Abstract
In the present study, an LC-MS/MS method allowing to quantify pretomanid and pyrazinamide simultaneously in rat plasma was developed. Chromatographic separation was achieved on an Agilent Eclipse plus C18 column (100 mm × 2.1 mm, 3.5 μm; Agilent, USA) and maintained at 30 °C. Multiple reaction monitoring (MRM) using positive-ion ESI mode to monitor ion transitions of m/z 360.1 → m/z 175.1 for pretomanid, m/z 124.1 → m/z 81.0 for pyrazinamide, m/z 172.1 → m/z 128.1 for metronidazole (IS). The calibration curves showed good linear relationships over the concentration range of 50–7,500 ng mL−1 for pretomanid and 500–75,000 ng mL−1 for pyrazinamide. The precision and accuracy were below 15% and within ±15% of the nominal concentrations, respectively. The selectivity, recovery and matrix effect of this method were all within acceptable limits of bioanalytics. The method was applied to the analysis of plasma samples from pharmacokinetic studies in rats. The results show that the main pharmacokinetic parameters of pyrazinamide, namely, Tmax, t1/2, and AUC(0–t), decreased in the combined group than in the alone group.
1 Introduction
Tuberculosis (TB) remains a major global public health threat, infecting and killing mainly in developing countries [1, 2]. The reason why TB is so difficult to treat is because of the TB bacterium's three “weapons”: persistence [3], drug resistance [4] and virulence [5]. Among them, the infection of drug-resistant Mycobacterium tuberculosis (MTB) is a major difficulty in TB control [6]. In 2018, the world health organization (WHO) estimates that at least 6.3 million new cases of TB caused by pyrazinamide and fluoroquinolone are reported, including 490,000 cases of multidrug-resistant (MDR) -TB [7]. In addition, due to the increasing incidence of MDR and extensively drug-resistant (XDR) -TB, there are limited antibiotic options available to treat TB [8]. Therefore, there is an urgent need to develop new drugs, find new targets and new combinations of treatment methods to cope with the growing trend of TB.
Due to the great resistance to first-line drugs, XDR-TB and MDR-TB are often difficult to cure completely [9]. Pretomanid is one of the preferred drugs evaluated in clinical trials for the treatment of drug-resistant MTB [10]. By inhibiting cell wall formation and respiratory toxicity, pretomanid shows significant bactericidal activity against both replicative and stationary MTB [11]. The combination of pretomanid, bedaquinoline and linezolid, which has synergistic or additive potential in killing bacteria and inhibiting drug resistance, is also described, and is expected to further improve current TB treatment outcomes [12].
Among the first-line anti-TB drugs, pyrazinamide can kill MTB and retain bacteria in the semi-dormant period under acidic conditions [13]. And pyrazinamide can shorten the treatment time of TB from 9–12 months to 6 months [14], so it plays an important role in shortening the treatment time in short-course chemotherapy of TB. Although pyrazinamide can completely eradicate TB, it has obvious disadvantages in clinical application. Compared with other first-line anti-TB drugs (such as isoniazid in vitro MIC value of 0.061 μg mL−1, rifampicin in vitro MIC Value is 60 μg mL−1), in vitro MIC value of pyrazinamide (50–120 μg mL−1) is high, so it must be administered in large doses to play a therapeutic role, long-term large doses of pyrazinamide will cause significant liver toxicity [15].
At present, there is experimental evidence suggests that the new drug pretomanid combined with pyrazinamide, may assist treatment shortening of drug-susceptible and XDR-TB [16]. In the current work, we conducted studies to characterize the potential for a pharmacokinetic interaction between pretomanid and pyrazinamide in rats using LC-MS/MS. We have developed and validated an LC-MS/MS method for the simultaneous determination of pretomanid and pyrazinamide in rat plasma. It was then applied to the pharmacokinetic interaction study in rats with simple protein precipitation method as sample preparation approach. Significant differences in pharmacokinetic properties of pyrazinamide were observed between the alone and combined groups after administration of equal dose of pretomanid and pyrazinamide.
