.05 was considered significant. Abbreviations MT: Matrine; E. coli : Escherichia coli ; AMO: Amoxicillin; PK: pharmacokinetics; PDDI: pharmacokinetic drug-drug interaction; MRM: multiple reaction monitoring; LC-MS/MS: liquid chromatography tandem mass spectrometry; LLOQ: lower limits of quantification; C max : maximum concentration; AUC 0 → 36 h : area under the curve from time 0 to 36 h; V d : apparent volume of distribution; Cl B : total body clearance; T max : time to maximum concentration; MRT: mean residence time; λ z : terminal elimination rate constant; t 1/2 : elimination half-life; ESI+: positive electrospray ionization; QC: quality control; RSD: relative standard deviation; RE: relative error; SD: standard deviation. Declarations Ethics approval and consent to participate All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Research Ethics Committee of the College of Animal Science, Fujian Agriculture and Forestry University (No. PZCASFAFU21031). Consent for publication Not applicable. Availability of data and materials The data and material used and analyzed during the current study are available from the corresponding author on reasonable request. Competing interests None of the authors has any financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of the paper. Funding This work was financially supported by the grants from the Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs) (No. KLPCAAB-2021-01) and the National Natural Science Foundation of China (No. 32172906). Authors' contributions RuoNan Li: data curation, investigation, methodology; DanNa Zhou: data curation, methodology, resources; HuiYu Hu: investigation, methodology, validation; FuHao Wang: data curation, investigation, validation; XiaoLing Lv: methodology, resources; Lei Sun: data curation, software; XueYan Sun: investigation; DaoJin Yu: conceptualization, data curation, supervision, validation, writing – original draft; Bo Yang: conceptualization, funding acquisition, supervision, writing original draft and writing – review & editing. All data were generated in-house, and no paper mill was used. All authors have participated in this research and approved the final manuscript. Acknowledgements Not applicable. Authors' information (optional) 1 University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province/Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China. 2 Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs), Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064, P.R. China. 3 Fujian Sunner Development Co., Ltd, Nanping, 354100, P.R. China. 4 National Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, 430000, P.R. China References Lillehoj H, Liu Y, Calsamiglia S, Fernandez-Miyakawa ME, Chi F, Cravens RL, Oh S, Gay CG. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. 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Matrine Targets Intestinal Lactobacillus acidophilus to Inhibit Porcine Circovirus Type 2 Infection in Mice. Int J Mol Sci. 2023;24(15):11878. Sun N, Wang ZW, Wu CH, Li E, He JP, Wang SY, Hu YL, Lei HM, Li HQ. Antiviral activity and underlying molecular mechanisms of Matrine against porcine reproductive and respiratory syndrome virus in vitro. Res Vet Sci. 2014;96(2):323–7. Burch DGS, Sperling D. Amoxicillin-current use in swine medicine. J Vet Pharmacol Ther. 2018;41(3):356–68. Wu X, Yamashita F, Hashida M, Chen X, Hu Z. Determination of matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies. Talanta. 2003;59(5):965–71. Yang Z, Gao S, Yin T, Kulkarni KH, Teng Y, You M, Hu M. Biopharmaceutical and pharmacokinetic characterization of matrine as determined by a sensitive and robust UPLC-MS/MS method. J Pharm Biomed Anal. 2010;51(5):1120–7. Gao G, Law FC. Physiologically based pharmacokinetics of matrine in the rat after oral administration of pure chemical and ACAPHA. Drug Metab Dispos. 2009;37(4):884–91. Jiang M, Wang L, Jiang W, Huang S. Simultaneous determination of 14-thienyl methylene matrine and matrine in rat plasma by high-performance liquid chromatography-tandem mass spectrometry and its application in a pharmacokinetic study. J Chromatogr B Analyt Technol Biomed Life Sci. 2015;974:126–30. Tang Z, Wang Q, He Z, Yin L, Zhang Y, Wang S. Liver, blood microdialysate and plasma pharmacokinetics of matrine following transdermal or intravenous administration. Pharmazie. 2017;72(3):167–70. Wang Y, Ma Y, Li X, Qin F, Lu X, Li F. Simultaneous determination and pharmacokinetic study of oxymatrine and matrine in beagle dog plasma after oral administration of Kushen formula granule, oxymatrine and matrine by LC-MS/MS. Biomed Chromatogr. 2007;21(8):876–82. Wu YJ, Chen JJ, Cheng YY. A sensitive and specific HPLC-MS method for the determination of sophoridine, sophocarpine and matrine in rabbit plasma. Anal Bioanal Chem. 2005;382(7):1595–600. Zhang XL, Xu HR, Chen WL, Chu NN, Li XN, Liu GY, Yu C. Matrine determination and pharmacokinetics in human plasma using LC/MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci. 2009;877(27):3253–6. Agersø H, Friis C. Bioavailability of amoxycillin in pigs. J Vet Pharmacol Ther. 1998;21(1):41–6. Agersø H, Friis C. Penetration of amoxycillin into the respiratory tract tissues and secretions in pigs. Res Vet Sci. 1998;64(3):245–50. Agersø H, Friis C, Nielsen JP. Penetration of amoxycillin to the respiratory tract tissues and secretions in Actinobacillus pleuropneumoniae infected pigs. Res Vet Sci. 1998;64(3):251–7. Agersø H, Friis C, Nielsen JP. Pharmacokinetics and tissue distribution of amoxicillin in healthy and Salmonella Typhimurium-inoculated pigs. Am J Vet Res. 2000;61(8):992–6. Dong X, Ding L, Cao X, Jiang L, Zhong S. A sensitive LC-MS/MS method for the simultaneous determination of amoxicillin and ambroxol in human plasma with segmental monitoring. Biomed Chromatogr. 2013;27(4):520–6. Armoudjian Y, Lin Q, Lammens B, Van Daele J, Annaert P. Sensitive and rapid method for the quantitation of amoxicillin in minipig plasma and milk by LC-MS/MS: A contribution from the IMI ConcePTION project. J Pharmacol Toxicol Methods. 2023;123:107264. EMEA. (2000). Points to consider on pharmacokinetics and pharmacodynamics in the development of antibacterial medicinal products. FDA. Guidance for Industry. Developing antimicrobial drugs—general considerations for clinical trials; 1998. National Research Council. Guide for the care and use of laboratory animals. 8th ed. Washington, DC: National Academies; 2011. DHHS US, FDA, CDER CVM. 2001. Guidance for industry, bioanalytical method validation. Rockville, MD, pp. 4–10. Wen A, Hang T, Chen S, Wang Z, Ding L, Tian Y, Zhang M, Xu X. Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS/MS: validation and application to pharmacokinetic study. J Pharm Biomed Anal. 2008;48(3):829–34. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4566584","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":319308482,"identity":"32381c94-2a47-48fd-8052-352fc4f392bd","order_by":0,"name":"RuoNan Li","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"RuoNan","middleName":"","lastName":"Li","suffix":""},{"id":319308483,"identity":"8a86d3e8-26c0-4a8e-89ec-a638f0d6d126","order_by":1,"name":"DanNa Zhou","email":"","orcid":"","institution":"Hubei Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"DanNa","middleName":"","lastName":"Zhou","suffix":""},{"id":319308484,"identity":"4e553cdb-0035-416a-85cf-d5f8ce8b0ff2","order_by":2,"name":"HuiYu Hu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"HuiYu","middleName":"","lastName":"Hu","suffix":""},{"id":319308485,"identity":"d381b1ce-e891-4f3c-8404-66a6a4847bb4","order_by":3,"name":"FuHao Wang","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"FuHao","middleName":"","lastName":"Wang","suffix":""},{"id":319308486,"identity":"f6d60c91-4835-49bd-a263-b3f40c5fa317","order_by":4,"name":"XiaoLing Lv","email":"","orcid":"","institution":"Fujian Sunner Development Co., Ltd","correspondingAuthor":false,"prefix":"","firstName":"XiaoLing","middleName":"","lastName":"Lv","suffix":""},{"id":319308487,"identity":"d79feab8-03e3-4087-88f2-08fbe832f8f0","order_by":5,"name":"Lei Sun","email":"","orcid":"","institution":"Huazhong Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lei","middleName":"","lastName":"Sun","suffix":""},{"id":319308488,"identity":"bd759471-eae5-412f-933c-9c9919dda55c","order_by":6,"name":"XueYan Sun","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"XueYan","middleName":"","lastName":"Sun","suffix":""},{"id":319308489,"identity":"b4ac9a10-53f2-4bc0-8e46-db1f7351dc58","order_by":7,"name":"DaoJin Yu","email":"","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":false,"prefix":"","firstName":"DaoJin","middleName":"","lastName":"Yu","suffix":""},{"id":319308490,"identity":"ddd7f5e9-a133-4416-9927-43a823b0176e","order_by":8,"name":"Bo Yang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYDACCQY2IGkDJknSkka6lsMkuMvgdo/Zw59t5+35pJsfMPyo2EaEljtnzI15224ntskcM2DsOXObCC03crdJM267ncAmkWDAzNhGpBbJn9vO2bNJpH8gXosE77YDjG0SOUTaInnn/Ddp3n/JiUAtBQeJ8gvf7bY0yR9n7OzlZ6RvfPCjgggtCgeQOAdwKEIF8g1EKRsFo2AUjIIRDQAHEjubPfg+7gAAAABJRU5ErkJggg==","orcid":"","institution":"Fujian Agriculture and Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-06-12 00:27:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4566584/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4566584/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59246449,"identity":"72c60e69-dd39-428d-a039-5fc2d61ee10d","added_by":"auto","created_at":"2024-06-28 06:39:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3011653,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structures of: (A) MT and (B) AMO.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/26fceac41f259d5dc3d45f6f.png"},{"id":59246909,"identity":"0ec73fbe-b42f-4997-990e-5caa6d1a9bb7","added_by":"auto","created_at":"2024-06-28 06:47:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2504570,"visible":true,"origin":"","legend":"\u003cp\u003eProduct ion spectra of protonated (A) MT and (B) AMO.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/6fce03e859ddfdbea46f8840.png"},{"id":59246910,"identity":"9814257c-7922-4cdc-ad27-44f8e6709bae","added_by":"auto","created_at":"2024-06-28 06:47:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1625118,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative MRM chromatograms of MT in porcine plasma samples. (A) blank plasma sample; (B) blank plasma sample spiked with MT at 5 μg/L (LLOQ); (C) plasma sample collected from a pig 8 h after a single gavage administration of MT (50 mg/kg).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/b17eafe2a9711167df3ee91e.png"},{"id":59246447,"identity":"b411d6dd-f359-4a09-a71c-cb3cc6b612eb","added_by":"auto","created_at":"2024-06-28 06:39:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1237274,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative MRM chromatograms of AMO in porcine plasma samples. (A) blank plasma sample; (B) blank plasma sample spiked with AMO at 5 μg/L (LLOQ); (C) plasma sample collected from a pig 8 h after a single gavage administration of AMO (50 mg/kg).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/f81415b25bcc2125975ad4d1.png"},{"id":59246451,"identity":"aaa70196-70f1-4ff5-803d-e4c790e24b80","added_by":"auto","created_at":"2024-06-28 06:39:35","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2839621,"visible":true,"origin":"","legend":"\u003cp\u003eThe plasma concentration-time profiles of (A) MT and (B) AMO in pigs (n = 8 samples per time point) after gavage administration.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/75a7c5814783da0c7306db25.png"},{"id":79341488,"identity":"9cb6bec7-52ea-4e39-bb02-f9947e62dc62","added_by":"auto","created_at":"2025-03-27 08:47:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":21202353,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4566584/v1/1e4cdec4-d376-43e8-b798-9a7834f3990c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pharmacokinetics of matrine and amoxicillin in pigs after gavage administration of each drug alone and in combination","fulltext":[{"header":"Background","content":"\u003cp\u003eBacterial and viral infectious diseases have caused huge economic losses to global pig industry. Some phytochemicals have been found to have unique antibacterial and/or antiviral activities, and their use alone or in combination with existing antibiotics may become a promising anti-infective treatment strategy [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMatrine (MT, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) is one of the major quinolizidine alkaloids isolated from \u003cem\u003eSophora flavescens\u003c/em\u003e Ait and \u003cem\u003eSophora alopecuroides\u003c/em\u003e [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Previous \u003cem\u003ein vitro\u003c/em\u003e experiments showed that MT could reverse the resistance of \u003cem\u003eHaemophilus parasuis\u003c/em\u003e to cefaclor [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], and restore the susceptibility of \u003cem\u003eEscherichia coli\u003c/em\u003e (\u003cem\u003eE. coli\u003c/em\u003e) to a variety of antibiotics [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Another study revealed the inhibitory effect of MT on biofilm formation of antibiotic-resistant \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which helped to increase the susceptibility of these resistant strains to antibiotics and decrease their pathogenicity to hosts. In addition, MT also exhibited activity against porcine circovirus type 2 [\u003cspan additionalcitationids=\"CR8 CR9\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and porcine reproductive and respiratory syndrome virus [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Such antibiotic resistance reversal and antiviral activity make MT a potential therapeutic or prophylactic drug for porcine colibacillosis, porcine circovirus associated disease and porcine reproductive and respiratory syndrome.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAmoxicillin (AMO, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) is a β-lactam antibiotic that was once widely used to treat porcine colibacillosis. Unfortunately, it is becoming ineffective due to increasing resistance to \u003cem\u003eE. coli\u003c/em\u003e [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], which poses a serious challenge for the treatment of porcine colibacillosis. Using AMO in combination with MT may be an effective strategy for addressing this challenge.