Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine

Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine | 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 Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine Dongxin Chen, Jie Chen, Yuxin Shen, Xiaohai Chen, Hailun Xia, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4116048/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 Almonertinib, a novel third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, was selected by the Chinese Society of Clinical Oncology as a first-line therapy for EGFR T790M mutated non-small cell lung cancer in 2021. Almonertinib is primarily metabolized by CYP3A4, so it could interact with a variety of drugs metabolized by CYP3A4, leading to the changes of systemic exposure. For the purpose of this experiment, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay with accuracy and simplicity was optimized and fully validated for the simultaneous quantitative determination of almonertinib and its metabolite HAS-719, and drug-drug interactions (DDIs) between almonertinib and nicardipine in vivo and in vitro was researched. After one-step precipitation of protein with acetonitrile, chromatographic separations of almonaitinib, HAS-719 and gefitinib (internal standard, IS) were achieved by gradient elution with 0.1% formic acid aqueous solution and acetonitrile. Detection of analytes was achieved by MS/MS coupled with multiple reaction monitoring (MRM) in the positive ion mode with ion transitions of m/z 526.01 → 72.04 for almonertinib, m/z 512.18 → 455.08 for HAS-719, and m/z 447.16 → 128.11 for IS. There was favorable linearity in the 0.5–200 ng/mL calibration range for almonertinib and 0.5–100 ng/mL for HAS-719. The lower limit of quantification (LLOQ) for both analytes were 0.5 ng/mL. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation were consistent with the requirements of FDA guideline. Then, the UPLC-MS/MS assay was employed successfully on the interactions of almonertinib and nicardipine in vivo and in vitro . The half-maximal inhibitory concentration (IC 50 ) was 1.19 µM in rat liver microsomes (RLM), where nicardipine inhibited the metabolism of almonertinib with a mixed inhibitory mechanism. In pharmacokinetic experiments of rats, it was observed that nicardipine could significantly alter the pharmacokinetic profiles of almonertinib, including AUC (0−∞), AUC (0−t) and C max , but had no effect on the metabolism of HAS-719. According to the findings, it was indicated that nicardipine could inhibit the metabolism of almonertinib in vitro and in vivo . almonertinib HAS-719 nicardipine UPLC-MS/MS rat plasma Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The epidermal growth factor receptor (EGFR) with cytoplasmic kinase activity is a transmembrane protein delivering growth factor signals from the extracellular environment to the cell. It plays an important role in tumorigenesis in breast cancer, lung cancer and glioblastoma in the pathological setting. Undesirable activated EGFR is mainly caused by amplification of point mutations and genomic motifs [ 1 ]. EGFR tyrosine kinase inhibitors (TKIs) are sensitive and effective in cancers with EGFR mutations [ 2 ], inhibiting EGFR-related tyrosine kinase activity and intracellular phosphorylation, and blocking the growth of EGFR-induced tumor cells by binding competitively to the adenine nucleoside triphosphate site on the structural domain of intracellular tyrosine kinase [ 3 ]. Generation II EGFR-TKIs, like dacomitinib and afatinib, fail to overcome drug resistance arising from T790M mutations [ 4 ]. Almonertinib is a novel third-generation EGFR TKI (Fig. 1 A). As a highly selective and potent irreversible inhibitor of wild-type and mutant EGFR, it is approved for treating EGFR-mutated non-small cell lung cancer (NSCLC) patients [ 5 , 6 ]. N-Desmethylated almonertinib (HAS-719) (Fig. 1 B) is the major metabolite of almonertinib. Regarding drug elimination, CYP3A4 is the main metabolizing enzyme of almonertinib. It is known that CYP3A4 plays an important role in drug metabolisms, and 40–60% of the drugs used in clinical practice are metabolized by CYP3A. Since CYP3A4 is the main metabolizing enzyme of almonertinib, clinical attentions should be paid to the interactions between almonertinib and other drugs, especially CYP3A4 inducers or inhibitors. Therefore, the possibility of drug-drug interactions (DDIs) should be considered before and during medication therapy. Therapeutic drug monitoring (TDM) could be performed if necessary. As a second-generation dihydropyridine class of calcium channel blockers (CCBs), nicardipine is an approved drug for treating hypertension, angina, and related cerebrovascular diseases [ 7 , 8 ]. It has been reported to be a substrate of CYP3A2 in rats [ 9 ], and CYP3A4 and P-glycoprotein (P-gp) in humans [ 10 , 11 ]. It was demonstrated that nicardipine inhibited tacrolimus metabolism by CYP3A4 [ 12 , 13 ]. Given the wide use of CCBs in the management of cardiovascular diseases as well as the well-recognised role of calcium signalling in cancer progression, there has been a longtime interest in the possible effects of nicardipine on the clinical outcomes in cancer patients [ 14 , 15 ]. In addition, NSCLC patients may also have cardiovascular diseases, so it is necessary to take nicardipine with almonertinib at the same time for treatment. In studies of metabolic stability, pharmacokinetics, DDIs and TDM of different analytes in different matrices, ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has become the reference tool because of its high selectivity and efficiency of quantification [ 16 – 18 ]. Reports of UPLC-MS/MS assays to investigate almonertinib have been published, while the determination of HAS-719 (the main metabolite of almonertinib) was not mentioned and the long run time (> 3.0 min) was not suitable for analyzing large numbers of samples in these studies [ 19 – 21 ]. However, only one methodological study involved the simultaneous detection of almonertinib and HAS-719, but this assay used isotope-labeled internal standard (IS), which was expensive and not commercially available in the common laboratory [ 22 ]. Thus, it was designed to optimize an UPLC-MS/MS analytical method with accurateness and simplicity to simultaneously determine almonertinib and HAS-719, and to study the interactions between almonertinib and nicardipine in vitro and in vivo . Experimental Chemicals and reagents Reference standards of almonertinib (over 98% purity), and HAS-719 (over 98% purity) were provided kindly by Jiangsu Hansoh Pharmaceutical Group Co. Ltd. (Lianyungang, China). Gefitinib (IS, over 98% purity) was supplied by Beijing Sunflower Technology Development Co. Ltd. (Beijing, China). The chemicals and solvents used for this study including methanol and acetonitrile (both LC–MS grade) were purchased from Merck Company (Darmstadt, Germany). Formic acid with LC–MS grade was supplied by Anaqua Chemicals Supply (ACS, American). Deionized water was generated using a Milli-Q water purifier from Millipore (Bedford, USA). UPLC-MS/MS conditions The UPLC–MS/MS system was performed on a Waters XEVO TQS triple quadruple mass spectrometer and an ultra-performance liquid chromatograph (Waters Corp., USA). The separations of the analytes and IS were conducted using an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm, Waters Corp., USA) at 40°C with a mobile phase flow rate of 0.4 mL/min. A gradient elution was carried out using acetonitrile (A) and 0.1% formic acid aqueous solution (B) as mobile phases. Linear gradient scheme was set and conducted as follows: 0–0.5 min, 10% A; 0.5–1.0 min, 10–90% A; 1.0–1.5 min, 90% A;1.5–1.6 min, 90 − 10% A; 1.5–2.0 min, 10% A. The injection volume was 1.0 µL and the temperature of the autosampler was 10°C. The mass spectrometry was performed using an electrospray ionization source in the positive ionization and multiple reaction monitoring (MRM) mode with ion transitions of m/z 526.01 → 72.04 for almonertinib, m/z 512.18 → 455.08 for HAS-719, and m/z 447.16 → 128.11 for IS. The optimized parameters for the mass spectra of almonertinib, HAS-719 and IS included: cone voltage was 10 V for each analyte, and collision energy were 26 V, 23 V and 22 V, respectively. The mass spectrograms of almonertinib, HAS-719 and IS were presented in Table 1 . Table 1 Specific mass spectrometric parameters and retention times (RTs) for the analytes and IS, including cone voltage (CV), and collision energy (CE). Analytes Precursor ion Product ion CV (V) CE (V) RT (min) Almonertinib 526.01 72.04 10 26 1.21 HAS-719 512.18 455.08 10 23 1.20 IS 447.16 128.11 10 22 1.14 Preparation of working solutions and quality control (QC) samples For the preparation of 1.0 mg/mL of standard stock solutions, almonertinib and HAS-719 were accurately weighed and dissolved in methanol, respectively. Then, a deeper dilution was continued with methanol to obtain other concentrations of the standard solution. The same procedures were used to prepare the IS working solution (200 ng/mL). Eight calibrators by spiking 10% of standard solution volume into blank rat plasma were prepared at nominal plasma concentrations of 0.5–200 ng/mL for almonertinib and 0.5–100 ng/mL for HAS-719. Quality control (QC) samples containing the lower limit of quantification (LLOQ) were formulated at four levels of 0.5, 1.0, 40 and 160 ng/mL for almonertinib and 0.5, 1.0, 40 and 80 ng/mL for HAS-719, respectively. The stock and working solutions were stored in refrigerators set 4°C. Sample processing Prior to the treatment of samples, the samples were transferred from the − 80°C refrigerator and thawed at room temperature. Then, 20 µL of IS solution, 100 µL of plasma and 300 µL of acetonitrile were added to a 1.5 mL centrifuge tube and thoroughly mixed by vortexing for 3 min. The mixture was centrifuged at 13,000 × g for 10 min after thorough precipitation of the proteins by acetonitrile, and then 100 µL supernatant was transferred to a new autosampler vial, where 1.0 µL of the injection volume was used for UPLC-MS/MS investigation. Method validation Methodological validation of the bioassay was undertaken in compliance with the FDA regulatory principles for the assessment of selectivity, LLOQ, calibration curve, accuracy, precision, matrix effect, recovery and stability of the analytes in matrix [ 19 ]. The half-maximal inhibitory concentration (IC 50 ) and inhibitory mechanism of almonertinib by nicardipine in RLM The rat liver microsomes (RLM) incubation system was set up, then the drug was added to the system and incubated for 45 min as to determine the metabolism of the drug by the microsomes. The system with a final volume of 200 µL contained 1 M PBS, almonertinib (0.1, 0.2, 0.5, 1.0, 2.0, 5, 10, 20 µM), RLM (0.3 mg/mL), and NADPH (1 mM) in incubation system. The system was used to detect the Michaelis-Menten constant (K m ) values of almonertinib in RLM. The mixture without NADPH was preincubated for 5 min at 37°C before adding 1 mM NADPH to activate the reaction. After incubation at 37°C for 40 min, the mixture was ceased by rapidly cooling to -80°C, and was prepared by adding with 20 µL of IS