Forced Degradation Studies of Topiroxostat Tablet by UV-Visible Spectroscopy as Per ICH Guidelines

Gout is a chronic inflammatory disease characterized by the deposition of monosodium urate crystals in joints and tissues, resulting from rolonged hyperuricemia, leading to severe joint pain and inflammation [1].

Topiroxostat is a selective Xanthine Oxidase inhibitor used to treat and control gout and hyperuricemia. Purine metabolism is regulated by xanthine oxidase, also known as xanthine oxidoreductase (XOR), and serum urate levels can be effectively decreased by inhibiting the enzyme. 4-(5-pyridine-4-yl-1H-1, 2, 4-triazol-3-yl) pyridine-2-Carbonitrile is the IUPAC name of Topiroxostat and 248.24 g/mol [2, 3].

Figure. 1 Structure of Topiroxostat

Stability-indicating methods are quantitative analytical techniques that utilize the unique structural, chemical, or biological properties of each active ingredient in a drug product. These methods enable the differentiation between the active ingredient and its degradation products, allowing for accurate measurement of the active content [4].

The ICH guideline Q1A on “Stability Testing of New Drug Substances and Products” gives indications for the testing parameters which are may be susceptible to change during long storage and are likely to affect quality, safety and efficacy must be done by validated stability indicating testing methods. It is mentioned that forced degradation studies or stress testing at temperatures in 10°C increments above the accelerated temperatures, and under acidic, basic, neutral hydrolysis, oxidative, and photolytic conditions have to be carried out on the drug substance so to set up the stability characteristics and degradation pathways to back up the suitability of the proposed analytical procedures [5].Instruments which measure the ratio, or a function of the ratio, of the intensity of two beams of light in the ultraviolet region are called UV-Visible Spectrophotometers. The technique of UV-Visible Spectrophotometry is one of the most frequently employed in Pharmaceutical Analysis. It involves the measurement of the amount of Ultraviolet (200-400nm) or visible (400-800nm) radiation absorbed by substance in solution. Absorption of light in both the UV and visible regions of the electromagnetic spectra occurs when the energy of light matches that required to induced in the molecular electronic transition and its associated vibrational and rotational transitions. It is thus convenient to consider the techniques of UV and visible spectrophotometry together [6, 7].

Figure. 2 UV-Visible Spectrophotometry

The present study aims to develop and validate a UV spectrophotometric method for the estimation of Topiroxostat and to evaluate its degradation behavior under different stress conditions as per ICH guidelines.

MATERIALS AND METHODS:

Chemical and Reagents: All reagents and solvents employed in this study were of analytical grade. Topiroxostat (bulk drug) and a commercial tablet formulation containing 20 mg of Topiroxostat were obtained from Alkem Laboratories, Alkem House, Mumbai, India. Methanol was procured from Rankem. Hydrochloric acid (HCl), Sodium hydroxide (NaOH), and Hydrogen Peroxide (H₂O₂) were used for forced degradation studies. Distilled water was used throughout the study.

Instruments: UV-Visible spectrophotometric analysis was carried out using a double beam UV-Visible spectrophotometer (Shimadzu UV-1900i) equipped with LabSolutions UV-Vis software and 1 cm pathlength quartz cuvettes. An analytical balance (REPTECH) and an ultrasonicator (Themis Medicare) was used.

Preparation of Standard Solution: An accurately weighed quantity of Topiroxostat (20mg) was transferred to a 50 ml volumetric flask, dissolved in methanol with sonication, and volume was made up to the mark to obtain a stock solution of 400 μg/ml. Further dilution of 1.25 ml of this solution to 10 ml with methanol yield a working standard solution of 50 μg/ml.

Determination of Wavelength of Maximum Absorbance (λmax): An aliquot of the working solution was suitably diluted to obtain a concentration of 10 μg/ml and scanned in the range of 200-400nm against methanol as blank. The maximum absorbance (λmax) was observed at 275 nm, which was selected for further analysis [8].

Figure 3. Absorbance Spectra of Topiroxostat (10 μg/ml)

Preparation of Calibration Curve: Standard solution in the concentration range of 6-14 μg/ml were prepared by appropriate dilution of the working standard solution. The absorbance of each solution was measured at 275 nm against methanol as blank. The calibration curve was constructed by plotting absorbance (A) versus Concentration (C, μg/ml).

Method Validation: The objective of method validation is to demonstrate that the method is suitable for its intended purpose. The method was validated for linearity, precision, accuracy, LOD, and LOQ as per ICH guidelines [9].

1. Linearity: Linearity was determined by analyzing standard solutions in the range of 6-14 μg/ml. Calibration curve of absorbance vs. concentration was plotted and correlation coefficient and regression line equations for Topiroxostat were calculated.
2. Precision: The repeatability of the method was checked by scanning 10 μg/ml of solution six times. Intra-day precision was determined by checking absorbance of 8-12 μg/ml on the same day. Inter-day precision was determined by checking the absorbance of 8-12 μg/ml on tree different days. The % RSD was calculated.
3. Accuracy: Accuracy study was conducted by spiking at three different concentration levels (80%, 100%, and 120%). At each level samples were scanned and from the absorbance % recovery was determined.
4. Limit of Detection (LOD): LOD is the lowest amount of an analyte in a sample that can be detected, but not necessarily quantified, under stated experimental conditions. Estimated from the set of 5 calibration curves that were used to determine linearity.                                LOD= 3.3 ×S.D./Slope
5. Limit of Quantitation (LOQ): LOQ is the lowest amount of an analyte in a sample that can be quantified under stated experimental conditions. Estimated from the set of 5 calibration curves that were used to determine linearity. LOD= 10 × S.D./Slope

Assay of Commercial Tablet Formulations: Tablet powder equivalent to 20 mg of Topiroxostat was accurately weighed, dissolved in methanol, sonicated for 5 minutes, and filtered through Whatmann filter paper. Appropriate dilutions were made to obtain a final concentration of 10 μg/ml, and absorbance was measured at 275 nm to determine drug content.

