1. INTRODUCTION:

Thalassemia syndromes are a diverse group of hemoglobin abnormalities. Both a- or b-thalassemia are types of thalassemia, depending on the molecular abnormalities on the defective globin chain.^1-2The chemical name of a deferiprone is a 3-Hydroxy-1, 2-dimethyl-4(1H)-pyridone or 1,2-dimethyl-3- hydroxypyridin-4-one. It has a molecular formula of C₇H₉NO₂ and molecular weight of 139.152g/mol. Deferiprone is slightly soluble in ethanol and chloroform and sparingly soluble in water and methanol.Deferiprone is a member of the family of chelating compounds known as alpha-ketohydroxypyridines.^3-5

Deferiprone is a second line iron chelating medication used as a backup treatment for thalassemia.Deferiprone is bidentate iron chelator that interacts with Fe³⁺ to generate a metal-drug complex with a binding ratio of 1:3 and eliminate the excess iron from body through urine. The [Fe(Deferiprone)₃] complex is a neutral hydrophilic compound that is stable over a wide pH range.^3-5

Figure 1: Chemical structure of deferiprone

Analytical QbD in method development is an important component of the pharmaceutical quality system, pharmaceutical development, and risk management.⁶ There are very few methods reported in the literature for analysis of deferiprone in bulk or its pharmaceutical formulation by UV and RP-HPLC, LC-MS and in a combination with other iron chelators. Bioanalytical RP- HPLC method was also found for deferiprone determination in its capsule dosage form and RP-HPLC method was also found for the estimation of deferiprone and its impurity.^7-14

In the literature review for the quantitative analysis of deferiprone in its pharmaceutical formulations, there were no reports of stability indicating HPLC method. Hence, the objective of this study was to develop and validate a stability indicating reliable, accurate RP-HPLC method for the analysis of deferiprone in its capsule dosage form.

2. MATERIALS AND METHODS:

2.1 Instrumentation:

For the spectrophotometric measurements Lab India UV3200+ Spectrophotometer, UV-Vis Analyst software was used. ThermoFisher Scientific separation module, using Chromelon Version 7.30 software with UV-Visible spectrophotometer detector LC instrument control was used for chromatographical analysis for analytical method development. Chromatographic analysis for force degradation study was performed on WATERS 2695 Alliance HPLC system equipped with WATERS 2996 Photo Diode Array Detector. Empower 2 software was used for data processing and evaluation. All weighing was done with a Sartorius Analytical Balance.

2.2 Chemicals and Reagent:

An analytically pure deferiprone working standard was procured from Central Drug Testing Laboratory, Mumbai with defined potency [99.24%) as is basis]. Deferiprone capsule formulation (KELFER containing 500 mg of drug content, CIPLA LTD, India) was received as a gift sample from Central Drug Testing Laboratory, Mumbai.

Phosphoric acid (H₃PO₄) and potassium dihydrogen phosphate (KH₂PO₄) from Clairofilt India Ltd, Triethylamine (C₆H₁₅N) from Sisco Research Laboratories, Methanol (HPLC grade) from Merck life science Pvt. Ltd, were used. The ultra-purified HPLC grade distilled water was obtained from the Milli-Q® system (Millipore, Milford, MA, USA) water purification unit. A high flownylon membrane filter (0.45μm) was purchased from Axiva Sichem Pvt. Ltd.

3. EXPERIMENTAL WORK:

3.1 Selection of suitable solvent:

Considering the chemical nature and solubility data of deferiprone, the suitable diluent was selected for standard and sample preparations.

3.2 Selection of wavelength of maximum absorbance of deferiprone:

Deferiprone standard 10mg was transferred to a 100ml volumetric flask and the volume was made up to the mark with water: methanol (50:50) to get concentration (100µg/ml) and further dilutions were made to get a concentration of 10µg/ml. This solution was scanned in 200-400nm region.

3.3 Preparation of standard solution:

Deferiprone standard 10mg was transferred to a 100ml volumetric flask and the volume was made up to the mark with water: methanol (50:50) to get concentration (100µg/ml).

