Development and Validation of RP-HPLC Method for the Determination of Pazopanib Hydrochloride (A Tyrosine Kinase Inhibitor)

in Pharmaceutical Dosage Form

 

P. Ravi Sankar*, K. Saisneha Latha, A. Bhavani Sailu, SK. Taheera, B. Madhuri

Department of Pharmaceutical Analysis, Vignan Pharmacy College, Vadlamudi, Guntur - 522 213,

Andhra Pradesh, India.

 

ABSTRACT:

A simple, precise, accurate, highly sensitive, rapid, and reproducible high-performance liquid chromatographic method was developed for the determination of Pazopanib Hydrochloride in bulk and tablet dosage form. Good quality chromatographic separation of Pazopanib Hydrochloride was carried out using Eclipse plus C₁₈ column (4.5mm i.d. X 150mm, 3.5µm particle size) (based on 99.999% ultra-high purity silica) using mobile phase that containing 0.1% Orthophosphoric acid: Acetonitrile (55:45% v\v) at a flow rate of 1.0mL/minute. The λ_max of Pazopanib Hydrochloride in the mobile phase was found to be 271.4nm. It had a retention time of 1.430 min. The calibration curve was in the linear function of the drug in the concentration range of 2-10µg/mL (r² = 0.999) for the HPLC method. The regression equation for Pazopanib Hydrochloride was found to be Y =70095 x + 28022 for HPLC. The Detection Limit (DL) and Quantitation Limit (QL) results of Pazopanib Hydrochloride were found to be 0.167548μg/mL 0.055290µg/mL for HPLC method, respectively. The developed methods are validated in pursuance of ICH Q2 (R1) guidelines. The method was linear, precise, accurate with recoveries in the range of 98 – 102% and minimum values of % RSD indicate the accuracy of the method. The % assay of the Pazopanib Hydrochloride was found to be 99.85±12, which was in good agreement with the labelled claim. The detailed quantitative results of the study show that this method is precise, accurate, cost-effective, and robust. Thus, the developed gradient RP-HPLC method can be successfully practiced for the routine quality control analysis of Pazopanib Hydrochloride in a pharmaceutical formulation.

 

 

 

INTRODUCTION:

The chemical name for Pazopanib hydrochloride is 5-[[4-[(2, 3-dimethylindazol-6-yl)-methyl amino] pyrimidin-2-yl] amino]-2-methylbenzenesulfonamide. It has a molecular formula C₂₁H₂₃N₇O₂S.HCl and a molecular weight of 473.991. Pazopanib hydrochloride is used to treat advanced Kidney cell Carcinoma, and it is also used to treat advanced soft tissue sarcoma that has been treated with other anticancer drugs. Pazopanib hydrochloride works by decreasing the blood supply to the cancer tumor to reduce tumor growth.

 

As per the Literature Survey, it is revealed that the drug has been estimated by LC-MS/MS^1-5, UPLC-Q-TOF/MS⁶, High-Performance Liquid Chromatography -UV^7-8, Ultra Violet^9-10 But only a few UV–Spectroscopic method and Liquid Chromatography analysis have been reported for the estimation in bulk and pharmaceutical dosage forms. Generally, HPLC has proven to be useful in diagnostic purposes and the pharmaceutical  industry^11-12. The aim and objective of the present work were to develop and validate a precise, sensitive HPLC for Pazopanib hydrochloride in its bulk and tablet dosage form. The chemical structure of Pazopanib Hydrochloride is shown in Fig. 1.

 

Fig.1: Chemical Structure of Pazopanib hydrochloride

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Sample of Pazopanib Hydrochloride was gifted from Hetero Labs Ltd., Hyderabad, India. Pazopanib Hydrochloride (Pazopanib Hydrochloride 200mg.) was purchased from a local pharmacy. HPLC grade Methanol, HPLC grade Acetonitrile, HPLC grade Water and AR grade HCl, AR grade Sodium Hydroxide, AR grade Hydrogen Peroxide were purchased from Merck specialties Pvt. Ltd., Mumbai, India. Other excipients were prepared in our laboratory.

 

Instrumentation:

For UV detection of the sample, Elico SL-210 UV spectrophotometer with 1cm matched quartz cells were used for all spectral and absorbance measurements, and solutions were prepared in methanol. For HPLC, the chromatographic system consists of Agilent HPLC quaternary-1260 Infinite- II series, Eclipse plus C₁₈ column, 1260 series with diode array detector was used for higher data quality for more confidence. For homogenizing the solution prepared, Ultra-Sonicator of Spectral labs, model UCB 40, was used. For the weighing of the sample and excipients, radwag analytical balance AS 82/220.R₂) (0.001g), was used. For measuring the pH of the prepared solutions, pH meter, Systronics model – 802 was used. The data was acquired and processed by utilizing EZ chrome elite software.

