INTRODUCTION:
Tovorafenib
Tovorafenib, sold under the brand name Ojemda, is a medication used for the treatment of glioma. It is a kinase inhibitor.¹ Chemically Tovorafenib (r)-2-(1-(6-Amino-5-chloropyrimidine-4-carboxamido)ethyl)-n-(5-chloro-4-(trifluoromethyl)pyridin-2-yl)thiazole-5-carboxamide. It is also be spelled as "Ojemda".²-⁵
It is available in the Indian market as tablet dosage form in the brand names Ojemda.⁶ Tovorafenib is a medicine used to treat the symptoms of Glioma.⁷ It is Highly soluble in Dimethylsulfoxide.⁸ Mode of action Tovorafenib is an orally available inhibitor of wild-type and certain mutant forms of A-Raf, B-Raf and C-Raf protein kinases, with potential antineoplastic activity. Upon administration, tovorafenib inhibits Raf-mediated signal transduction pathways, which may lead to an inhibition of tumor cell growth.⁹ Literature survey reveals that only Liquid chromatography and Mass Chromatography methods has been developed for estimation of Tovorafenib so there is no UV- Visible spectroscopic method available to estimate either from bulk drug or from the pharmaceutical dosage forms. The present investigation was proposed to estimate Tovorafenib in bulk and pharmaceutical dosage form by UV-Visible spectroscopic method which is rather simple, sensitive, specific, precise, accurate method. The present work was to develop and validate as per ICH guidelines so the method was developed validated according to ICH guidelines for accuracy, precision, reproductivity, repeatability and robustness.¹⁰
MATERIALS AND METHODS:
Materials and reagents:
A gift sample of Tovorafenib from Biopharmaceuticals, Inc. Swati Clover Shilaj Circle, Sardar Patel Ring Rd, Thaltej, Ahmedabad, Gujarat 380054. Sample bought from the local market. Other chemicals like Dimethylsulfoxide as were bought from SD fine chemicals, Mumbai, India.
APPARATUS AND EQIUPMENTS REQUIRED:
1. UV-Vis double beam spectrophotometer (Model; Shimadzu: 1700S, Japan), Electric Sonicator, Volumetric flasks (10ml, 50ml, 100ml), Calibrated analytical pipettes, Electronic digital balance (Techno, Mumbai)
Preparation of concentration range (Beer’s limit):
Determination of concentration range which obeys the Lambert and Beer’s law is necessary for accuracy and reproducibility in UV- visible Spectrophotometric analysis for quantitative determination of any drug. For this; Tovorafenib stock solution (100µg/ml) was prepared using pure drug in Dimethylsulfoxide. Further dilutions were made using 0.2ml, 0.4ml, 0.6ml, 0.8ml, 1.0ml, 1.2ml, 1.4ml, 1.6ml and 1.8ml of above solution was transferred to a series of 10ml volumetric flasks this gave a series of concentrations ranging from 2 to 18 µg/ml of Dimethylsulfoxide.The final volume was made up to 10ml mark using Dimethylsulfoxide, sonicated for 5 minutes. The resultant solutions were measured using a double beam uv-vis-spectrophotometer at wave length of 260.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance. From the graph it was clear that Beer’s law was obeyed in concentration range of 2-14µg/ml and deviation was observed above these concentrations.
Preparation of standard calibration curve:
100mg of pure Tovorafenib drug was dissolved in little quantity of Dimethylsulfoxide as in a 100ml volumetric flask, the volume was made up to the mark using Dimethylsulfoxide. The solution was sonicated for 10 minutes. This gave a Tovorafenib solution with concentration of 1mg/ml (1000µg/ml). 10ml of this solution was further diluted 100ml in volumetric flask using Dimethylsulfoxide as to obtain a concentration of 100µg/ml. Further dilutions were made using 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8ml solution was transferred to a series of 10ml volumetric flasks (to Obtain a series of concentrations ranging from 2-14 µg/ml of Tovorafenib). The final volume was made up to 10ml mark using Dimethylsulfoxide, sonicated for 5 minutes, the absorbances were measured at of 260.2nm against a reagent blank. A calibration curve was plotted with concentration against absorbance.
Estimation of Tovorafenib in tablet dosage forms:
Twenty tablets were weighed accurately and powdered. The tablet powder equivalent to 100 mg of Tovorafenib was transferred into a 100ml volumetric flask and dissolved in little quantity of Dimethylsulfoxide. Then the solution was sonicated for 30 minutes and filtered using whatman filter paper No#41. The filtrate so obtained was diluted with Dimethylsulfoxide as to produce 100ml. Further dilutions were made with Dimethylsulfoxide as to get required concentrations within Beer’s - Lambert limits. The resultant solutions were measured at wave length of 260.2nm against a reagent blank. The concentration of drug was calculated with the help of standard calibration curve.
