Related Substances Method Development and Validation of Axitinib, Zanubrutinib and Upadacitinib Using RP-HPLC and its Degradation Products were Characterized by Using LC-MS/MS

Background: Axitinib was used to treat carcinoma of renal cells and Upadacitinib was used to treat rheumatoid arthritis and Zanubrutinib used as an anti cancer medication for mantle cell lymphoma. In the current application, Zanubrutinib, Upadacitinib and Axitinib and their associated substances will be developed and validated. Materials and Methods: The optimised method includes the Zanubrutinib, Upadacitinib and Axitinib gradient elution and associated flow rates with dimensions of 1 mL/min and phenyl column X-bridge (150 x 4.6 mm, 3.5 μ). A mobile phase was employed with 1.2 g of Hexane sulphonic acid of pH-2.5 adjusted with ortho-phosphoric acid (buffer) and acetonitrile. Zanubrutinib, Upadacitinib and Axitinib were separated from their associated substances for a total run time of 60 min. The approach developed has been validated in accordance with the ICH guidelines. Results: The test concentration LOD and LOQ were established for both drugs and for their impurities. The diagrams that were drawn were straightforward with an R 2 regression coefficient > 0.999. As part of the method validation, recovery, specificity, linearity, accuracy, robustness was determined and the results were found to be within an acceptable range. Conclusion: HPLC was used to validate the method in terms of accuracy, linearity, method precision, accuracy, limit of detection, limit of quantification, robustness, and degradation, and LC-MS/MS was used to characterise the degradation products. The system was validated in all of these aspects.

inhibitors, biological DMARDs, 13 or immune suppressants 14 such as Azathioprine and Cyclosporine, which are known to depress the immune system.Infections such as pneumonia, 15 cellulitis, 16 TB, and shingles, 17 as well as other herpes disease infections, including life-threatening complications, are among the most serious side effects.
Pfizer is the company that created the small molecule tyrosine kinase inhibitor 18 known as Axitinib, which is sold under the brand name Inlyta.It has been shown to considerably suppress the development of breast cancer 19 in animal (xenograft) models 20 and to induce partial responses in clinical studies with renal cell carcinoma (RCC) 21 and a range of other kinds of tumors. 22t is hypothesised that its principal mechanism of action is the suppression of vascular endothelial growth factor 23 receptors 1 through 3, c-KIT, and PDGFR. 24This allows it to block angiogenesis, which is the development of new blood vessels by tumours. 25It was also hypothesised that it could function by activating autophagy, 26 similar to how certain other tyrosine kinase inhibitors, such as sorafenib do. 27In addition to this, it has been established 28 that it interacts to the BCR-ABL fusion protein, suppressing particularly the drug-resistant T315I mutant isoform by adopting a different conformation than VEGF does when it does so.More than 20 percent of patients have one or more of the following adverse effects: diarrhoea, hypertension, tiredness, reduced appetite, nausea, dysphonia, hand-foot syndrome, 29 weight loss, vomiting, asthenia, and constipation.These are the most commonly reported side effects.The structural depiction of Zanubrutinib, Upadacitinib, and Axitinib, together with other chemicals that are linked to them, may be Figure 1.
There has been no HPLC technique documented in the scientific literature so far.Thus, we devised a technique for simultaneously determining the concentrations of Zanubrutinib, Upadacitinib, Axitinib, and their related compounds.The HPLC technique established for the in vitro determination of the combination of medicines was used.
Analysis of pharmaceutical compounds such as Zanubrutinib, Upadacitinib, Axitinib, and their metabolites is the goal of this research.

Chemicals
At Merck India Ltd. in Mumbai, India, we were able to purchase acetonitrile with an HPLC mark as well as orthophosphoric acid with an HPLC mark.Spectrum Solution for Pharmacy Research Pvt, Ltd, Hyderabad provided the APIs for Zanubrutinib, Upadacitinib, and Axitinib, as well as their impurities as reference standards.

Instrumentation
For this study, the Waters Alliance Liquid Timers (model 2695) monitored with a soft ware of Empower 2.0 management system and a photo diode array detector (model 2998) were used.