3.4 Preparation of marketed sample solution:

10capsules of deferiprone weighed separately and net content of capsules were determined. A weight of powder of capsule equivalent to 100mg transferred to 100mL flask and volume was made up to the mark 100 ml with water: methanol (50:50) to get a concentration of 1000ug/ml and further dilutions were made to get a concentration of 100µg/ml.

3.5 Preparation of the mobile phase:

3.5.1 Preparation of buffer:

25mM potassium phosphate was prepared by weighing 3.4022gm of potassium dihydrogen phosphate transferring to a 1000ml mobile phase bottle and add 1 ml of trimethylamine and add distilled water up to 1000 ml, and sonicating for a few minutes using an ultra sonicator. Further, PH was adjusted to 3.5 with orthophosphoric acid and vacuum filtered through a 0.45 μm high flow nylon membrane filter.

3.5.2 Preparation of mobile phase:

25mM Potassium dihydrogen phosphate and 1ml TEA in 1000ml at (pH 3.5) containing buffer and methanol in the ratio of (60:40% v/v) were used as mobile phase for the present study and sonicated, degassed before use.

3.6 Method optimization:

Deferiprone is a weakly basic non-polar compound. Based on the chemical nature of the molecule, different mobile phase systems and HPLC columns were tried to obtain a proper separation of the molecule. The initial trials were carried out on Inertsil-C18 column with a mobile phase of 0.1% orthophosphoric acid: methanol with different ratio with an injection volume was 20μl and a flow rate of 1ml/ min. However, poor peak shape was observed.

Chromatogram with a good peak shape was found with the mobile phase composition of 25mM Potassium dihydrogen phosphate and 1ml TEA in 1000ml at PH 3.5: Methanol in the ratio of [60:40%] v/v on Zorbax SB C18 (4.6*250mm,5um) column.

3.7 Method Validation:

The validation parameters for optimised chromatographic method are specificity, linearity, range, accuracy, precision, sensitivity (LOQ and LOD), and robustness, system suitability.^15-19

3.7.1 Specificity:

Specificity is one of the key characteristics of HPLC, which describes the analytical method's capacity to distinguish between the analyte and the other components in the complex mixture. By injecting 20ul solutions of the standard, sample, blank separately, the specificity of the technique was assessed.

3.7.2 Linearity:

Different standard solutions were made by diluting the standard stock solution with the diluent in different concentrations of deferiprone: 10, 50, 75, 100, 125, and 150ug/ml. This was done to assess the linearity and range of the method. The same circumstances were used to analyse three injections from each concentration. The calibration curve's linearity was assessed using linear regression analysis utilizing the least squares method.

3.7.3 Accuracy:

Recovery studies at three concentration levels (110%, 120%, and 130% using standard addition method) were used to evaluate the method's accuracy. There were three injections of each concentration of samples.

3.7.4 Precision:

The degree of consistency of results from repeated measurements is referred to as precision.

3.7.4.1 System precision:

The HPLC system injected six replicate injections of a standard deferiprone solution (100ug/mL). The mean, SD, and %RSD were reported.

3.7.4.2 Method precision:

The method precision was established using six assays of the sample solution at 100ug/ml concentration levels, carried out on two different days. The mean, SD, and %RSD findings of the developed approach were calculated to evaluate the repeatability of the results. Freshly prepared 6 injections of standard solution and 6 injections of sample solution were injected at varied time intervals on the same day (intraday) and on two separate days (interday) to assess the repeatability of the developed method. The mean, SD, and %RSD were reported.

3.7.5 Robustness:

The proposed method's robustnes was evaluated by altering the flow rate (±0.2ml), temperature (±5°C), and the wavelength (±2nm). With these chromatographic conditions, deferiprone sample solutions were prepared and injected in triplicates. Six replicate injections of the effective standard solution were also performed. Mean, SD, and %RSD estimated percentages were reported.

3.7.6.LOD and LOQ:

In terms of LOD and LOQ, the method's sensitivity was calculated. The slope of the calibration curve and the standard deviation of the y-intercept were used to compute the limits of detection (LOD) and quantification (LOQ).

