Introduction

Non-small-cell lung cancer (NSCLC) accounts for the majority (80–85%) of lung cancer, which is the leading cause of cancer mortality, with 1.8 million deaths (18%) globally in 2020 (Sung et al. 2021). Over the past few decades, substantial advancements have been made in the development of targeted therapies and immunotherapies for NSCLC (Li et al. 2023a, b). Nevertheless, the emergence of treatment resistance remains a significant challenge in effectively managing the disease, especially in patients with activating epidermal growth factor receptor (EGFR) mutations. These mutations play a critical role in the development of NSCLC, with a particularly high prevalence in adenocarcinoma, the most common subtype of NSCLC (Midha et al. 2015).

The most prevalent EGFR-activating mutations (EGFR+) observed in NSCLC include exon 19 deletions and a single point mutation in exon 21 (L858R) of EGFR (Gazdar 2009). Patients with EGFR-mutated NSCLC frequently exhibit positive responses to first- and second-generation EGFR tyrosine kinase inhibitors (TKIs) such as afatinib, erlotinib, and gefitinib (Chang et al. 2023). The T790M mutation is a secondary alteration that can arise in NSCLC patients who initially respond to first- and second-generation EGFR TKIs but subsequently develop resistance to these medications by restoring a few of the EGFR protein activities (Belani 2010; Santoni-Rugiu et al. 2019). Third-generation TKI, such as osimertinib and lazertinib, can effectively inhibit EGFR+/T790M-positive tumors (Li et al. 2022). However, acquired resistance still emerges over time with C797S, another secondary mutation within the EGFR gene, limiting the clinical efficiency of the third-generation TKI (Lategahn et al. 2019; Li et al. 2023a, b). Recent reports indicate that approximately 40% of patients develop C797S mutations following treatment with third-generation EGFR TKIs (Duggirala et al. 2022). Pharmaceutical companies worldwide have focused on developing small molecules to target the EGFR+/T790M/C797S triple mutant NSCLC. Several compounds with varying scaffolds and binding interactions have been developed as fourth-generation small-molecule EGFR-TKIs (Mansour et al. 2023). Among these, BLU-945 is a highly potent drug candidate designed to selectively target and inhibit the EGFR T790M/C797S and T790M resistance mutations (Schalm et al. 2020a, b).

In enzymatic assays, BLU-945 exhibits excellent inhibitory activity against EGFR+(L858R, ex19del), EGFR+/T790M, and EGFR+/T790M/C797S mutants (Schalm et al. 2020a, b). In preclinical pharmacokinetic studies involving rats, dogs, and cynomolgus monkeys, BLU-945 exhibited low-to-moderate clearance, a moderate volume of distribution, and good oral bioavailability (Eno et al. 2022). Significant tumor regression was observed in mice carrying xenografts of NCI-H1975 (a resistant NSCLC cell line), mice with engineered Ba/F3 EGFR L858R/T790M/C797S and Ba/F3 ex19del/T790M/C797S osimertinib-resistant tumors, and in mice bearing a patient-derived cell line (osimertinib-resistant EGFR ex19del/T790M/C797S). Preclinical toxicity studies in rats and non-human primates have shown suitable safety margins for BLU-945. In a phase 1 dose-escalation study (NCT04862780), BLU-945 was evaluated in patients with EGFR-mutated NSCLC who had previously undergone at least one EGFR-targeted TKI treatment. The plasma concentration–time profile of BLU-945 (25 mg) showed low clearance and an extended plasma half-life (T1/2) of the candidate drug (Eno et al. 2022).

Liquid chromatography-tandem mass spectrometry (LC–MS/MS) was used to analyze BLU-945 in in vivo PK studies (Eno et al. 2022). However, details of the method, including compound extraction from plasma and MS/MS parameters, have not been reported, and the method has not been validated. The objective of this study was to develop and validate a bioanalytical method for quantifying BLU-945 using LC–MS/MS. The developed method was used to investigate the in vitro metabolic stability and in vivo oral pharmacokinetic profile of the compound in rats. These data will be valuable for future preclinical and clinical evaluations of BLU-945.

