RP-HPLC Method Development and Validation for The Estimation of Sevabertinib In Bulk and Pharmaceutical Dosage Form

Sevabertinib is an oral, targeted tyrosine kinase inhibitor (TKI) used to treat adults with advanced, non-squamous non-small cell lung cancer (NSCLC) harbouring HER2 (ERBB2) mutations. It is specifically approved for patients whose cancer has progressed after prior systemic therapy and is taken once or twice daily with food. ^[1-4] Till now no analytical method was reported for the estimation of Sevabertinib in bulk and pharmaceutical formulation. The Chemical structure of Sevabertinib is shown as Figure 01.

MATEIALS AND METHODS

Chemicals and Reagents

The working standard drug Sevabertinib (99.10% purity) & The formulation dosage form having brand name Hyrnuo containing 10 mg of Sevabertinib, were obtained from Zydus Life sciences Limited, Ahmedabad. HPLC grade Methanol, Water and Acetonitrile were purchased form Merk chemicals private limited, Mumbai. Formic acid used for the study were AR Grade and purchased from Merck Specialties Private Limited, Mumbai, India.

Preparation of Mobile Phase

Acetonitrile and 0.1% formic acid (P^H 2.8) were combined in a 40:60% v/v ratio. Before being used, the mobile phase was filtered through a 0.45 µm membrane filter after being sonicated for 15 minutes to eliminate dissolved gases

Preparation of standard drug solution

A 10 ml volumetric flask was filled with precisely weighed 10 mg of the standard sevabertinib. After that, an ultrasonicator was used to fully dissolve it in 5mL of acetonitrile. The solution was filtered using 0.45µm membrane filter paper after the final volume in the volumetric flask was adjusted using the same solvent. A standard stock solution of 1000 µg/mL was acquired. Using mobile phase as a diluent, the concentrations (10 µg/mL) needed for method development and validation parameters were made from the stock (1000 µg/mL) solution of sevabertinib.

Preparation of formulation solution

Twenty Sevabertinib tablets (label claim: 10 mg) were precisely weighed and ground into a fine powder. A 10 mL volumetric flask was filled with precisely weighed tablet powder equal to 10 mg of sevabertinib. To guarantee full drug extraction from the tablet matrix, around 7 mL of acetonitrile was added to the flask, and the mixture was sonicated for 15 minutes. After letting the solution cool to ambient temperature, the volume was adjusted using the same diluent. A sample stock solution with a concentration of 1000 µg/mL was obtained by thoroughly mixing the resultant solution. A 0.45 µm membrane filter was used to filter the sample stock solution, and the first few mL of the filtrate were discarded. To get a working sample solution of 100 µg/mL, 1.0 mL of the filtered sample stock solution was precisely pipetted into a 10 mL volumetric flask and diluted to volume with the diluent. To get the necessary 10 µg/mL concentration for assay and validation tests, further dilutions were prepared from the working sample solution using the same diluent.

Method Development

Selection of Wavelength

To select a suitable wavelength, the standard solutions of 10 μg /mL was prepared and scanned in the PDA detector. The obtained wavelength maximum was selected as suitable wavelength for the detection.

Table 01: Optimized Chromatographic Conditions

Method Validation

In accordance with the ICH criteria, the method was validated in terms of specificity, system appropriateness, LOD & LOQ, linearity, accuracy, precision, ruggedness, and robustness. Replicate injections of the sample and standard solutions into the column were used to conduct validation. ^[5]

RESULTS AND DISCUSSION

Method Development

Table 02: Results for Optimized Chromatogram

Method Validation

Specificity

       No inference of diluent & Placebo

Linearity

Table 03: Results for Linearity

LOD & LOQ

Sevabertinib's LOD was found to be 1.138µg/mL, while its LOQ was determined to be 3.019µg/mL.

Precision

Sevabertinib's system and method precision %RSD were determined to be 0.02 and 0.01, respectively. For both system and method precision, the %RSD was found to be within the acceptable level of less than 2. Thus, it was stated that the developed procedure was precise.

Table 04: Results for Precision

Accuracy

It was shown that the recovery percentage ranged from 98.50 to 99.06%. The% RSD was determined to be within the acceptable limit for sevabertinib at 50%, 100%, and 150% spiking levels. With an acceptance limit of 98–102% and a percentage RSD of less than two, the results demonstrated the accuracy of the proposed method.

Table 05: Results for Accuracy

Ruggedness (Intermediate Precision)

The %RSD used to express ruggedness had to be less than 2. sevabertinib 's % RSD in the developed technique was 0.02 (Table 06). The method's robustness is confirmed by results that fall within the acceptable range.

Robustness

Sevabertinib's percentage change in the developed method was found to be within the acceptable range of less than 2. Consequently, it was shown that the recommended method was suitable for the analysis of sevabertinib when the analytical conditions were slightly changed. This demonstrates that even a small alteration to the analytical conditions has no impact on the outcomes (Table 07). 

Assay

In formulation analysis, the assay percentage for sevabertinib was 98.90% (Table 08). The method was therefore shown to be suitable for the routine analysis of sevabertinib in both bulk and formulations drug.

Table 06: Results for Ruggedness

Table 07: Results for Robustness

Table 08: Results for Formulation