2 Experimental
2.1 Materials and reagents
Pretomanid, pyrazinamide and metronidazole (IS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Methanol and acetonitrile were of MS grade (Tedia Co. Inc., Ohio OH, USA). Milli-Q water was used throughout the whole experiments (Millipore, Bedford, MA, USA). Other solvents used for analysis were of analytical grade.
2.2 Animals
Sprague-Dawley rats (250 ± 20 g) were purchased from the Experimental Animal Center, Air Force Military Medical University (Shaanxi, China). All the experimental protocols were approved by the Guidelines for the Care and Use of Laboratory Animals. Rats were acclimated in the laboratory for one week prior to the experiments, housed separately in a metabolic cage at a temperature of 23 ± 2 °C with a relative humidity of 50% and a 12 h light/dark cycle, free access to standard water and diet. The rats were fasted for 12 h before oral administration with free access to water.
2.3 Drug administration and sampling
In the pharmacokinetic study, 24 rats were randomly divided into three groups (n = 8 per group), each group were orally administered Pretomanid (20 mg mL−1), pyrazinamide (150 mg mL−1), and Pretomanid (20 mg mL−1) plus pyrazinamide (150 mg mL−1), respectively. Blood samples (0.3 mL) were collected via retro-orbital puncture at 5, 15, 30, 60, 120, 180, 240, 360, 480, 720, 1,080, 1,440, 2,160 min post-dosing after the rats were orally administered. Then, each blood sample was immediately centrifuged at approximately 10,000 rpm, 4 °C for 10 min and a 100 µL aliquot of supernatant plasma layer was transferred into another tube and stored at −80 °C until analysis. An aliquot of 100 µL rat plasma sample was mixed with 10 µL of internal standard solution (1 μg mL−1), 800 µL acetonitrile were added. The sample was vortexmixed for 5 min, and the mixture was separated by centrifugating at 10,000 rpm for 10 min. An aliquot of 500 µL of the protein-free supernatant was then evaporated to dryness at 40 °C under nitrogen. The resulting extract was dissolved in 50 µL of methanol, and vortexmixed for 3 min. After centrifugation at 10,000 rpm for 10 min, an aliquot of 5 µL supernatant was injected into the LC-MS.
2.4 Preparation of stock solutions
All compounds were dissolved by methanol (1 mg mL−1). These stock solutions were diluted with methanol to obtain calibration solutions. Internal standard solution was prepared by dilution of stock solution to a concentration of 50 ng mL−1. All the solutions were stored at −20 °C and were brought to room temperature before use.
2.5 LC-MS/MS analysis
The liquid chromatography was performed on a Shimadzu HPLC, which included a LC-30AD binarypump, an online vacuum degasser, an autosampler, and a columnoven (Shimadzu, Kyoto, Japan). The LC system was coupled with an API 4,000 tandem mass spectrometer (Applied Biosystems/MDS SCIEX, USA) equipped with an electrospray ionization source. Data were collected and analyzed by Analyst software (Applied Biosystems/MDS SCIEX, version 1.6.2).
Chromatographic separation was achieved on an Agilent Eclipse plus C18 column (100 mm × 2.1 mm, 3.5 μm; Agilent, USA) and maintained at 30 °C. The mobile phase consisted of methanol-0.1% ammonia in water (80:20, v/v) with fast isocratic elution at a flow rate of 0.4 mL min−1 and run time of 5 min. The sample volume injected was 5 µL and the temperature of auto sampler was set at 4 °C.
The mass spectrometer was run in electrospray ionization (ESI) mode using multiple reaction monitoring (MRM) to monitor the mass transitions. The MRM transitions were chosen to be m/z 360.1 → m/z 175.1 for pretomanid, m/z 124.1 → m/z 81.0 for pyrazinamide, m/z 172.1 → m/z 128.1 for IS. The declustering potential values set for pretomanid, pyrazinamide and IS were 65 V, 38 and 50 V, respectively. The collision energy values set for pretomanid, pyrazinamide and IS were 35 eV, 26 and 19 eV. The electrospray ionization voltage in positive mode was set 3,500 V. The turbo spray temperature was maintained at 350 °C. The curtain gas, gas 1 (nebulizer gas), and gas 2 (turbo gas, nitrogen) were 15, 50 and 50 psi, respectively. The product ion mass spectra of [M+H]+ of pretomanid, pyrazinamide and IS were presented in Fig. 1.