ANALYTICAL METHOD VALIDATION:
Validation of an analytical procedure is the process by which it is established, by laboratory studies, that the performance characteristics of the procedure meet the requirements for its intended use. All analytical methods that are intended to be used for analyzing any clinical samples will need to be validated. Validation of analytical methods is an essential but time consuming activity for most analytical development laboratories. It is therefore important to wave length. Understand the requirements of method validation in more detail and the options that are available to allow for optimal utilization of analytical resources in a development laboratory.
RESULTS AND DISCUSSION:
Results of Determination of Beer's Limit:
Table:1 showing Beer’s range for Tovorafenib:
|
Sl.No. |
Concentration in µg/ml |
**Absorbance at λmax 260.2nm |
|
1. |
0.0 |
0.000 |
|
2. |
2 |
0.085 |
|
3. |
4 |
0.180 |
|
4. |
6 |
0.270 |
|
5. |
8 |
0.375 |
|
6. |
10 |
0.470 |
|
7. |
12 |
0.550 |
|
8. |
14 |
0.640 |
|
9. |
16 |
0.820 |
|
10. |
18 |
1.000 |
(** Average of three determinations)
Figure: 1 showing Beer’s limit for Tovorafenib pure drug:
Figure:1 Showing Beer's limit for Tovorafenib at 260.2nm)
Standard calibration Curve of Tovorafenib:
Table: 2 Showing absorbance of Tovorafenib at various concentrations:
|
Sl. No. |
Concentration in µg/ml |
Absorbance at λmax 260.2nm |
|
1. |
0.0 |
0.000 |
|
2. |
2 |
0.085 |
|
3. |
4 |
0.180 |
|
4. |
6 |
0.270 |
|
5. |
8 |
0.375 |
|
6. |
10 |
0.470 |
|
7. |
12 |
0.550 |
|
8. |
14 |
0.640 |
Figure 2 Showing standard calibration curve for Tovorafenib:
Results of determination of Absorption Maxima
Figure 3 Showing absorption maxima of Tovorafenib at 260.2nm:
Estimation of Tovorafenib in tablet dosage form:
Table 3 Showing absorption of drug from tablet dosage form:
|
Volume of stock Solution Used |
Amount of drug (Label claim) (µg/ml) |
Absorbance at 3260.2nm |
Amount of drug Found (µg/ml) |
Percentage purity Found ± S.D** (%w/w) |
|
0.2ml |
2 |
0.1093 |
2.019 |
100.75±0.16 |
|
0.6ml |
6 |
0.317 |
6.043 |
100.68±1.47 |
|
1.0ml |
10 |
0.514 |
9.985 |
99.85±0.66 |
|
1.4ml |
14 |
0.698 |
13.984 |
99.90±0.51 |
(** Average of three determinations)
Determination of Accuracy:
Table:4 Accuracy results for Tovorafenib:
|
Brands |
Initial amount (µg/ml) |
Amount of pure drug Added (µg/ml) |
Amount Recovered (µg/ml) |
% Recovery ±S.D** |
|
OJEMDA |
10 |
8(80%) |
8.031 |
100.37±0.147 |
|
10 |
10(100%) |
9.901 |
98.01±0.231 |
|
|
10 |
12(120%) |
12.030 |
100.25±0.421 |
(**Average of six determinations, n=6)
Determination of Precision:
Table:5 Precision results for Tovorafenib:
|
Sl |
Conc. in(µg/ml) |
Inter-day absorbance Mean±S.D** |
% C.V |
Intra-day absorbance Mean±S.D** |
% C.V |
|
1. |
8 |
0.417±0.009 |
0.42 |
0.416±0.021 |
0.69 |
|
2. |
10 |
0.516±0.030 |
0.41 |
0.516±0.025 |
0.30 |
|
3. |
12 |
0.609±0.023 |
0.67 |
0.612±0.031 |
0.31 |
(**Average of three determinations, n=3)
Determination of Ruggedness parameters:
Table:6 Showing Ruggedness parameters:
|
Parameters |
Laboratory |
Name of the instrument |
Manufacturer of the chemicals used |
|
Lab. 1 with analyst I |
M.M.U. College of Pharmacy, Ramanagara |
Shimadzu-(model: 1700S, Japan) double beam uv-vis spectrophotometer |
S.D Fine chemicals, Mumbai. |
|
Lab. 2 with analyst II |
Dr. H.L.T. College of Pharmacy, kengal, Channapatna |
SystronicUv-Vis Double beam spectrophotometer |
Loba chemicals, Mumbai |
Table:7 Showing Ruggedness results for Tovorafenib:
|
Sl. No |
Brand |
Label claim(mg) |
Lab. 1* with analyst I |
Lab. 2* with analyst II |
||
|
Amount Found (mg) |
% Recovery ± S.D** |
Amount Found (mg) |
% Recovery ± S.D** |
|||
|
1. |
OJEMDA 10 |
10.09 |
100.9± 0.749 |
9.97 |
99.7± 0.349 |
|
(Lab 1* MMU College of pharmacy, Lab 2*
Dr.HLT College of pharmacy, .**Average of six determinations, n=6)
Determination of Robustness:
Table 8 Showing Robustness results for Tovorafenib:
|
TYPE: PURE DRUG |
|||||
|
Sl. No.
