1. A stability-indicating analytical method for the determination, separation, identification, and characterization of Vimseltinib in the presence of degradation products and process-related impurities, comprising: subjecting a sample with Vimseltinib to one or more forced degradation conditions selected from acidic, alkaline, oxidative, reductive, photolytic, thermal, and hydrolytic conditions to form one or more degradation products, thereby obtaining a stressed sample; analyzing the stressed sample using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with photodiode array (PDA) detection; and characterizing the degradation products using liquid chromatography–tandem mass spectrometry (LC–MS/MS), wherein the RP-HPLC comprises an octadecylsilane-bonded silica stationary phase and a carrying phase comprising 0.1% TFA buffer solution with acetonitrile in a volumetric ratio of 60:40 (v/v), wherein the method is performed at a flow rate of 1.0 mL/min, detection wavelength of 257 nm, and run time of 5 min.

2. The method as claimed in claim 1, wherein the RP-HPLC is used for the quantitative determination of Vimseltinib in tablet dosage form, wherein the RP-HPLC comprises: preparing a sample solution from a tablet formulation; injecting the solution into the RP-HPLC; and determining the amount of Vimseltinib by comparison with a reference standard.

3. The method as claimed in claim 1, wherein the forced degradation condition comprises alkaline degradation by treating the sample of Vimseltinib with 1 N sodium hydroxide for 30 min to form a first degradation product.

4. The method as claimed in claim 3, wherein the first degradation product exhibits a mass-to-charge ratio (m/z) of 373.1 and has a molecular weight of 373.15 and molecular formula C₁₉H₂₀N₅NaO₂, and is identified as 3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.

5. The method as claimed in claim 1, wherein the forced degradation condition comprises oxidative degradation by treating the sample of Vimseltinib with 30% hydrogen peroxide for 30 min to form a second degradation product.

6. The method as claimed in claim 5, wherein the second degradation product exhibits a mass-to-charge ratio (m/z) of 447.20 and has a molecular weight of 447.20 and molecular formula C₂₃H₂₅N₇O₃, and is identified as 2-[hydroxy(propan-2-yl)amino]-3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-3,4-dihydropyrimidin-4-one.

7. The method as claimed in claim 1, wherein the forced degradation condition comprises thermal degradation by exposing the sample of Vimseltinib to a temperature of 105 °C for three hours to form a third degradation product.

8. The method as claimed in claim 7, wherein the third degradation product exhibits a mass-to-charge ratio (m/z) of 274.14 and has a molecular weight of 274.14 and molecular formula C₁₄H₁₈N₄O₂, and is identified as 5-(5-hydroxy-6-methylpyridin-2-yl)-3-methyl-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.

9. The method as claimed in claim 1, wherein the forced degradation condition comprises hydrolytic degradation by treating the sample of Vimseltinib with water for 30 min to form a fourth degradation product.

10. The method as claimed in claim 9, wherein the fourth degradation product exhibits a mass-to-charge ratio (m/z) of 357.10 and has a molecular weight of 357.10 and molecular formula C₁₆H₁₅N₅O₅, and is identified as 3-methyl-5-[6-methyl-5-(pyridin-4-yloxy)pyridin-2-yl]-2-nitro-3,4-dihydropyrimidine-4,4-diol.