\u003c/p\u003e \u003cp\u003eNevertheless, it remains unclear whether MT can also exert antibiotic resistance reversal and antiviral activity in pigs. The lack of pharmacokinetics (PK) data is one of the important reasons for the inability to accurately evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy of MT. To our knowledge, the PK profiles of MT have been described in rats [\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], dogs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], rabbits [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], and humans [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and the PKs of AMO have also been investigated in pigs [\u003cspan additionalcitationids=\"CR22 CR23\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], but no data are available on the PKs of MT or the combination of MT and AMO in pigs.\u003c/p\u003e \u003cp\u003eThe present study was intended to ascertain the PKs of MT and AMO in pigs after gavage administration of each drug alone and in combination. The pharmacokinetic drug-drug interaction (PDDI) between MT and AMO was also investigated. The results would provide theoretical bases for evaluating the \u003cem\u003ein vivo\u003c/em\u003e efficacies of MT and AMO-MT combination.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the qualitative ions were m/z 249.1\u0026rarr;176.0, m/z 249.1\u0026rarr;148.0 for MT and m/z 366.0\u0026rarr;208.1, m/z 366.0\u0026rarr;114.3 for AMO, respectively; the quantification ions were m/z 249.1\u0026rarr;148.0 for MT and m/z 366.0 \u0026rarr; 114.2 for AMO, respectively. Representative multiple reaction monitoring (MRM) chromatograms obtained from blank plasma samples, blank plasma samples spiked with MT or AMO, and plasma samples after administration of MT or AMO were presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The retention times of MT and AMO were approximately 1.558 and 2.630 min, respectively. There were no endogenous interfering peaks observed at the retention times of MT and AMO. The peak area ratios of the analytes at the concentration of 1, 25, 100 \u0026micro;g/L were: MT 13.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.26%, 12.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.14% and 12.82\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88%; AMO 40.21\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21%, 50.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70% and 54.10\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82%, respectively. There were significant matrix suppression effects in porcine plasma samples. The matrix-matched calibration curves showed good linearity (MT: y\u0026thinsp;=\u0026thinsp;4216.10x \u0026ndash; 8298.30, r\u0026thinsp;=\u0026thinsp;0.9990; AMO: y\u0026thinsp;=\u0026thinsp;62.817x\u0026thinsp;+\u0026thinsp;129.90, r\u0026thinsp;=\u0026thinsp;0.9949) over the concentration range from 1 to 100 \u0026micro;g/L. The accuracy and precision of MT and AMO were listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The accuracy ranged from \u0026minus;\u0026thinsp;18.59 to 14.37% for intra-day determination and \u0026minus;\u0026thinsp;15.18 to 13.54% for inter-day determination, respectively. The corresponding precision ranged from 1.99 to 7.87% and 0.71 to 7.62%, respectively. The developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods showed high sensitivity in determining MT and AMO with a lower limit of quantification (LLOQ) of 5 \u0026micro;g/L. The results of stability evaluation were listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Good stability was observed for MT and AMO under the conditions mentioned above.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrecision and accuracy of the LC-MS/MS methods for the determination of MT and AMO in pig plasma samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalyte\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNominal concentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eIntraday (n\u0026thinsp;=\u0026thinsp;6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eInterday (n\u0026thinsp;=\u0026thinsp;18)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetermined concentration (\u0026micro;g/l) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRE (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDetermined concentration (\u0026micro;g/l) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRE (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eMT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-17.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-15.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-13.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-14.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e96.27\u0026thinsp;\u0026plusmn;\u0026thinsp;3.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e-3.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e96.49\u0026thinsp;\u0026plusmn;\u0026thinsp;3.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-3.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e95.90\u0026thinsp;\u0026plusmn;\u0026thinsp;3.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-4.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e537.28\u0026thinsp;\u0026plusmn;\u0026thinsp;12.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e542.94\u0026thinsp;\u0026plusmn;\u0026thinsp;11.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e8.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e549.67\u0026thinsp;\u0026plusmn;\u0026thinsp;11.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e541.85\u0026thinsp;\u0026plusmn;\u0026thinsp;10.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"8\" rowspan=\"9\"\u003e \u003cp\u003eAMO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e7.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026ndash;12.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;12.43\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.07\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;18.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113.82\u0026thinsp;\u0026plusmn;\u0026thinsp;4.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e112.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e12.67\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e113.38\u0026thinsp;\u0026plusmn;\u0026thinsp;2.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.38\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110.80\u0026thinsp;\u0026plusmn;\u0026thinsp;3.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e571.83\u0026thinsp;\u0026plusmn;\u0026thinsp;22.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e14.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e567.68\u0026thinsp;\u0026plusmn;\u0026thinsp;4.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e13.54\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e567.38\u0026thinsp;\u0026plusmn;\u0026thinsp;28.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e563.83\u0026thinsp;\u0026plusmn;\u0026thinsp;28.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e12.