solution (200 ng/mL) and 400 µL of acetonitrile. Following centrifugation at 13000 × rpm for 10 min, an upper supernatant of 100 µL was taken for determination by UPLC-MS/MS. For the detection of possible DDIs between almonertinib and nicardipine, the concentration of almonertinib was 5.25 µM in RLM, according to the K m value, while the concentrations of nicardipine were set at 0.01, 0.1, 1.0, 10, 25, 50 and 100 µM. To investigate the potential inhibitory mechanism, the concentrations of almonertinib and nicardipine were identified based on the K m and IC 50 values, respectively. The concentrations of nicardipine were 0, 0.60, 1.19, 2.38 and 4.76 µM, while the concentrations of almonertinib were 1.31, 2.63, 5.25, 10.50 and 21.00 µM. The sample was prepared in accordance with the experiment mentioned above. Effect of nicardipine on the pharmacokinetics of almonertinib in rats Ten healthy male Sprague-Dawley (SD) rats weighing 200–220 g were obtained from the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China). The rats were permitted to feed and drink freely, with a laboratory temperature of 25°C and a light/dark cycle of 12 h. During the experiments, the animals were operated in accordance with the animal ethics guidelines formulated by the Animal Care and Use Committee of the First Affiliated Hospital of Wenzhou Medical University (WYYY-IACUC-AEC-2023-014). Almonertinib and nicardipine were formulated in 0.5% sodium carboxymethylcellulose (CMC-Na) solution. Before the experiment started, ten rats were randomly divided into two groups: Group A (orally given 11 mg/kg almonertinib alone); Group B (orally given 11 mg/kg almonertinib and 6 mg/kg nicardipine). Administration of the drug was preceded by a 12-h fast with no restriction on water. Ten rats were given almonertinib 30 min after oral administration of nicardipine or 0.5% CMC-Na solution. Pre-dose (marked as 0 h) and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h post-dose, 0.3 mL of blood samples were obtained through the caudal vein, which were collected into tubes containing heparin and centrifuged at 8000 × rpm at 4°C for 10 min. Afterwards, plasma was shifted to tubes and stored at -80°C until later investigation. Euthanasia of experimental animals was performed using the anesthesia method according to the AVMA Guidelines for the Euthanasia of Animals. After completion of the experiment, all experimental animals were euthanized by intravenous pentobarbital (150 mg/kg). After ensuring that the animals were free of life pointers, they were packaged and cremated. The entire experimental process of the animals strictly adhered to the regulations for the care and use of laboratory animals as reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China). Statistical Analysis The Michaelis–Menten, IC 50 , Lineweaver-Burk plots were obtained using GraphPad Prism 8.0 software. Non-compartmental analysis was performed by using Drugs and Statistics (DAS) software (version 3.0 software, Shanghai University of Traditional Chinese Medicine, China) to obtain pharmacokinetic parameters. Mean plasma concentration-time curves were generated with Origin 8.0, and independent samples t -test was used to statistically investigate the parameters between the two groups. All data were shown as mean ± SD and analyzed using SPSS 24.0. Results and discussions Method validation Selectivity The selectivity of this assay was examined by comparing analyte-free plasma (Fig. 2 A), blank plasma sample spiked with almonertinib, HAS-719 and IS (Fig. 2 B) and the real plasma sample after medication (Fig. 2 C). It was observed that the MRM mode revealed high-resolution symmetric peaks with distinguishable retention times of almonertinib (1.21 min), HAS-719 (1.20 min) and IS (1.14 min). These data clearly demonstrated an excellent selectivity. Linearity of calibration curve and LLOQ Relative peak areas (almonertinib/IS) and corresponding plasma concentrations were analyzed by linear regression using the least squares method. The fitted calibration curve in rat plasma at a range of 0.5–200 ng/mL for almonertinib and 0.5–100 ng/mL for HAS-719 showed good linearity, which was derived with the coefficients of determination ( r 2 ) > 0.99. Table 2 summarized the typical regression equation of analytes. The developed approach provided LLOQ values of 0.5 ng/mL for almonertinib and HAS-719. The relative standard deviation (RSD) and relative error (RE) were < 12.1% and − 2.7 to 9.0%, respectively, which were within ± 20% according to the bioanalytical method validation guidelines (shown in Table 3 ). Table 2 Calibration curves for the analytes of almonertinib and HAS-719 in rat plasma. Analytes Regression equation r 2 Linear range (ng/mL) LLOQ (ng/mL) Almonertinib y = 0.025158x + 0.0335944 0.999 0.5–200 0.5 HAS-719 y = 0.0219341x + 0.00687146 0.995 0.5–100 0.5 Table 3 The accuracy and precision of each analyte in rat plasma (n = 5). Analytes Concentration (ng/mL) Intra-day Inter-day Mean ± SD RSD% RE% Mean ± SD RSD% RE% Almonertinib 0.5 0.49 ± 0.03 6.2 -2.7 0.48 ± 0.06 12.1 -3.7 1.0 0.94 ± 0.07 7.2 -5.9 1.00 ± 0.10 10.1 -0.2 40 39.77 ± 0.73 1.8 -0.6 40.77 ± 1.08 2.6 1.9 160 162.02 ± 5.78 3.6 1.3 165.17 ± 6.11 3.7 3.2 HAS-719 0.5 0.55 ± 0.02 4.0 9.0 0.50 ± 0.05 9.2 -0.3 1.0 1.08 ± 0.05 4.6 7.6 1.02 ± 0.06 5.8 1.8 40 43.58 ± 1.32 3.0 8.9 43.39 ± 1.13 2.6 8.5 80 85.35 ± 2.42 2.8 6.7 85.57 ± 4.33 5.0 7.0 Accuracy and precision The accuracy and precision of the method at four different levels of QC samples containing LLOQ were evaluated. Intra-day accuracy and precision were assessed by repeating the measurements of five duplicate samples on the same day, and inter-day accuracy and precision were obtained by measuring the samples on three different consecutive days. The precision for almonertinib and HAS-719 were within 15%. The estimated accuracies were within ± 15%, as shown in Table 3 . It was found that the developed method was reproducible, accurate and could simultaneously quantify almonertinib and HAS-719. Recovery and Matrix effect Extraction recoveries were obtained from the analysis by comparing the peak areas of analytes in extracted and non-extracted samples. The matrix effect was evaluated by comparing the peak areas of the plasma sample and the pure solution at their respective concentration levels. Table 4 summarized the recoveries and matrix effects of almonertinib and HAS-719 in plasma. For QC samples, the extraction recoveries ranged from 85.1–100.5% (n = 5). The matrix effects ranged from 88.6-110.9% of almonertinib and HAS-719 at three QCs levels (n = 5). The results indicated that the assay had high recoveries in QC samples, and matrix effects can be neglected in daily testing. Table 4 Recovery and matrix effect of each analyte in rat plasma (n = 5). Analytes Concentration (ng/mL) Recovery (%) Matrix effect (%) Mean ± SD RSD (%) Mean ± SD RSD (%) 1.0 93.2 ± 9.5 10.2 106.7 ± 13.3 12.5 Almonertinib 40 88.6 ± 4.7 5.3 89.1 ± 4.6 5.2 160 96.0 ± 2.4 2.5 88.6 ± 13.0 14.7 1.0 100.5 ± 6.0 5.9 110.9 ± 11.1 10.0 HAS-719 40 85.1 ± 1.6 1.9 104.4 ± 1.5 1.5 80 91.2 ± 1.7 1.9 102.4 ± 2.3 2.3 Stability The stability of almonertinib and HAS-719 in rat plasma was evaluated in four storage settings including short-term (3 h at room temperature), three freeze-thaw cycles (-80°C to room temperature), long-term (-80°C for 21 days) and 4 h in an autosampler (10°C). According to the results, the analytes remained stable with small variations, with RSD < 15% independent of different storage conditions, as shown in Table 5 . Table 5 Stability results of each analyte in plasma under different conditions (n = 5). Analyte Added (ng/mL) Room temperature, 3 h Autosampler 10°C, 4 h Three freeze-thaw -80°C, 3 weeks RSD (%) RE (%) RSD (%) RE (%) RSD (%) RE (%) RSD (%) RE (%) Almonertinib HAS-719 1.0 3.3 -8.9 6.5 -7.7 3.6 -3.2 2.4 12.8 40 3.0 -8.3 2.8 0.2 2.2 -7.9 2.2 -0.6 160 1.0 40 80 2.9 1.0 1.5 2.8 -8.5 -9.6 0.5 -1.0 2.4 1.6 1.3 1.4 0.4 12.6 2.1 -0.9 3.5 5.1 3.3 2.9 -0.9 -12.4 10.7 12.4 2.8 2.0 3.7 1.7 -10.6 0.6 0.0 1.7 Effects of nicardipine on the metabolism of almonertinib in RLM The K m value of almonertinib in RLM was 5.25 µM (Fig. 3 A) and IC 50 by nicardipine was 1.19 µM (Fig. 3 B). The inhibitory mechanism of nicardipine on almonertinib in RLM was indicated as a mixed type of non-competitive and competitive inhibition with moderate inhibitory effect [ 23 ], as shown in Fig. 4 . The Ki and αKi value for nicardipine were 0.83 and 3.48 µM, respectively. Nicardipine Significantly Alters the Pharmacokinetic Profiles of Almonertinib in SD Rats The pharmacokinetic parameters of almonertinib and HAS-719 were shown in Tables 6 and 7 , with their mean concentration-time curves in vivo presented in Fig. 5 . When nicardipine and almonertinib were are co-administered orally, the values of AUC (0–∞) , AUC (0–t) and C max were 1.95, 1.87 and 2.01-fold of group A, respectively, when compared with the rats receiving oral almonertinib alone. However, the pharmacokinetic parameters of HAS-719 were not significantly different between the two groups of rats. Table 6 The main pharmacokinetic parameters of almonertinib in two rat groups (group A: 11 mg/kg almonertinib dosed orally; group B: 11 mg/kg almonertinib and 6 mg/kg nicardipine dosed orally) (n = 5, Mean ± SD). Parameters Group A Group B AUC 0→t (ng/mL•h) 450.75 ± 107.091 880.04 ± 96.52 *** AUC 0→∞ (ng/mL•h) 488.26 ± 100.95 913.11 ± 95.60 ** t 1/2 (h) 5.96 ± 1.48 4.75 ± 0.51 T max (h) 4.20 ± 1.10 3.40 ± 1.82 CLz/F (L/h/kg) 23.32 ± 4.83 12.15 ± 1.22 Vz/F (L/kg) 206.84 ± 84.36 82.74 ± 15.37 C max (ng/mL) 52.39 ± 13.56 105.45 ± 32.32 ** **p < 0.01; ***p < 0.001, compared with the group A. AUC: area under the plasma concentration-time curve; t 1/2 : elimination half time; T max : peak time; CLz/F: plasma clearance; C max : maximum plasma concentration. Table 7 The main pharmacokinetic parameters of HAS-719 in two rat groups (group A: 11 mg/kg almonertinib dosed orally; group B: 11 mg/kg almonertinib and 6 mg/kg nicardipine dosed orally) (n = 5, Mean ± SD). Parameters Group A Group B AUC 0→t (ng/mL•h) 224.03 ± 70.37 204.28 ± 36.17 AUC 0→∞ (ng/mL•h) 239.29 ± 73.02 220.38 ± 29.34 t 1/2 (h) 5.67 ± 1.67 5.83 ± 1.55 T max (h) 5.20 ± 1.09 5.60 ± 0.89 CLz/F (L/h/kg) 49.11 ± 12.98 50.55 ± 5.96 Vz/F (L/kg) 399.36 ± 135.78 432.29 ± 151.75 C max (ng/mL) 23.67 ± 4.82 19.99 ± 4.03 AUC: area under the plasma concentration-time curve; t 1/2 : elimination half time; T max : peak time; CLz/F: plasma clearance; C max : maximum plasma concentration. Discussion The chromatographic condition was optimized to quantify the levels of almonertinib and HAS-719 with gifitinib as IS. Referring to our previous experiences, favorable results were obtained by acetonitrile and 0.1% formic acid aqueous solution. The experimental results showed that it could increase the sensitivity and improve the peak shape of the sample by adding formic acid to the mobile phase. Moreover, gradient elution is more conducive to the separation of analyte peaks. In addition, pre-treatment of sample was carried out by one-step protein precipitation with acetonitrile, which could obtain a satisfactory recovery. The isotopic IS has the same chemical property and the