Introduction

Lung cancer remains the leading cause of cancer-related mortality worldwide, accounting for approximately 1.8 million deaths annually.1 Among lung cancer subtypes, non-small cell lung cancer (NSCLC) represents the majority of cases, and anaplastic lymphoma kinase (ALK) rearrangements have been identified in approximately 4–7% of patients with NSCLC.2,3 The discovery of ALK gene rearrangements has led to the development of targeted ALK tyrosine kinase inhibitors (ALK-TKIs), which have significantly improved the prognosis of patients with ALK-positive NSCLC compared with conventional chemotherapy.4–6

Ensartinib (Figure 1A) is a second-generation, orally administered ALK-TKI with potent and selective inhibitory activity against a broad spectrum of ALK variants.7 It was approved by the National Medical Products Administration (NMPA) in China as a first-line treatment for patients with ALK-positive locally advanced or metastatic NSCLC.8 Clinical studies have demonstrated that ensartinib exhibits superior systemic and intracranial efficacy compared with crizotinib, with a favorable safety profile and prolonged progression-free survival.9–11 In particular, ensartinib has shown enhanced blood–brain barrier penetration, making it a promising therapeutic option for patients with central nervous system metastases.12

Figure 1 Chemical structures of ensartinib (A) and its major metabolite M465 (B).

In terms of pharmacokinetics, ensartinib undergoes extensive hepatic metabolism. Ensartinib is metabolized in the liver largely via the CYP3A4 pathway, and liver injury might also be caused by production of a toxic or immunogenic intermediate of its metabolism.13 In vitro studies have further demonstrated that ensartinib exhibits strong inhibitory effects on CYP3A4 and CYP2C9, with moderate inhibition of other CYP isoforms.14 These findings suggest a complex metabolic profile and highlight the importance of accurately characterizing both the parent drug and its metabolites in biological matrices. In addition, studies have shown that ensartinib is a substrate of P-glycoprotein (P-gp), which plays a role in the development of ensartinib resistance.15

M465 (Figure 1B) has been identified as the major metabolite of ensartinib and exhibits considerably weaker inhibitory activity against ALK variants compared to the parent compound. Importantly, previous studies reported that M465 does not exhibit any inhibitory effect on CYP enzymes in vitro,16 suggesting that the observed enzyme inhibition can be primarily attributed to ensartinib itself. A clinical study demonstrated that ensartinib and its metabolite M465 are the major circulating components, contributing equally to the total plasma radioactivity (AUC0-24h pool). In addition, the mean Cmax and t1/2 values of ensartinib in plasma were 185 ng/mL and 18.3 h, respectively, indicating sustained presence in circulation. These findings suggest that the metabolite M465 may also play a non-negligible role in the overall pharmacokinetic profile.17 Characterizing the disposition of M465 in preclinical animal models is an important step toward understanding its formation, systemic exposure, and elimination characteristics prior to clinical evaluation.18 Although pharmacokinetic data obtained from animal models cannot directly predict human exposure, such information provides valuable support for subsequent pharmacokinetic, pharmacodynamic, and safety investigations. Several analytical methods for the quantification of ensartinib have been reported in recent years. However, these methods primarily focus on the determination of the parent drug alone or in combination with other co-administered drugs, while the major metabolite M465 has rarely been taken into consideration. Moreover, pharmacokinetic studies involving the simultaneous measurement of ensartinib and its metabolite remain limited. A comparison of previously reported analytical methods and the present method is summarized in Table 1.

Table 1 Comparison of Reported Analytical Methods for the Determination of Ensartinib and M465

Therefore, the development of a sensitive, reliable, and reproducible bioanalytical method is essential for the accurate quantification of ensartinib and its major metabolite M465. Such a method would provide a robust analytical basis for pharmacokinetic, metabolic, and safety evaluations and support further preclinical and clinical investigations of ensartinib.