Materials and methods

Materials

BLU-945 and ND207937 (internal standard, IS) were synthesized indoors. High-performance liquid chromatography (HPLC)-grade acetonitrile and methanol were purchased from Honeywell Burdick and Jackson (Ulsan, Korea). Formic acid, dimethyl sulfoxide (DMSO), PEG400 (Polyethylene glycol 400), and Kolliphor EL were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and solvents were of the highest quality or HPLC grade.

Instrumentation and analytical conditions

The LC–MS/MS system used for the BLU-945 determination consisted of a 4500 Triple Quad mass spectrometer and an Exion LC AD instrument (Applied Biosystems, Framingham, MA, USA). Electrospray ionization was performed in positive mode. The separation of compounds was performed using a Synergi Polar-RP 80 Å column (150 × 2.0 mm, 4 µm; Phenomenex (Torrance, CA, USA)) with an isocratic mobile phase comprising 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile (30:70, v:v). The flow rate of the mobile phase was set at 0.35 mL/min for 3 min of running time, and the injection volume was 1 µL. The autosampler was kept at 4 °C, while the column was maintained at 25 °C. The mass spectrometry parameters, including ion spray voltage, the temperature of the ion source, curtain gas, nebulizing gas, turbo gas, and entrance potential, were set at 5500 V, 550 °C, 35 psi, 50 psi, 40 psi, and 10 V, respectively, for both BLU-945 and IS. Other parameters were optimized for each compound: declustering potentials of 12 and 36 V, collision energies of 47 and 49 V, and collision cell exit potentials of 18 and 34 V for BLU-945 and IS, respectively. The multiple reaction monitoring (MRM) transitions were m/z 557.3 → 423.3 for BLU-945 and m/z 543.2 → 423.2 for IS (ND207937). The analytical data were processed using the Analyst software version 1.6.2 (Applied Biosystems-SCIEX).

Sample preparation

A primary stock solution of BLU-945 was prepared at a concentration of 1 mg/mL in DMSO and serially diluted with methanol to obtain working standards and quality control (QC) samples. Two µL of working standard solution or QC sample was spiked to 18 µL of blank plasma, followed by the addition of 130 µL acetonitrile containing IS (200 ng/mL ND207937) for the protein precipitation. After vortexing for 5 min and centrifuging at 4 °C for 5 min at 14,000 rpm, the aliquots of the supernatant were injected into the LC–MS/MS system for BLU-945 analysis. The standard concentrations were 1, 2, 5, 10, 20, 50, 100, 200, and 1000 ng/mL. QC samples were prepared at concentrations of 1 (lower limit of quantitation, LLOQ), 3 (Low QC, LQC), 30 (Middle QC1, MQC1), 500 (Middle QC2, MQC2), and 800 ng/mL (High QC, HQC).

Assay validation

Full validation of the analytical method, including selectivity, linearity, accuracy, precision, matrix effect, recovery, process efficiency, stability, and dilution integrity, was performed on rat and mouse plasma according to the guidelines of the United States Food and Drug Administration and European Medicines Agency (US-FDA 2018; ICH 2019).

Selectivity was evaluated by analyzing plasma from six individual sources (mouse or rat), including blank plasma (double blank), blank plasma spiked with IS only (zero), blank plasma spiked with the BLU-945 working solution and IS, and plasma samples after oral administration. The chromatographic retention times of BLU-945 and the IS were compared across samples to confirm the absence of interference from endogenous substances or impurities in the plasma.

Calibration curves for BLU-945 in rat and mouse plasma were obtained by calculating the peak ratios of the analyte to IS versus the nominal concentrations of the standards. Calibration curves were fitted using linear least-squares regression with a weighting factor of 1/x2. Linearity was assessed using correlation coefficients (r), with values ≥ 0.990 considered acceptable (Yoon et al. 2020).

QC samples (1, 3, 30, 500 and 800 ng/mL) were used to assess the accuracy and precision of the method. Intraday accuracy and precision were estimated using six replicates on the same day, whereas interday values were calculated using experiments conducted over three days. Precision was expressed as the relative standard deviation (CV%), and accuracy was calculated as the difference between the calculated and nominal values divided by the nominal value (RE%). The acceptance criteria for precision and accuracy were CV ≤  ± 15% and RE ≤  ± 15%, respectively, except for the LLOQ, the acceptance value was ≤ 20%.

