STABILITY INDICATING RP-UPLC METHOD FOR ESTIMATING IPTACOPAN IN PHARMACEUTICAL DOSAGE FORM AND USES THEREOF

Abstract

The present invention provides an easy, sensitive, specific and precise method for the estimation of Iptacopan in oral suspension pharmaceutical dosage form using RP-UPLC, comprising of dissolving Iptacopan using Ammonium Acetate: Methanol taken in the ratio 70:30%v/v; running chromatogram through column CSH C18, 1.7 um, 2.1 mm X 150 mm; optimizing conditions of column at flow rate 0.4ml/min, injection volume 5.0mL, detecting wavelength 308nm, temperature maintained at 48.0°C, and run time 2.5min; running the sample and recording chromatogram from the chromatograph for estimation of Iptacopan. The method for estimation of Iptacopan, wherein the concentration of Iptacopan sample solution 50µg/ml; Retention time 0.941 min; %RSD of the Repeatability precision of Iptacopan is 0.5; LOD value from regression equation of Iptacopan 0.35, LOQ value from regression equation of Iptacopan 1.06; Regression equation of Iptacopan is y=7155x + 42097 and %Recovery is 99.90%. The present invention provides a conduction of forced degradation studies of Iptacopan under different stress conditions. The developed method is simple and economical and can be used in regular Quality control test in Industries as routine analysis of Iptacopan.