Product ion spectra of [M + H]+ of pretomanid (A), pyrazinamide (B), metronidazole (IS) (C) and their fragmentation pathways
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Product ion spectra of [M + H]+ of pretomanid (A), pyrazinamide (B), metronidazole (IS) (C) and their fragmentation pathways
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Product ion spectra of [M + H]+ of pretomanid (A), pyrazinamide (B), metronidazole (IS) (C) and their fragmentation pathways
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
2.6 Method validation
The LC-MS/MS method was validated according to the US Food and Drug Administration guidelines on bioanalytical method validation (US Food and Drug Administration, 2001).
2.6.1 Specificity
The selectivity of the method was evaluated by analyzing six individual sources of blank plasma samples to ensure that there were no interfering peaks at the retention time of either pretomanid, pyrazinamide and IS.
2.6.2 Linearity and sensitivity
The standard calibration curve for spiked plasma samples containing pretomanid and pyrazinamide was linear over the range 50–7,500 ng mL−1 and 500–75,000 ng mL−1. The calibration curves for all plasma samples showed good linearity (r2 > 0.9967 for pretomanid, r2 > 0.9956 for pyrazinamide) over the concentration ranges tested. The LLOQ was 50 ng mL−1 (pretomanid) and 500 ng mL−1 (pyrazinamide), which was sensitive enough for the pharmacokinetic study of pretomanid and pyrazinamide for oral administration in rats.
2.6.3 Accuracy and precision
The precision and accuracy of the method were assessed by analyzing QC samples in six replicates at three QC levels on three validation days. The intra-day and inter-day precisions were calculated using one-way analysis of variance (ANOVA) in terms of RSD and they should not exceed 15%. The accuracy was calculated as percentage deviation of the measured concentration from the nominal concentration (often expressed as relative error, RE) and it should be within ±15%.
2.6.4 Stability
The stability of pretomanid (100, 1,000, 5,000 ng mL−1) and pyrazinamide (1,000, 8,000, 50,000 ng mL−1) in rat plasma was evaluated by six replicates at three different concentrations. The short-term stability was examined by keeping the replicates of samples at room temperature for 12 h. The long-term stability was assessed by carrying out the experiment after 45 days of storage at −20°C. Freeze-thaw stability was assessed over three freeze-thaw cycles. Post-preparation stability was assessed by analyzing the extracted samples kept in the autosampler at 4°C for 24 h.
2.6.5 Extraction recovery and matrix effect
The extraction recovery of pretomanid, pyrazinamide and IS from rat plasma were evaluated by comparing the response of the extracted (pre-spiked) QC samples with those of the blank plasma at an equivalent concentration. The extraction recovery of pretomanid and pyrazinamide was determined at three different concentrations (high, medium, low), while IS at a single concentration of 50 ng mL−1. The recovery was expressed by the mean ± standard deviation (mean ± SD). The matrix effect was evaluated by comparing the response of the spike-after-extraction with those of neat samples at an equivalent concentration. The matrix effect was determined at three different concentrations (high, medium, low) and expressed by mean ± SD. The assessment of a relative matrix effect was performed based on direct comparison of the responses (peak areas) of the analyte spiked into extracts originating from six different lots (sources) of biofluids. The variability in these responses, determined as RSD (%), was considered as measure of relative matrix effect for a given analyte.
2.7 Pharmacokinetic application
All data were subsequently processed by the Analyst software (Applied Biosystems/MDS SCIEX, version 1.6.2) and Statistics version 3.0 (DAS 3.0, Anhui Provincial Center for Drug Clinical Evaluation). Data were shown as mean ± standard deviation (mean ± SD). The difference between groups was analyzed using student's t-test. A P-value less than 0.05 was considered as statistically significant.
