Development and validation of a stability indicating related substances of Opicapone by reverse phase high performance liquid chromatography and its degradation

Chemicals and reagents

HPLC grade of triethylamine, water, and acetonitrile, all of which were acquired from Merck India Ltd in Mumbai, India, were used. The reference standards for Opicapone and its related impurities were provided by Candila health care ltd, Ahmedabad, India. A capsule dosage form of Ongentys (50 mg) was acquired from Zydus, Cadila healthcare Ltd, Ahmedabad-382210, Gujarat, India.

Instrumentation

Waters alliance liquid chromatography (model e-2695) was utilized in this investigation, with the aid of the model 2998 photo diode array detector and the Empower 2.0 data management system.

Mobile phase-A

Acetonitrile

Preparation of mobile phase-B

1 ml of triethylamine is dissolved in 1 l of HPLC grade water and filter through 0.45 μ filter paper.

Optimization of mobile phase

Different trails have conducted their experiments, with different buffers and different mobile phases being utilized in the development of the approach. No peaks have been appropriately divided in all paths. As a result of this approach, all the peaks are resolved, and the limits are fulfilled on all suitability factors.

Chromatographic conditions

The HPLC analysis was conducted on a reverse-phase HPLC system, which employed gradient elution (Table 1) with acetonitrile and 0.1% triethylamine as the mobile phase and an Agilent Eclipse C18 (150 × 4.6 mm, 3.5 μm) column. The retention time of Opicapone was observed at 13.2 minutes with a run time of 20 minutes. A wavelength of 225 nm was observed by using PDA detector.

From all the above results, it is clear that the Opicapone was separated from its five impurities with simple gradient method and having good resolution between each impurity.

The comparision between the previously published works with the current method were given in the form of a Table 2.

Green chemistry and AGREE tool

Green analytical chemistry aims to make analytical techniques less harmful to the environment and more human-friendly. The amount and toxicity of reagents used, generated waste, energy requirements, the number of procedural steps, miniaturization, and automation are just a few of the many parameters taken into account when determining the greenness of an analytical approach. The application of greenness assessment criteria necessitates the use of specialized software such as The Appraisal of Guidelines for REsearch & Evaluation (AGREE). As a result, for a thorough, adaptable, and clear assessment technique, we employed the Analytical GREEnness calculator, which offered a simply interpretable and useful result.

Table 1. Gradient program. [Click here to view]

Table 2. Comparison between new HPLC method in this paper and old methods. [Click here to view]

Diluent

Mobile phase was used as a diluent.

Validation procedure

International Conference on Harmonization (ICH) Q2 (R1) guidelines were used to verify the performance of several analytical parameters, including system appropriateness, precision, specificity, accuracy, linearity, robustness, LOD, LOQ, forced deterioration, and stability.

Standard stock solution. Load 50 mg of Opicapone into a 100 ml volumetric flask and mix in 70 ml of sonicated diluent for 10 minutes to dissolve the contents. Then bring the total volume to the mark with more diluent.

Sample stock solution. Use 100 ml volumetric flasks with diluent to transfer 129 mg (50 mg of Opicapone) of sample. Make sure you filter the solution with a 0.45 μ nylon syringe filter.

Impurity standard stock solution. Set up a 100 ml volumetric flask, weighing 5 mg of each imp-1, imp-2, imp-3, imp-4, and imp-5 inside of it. Pour in 70 ml of solvent, then sonicate to dissolve it.

Spiked standard solution. Place 5 ml of standard stock solution into a 50-ml volumetric flask, then add 40 ml of diluent. After that, add 5 ml of impurity standard stock solution, and mix until full. Filter the sample through a 0.45 μ syringe filter.

Spiked sample solution. Transfer 5 ml of sample stock into a 50 ml volumetric flask, add 40 ml of diluent, and also add 5 ml of impurity standard stock solution and makeup to the mark with diluent. Filter through 0.45 μ syringe filter.

Results and Discussion

One of the biggest analytical difficulties in the creation of a new technique was separating active pharmaceutical components. The chromatographic conditions were altered to provide an optimal performance.

Method validation

ICH guidelines on system appropriateness, linearity, accuracy, precision, robustness, and stability were achieved by the improved RP-HPLC technique.

System suitability

To evaluate the applicability of devices, 50 μg/ml of Opicapone, 5 μg/ml each of imp-1, imp-2, imp-3, imp-4, and imp-5 were injected into six replicates of spiked standard solution. The findings reveal that the fitness parameter of the machine conforms to the limit defined by ICH. Table 3 and Figure 2 present the system precision results and standard chromatogram, respectively.

Table 3. Results of system suitability. [Click here to view]

Figure 2. Standard chromatogram. [Click here to view]

Specificity

To test the interference, placebo solutions, samples, and standards were tested independently in this test procedure. In the diagram below, you can see that the active ingredient, its associated compounds, and excipients were clearly separated from the blank and that the placebo did not interfere with the major peak. Therefore, the process is specialized. Blank chromatogram was shown in Figure 3.