LOD= 3.3 σ /S and LOQ= 10 σ /S,

Where S is the slope obtained from the calibration curve and σ stands for the regression line's standard deviation.

3.7.7 System suitability:

The deferiprone standard solution five replicate injections were given at a working concentration of 100 ug/ml. The system suitability was tested by calculating and comparing %R.S.D of the chromatographic parameters such as peak area, retention time, theoretical plates, and tailing factor of a standard solution of deferiprone.

3.8 Forced degradation studies:

Deferiprone sample solution was exposed to thermal, oxidative, hydrolytic, photolytic, acidic, and basic stress conditions.^20-21

3.8.1 Acid degradationl:

1ml from deferiprone sample stock solution (1000 ug/ml) was pipetted out in 10ml volumetric flask. 3ml of 5N hydrochloric acid (HCL)was added into this solution and diluted with diluent. Finally, the volume was made upto 10ml to get concentration 100ug/ml. This prepared solution was heated to evaporate for 3 hours on a water bath at 60°C and the same concentration was left in HCL for cooling at room temperature. When this time was over, each solution had been neutralized and injected into HPLC system.

3.8.2 Base degradation:

1 ml from deferiprone sample stock solution (1000 ug/ml) was pipetted out in 10ml volumetric flask. 3ml of 5N sodium hydroxide (NaOH)was added into it and then this solution was diluted up to 10ml with the diluent to obtain concentration 100ug/ml. This resultant solution was heated to evaporate for 3 hours on a water bath at 60°C and the same concentration was left in NaOH for cooling at room temperature. After this period, each solution was neutralized, and this resultant solution was injected into the HPLC system.

3.8.3 Peroxide degradation:

1ml of the deferiprone sample stock solution (1000 ug/ml) was pipetted out in 10ml volumetric flask.1ml 3% H₂O₂ was added in it and diluted up to 10ml with the diluent to obtain concentration 100ug/ml. This prepared solution was allowed to stand for1 hour and thisprepared sample solution was injected into the HPLC system.

3.8.4 Thermal degradation:

The deferiprone sample was kept in a LOD bottle for dry heat at 60⁰C for 1 hour. Deferiprone sample which kept for dry heat containing 10mg was transferred to a 100ml volumetric flask and the volume was made up to the mark with diluent obtain concentration (100µg/ml). This sample solution was injected into the HPLC system.

3.8.5 Water degradation:

1ml of deferiprone sample stock solution (1000ug/ml) was pipetted out in 10ml volumetric flask and diluted with water up to 10ml to obtain concentration 100 ug/ml. The solution was kept under water bath at 60°C for 3hours and then injected into HPLC system.

3.8.6 Photolytic degradation:

1ml from deferiprone sample stock solution (1000 ug/ml) was pipetted out in 10ml volumetric flask. This solution was diluted with diluent up to 10ml to get concentration 100ug/ml. Then, the solution was exposed to UV light at 254nm for 48hours. Later, this solution was injected to the HPLC system.

4. RESULT AND DISCUSSION:

4.1 Selection of suitable solvent:

The Water: Methanol in the ratio of (50:50) was selected as a diluent for further study.

4.2 Selection of wavelength of maximum absorbance of deferiprone:

The deferiprone standard solution was examined UV spectroscopically at a wavelength of 281nm. By considering a literature review, the working wavelength for further chromatographic analysis of deferiprone was 280nm.

Figure 2: UV spectrum of deferiprone

4.3 Method optimization:

The RP-HPLC separation was done on C18 column with a short retention time, good resolution, the use of cost-effective solvents, and convenience of preparation.High chromatographic response peak and the best possible resolution achieved for quantitative analysis.