same retention time as the target analyte, counteracting matrix effect and eliminating differences in the pre-treatment process. However, some isotopic IS are difficult to be synthesized and expensive, which limits the applications. Compared to the isotope IS [ 22 ], gefitinib was selected as IS in the present research and methodological validation was proved to be successful with much lower cost. Almonertinib, mainly metabolized by CYP3A4, is a novel third-generation EGFR tyrosine kinase inhibitor. Nicardipine is an artery-selective calcium channel blocker that is effective of treating myocardial ischemia, hypertension and vasospasm [ 24 , 25 ], which is a substrate and inhibitor of CYP3A4 [ 26 , 27 ]. In addition, nicardipine was a relatively potent inhibitor of human CYP3A4 in vitro , suggesting that DDIs between nicardipine and other drugs metabolized mainly by CYP3A4 appear to occur in vivo [ 28 ]. In clinic, interaction between tacrolimus and nicardipine in the treatment of transplant recipients was found due to the inhibition of CYP3A4 responsible for the metabolism of tacrolimus, which could lead to tacrolimus overexposure in patients [ 12 , 13 , 29 , 30 ]. In order to prevent for tacrolimus toxicity, if nicardipine therapy is necessary, a 50% reduction in tacrolimus dose and daily TDM were recommend. In addition, in our another report, we also exhibited that nicardipine had inhibitory effect on alectinib metabolism with a mixture of non-competitive and anti-competitive inhibition in vitro and significantly increased the exposure of alectinib in vivo [ 31 ]. These literatures were consistent with the results of our this study, where the IC 50 value of almonertinib by nicardipine in RLM was 1.19 µM, with Ki and αKi values of 0.83 and 3.48 µM, respectively. In addition, the results of the current in vivo research have shown that when almonertinib and nicardipine were co-administrated orally, the values of AUC (0–∞), AUC (0–t) and C max were 1.95, 1.87 and 2.01-fold higher than almonertinib alone, increasing the systemic exposure of almonertinib. This is consistent with in vitro studies demonstrating that nicardipine has a great inhibitory effect on almonertinib metabolism. Recently, the DDIs study of almonertinib and Paxlovid in rats was also investigated, where Paxlovid had a marked inhibitory effect on the metabolism of almonertinib, increasing the exposure of almonertinib [ 19 ]. Therefore, the interaction between almonertinib and nicardipine or Paxlovid should be monitored in the clinical treatment. Finally, optimization of the UPLC-MS/MS approach in the study made it easier to detect the concentration of almonertinib for TDM and achieving individualized treatments in future. Conclusions In conclusions, the reliable and convenient UPLC-MS/MS method was optimized with operating time of only 2.0 min to simultaneously quantify almonertinib and its main metabolite (HAS-719) in vivo and in vitro . The study in RLM showed that nicardipine inhibited almonertinib with a mixed mechanism. Meanwhile, when almonertinib was co-administered with nicardipine in rats, a remarkable difference of the pharmacokinetic parameters was observed, suggesting a significant increase in the exposure of almonertinib. The outcomes of the research provided a possible explanation for dose adjustment or TDM to achieve individualized therapy especially when almonertinib and nicardipine are co-administrated orally. In clinical applications, further research should be done to confirm the result. Declarations Ethics approval and consent to participate Animal experiments were demonstrated to be ethically acceptable and were carried out according to the Guidelines of the Experimental Animal Care and Use of Laboratory Animals of The First Affiliated Hospital of Wenzhou Medical University. All animal procedures and experimental protocols were approved by the Laboratory Animal Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Ethics approval number: WYYY-IACUC-AEC-2023-014). Consent for publication Not applicable. Availability of data and materials The raw data supporting the conclusion of this article will be made available by the authors, without undue reservation. Competing interests The authors declare no competing interests. Funding No funding. Authors' contributions Dongxin Chen and Jie Chen conceived and designed the experiments; Yuxin Shen performed the experiments; Xiaohai Chen and Hailun Xia analyzed the data; Ya-nan Liu wrote the paper; Ren-ai Xu conceived the experiments. All authors read and approved the final manuscript. References Sigismund S, Avanzato D, Lanzetti LJMO: Emerging functions of the EGFR in cancer . 2018, 12 (1). Jaenne PA, Yang CH, Kim DW, Planchard D, Ohe Y, Ramalingam SS, Ahn MJ, Kim SW, Su WC, Horn LJTNEjom: AZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer . 2015(372-18). Jr RRJPR: Small molecule inhibitors targeting the EGFR/ErbB family of protein- tyrosine kinases in human cancers . 2019, 139 :395-411. Weiwei, Han, Yongli, Chemistry DJ, Biodiversity: Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors . 2017, 14 (7). Jiang T, Luo Y, Wang BJM: Almonertinib-induced interstitial lung disease: A case report . 2021, 100 (3):e24393. Yang CH, Camidge DR, Yang CT, Zhou J, Lu SJJotoopotIAftSoLC: Safety, Efficacy and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients with EGFR-mutated Advanced NSCLC: a Multicenter, Open-label, Phase I Trial . 2020. Sorkin EM, Clissold SP: Nicardipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, in the treatment of angina pectoris, hypertension and related cardiovascular disorders . Drugs 1987, 33 (4):296-345. Peacock WFt, Hilleman DE, Levy PD, Rhoney DH, Varon J: A systematic review of nicardipine vs labetalol for the management of hypertensive crises . Am J Emerg Med 2012, 30 (6):981-993. Guengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ: Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism . J Biol Chem 1986, 261 (11):5051-5060. Hu YP, Chapey C, Robert J: Relationship between the inhibition of azidopine binding to P-glycoprotein by MDR modulators and their efficiency in restoring doxorubicin intracellular accumulation . Cancer Lett 1996, 109 (1-2):203-209. Wang EJ, Casciano CN, Clement RP, Johnson WW: Two transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence . Biochim Biophys Acta 2000, 1481 (1):63-74. Hooper DK, Fukuda T, Gardiner R, Logan B, Roy-Chaudhury A, Kirby CL, Vinks AA, Goebel J: Risk of tacrolimus toxicity in CYP3A5 nonexpressors treated with intravenous nicardipine after kidney transplantation . Transplantation 2012, 93 (8):806-812. Sassi MB, Gaies E, Salouage I, Trabelsi S, Lakhal M, Klouz A: Involvement of CYP 3A5 In the Interaction Between Tacrolimus and Nicardipine: A Case Report . Curr Drug Saf 2015, 10 (3):254-256. Li X, Chen Y, Bai L, Zhao R, Wu Y, Xie ZR, Wu JM, Bowen NJ, Danaher A, Cook N et al : Nicardipine is a putative EED inhibitor and has high selectivity and potency against chemoresistant prostate cancer in preclinical models . Br J Cancer 2023, 129 (5):884-894. Masone MC: Nicardipine inhibits chemoresistance in prostate cancer . Nat Rev Urol 2023, 20 (9):519. Attwa MW, AlRabiah H, Alsibaee AM, Abdelhameed AS, Kadi AA: An UPLC-ESI-MS/MS Bioanalytical Methodology for the Quantification of Gilteritinib in Human Liver Microsomes: Application to In Vitro and In Silico Metabolic Stability Estimation . Separations 2023, 10 (5). Attwa MW, Abdelhameed AS, Alsibaee AM, Kadi AA: A Rapid and Sensitive UPLC-MS/MS Method for Quantifying Capmatinib in Human Liver Microsomes: Evaluation of Metabolic Stability by In Silico and In Vitro Analysis . Separations 2023, 10 (4). Attwa MW, Alsibaee AM, Aljohar HI, Abdelhameed AS, Kadi AA: Development of a Fast and Sensitive UPLC-MS/MS Analytical Methodology for Fenebrutinib Estimation in Human Liver Microsomes: In Vitro and In Silico Metabolic Stability Evaluation . Separations 2023, 10 (5). Tang PF, Bao SS, Gao NY, Shao CF, Xie WF, Wu XM, Zhao LP, Xiao ZX: Development and Validation of a UHPLC-MS/MS Method for Quantitation of Almonertinib in Rat Plasma: Application to an in vivo Interaction Study Between Paxlovid and Almonertinib . Front Pharmacol 2022, 13 :960311. Fu Y, Li Y, Ma Y, He X, Xun X, Cui Y, Fan L, Dong Z: Effects of voriconazole and fluconazole on the pharmacokinetics of almonertinib in rats by UPLC–MS/MS . Biomedical Chromatography 2022, 37 (1). Li Y, Meng L, Ma Y, Li Y, Xing X, Guo C, Dong Z: Determination of Osimertinib, Aumolertinib, and Furmonertinib in Human Plasma for Therapeutic Drug Monitoring by UPLC-MS/MS . Molecules 2022, 27 (14). Liu L, Yang L, Li W, Chen X: Simultaneous determination of almonertinib and its active metabolite HAS-719 in human plasma by LC-MS/MS: Evaluation of pharmacokinetic interactions . J Chromatogr B Analyt Technol Biomed Life Sci 2022, 1197 :123231. Jin C, He X, Zhang F, He L, Chen J, Wang L, An L, Fan Y: Inhibitory mechanisms of celastrol on human liver cytochrome P450 1A2, 2C19, 2D6, 2E1 and 3A4 . Xenobiotica 2015, 45 (7):571-577. Kishi Y, Okumura F, Furuya HJBJoA: Haemodynamic effects of nicardipine hydrochloride. Studies during its use to control acute hypertension in anaesthetized patients . (9):1003. Hysing, Chelly, Doursout, Anesthesiology CJ: Cardiovascular effects of and interaction between calcium blocking drugs and anesthetics in chronically instrumented dogs. III. Nicardipine and isoflurane . 1986. Higuchi S, Shiobara YJX: Metabolic fate of nicardipine hydrochloride, a new vasodilator, by various species in vitro . 1980, 10 (12):889-896. Guengerich FPJJoBC: Reactions and significance of cytochrome P-450 enzymes . 1991, 266 (16):10019-10022. Nakamura K, Ariyoshi N, Iwatsubo T, Fukunaga Y, Higuchi S, Itoh K, Shimada N, Nagashima K, Yokoi T, Yamamoto K et al : Inhibitory effects of nicardipine to cytochrome P450 (CYP) in human liver microsomes . Biol Pharm Bull 2005, 28 (5):882-885. Hurst AL, Clark N, Carpenter TC, Sundaram SS, Reiter PD: Supra-therapeutic tacrolimus concentrations associated with concomitant nicardipine in pediatric liver transplant recipients . Pediatr Transplant 2015, 19 (4):E83-87. Hooper DK, Carle AC, Schuchter J, Goebel J: Interaction between tacrolimus and intravenous nicardipine in the treatment of post-kidney transplant hypertension at pediatric hospitals . Pediatr Transplant 2011, 15 (1):88-95. Liu YN, Chen J, Wang J, Li Q, Hu GX, Cai JP, Lin G, Xu RA: Effects of drug-drug interactions and CYP3A4 variants on alectinib metabolism . Arch Toxicol 2023, 97 (8):2133-2142. 