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStability of MT and AMO in pig plasma samples under various conditions (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCondition\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eNominal concentration (\u0026micro;g/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eMT (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eAMO (n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDetermined concentration (\u0026micro;g/mL) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRE (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eDetermined concentration (\u0026micro;g/mL) \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eRE (%)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u0026ndash;20\u0026deg;C, 7 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;12.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;8.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.23\u0026thinsp;\u0026plusmn;\u0026thinsp;4.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;10.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.89\u0026thinsp;\u0026plusmn;\u0026thinsp;6.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;6.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e470.39\u0026thinsp;\u0026plusmn;\u0026thinsp;30.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;5.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e540.48\u0026thinsp;\u0026plusmn;\u0026thinsp;25.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e4.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eRoom temperature, 24 h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e8.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;6.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;4.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e92.07\u0026thinsp;\u0026plusmn;\u0026thinsp;6.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;7.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e107.59\u0026thinsp;\u0026plusmn;\u0026thinsp;7.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e7.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.59\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e479.25\u0026thinsp;\u0026plusmn;\u0026thinsp;36.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;4.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e553.42\u0026thinsp;\u0026plusmn;\u0026thinsp;30.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e10.68\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eThree freeze/thaw cycles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e4.65\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;7.08\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.59\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;7.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e89.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;10.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e93.44\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;6.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e458.97\u0026thinsp;\u0026plusmn;\u0026thinsp;28.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;8.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e487.27\u0026thinsp;\u0026plusmn;\u0026thinsp;40.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e8.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026ndash;2.55\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003ePKs of MT and AMO in pigs\u003c/h2\u003e \u003cp\u003eTwenty-four pigs were randomly distributed to three treatments, namely group A (MT, 50 mg/kg), group B (AMO, 50 mg/kg), and group C (MT, 50 mg/kg\u0026thinsp;+\u0026thinsp;AMO, 50 mg/kg). The time course of MT and AMO in porcine plasma were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, and the main PK parameters were summarized in Tables\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and \u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Compared with AMO, MT exhibited a poor absorption, extensive distribution and rapid elimination in pigs. The maximum concentration (C\u003csub\u003emax\u003c/sub\u003e), area under the curve from time 0 to 36 h (AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e), apparent volume of distribution (V\u003csub\u003ed\u003c/sub\u003e), and total body clearance (Cl\u003csub\u003eB\u003c/sub\u003e) for group A were 1389.73\u0026thinsp;\u0026plusmn;\u0026thinsp;320.05 \u0026micro;g/L, 4138.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1245.23 h*\u0026micro;g/L, 63.08\u0026thinsp;\u0026plusmn;\u0026thinsp;22.24 L/kg and 12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 L/h/kg, respectively, versus 21153.92\u0026thinsp;\u0026plusmn;\u0026thinsp;16967.88 \u0026micro;g/L (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 40239.83\u0026thinsp;\u0026plusmn;\u0026thinsp;22044.18 h*\u0026micro;g/L (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 9.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 L/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and 1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 L/h/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) for group B. MT was no longer detectable in porcine plasma at 16 h post-dosing, while three out of eight plasma samples still had enough AMO to be quantified using the LC-MS/MS method at the last time point (36 h post-dosing). The PK profile of MT in pigs after the combined administration of MT and AMO was significantly different from that after the administration of MT alone. Significantly greater time to maximum concentration (T\u003csub\u003emax\u003c/sub\u003e), Cl\u003csub\u003eB\u003c/sub\u003e, V\u003csub\u003ed\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and lower C\u003csub\u003emax\u003c/sub\u003e, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed in group A compared to those in group C. No significant differences in mean residence time (MRT), terminal elimination rate constant (λ\u003csub\u003ez\u003c/sub\u003e), and elimination half-life (t\u003csub\u003e1/2\u003c/sub\u003e) were found between the two groups (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). After the combined administration of MT and AMO, the T\u003csub\u003emax\u003c/sub\u003e of AMO was significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and the V\u003csub\u003ed\u003c/sub\u003e of AMO was significantly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared with those after the administration of AMO alone. No significant difference in C\u003csub\u003emax\u003c/sub\u003e, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e, MRT, Cl\u003csub\u003eB\u003c/sub\u003e, λ\u003csub\u003ez\u003c/sub\u003e, and t\u003csub\u003e1/2\u003c/sub\u003e were found between the group B and group C.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ethe PK parameters of MT in pigs after gavage administration of MT alone (group A, n\u0026thinsp;=\u0026thinsp;8) and in combination with AMO (group C, n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1389.73\u0026thinsp;\u0026plusmn;\u0026thinsp;320.05*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e2009.96\u0026thinsp;\u0026plusmn;\u0026thinsp;694.75*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh*\u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4138.