Method Development and Validation of Capmatinib in Solid Dosage Form by RP-HPLC Method

Pharmaceutical Analysis 

The thesis describes pharmaceutical analysis as the branch of analytical chemistry dealing with the qualitative and quantitative analysis of drug substances and pharmaceutical formulations. It highlights the growing importance of analytical techniques in ensuring drug quality, efficacy, and safety throughout product development and manufacturing.  

Importance of Chromatography 

The thesis explains chromatography as one of the most important separation techniques used in pharmaceutical industries. High-performance liquid chromatography (HPLC) has become the preferred analytical technique because of its: 

•      High sensitivity  

•      High specificity  

•      Excellent precision  

•      Accuracy  

•      Rapid analysis  

•      Low detection limits  

HPLC is extensively employed in routine quality control and stability testing of pharmaceutical formulations.  

Capmatinib 

Capmatinib belongs to the kinase inhibitor class. 

It is approved for treating adult patients with metastatic non-small cell lung cancer (NSCLC) possessing MET exon 14 skipping mutations. 

The thesis discusses: 

•           MET signalling pathway  

•           Mechanism of action  

•           Clinical importance  

•           Therapeutic application  

•           Role in targeted cancer therapy  

Need of Study 

The thesis indicates that although several analytical methods exist for Capmatinib, there remains a need for a method that is: 

•           Rapid  

•           Economical  

•           Highly accurate  

•           Precise  

•           Robust  

•           Suitable for routine industrial quality control  

This forms the rationale for the study.  

5.         Aim 

To develop and validate a simple, rapid, precise, accurate, and robust RP-HPLC method for estimation of Capmatinib in tablet dosage form according to ICH guidelines.  

 6.        Objectives 

The thesis supports the following objectives: 

•      Develop RP-HPLC analytical method.  

•      Optimize chromatographic conditions.  

•      Establish system suitability.  

•      Validate according to ICH Q2(R1).  

•      Apply method to marketed tablet assay.  

7.         Materials 

The thesis includes: 

Drug 

Capmatinib 

Chemicals 

•      Methanol  

•      Orthophosphoric Acid  

•      Water  

•      Analytical reagents  

Instruments 

•      HPLC system  

•      UV detector  

•      OpenLab EZChrome software  

•      Phenomenex C18 column  

 8.        Experimental Methodology 

The thesis contains detailed procedures for: 

•      Standard preparation  

•      Sample preparation  

•      Placebo preparation  

•      Assay procedure  

•      Filter study  

•      Stability study  

•      Validation studies  

9.         Optimized Chromatographic Conditions 

LINEARITY AND RANGE: 

 Preparation of linearity solution The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are directly proportional to the concentration (amount) of analyte in the sample. 5 levels of Linearity was performed from 50% to 150% of working concentration Linearity Capmatinib stock solution: Weighed 29.41 mg of Capmatinib HCl anhydrous (Equivalent to 25 mg of Capmatinib) and dissolved in 50 mL with methanol. Further diluted 5.0 ml of stock solution to 50 mL with methanol. (100 PPM) 

Table: Linearity level preparation for HPLC

Determination: 

 Each level injected in triplicate and mean area calculated. Calibration curve was plotted graphically as a function of analyte concentration in µg/mL on X-axis Vs mean area on y-Axis as given in results.  

Acceptance criteria: 

 Correlation Coefficient: NLT 0.98  

Intercept: To be report  

Slope: To be report  

Limit of Detection (LOD) and Limit of Quantitation (LOQ):  

Detection limit:  

The detection limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantitated as an exact value.  

Quantitation limit:  

The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determined with suitable precision and accuracy. As per ICH Q2R1 guidelines LOD and LOQ was determined by using the approach Based on the Calibration Curve in which residual standard deviation of a regression line was calculated and determined the LOD and LOQ by using following formula: 

Accuracy levels details:  

Refer Following table for each sample:  

Table : Accuracy of sample preparation

Precision (Repeatability) Sample details are as follows:  

Table: Repeatability Precision sample preparation Acceptance criteria:

% Assay: 90-110% for each sample and mean assay value  

% RSD for % assay of 6 samples: NMT 2%  

II.   Intermediate precision  

It is performed by doing analysis on another day to check reproducibility of results. Samples prepared in same manner as that of Repeatability parameter (6 Samples prepared).  

Intermediate Precision Sample details are as follows:  

Table: Intermediate Precision sample preparation

Procedure for preparation of Accuracy sample solution:  

Take clean and dried 9 volumetric flasks of 100 mL. Weighed approx. 186.98 mg of placebo and transferred in each 100 mL volumetric flask. Weighed Capmatinib HCl anhydrous API as per accuracy level and transferred in same 100 ml volumetric flask. Add 70-75 ml of Methanol sonicated it for 10 minutes with intermittent shaking. Allowed to cool the solution at room temperature and made the volume up to the mark with Methanol. Filter the solution through suitable 0.45 µ syringe filter discarding5 mL of filtrate. Further dilute 1.0 ml of filtrate to 25 ml with mobile phase.   Selection of analytical wavelength  

Fig.  UV spectrum of Capmatinib

Observation: The standard solution was scanned between 200 nm to 800 nm. Wavelength of maximum absorption was determined for drug. Capmatinib showed maximum absorbance at 233 nm. It is shown in Figure No.2. Therefore 233 nm considered as an analytical wavelength for further determination.  