 

Method development and optimization of chromatographic conditions:

For HPLC development, a variety of mobile phases containing HPLC grade water, acetonitrile, methanol in different ratios with or without buffers, and also various flow rates were performed. A good symmetrical peak was found when the mobile phase containing a mixture of 0.1% Orthophosphoric acid: Acetonitrile (55:45% v\v)

 

Selection of detection wavelength:

To estimate the maximum λ_max, Pazopanib Hydrochloride 100μg/ml of working standard solution was prepared and scanned in a UV wavelength range of 200 - 400nm utilizing as a blank. It was observed that the drug showed maximum absorbance at 273.0nm which was chosen as the detection wavelength for the determination of Pazopanib Hydrochloride. The overlay spectrum of Pazopanib Hydrochloride is shown in Fig. 2.

 

Fig. 2: Overlay Spectrum of Pazopanib Hydrochloride for UV method

 

Preparation of the mobile phase:

The mobile phase was prepared by mixing 0.1% Orthophosphoric acid and acetonitrile in the proportion of 55: 45 v/v. The prepared mobile phase was filtered through a 0.45µm nylon membrane filter and degassed by sonication.

 

Preparation of Stock and Working standard solution:

Precisely weighted 100mg of Pazopanib Hydrochloride was transferred into a 100mL volumetric flask, dissolved and diluted up to the mark with mobile phase to get a stock solution containing 1.0mg/mL of Pazopanib Hydrochloride. Aliquots of stock solution were diluted with the mobile phase to attain the standard calibration solutions over the range of 2, 4, 6, 8, and 10µg/mL.

 

Preparation of sample solution:

For the assay of pharmaceutical formulation, 20 tablets of Pazopanib Hydrochloride marketed formulation (Votrient 200mg) were weighed, the average weight was calculated, and a quantity of tablet powder equivalent to 100mg of Pazopanib Hydrochloride was accurately weighed and transferred into a 100mL volumetric flask containing 30mL of the mobile phase. The solution was ultrasonicated for about 15 minutes, filtered through a What man filter paper (0.45µm) nylon filter, and the filtrate was made up to volume with the mobile phase. The concentration was 1mg/mL. Transfer 1ml of the filtered sample solution to 10mL volumetric flask and made up to volume with mobile phase to get a solution of 100µg/mL. The solution is further diluted with the mobile phase to obtained the required concentrations. This is used as a working solution for the preparation of the assay. Then 0.2ml of this solution is transferred into a 10ml volumetric flask and made up to volume to obtain 2µg/mL, which is used for the assay. Solutions were injected as per the above chromatographic conditions, and peak areas were recorded. The quantifications were carried out by keeping these values to the straight-line equation of the calibration curve. The assay results are presented in Table 4.

 

Method development optimization:^13-16

The optimized HPLC conditions of several mobile phases with different compositions were tested to develop an optimization of chromatographic conditions like tailing factor, peak shape, and the number of theoretical plates. For the selection of the mobile phase, primarily methanol: acetonitrile, ethanol: water, acetonitrile: water has been tested for different compositions. Eventually, the gradient mode and mobile phase containing a mixture of 0.1% Orthophosphoric acid: Acetonitrile (55:45 % v/v) at a flow rate of 1mL/ minute was found to be satisfactory and proper system suitability parameters obtained. Optimized chromatographic conditions, system suitability parameters for estimation of Pazopanib Hydrochloride by the proposed gradient RP-HPLC method are depicted in Table 1.

 

Table1: Optimized chromatographic conditions and SSP for the proposed HPLC method

Parameter	Chromatographic conditions
Instrument	Agilent HPLC quaternary-1260 Infinite- II series
Column	Eclipse plus C18 column (4.5 mm i.d. X 150 mm, 3.5 µm particle size) (based on 99.999 % ultra high purity silica)
Detector	1260 Diode Array Detector.
Mobile phase	0.1 % Orthophosphoric acid: Acetonitrile (55:45 %v\v).
Flow rate	1 mL/minute
Detection wavelength	UV at 271.4nm
Run time	5 minutes
Temperature	Room temperature (25 oC).
The volume of the injection loop	10µL
System suitability parameters (SSP)
Retention time (Rt)	1.403 minutes
Theoretical plates [th.pl] (Efficiency)	3848
Theoretical plates per meter [t.p/m]	73112
Tailing factor (asymmetry)	1.122

 

METHOD VALIDATION:

Validation is a process of establishing documented evidence, which provides a high degree of assurance that a specific activity will consistently produce a desired result or product meeting its pre-determined specifications and quality characteristics. The method was validated as per ICH guidelines¹⁷.

