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INTRODUCTION
Chemical stability of pharmaceutical molecules is a matter of great concern as it affects the safety and efficacy of the drug. The FDA and ICH Guidance state the requirements of stability testing data to understand how the quality of a drug substances and drug products changes with time under the influences of various environmental factors. The behaviour of thymoquinone also demonstrated erratic degradation behaviour in various strains with which the drug was subjected to and is the result of the chemical form of the drug being a quinone-based molecule (Kelly et al., 2017). These observations reveal that thymoquinone is particularly sensitive to high temperatures and light that could lead to structure rearrangement, and photochemical reactions. This very distinction proves the stability-signalling nature of the developed approach to an analytical method and its versatility to stability testing and profiling of impurities. These degradation products were detected under some severe stress factors which included oxidative, thermal, photolytic stress, and the force of quinone structure of thymoquinone to redox, photochemical reaction. The structural clarification of the degradation products of thymoquinone was explained through the mass spectrometry and nuclear magnetic resonance spectroscopy. The stability behaviour of endoxifen displaying in the present study indicate typical degradation profile that convey impressively varied profile as it is the case in also largely affected by chemical stressor and not physical stress conditions. The present differing behaviour can provide worth in comprehending about the relative chemical stability of endoxifen and with the immediate implication on the designing of formulations, the designing of the analytical methods and the storage recommendations.
The extreme degradation of endoxifen disclosed its high acidity and alkalinity, which is an indication that the molecule has sensitive functional groups, when it comes to change of the pH. Endoxifen is a compound, whose ionizable groups and heteroatom-based functional group might be protonated or deprotonated at extremely polarized pH (Iriventi et al., 2020). In such ionization, certain chemical bonds might also be destabilized such that hydrolytic dissociation or restructuring might occur. During acidic reaction, the susceptible functional groups could attain an increased electrophilicity where the functional group could be hydrolyzed. In addition to this, the selection of excipients has to be selected carefully to exclude such excipients that are likely to alter the microenvironmental pH or that react with endoxifen in a manner that will lead to degradation. Protection against the oxidative environment is another significant problem in the development and packaging of endoxifen containing products. Antioxidant may be included where required to aid in the reduction of oxidative degradation (Barzani et al., 2025). It may also be improved by packaging the products in material that is less oxygen-permeable or airtight containers to protect the products. The above measures can particularly be applied to the stability and effectiveness of endoxifen formulations in the long term. the chemical stress factors which influence stability behaviour of endoxifen are largely acidic, basic and oxidative stresses with moderate insensitivity to thermal and photolytic stress. Such findings can be very beneficial in the formulation, package selection, and storage suggestions. The research links itself with questioning the significance of the assessment of the stability of pharmaceutical products that comprise endoxifenbased on their own features and the aspects that the existence of the necessity to practice pH regulation and protection against oxidative conditions so as to preserve the quality, safety, and effectiveness of the chosen pharmaceutical products.
MATERIALS AND METHODS
Material
Instruments Specification
- Instrumentation: HPLC LC-2010 CHT Liquid Chromatography.
- Detector: UV-PDA detector LC-2010 CHT.
- Column: Zodiac C18 (150 mm X 4.6 mm), 5 µ Make:
Zodiac Life sciences.
• Chromatographic software: Lab Solution.
Reagents and Materials Used
- Acetonitrile HPLC Merck, Rankem.
- Water HPLC Merck, Rankem.
- Ortho-phosphoric acid AR Merck, Rankem.
- Methanol HPLC Merck, Rankem.
Chromatographic Parameters
Zodiac Life sciences.
- Flow rate: 1.0 mL/min.
- Wave length: 278 nm.
- Injection Volume: 10 µL.
- Temperature: 30ºC.
- Diluent: can.
- Column: Zodiac C18 (150 mm × 4.6 mm), 5 µ make: Zodiac Life sciences.
- Buffer*: Trifluoro acetic acid 1 mL was added in 1000 mL water, mixed well. Filtered through 0.45 µ nylon filter and degassed before used. (0.1%).
- Mobile phase: Buffer: ACN (78:22) pH 3.5.
Stock Solution
Accurately weighed quantity of Endoxifen 20 mg was transferred into 100 mL volumetric flask and final volume of solution was made 100 mL with mobile phase to get stock solution containing 200 µg/mL of Endoxifen. 50 mL of aliquot from above stock solution was further diluted to 100 mL with mobile phase.
Preparation of Sample Solution
Powder equivalent to 10 mg of Endoxifen was The solution was filtered through 0.45 µ membrane filter. 10 mL of aliquot from above stock solution was further diluted to 100 mL with mobile phase. The sample solution of 10 mcg/mL was assayed as per proposed method and % assay was calculated. Powder equivalent to 10 mg of Endoxifen was weighed and transferred in a 100 mL volumetric flask and sufficient mobile phase was added to dissolve the drug. It was sonicated for 20 min and final volume was made to the mark with mobile phase. % assay was calculated.