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1245.23**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e8873.40\u0026thinsp;\u0026plusmn;\u0026thinsp;3229.87**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e63.08\u0026thinsp;\u0026plusmn;\u0026thinsp;22.24*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e30.31\u0026thinsp;\u0026plusmn;\u0026thinsp;15.31*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e4.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL/h/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e6.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eλ\u003csub\u003ez\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003et\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.21\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eC\u003csub\u003emax\u003c/sub\u003e maximum concentration, T\u003csub\u003emax\u003c/sub\u003e time to maximum concentration, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e area under the curve from time 0 to 36 h, V\u003csub\u003ed\u003c/sub\u003e apparent volume of distribution, MRT mean residence time, Cl\u003csub\u003eB\u003c/sub\u003e total body clearance, λ\u003csub\u003ez\u003c/sub\u003e terminal elimination rate constant, t\u003csub\u003e1/2\u003c/sub\u003e elimination half-life.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ** Highly significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ethe PK parameters of AMO in pigs after gavage administration of AMO (group B, n\u0026thinsp;=\u0026thinsp;8) and in combination with AMO (group C, n\u0026thinsp;=\u0026thinsp;8).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eUnit\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e21153.92\u0026thinsp;\u0026plusmn;\u0026thinsp;16967.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e16708.31\u0026thinsp;\u0026plusmn;\u0026thinsp;10686.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh*\u0026micro;g/L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e40239.83\u0026thinsp;\u0026plusmn;\u0026thinsp;22044.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e56259.82\u0026thinsp;\u0026plusmn;\u0026thinsp;23364.90\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eV\u003csub\u003ed\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e9.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e5.56\u0026thinsp;\u0026plusmn;\u0026thinsp;2.07*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMRT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.48\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e3.37\u0026thinsp;\u0026plusmn;\u0026thinsp;1.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCl\u003csub\u003eB\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL/h/kg\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eλ\u003csub\u003ez\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1/h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.220\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003et\u003csub\u003e1/2\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.26\u0026thinsp;\u0026plusmn;\u0026thinsp;3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e4.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.94\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003eC\u003csub\u003emax\u003c/sub\u003e maximum concentration, T\u003csub\u003emax\u003c/sub\u003e time to maximum concentration, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e area under the curve from time 0 to 36 h, V\u003csub\u003ed\u003c/sub\u003e apparent volume of distribution, MRT mean residence time, Cl\u003csub\u003eB\u003c/sub\u003e total body clearance, λ\u003csub\u003ez\u003c/sub\u003e terminal elimination rate constant, t\u003csub\u003e1/2\u003c/sub\u003e elimination half-life.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"4\"\u003e*Significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), ** Highly significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eTwo reliable LC-MS/MS methods were developed and validated for the determination of MT and AMO in porcine plasma samples. Critical analytical performance parameters such as specificity, linearity, accuracy, precision, sensitivity, and stability were well demonstrated by validation data provided here. Unlike previous reports [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], significant matrix suppression effects were observed when analysing porcine plasma samples. Matrix-matched calibration curves were therefore used to compensate for matrix effects and to ensure accurate quantification. Satisfactory linearities (\u003cem\u003er\u003c/em\u003e\u0026thinsp;\u0026ge;\u0026thinsp;0.999) were obtained within the concentration range from 1 to 100 \u0026micro;g/L. The plasma samples containing excessive concentrations of analytes (\u0026gt;\u0026thinsp;100 \u0026micro;g/L) were diluted with blank plasma extracts prior to analysis so that they fell within the linear range of the matrix matching standard curves.\u003c/p\u003e \u003cp\u003eIn this study, the PKs of MT and AMO in pigs were investigated after gavage administration of each drug alone and in combination. These results could be helpful to evaluate the \u003cem\u003ein vivo\u003c/em\u003e efficacy of MT. Judged by the relatively low C\u003csub\u003emax\u003c/sub\u003e and AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e, MT appeared to be poorly absorbed through porcine intestine tract, which was consistent with previously observations in rats [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and dogs [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Several \u003cem\u003ein vitro\u003c/em\u003e experiments further confirmed that first pass metabolism, bile excretion, permeability, water solubility, and stability were not responsible for the relatively low C\u003csub\u003emax\u003c/sub\u003e and AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. One reason for this result might be that most of administered MT dose remained unabsorbed in porcine intestine tract. Since porcine intestine tract was considered to be the main site infected by \u003cem\u003eE. coil\u003c/em\u003e and some other pathogens, the presence of high concentration of MT in porcine intestine tract provided a guarantee for its \u003cem\u003ein vivo\u003c/em\u003e efficacy. Another possible explanation for the relatively low C\u003csub\u003emax\u003c/sub\u003e and AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e was that MT could be extensively distributed in the body and accumulated in some organs after administration. This speculation was supported by the fact that the V\u003csub\u003ed\u003c/sub\u003e of MT in pigs (63.08\u0026thinsp;\u0026plusmn;\u0026thinsp;22.24 L/kg) was more than five times the volume of the whole body (10.