 

System suitability:

System suitability parameters can be defined as tests to ensure that the method can generate results of acceptable accuracy and precision. The requirements for system suitability are usually developed after method development and validation has been completed. The system suitability parameters like theoretical plates, retention time, tailing factor, were studied and found satisfactory.

 

Precision:

The precision of the assay was investigated by measurement of both repeatability and intermediate precision.

 

Repeatability

For RP-HPLC, six replicate injections of standard and blank were injected into the HPLC system. The % RSD values of the standard Pazopanib Hydrochloride (10µg/mL) are determined.

 

Method precision and Intermediate precision:

For method precision, six test preparations were analyzed as per the methodology representing a single batch, and the assay was determined for the same. The % RSD for assay of six test preparations should not be more than 2.0. The results are well within acceptance criteria, and the % RSD observed for assay values indicates the precision of the method. In the intermediate precision (Intra-day studies), standard and sample solutions prepared as described above were analyzed in triplicate on the three consecutive days at 100% of the test concentration, and the percentage of RSD was calculated. System precision, Method precision and Intermediate precision is tabulated in Table 3. Precision at different levels of the analytical method was determined in the concentration range of 50%, 100%, 150% levels, and eventually % of RSD was calculated.

 

Linearity:

The linearity of Pazopanib Hydrochloride was determined in the concentration range of 2 to 10µg/mL. According to ICH recommendations, at least five concentrations were chosen in the ranges 2-10µg/ml. The calibration curve of Pazopanib Hydrochloride is shown in Fig. 3.

 

Fig. 3: Calibration graph of Pazopanib Hydrochloride by RP- HPLC

 

Accuracy (Recovery studies):

Accuracy was assessed using nine determinations over three concentration levels covering the specified range (80, 100 and 120%). Accuracy was reported as percent recovery by the assay of the known added amount of analyte in the sample is shown in Table 3.

 

Robustness:

Robustness of an analytical procedure is a measure of its capacity to remain unaffected by small variations in method parameters and provides an indication of its reliability during normal usage. Robustness was tested by studying the effect of changing mobile phase composition, flow rate±0.2ml/min detection wavelength ± 5nm, had no significant effect on the chromatographic resolution of the method. The robustness of the analytical method was established by demonstrating its reliability against deliberate changes in the chromatographic conditions.

 

Stability of analytical solution:

Also, as part of the evaluation of robustness, solution stability was evaluated by monitoring the peak area response. The Pazopanib solutions were found to be stable for three days at 5°C and for a day at room temperature.

 

Table. 2: Robustness results of Pazopanib Hydrochloride.

Parameters	Optimized	Used	Retention time (min)	Plate count $	Peak asymmetry #	Remarks
Flow rate (± 0.2 mL/min)	1.0 mL/min	0.8 mL/min	1.510 min	4756	1.249	*Robust
		1.0 mL/min	1.403min	3848	1.127	*Robust
		1.2 mL/min	1.250min	2736	1.016	*Robust
Detection wavelength (± 5 nm)	271nm	260 nm	1.403min	3847	1.249	*Robust
		271nm	1.430min	3848	1.127	*Robust
		270 nm	1.403min	3846	1.016	*Robust
Mobile phase composition 0.1% orthophosphoric acid: ACN (55:45%v/v)	55:45 v/v	55:45v/v	1.403min	3848	1.127	*Robust
		50:50 v/v	1.403 min	3847	1.016	*Robust

Acceptance criteria (Limits): ^#Peak Asymmetry < 1.5, ^$ Plate count > 2000, * Significant change in Retention time

 

Table 3. Summary of the RP- HPLC of Sorafenib validation parameters

 

LOD and LOQ:

Limit of Detection is the lowest concentration in a sample that can be detected but not necessarily quantified under the stated experimental conditions. The limit of Quantitation is the lowest concentration of an analyte in a sample that can be determined with acceptable precision and accuracy. According to the ICH recommendations, the determination of limits of detection and quantitation was based on the standard deviation of the y-intercepts of regression lines and the slope of the calibration plots.

 

Analysis Pazopanib Hydrochloride in tablet formulation:

The proposed and validated method was successfully applied for the determination of Pazopanib Hydrochloride in their tablet dosage form. Six replicate determinations were performed. The assay result of Table 4 shows that satisfactory results were obtained for Pazopanib HCl in good agreement with label claim. The standard chromatogram of Pazopanib Hydrochloride is represented in Fig.4 and the representative sample chromatogram of Pazopanib Hydrochloride is shown in Fig. 5.