METHODOLOGY DEGRADATION STUDY OF ENDOXIFEN
Acid Degradation
To 5 mL stock Endoxifen, 2 mL of 2 N Hydrochloric acid was added and refluxed for 8 hr at 60ºC. The acidic degradation was performed under dark condition to exclude the possible photolytic degradation. The degradation sample was cooled at room temperature and neutralized the sample with same strength of NaOH. Suitable aliquots of resultant degradation samples were pipetted out and made up the volume with the mobile phase.
Base Degradation
To 5 mL stock solution of Endoxifen, 2 mL of 2 N Sodium hydroxide was added and refluxed for 8 hr at 60ºC. The basic degradation was performed under dark condition to exclude the possible photolytic degradation. The degradation sample was cooled at room temperature and neutralized the sample with same strength of HCl. Suitable aliquots of resultant degradation samples were pipetted out and made up the volume with the mobile phase.
Oxidative Degradation
To 5 mL stock solution of Endoxifen, 2 mL of 5% Hydrogen peroxide (H2 O2) was added separately. The solutions were refluxed for 6 hr at room temperature. The oxidative degradation performed under dark condition to exclude the possible photolytic degradation. The degradation sample was cooled at room temperature. Suitable aliquots of resultant degradation samples were pipetted out and made up the volume with the mobile phase.
Thermal Degradation
For dry heat degradation the sample were placed in the oven at 80ºC for 8 hr under the dark condition and then cooled at room temperature. Degradation sample were subjected to analysis after its dilution (10 μg/mL) with the mobile phase.
Photolytic Degradation
For Photolytic degradation samples were kept in Photolytic chamber for exposure of 1.2 Mill. Lux light and samples were subjected to analysis (10 μg/mL) after dilution with mobile phase. (10 μg/mL).
RESULTS AND DISCUSSION
Forced Degradation of Endoxifen
Parameter | Standard Area | Endoxifen Peak Area (mV) After Degradation | % Degradation |
|---|---|---|---|
Acid Degradation | 697438 | 620162 | 11.08 |
Base Degradation | 582849 | 16.43 | |
Peroxide Degradation (Oxidative) | 577200 | 17.24 | |
Thermal Degradation | 644502 | 7.59 | |
Photolytic Degradation | 633832 | 9.12 |
Endoxifen was much different when compared to thymoquinone with a higher sensitivity of the compound to stress conditions in the chemical environment. It also shows in Table 1 that the highest percentage of degradation was observed in the acidic, basic and oxidative environments with the percentage of degradation being Figure 1: Acid Degradation. 11.08%, 16.43% and 17.24% respectively (Table 1). The findings revealed that Endoxifen was heavily prone to alteration in pH and conditions of oxidation which can most likely be explained by the fact that functional groups are likely to exhibit vulnerability to hydrolytic and oxidative reactions (Pathan et al., 2011). The photolytic and thermo stress produced comparatively low level of degradation at 7.59% level and 9.12% level of degradation of Endoxifen. The chromatographic data of Endoxifen in various stress states are presented in that indicates the formation of various degradation peaks that were distinctly spotted by the Endoxifen parent drug peak. The absence of co-elution and total separation of RP-HPLC product of degradation testifies to the selectivity of a system developed. These results confirm the fact that the approach was meaningful to measure the Endoxifen well even in the presence of the degradation products that achieved regulatory requirements of analytical methods (Figures 1 2 3 4 5).
N-Desmethyl Endoxifen
Impurity Characterization of Endoxifen
The chromatogram analysis of the stressed Endoxifen samples indicated that it had numerous degradation peaks, which were sharply achieved when compared to the Endoxifen drug peak. These degradation products were detected under acidic, basic and oxidative conditions suggestive of the chemical vulnerability of Endoxifen to reactions of pH and oxidation. An analysis of degradation products of mass spectrometric proteins using the mass spectrometric technique has shown that there was a slight modification of the molecular weight and this was consistent with the process of hydrolysis or oxidation as a result of the interactions of functional groups within the molecule. These findings demonstrate how the analytical method can realize the identification and separation of the degradation products according to the regulatory requirements of the process of monitoring of impurities and testing of stability (Figures 6 7 8).
CONCLUSION
The rate of degradation endoxifen was relatively low in the presence of both thermal and photolytic degradation conditions which means that chemical stressors are more likely to cause endoxifen stability in the presence of chemical stressors than the physical ones (Pandya et al., 2025). Such findings emphasise the importance of pH balance during formulation and minimise contact with reducing agents during the production procedure, storage and handling. The variability in the stability profile of endoxifen can substantiate the fact that the average analytical processes cannot be viewed as generalizable but rather individual stability measurements must occur.