The matrix effect, extraction recovery, and process efficiency were investigated for BLU-945 at four QC levels (LQC, MQC1, MQC2, and HQC) and IS at a concentration of 200 ng/mL. Three sets of samples were prepared with four replicates for each concentration. Set 1 was defined as the response of the analyte from the extracted spiked plasma, whereas set 2 was the response of the analyte from the extracted blank plasma spiked with BLU-945 (post-extraction samples). Set 3 was defined as the response of the analyte to the neat standard solution. The matrix effect was calculated by dividing the mean peak area of set 2 by that of set 3. Extraction recovery was computed by dividing the data of set 1 by that of set 2, and process efficiency was assessed by comparing the data of sets 1 and 3 (Hyun et al. 2017).

The stock solution stability of BLU-945 (300 ng/mL) and the IS (200 ng/mL) was conducted by comparing the peak response from a freshly prepared solution in methanol and that of solutions subjected to short-term storage conditions (approximately 25 °C for 6 h) and long-term storage conditions (− 20 °C for 6 months). The stability of BLU-945 in rat and mouse plasma was evaluated by analyzing three replicates of LQC (3 ng/mL) and HQC (800 ng/mL). The short-term stability study was performed at room temperature (25 ℃) for 1 h before processing, while samples stored at − 20 °C for 8 weeks were analyzed to assess long-term stability. The processed sample stability was determined by analyzing samples in an autosampler at 4 °C for 12 h. Samples subjected to three freeze–thaw cycles were analyzed for freeze–thaw stability. All stability samples were evaluated in comparison to the freshly prepared ones, and a sample was considered stable with a difference in peak response of ≤ 15%.

Dilution integrity was investigated using dilution quality controls (DQC) at 4000 ng/mL (DQC1) and 5000 ng/mL (DQC2), which were further diluted five times with blank rat/mouse plasma to obtain samples at 800 ng/mL and 1000 ng/mL, respectively (Lee et al. 2022). Six replicates were tested in one run to determine accuracy and precision. The acceptance criteria for diluted samples were precision and accuracy within ± 15%.

In vitro metabolic stability

The liver microsomal and plasma stabilities of BLU-945 were investigated in humans, rats, and mice, as previously reported (Doan et al. 2020; Nguyen et al. 2022; Le et al. 2023). For liver microsomal stability, a mixture of liver microsomes (final protein concentration, 0.5 mg/mL) and 1 mM nicotinamide adenine dinucleotide phosphate in phosphate buffer (pH 7.4) was pre-incubated in a thermal shaker (200 rpm, 37 °C, 5 min). The reaction was initiated by adding 5 µL of BLU-945 (final concentration of 1 µM) to the pre-incubated mixtures. Aliquots of the reaction mixture were collected at 0, 15, 30, 60 and 120 min of incubation in a thermomixer. Verapamil and buspirone were used as controls in the microsomal stability assay. The plasma stability of BLU945 was investigated by spiking the compound into blank plasma (final concentration of 1 µM). Aliquots of plasma were collected at 0, 15, 30, 60, 120 and 240 min of incubation (37 °C, 200 rpm). Procaine was used as the control in the plasma stability assay. At each sampling point, 20 µL of the microsomal or plasma reaction mixture was pipetted into a microtube containing 130 µL of ice-cold IS in acetonitrile to terminate the reaction. The tubes were vortexed for 5 min and centrifuged at 14,000 rpm for 5 min. The supernatant was used to determine the amount of BLU-945 remaining using LC–MS/MS analysis. The area ratio of BLU-945 to IS was plotted against time to calculate the metabolic rate constant (ke). The elimination half-life (T1/2) was calculated using the following equation: T1/2 = ln2/ke (Maeng et al. 2019; Ji et al. 2020).