Methods Materials and Instruments Materials

Ensartinib and its metabolite M465 were supplied by Betta Pharmaceuticals Co, Ltd. (Zhejiang, China). Crizotinib (used as the internal standard, IS; purity 98%) was obtained from Shanghai Canspec Scientific Instruments Co, Ltd. (Shanghai, China). Other reagents used in the study included formic acid (Anaqua Chemicals Supply, ACS, USA) and LC-MS grade methanol and acetonitrile (Merck KGaA, Darmstadt, Germany). Ultrapure water was generated using a Milli-Q purification system (Millipore, Bedford, MA, USA).

Instrumental Conditions

Chromatographic separation of ensartinib, M465, and IS was performed on a Waters Acquity BEH C18 column (2.1 mm × 50 mm, 1.7 μm) maintained at 40°C. The mobile phase consisted of 0.1% formic acid in water (A) and acetonitrile (B), delivered at a flow rate of 0.40 mL/min, with a total run time of 2.0 min. The gradient elution program was as follows: 0–0.5 min (A: 90%); 0.5–1.0 min (A: 90–10%); 1.0–1.4 min (A: 10%); 1.4–1.5 min (A: 10–90%); and 1.5–2.0 min (A: 90%).

Mass spectrometric detection was conducted using a Waters Xevo TQ-S triple quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source operating in positive ion mode. Quantification was performed in multiple reaction monitoring (MRM) mode. The monitored ion transitions were m/z 561.30→257.10 for ensartinib, m/z 465.05→275.00 for M465, and m/z 450.06→260.07 for IS, respectively.

Calibration Standard Curves, and Quality Control (QC)

Stock solutions of ensartinib (1.00 mg/mL), M465 (1.00 mg/mL), and IS (1.00 mg/mL) were separately prepared in methanol. Working solutions were prepared by serial dilution of the stock solutions with methanol. The IS working solution was diluted to a concentration of 200 ng/mL. Calibration standards were prepared by spiking 10 μL of ensartinib working solution and 10 μL of M465 working solution into 80 μL of blank rat plasma. The final calibration concentrations were 0.1, 1, 5, 10, 20, 40, 80 and 100 ng/mL for ensartinib, and 0.05, 0.1, 0.5, 1, 2, 4, 8 and 10 ng/mL for M465, respectively. Quality control (QC) samples were prepared independently at three concentration levels: low, medium, and high (0.2, 40, and 80 ng/mL for ensartinib; 0.1, 4, and 8 ng/mL for M465). The lower limit of quantification (LLOQ) samples were prepared at 0.1 ng/mL for ensartinib and 0.05 ng/mL for M465, respectively. All solutions and spiked plasma samples were stored at −80°C.

Sample Preparation

A simple protein precipitation method was used for sample extraction. Briefly, 100 μL of plasma sample (or calibration standard/QC) was mixed with 10 μL of the IS working solution (200 ng/mL) and 300 μL of acetonitrile. The mixture was vortexed for 2 min and centrifuged at 13,000 rpm for 10 min at 4°C. An aliquot of 8.0 μL of the supernatant was injected into the UPLC–MS/MS system for analysis.

Method Development and Validation

The method was validated in accordance with the US Food and Drug Administration (FDA) Bioanalytical Method Validation Guidance for Industry.22 The validation parameters included selectivity, linearity, sensitivity, LLOQ, accuracy, precision, recovery, matrix effect, stability, carry-over, and dilution integrity for ensartinib and its metabolite M465.

Selectivity was assessed by comparing chromatograms of blank samples, matrix samples spiked with analytes and IS, and real samples, to verify the absence of endogenous interference at the retention times of ensartinib, M465, and IS. Calibration curves were constructed by plotting the concentration of each analyte against the peak area ratio of analyte to IS, using weighted least-squares regression. The LLOQ was defined as the lowest concentration on the calibration curve that met the criteria of acceptable accuracy and precision.

