INTRODUCTION:

I. Tepotinib, chemically known as (3-{1-[(3-{5-[(1-methylpiperidin-4-yl)methoxy]pyrimidin-2-yl}phenyl)methyl]-6-oxo-1,6-dihydropyridazin-3-yl}benzonitrile),(Fig 1) molecular formula is C₂₉H₂₈N₆O₂and molecular weight is 492.583. A mesenchymal-epithelial transition (MET) exon 14 skipping change is treated with the oral tyrosine kinase inhibitor tepotinib in adult patients with metastatic non-small cell lung cancer (NSCLC)¹.

This medication should be taken once day by mouth at a dosage of 450mg. 3-4% of cases of NSCLC had the MET exon 14 (METex14) skipping mutation, which is most common in this cancer type². Tepotinib inhibit their target’s Phosphotransferase activity by competes with ATP. Peripheral edema (63%), nausea (26%), diarrhea (22%), an increase in blood creatinine (18%), and hypoalbuminemia (16%) are among the frequent side effects of tepotinib³. Tepotinib received the first approval for advanced NSCLC with METex14 skipping in March 2020 in Japan. Tepotinib was then approved in the following countries in 2021: Singapore, Great Britain, the United States, Brazil, Canada, Taiwan, Republic of Korea, and Switzerland^. The goal of the current effort is to create and test an RP-HPLC method for evaluation of Tepotinibin bulk and pharmaceutical dosage form ^4-5. There is currently no information on the method development and validation for tepotinib in the literature review. As a result, the current work represents a novel method for determining tepotinib in bulk and pharmaceutical dosage form by RP-HPLC.

Fig 1: Tepotinib Chemical structure

METHODS:

Materials and Reagents:

All the chemicals and solvents used in the investigation, including the Tepotininb (API), Tepotinib tablets (Tepmetko), Milli-Q water, Acetonitrile, Phosphate buffer, Methanol, Potassium dihydrogen Ortho phosphate buffer, and Ortho phosphoric acid, were bought from Rankem.

Instrumentation and Chromatographic conditions:

The WATERS HPLC 2695 SYSTEM equipped with quaternary pumps and a photo diode array detector was used to carry out the chromatographic evaluation. Using empower software, output signals were tracked and analyzed. The mobile phase comprised 0.1% Ortho Phosphoric acid in a 70:30(v/v) acetonitrile mixture that had been degassed before review in order to obtain a sharp peak. It was satisfied that the chosen mobile phase produced a sharp peak with a low tailing factor of less than 2.0 and a plate count of more than 2,000^[6]. Injection volume was 10µl, flow rate was 1.0ml/min, and the eluent was detected at 308nm maintained column temperature at 30°c achieved the analysis on a KromosilC18 column (4.6x 150mm, 5µ). Tepotinib demonstrated a strong peak in these parameters, with a retention duration of 2.492 mins.

Buffer with 0.1% Ortho Phosphoric Acid preparation:

In order to create a 0.1% OPA buffer, 1ml of Ortho-phosphoric acid was put into 1000ml of HPLC grade water and diluted. The prepared solution was filtered before being sonicated for 15 mins to remove the gas.

Mobile phase preparation:

It included 0.1% OPA andACN in the ratio of (70:30) by volume. An isocratic programme was used to elute Tepotinib.

Diluent selection:

Acetonitrile and water were chosen as the diluent in a 50:50 ratio based on the solubility of the drug.

Preparation of standard stock solution:

In a volumetric flask with a capacity of 50ml, accurately measure and weigh out 11.25mg of Tepotinib, and then combine with 20ml of diluents. The solution should be entirely dissolved after ten minutes of sonication. Finish by adding more diluents to the flask.

Preparation of standard working solution (100% solution):

A 10ml volumetric flask was filled with diluent after 1ml from each stock solution was pipetted into it.

Preparation of sample stock solution:

The average weight of 10 tablets was estimated, and the weight equivalent to 1 tablet was put into a volumetric flask with 100ml of diluents, 50ml of diluent added, and the volume sonicated for 25minutes before being made up with diluent and filtered through HPLC filters.

