METHOD DEVELOPMENT AND VALIDATION OF TIVOZANIB BY USING RP-HPLC IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: The current investigation was pointed at developing and progressively validating novel, simple, responsive and stable RP-HPLC method for the measurement of active pharmaceutical ingredient of Tivozanib. Methods: A simple, selective, validated and well-defined stability that shows isocratic RP-HPLC methodology for the quantitative determination of Tivozanib. The chromatographic strategy utilized X-bridge phenyl column of dimensions 150x4.6 mm, 3.5 micron, using isocratic elution with a mobile phase of acetonitrile and 0.1 percent formic acid (50:50). A flow rate of 1 ml/min and a detector wavelength of 216 nm utilizing the PDA detector were given in the instrumental settings. Validation of the proposed method was carried out according to an international conference on harmonization (ICH) guidelines. Results: LOD and LOQ for the two active ingredients were established with respect to test concentration. The calibration charts plotted were linear with a regression coefficient of R 2 >0.999, means the linearity was within the limit. Recovery, specificity, linearity, accuracy, robustness, ruggedness were determined as a part of method validation and the results were found to be within the acceptable range. Conclusion: The proposed method to be fast, simple, feasible and affordable in assay condition. During stability tests, it can be used for routine analysis of the selected drugs.


INTRODUCTION
Tivozanib, sold under the brand name Fotivda, is a medication used for the treatment of relapsed [1,2] or refractory advanced renal cell carcinoma (RCC) [3][4][5]. It is an oral VEGF receptor tyrosine kinase inhibitor [6]. The most common side effects include fatigue [7], hypertension [8,9], diarrhea, decreased appetite [10], nausea, dysphonia [11], hypothyroidism [12,13], cough [14], and stomatitis [15]. Tivozanib must not be combined with St. John's Wort, an inducer of the liver enzyme [16,17] CYP3A4. It should not be taken during pregnancy as it is teratogenic [18,19], embryotoxic and fetotoxic in rats. Administration of a single dose of tivozanib with rifampicin, a strong inducer of the enzyme CYP3A4 [20,21], cuts the biological halflife and total exposure (AUC) of tivozanib in half, but has no relevant influence on highest concentrations in the blood. Combination with ketoconazole, a strong CYP3A4 inhibitor, has no relevant effects. The clinical significance of these findings is not known. A quinoline urea derivative, tivozanib suppresses angiogenesis [22,23] by being selectively inhibitory against vascular endothelial growth factor (VEGF) [24,25]. It is designed to inhibit all three VEGF receptors [26,27]. After tivozanib is taken by mouth, highest blood serum levels are reached after 2 to 24 h. The total AUC is independent of food intake. When in the bloodstream, over 99% of the substance are bound to plasma proteins, predominantly albumin. Although the enzymes CYP3A4 and CYP1A1 [28] and several UGTs are capable of metabolising the drug, over 90% circulate in unchanged form. The metabolites are demethylation, hydroxylation and N-oxidation products and glucuronides [29]. The biological half-life is 4.5 to 5.1 d; 79% being excreted via the faeces, mostly unchanged, and 12% via the urine, completely unchanged. Tivozanib is used in form of the hydrochloride monohydrate. The aim of the study is to estimate the pharma ingredient Tivozanib by using RP-HPLC.

Chemicals
Acetonitrile, HPLC-grade formic acid, water were purchased from Merck India Ltd, Mumbai, India. API of Tivozanib standard was procured from Glenmark, Mumbai.

The instrumentation
Waters alliance liquid chromatography (model 2695) monitored with empower 2.0 data handling system and a detector of photo diode array (model 2998) was used for this study [30].

Method optimization
To optimize the chromatographic conditions, different ratios of phosphate buffer and the acetonitrile in the mobile phase with isocratic mode was tested. However the mobile phase composition was modified at each trial to enhance the plate count and also to achieve acceptable retention times. Finally 0.1% formic acid buffer and acetonitrile with isocractic elution was selected because it results in a greater response of active pharmacy ingredient. During the optimization of the method various stationary phases such as C8, C18 and amino, phenyl columns were tested. From these trials the peak shapes were relatively good with X-bridge phenyl column of 150 x 4.6 mm, 3.5 µ with a PDA detector. The mobile phase flow rate has been done at 216 nm in order to obtain enough sensitivity. By using above conditions we get retention time of Tivozanib was about 4.07 min with a tailing factor of 1.21. The number of theoretical plates for Tivozanib was 4257 which indicate the column's successful output the % RSD for six replicate injections was around 1.35%, the proposed approach suggests that it is extremely precise. According to ICH guidelines, the method established was validated.
Till today there are no HPLC methods were reported in the literature. Hence we developed a method for the quantification of Tivozanib. The developed HPLC method was utilized for the estimation of the drug by in vitro method. Different extractions were tried using acetonitrile, methanol, and dimethylformamide.

Preparation of buffer
1 ml of formic acid is dissolved in 1 lt of HPLC grade water and filter through 0.45 µ filter paper.

Chromatographic conditions
The HPLC analysis was performed on reverse phase HPLC system with isocratic elution mode using a mobile phase of acetonitrile and 0.1% formic acid (50:50 v/v) and X-bridge phenyl (150x4.6 mm, 3.5 μ) column with a flow rate of 1 ml/min.