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 kg). Such distribution characteristics ensure a sufficient exposure of pathogens to MT, because most infections occurred in specific organs (such as trachea, lung, and heart) rather than in blood [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PDDI between MT and AMO. As mentioned above, the co-administered AMO promoted the absorption of MT in the porcine intestine tract, while the absorption of AMO did not appear to be affected by the co-administered MT. The promotion of absorption of MT meant a decrease in the amount of MT in porcine intestine tract. Nevertheless, this did not change the fact that MT was still present in high concentrations in porcine intestine tract. Previous \u003cem\u003ein vitro\u003c/em\u003e experiments showed that MT at concentrations of 0.3\u0026ndash;0.5 mg/mL reversed the resistance of \u003cem\u003eE. coli\u003c/em\u003e to ciprofloxacin [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. We believed that the maximum concentration of MT in porcine intestine tract would far exceed this range after gavage administration at dose of 50 mg/kg. The PDDI between MT and AMO was observed during the distribution phase too. The co-administered AMO affected the distribution of MT, and vice versa. However, the V\u003csub\u003ed\u003c/sub\u003e of MT in pigs (30.31\u0026thinsp;\u0026plusmn;\u0026thinsp;15.31 L/kg) was still approximately three times the volume of the whole body after the combined administration. This indicated that the characteristics of MT distribution in the body did not change. It should be noted that the accumulation of MT in porcine organs might pose a food safety risk to consumers. Further research on the tissue residue depletion of MT in pigs should be undertaken. Finally, administering MT in combination with AMO delayed the elimination of MT. As shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, the Cl\u003csub\u003eB\u003c/sub\u003e of MT in pigs was significantly decreased from 12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 L/h/kg to 6.45\u0026thinsp;\u0026plusmn;\u0026thinsp;2.88 L/h/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) after the combined administration. The significant decrease in Cl\u003csub\u003eB\u003c/sub\u003e could increase the concentration of MT in target organs, which might be helpful for MT to exert its resistance reversal activity and antiviral activity \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eCompared with AMO, MT was poorly absorbed, extensively distributed and rapid eliminated in pigs. The PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PDDI between MT and AMO. To our knowledge, this is the first to investigate the PK profiles of MT and the combination of MT and AMO in pigs. These results could be helpful to evaluate their \u003cem\u003ein vivo\u003c/em\u003e efficacies.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eThe standards of MT and AMO (greater than 98.0% purity) were purchased from Shanghai yuanye Biotechnology Co., Ltd. (Shanghai, China) and Dr. Ehrenstorfer GmbH (Augsburg, Germany), respectively. The standard stock solutions (1000 \u0026micro;g/mL) of MT and AMO were prepared separately in acetonitrile and stored at \u0026minus;\u0026thinsp;20\u0026deg;C. The working standard solutions (100 \u0026micro;g/mL) of each compound were prepared weekly by dilution of the stock standard solutions in acetonitrile and stored at 4\u0026deg;C. AMO soluble powder (30%) for animal study was purchased from Wuhan Hvsen Biotechnology Co., Ltd. (Wuhan, Hubei Province, China). LC-MS grade acetonitrile and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Deionized water (18.25 MΩ cm) produced by a Milli-Q system (Millipore Co., Bedford, MA, USA) was used in the whole study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eTwenty-four six weeks old crossbred (Landrace \u0026times; Large White) healthy pigs weighing 10.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67 kg were purchased from Fujian Minlv Three Dimensional Agricultural Comprehensive Development Co., Ltd. (Ningde, Fujian Province, China). The pigs were randomly distributed to three treatments with eight replicates per treatment, namely group A (MT, 50 mg/kg); group B (AMO, 50 mg/kg); and group C (MT, 50 mg/kg\u0026thinsp;+\u0026thinsp;AMO, 50 mg/kg). The three groups were raised separately in three pig beds to acclimatize for seven days prior to the experiment. The environment temperature and humidity were maintained at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;6 \u003csup\u003e◦\u003c/sup\u003eC and 72\u0026ndash;88%, respectively, and good ventilation was ensured. During the experiment, the pigs were fed with drug-free feed and water. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and were approved by the Research Ethics Committee of the College of Animal Science, Fujian Agriculture and Forestry University (No. PZCASFAFU21031).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eAnalytical method\u003c/h2\u003e \u003cp\u003eThe concentrations of MT and AMO in porcine plasma samples were determined using LC-MS/MS methods. Briefly, 500 \u0026micro;L of plasma sample was deproteinized with 500 \u0026micro;L of acetonitrile. After vortexing and centrifuging at 10000 rpm for 5 min, the supernatant was transferred to a clean centrifuge tube. For the determination of MT, the supernatant was filtered through 0.22-\u0026micro;m PTFE syringe filters (Lizhu Biological Technology Co., Guangzhou, Guangdong Province, China) prior to analysis. For the determination of AMO, the supernatant was diluted two times with deionized water, and then filtered through 0.22-\u0026micro;m PTFE syringe filters for LC-MS/MS analysis.\u003c/p\u003e \u003cp\u003eThe sample that contained MT was analyzed using an Agilent 6460 Triple Quadrupole LC-MS system (Agilent Technologies Inc., Santa Clara, CA, USA). Chromatographic separation was performed at 35 \u003csup\u003e◦\u003c/sup\u003eC using a ChromCore C\u003csub\u003e18\u003c/sub\u003e column (2.1 \u0026times; 100 mm, 3 \u0026micro;m) from NanoChrom Technologies (Suzhou) Co., Ltd, (Suzhou, Jiangsu Province, China). The mobile phases were (A) 1% formic acid in water and (B) acetonitrile, and the corresponding gradient profile was 80% A for 0.2 min, then a linear gradient to 50% A at 2 min and return to 80% A in 0.5 min. The flow rate was 0.2 mL/min, and the injection volume was 2 \u0026micro;L. The mass spectrometer was operated in positive electrospray ionization (ESI+) mode with MRM resolution. Nitrogen was used as the desolvation gas at a flow rate of 720 L/h. Others parameters were listed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe optimized MS/MS parameters for MT and AMO\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"9\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompounds\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQualitative ions (m/z)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eQuantification ions (m/z)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDwell time (s)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCone voltage (V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCollision energy (V)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eCapillary voltage (Kv)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eSource temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eDesolvation temperature (\u0026deg;C)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eMT\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e249.1 \u0026rarr; 176.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e249.1 \u0026rarr; 148.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e350\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e249.1 \u0026rarr; 148.