Specification

Description:Technical Field of the Invention
The present invention relates to RP-UPLC method for estimation of Iptacopan. This invention is more specifically related to RP-UPLC method for estimation of Iptacopan in bulk and pharmaceutical dosage form. The present invention is used to develop an accurate, precise, sensitive, selective, reproducible and rapid analytical technique for estimation of Iptacopan in oral suspension dosage form.
Background of the Invention
Iptacopan is the first oral medicine for the treatment of paroxysmal nocturnal hemoglobinuria (PNH) in adults. The disease is a rare hematological disorder characterized by the erythrocytes hemolysis and leading to hemolytic anemia, impaired bone marrow (BM) function, and potentially life-threatening thrombotic events. The clinical trials study shows that Iptacopan increased the hemoglobin levels of most of the patients, and none of the treated patients received blood transfusions, showing a significant therapeutic effect [Jang, J.H., Wong, L., Ko, B.S., Yoon, S.S., Li, K., Baltcheva, I., Nidamarthy, P.K., Chawla, R., Junge, G. and Yap, E.S., 2022. Iptacopan monotherapy in patients with paroxysmal nocturnal hemoglobinuria: a 2-cohort open-label proof-of-concept study. Blood advances, 6(15), pp.4450-4460]. Many methods have been proposed on the quantitative and qualitative estimation of Iptacopan in bulk and its pharmaceutical formulation [James, A.D., Kulmatycki, K., Poller, B., Romeo, A.A., Van Lier, J.J., Klein, K. and Pearson, D., 2023. Absorption, distribution, metabolism, and excretion of [14C] iptacopan in healthy male volunteers and in in vivo and in vitro studies. Drug Metabolism and Disposition, 51(7), pp.873-883; Li, J., Liu, S., Jia, C., Li, J., Zhang, Z., Chen, J., Cao, Y. and Ma, C., Pharmacokinetic study of iptacopan and its two acyl glucuronide metabolites in monkey plasma by liquid chromatography combined with electrospray ionization tandem mass spectrometry. Biomedical Chromatography, p.e6002; Gao, H., Deng, S. and Obach, R.S., 2010. A simple liquid chromatography-tandem mass spectrometry method to determine relative plasma exposures of drug metabolites across species for metabolite safety assessments. Drug metabolism and disposition, 38(12), pp.2147-2156]. However, little work has been done on RP-UPLC method for estimation of Iptacopan in pharmaceutical dosage form and stability studies. Therefore, an accurate, precise, selective, reproducible and rapid RP-UPLC method for estimation of Iptacopan in pharmaceutical dosage form and stability studies is provided.
Objects of the Invention
The main object of the present invention is to provide a RP- UPLC method for estimation of Iptacopan in bulk and pharmaceutical dosage form.
Another object of the present invention is to develop accurate, precise, sensitive, selective, reproducible and rapid analytical method for estimation of Iptacopan in oral suspension dosage form.
Yet another object of the present invention is to provide a conduction of forced degradation studies of Iptacopan under stress conditions.
Summary of the Invention
The present invention provides a Simple, sensitive, specific and precise method for the estimation of Iptacopan in pharmaceutical dosage form. In an embodiment of the present invention, the method for estimation of Iptacopan in pharmaceutical dosage form using RP-UPLC, comprising: a) dissolving Iptacopan using Ammonium Acetate: Methanol taken in the ratio 70:30 %v/v; b) running chromatogram through column C18, 1.7 um, 2.1 mm X 150 mm using mobile phase of step a); c) optimizing conditions of column of step b) at flow rate 0.4ml/min, detecting wavelength at 308nm, temperature maintained at 48.0°C and run time 2.5min; d) running the sample of step c) and recording chromatogram from the chromatograph for estimation of Iptacopan. The method for estimation of Iptacopan, wherein the concentration of Iptacopan sample solution 50µg/ml. The method for estimation of Iptacopan, wherein the Retention time of Iptacopan is 0.941min; %RSD of the Repeatabily precision of Iptacopan is 0.5. The method for estimation of Iptacopan, wherein the LOD value obtained from regression equation of Iptacopan is 0.35, LOQ value obtained from regression equation of Iptacopan is 1.06; Regression equation of Iptacopan is y=7155x + 42097 and %Recovery is 99.90%. The method for estimation of Iptacopan, wherein the invention provides a conduction of forced degradation studies of Iptacopan under different stress conditions. In an embodiment of the present invention, the process for the preparation of sample stock solutions, comprising: a) dissolving a volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan using about 3/4th ml of solvent in 100-ml volumetric flask; b) sonicating for 10 min to obtain clear solution; c) adjusting the volume with the same solvent and filtering using 0.45-μ membrane filter to obtain 500µg/ml of Iptacopan. process for the preparation of sample stock solutions, the process for the preparation of sample working solution, comprising: a) transferring 1ml of filtered sample stock solution to 10ml volumetric flask; and b) making up volume with diluent to obtain 50µg/ml of Iptacopan. In an embodiment of the present invention, the method for estimation of Iptacopan, wherein the process for the preparation of standard stock solution comprising: a) weighing accurately 25mg of Iptacopan and transferring to 50ml volumetric flask; b) adding 3/4th of diluent to the flask and sonicating for 10 minutes and making up the flask with diluents and labeled as Standard stock solution of 500µg/ml of Iptacopan. In an embodiment of the present invention, the method for estimation of Iptacopan, wherein the process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by pipetting out 1ml from stock solution and taking into a 10ml volumetric flask and making up with diluent to obtain 50µg/ml of Iptacopan. The method for estimation of Iptacopan, wherein the developed method is simple and economical and can be used in regular Quality control test in Industries as a routine analysis of Iptacopan.