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-4116048","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282964000,"identity":"3066561a-6461-4109-ad2d-007d25bea1d5","order_by":0,"name":"Dongxin Chen","email":"","orcid":"","institution":"Ningbo University","correspondingAuthor":false,"prefix":"","firstName":"Dongxin","middleName":"","lastName":"Chen","suffix":""},{"id":282964001,"identity":"442ea6e9-7ad0-4d8a-965c-00ffd2e7d454","order_by":1,"name":"Jie Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Chen","suffix":""},{"id":282964002,"identity":"badd2dbc-baa7-4b04-9bd4-e1013af347c0","order_by":2,"name":"Yuxin Shen","email":"","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxin","middleName":"","lastName":"Shen","suffix":""},{"id":282964003,"identity":"6a429765-f734-4be3-b414-d29bd344f588","order_by":3,"name":"Xiaohai Chen","email":"","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaohai","middleName":"","lastName":"Chen","suffix":""},{"id":282964004,"identity":"294db8e3-3ddd-41d5-a830-6c52badfcc9f","order_by":4,"name":"Hailun Xia","email":"","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hailun","middleName":"","lastName":"Xia","suffix":""},{"id":282964005,"identity":"e5153144-0bae-4778-9f4f-88a28ce7440e","order_by":5,"name":"Ya-nan Liu","email":"","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Ya-nan","middleName":"","lastName":"Liu","suffix":""},{"id":282964006,"identity":"881f2899-ea12-4ea7-af06-468c24637376","order_by":6,"name":"Ren-ai Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDADNvYeMM3DR7wWnjMMDAeAWtiIt0YiB6yFgaAWg/NnzKQrKu7I80m+Pfj4Y46dDBsD88NHN/BpuZGWJnnmzDPDNum8ZIOD25KBDmMzNs7Bq4X5mGRj22HGNukcM4mD25iBWnjYpPFqOX+wTbLx32H7NskzIC31RGg5kAy0peFwYpsED0jLYcJaJG+kJVs2HDuc3MaTY2xwdttxHjZmAn7hO3/G8GZDzWHb+e1nDB9Ubqu252dvfvgYnxaFAxhCzHiUg4B8AwEFo2AUjIJRMAoYAEutR0rqzO11AAAAAElFTkSuQmCC","orcid":"","institution":"The First Affiliated Hospital of Wenzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ren-ai","middleName":"","lastName":"Xu","suffix":""}],"badges":[],"createdAt":"2024-03-17 08:29:17","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-4116048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4116048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53541290,"identity":"f4b7fc51-23f3-4a41-b9f3-0ceaa8cd9bfa","added_by":"auto","created_at":"2024-03-27 08:48:15","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":627862,"visible":true,"origin":"","legend":"\u003cp\u003eThe chemical structures and product mass spectra of [M + H] + ions of almonertinib (A), HAS-719 (B), and gifitinib (C).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/a425a4763179b02c468b0b2c.png"},{"id":53541889,"identity":"c078e1a8-96c2-4c14-bb7f-a0abe383ce98","added_by":"auto","created_at":"2024-03-27 08:56:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1585079,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative MRM chromatograms of almonertinib, HAS-719 and IS in SD rat sample: blank plasma (A), blank plasma spiked with standard solutions (B) and real plasma sample collected from a rat after 1.0 h oral administration of 11 mg/kg almonertinib (C).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/700a52f305cbb38a9382c8a8.png"},{"id":53541292,"identity":"58e8b619-a502-4986-ad99-76c5af30cf4c","added_by":"auto","created_at":"2024-03-27 08:48:15","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":126701,"visible":true,"origin":"","legend":"\u003cp\u003eThe Michaelis-Menten constant (K\u003csub\u003em\u003c/sub\u003e) value of almonertinib in RLM (A). The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of almonertinib by nicardipine in RLM (B).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/3924ed4a6842f89f43236a50.png"},{"id":53541289,"identity":"35ea93cc-1e6e-4bf1-abca-57142cae197a","added_by":"auto","created_at":"2024-03-27 08:48:15","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":246693,"visible":true,"origin":"","legend":"\u003cp\u003eLineweaver-Burk plot and the secondary plot for Ki in the inhibition of almonertinib metabolism by nicardipine with various concentrations in RLM.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/81917d29c5e6ec3464b8bd53.png"},{"id":53541293,"identity":"007ff278-7674-481d-a0f2-7473da9b5747","added_by":"auto","created_at":"2024-03-27 08:48:15","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":183064,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative almonertinib (A) and HAS-719 (B) concentration versus time profiles in two groups of SD rats. (n = 5, Mean ± SD).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/2dea4de50584c0f84e9a864f.png"},{"id":53914587,"identity":"0cca6fc5-8e2d-4404-ae01-632c10955ddb","added_by":"auto","created_at":"2024-04-02 07:27:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3143808,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4116048/v1/f4c7903b-fc1c-49e0-91fb-8870e037e022.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe epidermal growth factor receptor (EGFR) with cytoplasmic kinase activity is a transmembrane protein delivering growth factor signals from the extracellular environment to the cell. It plays an important role in tumorigenesis in breast cancer, lung cancer and glioblastoma in the pathological setting. Undesirable activated EGFR is mainly caused by amplification of point mutations and genomic motifs [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. EGFR tyrosine kinase inhibitors (TKIs) are sensitive and effective in cancers with EGFR mutations [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], inhibiting EGFR-related tyrosine kinase activity and intracellular phosphorylation, and blocking the growth of EGFR-induced tumor cells by binding competitively to the adenine nucleoside triphosphate site on the structural domain of intracellular tyrosine kinase [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Generation II EGFR-TKIs, like dacomitinib and afatinib, fail to overcome drug resistance arising from T790M mutations [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlmonertinib is a novel third-generation EGFR TKI (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As a highly selective and potent irreversible inhibitor of wild-type and mutant EGFR, it is approved for treating EGFR-mutated non-small cell lung cancer (NSCLC) patients [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. N-Desmethylated almonertinib (HAS-719) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) is the major metabolite of almonertinib. Regarding drug elimination, CYP3A4 is the main metabolizing enzyme of almonertinib. It is known that CYP3A4 plays an important role in drug metabolisms, and 40\u0026ndash;60% of the drugs used in clinical practice are metabolized by CYP3A. Since CYP3A4 is the main metabolizing enzyme of almonertinib, clinical attentions should be paid to the interactions between almonertinib and other drugs, especially CYP3A4 inducers or inhibitors. Therefore, the possibility of drug-drug interactions (DDIs) should be considered before and during medication therapy. Therapeutic drug monitoring (TDM) could be performed if necessary.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAs a second-generation dihydropyridine class of calcium channel blockers (CCBs), nicardipine is an approved drug for treating hypertension, angina, and related cerebrovascular diseases [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. It has been reported to be a substrate of CYP3A2 in rats [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], and CYP3A4 and P-glycoprotein (P-gp) in humans [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It was demonstrated that nicardipine inhibited tacrolimus metabolism by CYP3A4 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Given the wide use of CCBs in the management of cardiovascular diseases as well as the well-recognised role of calcium signalling in cancer progression, there has been a longtime interest in the possible effects of nicardipine on the clinical outcomes in cancer patients [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In addition, NSCLC patients may also have cardiovascular diseases, so it is necessary to take nicardipine with almonertinib at the same time for treatment.\u003c/p\u003e \u003cp\u003eIn studies of metabolic stability, pharmacokinetics, DDIs and TDM of different analytes in different matrices, ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) has become the reference tool because of its high selectivity and efficiency of quantification [\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Reports of UPLC-MS/MS assays to investigate almonertinib have been published, while the determination of HAS-719 (the main metabolite of almonertinib) was not mentioned and the long run time (\u0026gt;\u0026thinsp;3.0 min) was not suitable for analyzing large numbers of samples in these studies [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, only one methodological study involved the simultaneous detection of almonertinib and HAS-719, but this assay used isotope-labeled internal standard (IS), which was expensive and not commercially available in the common laboratory [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThus, it was designed to optimize an UPLC-MS/MS analytical method with accurateness and simplicity to simultaneously determine almonertinib and HAS-719, and to study the interactions between almonertinib and nicardipine \u003cem\u003ein vitro and in vivo\u003c/em\u003e.\u003c/p\u003e"},{"header":"Experimental","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eChemicals and reagents\u003c/h2\u003e \u003cp\u003eReference standards of almonertinib (over 98% purity), and HAS-719 (over 98% purity) were provided kindly by Jiangsu Hansoh Pharmaceutical Group Co. Ltd. (Lianyungang, China). Gefitinib (IS, over 98% purity) was supplied by Beijing Sunflower Technology Development Co. Ltd. (Beijing, China). The chemicals and solvents used for this study including methanol and acetonitrile (both LC–MS grade) were purchased from Merck Company (Darmstadt, Germany). Formic acid with LC–MS grade was supplied by Anaqua Chemicals Supply (ACS, American). Deionized water was generated using a Milli-Q water purifier from Millipore (Bedford, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eUPLC-MS/MS conditions\u003c/h2\u003e \u003cp\u003eThe UPLC–MS/MS system was performed on a Waters XEVO TQS triple quadruple mass spectrometer and an ultra-performance liquid chromatograph (Waters Corp., USA). The separations of the analytes and IS were conducted using an Acquity UPLC BEH C18 column (2.1 mm × 50 mm, 1.7 µm, Waters Corp., USA) at 40°C with a mobile phase flow rate of 0.4 mL/min. A gradient elution was carried out using acetonitrile (A) and 0.1% formic acid aqueous solution (B) as mobile phases. Linear gradient scheme was set and conducted as follows: 0–0.5 min, 10% A; 0.5–1.0 min, 10–90% A; 1.0–1.5 min, 90% A;1.5–1.6 min, 90 − 10% A; 1.5–2.0 min, 10% A. The injection volume was 1.0 µL and the temperature of the autosampler was 10°C. The mass spectrometry was performed using an electrospray ionization source in the positive ionization and multiple reaction monitoring (MRM) mode with ion transitions of \u003cem\u003em/z\u003c/em\u003e 526.01 → 72.04 for almonertinib, \u003cem\u003em/z\u003c/em\u003e 512.18 → 455.08 for HAS-719, and \u003cem\u003em/z\u003c/em\u003e 447.16 → 128.11 for IS. The optimized parameters for the mass spectra of almonertinib, HAS-719 and IS included: cone voltage was 10 V for each analyte, and collision energy were 26 V, 23 V and 22 V, respectively. The mass spectrograms of almonertinib, HAS-719 and IS were presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\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\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\u003eSpecific mass spectrometric parameters and retention times (RTs) for the analytes and IS, including cone voltage (CV), and collision energy (CE).