LC-MS/MS CHARACTERIZATION OF FORCED DEGRADATION PRODUCTS OF TUCATINIB, A NOVEL TYROSINE KINASE INHIBITOR: DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD

Objective: The current study focused on the development, validation, and characterization of forced degradation products using LC-MS/MS. Methods: A simple, selective, validated and well-defined isocratic HPLC methodology for the quantitative determination of Tucatinib at a wavelength of 239 nm. An isocratic elution of samples was performed on an Inertsil ODS (250x4.6 mm, 5m) column with a mobile phase of 70:30v/v Acetonitrile and formic acid (0.1%) delivered at a flow rate of 1.0 ml/min. MS/MS was used to characterize degradation products formed in the forced degradation study. The validation and characterization of forced degradation products were performed in accordance with ICH guidelines. Results: Over the concentration range of 5-100 μ g/ml, a good linear response was obtained. Tucatinib's LOD and LOQ were determined to be 0.05 and 0.5, respectively. According to standard guidelines, the method was quantitatively evaluated in terms of system suitability, linearity, precision, accuracy, and robustness, and the results were found to be within acceptable limits. The drug was degraded under acidic, alkaline, and reduction conditions in forced degradation studies. Conclusion: The method was found to be applicable for routine tucatinib analysis. Because no LC-MS/MS method for estimating tucatinib and its degradation products has been reported in the literature. There is a need to develop a method for studying the entire tucatinib degradation pathway.

Tucatinib's molecular formula and molecular weight are C26H24N8O2 and 480.5212 μg/mol, respectively. Tucatinib, sold under the brand name Tukysa, is a small molecule inhibitor of HER2 for the treatment of HER2-positive breast cancer [3,4]. It works by preventing the abnormal protein from signalling cancer cells [5,6] to multiply. This aids in the prevention or slowing of the spread of cancer cells. Tucatinib, in combination with trastuzumab and capecitabine, was approved by the FDA on April17, 2020 for the treatment of patients with advanced unresectable or metastatic HER2-positive breast cancer.
Tucatinib inhibits HER2 and HER3 phosphorylation [7,8] in vitro, resulting in inhibition of downstream MAPK [9,10] and AKT [11,12] signalling and cell proliferation, and has anti-tumor activity in HER2 expressing tumour cells. Tukysa is the brand name for tucatinib. Array BioPharma developed it. Patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer who progress after multiple HER2-targeted agents have few treatment options. Common side effects are diarrhea, palmarplantar erythrodysesthesia [13,14] (burning or tingling discomfort in the hands and feet), nausea, fatigue, hepatotoxicity [15] (liver damage), vomiting, stomatitis [16] (inflammation of the mouth and lips), decreased appetite [17], abdominal pain [18], headache, anemia and rash. Pregnant or breastfeeding women should not take Tucatinib because it may cause harm to a developing fetus or newborn baby.
There are only a few methods available for tucatinib. There are preclinical studies available, but no analytical methods are reported. The purpose of this research is to create, validate, and characterize forced degradation products using LC-MS/MS.

Chemicals and reagents
Shree Icon Lab provided the pure Tucatinib standard (Vijayawada, India). Finar Chemicals supplied HPLC-grade acetonitrile and formic acid (Ahmedabad, India). Filtration through a Millipore MilliQ plus system produced HPLC grade water (Millipore, USA). Merck supplied analytical grade reagents of sodium hydroxide, hydrochloric acid, 30% hydrogen peroxide, and sodium bisulphate (Mumbai, India).

HPLC
Waters alliance model e2695 liquid chromatography instrument, Waters (2998) Photodiode array Detector, Waters (2700) Auto sample injector, Solvent degasser, Quaternary pump, Temperaturecontrolled compartment. Using a sonicator, all of the solutions were sonicated for effective mixing and degassing (Unichrome associates 701). Weighing is done with an analytical balance (Denver). A hot air oven was used to conduct a thermal degradation study (KEMI).