Intra- and inter-day precision and accuracy were evaluated by analyzing QC samples at four concentration levels (including LLOQ) in five replicates over three consecutive days, with fresh calibration curves prepared on each day. Precision was expressed as the relative standard deviation (RSD%), and accuracy as the relative error (RE%). Values within ±15% (±20% for LLOQ) were considered acceptable.

The extraction recoveries of ensartinib and M465 were evaluated by comparing the peak areas of analytes spiked before extraction with those of analytes spiked after extraction at equivalent concentrations. Blank plasma samples from six individual male Sprague–Dawley rats were collected and used for the evaluation of matrix effects. Equal volumes of plasma from each individual animal were pooled to prepare pooled matrix samples. After extraction, ensartinib and M465 were spiked into the processed plasma samples and compared with standard solutions prepared in methanol at the same concentrations.

Stability of ensartinib and M465 was investigated under various conditions, including short-term stability at room temperature, long-term stability after storage at −80°C, three freeze-thaw cycles between −80°C and room temperature, and autosampler stability after 4 h at 10°C. All stability evaluations were performed using QC samples at three concentration levels. Values within ±15% were considered acceptable. In addition, the stability of ensartinib and M465 standard solutions was evaluated after storage at room temperature for 6 h and at 4°C for 24 h. The stored solutions were compared with freshly prepared standard solutions at equivalent concentrations. The solutions were considered stable if the deviation was within ±10%.

Carry-over was evaluated by injecting a blank rat plasma sample immediately after the upper limit of quantitation (ULOQ) sample. The responses of ensartinib, M465, and IS in the blank sample were examined to determine whether residual analytes remained in the analytical system. Carry-over was considered acceptable if the response in the blank sample was less than 20% of the LLOQ for analytes and less than 5% of the IS response.

Dilution integrity was evaluated by diluting dilution quality control (DQC) samples 5-fold and 10-fold with blank rat plasma to assess the accuracy and precision of each analyte. The nominal concentrations of the DQC samples were 400 ng/mL for ensartinib and 40 ng/mL for M465. Values within ±15% were considered acceptable.

Pharmacokinetic Study in Rats

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee (approval number: WYYY-IACUC-AEC-2026-005) and were conducted in strict accordance with the national standard for the welfare of laboratory animals (GB/T 42011-2022). Five male Sprague–Dawley rats (200 ± 10 g) were obtained from the Experimental Animal Center of the First Affiliated Hospital of Wenzhou Medical University (Zhejiang, China). Prior to the experiment, all animals were housed in a standard laboratory animal facility and allowed to acclimatize under controlled environmental conditions, including a 12 h light/dark cycle, a constant temperature of 22–25°C, and a relative humidity of 45–65%, with free access to standard laboratory chow and water. The sample size (n = 5) was based on prior pharmacokinetic and DDI studies in rats, demonstrating adequacy for detecting meaningful differences.

The rats were fasted for 12 h before dosing, while water was available ad libitum. Ensartinib was formulated as an oil-based solution at a concentration of 8 mg/mL and administered as a single oral dose. All rats received ensartinib by gavage at a dose of 22.5 mg/kg. The dosing volume was 2.8 mL/kg. The selected dose was determined based on human equivalent dose conversion using FDA-recommended guidelines. Blood samples were collected from the tail vein at 0.25, 0.5, 1, 1.5, 2, 4, 6, 12, 24, and 48 h after administration. Each blood sample (0.3 mL) was centrifuged at 13,000 rpm for 8 min, and 100 μL of the resulting plasma was separated and stored at −80°C until analysis.

Euthanasia of experimental animals was performed using the anesthetic 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 confirming the absence of vital signs, the animal carcasses were placed in appropriate bags and incinerated. 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 mean plasma concentration–time curves of ensartinib and its metabolite M465 were generated using GraphPad Prism 9.5 software to visualize their systemic exposure profiles. Pharmacokinetic parameters of ensartinib and M465, including the area under the concentration–time curve (AUC), elimination half-life (t1/2), time to reach maximum concentration (Tmax), apparent clearance (CLz/F), and maximum plasma concentration (Cmax) were calculated based on the observed plasma concentration–time data using non-compartmental analysis with DAS 3.0 software (Drug and Statistical Software, Shanghai Mathematical Pharmacology Professional Committee, China).