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalytes\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrecursor ion\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProduct ion\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCV (V)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCE (V)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRT (min)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlmonertinib\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e526.01\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e72.04\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e26\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.21\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHAS-719\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e512.18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e455.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.20\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIS\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e447.16\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e128.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e22\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.14\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003ePreparation of working solutions and quality control (QC) samples\u003c/h2\u003e \u003cp\u003eFor the preparation of 1.0 mg/mL of standard stock solutions, almonertinib and HAS-719 were accurately weighed and dissolved in methanol, respectively. Then, a deeper dilution was continued with methanol to obtain other concentrations of the standard solution. The same procedures were used to prepare the IS working solution (200 ng/mL). Eight calibrators by spiking 10% of standard solution volume into blank rat plasma were prepared at nominal plasma concentrations of 0.5–200 ng/mL for almonertinib and 0.5–100 ng/mL for HAS-719. Quality control (QC) samples containing the lower limit of quantification (LLOQ) were formulated at four levels of 0.5, 1.0, 40 and 160 ng/mL for almonertinib and 0.5, 1.0, 40 and 80 ng/mL for HAS-719, respectively. The stock and working solutions were stored in refrigerators set 4°C.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eSample processing\u003c/h2\u003e \u003cp\u003ePrior to the treatment of samples, the samples were transferred from the − 80°C refrigerator and thawed at room temperature. Then, 20 µL of IS solution, 100 µL of plasma and 300 µL of acetonitrile were added to a 1.5 mL centrifuge tube and thoroughly mixed by vortexing for 3 min. The mixture was centrifuged at 13,000 × g for 10 min after thorough precipitation of the proteins by acetonitrile, and then 100 µL supernatant was transferred to a new autosampler vial, where 1.0 µL of the injection volume was used for UPLC-MS/MS investigation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eMethod validation\u003c/h2\u003e \u003cp\u003eMethodological validation of the bioassay was undertaken in compliance with the FDA regulatory principles for the assessment of selectivity, LLOQ, calibration curve, accuracy, precision, matrix effect, recovery and stability of the analytes in matrix [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) and inhibitory mechanism of almonertinib by nicardipine in RLM\u003c/h2\u003e \u003cp\u003eThe rat liver microsomes (RLM) incubation system was set up, then the drug was added to the system and incubated for 45 min as to determine the metabolism of the drug by the microsomes. The system with a final volume of 200 µL contained 1 M PBS, almonertinib (0.1, 0.2, 0.5, 1.0, 2.0, 5, 10, 20 µM), RLM (0.3 mg/mL), and NADPH (1 mM) in incubation system. The system was used to detect the Michaelis-Menten constant (K\u003csub\u003em\u003c/sub\u003e) values of almonertinib in RLM. The mixture without NADPH was preincubated for 5 min at 37°C before adding 1 mM NADPH to activate the reaction. After incubation at 37°C for 40 min, the mixture was ceased by rapidly cooling to -80°C, and was prepared by adding with 20 µL of IS solution (200 ng/mL) and 400 µL of acetonitrile. Following centrifugation at 13000 × rpm for 10 min, an upper supernatant of 100 µL was taken for determination by UPLC-MS/MS.\u003c/p\u003e \u003cp\u003eFor the detection of possible DDIs between almonertinib and nicardipine, the concentration of almonertinib was 5.25 µM in RLM, according to the K\u003csub\u003em\u003c/sub\u003e value, while the concentrations of nicardipine were set at 0.01, 0.1, 1.0, 10, 25, 50 and 100 µM. To investigate the potential inhibitory mechanism, the concentrations of almonertinib and nicardipine were identified based on the K\u003csub\u003em\u003c/sub\u003e and IC\u003csub\u003e50\u003c/sub\u003e values, respectively. The concentrations of nicardipine were 0, 0.60, 1.19, 2.38 and 4.76 µM, while the concentrations of almonertinib were 1.31, 2.63, 5.25, 10.50 and 21.00 µM. The sample was prepared in accordance with the experiment mentioned above.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eEffect of nicardipine on the pharmacokinetics of almonertinib in rats\u003c/h2\u003e \u003cp\u003eTen healthy male Sprague-Dawley (SD) rats weighing 200–220 g were obtained from the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China). The rats were permitted to feed and drink freely, with a laboratory temperature of 25°C and a light/dark cycle of 12 h. During the experiments, the animals were operated in accordance with the animal ethics guidelines formulated by the Animal Care and Use Committee of the First Affiliated Hospital of Wenzhou Medical University (WYYY-IACUC-AEC-2023-014). Almonertinib and nicardipine were formulated in 0.5% sodium carboxymethylcellulose (CMC-Na) solution. Before the experiment started, ten rats were randomly divided into two groups: Group A (orally given 11 mg/kg almonertinib alone); Group B (orally given 11 mg/kg almonertinib and 6 mg/kg nicardipine). Administration of the drug was preceded by a 12-h fast with no restriction on water. Ten rats were given almonertinib 30 min after oral administration of nicardipine or 0.5% CMC-Na solution. Pre-dose (marked as 0 h) and 0.33, 0.67, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h post-dose, 0.3 mL of blood samples were obtained through the caudal vein, which were collected into tubes containing heparin and centrifuged at 8000 × rpm at 4°C for 10 min. Afterwards, plasma was shifted to tubes and stored at -80°C until later investigation.\u003c/p\u003e \u003cp\u003e Euthanasia of experimental animals was performed using the anesthesia method according to the AVMA Guidelines for the Euthanasia of Animals. After completion of the experiment, all experimental animals were euthanized by intravenous pentobarbital (150 mg/kg). After ensuring that the animals were free of life pointers, they were packaged and cremated. The entire experimental process of the animals strictly adhered to the regulations for the care and use of laboratory animals as reviewed and approved by the Ethics Committee of The First Affiliated Hospital of Wenzhou Medical University (Wenzhou, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eThe Michaelis–Menten, IC\u003csub\u003e50\u003c/sub\u003e, Lineweaver-Burk plots were obtained using GraphPad Prism 8.0 software. Non-compartmental analysis was performed by using Drugs and Statistics (DAS) software (version 3.0 software, Shanghai University of Traditional Chinese Medicine, China) to obtain pharmacokinetic parameters. Mean plasma concentration-time curves were generated with Origin 8.0, and independent samples \u003cem\u003et\u003c/em\u003e-test was used to statistically investigate the parameters between the two groups. All data were shown as mean ± SD and analyzed using SPSS 24.0.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussions","content":"\u003ch2\u003eMethod validation\u003c/h2\u003e\u003ch2\u003eSelectivity\u003c/h2\u003e\u003cp\u003eThe selectivity of this assay was examined by comparing analyte-free plasma (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), blank plasma sample spiked with almonertinib, HAS-719 and IS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and the real plasma sample after medication (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). It was observed that the MRM mode revealed high-resolution symmetric peaks with distinguishable retention times of almonertinib (1.21 min), HAS-719 (1.20 min) and IS (1.14 min). These data clearly demonstrated an excellent selectivity.\u003c/p\u003e\u003ch2\u003eLinearity of calibration curve and LLOQ\u003c/h2\u003e\u003cp\u003eRelative peak areas (almonertinib/IS) and corresponding plasma concentrations were analyzed by linear regression using the least squares method. The fitted calibration curve in rat plasma at a range of 0.5–200 ng/mL for almonertinib and 0.5–100 ng/mL for HAS-719 showed good linearity, which was derived with the coefficients of determination (\u003cem\u003er\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e) \u0026gt; 0.99. Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarized the typical regression equation of analytes. The developed approach provided LLOQ values of 0.5 ng/mL for almonertinib and HAS-719. The relative standard deviation (RSD) and relative error (RE) were \u0026lt; 12.1% and − 2.7 to 9.0%, respectively, which were within ± 20% according to the bioanalytical method validation guidelines (shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\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=\".\" 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\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\u003eCalibration curves for the analytes of almonertinib and HAS-719 in rat plasma.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnalytes\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRegression equation\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003er\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLinear range (ng/mL)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLLOQ (ng/mL)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlmonertinib\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey = 0.025158x + 0.0335944\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.999\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5–200\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHAS-719\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ey = 0.0219341x + 0.00687146\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.995\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.5–100\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cdiv class=\"gridtable\"\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\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 accuracy and precision of each analyte in rat plasma (n = 5).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalytes\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c5\" namest=\"c3\"\u003e \u003cp\u003eIntra-day\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003eInter-day\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean ± SD\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\u003eMean ± SD\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=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAlmonertinib\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.49 ± 0.03\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e6.