Forced Degradation Studies: Forced degradation studies were performed to evaluate the stability of Topiroxostat under different stress conditions, following ICH Q1A (R2) guidelines [10].

For all conditions, Topiroxostat equivalent to 20mg was weighed accurately and exposed to the following environments:

1. Acidic Degradation: 20 mg Topiroxostat in 2ml of 0.1N HCl and allowed to rest for 30 minutes. After that sample was neutralized with 2ml of 0.1 N NaOH.
2. Basic Degradation: 20 mg Topiroxostat in 2ml of 0.1 N NaOH and allowed to rest for 30 minutes. After that sample was neutralized with 2ml of 0.1 N HCl.
3. Neutral Degradation: 20 mg Topiroxostat dissolved in 2 ml Distilled water and allowed to rest for 30 minutes.
4. Oxidative Degradation: 20 mg Topiroxostat treated with 2 ml 3% hydrogen peroxide solution and allowed to rest for 30 minutes.
5. Photolytic Degradation: 20 mg Topiroxostat exposed to direct sunlight for 30 minutes [11].
6. Thermal Degradation: 20 mg Topiroxostat placed in hot air oven at 45-50°C for 30 minutes.

After each stress treatment, samples were diluted with methanol to yield a concentration of 10 μg/ml. The absorbance of each sample was measured at 275 nm, and the percentage of degradation was calculated using the initial absorbance of the unstressed sample.

RESULTS AND DISCUSSION:

Linearity and Calibration Curve: The developed method exhibited excellent linearity within the validated concentration range of 6-14 μg/ml. The absorbance values for each concentration was measured at 275 nm and are presented in Table 1. The calibration curve of absorbance vs. concentration showed a straight line with a regression equation: y= 0.0634x – 0.0231 with regression coefficient (R²) 0.9956 and correlation coefficient (r) 0.9977.

Table 1. Linearity of Topiroxostat

The absorbance values were below 1.0, ensuring reliable quantitative analysis.

Calibration Curve of Topiroxostat was presented in Figure 4:

Figure. 4 Calibration Curve of Topiroxostat

Precision: The method demonstrated acceptable precision with low %RSD values for repeatability, intraday, and interday studies and are presented in Tables.

Table 2. Repeatability of Topiroxostat

Table 3. Intraday Precision of Topiroxostat

Table 4. Interday Precision of Topiroxostat

The %RSD values were found to be within acceptable limits (<2%), confirming the reliability and reproducibility of the method.

Accuracy: Accuracy was evaluated by recovery studies at 80%, 100%, and 120% levels. The percentage recovery values were found to be within the acceptable range of 98-102%, indicating the method is accurate and free from interference.

Table 5. Accuracy of Topiroxostat

LOD and LOQ: The LOD and LOQ for Topiroxostat were found to be 0.55 μg/ml and 1.69 μg/ml.

Assay of Topiroxostat tablets: The developed method was successfully applied to the analysis of Topiroxostat (20mg) in Tablet dosage form. The assay results were found to be within acceptable limits, confirming the applicability of the method for routine quality control analysis.

Table 6. Assay of Topiroxostat tablets

Table 7. Summary of UV- spectroscopic method

The test result of proposed method found to within acceptable limit.

Forced degradation studies: Forced degradation testing was carried out to evaluate the stability behavior of Topiroxostat under a variety of stress conditions including acid, base, neutral hydrolysis, oxidative, thermal, and photolytic degradation.

Figure. 5 Control Absorbance (10μg/ml)

Table 8. Control Condition for Topiroxostat

1. Acid Degradation:

Spectra of acid degradation is shown in figure 6 and Table 9 shows % degradation in acidic conditions.

Figure. 6 Spectra of acid hydrolysis

Table 9. Acid degradation condition

The degradation in acidic condition was found to be 28.10 %.

2. Base Degradation:

Spectra of base degradation is shown in figure 7 and table 10 shows % degradation in basic condition:

Figure. 7 Spectra of base hydrolysis

Table 10. Base degradation condition

The degradation in basic condition was found to be 6.75 %.

3. Neutral Degradation:

Spectra of neutral degradation is shown in figure 8 and table 11 shows % degradation in neutral condition:

Figure. 8 Spectra of neutral hydrolysis

Table 11. Neutral degradation condition

The degradation in neutral condition was found to be 15.94 %.

4. Oxidative Degradation:

Spectra of oxidative degradation is shown in figure 9 and table 12 shows % degradation in oxidative condition:

Figure. 9 Spectra of oxidative degradation

Table 12. Oxidative degradation condition

The degradation in oxidative condition was found to be 26.68 %.

5. Photolytic Degradation:

Spectra of photolytic degradation is shown in figure 10 and table 13 shows % degradation in photolytic condition:

Figure. 10 Spectra of photolytic degradation

Table 13. Photolytic degradation condition

The degradation in photolytic condition was found to be 54.10 %.

6. Thermal Degradation:

Spectra of thermal degradation is shown in figure 11 and table 14 shows % degradation in thermal condition:

Figure. 11 Spectra of thermal degradation

Table 14. Thermal degradation condition

The degradation in thermal condition was found to be 24.63 %.

Table 15 Summary of Forced degradation study