Diluent
Mobile phase was used as diluent.

Preparation of the standard solution
For standard stock solution preparation, add 70 ml of diluents to 13.4 mg of Tivozanib taken in a 100 ml volumetric flask and sonicate for 10 min to fully dissolve the contents and then make up to the mark with diluent. 5 ml of solution is drawn from the above normal stock solution into a 50 ml volumetric flask and diluted up to the level.

Preparation of the sample solution
For sample solution preparation, add 70 ml of diluents to 52.8 mg of Tivozanib sample (each tablet contains 1.34 mg of Tivozanib) taken in a 10 ml volumetric flask and sonicate it for 20 min to fully dissolve the contents and then make up to the mark with diluent. 1 ml of solution is drawn from the above sample stock solution into a 10 ml volumetric flask and diluted up to the level.

RESULTS AND DISCUSSION
The main analytical challenge during development of a new method was to separate active Pharma ingredients. In order to provide a good performance the chromatographic conditions were optimized.

System suitability
In System suitability injecting standard solution and reported USP tailing and plate count values are tabulated in table 1 [35].  In this test method placebo, standard and sample solutions were analyzed individually to examine the interference. The below fig. shows that the active ingredient was well separated from blank and their excipients and there was no interference of placebo with the principal peak. Hence the method is specific.

Linearity
The area of the linearity peak versus different concentrations has been evaluated for Tivozanib, as 10, 25, 50, 75, 100, 125, 150 percent respectively. Linearity was performed in the range of 1.34-20.1µg/ml of Tivozanib. The correlation coefficient achieved was greater than 0.9991.  In this method, Accuracy was conducted in triplicate by analyzing active pharma ingredient sample solution at three kinds of concentration levels of 50, 100 and 150% of each at a specified limit. The percentage recovery was measured and found to be within the limit. The accuracy and reliability of the developed method was established. The percentage recovery values were found to be in the range of 99.65-100.93% for Tivozanib. The results are given in table 3.

Intraday precision
Six replicates of a sample solution containing Tivozanib (13.4 μg/ml) were analysed on the same day. Peak areas were calculated, which were used to calculate mean, SD and %RSD values. These results are given below table 4.

Intermediate precision
Six replicates of the sample solutions were studied by various researchers, and on separate days different instruments were tested.
The peak regions used to determine mean percent RSD values have been calculated. The results are given in the following table.

Inter-day precision
Six replicates of a sample solution containing Tivozanib (13.4 μg/ml) were analysed on a different day. Peak areas were calculated which were used to calculate mean, SD and %RSD values. The present method was found to be precise as the RSD values were less than 2% and also the percentage assay values were close to be 100%. The results are given in table 5 [36].

LOD and LOQ
The LOD concentration for Tivozanib was 0.017 µg/ml and s/n values is 6. The LOQ concentration for Tivozanib was 0.055 µg/ml and the s/n value was 27. The method is validated as per the ICH guidelines [37]. Results of LOD and LOQ were shown in table 6.

Robustness
The conditions of the experiment were designed to test the robustness of established system intentionally altered, such as flow rate, mobile phase in organic percentage in all these varied conditions. Robustness results for Tivozanib found to be within the limit and results are tabulated in table 7 [38].

Stability
The sample solution was kept at room temperature and at 2-8 °C up to 24 h. Then these solutions were pumped into the device and calculate the % of deviation from initial to 24 h [39]. There was no significant deviation observed and confirmed that the solutions were stable up to 24 h percentage of the assay was not quite 2%.
There is no effect in storage conditions for Tivozanib drug. The results are given below table 8.

Degradation studies
The Tivozanib sample was subjected into various forced degradation conditions to effect partial degradation of the drug. Studies of forced degradation have carried out to find out that the method is suitable for products of degradation [40]. In addition, the studies provide details about the conditions during which the drug is unstable, in order that the measures are often taken during formulation to avoid potential instabilities [41].

Acid degradation
Acid degradation was done by using 1N HCl and 15.2% of Tivozanib degradation was observed.

Alkali degradation
Alkali degradation was done at 1N NaOH and 16.5% of Tivozanib degradation was observed.

Peroxide degradation
Peroxide degradation was performed with 30% hydrogen peroxide and 14.8% Tivozanib degradation was observed.

Reduction degradation
Reduction degradation was performed with 30% sodium bi sulphate solution, 13.9% Tivozanib degradation was observed.

Thermal degradation
In thermal degradation the sample was degraded to 12.7% of Tivozanib.

Photo degradation
In photo degradation the sample was degraded to 11.9% of Tivozanib.
All degradation results are tabulated in table 9.

CONCLUSION
We present in this article simple, selective, validated and welldefined stability that shows isocratic RP-HPLC methodology for the quantitative determination of Tivozanib. All the products of degradation formed during the stress conditions and the active pharma ingredient were well separated and peaks were well resolved from each other and separate with an appropriate retention time indicating that the proposed method to be fast, simple, feasible and affordable in assay condition. Therefore the developed method during stability tests, it can be used for routine analysis of production standards and to verify the quality of drug standards during stability studies.