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAMO\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e366.0 \u0026rarr; 208.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e366.0 \u0026rarr; 114.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e150\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e366.0 \u0026rarr; 114.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.036\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe sample that contained AMO was analyzed using a Waters UPLC-Xevo TQ-S Micro system (Waters, Milford, MA, USA). Chromatographic separation was performed at 35 \u003csup\u003e◦\u003c/sup\u003eC on a Waters ACQUITY UPLC BEH C\u003csub\u003e18\u003c/sub\u003e column (2.1 \u0026times; 100 mm, 1.7 \u0026micro;m; Waters, Milford, MA, USA). The mobile phase composition was the same as that for the analysis of MT, and the corresponding gradient profile was 98% A for 0.2 min, then a linear gradient to 20% A at 3.5 min and return to 80% A in 1.5 min. The flow rate was 0.3 mL/min, and the injection volume was 3 \u0026micro;L. The MS was operated in ESI\u0026thinsp;+\u0026thinsp;with MRM resolution. The desolvation gas (N\u003csub\u003e2\u003c/sub\u003e) flow was 800 L/h. Others parameters were listed in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eThe analytical performances of the two LC-MS/MS methods were evaluated according to USFDA guidelines [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Five batches of blank porcine plasma samples from different sources and twenty-five blank porcine plasma samples spiked with MT (or AMO) at LLOQ were analyzed as mentioned above. The MRM chromatograms of blank samples and analyte-spiked blank samples were compared to evaluate the analytical specificity. Matrix effect was investigated as described in a previous study [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Three different concentrations of analytes (1, 25, 100 \u0026micro;g/L) were investigated by analyzing five replicates for each concentration. Matrix-matched calibration curves were established by spiking blank plasma extracts with known concentrations of MT or AMO. These curves were used to calculated the concentrations of MT and AMO in porcine plasma samples. The linearity of the matrix-matched calibration curves was represented by the correlation coefficient, and a correlation coefficient greater than 0.98 was acceptable. Three consecutive analytical batches including three concentrations of quality control (QC) samples (spiked at 5, 100 and 500 \u0026micro;g/L) with six replicates for each were performed to evaluate the analytical precision and accuracy. The relative standard deviation (RSD) and relative error (RE) were used as indicators for the evaluation of analytical precision and accuracy, respectively. The blank samples spiked with MT (or AMO) at 1, 5, 25, and 100 \u0026micro;g/L, with six replicates for each, were analyzed as mentioned above. The LLOQ, defined as the lowest concentration with RSD and RE\u0026thinsp;\u0026le;\u0026thinsp;20% was used as the indicator for the evaluation of analytical sensitivity. The stabilities of MT and AMO in porcine plasma were evaluated under various conditions. The analyte-spiked blank samples at 5, 100, 500 \u0026micro;g/L were subjected to short-term condition (room temperature, 24 h), to long-term condition (\u0026ndash;20\u0026deg;C, seven days), and to three cycles of freeze-thaw stability studies (freezing at \u0026minus;\u0026thinsp;20\u0026deg;C for 24 h then thawing at room temperature for 12 h). Then, these samples were analyzed as mentioned above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eThe drug solutions for gavage administration included the MT solution (25 mg/mL, measured as MT), AMO solution (25 mg/mL, measured as AMO), and MT-AMO mixed solution (25 mg/mL, measured as MT, and 25 mg/mL, measured as AMO), which were prepared by dissolving appropriate amount of MT standard, AMO soluble power, and a mixture of both in sterile water, respectively. During the experiment, the pigs in group A received a single dose of MT at 50 mg/kg, the pigs in group B received a single dose of AMO at 50 mg/kg, and the pigs in group C received a single dose of MT at 50 mg/kg and AMO at 50 mg/kg, respectively. Blood (approximately 3 mL) were collected via the anterior vena cava at 0 (pre-dosing), 0.25, 0.5, 1, 2, 3, 4, 5, 8, 12, 16, 24, and 36 h post-dosing. Then, plasma was prepared and stored at \u0026minus;\u0026thinsp;20 \u003csup\u003e◦\u003c/sup\u003eC until analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eData analysis\u003c/h2\u003e \u003cp\u003eThe PK parameters for each subject were determined with WinNonlin version 5.2.1 (Pharsight Co., Mountain View, CA, USA) by non-compartmental analysis. These parameters were as follows: C\u003csub\u003emax\u003c/sub\u003e, T\u003csub\u003emax\u003c/sub\u003e, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e, V\u003csub\u003ed\u003c/sub\u003e, MRT, Cl\u003csub\u003eB\u003c/sub\u003e, λ\u003csub\u003ez\u003c/sub\u003e, and t\u003csub\u003e1/2\u003c/sub\u003e. The plasma drug concentrations, PK parameters, and other data involve in this study were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The differences in PK parameters between the treatment groups were determined by a two-tailed t-test using SPSS version 21 (IBM Co., Armonk, NY, USA), and a \u003cem\u003eP\u003c/em\u003e-value of \u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMT: Matrine; \u003cem\u003eE.\u003c/em\u003e\u003cem\u003e\u0026nbsp;coli\u003c/em\u003e:\u003cem\u003e\u0026nbsp;Escherichia coli\u003c/em\u003e;\u0026nbsp;AMO: Amoxicillin;\u0026nbsp;PK: pharmacokinetics;\u0026nbsp;PDDI: pharmacokinetic drug-drug interaction;\u0026nbsp;MRM: multiple reaction monitoring;\u0026nbsp;LC-MS/MS: liquid chromatography tandem mass spectrometry;\u0026nbsp;LLOQ: lower limits of quantification;\u0026nbsp;C\u003csub\u003emax\u003c/sub\u003e: maximum concentration;\u0026nbsp;AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e: area under the curve from time 0 to 36 h; V\u003csub\u003ed\u003c/sub\u003e: apparent volume of distribution; Cl\u003csub\u003eB\u003c/sub\u003e: total body clearance; T\u003csub\u003emax\u003c/sub\u003e: time to maximum concentration; MRT: mean residence time;\u0026nbsp;\u0026lambda;\u003csub\u003ez\u003c/sub\u003e: terminal elimination rate constant; t\u003csub\u003e1/2\u003c/sub\u003e: elimination half-life; ESI+: positive electrospray ionization; \u0026nbsp;QC: quality control; RSD: relative standard deviation; RE: relative error; SD: standard deviation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAll animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the\u0026nbsp;Research Ethics Committee of the College of Animal Science, Fujian Agriculture and Forestry University (No. PZCASFAFU21031).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe data and material used and analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNone of the authors has any financial or personal relationships with other people or organizations that could inappropriately influence or bias the content of the paper.