INTRODUCTION:

Regorafenib (BAY 73-4506, Commercial name Stivarga) is an oral multi-kinase inhibitor developed by Bayer which targets angiogenic, stromal and oncogenic receptor tyrosine kinase (RTK). Regorafenib is an orally-administered inhibitor of multiple kinases. It is used for the treatment of metastatic colorectal cancer and advanced gastrointestinal stromal tumors. FDA approved on September 27, 2012. Approved use of Regorafenib¹ was expanded to treat Hepatocellular Carcinoma in April, 2017.

Regorafenib is indicated for the treatment of patients with metastatic colorectal cancer (CRC) who have been previously treated with fluoropyrimidine-, oxaliplatin-and irinotecan-based chemotherapy, an anti-VEGF therapy, and, if KRAS wild type, an anti-EGFR therapy. Regorafenib is also indicated for the treatment of patients with locally advanced, unresectable or metastatic gastrointestinal stromal tumor (GIST) who has been previously treated with imatinib mesylate and sunitinib malate.

Regorafenib is a small molecule inhibitor of multiple membrane-bound and intracellular kinases involved in normal cellular functions and in pathologic processes such as oncogenesis, tumor angiogenesis, and maintenance of the tumor microenvironment. In in vivo models, regorafenib demonstrated anti-angiogenic activity in a rat tumor model, and inhibition of tumor growth as well as anti-metastatic activity in several mouse xenograft models including some for human colorectal carcinoma.

The IUPAC²Name for Regorafenib is 4-[4-({[4-chloro-3-(Trifluoro methyl) phenyl] carbamoyl} amino)-3-fluorophenoxy]-N-methylpyridine-2-carboxamide. The molecular formula³ of Regorafenib is C₂₁H₁₅ClF₄N₄O₃. The molecular weight is 482.815 g/mol. The chemical structure⁴of Regorafenib shown in following Fig-1.

Fig-1: Structure of Regorafenib

MATERIALS AND METHODS:

Experimental:

Pharmaceutical grade working standard Regorafenib was obtained from Syncorp Clincare Technologies Pvt. Ltd. Laboratories, Hyderabad, India. All chemicals and reagents were HPLC grade and were purchased from S D Fine-Chem Limited and Loba Chemie Pvt. Ltd, Mumbai, India.

Instrumentation:

The analysis was performed using HPLC (Waters-717 series) with UV detector and data handling system Empower software, UV-Visible double beam spectrophotometer (Labindia), analytical balance 0.1mg Sensitivity (Labindia), pH meter (Labindia), Vaccum filtration, Ultra sonicator. The column used is Symmetry C18 Column, 250 mm x 4.6 mm i.d. and 5µm particle sizewith the flow rate 1.0ml/min (isocratic).

Preparation of Phosphate buffer:

About 6.8 grams of Potassium dihydrogen orthophosphate was weighed and transferred into a 1000ml beaker, dissolved and diluted to 1000ml with HPLC water. The pH⁵ was adjusted to 4.80 with Orthophosphoric acid.

Preparation of mobile phase:

Mobile phase was prepared by taking Methanol: phosphate buffer (pH-4.80) (70:30 v/v). Mobile phase was filtered⁶ through 0.45 mm membrane filter and degassed under ultrasonic bath prior to use. The mobile phase was pumped⁷ through the column at a flow rate of 1.0 ml/min.

Standard Preparation for the Analysis:

25 mg of Regorafenib working standard was transferred into 25 ml volumetric flask, dissolved and make up to volume with mobile phase.

Further dilution⁸ was done by transferring 0.1 ml of the above solution into a 10ml volumetric flask and make up to volume with mobile phase.

Sample Preparation for the Analysis:

Twenty tablets were taken and the I.P. method was followed to determine the average weight. Finally the weighed tablets are powdered and triturated well by using mortar and pestle. A quantity of powder which is equivalent to the 100mg of drug was transferred to a clean and dry 100ml of volumetric flask and add 70 ml of diluent and the resulted solution was sonicated for 15 minutes by using ultra Sonicator. Then the final volume was make up to the mark with the same diluent. The final solution was filtered through a selected membrane filter (0.45 µm). From this above stock solution (1 ml) was transferred to five different 10 ml volumetric flask and volume was made up to 10ml with same solvent system. The prepared solution was injected in six replicates into the HPLC system and the observations were recorded.

Diluent:

Mobile phase can be used as diluent.

Study of Spectra and selection of wavelength:

Regorafenib working standard solution was scanned between the range 200-400 nm in 1cm cell against blank. Maximum absorbing wavelength of Regorafenib was selected from spectral data and wavelength selected from spectra of UV spectrophotometer. The λmax for Regorafenib was found to be 268nm. UV spectrum and typical standard chromatogram of Regorafenib are shown in Fig-2.