3 Results and discussion
3.1 Method validation
3.1.1 Specificity
The selectivity of the method was assessed by LC-MS/MS derived from blank plasma samples. Figure 2 shows the typical chromatograms of a blank plasma sample, a blank plasma sample spiked with pretomanid, pyrazinamide and IS, a plasma sample from a rat 1 h after an oral administration. Under the given condition, pretomanid, pyrazinamide and IS eluted at retention time of 4.08 min, 3.64 min and 3.60 min. The result demonstrated that there was no interference with the determination of the pretomanid, pyrazinamide and IS, granting good method selectivity.
Representative MRM chromatograms for pretomanid, pyrazinamide and IS: (A) a blank plasma sample; (B) a blank plasma sample spiked with pretomanid (20 ng mL−1), pyrazinamide (150 ng mL−1) and IS (50 ng mL−1); (C) a plasma sample from a healthy rat 1h after an oral administration of pretomanid (20 mg kg−1) and pyrazinamide (150 mg kg−1)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Representative MRM chromatograms for pretomanid, pyrazinamide and IS: (A) a blank plasma sample; (B) a blank plasma sample spiked with pretomanid (20 ng mL−1), pyrazinamide (150 ng mL−1) and IS (50 ng mL−1); (C) a plasma sample from a healthy rat 1h after an oral administration of pretomanid (20 mg kg−1) and pyrazinamide (150 mg kg−1)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Representative MRM chromatograms for pretomanid, pyrazinamide and IS: (A) a blank plasma sample; (B) a blank plasma sample spiked with pretomanid (20 ng mL−1), pyrazinamide (150 ng mL−1) and IS (50 ng mL−1); (C) a plasma sample from a healthy rat 1h after an oral administration of pretomanid (20 mg kg−1) and pyrazinamide (150 mg kg−1)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
3.1.2 Linearity and sensitivity
The calibration curves were obtained over the concentration range 50–7,500 ng mL−1 for pretomanid and 500–75,000 ng mL−1 for pyrazinamide in plasma in all biosamples with a correlation coefficient (y = 0.642x + 0.024, r2 > 0.9967 for pretomanid, y = 0.046x + 0.011, r2 > 0.9956 for pyrazinamide). The lowest limit of quantification (LLOQ) was defined as the lower concentration could be determined with both accurately and precisely.
3.1.3 Accuracy and precision
The accuracy and precision were estimated by RE and RSD. The results of intra- day and inter-day precision and accuracy for the assay of pretomanid and pyrazinamide in rat plasma were summarized in Table 1. The intra- day and inter-day RSD (%) were both no more than 9.87%, while the RE (%) of assay accuracies ranged from −6.12 to 11.63%. These results suggest that the method was reliable and reproducible for the quantitative pretomanid and pyrazinamide in rat plasma. The LLOQ for analytes at which both precision and accuracy were less than 20% and within ±20%, respectively.
Precision and accuracy for pretomanid and pyrazinamide in rat plasma (n = 6, Mean ± SD)
| Spiked conc. (ng mL−1) | Intra-day (n = 6) | Inter-day (n = 3 × 6) | |||
| Precision | Accuracy | Precision | Accuracy | ||
| (% RSD) | (% RE) | (% RSD) | (% RE) | ||
| Pretomanid | 50 | 6.45 | 9.45 | 6.34 | −6.12 |
| 100 | 7.12 | 7.14 | 5.36 | 8.34 | |
| 1,000 | 7.95 | 6.25 | 2.47 | 6.45 | |
| 5,000 | 6.27 | −5.63 | 6.97 | 7.12 | |
| Pyrazinamide | 500 | 9.87 | 9.64 | 8.39 | 6.39 |
| 1,000 | 3.96 | 7.69 | 4.95 | 8.42 | |
| 8,000 | 5.12 | 11.63 | 6.87 | 3.24 | |
| 50,000 | 7.65 | 5.27 | 3.87 | 5.73 | |
3.1.4 Stability
The data of stability which summarized in Table 2 indicated that pretomanid and pyrazinamide were stable in frozen for 45 days, three freeze–thaw cycles, room temperature for 12 h, prepared samples in Freezer (4°C) for 24 h. The results proved that the method for sample extraction, storage and intermittent analysis were validated and suited for large scale sample analysis.