Application in pharmacokinetics studies

Male Sprague Dawley (SD) rats, aged 7 weeks and weighing 240–270 g, were purchased from Orient Bio Inc. (Seongnam, Republic of Korea) and allowed to acclimatize before the experiments. Animal studies were performed following the Guidelines for Animal Care and Use of Gachon University (approval number: GUI-2023-IA0040-00). The animals were provided ad libitum access to both food and water in plastic cages under the specific conditions: a 12/12 h light/dark cycle, room temperature maintained at 20–25 °C, and relative humidity set at 40–60%. Before oral administration, the rats were fasted for 14 h. Pharmacokinetic studies were performed as reported previously (Yoon et al. 2020; Vo et al. 2022; Le et al. 2023). Anesthesia was performed via intraperitoneal injection with Rompun® (Bayer AG, Leverkusen, Germany) and Zoletil® (Vibrac, Westlake, TX, USA). For blood sampling, the rat femoral arteries were catheterized using polyethylene tubes (Clay Adams, Parsippany, NJ, USA). BLU-945 was dissolved in 70% saline (2.5 mg/mL) and administered intravenously to rats at 5 mg/kg. The blood sampling times were 1, 2, 5, 15, 30, 60, 120, 240, 480, 720 and 1440 min. For oral administration, a vehicle mixture containing 5% DMSO, 20% PEG400, 5% Kolliphor EL, and 70% distilled water was prepared to dissolve the drug. The rats were orally administered doses of 5, 10 and 20 mg/kg. The blood sampling times were 15, 30, 60, 120, 240, 480, 720 and 1440 min. Samples above the upper limit of quantitation were diluted with blank plasma using the same dilution factor used in the dilution integrity test. Plasma obtained after centrifugation of blood (14,000 rpm, 15 min) was stored at − 20 °C for further LC–MS/MS analyses. For sample preparation, 130 µL of IS in acetonitrile was added to 20 µL of rat plasma for deproteinization. After vortexing and centrifugation, the supernatants were analyzed for drug quantification. The plasma peak concentration (Cmax) and time to reach Cmax (Tmax) after oral administration were determined directly from the plasma concentration–time profile. Other pharmacokinetic parameters, including terminal elimination half-life (T1/2), area under the time curve (AUC) from zero to the last time (AUClast) and to infinity (AUCinf), mean residence time (MRT), total clearance (CL), and apparent distribution volume at steady state (Vss), for each rat were analyzed by non-compartmental analysis using Phoenix® 8.2 software (Certara L.P., Princeton, NJ, USA).

Statistical analyses

Statistical significance was assessed using the analysis of variance (ANOVA) tests followed by Duncan’s test. A p-value of < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism v. 8.4.2 software (San Diego, CA, USA).

Results and discussion

LC–MS/MS method development

We have developed a highly sensitive LC–MS/MS method to quantify BLU-945 in rat and mouse plasma samples. This method employs a straightforward protein precipitation technique for compound extraction. BLU-945 and the IS were protonated in the positive electrospray ionization mode. The protonated molecular ions ([M+H]+) of the target and IS were detected at m/z 557.3 and 543.2, respectively. As shown in Fig. 1, the most prominent fragments in the product ion spectra were at m/z 423.2 for both BLU-945 and ND207937. These product ions were formed, likely due to the breaking of the azetidine ring of the precursor ions. Quantitative analyses were performed in MRM mode with m/z 557.43 → 423.2 and 543.23 → 423.4 for BLU-945 and ND207937, respectively. The operational parameters of the LC–MS/MS system, including the declustering potential, collision energy, and collision cell exit potential, were fine-tuned to maximize the responses of both BLU-945 and IS. The chromatographic separation was optimized by evaluating the mobile phase composition and column. The final LC conditions involved the use of a polar reversed-phase column with a typical mobile phase consisting of 0.1% formic acid in distilled water and 0.1% formic acid in acetonitrile (30:70, v/v).

Fig. 1
figure 1

Representative product ion spectra of BLU-945 (A) and ND207937 (B) in positive ionization mode

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Method validation

Under optimized LC–MS/MS conditions, successful separation of BLU-945 and the IS from the endogenous components of blank rat and mouse plasma was achieved (Figs. 2 and 3). No interference was observed in the blank rat and mouse plasma at the retention times of BLU-945 and the IS. In the case of the zero-calibrator, only the IS peak was observed. In the chromatogram of LLOQ, symmetrical peaks were observed at about 1.9 and 1.7 min for BLU-945 and IS, respectively. The retention times of BLU-945 and the IS were consistent and identical to those of rat plasma samples from the pharmacokinetic study. These results confirmed the specificity of the validated analysis method for BLU-945 in rat and mouse plasma samples.