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-2.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.48 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e12.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-3.7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.94 ± 0.07\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-5.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.00 ± 0.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e39.77 ± 0.73\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-0.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e40.77 ± 1.08\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e162.02 ± 5.78\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e165.17 ± 6.11\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eHAS-719\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.55 ± 0.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e9.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.50 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e9.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.08 ± 0.05\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.02 ± 0.06\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e1.8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e43.58 ± 1.32\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e43.39 ± 1.13\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e8.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85.35 ± 2.42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e85.57 ± 4.33\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e7.0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eAccuracy and precision\u003c/h2\u003e\u003cp\u003eThe accuracy and precision of the method at four different levels of QC samples containing LLOQ were evaluated. Intra-day accuracy and precision were assessed by repeating the measurements of five duplicate samples on the same day, and inter-day accuracy and precision were obtained by measuring the samples on three different consecutive days. The precision for almonertinib and HAS-719 were within 15%. The estimated accuracies were within ± 15%, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It was found that the developed method was reproducible, accurate and could simultaneously quantify almonertinib and HAS-719.\u003c/p\u003e\u003ch2\u003eRecovery and Matrix effect\u003c/h2\u003e\u003cp\u003eExtraction recoveries were obtained from the analysis by comparing the peak areas of analytes in extracted and non-extracted samples. The matrix effect was evaluated by comparing the peak areas of the plasma sample and the pure solution at their respective concentration levels. Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e summarized the recoveries and matrix effects of almonertinib and HAS-719 in plasma. For QC samples, the extraction recoveries ranged from 85.1–100.5% (n = 5). The matrix effects ranged from 88.6-110.9% of almonertinib and HAS-719 at three QCs levels (n = 5). The results indicated that the assay had high recoveries in QC samples, and matrix effects can be neglected in daily testing.\u003c/p\u003e\u003cdiv class=\"gridtable\"\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=\"±\" 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\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\u003eRecovery and matrix effect of each analyte in rat plasma (n = 5).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eAnalytes\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003cp\u003e(ng/mL)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eRecovery (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eMatrix effect (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean ± SD\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eMean ± SD\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e93.2 ± 9.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e106.7 ± 13.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e12.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlmonertinib\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e88.6 ± 4.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e89.1 ± 4.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5.2\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e96.0 ± 2.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e88.6 ± 13.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14.7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e100.5 ± 6.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e5.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e110.9 ± 11.1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10.0\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHAS-719\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e85.1 ± 1.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e104.4 ± 1.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e91.2 ± 1.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e102.4 ± 2.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2.3\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eStability\u003c/h2\u003e\u003cp\u003eThe stability of almonertinib and HAS-719 in rat plasma was evaluated in four storage settings including short-term (3 h at room temperature), three freeze-thaw cycles (-80°C to room temperature), long-term (-80°C for 21 days) and 4 h in an autosampler (10°C). According to the results, the analytes remained stable with small variations, with RSD \u0026lt; 15% independent of different storage conditions, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\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\u003eStability results of each analyte in plasma under different conditions (n = 5).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"10\"\u003e\u003c/colgroup\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\u003eAdded (ng/mL)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e \u003cp\u003eRoom temperature, 3 h\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003eAutosampler 10°C, 4 h\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003eThree freeze-thaw\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e \u003cp\u003e-80°C, 3 weeks\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eRE (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eRE (%)\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\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003eRSD (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\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\u003eAlmonertinib\u003c/p\u003e \u003cp\u003eHAS-719\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e6.5\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e-7.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-3.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e12.8\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-7.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-0.6\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e160\u003c/p\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e40\u003c/p\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.9\u003c/p\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e1.5\u003c/p\u003e \u003cp\u003e2.8\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-8.5\u003c/p\u003e \u003cp\u003e-9.6\u003c/p\u003e \u003cp\u003e0.5\u003c/p\u003e \u003cp\u003e-1.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.4\u003c/p\u003e \u003cp\u003e1.6\u003c/p\u003e \u003cp\u003e1.3\u003c/p\u003e \u003cp\u003e1.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.4\u003c/p\u003e \u003cp\u003e12.6\u003c/p\u003e \u003cp\u003e2.1\u003c/p\u003e \u003cp\u003e-0.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3.5\u003c/p\u003e \u003cp\u003e5.1\u003c/p\u003e \u003cp\u003e3.3\u003c/p\u003e \u003cp\u003e2.9\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e-0.9\u003c/p\u003e \u003cp\u003e-12.4\u003c/p\u003e \u003cp\u003e10.7\u003c/p\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e2.8\u003c/p\u003e \u003cp\u003e2.0\u003c/p\u003e \u003cp\u003e3.7\u003c/p\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e-10.6\u003c/p\u003e \u003cp\u003e0.6\u003c/p\u003e \u003cp\u003e0.0\u003c/p\u003e \u003cp\u003e1.7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch2\u003eEffects of nicardipine on the metabolism of almonertinib in RLM\u003c/h2\u003e\u003cp\u003eThe K\u003csub\u003em\u003c/sub\u003e value of almonertinib in RLM was 5.25 µM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and IC\u003csub\u003e50\u003c/sub\u003e by nicardipine was 1.19 µM (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The inhibitory mechanism of nicardipine on almonertinib in RLM was indicated as a mixed type of non-competitive and competitive inhibition with moderate inhibitory effect [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. The Ki and αKi value for nicardipine were 0.83 and 3.48 µM, respectively.\u003c/p\u003e\u003ch2\u003eNicardipine Significantly Alters the Pharmacokinetic Profiles of Almonertinib in SD Rats\u003c/h2\u003e\u003cp\u003eThe pharmacokinetic parameters of almonertinib and HAS-719 were shown in Tables\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e and \u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e, with their mean concentration-time curves \u003cem\u003ein vivo\u003c/em\u003e presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. When nicardipine and almonertinib were are co-administered orally, the values of AUC\u003csub\u003e(0–∞)\u003c/sub\u003e, AUC\u003csub\u003e(0–t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e were 1.95, 1.87 and 2.01-fold of group A, respectively, when compared with the rats receiving oral almonertinib alone. However, the pharmacokinetic parameters of HAS-719 were not significantly different between the two groups of rats.\u003c/p\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe main pharmacokinetic parameters of almonertinib in two rat groups (group A: 11 mg/kg almonertinib dosed orally; group B: 11 mg/kg almonertinib and 6 mg/kg nicardipine dosed orally) (n = 5, Mean ± SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0→t\u003c/sub\u003e (ng/mL•h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e450.75 ± 107.091\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e880.04 ± 96.52\u003csup\u003e***\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0→∞\u003c/sub\u003e (ng/mL•h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e488.26 ± 100.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e913.11 ± 95.60\u003csup\u003e**\u003c/sup\u003e\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 (h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e5.96 ± 1.48\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e4.75 ± 0.51\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e (h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e4.20 ± 1.