Stability of pretomanid and pyrazinamide in rat plasma under different storage conditions (n = 6, Mean ± SD)
| Analytes | Spiked conc. (ng mL−1) | Post-preparation 12 h at room temperature | Frozen (−20 °C) for 45 days | Three freeze-thaw cycles | Auto-sampler stability at 4 °C for 24 h |
| Accuracy (%) | Accuracy (%) | Accuracy (%) | Accuracy (%) | ||
| Pretomanid | 100 | 6.34 | 8.95 | 6.46 | 8.45 |
| 1,000 | 7.64 | 7.64 | −5.16 | 9.15 | |
| 5,000 | 3.94 | 2.96 | 7.91 | −7.11 | |
| Pyrazinamide | 1,000 | 5.66 | 6.79 | 6.15 | 6.37 |
| 8,000 | 10.94 | −9.15 | 7.15 | 7.89 | |
| 50,000 | −8.74 | 3.57 | 4.88 | 4.48 |
3.1.5 Extraction recovery and matrix effect
The extraction recoveries of pretomanid, pyrazinamide and IS shown in Table 3. It was proved that the method was consistent, reproducible, and acceptable. The results of matrix effects of pretomanid, pyrazinamide and IS were summarized in Table 3. It was indicated that the method was acceptable in different matrix.
Recovery and matrix effect of pretomanid and pyrazinamide in rat plasma (n = 6, Mean ± SD)
| Analytes | Spiked conc. | Recovery | Matrix effect | ||
| (ng mL−1) | (Mean ± SD %) | RSD (%) | (Mean ± SD %) | RSD (%) | |
| Pretomanid | 100 | 97.42 ± 5.56 | 5.71 | 93.38 ± 3.64 | 3.89 |
| 1,000 | 95.83 ± 1.70 | 1.78 | 95.21 ± 2.96 | 3.10 | |
| 5,000 | 96.15 ± 3.28 | 3.42 | 97.26 ± 2.11 | 2.17 | |
| Pyrazinamide | 1,000 | 93.93 ± 3.24 | 3.46 | 96.37 ± 1.67 | 1.73 |
| 8,000 | 103.46 ± 3.72 | 3.59 | 98.05 ± 3.35 | 3.42 | |
| 50,000 | 96.79 ± 2.03 | 2.09 | 97.67 ± 4.33 | 3.42 | |
| IS | 50 | 91.52 ± 5.49 | 6.05 | 91.76 ± 8.72 | 9.50 |
3.2 Optimization of LC–MS/MS conditions
It was a challenge to establish a suitable biological analysis method for simultaneous determination of pretomanid and pyrazinamide in plasma. After reviewing the literature [17–19], we decided to use LC-MS/MS to complete the simultaneous determination of the two drugs. Both positive and negative ion modes were used in our attempts to optimize the LC-MS response to pretomanid and pyrazinamide. Detection in positive ion mode was chosen due to stronger and more sensitive responses for both drugs and IS. The MRM transitions were chosen to be m/z 360.1 → m/z 175.1 for pretomanid, m/z 124.1 → m/z 81.0 for pyrazinamide, m/z 172.1 → m/z 128.1 for IS. Initially, we chose an Inertsil ODS3 C18 column to complete the separation, but the experimental results were always unsatisfactory because of the poor peak shape of metformin. Subsequently, good peak shape and successful separation were achieved on an Agilent Eclipse plus C18 column. The best resolution in terms of peak shape and strong signal was obtained using mobile phase A was methanol (A) and mobile phase B was 0.1% ammonia in water. A flow rate of 0.4 mL min−1 and operation time of 5.0 min were used for isocratic elution (A: B = 80:20, v/v).