Method Development by RP – HPLC  

Optimization of HPLC method  

Fig. Typical chromatogram of Trial 1

Observation: Capmatinib eluted with unacceptable chromatography. (Asymmetry: 2.41 & Theoretical plate: 720) Conclusion: Method rejected.  

Fig.  Typical chromatogram of Trial 2

Observation: Capmatinib eluted with unacceptable chromatography. Peak shape is not sharp.   (Asymmetry: 2.29 & Theoretical plate: 813)  

Conclusion: Method rejected Trial 3:  

Chromatogram:  

Observation: Capmatinib eluted at R.T. 3.13 min, with good chromatograph. (Asymmetry:   1.23 & Theoretical plate: 8957) Conclusion: Method Accepted.  

Conclusion: From the observations of trials first to three, it was concluded that chromatographic conditions in trial three gives better peak, good retention time and tailing factor therefore chromatographic conditions in trial three was subjected for method validation  

Table: Optimized Chromatographic Conditions

System suitability test  

Results for System Suitability Test of Capmatinib  

Table: Results for System Suitability Test of Capmatinib

 System Suitability Acceptance Criteria:  

1.    Relative standard deviation of the area of analyte peaks in standard chromatograms should not be more than 2.0 %.  

2.    Theoretical plates of analyte peak in standard chromatograms should not be less than 2000.  

3.    Tailing Factor (Asymmetry) of analyte peaks in Standard Chromatograms should be less than 2.0  

Data interpretation: It was observed from the data tabulated above; the method complies with system suitability parameters. Hence, it can be concluded that the chromatographic method is adequate for intended analysis.  

Fig.  Typical chromatogram Standard solution 1 of system suitability solution.

 Analysis of Marketed Test samples (Assay)  

a) Rahika 200 mg Tablet:  

Weight of 20 tablets = 19.6640 gm  

Average weight of tablet = 19.6640 /20 = 0.9832 gm = 983.2 mg  

Assay results of Rahika 200 mg Tablet  

Fig.  Typical chromatogram Rahika 200 mg Tablet sample.

Acceptance criteria:  

1) % Assay found should be in the range of 90-110%.  

Data interpretation:  

From the above results, it can be concluded that the assay result is within the limit for selected marketed test sample and sample can be used for validation.  

VALIDATION OF RP-HPLC METHOD 1) FILTRATION STUDY:  

Filtration study of an analytical procedure checks the interference of extraneous components from filter, deposition on filter bed and compatibility of filter with sample. Performed on Tablet test sample.  

Results of Filter study  

Table: Results of Filter study   Chromatograms:

Fig. Typical chromatogram of sample filtered through 0.45µ Nylon filter.

SOLUTION STABILITY: Stability study was conducted for Standard as well as Test Sample. Stability study was performed at normal laboratory conditions. The solution was stored at normal illuminated laboratory conditions and analyzed at initial, after 12 hours and 24 hours.  

Results of Solution stability.  

Table: Results of Solution Stability

Fig. Typical chromatogram of Standard solution Initial.

Fig. Typical chromatogram of Standard solution After 24 Hrs.

Fig.  Typical chromatogram of Test solution Initial.

Fig. Typical chromatogram of Test solution After 24 Hrs.  Results of Specificity.

Table: Results of Specificity

Fig.  Typical chromatogram of Placebo solution.

Linearity and Range  

Linearity of an analytical method is its ability to elicit test results that are proportional to the concentration of analyte in samples within a given range.  

Linearity Data for Capmatinib:  

Table: linearity Data for Capmatinib

Fig. Calibration curve of Capmatinib

Data of linearity of Capmatinib:  

Table: Linearity summary of Capmatinib

The respective linear equation for Capmatinib was  

Y = M X + C  

Y = 445397.6639 X + -24820.600  

Where, X= concentration of Analyte in µg/ml Y = is area of peak.  

M = Slope  

C= Intercept  

Fig. Typical chromatogram of Linearity 50%.

Fig. Typical chromatogram of Linearity 125%.

Fig. Typical chromatogram of Linearity 150%.

Results and statistical data of Accuracy of capmatinib:  

Table: Result and statistical data of Accuracy of Capmatinib

Overall Recovery: 99.68%  

% RSD for Overall Recovery: 0.805  

Fig. Typical chromatogram of Accuracy 50%.

Fig. Typical chromatogram of Accuracy 100%.

Fig.  Typical chromatogram of Accuracy 150%.

Result of Intra- day and Inter- Day Precision for Capmatinib test sample assay:  

Table. Result of Intra- day and Inter- Day Precision for Capmatinib Chromatograms:  

Fig.  Typical chromatogram of Repeatability precision (Sample 1).

Fig. Typical chromatogram of Inter-day precision (Sample 1). 

Acceptance criteria: 

Result of Robustness study of Capmatinib  

Table. Result of Robustness study

Fig.  Typical chromatogram of Standard +3 NM.

B. Fig.  Typical chromatogram of Standard -3 NM

C.        Change in Flow rate by + 10% (1.1 mL/min)

Fig.  Typical chromatogram of Standard +10 F.R.%.  

D.        Change in Flow rate by - 10% (0.9 mL/min)

Fig. Typical Chromatogram of standard -10 F.R.%

5. Change in Column Oven temperature by +2°C:

Fig. Typical chromatogram of Standard +2°C C.O.T

Change in Column Oven temperature by -2°C:

Fig. Typical chromatogram of Standard -2ºC C.O.T.