Description:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of pharmaceutical analysis, specifically to a stability-indicating reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the determination of Vimseltinib and the identification and structural characterization of its degradation products using photodiode array detection and liquid chromatography–tandem mass spectrometry, wherein four degradation products formed under stress conditions are characterized by their respective mass-to-charge (m/z) values of 373.15, 447.20, 274.14, and 357.10.
Background of the invention:
[0002] Vimseltinib is an orally administered small-molecule tyrosine kinase inhibitor developed for selective inhibition of colony-stimulating factor 1 receptor (CSF1R), which is implicated in the pathogenesis of Tenosynovial Giant Cell Tumor (TGCT). TGCT is a locally aggressive proliferative disorder of synovial tissue characterized by overexpression of colony-stimulating factor 1 (CSF1), resulting in activation of CSF1R signalling pathways and abnormal macrophage accumulation. Consequently, targeted inhibition of CSF1R has emerged as a promising therapeutic strategy for the management of TGCT, particularly in patients for whom surgical intervention is not feasible or may result in significant morbidity.
[0003] Like other active pharmaceutical ingredients, Vimseltinib is susceptible to chemical degradation when exposed to environmental and stress conditions such as acidic, alkaline, oxidative, reductive, thermal, photolytic, and hydrolytic environments. Degradation of the drug substance may result in formation of structurally related impurities, which can influence stability, safety, and regulatory acceptance. Regulatory authorities require identification, separation, and structural characterization of such degradation products in accordance with ICH guidelines to establish stability-indicating capability of analytical methods.
[0004] Although analytical techniques such as high-performance liquid chromatography (HPLC) and mass spectrometry are widely used for impurity profiling, no prior report has comprehensively disclosed a stability-indicating chromatographic method capable of effectively separating Vimseltinib from all of its stress-induced degradation products within a short analytical run time while simultaneously enabling structural elucidation of the degradation impurities formed under multiple stress conditions. In particular, the structural characterization of specific low-level degradation products formed under alkaline, oxidative, thermal, and hydrolytic conditions has not been fully established.
[0005] Existing analytical methods described for kinase inhibitors generally focus on assay determination or limited impurity detection and do not provide a systematic stress degradation profile combined with mass-spectrometric fragmentation pathway analysis for each identified degradant. Furthermore, previously reported approaches do not demonstrate complete resolution of degradation peaks with confirmed peak purity under forced degradation conditions using a single optimized chromatographic system.
[0006] Accordingly, there remains a need for a validated, stability-indicating analytical method that provides reliable separation of Vimseltinib from its degradation products, enables structural characterization of stress-induced degradants through LC–MS/MS fragmentation analysis, and ensures reproducible impurity profiling suitable for regulatory compliance and routine quality control applications.
Objectives of the invention:
[0007] The primary object of the present invention is to provide a novel, rapid, and precise reverse-phase high-performance liquid chromatographic (RP-HPLC) method for the determination of Vimseltinib in bulk drug and pharmaceutical compositions, which is stability-indicating and suitable for routine quality control.
[0008] Another object of the present invention is to develop an analytical method capable of effectively separating Vimseltinib from its degradation products and process-related impurities under various forced degradation conditions, including alkaline, oxidative, thermal, hydrolytic, acidic, reductive, and photolytic stress conditions.
[0009] Another object of the present invention is to validate the developed analytical method in accordance with applicable regulatory guidelines, such as ICH Q2(R1), to ensure its accuracy, precision, specificity, and reproducibility for routine quality control and stability testing.
[0010] Another object of the present invention is to identify, quantify, and structurally characterize previously unreported degradation products of Vimseltinib, specifically first degradation product, second degradation product, third degradation product, and fourth degradation product, formed under alkaline, oxidative, thermal, and hydrolytic stress conditions, using RP-HPLC coupled with liquid chromatography–tandem mass spectrometry (LC-MS/MS).
[0011] Another object of the present invention is to demonstrate the novelty of the degradation products first degradation product, second degradation product, third degradation product, and fourth degradation product, which have not been disclosed in prior art, thereby contributing to the inventive merit of the present invention and advancing the understanding of Vimseltinib's stability behavior.
[0012] Another object of the present invention is to provide an analytical method that is cost-effective, robust, and environmentally friendly, minimizing solvent consumption and ensuring sustainability while maintaining high analytical performance and regulatory compliance.
Summary of the invention:
[0013] The present invention relates to a stability-indicating chromatographic method for Vimseltinib degradation impurities characterization. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.
[0014] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a rapid, accurate, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method, supported by photodiode array detection (PDA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), for the determination of Vimseltinib in the presence of its degradation products.
[0015] According to an aspect, the invention provides a method of a stability-indicating analytical method for the determination, separation, identification, and characterization of Vimseltinib in the presence of one or more degradation products. At first, Vimseltinib is subjected to forced degradation under one or more stress conditions selected from acidic, alkaline, oxidative, reductive, photolytic, thermal, and hydrolytic conditions to obtain stressed samples.
[0016] In one embodiment, the forced degradation condition comprises alkaline degradation by treating the sample comprising Vimseltinib with about 1 N sodium hydroxide for about 30 minutes to form a first degradation product having a mass-to-charge ratio (m/z) of about 373.1. The first degradation product has a molecular weight of about 373.15 and molecular formula C₁₉H₂₀N₅NaO₂, and is identified as 3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
[0017] In one embodiment, the forced degradation condition comprises oxidative degradation by treating the sample comprising Vimseltinib with about 30% hydrogen peroxide for about 30 minutes to form a second degradation product having an m/z of about 447.20. The second degradation product has a molecular weight of about 447.20 and molecular formula C₂₃H₂₅N₇O₃, and is identified as 2-[hydroxy(propan-2-yl)amino]-3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-3,4-dihydropyrimidin-4-one.
[0018] In one embodiment, the forced degradation condition comprises thermal degradation by exposing the sample comprising Vimseltinib to a temperature of about 105 °C for about three hours to form a third degradation product having an m/z of about 274.14. The third degradation product has a molecular weight of about 274.14 and molecular formula C₁₄H₁₈N₄O₂, and is identified as 5-(5-hydroxy-6-methylpyridin-2-yl)-3-methyl-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
[0019] In one embodiment, the forced degradation condition comprises hydrolytic degradation by treating the sample comprising Vimseltinib with water for about 30 minutes to form a fourth degradation product having an m/z of about 357.10. The fourth degradation product has a molecular weight of about 357.10 and molecular formula C₁₆H₁₅N₅O₅, and is identified as 3-methyl-5-[6-methyl-5-(pyridin-4-yloxy)pyridin-2-yl]-2-nitro-3,4-dihydropyrimidine-4,4-diol.
[0020] Next, the stressed samples are analysed using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with photodiode array (PDA) detection. In one embodiment, the RP-HPLC method is used for the quantitative determination of Vimseltinib in tablet dosage form. The RP-HPLC method comprises a sample solution is prepared from the tablet formulation. Next, the solution is injected into the RP-HPLC system. Later, the amount of Vimseltinib is determined by comparison with a reference standard. Later, the degradation products are characterized using liquid chromatography–tandem mass spectrometry (LC–MS/MS).The method is stability-indicating, rapid, specific, sensitive, accurate, and reproducible.
[0021] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0022] The specification includes the accompanying drawings, which show an embodiment of the invention and, when combined with the description, provide an explanation of its concepts.