3.3 Pharmacokinetic application
In the present study, the LC–MS/MS method was applied to the determination of pretomanid, pyrazinamide, and their combination in rat plasma, then we investigate and compared the pharmacokinetic parameters of rats which were administered pretomanid, pyrazinamide and pretomanid plus pyrazinamide, respectively. The main pharmacokinetic parameters calculated using non-compartmental analysis were shown in Table 4. Mean plasma concentration–time curves (n = 8) were presented in Figs 3 and 4. For pretomanid, there were little changes in the main pharmacokinetic parameters of pretomanid between alone and combined groups. Meanwhile, significant differences of AUC(0–t), tmax and t1/2 of pyrazinamide were observed between the alone and combined groups after administration to rats. The AUC(0–t), t1/2, tmax of pyrazinamide were decreased when the drug was co-administered with pretomanid, but the Cmax of pyrazinamide almost no change at all. The results show that there is an obvious pharmacokinetic interaction between pretomanid and pyrazinamide, which is manifested in the changes of the pharmacokinetic parameters of pyrazinamide.
Main pharmacokinetic parameters of pretomanid and pyrazinamide after oral administration of pretomanid (20 mg kg−1) and pyrazinamide (150 mg kg−1) alone and their combination in SD rats (n = 8, Mean ± SD)
| Parameter | Pretomanid | Pyrazinamide | ||
| Pretomanid | Pretomanid + Pyrazinamide | Pyrazinamide | Pretomanid + Pyrazinamide | |
| AUC(0-t) (ng mL−1·min−1) | 3401550.2 ± 354614.3 | 3527401.5 ± 1129228.5 | 32302911.5 ± 2502411.6 | 26919693.1 ± 2460902.1 |
| AUC(0-∞) (ng mL−1·min−1) | 3661748.5 ± 332917.7 | 3933291.7 ± 1463467.8 | 38707718.5 ± 4465558.3 | 29172451.1 ± 3191819.7 |
| tmax (min) | 360 ± 64.1 | 375 ± 76.9 | 210 ± 64.1 | 153.7 ± 54.2 |
| t1/2 (min) | 499.8 ± 155.4 | 556.2 ± 163.2 | 803.5 ± 215.7 | 581.8 ± 196.6 |
| CLz/F (L min−1 kg−1) | 0.006 ± 0.001 | 0.006 ± 0.001 | 0.004 ± 0.001 | 0.005 |
| Cmax (ng mL−1) | 3753.7 ± 729.2 | 3542.8 ± 799.8 | 33962.8 ± 3063.5 | 35750.3 ± 3224.7 |
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pretomanid (20 mg mL−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pretomanid (20 mg mL−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pretomanid (20 mg mL−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pyrazinamide (150 mg kg−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pyrazinamide (150 mg kg−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
Mean plasma concentration–time profiles (n = 8, Mean ± SD) of pyrazinamide (150 mg kg−1) alone and pretomanid (20 mg mL−1) plus pyrazinamide (150 mg kg−1) after oral administration to rats
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01087
4 Conclusion
In summary, a simple, sensitive and accurate LC-MS/MS method was developed and validated for the simultaneous determination of pretomanid and pyrazinamide in rat plasma, and it was applied to investigate the pharmacokinetics of these two drugs in rats. The drug-drug interaction of pretomanid and pyrazinamide in rats was reported for the first time. We studied the differences in the pharmacokinetic parameters of the two drugs used alone and in combination to investigate whether there is drug-drug interaction between the two drugs. Significant changes in AUC(0–t), tmax and t1/2 of pyrazinamide occurred after the two drugs were given at the same time. The pharmacokinetic drug-drug interaction results indicated that the pharmacokinetic parameters of pyrazinamide would affected by combined medication. It is hoped that our work would provide some reliable evidence for the safety of clinical medications.
Acknowledgment
This research was supported by the Basic Research plan of Natural Science of Shaanxi Province, China (Grant No: 2021JQ-887) and Scientific Research Program Funded by Education Department of Shaanxi Provincial Government (Grant No: 21JK0578).
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