Fig. 2
figure 2

MRM LC–MS/MS chromatograms of BLU-945 (left) and ND207937 (right) after deproteinization of blank rat plasma (A), zero calibrator (B), LLOQ (C), and rat plasma sample 60 min after PO administration of BLU-945 at a dose of 5 mg/kg (D)

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Fig. 3
figure 3

MRM LC–MS/MS chromatograms of BLU-945 (left) and ND207937 (right) after deproteinization of blank mouse plasma (A), zero calibrator (B), and LLOQ (C)

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The calibration curves of BLU-945 were established within the range of 1–1000 ng/mL in the rat and mouse plasma samples. Using a 1/x2 weighting factor, the resulting linear equations were determined to be y = 0.00140x + 0.00057 (r2 = 0.9973) and y = 0.00126x + 0.00077 (r2 = 0.9971) for the rat and mouse plasma, respectively, where y denotes the peak area ratio and x refers to the concentration ratio of the target and IS. In rat plasma, the accuracy and precision of all standards were 0.267–11.33% and 2.834–7.654%, respectively. Meanwhile, these values ranged from 0.600 to 9.867% for accuracy and from 1.868 to 7.399% for precision in mouse plasma. Thus, the peak ratios of BLU-945 and the IS were directly proportional to their plasma concentration ratios with acceptable accuracy and precision, indicating the linearity of the assay in the investigated range.

The intra- and inter-day accuracy and precision of the QC samples are presented in Table 1. These values for the rat plasma ranged from 0.438 to 7.900% for accuracy and 2.916–8.018% for precision. In mouse plasma, the intra- and inter-day accuracy and precision values ranged from 0.667 to 4.878% and 3.576 to 10.19%, respectively. The dilution integrity, as indicated by the accuracy and precision values of DQC after five dilutions in blank rat and mouse plasma, is also displayed in Table 1. In rat plasma, the accuracy and precision values were 3.217–5.650% and 1.230–2.260%, respectively. Meanwhile, in mouse plasma, the accuracy ranged from 4.967 to 8.483%, and the precision ranged from 1.267 to 1.813%. These data indicate the reliability of the validated method in analyzing samples exceeding the upper limit of quantification after dilution. Overall, these results satisfied all criteria, indicating the reliability of the developed method for BLU-945 analysis in rat and mouse plasma.

Table 1 Precision and accuracy of BLU-945 quantification in rat and mouse plasma
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Matrix effect, extraction recovery, and process efficiency were investigated for the QC and IS samples (Table 2). The matrix effect values in rat and mouse plasma ranged from 100.2 to 106.9% and 101.8 to 109.3%, respectively. The CV% for the matrix effect met the required criterion of < 15% across all samples. These data suggest the absence of matrix effects for BLU-945 and the IS in both rat and mouse plasma. For the extraction recovery, values for BLU-945 and IS in rat and mouse plasma ranged from 94.48 to 102.0% and 85.91 to 95.05%, respectively. In addition, the overall process efficiency values in rat and mouse plasma were within the acceptable range, 97.22–103.1% and 92.71–100.4%, respectively. These results demonstrated that the protein precipitation method is suitable for compound extraction from rat and mouse plasma samples.

Table 2 Matrix effect, extraction recovery, and process efficiency of the assay in rat and mouse plasma
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The short-term and long-term stabilities of stock solution were 93.03 ± 0.907% and 103.4 ± 2.030% for BLU-945 and 99.35 ± 0.812% and 100.6 ± 2.207% for the IS, respectively. The stability of BLU-945 at LQC and HQC levels in rat and mouse plasma is shown in Table 3. BLU-945 was stable after short- and long-term storage, with a bias in the peak area of stored samples and freshly prepared samples of < 15%. The stability of the compound in processed samples stored in the autosampler for 24 h or in QC samples subjected to three freeze–thaw cycles was within the acceptance criteria. These data show that BLU-945 remained stable under specific sample handling and storage conditions.