Linearity

To measure linearity, a calibration curve was created in which the peak area of each concentration was plotted against its concentration. The calibration curve between 5–75 μg/ml of Opicapone and 0.5–7.5 μg/ml of each of imp-1, imp-2, imp-3, imp-4, and imp-5 revealed that the calibration curve was linear. The regression equations for calibration curve were Y = 379,503.15x + 450,638. (R² = 0.9992) for Opicapone, Y = 50,691.95x + 2,263.91 (R² = 0.9993) for imp-1, Y = 54,806.35x + 907.52 (R² = 0.9994) for imp-2, Y = 23,404.21x + 1,816.31 (R² = 0.9995) for imp-3, and Y = 54,131.96x + 1,681.90 (R² = 0.9999) for imp-4, Y = 114,430.96x + 6,088.62 (R² = 0.9996) for imp-5. Table 4 provide the results of linearity testing, and Figure 4 illustrates the calibration plots of Opicapone and its related substances.

Table 4. Linearity results of Opicapone and its impurities. [Click here to view]

Figure 3. Chromatogramof blank. [Click here to view]

Accuracy

The system’s precision was gained by doing the recovery trials at three different points (50%, 100%, and 150%). Active pharmaceutical ingredient (API) stock solutions with a 25, 50, and 75 μg/ml concentration of Opicapone were developed. To administer the test for each injection step, a different test solution was injected three times and was carried out according to the defined protocol. Both the recovery results and Relative Standard Deviation (RSD) values were 100% and under ±2%, respectively. The mean and standard deviation were found for the % recovery. The results of accuracy studies are displayed in Table 5.

Table 5. Results of accuracy. [Click here to view]

Table 6. Intraday precision results of allantoin and permethrin. [Click here to view]

Precision

Measurements produced from several homogeneous mixture samplings can be highly precise, depending on the degree of precision of the analytical procedure. Six measurements of Opicapone and its related compounds were injected to calculate the precision of the medication delivery procedure. Method precision results were shown in Table 6 and sample chromatogram was shown in Figure 5.

Intermediate precision

The samples were evaluated by six different researchers, using different equipment, and were tested on days that varied. It is clear that the average percentage of RSD values is estimated based on the regional maximums. The findings are shown in Table 7.

Table 7. Inter-day precision results. [Click here to view]

Figure 4. Calibration plots of Opicapone and its impurities. [Click here to view]

LOD and LOQ

The calibration curve technique was used to find LOD and LOQ independently. Using the RP-HPLC method devised for the chemical, the LOD and LOQ were evaluated by injecting a series of standard solution concentrations. The concentration levels of LOD and LOQ, as well as their respective Signal to noise ratio (s/n) values, were provided in Table 8.

Table 8. Results of LOD and LOQ. [Click here to view]

Robustness

The experimental circumstances were varied to assess the robustness of variables that were modified on purpose, such as flow rate, mobile phase organic percentage, and more. As seen in Table 9, the Opicapone impurities as well as the material itself met the specifications for robustness.

Stability

At room temperature and 2°C–8°C, normal solution can be stored for up to 24 hours. The solutions were then fed into the system and the % variation was measured from the initial to 24 hours. It was confirmed that no significant variation occurred, and that the solutions were stable for up to 24 hours. The assay’s percentage did not reach 2%. There is no impact on Opicapone and its byproducts regardless of storage circumstances. The results of the stability testing were shown in Table 10.

Figure 5. Chromatogram of sample. [Click here to view]

Degradation studies

To study the effects of partially degraded Opicapone, the chemical was exposed to a number of experimental situations. For research purposes, degradation trials were forced on material to prove that the technique was appropriate for being used in degraded goods. Another important aspect of the studies is information on the environment in which the medicine is unstable, therefore methods to reduce potential instabilities are also implemented during formulation. Degradation results were shown in Table 11.

Acid degradation. One millilitre of sample stock solution and 1 ml of impurity stock solution were transferred to a 10-ml volumetric flask, with 1 ml of 1 N HCl added and allowed to sit for 15 minutes. Make the diluent to the mark and add 1 ml of 1 N NaOH after waiting 15 minutes. Use a syringe filter to filter the solution, then inject it into the HPLC system.

Table 9. Robustness results. [Click here to view]

Table 10. Stability results of Opicapone. [Click here to view]

Alkali degradation. One millilitre of sample stock solution 1 ml of impurity stock solution were transferred to a 10-ml volumetric flask, with 1 ml of 1 N NaOH added and allowed to sit for 15 minutes. Make the diluent to the mark and add 1 ml of 1 N HCl after waiting 15 minutes. Use a syringe filter to filter the solution, then inject it into the HPLC system.

Peroxide degradation. Add 1 ml of the impurity stock solution to a 10-ml volumetric flask and transfer 1 ml of the sample stock solution to the same flask. Finally, add 1 ml of 30% hydrogen peroxide solution to the volumetric flask and then dilute to the mark with a suitable solvent. Use a syringe filter to filter the solution, then inject it into the HPLC system.

Reduction degradation. To prepare a 10 ml volumetric flask, move 1 ml of sample stock solution and 1 ml of impurity stock solution. Add 1 ml of 30% sodium bi sulphate solution and top off with diluents. Use a syringe filter to filter the solution, then inject it into the HPLC system.

Thermal degradation. The sample solution was set in an oven at 105°C for 6 hours. The resultant solution was injected into HPLC system.

Hydrolysis degradation. To prepare a 10 ml volumetric flask, move 1 ml of sample stock solution and 1 ml of impurity stock solution. Add 5 ml of HPLC water and top off with diluents. Use a syringe filter to filter the solution, then inject it into the HPLC system.

Table 11. Forced degradation results. [Click here to view]