 

Table 4: Assay results of Pazopanib Hydrochloride by HPLC

S. No.	Formulation	Labeled claim	Amount found*	Mean % recovery ± SD	% RSD
1	Pazopanib Hydrochloride tablets (Votrient)	200 mg/tablet	199.7 mg/tablet	99.85 ± 12	1.12

*Average of six determinations, SD denotes standard deviation; RSD denotes % relative standard deviation

 

RESULTS AND DISCUSSION:

Since the above mentioned Pazopanib Hydrochloride is relatively polar, an RP-HPLC method was used. The column for the separation was a C₁₈ column that has an internal diameter of 4.5mm, length of 150mm, and 3.5µ particle size. The number of trials was performed using various buffer solutions with various compositions of methanol, ethanol, acetonitrile, and HPLC grade water and variable flow rates. Eventually, the optimum separation was obtained with a mixture of 0.1% Orthophosphoric acid: Acetonitrile (55:45 %v/v). The mobile phase flow rate was adjusted at 1mL/min, and the detection wavelength was set at 271.4nm. Thus, a proper chromatographic peak was obtained with excellent symmetry and least peak tailing. Overlay spectra of Pazopanib Hydrochloride is shown in Fig.2

 

System suitability was conducted as per the methodology system suitability solution, and six replicate of standard preparation was injected into HPLC. The tailing factor was found to be 1.122. The number of theoretical plates was 3848; the number of theoretical plates per meter was 73,112. The retention time was found out to be 1.430 minutes, and the %RSD was calculated to 0.112. The results were well within the acceptance criteria, and the study concludes the suitability of the analytical system for analysis.

 

The precision of the method was examined by using System precision, Method, and Intermediate precisions. Various levels of concentration were taken in six replicate samples. For Method and Intermediate precisions, the %RSD was found to be 0.540878 and 0.606061. The %RSD of the System precision was found to be 0.011683. The precision at different levels was mentioned in Table The results are well within the acceptance criteria, and the %RSD observed for the replicate injections indicates the precision of the HPLC used, assay values indicate the precision of the method.

 

The linearity of Pazopanib Hydrochloride was determined in the concentration range of 2µg/mL to 10 µg/mL of the test concentration. The squared correlation coefficient value was found to be 0.999, which is well within the limit. To determine the accuracy of the Pazopanib Hydrochloride, the drug was spiked with a placebo at three different levels in triplicate preparations. The mean % recovery at each level was found out to be within limits, i.e., 98.0% to 102.0%

 

The robustness of the HPLC was determined for the suitability and assay value under multiple variable conditions like Flow rate change, Wavelength change, and change in mobile phase composition. The results are mentioned in Table 2.

 

The Limit of Detection and Limit of Quantitation of Pazopanib Hydrochloride were found out to be 0.167548 μg/mL and 0.055290μg/mL, respectively.

 

The % assay of the Pazopanib Hydrochloride was found to be 99.85±12, which was in good agreement with the labelled claim. The method was specific and has no interference observed when the Pazopanib Hydrochloride were determined in the presence of excipients. The Summary of the RP- HPLC of Sorafenib validation parameters is shown in Table 3.

 

Fig. 4: Standard chromatogram of Pazopanib Hydrochloride (10 µg/mL)

 

Fig.5: Sample chromatogram of Pazopanib Hydrochloride (Votrient)

 

CONCLUSION:

The present study demonstrated a validated Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the estimation of Pazopanib Hydrochloride available as the tablet dosage form. The scope of the present work is to build up the linearity and optimization of the chromatographic conditions, to develop the RP-HPLC method for the estimation of the drug in the tablet dosage form. The method was completely validated and showed satisfactory results. The method was free from the interference of the other active ingredients and additives used in the formulation. The RP-HPLC method for the estimation of Pazopanib Hydrochloride has various advantages like less solvent consumption, low retention time, good peak symmetry, precise, accurate, and robust. The results of the study indicate that the developed method was found to be accurate, precise, linear, sensitive, simple, economical, and reproducible, which has a short run time, which makes the method rapid. Hence it can be concluded that this method may be employed for the routine quality control analysis of Pazopanib Hydrochloride in active pharmaceutical preparations.

 

ACKNOWLEDGEMENT:

The authors are thankful to Hetero Labs for providing the sample of Pazopanib Hydrochloride. We are very grateful to Dr. L. Rathaiah, esteemed chairman of the Vignan group of institutions, Vadlamudi, Guntur, for providing us the necessary laboratory facilities to carry out the present investigation.

 

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

 

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Accepted on 12.05.2020           © RJPT All right reserved

Research J. Pharm. and Tech 2021; 14(3):1549-1554.