Preparation of buffer
After dissolving 1.2 g of hexane sulfonic acid in one litre of HPLCgrade water, the pH of the solution was brought down to 2.5 using orthophosphoric acid.After that, filter the buffer solution using filter paper with a 0.45 micron pore size.

Chromatographic conditions
The HPLC analysis was conducted with a gradient elution mode in the reverse phase HPLC system using a 1 mL/min motive stage of acetonitrile and Hexane sulfonic acid (HSA) and phenyl X-bridge (150 x4.6 mm, 3.5 μ).The gradient programme is Table 1.

Diluents
Mobile phase.

RESULTS
When developing a new method, the main analytical challenge was the separation of active pharmaceutical ingredients.The chromatographic conditions were optimised to ensure good performance.

Method optimization
Diverse phosphate buffer and acetonitrile ratios in mobile phase were tested with isocratic and gradient modes to maximise the chromatographic conditions.However, each test modified the mobile phase composition to improve the resolution and also to reach acceptable retention times.Finally, HSA and acetonitrile with gradient elution were selected because the active pharmacological ingredients and their impurities were more responsive.Various stationary phases such as C 8 , C 18 and amino columns were tested during the optimisation of the method.The top forms of the X-bridge phenyl 150 x 4.6 mm, 3,5 μ with a PDA detector were relatively good from these tests.In order to achieve sufficient sensitivity, the Mobile Flow rate was achieved at 220nm.In the above circumstances, It is obtained a time limit of about

System suitability
Device fitness parameters such as USP, USP tailings and percent RSD have been assessed.Results of system suitability were shown in Table 2 and the chromatogram was Figure 2.

Specificity
According to the placebo test method, the interference was individually analysed with the test samples and standard solutions.The accompanying Figure demonstrates that the active components could be easily recognised from the blank and its excipients, whereas the placebo had no effect on the primary peak.Because of this, the procedure is rather particular.The blank chromatogram is Figure 3.

Linearity
For Upadacitinib, Zanubrutinib, Axitinib and their related substances, the linearity peak area against different concentrations was evaluated.Test solutions are developed from 10 to 150 percent for the related impurity stock solution substance method at different concentration levels.We have linear connections between peak areas and the relevant pitch concentration in optimal chromatographic conditions.For all components, the correlation coefficients were below the limit.The linearity results were shown in Table 3 and the calibration plots were shown in Figure 4.

Accuracy
Accuracy in 3 different types of concentrations of 50, 100 and 150 per cent each at a given limit was carried out through analysis of active pharmaceuticals solution spotted with known amounts from all impurities.It was determined that the recovery percentages were within acceptable limits for all contaminants.Accuracy results were in Table 4.

Precision
A method's precision is determined by how close a series of measurements from several homogenous mixture samples are to one another.Six individual injection determinations of Upadacitinib, Zanubrutinib, Axitinib and related substances were injected to determine the exactness of the process of associated substances.The percent RSD of each impurity has been determined and the results showed that under the specified experimental conditions the technology is accurate.Intraday precision results were in Table 5 and Figure 5 gives the sample chromatogram.

Ruggedness
In all, six duplicates of the solution standard were evaluated by six different analyzers and on distinct days, using different tools.The drug responses were measured for a medium percentage of RSD

LOD and LOQ
LOD and LOQ of the compounds were continuously injected into the lower compounds.LOD and LOQ results were given in Table 7 and the Figure 6 shows the LOD and LOQ chromatograms.

Robustness
All of these diverse circumstances, such as fluid rate and organic mobile phase %, have been created to evaluate the durability of the constructed system.Retention duration, count, and tailing were not substantially affected by contaminants, and neither were active medicinal components.This was therefore a robust method.Table 8 gives the robustness data.

Degradation studies
In order to achieve just a partial breakdown of the medication, the normal preparations of Upadacitinib, Zanubrutinib, and Axitinib were treated to numerous circumstances that accelerated their degradation.Studies of forced degradation have been carried out in order to determine whether or not the procedure is suitable for the products of degradation.Using LC-MS, the generated degradation products were analysed in order to characterise them.Degradation data was in Table 9 and the mass spectr as were Figure 7.