Brief Description of drawings
In the drawings accompanying the specification, Figure 1 shows Calibration curve of Iptacopan.
Detailed description of the Invention
The present invention provides a method for estimation of Iptacopan in pharmaceutical dosage form using RP-UPLC. The method for estimation of Iptacopan in pharmaceutical dosage form using RP-UPLC, comprising: a) dissolving Iptacopan using Ammonium Acetate: Methanol taken in the ratio 70:30 %v/v; b) running chromatogram through column C18, 1.7 um, 2.1 mm X 150 mm using mobile phase of step a); c) optimizing conditions of column of step b) at flow rate 0.4ml/min, detecting wavelength at 308nm, temperature maintained at 48.0°C and run time 2.5min; d) running the sample of step c) and recording chromatogram from the chromatograph for estimation of Iptacopan. The method for estimation of Iptacopan, wherein the concentration of Iptacopan sample solution 50µg/ml. The method for estimation of Iptacopan, wherein the Retention time of Iptacopan is 0.941min; %RSD of the Repeatability precision of Iptacopan is 0.5. The method for estimation of Iptacopan, wherein the LOD value obtained from regression equation of Iptacopan is 0.35, LOQ value obtained from regression equation of Iptacopan is 1.06; Regression equation of Iptacopan is y=7155x + 42097 and %Recovery is 99.90%. The method for estimation of Iptacopan, wherein the invention provides a conduction of forced degradation studies of Iptacopan under different stress conditions. The present invention provides a process for the preparation of sample stock solutions. The process for the preparation of sample stock solutions, comprising: a) dissolving a volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan using about 3/4th ml of solvent in 100-ml volumetric flask; b) sonicating for 10 min to obtain clear solution; c) adjusting the volume with the same solvent and filtering using 0.45-μ membrane filter to obtain 500µg/ml of Iptacopan. The present invention provides a process for the preparation of sample working solution. The process for the preparation of sample working solution, comprising: a) transferring 1ml of filtered sample stock solution to 10ml volumetric flask; and b) making up volume with diluent to obtain 50µg/ml of Iptacopan. The present invention provides a process for the preparation of standard stock solution The method for estimation of Iptacopan, wherein the process for the preparation of standard stock solution comprising: a) weighing accurately 25mg of Iptacopan and transferring to 50ml volumetric flask; b) adding 3/4th of diluent to the flask and sonicating for 10 minutes and making up the flask with diluents and labeled as Standard stock solution of 500µg/ml of Iptacopan. The present invention provides a process for the preparation of standard working solution. The method for estimation of Iptacopan, wherein the process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by pipetting out 1ml from stock solution and taking into a 10ml volumetric flask and making up with diluent to obtain 50µg/ml of Iptacopan. The method for estimation of Iptacopan, wherein the developed method is simple and economical and can be used in regular Quality control test in Industries as a routine analysis of Iptacopan.
MATERIALS AND METHODS
Materials:
Iptacopan pure drug (API), Iptacopan Oral suspension, Distilled water, Acetonitrile, Phosphate buffer, Methanol, Potassium dihydrogen ortho phosphate buffer, Ortho-phosphoric acid. All the above chemicals and solvents are from Rankem.
Methods:
Diluent: Based up on the solubility of the drugs, diluent was selected, Methanol and Water taken in the ratio of 50:50.
Preparation of buffer:
0.01N Ammonium Aceatate Buffer: Accurately weighed 0.77gm of Ammonium Aceatate in a 1000ml of Volumetric flask add about 900ml of milli-Q water added and degas to sonicate and finally make up the volume with water then PH adjusted to 5.4 with dil. Orthophosphoric acid solution.
Preparation of Standard stock solutions: Accurately weighed 25mg of Iptacopan transferred to 50ml volumetric flask. 3/4 th of diluents was added to the flask and sonicated for 10 minutes. Flask was made up with diluents and labeled as Standard stock solution. (500µg/ml of Iptacopan)
Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (50µg/ml of Iptacopan).
Preparation of Sample stock solutions: A volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan was dissolved using about 3/4th ml of solvent in 100-ml volumetric flask then sonicated for 10 min to obtain clear solution. A volume was adjusted with the same solvent and filtered using 0.45-μ membrane filter. (500µg/ml of Iptacopan)
Preparation of Sample working solutions (100% solution):1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (50µg/ml of Iptacopan)
Validation
System suitability parameters:
The system suitability parameters were determined by preparing standard solution of Iptacopan (40ppm) and the solution were injected six times and the parameters like peak tailing, resolution and USP plate count were determined.
The % RSD for the area of six standard injections results should not be more than 2%.
Specificity: Checking of the interference in the optimized method. We should not find interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.
Precision:
Preparation of Sample stock solutions: A volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan was dissolved using about 3/4th ml of solvent in 100-ml volumetric flask then sonicated for 10 min to obtain clear solution. A volume was adjusted with the same solvent and filtered using 0.45-μ membrane filter. (500µg/ml of Iptacopan).
Preparation of Sample working solutions (100% solution):1ml of filtered sample stock solution was transferred to 10ml volumetric flask and made up with diluent. (50µg/ml of Iptacopan).
The precision were determined by preparing sample solution of Iptacopan (50ppm) and the solution were injected six times, The % RSD for the area of six standard injections results should not be more than 2%.
Linearity:
Preparation of Standard stock solutions: Accurately weighed 25mg of Iptacopan transferred to 50ml volumetric flask. 3/4 th of diluents was added to the flask and sonicated for 10 minutes. Flask was made up with diluents and labeled as Standard stock solution. (500µg/ml of Iptacopan).
25% Standard solution: 0.25ml from standard stock solution was pipetted out and made up to 10ml. (12.5µg/ml of Iptacopan).
50% Standard solution: 0.5ml from standard stock solutions was pipetted out and made up to 10ml. (25µg/ml of Iptacopan).
75% Standard solution: 0.75ml from standard stock solutions was pipetted out and made up to 10ml. (37.5µg/ml of Iptacopan).
100% Standard solution: 1.0ml from standard stock solutions was pipetted out and made up to 10ml. (50µg/ml of Iptacopan).
125% Standard solution: 1.25ml from standard stock solutions was pipetted out and made up to 10ml. (62.5µg/ml of Iptacopan).
150% Standard solution: 1.5ml from standard stock solutions was pipetted out and made up to 10ml (75µg/ml of Iptacopan).
Accuracy:
Preparation of Sample stock solutions: A volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan was dissolved using about 3/4th ml of solvent in 100-ml volumetric flask then sonicated for 10 min to obtain clear solution. A volume was adjusted with the same solvent and filtered using 0.45-μ membrane filter. (500µg/ml of Iptacopan).
Preparation of Standard working solutions (100% solution): 1ml from each stock solution was pipetted out and taken into a 10ml volumetric flask and made up with diluent. (50µg/ml of Iptacopan).
Preparation of 50% Spiked Solution: 0.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.
Preparation of 100% Spiked Solution: 1.0ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.
Preparation of 150% Spiked Solution: 1.5ml of sample stock solution was taken into a 10ml volumetric flask, to that 1.0ml from each standard stock solution was pipetted out, and made up to the mark with diluent.
Acceptance Criteria:
The % Recovery for each level should be between 98.0 to 102.
Robustness: Small deliberate changes in method like Flow rate, mobile phase ratio, and temperature are made but there were no recognized change in the result and are within range as per ICH Guide lines.
Robustness conditions like Flow minus (0.3ml/min), Flow plus (0.5ml/min), mobile phase minus, mobile phase plus, temperature minus (42.2°C) and temperature plus (53.2°C) was maintained and samples were injected in Triplicate manner. System suitability parameters were not much effected and all the parameters were passed. %RSD was within the limit.
LOD sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flasks and made up with diluents. From the above solutions 0.1ml each of Iptacopan, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluents
LOQ sample Preparation: 0.25ml each from two standard stock solutions was pipetted out and transferred to two separate 10ml volumetric flask and made up with diluent. From the above solutions 0.3ml each for Iptacopan, solutions respectively were transferred to 10ml volumetric flasks and made up with the same diluent.
Degradation studies:
Oxidation:
To 1 ml of stock solution of Iptacopan, 1 ml of 20% hydrogen peroxide (H2O2) was added separately. The solution was kept for 30 min at 600c. For UPLC study, there resultant solution was diluted to obtain 50µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Acid Degradation Studies:
To 1 ml of stock solution Iptacopan, 1ml of 2N Hydrochloric acid was added and refluxed for 30mins at 600c.The resultant solution was diluted to obtain 50µg/ml solution and 10µlsolution were injected in to the system and the chromate grams were recorded to assess the stability of sample.
Alkali Degradation Studies:
To 1 ml of stock solution Iptacopan, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 600c. The resultant solution was diluted to obtain 50µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Dry Heat Degradation Studies:
The standard drug solution was placed in oven at 105°C for 6h to study dry heat degradation. For UPLC study, the resultant solution was diluted to 50µg/ml solution and10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.
Photo Stability studies:
The photochemical stability of the drug was also studied by exposing the 500µg/ml solution to UV Light by keeping the beaker in UV Chamber for 7days or 200 Watt hours/m2 in photo stability chamber. For UPLC study, the resultant solution was diluted to obtain 50µg/ml solutions and 10µl were injected into the system and the chromatograms were recorded to assess the stability of sample.
Neutral Degradation Studies:
Stress testing under neutral conditions was studied by refluxing the drug in water for 6hrs at a temperature of 60º. For UPLC study, the resultant solution was diluted to 50µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.
RESULTS AND DISCUSSION
Method development: Method development was done by changing various, mobile phase ratios, buffers etc.
 