Results Method Validation Selectivity

Representative chromatograms of blank plasma, blank plasma spiked with analytes and IS, and plasma samples obtained after oral administration are shown in Figure 2. Under the established chromatographic conditions, the retention times of ensartinib, M465, and IS were 1.23, 1.36, and 1.16 min, respectively. No significant endogenous interference was observed at the retention times of the analytes or the IS, indicating that the method exhibited good selectivity in rat plasma.

Figure 2 Representative chromatograms of ensartinib, M465 and crizotinib (IS) in rat plasma: blank plasma (A), blank plasma with analytes at LLOQ and IS (B), and plasma sample from a rat after oral administration of 22.5 mg/kg ensartinib (C).

Linearity and LLOQ

Under the established analytical conditions, ensartinib exhibited good linearity over the concentration range of 0.1–100 ng/mL, with a regression equation of Y = 0.0449172X + 0.00735752 (r2 = 0.997). M465 also showed good linearity over the range of 0.05–10 ng/mL, with a regression equation of Y = 0.0564485X + 0.00355316 (r2 = 0.992). Calibration curves were calculated by using 1/x2 weighted linear least squares regression to plot the peak area ratio of analyte to IS and the nominal concentration of analyte. High correlation coefficients were obtained for both analytes within their respective calibration ranges, indicating reliable and consistent quantitative performance. The LLOQs for ensartinib and M465 were determined to be 0.1 ng/mL and 0.05 ng/mL, respectively, demonstrating the high sensitivity of the method. As summarized in Tables S1 and S2, the relative errors of ensartinib calibration standards ranged from −8.0% to 8.7%, and all values obtained for M465 were also within the acceptable limits. All calibration standards fulfilled the acceptance criterion of a deviation within ±15% from the nominal concentrations.

Precision and Accuracy

The intra- and inter-day precision and accuracy of the method were systematically evaluated by analyzing QC samples at different concentration levels over three consecutive days. As shown in Table 2, the intra-day RSD% values for ensartinib and M465 ranged from 3.5% to 16.4% and 2.2% to 14.9%, respectively, while the inter-day RSD% values ranged from 3.2% to 13.7% and 5.2% to 13.9%, respectively. The relative error (RE%) for all analytes ranged from −12.3% to 8.0%. These results indicated that the precision and accuracy of the method met the acceptance criteria of the FDA bioanalytical guidelines (RSD within 15%, and within 20% at the LLOQ).

Table 2 Precision and Accuracy of Ensartinib and Its Metabolite M465 in Rat Plasma (n = 5)

Extraction Recovery and Matrix Effect

The extraction recovery and matrix effects were quantitatively evaluated by comparing the responses of analytes in pre-extraction spiked samples, post-extraction spiked samples, and neat solutions (Table 3). The extraction recoveries of ensartinib and M465 ranged from 98.0% to 99.6% and 96.5% to 99.3%, respectively, with RSD% values below 12.0%, indicating high extraction efficiency and good reproducibility. The matrix effects for ensartinib and M465 ranged from 92.9% to 110.7% and 89.3% to 91.8%, respectively, suggesting that endogenous plasma components had no significant impact on analyte ionization.

Table 3 Recovery and Matrix Effect of Ensartinib and M465 in Rat Plasma (n = 5)

Stability

The stability of ensartinib and M465 in rat plasma was systematically investigated under various storage and analytical conditions. As shown in Table 4, all QC samples exhibited RSD% and RE% values within ±11.7% after storage at room temperature for 3 h, three freeze–thaw cycles, long-term storage at −80°C for 21 days, and storage in the autosampler at 10°C for 4 h, indicating both analytes had good stability. As shown in Table S3, the standard solutions stored at room temperature for 6 h and at 4°C for 24 h showed acceptable accuracy and precision. The RE% values ranged from −4.5% to 3.2% for ensartinib and were within 6.3% for M465, while all RSD% values were within 7.2%. These results demonstrated that ensartinib and M465 standard solutions were stable under the tested conditions.

