Choppala Asha Deepti and Alapati Sahithi*
Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India
*Corresponding Author: Alapati Sahithi, Department of Pharmaceutical Analysis, GITAM School of Pharmacy, GITAM (Deemed to be University), Visakhapatnam, India.
Received: October 14, 2024; Published: October 26, 2024
Citation: Choppala Asha Deepti and Alapati Sahithi. “New Stability Indicating RP-UPLC Method for the Estimation of Nintedanib Capsules". Acta Scientific Pharmaceutical Sciences 8.11 (2024):25-30.
Nintedanib is used to treat idiopathic pulmonary fibrosis. It is also used to treat people with a chronic interstitial lung disease. A new RP-UPLC method has been developed for the estimation of Nintedanib in pharmaceutical formulations and the method was and validated. UPLC Acquity Waters – Empower software 2.0 versions equipped with photodiode array detector with Phenomenex C18 (50 x 2.1 mm, 1.6 μm) was used with a mobile phase consisting of Acetonitrile: dihydrogen phosphate buffer (pH adjusted to 3.0 with ortho phosphoric acid) (30:70) and flow rate of 0.2 mL/min (UV detection at 271nm) with a run time of 2.0 mins. Nintedanib has shown linearity 25–150 μg/mL with linear regression equation y = 28788.86 x + 24028.04 (r2 = 0.99958). The LOQ was found to be 1.0 μg/mL and the LOD was found to be 0.30 μg/mL. Nintedanib was subjected to forced degradation and the method was validated as per ICH guidelines.
Keywords: Nintedanib; RP-UPLC; Stability INDICATING; Validation; ICH GUIDELINES
Nintedanib (Figure 1), chemically methyl (3Z)-3- [[4- [methyl- [2-(4-methyl piperazine-1-yl) acetyl] amino] aniline]-phenyl methylidene]-2-oxo-1H-indole-6-carboxylate is a multiple tyrosine kinase inhibitor for platelet-derived growth factor receptor, fibroblast growth factor receptor, and vascular endothelial growth factor receptor [1]. Nintedanib is an yellow crystalline solid that melts at 244 °C to 251 °C. It has poor solubility in water, and somewhat better solubility in dimethyl sulfoxide at 25 g/L. The molecular formula of Nintedanib is C31H33N5O4 and Molecular weight of 539.636 g/mol. Literature survey reveals that Nintedanib was estimated by HPLC [2-6] in pharmaceutical dosage forms and also in biological fluids.
Chromatographic separation was achieved by UPLC Acquity Waters – Empower software 2.0 versions equipped with photodiode array detector with Phenomenex C18 (50 x 2.1 mm, 1.6 µm) was used for the present study.
1.36 grams of potassium dihydrogen phosphate (KH2PO4) was dissolved in water in a 1L volumetric flask, pH was adjusted to 3.0 with OPA and finally filtered through 0.22 μ membrane filter paper.
Accurately weigh and transfer 10mg of Nintedanib, working standard into a 10ml volumetric flask, add 7ml of diluent and sonication are used to thoroughly dissolve the sample and bring the volume to the desired level. From this stock solution 1 mL was taken in to a 10 mL volumetric flask, and diluent was added to make remaining volume This solution of 100 µg/mL of Nintedanib used as standard and future diluted as required.
Accurately weighed and transfer 18 mg of Nintedanib sample into a 10 mL clean dry volumetric flask add diluent and sonicate it up to 30 mins to dissolve, and centrifuge for 30 min. to dissolve it completely and make volume up to the mark with the same solvent. Then it is filtered through 0.45-micron Injection filter. Further pipette 1 mL of the above stock solutions into 10 ml volumetric flask and dilute up to the mark with diluents.
A series of 25-150 µg/mL Nintedanib solutions were prepared from the stock solution with mobile phase and 20 μL of each of these solutions were injected in to the UPLC system. The mean peak area of Nintedanib were calculated from the chromatograms and a calibration curve was drawn by taking the concentration of the Nintedanib solutions on the x-axis and the corresponding mean peak area values on the y-axis.
Intraday and inter-day precision were studied using three different concentrations of Nintedanib on the same day and on three consecutive days respectively and the % RSD was calculated. The accuracy of the assay method was evaluated in triplicate at three concentration levels (50, 100 and 150%), and the percentage recoveries were calculated. Standard addition and recovery experiments were conducted to determine the accuracy of the method for the quantification of Nintedanib in the drug product and the percentage recovery was calculated. The robustness of the method was assessed by exposing the drug solution to different analytical conditions purposely changing from the original optimized conditions. The effects so obtained were summarized to calculate the % RSD and has to be less than 2.0% specifying that the proposed method was robust.