Optimization of HPLC Method:

The chromatographic conditions⁹ were optimized by different means. (Using different column, different mobile phase, different flow rate, different detection wavelength and different diluents for sample preparation etc. The selected and optimized mobile phase was Methanol: Potassium dihydrogen orthophosphate buffer (pH-4.80) (70:30v/v) and conditions optimized were: flow rate (1.0 ml/minute), wavelength (268 nm, UV-detector), run time was 7.0 mins and injection volume was 20 µl.

Method Validation

Recovery study:

To determine the accuracy of the proposed method, recovery studies were carried out by adding different amounts (80%, 100%, and 120%) of pure drug of Regorafenib were taken and added to the pre-analyzed formulation of concentration 10mg/ml. From that percentage recovery values¹⁰were calculated. The results were shown in Table-1.

Precision:

Repeatability:

The precision of each method was ascertained separately from the peak areas and retention times obtained by actual determination of six replicates of a fixed amount of drug. Regorafenib (API). The percent relative standard deviation¹¹ was calculated for Regorafenib are presented in the Table-2.

Intermediate precision:

Intra-assay and inter-assay:

The intra and inter day variation of the method was carried out and the high values of mean assay and low values of standard deviation and % RSD (%RSD<2%) within a day and day to day variations¹² for Regorafenib revealed that the proposed method is precise.

Linearity and Range:

The calibration standard solution of Regorafenib was injected into the HPLC system and the chromatograms were recorded at 268nm and a calibration graph was obtained by plotting peak area versus concentration of Regorafenib. The linearity data is presented in figure-3 and table-4.

Method Robustness:

Influence of small changes in chromatographic conditions such as change in flow rate (±0.1ml/min), Temperature (±2⁰C), Wavelength of detection (±2nm) and Acetonitrile content in mobile phase (±2%) studied to determine the robustness¹³ of the method are also in favour of (Table-5, % RSD<2%) the developed RP-HPLC method for the analysis of Regorafenib (API).

LOD and LOQ:

The minimum concentration of the analyte can be used to detect the sample by using all experimental conditions and the minimum concentration of the analyte can be used to quantify the sample by using all experimental conditions

The LOD and LOQ were calculated by the use of the equations

LOD=3.3×σ/S

and

LOQ=10×σ/S

Where,

σ is the standard deviation of intercept of Calibration plot and S is the average of the slope of the corresponding Calibration plot.

System Suitability Parameter:

System suitability testing is an integral part of many analytical procedures. The tests are based on the concept that the equipment, electronics, analytical operations and samples to be analyzed constitute an integral system that can be evaluated as such. Following system suitability¹⁴ test parameters were established. The data are shown in Table-6.

Estimation of Regorafenib in Tablet Dosage Form:

Label claim: each tablet contains 40 mg

Twenty tablets were taken and the I.P. method was followed to determine the average weight. Above weighed tablets were finally powdered and triturated well. A quantity of powder equivalent to 25 mg of drugs were transferred to 25 ml volumetric flask, make and solution was sonicated for 15 minutes, there after volume was made up to 25 ml with same solvent. Then 10 ml of the above solution was diluted to 100 ml with mobile phase. The solution was filtered through a membrane filter (0.45 mm) and sonicated to degas. The solution prepared was injected in five replicates into the HPLC system and the observations were recorded.

A duplicate injection¹⁵ of the standard solution was also injected into the HPLC system and the peak areas were recorded. The data are shown in Table-7.

ASSAY:

AT WS DT P

Assay % =----- X -------- X ------- X -------- X Avg. Wt.

AS DS WT 100

= mg/ Tab

Where:

AT=Peak Area of drug obtained with test preparation

AS=Peak Area of drug obtained with standard preparation

WS=Weight of working standard taken in mg

WT=Weight of sample taken in mg

DS=Dilution of Standard solution

DT=Dilution of sample solution

P=Percentage purity of working standard

RESULTS AND DISCUSSION:

Optimized method:

To develop a new, simple, accurate, precise, robust and isocratic RP-HPLC method has been developed and subsequently validated for the determination of Regorafenib in pure form and pharmaceutical dosage forms as per ICH guidelines. The separation achieved on a Symmetry C₁₈ Column, 250 mm x 4.6 mm i.d. and 5µm particle size column as a stationary phase and Methanol: Phosphate buffer (pH adjusted to 4.80 with phosphoric acid) in the ratio of 70:30v/v used as mobile phase at a flow rate of 1.0 ml/min. The UV detection was performed at 268nm. The retention time for Regorafenib was found to be 3.544 minutes.