Preparation of sample working solution (100% solution):

A 10ml volumetric flask was filled with diluent after receiving 0.5ml of filtered sample stock solution.

Wavelength selection:

Precisely weigh 11.25mg of Tepotinib and pass on into 50ml volumetric flask, dissolved by using diluent up to the mark. 1ml was pipetted out of the aforementioned solution and diluted with diluent in a 10ml volumetric flask. Using PDA spectroscopy, the scanning was carried out between 200 and 400nm.

Analytical method validation:

System suitability parameter. Device suitability parameters, such as theoretical plate number, duration, peak area, tailing factor, and resolution, were gathered from Empower software following six injections of a normal solution.

Specificity. Evaluation of Standard and sample injections used as the method for determining specificity. Comparison of the chromatograms of the standard and sample revealed high correlation and the absence of excipient interference with the medication⁷.

Linearity. Aliquots of 0.25, 0.5, 0.75, 1.0, 1.25, and 1.5ml from the standard stock solutions were collected in a 10ml volumetric flask and diluted up to the mark with diluent to provide a final concentration in the range of 5.625-33.75µg/ml. By graphing the peak area v/s concentration, a calibration curve was created.

Precision: The degree of scatter between a set of measurements obtained from multiple sampling of the same homogeneous sample under the specified conditions is referred to as precision, and it expresses how closely measurements agree with one another (degree of accuracy). System precision, Method precision and Intermediate precision are the three types of precision. Six injections from a single working standard solution were taken to achieve system precision. Method precision was achieved by repeated sampling from the sample stock solution to create 6 separate working sample solutions of the same concentration. By multiple sampling from the sample stock solution to create six separate working sample solutions of the same concentration to achieve intermediate precision, and these samples were injected the next day after preparation. Percentage relative standard deviation (%RSD) was used to express the precision results.

Accuracy. The recovery research was used to determine accuracy, and three distinct levels of Tepotinib known standard concentration spikes-50%, 100%, and150% The trial was conducted in triplicate for each level. In order to obtain a final concentration in the range of 50%, 100%, and 150%, aliquots of 0.5, 1.0, and 1.5 ml from the sample stock solution were added to all of the aliquots, along with 1.0 ml of the standard stock solution was added to all of the aliquots and diluting them up to the mark with diluent. Standard addition technique was used to create the samples for preparing these concentrations. While the RSD rate was under2%, the recovery rate was identical at 100%. Along with the percentage of recoverable data, the average and relative standard deviations was also calculated⁸.

Specificity. In this technique, we should not detect interfering peaks in the placebo and blank groups at these retention time of the Tepotinib. Consequently, it was claimed that this approach was specific.

Limit of detection (LOD) and limit of quantification (LOQ). Pipetting 0.1 ml of the 25% linearity solution into a 10ml volumetric flask allowed it to be diluted to the proper amount and brought up to the required level (LOD). Pipetting 0.3 ml of the 25% linearity solution into a 10ml volumetric flask allowed it to be diluted to the proper amount and brought up to the required level (LOQ).

Robustness. Small, purposeful changes were made to a select few parameters to examine the method's robustness. Flow rate, mobile phase, and temperature were all altered by ±0.5 ml/min, ±20% and ±10 °C, respectively. The samples were administered in six replicate manner. The system suitability requirements did not alter at all. It was therefore asserted that the provided approach was robust.

RESULTS AND DISCUSSION:

The objective of the study is to create a single, precise, isocratic HPLC method for the quantitative determination of Tepotinib in bulk and pharmaceutical dosage form. Using isocratic development techniques, developers evaluated acidic buffers, acetonitrile, and methanol. To achieve better outcomes, different mobility phases were altered at each trial. Kromosil C₁₈ column (150 x 4.6mm, 5µm) and moving phase of 0.1% OPA: Acetonitrile (70:30 v/v) with a PDA detector set to monitor at 308nm were utilized. It lasted five minutes throughout the duration of the show. Enhanced chromatographic circumstances (Table 1).