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e3.40 ± 1.82\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLz/F (L/h/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e23.32 ± 4.83\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e12.15 ± 1.22\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVz/F (L/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e206.84 ± 84.36\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e82.74 ± 15.37\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (ng/mL)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e52.39 ± 13.56\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e105.45 ± 32.32\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e**p \u0026lt; 0.01; ***p \u0026lt; 0.001, compared with the group A. AUC: area under the plasma concentration-time curve; t\u003csub\u003e1/2\u003c/sub\u003e: elimination half time; T\u003csub\u003emax\u003c/sub\u003e: peak time; CLz/F: plasma clearance; C\u003csub\u003emax\u003c/sub\u003e: maximum plasma concentration.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003cdiv class=\"gridtable\"\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\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe main pharmacokinetic parameters of HAS-719 in two rat groups (group A: 11 mg/kg almonertinib dosed orally; group B: 11 mg/kg almonertinib and 6 mg/kg nicardipine dosed orally) (n = 5, Mean ± SD).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup A\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup B\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0→t\u003c/sub\u003e (ng/mL•h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e224.03 ± 70.37\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e204.28 ± 36.17\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0→∞\u003c/sub\u003e (ng/mL•h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e239.29 ± 73.02\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e220.38 ± 29.34\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 (h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e5.67 ± 1.67\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e5.83 ± 1.55\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e (h)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e5.20 ± 1.09\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e5.60 ± 0.89\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCLz/F (L/h/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e49.11 ± 12.98\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e50.55 ± 5.96\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVz/F (L/kg)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e399.36 ± 135.78\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e432.29 ± 151.75\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (ng/mL)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e23.67 ± 4.82\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e19.99 ± 4.03\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003eAUC: area under the plasma concentration-time curve; t\u003csub\u003e1/2\u003c/sub\u003e: elimination half time; T\u003csub\u003emax\u003c/sub\u003e: peak time; CLz/F: plasma clearance; C\u003csub\u003emax\u003c/sub\u003e: maximum plasma concentration.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe chromatographic condition was optimized to quantify the levels of almonertinib and HAS-719 with gifitinib as IS. Referring to our previous experiences, favorable results were obtained by acetonitrile and 0.1% formic acid aqueous solution. The experimental results showed that it could increase the sensitivity and improve the peak shape of the sample by adding formic acid to the mobile phase. Moreover, gradient elution is more conducive to the separation of analyte peaks. In addition, pre-treatment of sample was carried out by one-step protein precipitation with acetonitrile, which could obtain a satisfactory recovery.\u003c/p\u003e \u003cp\u003eThe isotopic IS has the same chemical property and the same retention time as the target analyte, counteracting matrix effect and eliminating differences in the pre-treatment process. However, some isotopic IS are difficult to be synthesized and expensive, which limits the applications. Compared to the isotope IS [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], gefitinib was selected as IS in the present research and methodological validation was proved to be successful with much lower cost.\u003c/p\u003e \u003cp\u003eAlmonertinib, mainly metabolized by CYP3A4, is a novel third-generation EGFR tyrosine kinase inhibitor. Nicardipine is an artery-selective calcium channel blocker that is effective of treating myocardial ischemia, hypertension and vasospasm [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], which is a substrate and inhibitor of CYP3A4 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In addition, nicardipine was a relatively potent inhibitor of human CYP3A4 \u003cem\u003ein vitro\u003c/em\u003e, suggesting that DDIs between nicardipine and other drugs metabolized mainly by CYP3A4 appear to occur \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In clinic, interaction between tacrolimus and nicardipine in the treatment of transplant recipients was found due to the inhibition of CYP3A4 responsible for the metabolism of tacrolimus, which could lead to tacrolimus overexposure in patients [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In order to prevent for tacrolimus toxicity, if nicardipine therapy is necessary, a 50% reduction in tacrolimus dose and daily TDM were recommend. In addition, in our another report, we also exhibited that nicardipine had inhibitory effect on alectinib metabolism with a mixture of non-competitive and anti-competitive inhibition \u003cem\u003ein vitro\u003c/em\u003e and significantly increased the exposure of alectinib \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese literatures were consistent with the results of our this study, where the IC\u003csub\u003e50\u003c/sub\u003e value of almonertinib by nicardipine in RLM was 1.19 \u0026micro;M, with Ki and αKi values of 0.83 and 3.48 \u0026micro;M, respectively. In addition, the results of the current \u003cem\u003ein vivo\u003c/em\u003e research have shown that when almonertinib and nicardipine were co-administrated orally, the values of AUC\u003csub\u003e(0\u0026ndash;\u0026infin;),\u003c/sub\u003e AUC\u003csub\u003e(0\u0026ndash;t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e were 1.95, 1.87 and 2.01-fold higher than almonertinib alone, increasing the systemic exposure of almonertinib. This is consistent with \u003cem\u003ein vitro\u003c/em\u003e studies demonstrating that nicardipine has a great inhibitory effect on almonertinib metabolism. Recently, the DDIs study of almonertinib and Paxlovid in rats was also investigated, where Paxlovid had a marked inhibitory effect on the metabolism of almonertinib, increasing the exposure of almonertinib [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, the interaction between almonertinib and nicardipine or Paxlovid should be monitored in the clinical treatment. Finally, optimization of the UPLC-MS/MS approach in the study made it easier to detect the concentration of almonertinib for TDM and achieving individualized treatments in future.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusions, the reliable and convenient UPLC-MS/MS method was optimized with operating time of only 2.0 min to simultaneously quantify almonertinib and its main metabolite (HAS-719) \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The study in RLM showed that nicardipine inhibited almonertinib with a mixed mechanism. Meanwhile, when almonertinib was co-administered with nicardipine in rats, a remarkable difference of the pharmacokinetic parameters was observed, suggesting a significant increase in the exposure of almonertinib. The outcomes of the research provided a possible explanation for dose adjustment or TDM to achieve individualized therapy especially when almonertinib and nicardipine are co-administrated orally. In clinical applications, further research should be done to confirm the result.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnimal experiments were demonstrated to be ethically acceptable and were carried out according to the Guidelines of the Experimental Animal Care and Use of Laboratory Animals of\u0026nbsp;The First Affiliated Hospital of\u0026nbsp;Wenzhou Medical University. All animal procedures and experimental protocols were approved by the Laboratory Animal Ethics Committee of\u0026nbsp;The First Affiliated Hospital of\u0026nbsp;Wenzhou Medical University (Ethics approval number: WYYY-IACUC-AEC-2023-014).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting the conclusion of this article will be made available by the authors, without undue reservation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDongxin Chen and Jie Chen conceived and designed the experiments; Yuxin Shen performed the experiments; Xiaohai Chen and Hailun Xia analyzed the data; Ya-nan Liu wrote the paper; Ren-ai Xu conceived the experiments. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSigismund S, Avanzato D, Lanzetti LJMO: \u003cstrong\u003eEmerging functions of the EGFR in cancer\u003c/strong\u003e. 2018, \u003cstrong\u003e12\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eJaenne PA, Yang CH, Kim DW, Planchard D, Ohe Y, Ramalingam SS, Ahn MJ, Kim SW, Su WC, Horn LJTNEjom: \u003cstrong\u003eAZD9291 in EGFR Inhibitor-Resistant Non-Small-Cell Lung Cancer\u003c/strong\u003e. 2015(372-18).\u003c/li\u003e\n\u003cli\u003eJr RRJPR: \u003cstrong\u003eSmall molecule inhibitors targeting the EGFR/ErbB family of protein- tyrosine kinases in human cancers\u003c/strong\u003e. 2019, \u003cstrong\u003e139\u003c/strong\u003e:395-411.\u003c/li\u003e\n\u003cli\u003eWeiwei, Han, Yongli, Chemistry DJ, Biodiversity: \u003cstrong\u003eRecent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors\u003c/strong\u003e. 2017, \u003cstrong\u003e14\u003c/strong\u003e(7).\u003c/li\u003e\n\u003cli\u003eJiang T, Luo Y, Wang BJM: \u003cstrong\u003eAlmonertinib-induced interstitial lung disease: A case report\u003c/strong\u003e. 2021, \u003cstrong\u003e100\u003c/strong\u003e(3):e24393.\u003c/li\u003e\n\u003cli\u003eYang CH, Camidge DR, Yang CT, Zhou J, Lu SJJotoopotIAftSoLC: \u003cstrong\u003eSafety, Efficacy and Pharmacokinetics of Almonertinib (HS-10296) in Pretreated Patients with EGFR-mutated Advanced NSCLC: a Multicenter, Open-label, Phase I Trial\u003c/strong\u003e. 2020.\u003c/li\u003e\n\u003cli\u003eSorkin EM, Clissold SP: \u003cstrong\u003eNicardipine. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficacy, in the treatment of angina pectoris, hypertension and related cardiovascular disorders\u003c/strong\u003e. \u003cem\u003eDrugs \u003c/em\u003e1987, \u003cstrong\u003e33\u003c/strong\u003e(4):296-345.\u003c/li\u003e\n\u003cli\u003ePeacock WFt, Hilleman DE, Levy PD, Rhoney DH, Varon J: \u003cstrong\u003eA systematic review of nicardipine vs labetalol for the management of hypertensive crises\u003c/strong\u003e. \u003cem\u003eAm J Emerg Med \u003c/em\u003e2012, \u003cstrong\u003e30\u003c/strong\u003e(6):981-993.