[0023] FIG. 1 illustrates a flowchart in accordance to an exemplary embodiment of the invention for Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0024] FIG. 2 refers to a graph depicting calibration curve obtained from linearity for Vimseltinib using the developed RP-HPLC method in the validation, in accordance to an exemplary embodiment of the invention.
[0025] FIGs. 3A – 3C illustrate graphs determining Vimseltinib using a RP-HPLC, UV, and LC-MS/MS, in accordance to an exemplary embodiment of the invention.
[0026] FIG. 4 illustrates a flowchart of subjecting to stress study for Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0027] FIG. 5 illustrates a graph of an acid degradation chromatogram of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0028] FIG. 6A illustrates a graph of an alkali degradation chromatogram of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0029] FIG. 6B illustrates a graph of an alkali degradation Mass spectrum of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0030] FIG. 6C illustrates a scheme for the proposed fragmentation pathways for the degradation products of Vimseltinib in acidic condition, in accordance to an exemplary embodiment of the invention.
[0031] FIG. 7A illustrates a graph of an oxidative degradation chromatogram of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0032] FIG. 7B illustrates a graph of an oxidative degradation Mass spectrum of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0033] FIG. 7C illustrates a graph of an oxidative degradation fragmentation path way of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0034] FIG. 8 illustrates a graph of a reduction degradation chromatogram of Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0035] FIG. 9A illustrates a graph of a thermal degradation chromatogram of Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0036] FIG. 9B illustrates a graph of a thermal degradation Mass spectrum of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0037] FIG. 9C illustrates a graph of a thermal degradation fragmentation path way of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0038] FIG. 10 illustrates a graph of a photolytic degradation chromatogram of Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0039] FIG. 11A illustrates a graph of a hydrolytic degradation chromatogram of Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0040] FIG. 11B illustrates a graph of a hydrolytic degradation Mass spectrum of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0041] FIG. 11C illustrates a graph of a hydrolytic degradation fragmentation path way of the Vimseltinib, in accordance to an exemplary embodiment of the invention.
[0042] FIG. 12 illustrates a flow chart of characterization of four degradation impurities of the Vimseltinib based on spectral data, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0043] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.
[0044] The present disclosure has been made with a view towards solving the challenge with the previous art defined above, and it is an object of the present invention to provide a Novel method for determining a Vimseltinib and characterizing its degradation products utilizing a RP-HPLC and LC-MS/MS.
[0045] According to an exemplary embodiment of the invention, FIG. 1 refers to a flow chart 100 of a stability-indicating analytical method for the determination, separation, identification, and characterization of Vimseltinib in the presence of one or more degradation products. At step 102, Vimseltinib is subjected to forced degradation under one or more stress conditions selected from acidic, alkaline, oxidative, reductive, photolytic, thermal, and hydrolytic conditions to obtain stressed samples.
[0046] In one embodiment, the forced degradation condition comprises alkaline degradation by treating the sample comprising Vimseltinib with about 1 N sodium hydroxide for about 30 minutes to form a first degradation product (DP-1) having a mass-to-charge ratio (m/z) of about 373.1. The first degradation product has a molecular weight of about 373.15 and molecular formula C₁₉H₂₀N₅NaO₂, and is identified as 3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
[0047] In one embodiment, the forced degradation condition comprises oxidative degradation by treating the sample comprising Vimseltinib with about 30% hydrogen peroxide for about 30 minutes to form a second degradation product (DP-2) having an m/z of about 447.20. The second degradation product has a molecular weight of about 447.20 and molecular formula C₂₃H₂₅N₇O₃, and is identified as 2-[hydroxy(propan-2-yl)amino]-3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-3,4-dihydropyrimidin-4-one.
[0048] In one embodiment, the forced degradation condition comprises thermal degradation by exposing the sample comprising Vimseltinib to a temperature of about 105 °C for about three hours to form a third degradation product (DP-3) having an m/z of about 274.14. The third degradation product has a molecular weight of about 274.14 and molecular formula C₁₄H₁₈N₄O₂, and is identified as 5-(5-hydroxy-6-methylpyridin-2-yl)-3-methyl-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
[0049] In one embodiment, the forced degradation condition comprises hydrolytic degradation by treating the sample comprising Vimseltinib with water for about 30 minutes to form a fourth degradation product (DP-4) having an m/z of about 357.10. The fourth degradation product has a molecular weight of about 357.10 and molecular formula C₁₆H₁₅N₅O₅, and is identified as 3-methyl-5-[6-methyl-5-(pyridin-4-yloxy)pyridin-2-yl]-2-nitro-3,4-dihydropyrimidine-4,4-diol.
[0050] At step 104, the stressed samples are analysed using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with photodiode array (PDA) detector. In one embodiment, the RP-HPLC method is used for the quantitative determination of Vimseltinib in tablet dosage form. The RP-HPLC method comprises a sample solution is prepared from the tablet formulation. Next, the solution is injected into the RP-HPLC system. Later, the amount of Vimseltinib is determined by comparison with a reference standard. At step 106, the degradation products are characterized using liquid chromatography–tandem mass spectrometry (LC–MS/MS). The method is stability-indicating, rapid, specific, sensitive, accurate, and reproducible.
[0051] For therapeutic medication monitoring, the RP-HPLC technique's high sensitivity and ability to precisely quantify with reduced sample amounts are essential. By separating Vimseltinib from degradation products according to their physical and chemical characteristics, the RP-HPLC technology reduces interference and improves accuracy. Clinical decisions and medication changes can be made more quickly because to the RP-HPLC technique's reduction in analytical time.
[0052] In one embodiment, one liter of high-performance liquid chromatography-grade water is used to dissolve one mL of Trifluoroacetic acid, which is then filtered using 0.45 filter paper. Mixing the obtained filtered buffer solution with acetonitrile in a volumetric ratio of 60:40 (v/v) to prepare a mobile-phase mixture, and filtering the mobile-phase mixture through a 0.22 membrane filter, and using the mobile phase as a diluent for subsequent preparations.
[0053] In one embodiment, weighed 7 mg of Vimseltinib standard into a 10 mL volumetric flask, added diluent, sonicated for 30 min to dissolved, and made up to volume. Pipette 1.0 mL of this stock into a 10 mL flask, diluted to volume with diluent, and filter through a 0.45 µm injection filter. Similarly, weighed 7 mg of Vimseltinib sample into a 10 mL volumetric flask, add diluent, sonicated for up to 30 min, make up to volume, diluted 1.0 mL to 10 mL with diluent, and filtered through a 0.45 µm injection filter.
[0054] An octadecylsilane-bonded silica stationary phase column having dimensions of about 150 mm × 4.6 mm and a particle size of about 3.5 µm is used for chromatographic separation. The working standard and sample solutions are injected using a mobile phase of Acetonitrile and (0.1%) Tri fluoro acetic acid buffer 40:60 (v/v) at a flow rate of 1.0 mL/min. The injection volume is 10 µL and diluent is ethanol, and the analysis is performed at room temperature under isocratic conditions within 5 minutes of run time. A symmetric Vimseltinib peak is obtained at 257 nm.
[0055] In one embodiment, the HPLC system is coupled to a mass spectrometer operated in positive electrospray ionization (ESI+) mode. Multiple reactions monitoring (MRM) is used to quantify Vimseltinib and its stress degradation products. The main MS parameters are: nitrogen as a drying and nebulizing gas (45 psi), highly filtered nitrogen gas as a collision gas, the capillary at 3000 V, the drying gas flow stream at 5 mL/min, the skimmer at 60 V, the fragmented voltage at 80 V, the collision energy at 14 V, and the ion spray voltage at 5500 V and dwell time 1 s.
[0056] After obtained best optimized chromatographic conditions validated ICH guidelines and table 1 depicts the optimized chromatographic conditions.
[0057] Table 1:
Stationary phase: Octadecylsilane-bonded silica stationary phase column (150 x 4.6 x 3.5 µm)
Carrying phase: 0.1% TFA buffer solution with acetonitrile in a volumetric ratio of 60:40 (v/v)
Injection volume: 10 µL
Flow rate: 1.0 ml/min
Column temperature: 25 °C
Wavelength: 257 nm
Run time: 5 min
RT of Vimseltinib: 3.014 min
Diluent Ethanol