Table 1: Optimized Chromatographic Conditions

Parameter	Condition
Mobile Phase	OPA:Acetonitrile(70:30 v/v)
Run Rate	1.0ml/min
Column	Kromosil C₁₈ column (150 x 4.6mm, 5µm)
Wavelength Detection	308nm
Temperature of the Column	30ºC
Volume of Injection	10µL
Travel Time	5min
Tepotinib Elution Time	2.492

System appropriateness. Following six injections of a standard solution, empower software was used to gather device suitability parameters like theoretical plate number, duration, peak area and tailing factor. The system suitability findings (Table 2)

Table 2: Results of System Suitability

Parameter	Tepotinib
Theoretical plate count	7,741
Tailing	1.2
Elution Time of Tepotinib	2.492

Specificity. During the time the eluent came into touch with the blank and placebo, there was no Tepotinib to be found.

Linearity. By plotting a calibration curve of the peak concentration area against its corresponding concentration (25%,50%,75%,100%,125%, and 150%), linearity was found. It was feasible to deduce from this calibration curve that the graph reflected a straight line between 5.625-33.75 µg/ml of Tepotinib. Y is calculated as 67,266X + 8797.9 (R2 = 0.9995). (Table 3) displays the results.

Table 3: Linearity Results

Tepotinib
S. No	Concentration (µg/ml)	Area
1	5.625	381470
2	11.25	761860
3	16.875	1152516
4	22.5	1552231
5	28.125	1911005
6	33.75	2248334
CC		0.9995
Slope		67,266
Intercept		8797.9

Precision. This process was investigated to assess intraday and intermediate intraday precision. The intraday experiments were carried out by analyzing the Tepotinib sample solution six times on the same day under the identical circumstances. In the same facility, this system was tested for accuracy by analyzing the data using a variety of instruments and examiners. Relative standard deviation (RSD) percentage values of less than 2% demonstrate its excellent precision. Each time the process was run at a higher concentration, there was a meaningful recovery of usable medication, suggesting that the method was accurate. The method precision findings (Table 4) in high-quality, Intermediate Precision outcomes (Table 5)

Table 4: Method precision outcomes

S. No	Area of Tepotinib
1	1520654
2	1539198
3	1544561
4	1542055
5	1536575
6	1526021
Mean	1534844
Std. Dev	9461.2
% RSD	0.6

Table 5: Intermediate Precision outcomes

S. No	Area of Tepotinib
1	1564426
2	1551226
3	1569016
4	1574643
5	1552693
6	1543231
Mean	1559206
Std. Dev	12025.7
% RSD	0.8

Accuracy. The recovery studies were measured in three stages at 50%, 100%, and150%, shown in the Fig 2,3,4) which provided the precision. Levels of Tepotinib of 11.25, 22.5, and 33.75µg/ml were used in Pharmaceutically Active Substance (APIs). The three injections of each spike's test solution was run, and the test procedure was carried out. The RSD rate was under 2%, the recovery rate was identical at 100%. In addition to calculating the average and relative standard deviations, the percentage of recoverable data was also calculated. The recovery values fell within the parameters, making the strategy successful. The accuracy data is given (Table 6).

Table 6: Accuracy Data

Accuracy	Tepotinib amount	% Recovery
50^a	50	100.94
100^a	100	99.78
150^a	150	100.36

Limit of Detection (LOD)and Limit of Quantification (LOQ). Tepotinib LOD and LOQ were determined using the formulas LOD = 3.3 N/B and LOQ = 10 N/B, respectively, for a signal-to-noise (S/N) ratio of 3:1 and10:1, respectively, where "N" stands for the standard deviation of the drug peak area (n=3), which is used to measure noise, and "B" stands for the slope of the related calibration plot. The ratio of signal to noise was calculated. A 0.05 and 0.16µg/ml LOD and LOQ were discovered, respectivelyshown in the(Fig 5,6).