Method Validation:

Accuracy:

Table-1: Accuracy Readings

Sample ID	Concentration (mg/ml)		Peak Area	% Recovery of Pure drug	Statistical Analysis
Sample ID	Amount Added	Amount Found	Peak Area	% Recovery of Pure drug	Statistical Analysis
S₁ : 80 %	8	8.069	485317	100.862	Mean=100.5413% S.D.=0.947606 %R.S.D.=0.942503
S₂ : 80 %	8	7.958	478751	99.475
S₃ : 80 %	8	8.103	487312	101.287
S₄: 100 %	10	10.048	601947	100.48	Mean=100.2367% S.D.=0.650103 % R.S.D.=0.648568
S₅ : 100 %	10	10.073	603395	100.73
S₆ : 100 %	10	9.950	596176	99.50
S₇: 120 %	12	11.985	716127	99.875	Mean=100.3607% S.D.=0.555257 % R.S.D.=0.553262
S₈ : 120 %	12	12.116	723840	100.966
S₉ : 120 %	12	12.029	718706	100.241

Precision

Repeatability:

Table-2: Repeatability Readings

HPLC Injection Replicates of Regorafenib	Retention Time	Peak Area
Replicate–1	3.545	661022
Replicate–2	3.537	683137
Replicate–3	3.543	671941
Replicate–4	3.538	682245
Replicate–5	3.542	671941
Replicate–6	3.550	692444
Average	3.5425	677121.7
Standard Deviation	0.004764	11046.13
% RSD	0.134494	1.631336

Intermediate Precision:

Intra-Assay and Inter-Assay:

Table-3: Results of intra-assay and inter-assay

Conc. Of Regorafenib (API) (µg/ml)	Observed Conc. Of Regorafenib (µg/ml) by the proposed method
	Intra-Day		Inter-Day
	Mean (n=6)	% RSD	Mean (n=6)	% RSD
8	7.76	0.82	8.28	0.98
10	10.16	0.42	9.59	0.23
12	11.68	0.13	12.19	0.33

Linearity and Range:

The calibration curve showed good linearity¹⁶ in the range of 0–16 µg/ml, for Regorafenib (API) with correlation coefficient¹⁷ (r²) of 0.999 (Fig-4). A typical calibration curve has the regression equation of y=58945x+9634 for Regorafenib.

Fig-3: Calibration curve of Regorafenib (API)

Table-4: Linearity Results

CONC.(µg/ml)	MEAN AUC (n=6)
0	0
6	370200
8	490231
10	602707
12	717538
14	829248
16	947852

Method Robustness:

Table-5: Result of method robustness test

Change in parameter	% RSD
Flow (1.1 ml/min)	0.52
Flow (0.9 ml/min)	0.56
Temperature (27⁰C)	0.52
Temperature (23⁰C)	0.49
Wavelength of Detection (270 nm)	0.97
Wavelength of detection (266 nm)	0.98

LOD and LOQ:

The Minimum concentration level at which the analyte can be reliable detected (LOD) and quantified ¹⁸ (LOQ) were found to be 0.90 and 2.90 µg/ml respectively.

System Suitability Parameter

Table-6: Data of System Suitability Parameter

S. No.	Parameter	Limit	Result
1	Resolution	Rs>2	9.41
2	Asymmetry	T£2	Regorafenib=0.25
3	Theoretical plate	N>2000	Regorafenib=3985

Estimation of Regorafenib in Tablet Dosage Form

Table-7: Recovery Data for estimation Regorafenib in Nublexa Tablets

Brand name of Tablets	Labelled amount of Drug (mg)	Mean (±SD) amount (mg) found by the proposed method (n=6)	Assay % (±SD)
Nublexa (40mg) (Silverline Medicare Private Limited)	40	39.91 (±0.498)	99.51 (±0.343)

The amount of drug found in Regorafenib Tablets was found to be 39.91 (±0.498) mg/tab for Regorafenib and % assay¹⁹ was 99.51 (±0.343).

CONCLUSION:

The result shows that the developed method is a sensitive and selective RP-HPLC method has been developed and validated for the determination of Regorafenib in pure form and Pharmaceutical dosage form. The UV detection was performed at 268nm. The Retention time of Rilpivirine was found to be 3.544minutes. The Percentage Standard Deviation (%RSD) of the Rilpivirine was and found to be 1.63%. The detector response was linear in the concentration range of 0-16µg/ml. The respective linear regression equation being Y=58945.x+9634 with R2=0.999. The percentage recovery values of Regorafenib in pharmaceutical dosage form were found to be within the limits. The limit of detection and the limit of quantification for Regorafenib were found to be 0.90µg/ml and 2.90µg/ml respectively. Further the proposed RP-HPLC method has excellent sensitivity, precision and reproducibility, accuracy. The result shows the developed method is yet another suitable method for assay, purity which can help in the analysis of Regorafenib in different marketed formulations. The developed method was validated as per ICH guidelines for accuracy calculated as % recovery was in the range of 98.0% to 102.0%. The statistical analysis of the data showed that the method is reproducible and selective for the estimation of Regorafenib in pure form and marketed Pharmaceutical dosage form during routine analysis. The results of the study showed that, the proposed RP-HPLC method was simple, rapid, precise, accurate which can be used for the routine determination of Regorafenib in pure form and marketed pharmaceutical dosage forms.

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Received on 12.06.2018 Modified on 18.06.2018

Asian J. Research Chem. 2018; 11(4):763-767.

DOI: 10.5958/0974-4150.2018.00134.7