Development and Validation of rp-HPLC Method of Cabozantinib in Active Pharmaceutical Ingredient and Pharmaceutical Dosage form

A specific, accurate rp-HPLC (reversed-phase high performance liquid chromatographic) method was developed for the quantification of Cabozantinib. The effective separation was achieved through reversed-phased C18 column 4.6 x 250 mm, 5μm using a mobile phase Methanol: phosphate buffer (ph. 3.00) with orthophosphoric acid (OPA) (55:45 % v/v). The flow rate of the mobile phase was found to be 0.8 mL/min. The detection was carried at a wavelength of 244 nm. The retention time of Cabozantinib was found to be 3.702 min. The correlation coefficient was found to be 0.9999. The developed method was accurately validated in the terms of accuracy, linearity range, precision, system suitability, robustness, limit of detection and limit of quantification. The details presented in this test will be useful for industrial application for determining Cabozantinib in active pharmaceutical ingredient and pharmaceutical dosage form.


Preparation of Solution
Preparation of mobile phase: Methanol and phosphate buffer with OPA: taken in the ratio 55:45. 0.01N Phosphate Buffer: Accurately weighed 1.36 gm of Potassium dihydrogen Orthophosphate in a 1000ml of volumetric flask add about 900 ml of milli-Q water added and degas to sonic ate and finally made up the volume with water then pH adjusted to 3.32 with dil. Orthophosphoric acid solution. 10 Mm Na2Hpo42 h20 (1.7799 gm in 1000 ml water) ph adjust 3 with OPA Preparation of diluent: Mobile Phase (Methanol and phosphate buffer with OPA: taken in the ratio 55:45) is used as diluent.
Preparation of standard stock solution: Accurately weighed 10 mg Cabozantinib working standard was transferred into 10 mL volumetric flasks. Drug was dissolved in 10 mL diluent and diluted up to mark with diluent, to achieve final concentration of 1000 µg/mL. Label it as stock solution 1. Preparation of sample solution: 20 tablets (COMETRIQ) were triturated and weigh their average weight. The total weight of 20 powered tablets was 4.184gm and the average powder weight is 0.2092 gm/tablet. The tablet powder equivalent to 10 mg (i.e., 10×209.2÷80 = 26.15mg) is 26.15mg. Take 26.15mg of tablet powder into a 1000 mL volumetric flask and dissolved in 10 ml diluent to produce 1000µgm/ml of Cabozantinib. Sonicate for 20 min and then the solution was filtered through 0.45 µm membrane filter and the residues were washed thoroughly with diluent. The filtrate and washings were combined in a 1000 mL volumetric flask and diluted to the mark with diluent to get a final concentration of 1000 μg/mL of Cabozantinib. The concentration of sample solution was found from regression equation of CABO. Label it as stock solution 2.

Method Validation
The developed rp-HPLC method was accurately validated as per ICH guidelines in terms of accuracy, linearity and range, precision, system suitability, robustness, limit of detection and limit of quantification.

Linearity and Range
In the chromatographic technique, linearity produces test result which is directly proportionate to the concentration of an analyte within the given range. The range refers to the interval between the upper limit and the lower limit of the analytes present in the samples. The linearity of Cabozantinib has the range of 10-60µg/ml. The chromatograms and the area for each concentration were recorded. The correlation coefficient was found to be 0.9999.

Method precision (% repeatability)
The repeatability studies were carried out by determining the response of concentration of Cabozantinib (20 µg/ml). The result was evaluated in terms of relative standard deviation (%RSD).

Intermediate precision (reproducibility)
The intra-day and inter-day precision (intermediate precision) were carried out by determining the responses three times on the same day and the different days of three different concentration of Cabozantinib (20, 30, 40 µg/ml). The results were evaluated in terms of relative standard deviation (%RSD).

Accuracy (recovery study)
Recovery studies (% recovery) were determined to validate the accuracy of developed technique. To pre-analyzed tablet solution, a definite concentration standard drug (80%, 100%, and 120%) was added and then its recovery was determined. Statistical validation of recovery studies was carried out.