Table 4 Stability Results of Ensartinib and M465 in Rat Plasma in Different Conditions (n = 5)

Dilutional Integrity and Carry-Over Studies

As shown in Table S4, the dilution integrity of ensartinib and M465 was acceptable at 5-fold and 10-fold dilution levels. The RSD% values were within 3.1%, and the RE% values ranged from −0.4% to 1.9%. These results demonstrated that the dilution procedure did not affect the accuracy and precision of the method. No significant carry-over was observed after injection of the ULOQ samples, as no detectable peaks of ensartinib, M465, and the IS were observed in the subsequent blank plasma samples.

Animal Study

Based on the validated analytical method, the pharmacokinetic profiles of ensartinib and its major metabolite M465 were systematically investigated in rats. As shown in Figure 3 and Table 5, ensartinib was moderately absorbed after oral administration and reached its maximum plasma concentration (Cmax) of 64.7 ± 14.3 ng/mL at approximately 4.9 h (Tmax), indicating a clear absorption process and measurable systemic exposure. In contrast, M465 exhibited significantly lower systemic exposure, with a Cmax of 6.3 ± 1.7 ng/mL. The elimination half-life (t1/2) of M465 was 5.9 ± 2.4 h. Overall, ensartinib predominantly existed in its parent form in vivo, while M465 showed limited plasma exposure and a relatively minor contribution to the overall pharmacokinetic behavior. For M465, pharmacokinetic parameters were derived from its observed plasma concentration–time profile following oral administration of ensartinib. It should be noted that M465 was not administered directly, and thus its concentration–time profile reflects the combined processes of formation from the parent compound and subsequent elimination.

Table 5 The Main Pharmacokinetic Parameters of Ensartinib and M465 in Sprague-Dawley Rats (n = 5)

Figure 3 Mean plasma concentration-time curves of ensartinib (A), M465 (B). Data are presented as the means ± SD, n = 5.

Discussion

In recent years, UPLC–MS/MS has been widely applied to the bioanalysis of small-molecule targeted anticancer agents.23–25 However, most previously published bioanalytical methods have primarily focused on the quantification of parent drugs, while systematic quantitative evaluation of metabolites has received relatively limited attention. For example, although several UPLC–MS/MS methods have been reported for the determination of novel oral targeted anticancer agents primarily used in non-small cell lung cancer, most of these methods focused exclusively on the parent drugs, and simultaneous quantitative analysis of both the parent compound and its metabolites was rarely performed.20,26 In contrast, parent drugs and their major metabolites often exhibit markedly different exposure levels, clearance characteristics, and potential safety implications in vivo. Therefore, the establishment of simultaneous quantitative analytical methods is of substantial importance for a comprehensive characterization of drug disposition.

In the limited studies that have attempted the simultaneous quantitative analysis of parent drugs and their metabolites, several methodological challenges have been consistently highlighted, particularly with respect to chromatographic separation, sensitivity matching among analytes, and control of matrix effects. Jiang et al systematically demonstrated that coexisting parent compounds and metabolites may cause significant signal interference during LC–ESI–MS analysis, arising from incomplete chromatographic resolution, ion suppression or enhancement, and mismatched ionization efficiencies, all of which can compromise quantitative accuracy.27 Their work emphasized that such interference is a critical but often underestimated issue in multi-analyte bioanalysis. In addition, effective control of matrix effects has been recognized as a key prerequisite for reliable metabolite quantification. Dubbelman et al proposed the use of postcolumn infusion of standards as a practical strategy to visualize and correct matrix-induced signal variability, thereby improving quantitative robustness in LC-MS-based metabolomic and bioanalytical assays.28 Collectively, these studies underscore that the development of a robust method for synchronous quantification of parent drugs and metabolites requires careful optimization of chromatographic behavior, ionization response, and matrix effect control, highlighting the technical complexity and methodological significance of such analyses.