|
Conc. In (µg/ml) |
Change in temperature |
Change in PH |
||
|
+50C |
-50C |
2drops of 0.1N NaOH |
2drops of 0.1N HCl |
||
|
|
|
Absorbance at 260.2nm Mean±S.D** |
|||
|
1 |
8 |
0.411± 0.032 |
0.412± 0.022 |
0.410± 0.017 |
0.412± 0.017 |
|
2 |
10 |
0.521± 0.028 |
0.519± 0.023 |
0.520± 0.016 |
0.521± 0.033 |
|
3 |
12 |
0.615± 0.028 |
0.616± 0.032 |
0.615± 0.055 |
0.614± 0.054 |
(**Average of three determinations, n=3)
Table:9 showing calibration data for Tovorafenib at 260.2nm:
|
|
Calibration data at 260.2nm |
|
λmax |
260.2nm |
|
Beer’s law limit (µg/ ml) |
2 -14µg/ml |
|
Molar Absorptivity |
1.1161Lmol-1cm-1 |
|
Regression Equation (Y=a+bc) |
Y= 0.055X+0.016 |
|
Slope (b) |
0.01426 to 0.01472 |
|
Intercept(a) |
- 0.004520 to 0.0016757 |
|
Correlation Coefficient (R2) |
0.998 |
|
Limit of detection (LOD) |
1.226µg/ml |
|
Limit of quantitation (LOQ) |
5.226µg/ml |
Determination of Beer’s limit:
The Beer’s limit felled in the range of 2-14 µg/ml under given experimental conditions.
Determination of absorption maxima:
100µg/ml stock solution of Tovorafenib was prepared and absorbances were measured from 200nm to 340nm. The optimum wave length was found to be 260.2nm
Assay:
Marketed tablets contained Tovorafenib were used for the assay. After extraction, proper dilution, measurement, the concentration was determined using standard calibration curve. The amount of drug found in the range of 99.80 – 100.70%w/w
Method validation:
The proposed method was validated in accordance to ICH guidelines.
a) Accuracy: Percentage of recoveries of Abrocitinib in tablets was found in the range of 99.01 – 100.37% w/w
b) Precision: The percent coefficient of variations (% C.V) was between 0.34-0.60 for intra-day and 0.42-0.67 for inter-day absorbances.
c) Repeatability: Repeatability was determined by analyzing the sample at the given concentration wavelength for at least six times and it was found that the variability in the results was not more than 0.5%.
d) Reproducibility: The standard solution of Tovorafenib by analyst-I and analyst-II separately. The values obtained were evaluated using F-test and t-test to verify their reproducibility. Calculated value for t-test was found to be less than the tabulated (standard) value it can calculated that no significant difference was observed in the result of analysis.
e) Ruggedness: Ruggedness of the developed method was determined by changing the analytical tools such as laboratory, instruments, analyst and chemicals. The result (in terms of %RSD) of six determinations indicated that there were no significant variations in the data.
f) Robustness: Robustness of the method was established by slightly changing the temperature and PH of the reaction mixture. The data so obtained showed no significant variation in the absorption pattern.
g) Limit of detection and limit of quantitation: were determined from the standard deviation of y – intercepts of six calibration curves and average slope of six calibration curves. LOD and LOQ of Finerenone was found to be 1.226µg/ml and 5.226 µg/ml respectively.
CONCLUSION:
A new UV-Visible spectrophotometric method was developed to estimate Tovorafenib in pure and tablet dosage forms. Dimethylsulfoxide as solutions of Tovorafenib was estimated by using UV-Visible spectrophotometer (Shimadzu 1700S, Japan) with matched 1cm quartz cell. It showed maximum absorption at the range of 2-14µg/ml at 260.2nm with coefficient of correlation (R2) of 0.998. The method so developed was validated according to ICH guide lines for accuracy, precision (inter and intra-day precisions), repeatability, reproducibility, ruggedness, robustness etc.
The proposed method was found to be simple, accurate, sensitive, precise, reproducible and rapid.
This method can be successfully employed for routine quantitative analysis of Tovorafenib in bulk and tablet dosage form.
ACKNOWLEDGMENT:
The authors are thankful to Head of the Department of Chemistry.
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Received on 17.06.2024 Revised on 15.10.2024 Accepted on 02.12.2024 Published on 28.02.2025 Available online from March 04, 2025 Asian Journal of Pharmaceutical Analysis. 2025;15(1):20-24. DOI: 10.52711/2231-5675.2025.00004 ©Asian Pharma Press All Right Reserved
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