Acid degradation
1 millilitre of standard stock solution is transferred to a volumetric flask containing 10 millilitres; then, 1 millilitre of 1N HCl is added, and the mixture is let to stand for 15 min.After waiting 15 min, add one millilitre of 1N sodium hydroxide and make it up to the mark using diluents.DP1 and DP2 are the two degradation products that were produced as a result of acid degradation.

Alkali degradation
After adding 1 ml of 1N sodium hydroxide and waiting 15 min, 1 ml of standard stock solution was placed in a volumetric pipette that had a capacity of 10 ml.After 15 min, add 1 ml of 1N HCl and bring the total volume up to the mark using diluents.DP3 and DP4 are the names of the two degradation products that were produced as a result of alkali degradation.

Peroxide degradation
In a volumetric flask with a capacity of 10 millilitres, one millilitre of standard stock solution was transferred, 0.3 millilitres of hydrogen peroxide at a concentration of 30 percent was added, and the volume was brought up to the appropriate level using diluents.The breakdown of peroxide results in the formation of two different degradation products, designated DP5 and DP6, respectively.

Reduction degradation
After transferring 1 ml of standard stock solution into a 10 ml volumetric flask, 1 ml of a solution containing 30% sodium bi   sulphate was added, and the volume was brought up to the appropriate level using diluents.Through the process of reduction degradation, just one degradation product, designated as DP7, was produced.

Thermal degradation
In order to establish the standard solution, it was baked in an oven at 105 degrees for six hours.The produced solution was then put through HPLC for analysis.The heat degradation process did not result in the formation of any degradation products.

Hydrolysis degradation
After transferring 1 ml of standard stock to a volumetric flask containing 10 ml, 1 ml of water was added, and the solution was diluted up to the mark.The hydrolysis method of degradation did not result in the formation of any degradation products.

Photolytic degradation
After transferring 1 ml of standard stock to a volumetric flask containing 10 ml, 1 ml of water was added, and the solution was diluted up to the mark.This solution was left out in the sun for three hours, and then the results of the exposed standard were evaluated.The process of photo degradation resulted in the formation of only one DP8 degradation product.

Stability
At room temperature and at 2-8°C, the standard solution was kept up to 24 hr.It was then necessary to run the system for 24 hr with this solution and determine the deviation %.The results were consistent for up to 24 hr, and no significant changes were seen.Table 10 displays the stability data.

DISCUSSION
The mobile phase with composition of Hexane sulphonic acid (pH-2.5 adjusted with Ortho phosphoric acid) and Acetonitrile with gradient elusion proven effective and quick separation of chosen pharmacological compounds, its associated impurities and their degradants generated during forced degradation research.Method's system adequacy was assessed by analysing the characteristics such as, retention time, area, theoretical plates, resolution and tailing factor.The present experiment was done on X-bridge phenyl (150mm × 4.6mm, Agilent 5μm) column at ambient temperature which demonstrated greater repeatability.The result of system suitability parameters were determined to be within acceptable stated ranges as per recommendations of International Council on Harmonization (ICH) guideline.The straight baseline was discovered in the blank chromatogram of mobile phase and both medicines, its associated impurities were efficiently resolved in active ingredient form.The calculated detection and quantification limit values revealed that approach was adequately sensitive for the detection of drug in the presence of their degradants.Also, % recoveries of the medicines were reported to be within permissible levels.Method's Specificity was tested by injecting blank, standard, sample and undergoing forced degradation.The statistical data gathered during robustness research show that the approach which is created is resilient.In the degradation investigation, the produced degradants were characterised by utilising LC-MS/MS.

CONCLUSION
It is proposed that the stability that is straightforward, selective, verified, and well stated.It demonstrates that Upadacitinib, Zanubrutinib and Axitinib and the chromatographic impurities of their gradients were determined in a quantitative RP-HPLC method.All degradation products formed under stress conditions and the associated uncleanness of the active ingredients in pharmaceuticals were clearly separated and peaks were well resolved, with a suitable retention time that indicates that the method proposed is quick, simple, feasible and inexpensive in an RS situation.Stability studies and regular examination of manufacturing samples might benefit from this technology since it can be utilised in both cases.

Figure 7 :
Figure 7: Mass spectras of degradation products formed in various degradation conditions.

Table 7 : Results of LOD and LOQ.
The results are given in the table below.Table6gives the inter-day precision results.