\u003c/li\u003e\n\u003cli\u003eGuengerich FP, Martin MV, Beaune PH, Kremers P, Wolff T, Waxman DJ: \u003cstrong\u003eCharacterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism\u003c/strong\u003e. \u003cem\u003eJ Biol Chem \u003c/em\u003e1986, \u003cstrong\u003e261\u003c/strong\u003e(11):5051-5060.\u003c/li\u003e\n\u003cli\u003eHu YP, Chapey C, Robert J: \u003cstrong\u003eRelationship between the inhibition of azidopine binding to P-glycoprotein by MDR modulators and their efficiency in restoring doxorubicin intracellular accumulation\u003c/strong\u003e. \u003cem\u003eCancer Lett \u003c/em\u003e1996, \u003cstrong\u003e109\u003c/strong\u003e(1-2):203-209.\u003c/li\u003e\n\u003cli\u003eWang EJ, Casciano CN, Clement RP, Johnson WW: \u003cstrong\u003eTwo transport binding sites of P-glycoprotein are unequal yet contingent: initial rate kinetic analysis by ATP hydrolysis demonstrates intersite dependence\u003c/strong\u003e. \u003cem\u003eBiochim Biophys Acta \u003c/em\u003e2000, \u003cstrong\u003e1481\u003c/strong\u003e(1):63-74.\u003c/li\u003e\n\u003cli\u003eHooper DK, Fukuda T, Gardiner R, Logan B, Roy-Chaudhury A, Kirby CL, Vinks AA, Goebel J: \u003cstrong\u003eRisk of tacrolimus toxicity in CYP3A5 nonexpressors treated with intravenous nicardipine after kidney transplantation\u003c/strong\u003e. \u003cem\u003eTransplantation \u003c/em\u003e2012, \u003cstrong\u003e93\u003c/strong\u003e(8):806-812.\u003c/li\u003e\n\u003cli\u003eSassi MB, Gaies E, Salouage I, Trabelsi S, Lakhal M, Klouz A: \u003cstrong\u003eInvolvement of CYP 3A5 In the Interaction Between Tacrolimus and Nicardipine: A Case Report\u003c/strong\u003e. \u003cem\u003eCurr Drug Saf \u003c/em\u003e2015, \u003cstrong\u003e10\u003c/strong\u003e(3):254-256.\u003c/li\u003e\n\u003cli\u003eLi X, Chen Y, Bai L, Zhao R, Wu Y, Xie ZR, Wu JM, Bowen NJ, Danaher A, Cook N\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eNicardipine is a putative EED inhibitor and has high selectivity and potency against chemoresistant prostate cancer in preclinical models\u003c/strong\u003e. \u003cem\u003eBr J Cancer \u003c/em\u003e2023, \u003cstrong\u003e129\u003c/strong\u003e(5):884-894.\u003c/li\u003e\n\u003cli\u003eMasone MC: \u003cstrong\u003eNicardipine inhibits chemoresistance in prostate cancer\u003c/strong\u003e. \u003cem\u003eNat Rev Urol \u003c/em\u003e2023, \u003cstrong\u003e20\u003c/strong\u003e(9):519.\u003c/li\u003e\n\u003cli\u003eAttwa MW, AlRabiah H, Alsibaee AM, Abdelhameed AS, Kadi AA: \u003cstrong\u003eAn UPLC-ESI-MS/MS Bioanalytical Methodology for the Quantification of Gilteritinib in Human Liver Microsomes: Application to In Vitro and In Silico Metabolic Stability Estimation\u003c/strong\u003e. \u003cem\u003eSeparations \u003c/em\u003e2023, \u003cstrong\u003e10\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eAttwa MW, Abdelhameed AS, Alsibaee AM, Kadi AA: \u003cstrong\u003eA Rapid and Sensitive UPLC-MS/MS Method for Quantifying Capmatinib in Human Liver Microsomes: Evaluation of Metabolic Stability by In Silico and In Vitro Analysis\u003c/strong\u003e. \u003cem\u003eSeparations \u003c/em\u003e2023, \u003cstrong\u003e10\u003c/strong\u003e(4).\u003c/li\u003e\n\u003cli\u003eAttwa MW, Alsibaee AM, Aljohar HI, Abdelhameed AS, Kadi AA: \u003cstrong\u003eDevelopment of a Fast and Sensitive UPLC-MS/MS Analytical Methodology for Fenebrutinib Estimation in Human Liver Microsomes: In Vitro and In Silico Metabolic Stability Evaluation\u003c/strong\u003e. \u003cem\u003eSeparations \u003c/em\u003e2023, \u003cstrong\u003e10\u003c/strong\u003e(5).\u003c/li\u003e\n\u003cli\u003eTang PF, Bao SS, Gao NY, Shao CF, Xie WF, Wu XM, Zhao LP, Xiao ZX: \u003cstrong\u003eDevelopment and Validation of a UHPLC-MS/MS Method for Quantitation of Almonertinib in Rat Plasma: Application to an in vivo Interaction Study Between Paxlovid and Almonertinib\u003c/strong\u003e. \u003cem\u003eFront Pharmacol \u003c/em\u003e2022, \u003cstrong\u003e13\u003c/strong\u003e:960311.\u003c/li\u003e\n\u003cli\u003eFu Y, Li Y, Ma Y, He X, Xun X, Cui Y, Fan L, Dong Z: \u003cstrong\u003eEffects of voriconazole and fluconazole on the pharmacokinetics of almonertinib in rats by UPLC\u0026ndash;MS/MS\u003c/strong\u003e. \u003cem\u003eBiomedical Chromatography \u003c/em\u003e2022, \u003cstrong\u003e37\u003c/strong\u003e(1).\u003c/li\u003e\n\u003cli\u003eLi Y, Meng L, Ma Y, Li Y, Xing X, Guo C, Dong Z: \u003cstrong\u003eDetermination of Osimertinib, Aumolertinib, and Furmonertinib in Human Plasma for Therapeutic Drug Monitoring by UPLC-MS/MS\u003c/strong\u003e. \u003cem\u003eMolecules \u003c/em\u003e2022, \u003cstrong\u003e27\u003c/strong\u003e(14).\u003c/li\u003e\n\u003cli\u003eLiu L, Yang L, Li W, Chen X: \u003cstrong\u003eSimultaneous determination of almonertinib and its active metabolite HAS-719 in human plasma by LC-MS/MS: Evaluation of pharmacokinetic interactions\u003c/strong\u003e. \u003cem\u003eJ Chromatogr B Analyt Technol Biomed Life Sci \u003c/em\u003e2022, \u003cstrong\u003e1197\u003c/strong\u003e:123231.\u003c/li\u003e\n\u003cli\u003eJin C, He X, Zhang F, He L, Chen J, Wang L, An L, Fan Y: \u003cstrong\u003eInhibitory mechanisms of celastrol on human liver cytochrome P450 1A2, 2C19, 2D6, 2E1 and 3A4\u003c/strong\u003e. \u003cem\u003eXenobiotica \u003c/em\u003e2015, \u003cstrong\u003e45\u003c/strong\u003e(7):571-577.\u003c/li\u003e\n\u003cli\u003eKishi Y, Okumura F, Furuya HJBJoA: \u003cstrong\u003eHaemodynamic effects of nicardipine hydrochloride. Studies during its use to control acute hypertension in anaesthetized patients\u003c/strong\u003e. (9):1003.\u003c/li\u003e\n\u003cli\u003eHysing, Chelly, Doursout, Anesthesiology CJ: \u003cstrong\u003eCardiovascular effects of and interaction between calcium blocking drugs and anesthetics in chronically instrumented dogs. III. Nicardipine and isoflurane\u003c/strong\u003e. 1986.\u003c/li\u003e\n\u003cli\u003eHiguchi S, Shiobara YJX: \u003cstrong\u003eMetabolic fate of nicardipine hydrochloride, a new vasodilator, by various species in vitro\u003c/strong\u003e. 1980, \u003cstrong\u003e10\u003c/strong\u003e(12):889-896.\u003c/li\u003e\n\u003cli\u003eGuengerich FPJJoBC: \u003cstrong\u003eReactions and significance of cytochrome P-450 enzymes\u003c/strong\u003e. 1991, \u003cstrong\u003e266\u003c/strong\u003e(16):10019-10022.\u003c/li\u003e\n\u003cli\u003eNakamura K, Ariyoshi N, Iwatsubo T, Fukunaga Y, Higuchi S, Itoh K, Shimada N, Nagashima K, Yokoi T, Yamamoto K\u003cem\u003e et al\u003c/em\u003e: \u003cstrong\u003eInhibitory effects of nicardipine to cytochrome P450 (CYP) in human liver microsomes\u003c/strong\u003e. \u003cem\u003eBiol Pharm Bull \u003c/em\u003e2005, \u003cstrong\u003e28\u003c/strong\u003e(5):882-885.\u003c/li\u003e\n\u003cli\u003eHurst AL, Clark N, Carpenter TC, Sundaram SS, Reiter PD: \u003cstrong\u003eSupra-therapeutic tacrolimus concentrations associated with concomitant nicardipine in pediatric liver transplant recipients\u003c/strong\u003e. \u003cem\u003ePediatr Transplant \u003c/em\u003e2015, \u003cstrong\u003e19\u003c/strong\u003e(4):E83-87.\u003c/li\u003e\n\u003cli\u003eHooper DK, Carle AC, Schuchter J, Goebel J: \u003cstrong\u003eInteraction between tacrolimus and intravenous nicardipine in the treatment of post-kidney transplant hypertension at pediatric hospitals\u003c/strong\u003e. \u003cem\u003ePediatr Transplant \u003c/em\u003e2011, \u003cstrong\u003e15\u003c/strong\u003e(1):88-95.\u003c/li\u003e\n\u003cli\u003eLiu YN, Chen J, Wang J, Li Q, Hu GX, Cai JP, Lin G, Xu RA: \u003cstrong\u003eEffects of drug-drug interactions and CYP3A4 variants on alectinib metabolism\u003c/strong\u003e. \u003cem\u003eArch Toxicol \u003c/em\u003e2023, \u003cstrong\u003e97\u003c/strong\u003e(8):2133-2142.\u003c/li\u003e\n\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":"almonertinib, HAS-719, nicardipine, UPLC-MS/MS, rat plasma","lastPublishedDoi":"10.21203/rs.3.rs-4116048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4116048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlmonertinib, a novel third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, was selected by the Chinese Society of Clinical Oncology as a first-line therapy for EGFR T790M mutated non-small cell lung cancer in 2021. Almonertinib is primarily metabolized by CYP3A4, so it could interact with a variety of drugs metabolized by CYP3A4, leading to the changes of systemic exposure. For the purpose of this experiment, an ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) assay with accuracy and simplicity was optimized and fully validated for the simultaneous quantitative determination of almonertinib and its metabolite HAS-719, and drug-drug interactions (DDIs) between almonertinib and nicardipine \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e was researched. After one-step precipitation of protein with acetonitrile, chromatographic separations of almonaitinib, HAS-719 and gefitinib (internal standard, IS) were achieved by gradient elution with 0.1% formic acid aqueous solution and acetonitrile. Detection of analytes was achieved by MS/MS coupled with multiple reaction monitoring (MRM) in the positive ion mode with ion transitions of \u003cem\u003em/z\u003c/em\u003e 526.01 \u0026rarr; 72.04 for almonertinib, \u003cem\u003em/z\u003c/em\u003e 512.18 \u0026rarr; 455.08 for HAS-719, and \u003cem\u003em/z\u003c/em\u003e 447.16 \u0026rarr; 128.11 for IS. There was favorable linearity in the 0.5\u0026ndash;200 ng/mL calibration range for almonertinib and 0.5\u0026ndash;100 ng/mL for HAS-719. The lower limit of quantification (LLOQ) for both analytes were 0.5 ng/mL. The precision, accuracy, stability, matrix effect, and extraction recovery required for methodological validation were consistent with the requirements of FDA guideline. Then, the UPLC-MS/MS assay was employed successfully on the interactions of almonertinib and nicardipine \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. The half-maximal inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) was 1.19 \u0026micro;M in rat liver microsomes (RLM), where nicardipine inhibited the metabolism of almonertinib with a mixed inhibitory mechanism. In pharmacokinetic experiments of rats, it was observed that nicardipine could significantly alter the pharmacokinetic profiles of almonertinib, including AUC\u003csub\u003e(0\u0026minus;\u0026infin;),\u003c/sub\u003e AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e, but had no effect on the metabolism of HAS-719. According to the findings, it was indicated that nicardipine could inhibit the metabolism of almonertinib \u003cem\u003ein vitro and in vivo\u003c/em\u003e.\u003c/p\u003e","manuscriptTitle":"Optimization of a sensitive and reliable UPLC-MS/MS method to simultaneously quantify almonertinib and HAS-719 and its application to study the interaction with nicardipine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 08:48:10","doi":"10.21203/rs.3.rs-4116048/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":"6d0e95c4-35e4-40d5-a6ad-e73ddf6ee50f","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-04-02T07:18:52+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-27 08:48:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4116048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4116048","identity":"rs-4116048","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}