[0058] In one embodiment, the specificity of the HPLC method is evaluated to ensure that there is no interference from the degradation products, excipients, or other impurities in the region of actives. The specificity is studied by injecting the unstressed and stressed standard solutions, samples, and blanks.
[0059] In one embodiment, the purpose of the system suitability test is to ensure that the complete testing system, including instruments, reagents, columns, analysts etc., is adequate for the intended analysis. The following parameters are usually determined: theoretical plate count, tailing factors, resolution, and reproducibility. These results are tabulated in table 2.
[0060] Table 2:
Parameter Vimseltinib
Injection Area
1 2514563
2 2571458
3 2553026
4 2586954
5 2578415
6 2592317
Mean 2566122
SD 28738.74
% RSD 1.12
USP plate count 7265
Tailing factor 0.94
Resolution NA
RT 3.027

[0061] In one embodiment, six distinct concentrations between 17.5 and 105.0µg/ml of the nominal standard value are tested for linearity. The suggested method's linearity is assessed by calculating the intercept, slope, and coefficient of correlation using a calibration curve. Table 3 presents a tabulation of these findings. B1 and 1 B2 provide a great illustration of the invention. By generating a calibration curve graph for a specific concentration range for the Vimseltinib, linear regression analysis shows the linearity. The linearity of Vimseltinib is evaluated in the range of 17.50 to 105.00µg/ml. Vimseltinib’s R2 is higher than 0.999. Table 3 shows the linearity equations and correlation coefficients for this drug.
[0062] Table 3:
Linearity level Vimseltinib
Concentration (µg/ml) Peak’s area Slope Intercept CC
1 17.50 633821 35950.33 17125.46 0.99964
2 35.00 1287459
3 52.50 1945420
4 70.00 2564712
5 87.50 3094586
6 105.00 3805626

