The compound ponatinib was obtained from MCE China (Shanghai, China). imp-A, and imp-C were obtained from Shanghai Macklin Biochemical (Shanghai, China), while imp-B was homemade by the laboratory of the authors. Merck (Darmstadt, Germany) provided HPLC-grade ACN and methanol (MeOH). Other analytical grade chemical reagents were purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd., (Beijing, China).

For this investigation, a RP-HPLC device (Agilent 1200, Agilent, United States) and a UV-visible spectrophotometer (Cary100, Varian, United States) were utilized. Furthermore, a Milli-Q water purification system was employed.

For the separation of ponatinib and its related impurities, a 5HC-C₁₈ column from Agilent (4.6 mm × 250 mm, 5 μm) was employed. Mobile phase A consisted of a water and acetonitrile (ACN) blend in a 9:1 v/v ratio. The aqueous solution contained 2 mM potassium dihydrogen phosphate solution (KH₂PO₄) and 0.4% triethylamine. Phosphoric acid adjusted the pH to 2.4. ACN was the only component of mobile phase B. The gradient elution was conducted as follows: mobile phase B maintained a constant concentration of 16% from 0 to 2 min, followed by an increase in concentration from 16% to 30% from 2 to 22 min. Subsequently, the concentration of mobile phase B further increased from 30% to 34% from 22 to 32 min. From 32 to 35 min, the concentration of mobile phase B increased to 55%, remaining constant until 42 min. Between 42 and 42.1 min, the concentration of mobile phase B decreased from 55% to 16%. Finally, from 42.1 to 50 min, mobile phase B maintained a constant concentration of 16%. The wavelength used for UV detection was 250 nm, with a flow rate of 1.0 mL/min and a sample volume of 10 µL.

To prepare imp-B, 200 mg of ponatinib was weighed and transferred into a 100 mL flask. Methanol (20 mL) was added to fully dissolve the powder. Subsequently, 30 mL of a 30% H₂O₂ solution was added, and the mixture underwent oxidative degradation. The reaction mixture was stirred at room temperature for 20 h before terminating the reaction with manganese dioxide. After centrifugation, the supernatant was decanted, and the crude product was obtained by freeze drying.

The imp-B crude product was purified using a preparative column (Agilent Eclipse XDB-C₁₈, 250 mm × 9.4 mm, 5 μm). The mobile phase consisted of a 75% methanol-water solution. The elution time was 6 min, and the flow rate was set at 4 mL/min.

To determine the structure of imp-B, nuclear magnetic resonance (¹H NMR, ¹³C NMR and 2D NMR) and high-resolution mass spectrometry (HRMS) were employed for analysis. The procedure is as follows: dissolve 10 mg of imp-B or 20 mg of imp-B in DMSO-d6 for ¹H NMR, ¹³C NMR and 2D NMR analysis. Prior to mass spectrometry analysis, the imp-B sample was diluted to a concentration of 100 ng/mL with a 50% methanol-water solution before being injected into the mass spectrometer.

To formulate a composite solution of ponatinib bulk drug and its related substances, approximately 10 mg of the ponatinib bulk drug was carefully measured and transferred into a 20 mL volumetric flask. Subsequently, 0.2 mL of reference standard stock solutions containing imp-A, imp-B, and imp -C were introduced. Following this, the solution was further diluted to a final volume of 20 mL by combining equal volumes of methanol and water in a proportion of 50:50 (v/v).

The system suitability solution was prepared by combining equal volumes of acidic and oxidative degradation solutions. The specific steps are as follows: Initially, accurately weigh 10 mg of ponatinib and transfer it into a 20 mL volumetric flask. Subsequently, add 10 mL of methanol to the flask to dissolve the ponatinib. Following this, add 5 mL of 1 M hydrochloric acid solution to the mixture and maintain the reaction at 70°C for 12 h. Upon completion of the reaction, neutralize the solution by adjusting the pH to 7 with 1 M sodium hydroxide. Lastly, dilute the reaction mixture to 20 mL with a 50:50 mixture of methanol (MeOH) and water (H₂O) (v/v).

A 10 mg measurement of ponatinib bulk drug was accurately added to a 20 mL volumetric bottle and dissolved in 10 mL of methanol. It was then combined with a 1 M hydrochloric acid solution and reacted at 70°C for 12 h. Subsequently, it was diluted to a total volume of 20 mL using a mixture of MeOH and H₂O in a 50/50 ratio (v/v). Likewise, an additional 10 mg of ponatinib was weighed and then mixed with 10 mL of methanol. Subsequently, it was introduced into a 10 mL solution of H₂O₂ (3%) and underwent a reaction at ambient temperature for a duration of 20 h. After the reaction was completed, any residual H₂O₂ was decomposed using MnO₂. The MnO₂ was then separated through filtration, and the resulting solution was subjected to freeze-drying. Ultimately, the oxidative degradation product was isolated and dissolved in a 50:50 (v/v) mixture of methanol (MeOH) and water (H₂O).