Optimized method:
Chromatographic conditions:
Mobile phase : Methanol : 0.01N AmmoniumAceatate(30:70 v/v)
Flow rate : 0.4 ml/min
Column : CSH C18 Column1.7 um, 2.1 mm X 150 mm
Detector wave length : 308nm
Column temperature : 48.0°C
Injection volume : 5.0mL
Run time : 2.5 min
Results : Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated.
Fig 1 shows Optimized chromatogram.
Observation: Iptacopan were eluted at 0.941min with good resolution. Plate count and tailing factor was very satisfactory, so this method was optimized and to be validated.
System suitability: All the system suitability parameters were within the range and satisfactory as per ICH guidelines.
Table: 1. System suitability parameters for Iptacopan
S no Iptacopan
Inj RT (min) USP Plate Count Tailing
1 1.181 3169 1.57
2 1.182 3154 1.56
3 1.184 3151 1.56
4 1.184 3145 1.56
5 1.186 3148 1.55
6 1.187 3148 1.54

Discussion: According to ICH guidelines plate count should be more than 2000, tailing factor should be less than 2 and resolution must be more than 2. All the system suitable parameters were passed and were within the limits.
Validation:
Specificity:
Discussion: Retention time of Iptacopan was 0.941 min. We did not found and interfering peaks in blank and placebo at retention times of these drugs in this method. So this method was said to be specific.
Linearity:
Table 2. Linearity table for Iptacopan
Iptacopan
Conc (μg/mL) Peak area
0 0
12.5 924501
25 1827185
37.5 2783693
50 3633004
62.5 4465826
75 5338578
Discussion: Six linear concentrations of Iptacopan (12.5-75µg/ml) were injected in a Triplicate manner. Average areas were mentioned above and linearity equations obtained for Iptacopan was y = 71155x + 42091.5 Correlation coefficient obtained was 0.999 for the two drugs.
 
System Precision:
Table 3. System precision table of Iptacopan
S. No Area of Iptacopan
1. 2413367
2. 2472139
3. 2488910
4. 2415511
5. 2460335
6. 2467573
Mean 2452973
S.D 31299.2
%RSD 1.3
Discussion: From a single volumetric flask of working standard solution six injections were given and the obtained areas were mentioned above. Average area, standard deviation and % RSD were calculated % RSD obtained as 1.3% for Iptacopan. As the limit of Precision was less than "2" the system precision was passed in this method.
Repeatability:
Table 4. Repeatability table of Iptacopan
S. No Area of Iptacopan
1. 2464351
2. 2440587
3. 2450668
4. 2469069
5. 2476286
6. 2456332
Mean 2459549
S.D 12981.5
%RSD 0.5
Discussion: Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated and obtained as 0.5% for Iptacopan. As the limit of Precision was less than "2" the system precision was passed in this method.
Intermediate precision (Day_ Day Precision):
Table 5. Intermediate precision table of Iptacopan
S. No Area of Iptacopan
1. 2486807
2. 2411176
3. 2448682
4. 2430573
5. 2435702
6. 2449418
Mean 2443726
S.D 25337.0
%RSD 1.0