Method optimization
Several parameters, including mobile phase, stationary phase, flow rate, and detector wavelength, were considered when developing and optimising the chromatographic separation conditions. Isocratic elution with mobile phase 70:30v/v Acetonitrile and formic acid (0.1 percent) pumped from a solvent reservoir at a flow rate of 1.0 ml/min to the analytical column of the Inertsil ODS (250x4.6 mm,5m) using column back pressure of 1570-1620 at the maximum detector wavelength of 239 nm was used to obtain a chromatographic separation. Detector performance was evaluated using Empower-2 software to determine the height peak area and other device suitability parameters. The injection volume was set at 10l, the ambient column temperature was maintained throughout the analysis.

Preparation of mobile phase
Acetonitrile and formic acid were mixed in a 70:30v/v ratio then filtered through 0.45μm filter paper and sonicated to remove any gas.

Chromatographic conditions
Tucatinib was separated chromatographically in an Inertsil ODS (250x4.6 mm, 5m) column. The mobile phase was composed of acetonitrile and formic acid (70:30v/v) and was pumped at room temperature at a flow rate of 1 ml/min with a UV detection wavelength of 239 nm and filtered through a 0.45 m nylon membrane filter under vacuum filtration. The injection volume was ten μ litres. Tucatinib had a retention time of 3.734 min, while the run lasted 6 min.

Diluents
Mobile phase was used as diluents.

Preparation of standard solution
Tucatinib standard stock solution was prepared by dissolving 100 mg of Tucatinib in 100 ml of diluents in a 100 ml clean and dry volumetric flask, and the standard solution was filtered through a 0.45m membrane filter and degassed with a sonicator to obtain a concentration of 1000g/ml of Tucatinib.

Selection of wavelength
A UV-Visible spectrophotometer was used to scan the maximum absorption wavelength of 50g/ml Tucatinib against a blank mobile phase in the wavelength range of 200-400 nm. As shown in fig. 2, the maximum wavelength was discovered to be 239 nm.

Acid degradation
Tucatinib solution was treated with 1N HCl, heated for 6 h, and refluxed for 5 h before adding 1 ml of 1N NaOH and diluting volume with diluents. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Alkali degradation
Tucatinib solution was treated with 1N NaOH for alkali degradation, heated for 6 h, and refluxed for 5 h before adding 1 ml of 1N HCl and diluting volume with diluents. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h. Tucatinib solution was treated with 30% H2O2 and refluxed for 5 h, then heated for 3 h before cooling to room temperature and diluting with diluents. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Reduction degradation
Tucatinib solution was treated with 30% sodium bi sulphate solution, refluxed for 5 h, heated to 3 h, cooled to room temperature, and then diluted volume with diluent. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Hydrolysis degradation
Tucatinib solution was treated with H2O and refluxed for 30 min before being heated for 3 h, cooled to room temperature, and diluted volume with diluent. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Thermal degradation
100 mg of sample was exposed at 105 °C for 6 h before being analysed. A sample of 62 mg was transferred to a 100 ml volumetric flask. Add 70 ml of diluent, sonicate to dissolve, and dilute to volume with a diluent before mixing. Dilute 5 ml to 50 ml more with diluent. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Photolytic degradation
100 mg of sample was exposed at UV light for 6 h before being analysed. A sample of 62 mg was transferred to a 100 ml volumetric flask. Add 70 ml of diluent, sonicate to dissolve, and dilute to volume with a diluent before mixing. Dilute 5 ml to 50 ml more with diluent. Inject the solution into the HPLC system several times over the course of 6 h, 12 h, 18 h, and 24 h.