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis work was financially supported by the grants from the Key Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs) (No. KLPCAAB-2021-01)\u0026nbsp;and the National Natural Science Foundation of China (No. 32172906).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eRuoNan Li: data curation, investigation, methodology; DanNa Zhou: data curation, methodology, resources;\u0026nbsp;HuiYu Hu:\u0026nbsp;investigation, methodology, validation; FuHao Wang: data curation, investigation, validation; XiaoLing Lv: methodology, resources; Lei Sun: data curation, software; XueYan Sun: investigation; DaoJin Yu: conceptualization, data curation, supervision, validation, writing \u0026ndash; original draft; Bo Yang:\u0026nbsp;conceptualization, funding acquisition, supervision, writing original draft and writing \u0026ndash; review \u0026amp; editing.\u0026nbsp;All data were generated in-house, and no paper mill was used. All authors have participated in this research and approved the final manuscript.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAuthors\u0026apos; information (optional)\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eUniversity Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province/Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, Fujian Agriculture and Forestry University, Fuzhou 350002, P.R. China.\u0026nbsp;\u003csup\u003e2\u003c/sup\u003eKey Laboratory of Prevention and Control Agents for Animal Bacteriosis (Ministry of Agriculture and Rural Affairs), Hubei Provincial Key Laboratory of Animal Pathogenic Microbiology, Institute of Animal Husbandry and Veterinary, Hubei Academy of Agricultural Sciences, Wuhan, 430064, P.R. China.\u0026nbsp;\u003csup\u003e3\u003c/sup\u003eFujian Sunner Development Co., Ltd, Nanping, 354100, P.R. China.\u0026nbsp;\u003csup\u003e4\u003c/sup\u003eNational Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, 430000, P.R. China\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLillehoj H, Liu Y, Calsamiglia S, Fernandez-Miyakawa ME, Chi F, Cravens RL, Oh S, Gay CG. Phytochemicals as antibiotic alternatives to promote growth and enhance host health. 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Liver, blood microdialysate and plasma pharmacokinetics of matrine following transdermal or intravenous administration. Pharmazie. 2017;72(3):167\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Ma Y, Li X, Qin F, Lu X, Li F. Simultaneous determination and pharmacokinetic study of oxymatrine and matrine in beagle dog plasma after oral administration of Kushen formula granule, oxymatrine and matrine by LC-MS/MS. Biomed Chromatogr. 2007;21(8):876\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu YJ, Chen JJ, Cheng YY. A sensitive and specific HPLC-MS method for the determination of sophoridine, sophocarpine and matrine in rabbit plasma. Anal Bioanal Chem. 2005;382(7):1595\u0026ndash;600.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang XL, Xu HR, Chen WL, Chu NN, Li XN, Liu GY, Yu C. Matrine determination and pharmacokinetics in human plasma using LC/MS/MS. 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Developing antimicrobial drugs\u0026mdash;general considerations for clinical trials; 1998.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Research Council. Guide for the care and use of laboratory animals. 8th ed. Washington, DC: National Academies; 2011.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDHHS US, FDA, CDER CVM. 2001. Guidance for industry, bioanalytical method validation. Rockville, MD, pp. 4\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWen A, Hang T, Chen S, Wang Z, Ding L, Tian Y, Zhang M, Xu X. Simultaneous determination of amoxicillin and ambroxol in human plasma by LC-MS/MS: validation and application to pharmacokinetic study. J Pharm Biomed Anal. 2008;48(3):829\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pharmacokinetics, Matrine, Amoxicillin, Pig, Drug-drug interaction","lastPublishedDoi":"10.21203/rs.3.rs-4566584/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4566584/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e \u003cp\u003eMatrine (MT) exhibits antibiotic resistance reversal and antiviral activities \u003cem\u003ein vitro\u003c/em\u003e. However, the \u003cem\u003ein vivo\u003c/em\u003e efficacies of MT and amoxicillin (AMO)-MT combination cannot be accurately evaluated due to the lack of pharmacokinetics (PK) data. The aim of this study was to investigate the PKs of MT and AMO in pigs after gavage administration of each drug alone and in combination.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTwenty-four pigs were randomly distributed to three treatments, namely group A (MT, 50 mg/kg), group B (AMO, 50 mg/kg), and group C (MT, 50 mg/kg\u0026thinsp;+\u0026thinsp;AMO, 50 mg/kg). The maximum concentration (C\u003csub\u003emax\u003c/sub\u003e), area under the curve from time 0 to 36 h (AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e), apparent volume of distribution (V\u003csub\u003ed\u003c/sub\u003e) and total body clearance (Cl\u003csub\u003eB\u003c/sub\u003e) for group A were 1389.73\u0026thinsp;\u0026plusmn;\u0026thinsp;320.05 \u0026micro;g/L, 4138.16\u0026thinsp;\u0026plusmn;\u0026thinsp;1245.23 h*\u0026micro;g/L, 63.08\u0026thinsp;\u0026plusmn;\u0026thinsp;22.24 L/kg and 12.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 L/h/kg, respectively, versus 21153.92\u0026thinsp;\u0026plusmn;\u0026thinsp;16967.88 \u0026micro;g/L (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 40239.83\u0026thinsp;\u0026plusmn;\u0026thinsp;22044.18 h*\u0026micro;g/L (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), 9.93\u0026thinsp;\u0026plusmn;\u0026thinsp;3.86 L/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and 1.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 L/h/kg (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01) for group B. Significantly greater T\u003csub\u003emax\u003c/sub\u003e, Cl\u003csub\u003eB\u003c/sub\u003e, V\u003csub\u003ed\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and lower C\u003csub\u003emax\u003c/sub\u003e, AUC\u003csub\u003e0 \u0026rarr; 36 h\u003c/sub\u003e (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were observed in group A compared to those in group C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e \u003cp\u003eCompared with AMO, MT was poorly absorbed, extensively distributed and rapid eliminated in pigs. The PK profiles of MT and AMO changed significantly after the combined administration of these two drugs, indicating the occurrence of PK drug-drug interaction between MT and AMO.\u003c/p\u003e","manuscriptTitle":"Pharmacokinetics of matrine and amoxicillin in pigs after gavage administration of each drug alone and in combination","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-28 06:39:29","doi":"10.21203/rs.3.rs-4566584/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08807046-ecb2-409f-af8b-ab596bfe10f4","owner":[],"postedDate":"June 28th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-27T08:38:38+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-28 06:39:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4566584","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4566584","identity":"rs-4566584","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}