Discussion: Multiple sampling from a sample stock solution was done and six working sample solutions of same concentrations were prepared, each injection from each working sample solution was given on the next day of the sample preparation and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated and obtained as 1.0% for Iptacopan. As the limit of Precision was less than "2" the system precision was passed in this method.
 
Accuracy:
Table 6. Accuracy table of Iptacopan
% Level Amount Spiked
(μg/mL) Amount recovered
(μg/mL) % Recovery Mean %Recovery
50% 25 25.02 100.09 99.90%
25 24.98 99.92
25 24.79 99.15
100% 50 50.40 100.80
50 49.64 99.28
50 49.55 99.11
150% 75 75.41 100.55
75 74.70 99.60
75 75.47 100.63

Discussion: Three levels of Accuracy samples were prepared by standard addition method. Triplicate injections were given for each level of accuracy and mean %Recovery was obtained as 99.90% for Iptacopan.
Sensitivity:
Table 7. Sensitivity table of Iptacopan
Molecule LOD LOQ
Iptacopan 0.35 1.06

 
Robustness:
Table 8. Robustness data for Iptacopan
S.no Condition %RSD of Iptacopan
1 Flow rate (-) 0.25ml/min 0.8
2 Flow rate (+) 0.35ml/min 0.5
3 Mobile phase (-) 65B:35A 0.8
4 Mobile phase (+) 55B:45A 0.5
5 Temperature (-) 25°C 1.5
6 Temperature (+) 35°C 0.9

Discussion: Robustness conditions like Flow minus (0.25ml/min), Flow plus (0.35ml/min), mobile phase minus (55:45A), mobile phase plus (65B:35A), temperature minus (25°C) and temperature plus(35°C) was maintained and samples were injected in Triplicate manner. System suitability parameters were not much affected and all the parameters were passed. %RSD was within the limit.
Assay: bearing the label claim Iptacopan 100mg.Assay was performed with the above formulation. Average % Assay for Iptacopan obtained was 100.17%.
Table 9. Assay Data of Iptacopan
S.no Standard Area Sample area % Assay
1 2413367 2464351 100.36
2 2472139 2440587 99.40
3 2488910 2450668 99.81
4 2415511 2469069 100.56
5 2460335 2476286 100.85
6 2467573 2456332 100.04
Avg 2452973 2459549 100.17
Stdev 31299.2 12981.5 0.5287
%RSD 1.3 0.5 0.53


DEGRADATION
Degradation Studies: Degradation studies were performed with the formulation and the degraded samples were injected. Assay of the injected samples was calculated and all the samples passed the limits of degradation.
Table 10. Degradation Data of Iptacopan
S.NO Degradation Condition % Drug Degraded % Drug Un Degraded
1 Acid 97.47 2.53
2 Alkali 94.86 5.14
3 Oxidation 93.45 6.55
4 Thermal 97.61 2.39
5 UV 98.01 1.99
6 Water 99.31 0.69

Discussion: Regarding the pH adjustment in mobile phase for the acid and base degradation studies have movement in retention time of drugs. But due to neutralized acid sample with 2N Base solution and base sample with 2N Acid solution there will be no change in retention time.
SUMMARY AND CONCLUSION
Summary Table
Parameters Iptacopan Limit
Linearity
Range(µg/ml) 12.5-75µg/ml



R< 1
Regression coefficient 0.999
Slope(m) 71155
Intercept(c) 42097
Regression equation
(Y=mx+c) y = 71155x + 42097
Assay (% mean assay) 100.17% 90-110%
Specificity Specific No interference of any peak
System precision %RSD 1.3 NMT 2.0%
Method precision %RSD 0.5 NMT 2.0%
Accuracy %recovery 99.90% 98-102%