[0063] In one embodiment, LOD is the lowest amount of analyte in a sample that can be detected but not necessarily quantifies under the stated experimental conditions. On the other hand, LOQ is the lowest amount of analyte in a sample that may be determined with acceptable accuracy and precision. These results are tabulated in table 4.
[0064] In one embodiment, the accuracy of an analytical method expresses the nearness between the expected value and the value found. Successive analysis (n = 3) for three different concentrations of standard mixture (80, 100, and 120% of nominal concentration) is carried out to determine the accuracy of the proposed method. These results are tabulated in table 4.
[0065] In one embodiment, the precision of the assay is assessed with respect to repeatability and reproducibility. The precision of the proposed method is checked by intra- and inter-day repeatability of responses on different columns and different HPLC machines after replicate injections and expressed as %RSD among responses using the formula [%RSD = (standard deviation/mean) × 100%] these results are tabulated in table 4.
[0066] In one embodiment, robustness is an indication of reliability of the analytical method during normal usage. The effect of the following deliberate changes in chromatographic conditions is monitored: flow rate ±5%, solvent ratio ±10%. These results are tabulated in table 4.
[0067] Table 4:
Parameter Details Acceptance Criteria Results
Vimseltinib
Specificity Retention time Different retention time No interference
Linearity Range (μg/mL) Range (μg/mL) 17.50-105.00
Range 25%–150% R2 > 0.999 0.99964
Slope NA 35950.33
Intercept 17125.46
Precision Determinations on same day-Repeatability (% Assay) 99.2
% RSD < 2% 0.79
Determinations on different day-Intermediate precision (% Assay) 99.4
%RSD < 2% 0.68
Accuracy
(% Recovery) Determinations at 80% % of Recovery
98 – 102 % 99.16
Determinations at 100% 99.43
Determinations at 120% 99.26
Sensitivity LOD: lowest detectable conc. S/N ratio > 3 0.63
LOQ: lowest quantitatable conc. S/N ratio > 10 2.1
Robustness Std. Flow Rate:1.0 mL /Min. & Std. Mobile Phase: 0.1% TFA and CH3CN: 60:40
Change Actual Change Limit Vimseltinib
FR: -10% Flow rate (mL/Min): 0.18 % RSD < 2 0.50
FR: +10% Flow rate (mL/Min): 0.22 0.63
MP: -5% MP: 0.1% TFA and CH3CN:-65:35 0.32
MP: +5% MP: 0.1% TFA and CH3CN:- 55:45 0.60