Robustness
The robustness of a method is the ability of a method to resist change without adapting its initial stable configuration. The robustness of a method was carried out by deliberately changing the experimental conditions like flow rate and wavelength.

System Suitability
The determination of system suitability was significant part of the method development to evaluate the suitable adequate performance of the chromatography system. The determination of system suitability was carried out before validation runs. Retention time, Theoretical Plates, Tailing Factor were evaluated.

Limit of detection (LOD)
The LOD is the lowest limit that can be detected. The LOD can be determined by the Standard Deviation (SD) of the response and the slope (S).

Limit of quantitation
The LOQ is the lowest concentration that can be quantitatively measured. The LOQ can be determined by the Standard Deviation (SD) of the response and the slope (S).

Analysis of Tablet Formulation
Pipette out 0.3ml from the above stock solution 2 into a 10 ml volumetric flask and dilute up to the mark with the mobile phase. The accurate chromatogram of test Cabozantinib is shown in (Fig. 2). The amount of Cabozantinib per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated two times with tablet formulation. Tablet Assay for % Label claim and for % RSD is calculated.

Linearity and Range
The linearity was evaluated for concentration 10 60 µg/ml. The correlation coefficient was found to be 0.9999. The linearity results were performed in the Table 1 and co-relation graph is shown in Figs. 2,3.

Analysis of Tablet Formulation
Pipette out 0.3ml from the above stock solution 2 into a 10 ml volumetric flask and dilute up to the mark with the mobile phase. The accurate chromatogram of test Cabozantinib is shown in . The amount of Cabozantinib per tablet were calculated by extrapolating the value of area from the calibration curve. Analysis procedure was repeated two times with tablet formulation. Tablet Assay for % Label claim and

AND DISCUSSION
The linearity was evaluated for concentration 10-60 µg/ml. The correlation coefficient was found to be 0.9999. The linearity results were performed relation graph is shown in

Precision
Method Precision (% Repeatability): The RSD value for repeatability was found to be 0.08% ( Table 2). The relative standard deviation (RSD) (less than 2%) indicates that the determined method is repeatable. Chromatograph of precision is shown in Fig. 4.
Intermediate Precision (Reproducibility): The RSD of inter-day was found to be 0.05-0.17% and the RSD of intra-day was found to be 0.06-0.44%. The results indicates that the determined method is precise. (Table 3,4)

Accuracy
The % mean recovery of accuracy was found to be 99.97-100.06 %. (Table 5). The results of accuracy indicates that the determined method is accurate. (Figs. 5,6,7)

Robustness
The mobile phase composition was changed in (±1 ml/min1) proportion, the flow rate was varied by (±1ml/min-1), and wavelength change (±2 ml/min-1) of optimized chromatographic condition. The results of robustness studies are shown in (Table 6). Robustness studies were also found satisfactory and hence the analytical method would be concluded.

System Suitability
The retention time was found to be 3.702min, the theoretical plates was found to be 3663 and the tailing factor was found to be 0.72.
Limit of Detection (LOD) and Limit of Quantitation (LOQ): The LOD and LOQ were determined theoretically. The LOD was found to be 0.2085 (µg/ml) and the LOQ was found to be = 0.6321 (µg/ml).    accurate, and precise. (Fig 8)   Fig. 7. Chromatogram of accuracy (120%

CONCLUSION
A simple, linear, accurate and precise HPLC method was developed and validated for the analysis of Cabozantinib in active pharmaceutical ingredient and pharmaceutical dosage form. The results of rp-HPLC method development and its validation indicates that the method carried out is accurate and precise. The system developed could be used effectively for the purpose of analysis and quality control of the pharmaceutical dosage type.

DISCLAIMER
The products used for this research are commonly and predominantly use products in our area of research and country. There is absolutely no conflict of interest between the authors and producers of the products because we do not intend to use these products as an avenue for any litigation but for the advancement of knowledge. Also, the research was not funded by the producing company rather it was funded by personal efforts of the authors.

CONSENT
It is not applicable.

ETHICAL APPROVAL
It is not applicable.