Robustness. Variations in flow rate, moving phase composition and temperature were taken into consideration when evaluating the chromatographic process. The RSD percentage was found to fall within acceptable bounds (Table 7) displays robustness findings.

Table 7: Robustness outcomes

Condition	Tepotinib %RSD
Flow (-) 0.9ml/min	0.8
Flow (+) 1.1ml/min	1.0
Mobile phase (-) 65B:35A	0.5
Mobile phase (+) 75B:25A	0.4
Temperature (-) 27°C	0.6
Temperature (+) 33°C	0.8

Stress testing studies. These investigations are done to evaluate the drug's stability under specific stress situations. Studies are carried out under stressful conditions such as acid, base, peroxide hydrolysis, UV, thermal, and neutral degradation. The limits of degradation are met by every result from the assay of the injected components (Table 8) describes the outcomes of the stress degradation studies

Table 8: Stress testing outcomes

Degradation Parameters	Tepotinib % Degradation
Acid	5.03
Base	4.26
Peroxide	7.71
Thermal	2.72
UV	1.16
Hydrolysis	0.84

CONCLUSION:

In this work, an innovative HPLC method was created to assess Tepotinib in API and pharmaceutical dosage that was quick, affordable, sensitive, and simple to use. The primary benefit of this strategy is that no methods have been created for the estimate of Tepotinib; instead, an effort has been made to create a new stability-indicating Reverse phase-HPLC method for the estimation of Tepotinib in bulk and tablet dosage forms. A straightforward and accurate reversed phase isocratic RP-HPLC method has been created, and it has been improved and validated in accordance with ICH recommendations. All metrics, including linearity, accuracy, specificity, robustness, and process precision, were evaluated and determined to be within acceptable bounds. The parameters RSD values were less than2%, demonstrating a precise process and a generally consistent outcome from that method. To estimate Tepotinib in bulk and pharmaceutical formulations as well as for routine analysis for quality control objectives, the current analytical approach was devised.

REFERENCES:

1. Afroz Patan, Reeyaz Basha S., et.al. Development and Validation of New RP- HPLC Method for theSimultaneous Estimation of Metformin Hydrochloride and Repaglinide in Pure and Pharmaceutical Formulations. Research Journal of. Pharmacy and Technology. 2021; 14(3): 1323-1328. doi: 10.5958/0974-360X.2021.00235.3

2. Brazel, D., Zhang, S., Nagasaka, M., Spotlight on Tepotinib and Capmatinib for Non-Small Cell Lung Cancer with MET Exon 14 Skipping Mutation. Lung Cancer: Targets and Therapy. 2022; 13: 33-35.

3. Drugbank.com, Tepotinib.https://go.drugbank.com/drugs/DB08828

4. Le X., et.al. Tepotinib Efficacy and Safety in Patients with MET Exon 14 Skipping NSCLC: Outcomes in Patient Subgroups from the VISION Study with Relevance for Clinical Practice VISION: Tepotinib Outcomes in METex14 Skipping NSCLC. Clinical Cancer Research. 2022; 28: 117-1126.

5. Takamori S,et.al. Dramatic intracranial response to tepotinib in a patient with lung adenocarcinoma harboring MET exon 14 skipping mutation. Thoracic Cancer. 2021; 12: 978-980.

6. Monika Maheshwari and Parul Soni. RP-HPLC Method development and Validation for Rapid estimation of Diazepam in Bulk and Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology. 2022; 15(5): 1938-2. doi: 10.52711/0974-360X.2022.00322

7. ICH, Q2 (R1) Validation of Analytical Procedures: Text and Methodology, Proceedings of the International Conference on Harmonization, November 2005.

8. ICH, Q1A (R2) Stability Testing of New Drug Substances and Products, Proceedings of the International Conference on Harmonization, November 2003.

Received on 03.02.2024 Revised on 15.06.2024

Accepted on 18.08.2024 Published on 28.01.2025

Available online from February 27, 2025

Research J. Pharmacy and Technology. 2025;18(2):557-562.

DOI: 10.52711/0974-360X.2025.00083

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