[0068] An accurately weighed quantity of commercially available tablet powder (marketed as Romvimza®) equivalent to 29mg of Vimseltinib (label claim 30mg, average tablet weight 124mg) is transferred into a 10 mL volumetric flask, dissolved in diluent with sonication to ensure complete extraction, and diluted to volume. From this solution, 1.0 mL is further diluted to 10 mL to obtain a final concentration of 70.16 µg/mL of Vimseltinib. The sample solution is analyzed using the developed stability-indicating RP-HPLC method, and duplicate injections produced peak areas of 2,551,473 and 2,561,285, with a mean area of 2,556,379. Quantification is performed using the external standard method, and the percentage assay of Vimseltinib in the Romvimza tablet powder is found to be 99.6%, confirming compliance with the labelled claim and demonstrating the suitability and accuracy of the method for routine quality control analysis.
[0069] According to an exemplary embodiment of the invention, FIG. 2 refers to a graph 200 depicting calibration curve obtained for Vimseltinib using the developed RP-HPLC method. In an exemplary embodiment, 7 milligrams of Vimseltinib are accurately weighed and transferred into a 10 mL volumetric flask, after which the mobile phase is added to bring the volume close to the mark, and the mixture is sonicated for 5 minutes to ensure complete dissolution of the drug. The resulting solution is then filtered through a 0.22μ membrane filter to remove any particulate matter, ensuring clarity and suitability for instrumental analysis. The filtrate is further diluted with the mobile phase to achieve a final working concentration of 70µg/mL, and this solution is injected into the LC-MS/MS system under optimized chromatographic conditions for quantification. After complete separation, chromatograms are recorded, peak areas are identified, and the mass of Vimseltinib is calculated based on the response from the LC-MS/MS detector. Additionally, UV-visible spectrophotometric analysis is carried out to determine the absorption characteristics of the drug, and the λmax of Vimseltinib is found to be 257 nm, indicating its optimal wavelength for measurement. After obtained best optimized validated chromatographic conditions determined the Vimseltinib by using UV, HPLC and LC-MS/MS.
[0070] According to another exemplary embodiment of the invention, FIGs. 3A – 3C refer to graphs (300, 302, 304) demonstrating the determination of Vimseltinib using RP-HPLC, UV spectroscopy, and LC–MS/MS techniques. FIG. 3A shows a representative RP-HPLC chromatogram 300 indicating a sharp and well-resolved peak of Vimseltinib at the optimized retention time, confirming specificity of the developed method. FIG. 3B presents the UV absorption spectrum 302 of Vimseltinib, showing a maximum absorbance at approximately 257 nm, supporting the selected detection wavelength. FIG. 3C depicts the LC–MS/MS spectrum 304 of Vimseltinib, confirming its molecular ion peak and characteristic fragmentation pattern. Collectively, these graphs (300, 302, 304) establish the analytical reliability and structural confirmation of Vimseltinib prior to and during degradation studies.
[0071] According to another exemplary embodiment of the invention, FIG. 4 refers to a diagram 400 representing the forced-degradation (stress) study conducted on Vimseltinib using optimized chromatographic conditions following ICH guidelines. In this study, Vimseltinib and corresponding blank solutions are exposed to different stress conditions to evaluate its stability and confirm method specificity. The stress conditions applied included acidic (1 N HCl, 25°C, 30 min), alkaline (1 N NaOH, 25°C, 30 min), reduction (sodium bisulfite under reflux, 30 min), neutral (water, 25 °C, 30 min), strong oxidation (30% H₂O₂, 25 °C, 30 min), and photolytic exposure (6 lux·hours). After treatment, all samples are analyzed using the optimized chromatographic method to identify degradation and ensure clear separation of Vimseltinib from its degradation products.
[0072] The study investigates potential degradation pathways under various stress environments including acid degradation 402, base degradation 404, oxidative degradation 406, reduction degradation 408, photolytic degradation 410, thermal degradation 412, and hydrolytic degradation 414. These stress conditions were applied to assess the intrinsic stability profile of the active pharmaceutical ingredient and the formulated composition, and to identify degradation susceptibilities under forced degradation conditions.
[0073] According to another exemplary embodiment of the invention, FIG. 5 refers to a graph 500 of the Vimseltinib acid degradation chromatogram. It is discovered that Vimseltinib is stable to acid hydrolysis in one of its embodiments. Using 0.1N HCl results in less than 10% degradation of the Vimseltinib. For degradation studies, 1N HCl is chosen by performing at 25°C for 30 minutes because it produced no major or minor degradation products and only 4.1% degradation in relation to Vimseltinib. A summary of the stress study's findings is provided in Table 5.
[0074] According to another exemplary embodiment of the invention, FIG. 6A refers to a graph 600 of the Vimseltinib base degradation chromatogram. It is discovered that Vimseltinib is stable to base hydrolysis in one of its embodiments. Using 0.1N NaOH results in less than 10% degradation of the Vimseltinib. For degradation studies, 1N NaOH is chosen by performing at 25°C for 30 minutes because it produced one major degradation product and 13.6% degradation in relation to Vimseltinib. A summary of the stress study's findings is provided in Table 5.
[0075] According to another exemplary embodiment of the invention, FIG. 6B refers to a mass spectrum 602 for the Vimseltinib base degradation chromatogram. The drug on basic hydrolysis formed one degradation product, namely DP-1 (Rt =2.235min). Extracted ion chromatograms for these peaks revealed their m/z values to be 373.15. The suggested name for the degradation product DP-1 is 3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.The mass spectrum of degradation product DP-1 is shown in FIG. 6B. MS2 scan of degradation product DP-1 formed fragment ions at m/z 296.1249, 189.0878 and 110.048.
[0076] According to another exemplary embodiment of the invention, FIG. 6C refers to a image 604 of a scheme 1 for the proposed fragmentation pathways for the degradation products of Vimseltinib in acidic condition. Scheme 1 shows the fragmentation mechanism of DP-1, and the ESI spectrum showed the most intense [M+H]+ ion of m/z-373.1515, which is observed under alkali degradation condition. The MS/MS spectrum of DP-1 displayed abundant product ions at m/z-296.1249 (loss of C5H5N from m/z-373.1515), m/z-189.0878 (loss of C6H7NO from m/z 296.1249) and m/z-110.0480 (loss of C3H8NNa from m/z 189.0878) The MS/MS experiments combined with accurate mass measurements have confirmed the proposed scheme.
[0077] According to another exemplary embodiment of the invention, FIG. 7A refers to a graph 700 of the Vimseltinib oxidation chromatogram. It has been discovered that Vimseltinib is unstable to oxidation degradation in one of its forms. For the degradation studies, 30% H2O2is chosen and tested at 25°C for 30 minutes, yielding one degradation products and only 12.1% degradation in relation to Vimseltinib. A summary of the stress study's findings is provided in Table 5.