Table 3 Stability of BLU-945 in rat and mouse plasma
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In vitro stability

BLU-945 was stable in human, rat, and mouse plasma after 4 h of incubation (Fig. 4A), suggesting that the drug was not metabolized by enzymatic degradation in the plasma of these species. In contrast, BLU-945 was unstable in a microsomal stability study. Following After 2-h incubation, the drug’s residual percentage (%remaining) gradually decreased to 42.60, 26.97, and 2.17% in human, rat, and mouse microsomes, respectively (Fig. 4B). Moreover, the calculated half-life, derived from the slopes of the corresponding curves were 110.0, 66.00, and 23.41 min for humans, rats, and mice, respectively. These data suggest that the primary metabolic route for BLU-945 is a phase I reaction involving hepatic enzymes, particularly CYP450, which exists in liver microsomal fractions.

Fig. 4
figure 4

Stability of BLU-945 in human (●), rat (■), and mouse (▲) plasma (A) and microsome (B) at 37 °C. Data are expressed as means ± SDs (n = 3)

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In vivo pharmacokinetic studies

The established bioanalytical method was subsequently used in a rat pharmacokinetic study. The average plasma concentration–time profiles of BLU-945 following intravenous (5 mg/kg) and oral (5, 10, and 20 mg/kg) administration are shown in Fig. 5. The pharmacokinetic parameters obtained from these profiles are summarized in Table 4. The drug concentration was readily measurable in the rat plasma collected up to 24 h after drug administration. The ratios of AUClast to AUCinf were 99.2% for intravenous and 88.2–93.5% for oral administration, indicating that the sensitivity of the developed method is adequate to measure the exact terminal phase concentration of BLU-945. The drug was slowly eliminated from the rat plasma, with a relatively long half-life (4.6 h) and a moderate CL of 1506.6 mL/h/kg, compared to the regular hepatic blood flow rate (3300 mL/h/kg) (El-Kattan 2017). Moreover, it showed 2.584 h of MRT and moderate volume distribution (3751.3 mL/kg) (Smith et al. 2015).

Fig. 5
figure 5

Plasma concentration–time curves after IV administration (A) 5 mg/kg (○), and PO administration (B) at three different doses: 5 mg/kg (●), 10 mg/kg (■), and 20 mg/kg (▲). Data are expressed as means ± SDs (n = 5)

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Table 4 Pharmacokinetic parameters of BLU-945 after intravenous and oral administration to rats
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After a single oral dose of 5, 10, and 20 mg/kg to the rats, BLU-945 rapidly reached maximum concentration (Cmax) in plasma at 1.4–2.8 h. Cmax and AUC elevated more than the increase in the administered dose. The dose-normalized AUCinf values differed among the three dose groups, suggesting that BLU-945 may follow nonlinear pharmacokinetics up to an oral dose of 20 mg/kg. When increasing the dose from 5 to 10 mg/kg, the AUCinf/Dose increased, but without reaching statistical significance (p > 0.05). However, the differences were significant when the dose was further increased to 20 mg/kg. In particular, the p-value was < 0.001 when comparing doses of 5 mg/kg and 20 mg/kg, indicating a highly significant difference. Similarly, the p-value for comparing doses of 10 mg/kg and 20 mg/kg was < 0.05, indicating a statistically significant difference. The absolute bioavailability increased with dose (55.91%, 70.35%, and 105.6% for 5, 10, and 20 mg/kg doses, respectively), indicating that BLU-945 may exhibit a high oral bioavailability in rats.

Overall, our findings suggest that the developed LC–MS/MS assay is appropriate for determining BLU-945 levels in pharmacokinetic studies.

Conclusion

This study describes a simple and sensitive LC–MS/MS method for quantifying BLU-945 in rat and mouse plasma. The developed bioanalytical method was fully validated according to the guidelines of the United States Food and Drug Administration and the European Medicines Agency and showed good reproducibility and reliability. BLU-945 is stable in human, rat, and mouse plasma. Moreover, it was moderately or completely metabolized in the human, rat, and mouse hepatic microsomal fractions after 2-h of incubation. Finally, the developed method was successfully applied to the pharmacokinetic study of BLU-945 to determine its drug concentration in rat plasma. The established analytical method and the outcomes of our research will be helpful for further investigations and clinical studies of BLU-945.