Forced degradation studies were performed to determine the ability of the drug to withstand its properties in the applied stress conditions. Nintedanib was exposed to different stress conditions.
Pipette 1 ml of the afore mentioned solution was added to a 10 ml vacuum flask, followed by 1 ml of 1N HCl. The vacuum flask was then maintained at 60°C for 1 hour before being neutralised with 1 N NaOH and diluted to 10ml with diluent. Filter the solution using 0.22-micron syringe filters and transfer to bottles.
Pipette 1 ml of above solution into a 10ml volumetric flask and add 1ml of 1N NaOH was added. Then, the volumetric flask was kept at 60ºC for 1 hour and then neutralized with 1N HCl and make up to 10ml with diluent. Filter the solution with 0.22 microns’ syringe filters and place in vials.
50 mg of Nintedanib standard was taken in petridish and kept in hot air oven at 1050 C for 3 hours. Then the analyzed standard was taken and diluted with diluents and injected into UPLC and analysed.
Pipette 1 ml above stock solution was added to a 10 ml vacuum flask, 1 ml of 3 percent w/v hydrogen peroxide was added to the flask and the volume was built up to the mark using diluent. The vacuum flask was then maintained at 60oC for 1hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22micron syringe filters and transfer to bottles.
Pipette 1ml of above-stock solution was added to a 10ml vacuum flask, 1ml of 10% Sodium bisulphite was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60oC for 1 hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22 μ syringe filters and transfer to bottles.
Nintedanib sample was placed in Photo stability chamber for 3 hours. Then the sample was taken and diluted with diluents and injected into UPLC and analysed.
Pipette 1ml of above-stock solution was added to a 10ml vacuum flask, 1ml of HPLC grade water was added to a flask and the volume was built up to the required volume with diluent. The vacuum flask was then maintained at 60°C for 1 hour. After that, the vacuum flask was left at room temperature for 15 minutes. Filter the solution using 0.22micron syringe filters and transfer to bottles.
Twenty capsules were procured and powder equivalent to 25 mg Nintedanib was extracted using the mobile phase in a 25 ml volumetric flask. The solution was sonicated for half an hour and filtered through 0.45 mm membrane filter and 20 µL of this solution was injected in to the UPLC system and the peak area was noted at its retention time from the resultant chromatogram.
A new RP-UPLC method has been developed for the estimation of Nintedanib in pharmaceutical formulations and the method was and validated. UPLC Acquity Waters-Empower software 2.0 versions equipped with photodiode array detector with Phenomenex C18 (50 x 2.1 mm, 1.6 µm) was used with a mobile phase consisting of Acetonitrile: dihydrogen phosphate buffer (pH adjusted to 3.0 with ortho phosphoric acid) (30:70) and flow rate of 0.2 mL/min (UV detection at 271nm) with a run time of 2.0 mins. The optimized chromatographic conditions were shown in Table 1 and the system suitability was shown in Table 2. The chromatograms of blank and the standard Nintedanib were shown in Figure 2A and Figure 2B respectively.
Nintedanib has shown linearity 25–150 μg/mL with linear regression equation y = 28788.86 x + 24028.04 with correlation coefficient 0.99958 (Table 3). The LOD and LOQ of Nintedanib were found to be 0.30 µg/mL and 1.00µg/ml. The calibration curve was shown in Figure 3.
The method precision and intermediate precision studies were performed and the % RSD was found to be less than 2% (Table 4). The accuracy of the method was proved by the standard addition method and the results were shown in Table 5. The robustness of the assay method was established by introducing small changes in the chromatographic conditions which include detection wavelength, percentage of acetonitrile in the mobile phase and flow rate and the results were shown in Table 6.
The purity of marketed capsule formulation was determined by utilizing optimized method and assay was found to be 100.7 % w/w.
Nintedanib was eluted at 0.968 min. Nintedanib has undergone acidic hydrolysis, thermal degradation, alkaline hydrolysis, peroxide degradation, hydrolysis, photolysis and the results were shown in Table 7 and the resultant chromatograms were shown in Figure 4.
The RP-UPLC techniques were validated as per ICH guidelines and found to be simple, economical and robust for the quantification of Nintedanib capsules.
The authors are grateful to M/s GITAM (Deemed to be University), Visakhapatnam for providing the research facilities and Glenmark Pharmaceuticals (India) for supplying gift samples of Nintedanib. The authors have no conflict of interest.
Copyright: © 2024 Choppala Asha Deepti and Alapati Sahithi. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.