LOD 0.35 NMT 3
LOQ 1.06 NMT 10

Robustness
FM 0.8 %RSD NMT 2.0
FP 0.5
MM 0.8
MP 0.5
TM 1.5
TP 0.9

Conclusion
An easy, sensitive, specific and precise RP-UPLC method for the pharmaceutical dose estimation of Iptacopan in Oral suspension. Retention time of Iptacopan was found to be 0.941 min. %RSD of the Iptacopan were and found to be 1.3. %RSD of Repeatability precision of Iptacopan was found to be 0.5. %Recovery was obtained as 99.90% for Iptacopan. %Assay was obtained as 100.17% for Iptacopan. LOD, LOQ values obtained from regression equation of Iptacopan were 0.35, 1.06. Regression equation of Iptacopan is y = 7155x + 42097 Retention times were decreased and that run time was decreased, so the method developed was simple and economical that can be adopted in regular Quality control test in Industries.
 
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, Claims:1. A method for estimation of Iptacopan in pharmaceutical dosage form using RP-UPLC, comprising:
a) dissolving Iptacopan using Ammonium Acetate: Methanol taken in the ratio 70:30 %v/v;
b) running chromatogram through column C18, 1.7 um, 2.1 mm X 150 mm using mobile phase of step a);
c) optimizing conditions of column of step b) at flow rate 0.4ml/min, detecting wavelength at 308nm, temperature maintained at 48.0°C and run time 2.5min;
d) running the sample of step c) and recording chromatogram from the chromatograph for estimation of Iptacopan.
2. The method for estimation of Iptacopan as claimed in claim 1, wherein the concentration of Iptacopan sample solution 50µg/ml.
3. The method for estimation of Iptacopan as claimed in claim 1, wherein the Retention time of Iptacopan is 0.941min; %RSD of the Repeatability precision of Iptacopan is 0.5.
4. The method for estimation of Iptacopan as claimed in claim 1, wherein the LOD value obtained from regression equation of Iptacopan is 0.35, LOQ value obtained from regression equation of Iptacopan is 1.06; Regression equation of Iptacopan is y=7155x + 42097 and %Recovery is 99.90%.
5. The method for estimation of Iptacopan as claimed in claim 1, wherein the invention provides a conduction of forced degradation studies of Iptacopan under different stress conditions.
6. A process for the preparation of sample stock solutions, comprising:
a) dissolving a volume of ml of oral suspension containing amount equivalent to 25 mg Iptacopan using about 3/4th ml of solvent in 100-ml volumetric flask;
b) sonicating for 10 min to obtain clear solution;
c) adjusting the volume with the same solvent and filtering using 0.45-μ membrane filter to obtain 500µg/ml of Iptacopan.
7. A process for the preparation of sample working solution, comprising:
a) transferring 1ml of filtered sample stock solution to 10ml volumetric flask; and b) making up volume with diluent to obtain 50µg/ml of Iptacopan.
8. The method for estimation of Iptacopan as claimed in claim 1, wherein the process for the preparation of standard stock solution comprising:
a) weighing accurately 25mg of Iptacopan and transferring to 50ml volumetric flask;
b) adding 3/4th of diluent to the flask and sonicating for 10 minutes and making up the flask with diluents and labeled as Standard stock solution of 500µg/ml of Iptacopan;
9. The method for estimation of Iptacopan as claimed in claim 1, wherein the process for the preparation of standard working solution comprising of preparing Standard working solutions of 100% solution by pipetting out 1ml from stock solution and taking into a 10ml volumetric flask and making up with diluent to obtain 50µg/ml of Iptacopan.
10. The method for estimation of Iptacopan as claimed in claim 1, wherein the developed method is simple and economical and can be used in regular Quality control test in Industries as a routine analysis of Iptacopan.

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