Method development and optimization
Initially, the RP-HPLC system was optimized using an Inertsil ODS (250x4.6 mm, 5m) column. To satisfy the system suitability parameters, a mixture of acetonitrile and formic acid (70:30v/v) as the mobile phase (Flow rate 1.0 ml/min) was found to be more appropriate. Table 1 summarizes the optimized chromatographic conditions. At 3.734 min, Tucatinib eluted as a sharp peak fig. 3.  . 4). The LOD and LOQ values are discovered to be 0.05 and 0.5, respectively. Using three different concentrations of tucatinib, the percentage relative standard deviation (RSD) was found to be 1.01 and0.83, respectively (2.0 percent), demonstrating that the method is precise. The method's accuracy [27] was demonstrated using the standard addition method, and the recovery values were calculated. The percentage RSD was found to be 0.5(2.0%), with a recovery rate of 98.4-101.3 percent. In the robustness study [28], the percentage RSD was found to be 0.2-1.21 (2.0 percent). The suitability of the system [29] and the stability of the solution was assessed, and the percentage RSD was 2%. The results are shown in table 2.       [31,32] in all of the degradation studies, indicating that the method is selective and specific. Fig. 5 depicts typical chromatograms obtained during a stress degradation study of tucatinib.

LC-MS/MS studies of forced degradation products
Four degradation products, DP1, DP2, DP3, and DP4 were identified and characterized by tandem mass spectrometric analysis (LC-MS/MS) and accurate mass measurement shown in table 4. DP1 and DP2 were degraded in acidic conditions, whereas DP3 was degraded in alkaline conditions, DP4 was degraded in reduction condition. The Identification of active drug and its novel degradation product was studied on Agilent Q-TOF of Mass spectrometric (MS) technique with ABSCIEX. Mass measurements of Tucatinib was shown in table 8.

MS/MS of DP1
The LC-ESI-MS/MS spectrum of DP1 with m/z 363 (Rt= 1.503 min) was examined in the proposed fragmentation pathway (fig. 7). The degradation ions of m/z 257 (loss of C7H8O from the parent ion at m/z 363), m/z 145 (loss of C5H10N2O from the parent ion at m/z 257), m/z 114 (loss of C8H7N3 from the parent ion at m/z 257). Accordingly, data obtained from MS/MS, elemental composition, and precise mass measurements are shown in (fig. 7).

MS/MS of DP2
The LC-ESI-MS/MS spectrum of DP2 with m/z 321 (Rt= 3.351 min) was examined in the proposed fragmentation pathway (fig. 8). The degradation ions of m/z 175 (loss of C5H9ClNO2 from the parent ion at m/z 321), m/z 148 (loss of C8H5N3O2 from the parent ion at m/z 321). Accordingly, data obtained from MS/MS, elemental composition, and precise mass measurements are shown in (fig. 8).

MS/MS of DP3
The LC-ESI-MS/MS spectrum of DP3 with m/z 270 (Rt= 1.336 min) was examined in the proposed fragmentation pathway ( fig. 9). The degradation ions of m/z 153 (loss of C6H5N3 from the parent ion at m/z 270), m/z 119 (loss of C7H7NO3 from the parent ion at m/z 270). Accordingly, data obtained from MS/MS, elemental composition, and precise mass measurements are shown in (fig. 9).

MS/MS of DP4
The LC-ESI-MS/MS spectrum of DP4 with m/z 479 (Rt= 1.165 min) was examined in the proposed fragmentation pathway ( fig. 10). The degradation ions of m/z 362 (loss of C6H5N3 from the parent ion at m/z 479), m/z 241 (loss of C7H9NO from the parent ion at m/z 362), m/z 145 (loss of H2O4S from the parent ion at m/z 241). Accordingly, data obtained from MS/MS, elemental composition, and precise mass measurements are shown in (fig. 10).

CONCLUSION
The validated stability indicting method developed for the determination of novel kinase inhibitors is specific and selective and more economical. The developed method is compatible for both LC and MS Techniques. Tucatinib degradation was investigated under various stress conditions (hydrolysis, oxidation, photolysis, and thermal). Because the developed method can effectively separate the drug from potential degradants, it can be used to assess the quality of tucatinib in bulk and stability samples. The drug was discovered to be unstable in hydrolytic conditions (basic and acid) and reduction, but stable in oxidation, hydrolysis, photolysis, and thermal stress. Using online LC-ESI-MS-MS experiments combined with precise mass measurements, four previously unknown DPs were identified and characterized. This research could aid in the investigation and characterization of other process-related impurities.