During method development in the present study, chromatographic conditions, mass spectrometric parameters, and sample pretreatment procedures were carefully optimized to achieve high sensitivity and selectivity for both ensartinib and M465. Protein precipitation using acetonitrile was selected as the sample preparation strategy due to its operational simplicity, good reproducibility, and high extraction efficiency. This approach has been widely applied in the bioanalysis of small-molecule drugs and their metabolites,29–31 and the present results further confirmed its suitability for the ensartinib–M465 analytical system.

Comprehensive method validation was conducted in accordance with FDA bioanalytical guidelines. The method demonstrated good linearity over wide concentration ranges, with LLOQ for both analytes. Precision, accuracy, recovery, matrix effect, stability, carry-over and dilution integrity results met the acceptance criteria, indicating that the method was robust and reproducible.

The validated method was successfully applied to a pharmacokinetic study following oral administration of ensartinib in rats. Ensartinib was absorbed at a moderate rate and reached its Cmax at approximately 4.85 h post-dose. However, the Tmax of M465 was 1.45 h, and the relatively large variability observed may be associated with differences in absorption processes and inter-individual variations in metabolic activity. In contrast, the systemic exposure of M465 was substantially lower, with a markedly lower Cmax, suggesting limited systemic exposure and extensive clearance of the metabolite. As a major metabolite of ensartinib, the formation of M465 may be mediated by CYP3A4. However, the relatively low exposure of M465 may be explained by the fact that its plasma exposure is determined by the balance between formation from the parent drug and subsequent elimination processes.32 Therefore, the lower exposure of M465 may be attributed to limited metabolic conversion from ensartinib and subsequent rapid elimination of the metabolite. Interestingly, previous studies have shown that the exposure of M465 was comparable to that of unchanged ensartinib when expressed as a proportion of total plasma radioactivity.17 This discrepancy may be due to species differences in the expression and activity of metabolic enzymes between rats and humans, which could contribute to variations in the formation and systemic exposure of M465.33

It should be noted that these pharmacokinetic results were obtained in a rat model. Given the potential species differences in metabolic enzyme expression and metabolic pathways, caution should be exercised when extrapolating these findings to humans. Further studies integrating in vitro metabolism experiments and clinical pharmacokinetic data are warranted.

Overall, this study provided a validated bioanalytical method and the first in vivo quantitative pharmacokinetic data for ensartinib and its metabolite M465 in rats. These findings offered important methodological and experimental support for further preclinical research and may contribute to a deeper understanding of the pharmacokinetic properties of ensartinib in subsequent translational and clinical studies.

Conclusion

In summary, the present study established and fully validated a UPLC–MS/MS method capable of the simultaneous determination of ensartinib and its major metabolite M465 in rat plasma, which demonstrated satisfactory analytical performance in terms of sensitivity, linearity, precision, accuracy, and matrix effect under the validated conditions. On the basis of this method, a pharmacokinetic study was successfully conducted in Sprague-Dawley rats, allowing for the quantitative characterization of the in vivo disposition of both the parent drug and its major metabolite. However, the relatively small sample size in the in vivo study (n = 5) may have limited the statistical power and increased the variability among individuals. Future studies with larger sample sizes are warranted to further validate and strengthen these findings. These results provide useful analytical support for preclinical pharmacokinetic investigations. Nevertheless, due to potential species differences, extrapolation of these findings to humans should be performed with caution, and further clinical studies are necessary to elucidate the pharmacokinetic behavior and clinical relevance of ensartinib and M465.

Disclosure

The authors report no conflicts of interest in this work.

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