[0078] According to another exemplary embodiment of the invention, FIG. 7B refers to a graph 702 of mass spectrum of degradation product. The drug on oxidation formed one degradation product, namely DP-2 (Rt =1.763min). Extracted ion chromatograms for these peaks revealed their m/z values to be 447.20. The suggested name for the degradation product DP-2 is 2-[hydroxy(propan-2-yl)amino]-3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-3,4-dihydropyrimidin-4-one. The mass spectrum of degradation product DP-2 is shown in FIG. 7B. MS2 scan of degradation product DP-2 formed fragment ions at m/z 367.1644, 274.1434, 183.1008 and 75.0684.
[0079] According to another exemplary embodiment of the invention, FIG. 7C refers to schemes 704 for the proposed fragmentation pathways for the degradation products of Vimseltinib in oxidation condition. Scheme 2 shows the fragmentation mechanism of DP-2, and the ESI spectrum showed the most intense [M+H]+ ion of m/z-447.2019, which is observed under peroxide degradation condition. The MS/MS spectrum of DP-2 displayed abundant product ions at m/z-367.1644 (loss .of C4H6N2 from m/z-447.2019), m/z-274.1430 (loss of C5H5NO from m/z 367.1644), m/z-183.1008 (loss of C6H7N from m/z 274.1430) and m/z-75.0684 (loss of C5H6N2O from m/z 183.1008). The MS/MS experiments combined with accurate mass measurements have confirmed the proposed scheme.
[0080] According to another exemplary embodiment of the invention, FIG. 8 refers to a graph 800 of the Vimseltinib reduction degradation chromatogram. It has been observed that Vimseltinib is stable against reduction in one of its variations. For the degradation experiments, 10% sodium bisulphite is utilized at 25°C for 30 minutes, which yielded no significant or minor degradation products and only 2.8% degradation relative to Vimseltinib. A summary of the stress study's results can be found in Table 5.
[0081] According to another exemplary embodiment of the invention, FIG. 9A refers to a graph 900 of the Vimseltinib thermal degradation chromatogram. It has been found that Vimseltinib is unstable to thermal in one of its embodiments. For degradation studies, 40 mg of Vimseltinib standard is taken in Petri dish and kept in hot air oven at 1050 C for 3 hours. Then weighed 7 mg of the analyzed standard and transferred in to 10 ml volumetric flask and diluted with diluents. Further diluted 1 ml to 10 ml with diluents and injected into HPLC and analyzed, which produced one significant degradation product and only 11.5% degradation concerning Vimseltinib. A summary of the findings from the stress study is included in Table 5.
[0082] According to another exemplary embodiment of the invention, FIG. 9B refers to a graph 902 of mass spectrum of degradation product. The drug on thermal condition formed one degradation product, namely DP-3 (Rt =3.806min). Extracted ion chromatograms for these peaks revealed their m/z values to be 274.1437. The suggested name for the degradation product DP-3 is 5-(5-hydroxy-6-methylpyridin-2-yl)-3-methyl-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one. The mass spectrum of degradation product DP-3. MS2 scan of degradation product DP-3 formed fragment ions at m/z 217.0857, and 109.0535.
[0083] According to another exemplary embodiment of the invention, FIG. 9C refers to schemes 904 for the proposed fragmentation pathways for the degradation products of Vimseltinib in thermal condition. Scheme 3 shows the fragmentation mechanism of DP-3, and the ESI spectrum showed the most intense [M+H]+ ion of m/z-274.1430, which is observed under Thermal degradation condition. The MS/MS spectrum of DP-3 displayed abundant product ions at m/z-217.0851 (loss of C3H9N from m/z-274.1430) and m/z-109.0528 (loss of C5H6N2O from m/z 217.0851) The MS/MS experiments combined with accurate mass measurements have confirmed the proposed scheme.
[0084] According to another exemplary embodiment of the invention, FIG. 10 refers to a graph 1000 of the Vimseltinib photo degradation chromatogram. It has been observed that Vimseltinib is stable against reduction in one of its variations. For the degradation experiments, 40 mg of Vimseltinib sample is placed in photo stability chamber for 6 hours. Then weighed 7 mg of the analyzed sample and transferred in to 10 ml volumetric flask and diluted with diluents. Further diluted 1 ml to 10 ml with diluents and injected into HPLC and analyzed, which yielded no significant or minor degradation products and only 2.3% degradation relative to Vimseltinib. A summary of the stress study's results can be found in Table 5.
[0085] According to another exemplary embodiment of the invention, FIG. 11A refers to a graph 1100 of the Vimseltinib hydrolytic degradation chromatogram. It has been observed that Vimseltinib is unstable against hydrolytic degradation in one of its variations. For the degradation experiments, accurately weighed and transferred 7 mg of the Vimseltinib sample in to a 10 ml vacuum flask, add 1 ml of HPLC water. The vacuum flask is then leaved for 30 min. After 30 min diluted to 10ml with diluent. Further dilute 1 ml to 10 ml with diluents, which yielded one significant degradation product and only 9.7% degradation relative to Vimseltinib. A summary of the stress study's results can be found in Table 5.
[0086] According to another exemplary embodiment of the invention, FIG. 11B refers to a mass spectrum 1102 of degradation product. The drug on hydrolytic condition formed one degradation product, namely DP-4 (Rt =2.057min). Extracted ion chromatograms for these peaks revealed their m/z values to be 274.1437. The suggested name for the degradation product DP-4 is 3-methyl-5-[6-methyl-5-(pyridin-4-yloxy)pyridin-2-yl]-2-nitro-3,4-dihydropyrimidine-4,4-diol. The mass spectrum of degradation product DP-4. MS2 scan of degradation product DP-4 formed fragment ions at m/z 280.8080, 173.0437, and 109.0528.
[0087] According to another exemplary embodiment of the invention, FIG. 11C refers to schemes 1104 the proposed fragmentation pathways for the degradation products of Vimseltinib in hydrolytic condition. Scheme 4 shows the fragmentation mechanism of DP-4, and the ESI spectrum showed the most intense [M+H]+ ion of m/z-357.1073, which is observed under Hydrolysis degradation condition. The MS/MS spectrum of DP-4 displayed abundant product ions at m/z-280.0808 (loss of C5H5N from m/z-357.1073), m/z-173.0437 (loss of C6H7NO from m/z-280.0808) and m/z-109.0528 (loss of C5H7N3O4 from m/z 280.0808) The MS/MS experiments combined with accurate mass measurements have confirmed the proposed scheme.
[0088] According to another exemplary embodiment of the invention, FIG. 12 illustrates a flowchart 1200 of characterization of four degradation impurities of the Vimseltinib based on spectral data. In one embodiment herein, the degradation results of Vimseltinib are shown in Table 5.
[0089] Table 5:
Stress Conditions Area % Degradation Peak Purity No of DPs Formed
Purity Angle Purity Threshold
CONTROL 2566481 0 1.826 8.778 -
Acid 2462154 4.1 1.887 8.763 -
Alkali 2218145 13.6 1.861 8.719 DP-1
Peroxide 2256521 12.1 1.896 8.75 DP-2
Reduction 2495126 2.8 1.803 8.728 -
Thermal 2272352 11.5 1.813 8.733 DP-3
Photolytic 2506948 2.3 1.847 8.781 -
Hydrolysis 2316936 9.7 1.858 8.741 DP-4