To prepare the sample solution, accurately weigh approximately 10 mg of ponatinib bulk drug and dissolve it in 20 mL of a 50:50 methanol-water solution (v/v). This procedure results in a sample solution with an estimated concentration of 0.5 mg/mL.

The synthesis of ponatinib, a potent TKI inhibitor, follows the pathway of patent W020110539338 as depicted in Supplementary Figure S1. Various starting materials and intermediates are utilized in the synthetic pathway of ponatinib. Forced degradation tests revealed three primary compounds associated with ponatinib: imp-A, imp-B, and imp-C. The chemical structures of ponatinib, imp-A, imp-B, and imp-C are depicted in Figure 1. imp-A is a process-related impurity, whereas imp-C serves not only as a process impurity but also as an alkaline degradation product of ponatinib. Furthermore, imp-B is a novel compound resulting from the oxidative degradation of ponatinib. Its structure was unequivocally established using nuclear magnetic resonance and mass spectrometry. A method employing reversed phase high performance liquid chromatography (RP-HPLC) was developed to determine the concentrations of ponatinib-related substance.

To optimize the method, we systematically investigated the effects of solvent, detection wavelength, mobile phase composition, and elution method on sample separation. To choose an appropriate solvent, ponatinib was dissolved in methanol (MeOH) and acetonitrile (ACN). The findings indicated that ponatinib exhibited low solubility in ACN but was completely soluble in MeOH. Consequently, we conducted additional research on ponatinib in a 1:1 MeOH/H₂O (v/v) solution and observed that it can dissolve at a concentration of 0.5 mg/mL without any evidence of turbidity. Subsequently, we investigated the influence of the MeOH/H₂O (v/v 1:1) solvent on the stability, peak shape, and resolution of ponatinib. The results demonstrated that the use of MeOH/H₂O (v/v 1:1) as the solvent did not result in any significant changes in the impurities of ponatinib over a 72-h period. Additionally, the symmetry factor of the ponatinib peak was 1.12 and the minimum resolution between it and nearby impurity peaks was greater than 1.5. Consequently, MeOH/H₂O (v/v 1:1) was ultimately selected as the solvent for this study.

To determine the optimal absorption wavelength, a solution of ponatinib standard stock and its associated compounds were diluted by a factor of 50 and then analyzed using UV-VIS scanning within the 200–400 nm range. Supplementary Figure S2 displays the acquired ultraviolet spectra, which unveil significant absorption around 250 nm for ponatinib and its associated compounds. Therefore, the wavelength of 250 nm was selected for detection in this research.

Due to the significant presence of difficult-to-separate impurities in the system suitability solution, this solution was chosen for method development. Initially, different proportions of mobile phase B to mobile phase A (80:20; 60:40) were employed for isocratic elution. The results indicated that the impurities overlapped on the chromatogram, with a limited number of impurities, and the separation degrees both among impurities and between the impurities and the main peak failed to meet the requirements (Figures 2A, B).

Owing to the number of impurities in the system suitability solution, achieving separation of individual impurities using isocratic elution proves challenging. Consequently, the adoption of a gradient elution method was deemed suitable. Initially, gradient condition 1 (0∼2 min, 10%B→10%B; 2∼20 min, 10%B→90%B; 20∼30 min, 90%B→90%B; 30∼30.1 min, 90%B→10%B; 30.1∼40 min, 10%B→10%B) was utilized for analysis, as depicted in Figure 2C. The minimum separation degree between impurities was 1.15, and between impurities and the main peak was 1.85, which did not meet the specified requirements. Subsequently, the gradient rate was optimized by decreasing the rate of change. Sample analysis was conducted using gradient conditions 2 (0∼2 min, 10%B→10%B; 2∼30 min, 10%B→90%B; 30∼35 min, 90%B→90%B; 35∼35.1 min, 90%B→10%B; 35.1∼45 min, 10%B→10%B) and conditions 3 (0∼2 min, 20%B→20%B; 2∼30 min, 20%B→75%B; 30∼35 min, 75%B→75%B; 35∼35.1 min, 75%B→20%B; 35.1∼45 min, 20%B→20%B), resulting in notable improvement in separation degrees between impurities and between impurities and the main peak on the chromatogram. However, these improvements did not suffice to meet the specified requirements (Figures 2D, E). After several rounds of optimization, the conditions described in Section 2.3 were identified as the definitive chromatographic conditions for the product. These conditions achieved a minimum separation degree of 1.62 among impurities and 1.8 between the impurities and the main peak, thereby meeting the required detection standards.

Imp-B was prepared following the procedures outlined in Section 2.4 of the study. The resulting chromatogram in Supplementary Figure S3 showed a retention time of 8.2 min with a purity of 97.5%. Figures 5A–C present the high-resolution mass spectrum, 1D proton nuclear magnetic resonance (NMR) spectrum, and 1D carbon NMR spectrum of imp-B, respectively. Additionally, the 2D NMR spectrum is illustrated in Supplementary Figure S4. Using mass spectrometry and NMR spectroscopy techniques, we successfully elucidated the structure of imp-B (Figure 5A), which possesses a molecular weight of 564. This result was in agreement with previously reported in the literature (Golla et al., 2023).