[0090] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, a rapid, accurate, and reproducible reverse-phase high-performance liquid chromatography (RP-HPLC) method, supported by photodiode array detection (PDA) and liquid chromatography–tandem mass spectrometry (LC–MS/MS), for the determination of Vimseltinib in the presence of its degradation products and process-related impurities.
[0091] The proposed method provides a validated stability-indicating analytical procedure capable of effectively separating Vimseltinib from its degradation products under multiple stress conditions. The proposed method enables identification and structural characterization of previously unreported degradation products using LC–MS/MS fragmentation analysis. The proposed method demonstrates excellent linearity, precision, accuracy, and reproducibility, making it suitable for quantitative determination of Vimseltinib in bulk drug and pharmaceutical dosage forms. The chromatographic conditions allow short run time with efficient resolution, improving analytical throughput and laboratory efficiency. The method supports comprehensive impurity profiling in accordance with regulatory requirements, including ICH guidelines for stability studies. The approach reduces analytical complexity by enabling separation and characterization using a single optimized chromatographic system. The method is suitable for routine quality control, stability testing, and lifecycle management of Vimseltinib.
[0092] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
, Claims:CLAIMS:
I/We Claim:
1. A stability-indicating analytical method for the determination, separation, identification, and characterization of Vimseltinib in the presence of degradation products and process-related impurities, comprising:
subjecting a sample with Vimseltinib to one or more forced degradation conditions selected from acidic, alkaline, oxidative, reductive, photolytic, thermal, and hydrolytic conditions to form one or more degradation products, thereby obtaining a stressed sample;
analyzing the stressed sample using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with photodiode array (PDA) detection; and
characterizing the degradation products using liquid chromatography–tandem mass spectrometry (LC–MS/MS),
wherein the RP-HPLC comprises an octadecylsilane-bonded silica stationary phase and a carrying phase comprising 0.1% TFA buffer solution with acetonitrile in a volumetric ratio of 60:40 (v/v),
wherein the method is performed at a flow rate of 1.0 mL/min, detection wavelength of 257 nm, and run time of 5 min.
2. The method as claimed in claim 1, wherein the RP-HPLC is used for the quantitative determination of Vimseltinib in tablet dosage form, wherein the RP-HPLC comprises:
preparing a sample solution from a tablet formulation;
injecting the solution into the RP-HPLC; and
determining the amount of Vimseltinib by comparison with a reference standard.
3. The method as claimed in claim 1, wherein the forced degradation condition comprises alkaline degradation by treating the sample of Vimseltinib with 1 N sodium hydroxide for 30 min to form a first degradation product.
4. The method as claimed in claim 3, wherein the first degradation product exhibits a mass-to-charge ratio (m/z) of 373.1 and has a molecular weight of 373.15 and molecular formula C₁₉H₂₀N₅NaO₂, and is identified as 3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
5. The method as claimed in claim 1, wherein the forced degradation condition comprises oxidative degradation by treating the sample of Vimseltinib with 30% hydrogen peroxide for 30 min to form a second degradation product.
6. The method as claimed in claim 5, wherein the second degradation product exhibits a mass-to-charge ratio (m/z) of 447.20 and has a molecular weight of 447.20 and molecular formula C₂₃H₂₅N₇O₃, and is identified as 2-[hydroxy(propan-2-yl)amino]-3-methyl-5-(6-methyl-5-{[2-(1-methyl-1H-pyrazol-4-yl)pyridin-4-yl]oxy}pyridin-2-yl)-3,4-dihydropyrimidin-4-one.
7. The method as claimed in claim 1, wherein the forced degradation condition comprises thermal degradation by exposing the sample of Vimseltinib to a temperature of 105 °C for three hours to form a third degradation product.
8. The method as claimed in claim 7, wherein the third degradation product exhibits a mass-to-charge ratio (m/z) of 274.14 and has a molecular weight of 274.14 and molecular formula C₁₄H₁₈N₄O₂, and is identified as 5-(5-hydroxy-6-methylpyridin-2-yl)-3-methyl-2-[(propan-2-yl)amino]-3,4-dihydropyrimidin-4-one.
9. The method as claimed in claim 1, wherein the forced degradation condition comprises hydrolytic degradation by treating the sample of Vimseltinib with water for 30 min to form a fourth degradation product.
10. The method as claimed in claim 9, wherein the fourth degradation product exhibits a mass-to-charge ratio (m/z) of 357.10 and has a molecular weight of 357.10 and molecular formula C₁₆H₁₅N₅O₅, and is identified as 3-methyl-5-[6-methyl-5-(pyridin-4-yloxy)pyridin-2-yl]-2-nitro-3,4-dihydropyrimidine-4,4-diol.