Table 1:- Optimized chromatographic conditions

Chromatographic parameters	Optimized conditions
HPLC System	ThermoFisher Scientific separation module
Column	Zorbax SB C18 column (4.6*250 mm,5 um)
Mobile phase	25 mM Potassium dihydrogen phosphate and 1ml TEA in 1000 ml (PH 3.5): Methanol [60:40%] v/v
Diluent	water: methanol (50:50) v/v
Chromatographic program	Isocratic
Flow rate	0.6 ml/min
Wavelength	280 nm
Injection volume	20µl
Column oven temperature	40°C
Detector	UV-Visible
Run time	10 min
Retention time	4.7 min

4.4 Method validation:

4.4.1 Specificity:

The chromatograms of the blank, standard, and sample solutions are represented in Figure 3. There are no co-eluting peaks seen at the retention time of deferiprone. This implies that the analyte peak was pure, which confirms the method's specificity

4.4.2 Linearity:

The calibration curve was created by plotting a graph of mean of peak area vs concentration. The calibration curve's linearity was evaluated using linear regression analysis. Linearity experiments revealed a linear relationship over the concentration range of 10-150 μg/mL is reported in Table 2. The regression equation was as follows: Y = 2782.3x+701.27. The co-relation coefficient was 0.9996, which met the approval criteria for analytical method validation as represented in Figure 4.

Table 2: linearity studies of deferiprone

Concentration	Area
10	31260
50	139654
75	203734
100	279007
125	350597
150	418935

Figure 4: linearity curve of deferiprone

4.4.3 Accuracy:

Deferiprone sample solutions had a mean % recovery of 99.37%, which was within the limit of 98-102% is given in Table 3.

Table 3: Accuracy studies of deferiprone

% Level	Std spiked (ml)	Amount recovered (%)	% Recovery	Mean % recovery	SD	% RSD
100	0	99.0	99.0	99.0	0.045	0.045
100	0	99.0	99.0
100	0	99.1	99.1
110	1	108.5	98.7	98.8	0.113	0.114
110	1	108.7	98.8
110	1	108.8	98.9
120	2	119.9	100.0	99.9	0.030	0.030
120	2	119.9	99.9
120	2	119.9	99.9
130	3	129.7	99.8	98.8	0.039	0.039
130	3	129.8	99.8
130	3	129.7	99.7

4.4.4 Precision:

Table 4: System precision data of deferiprone

Sample No.	Peak area
1	273657.49
2	273382.04
3	273389.30
4	273286.61
5	275086.45
6	276843.56
Mean	274274.24
SD	1428.01
% RSD (NMT 2%)	0.52

Table 5: Method precision studies of deferiprone

Sr. No.	Intraday precision		Interday precision
Sr. No.	Afternoon	Evening	Day 1	Day 2
1	99.08	99.08	99.08	99.97
2	99.37	98.65	99.37	99.96
3	99.38	99.37	99.38	100.00
4	99.08	99.17	99.08	99.71
5	99.85	99.85	99.85	99.81
6	99.13	99.38	99.13	99.81
Mean	99.32	99.25	99.32	99.88
SD	0.272	0.396	0.272	0.12
% RSD (NMT 2%)	0.27	0.399	0.27	0.117

The % RSD for peak regions of deferiprone standard solution was determined to be 0.52 in system precision is shown in Table 4. The mean assay percentage results of deferiprone sample solutions were found to be within the acceptable range. The intraday and interday % RSD was reported in Table 5.

4.4.5 Robustness:

The % RSD of the test with changes in method parameters was less than 2.0%, and the results were unaffected, and summarized in Table 6.

Table 6: Robustness studies of deferiprone

Para meter	Change in parameter	% Estimation	Mean	SD	% RSD
Wavelength (nm)	278	105.40	104.8	0.65	0.62
	280	104.10
	282	104.79
Flow rate (mL/min)	0.4	102.1308	102.92	0.71	0.68
	0.6	103.4785
	0.8	103.1637
Temperature ( ̊C)	35	104.46	104.3	0.19	0.18
	40	104.09
	45	104.31

4.4.6 LOD and LOQ:

The LOD and LOQ were determined by slope of a calibration curve obtained from linearity. As a result, the approach is found to be sensitive throughout a wide range.

Where, Regression equation: y= 2782.3x+ 701.27, Standard Error: 2836

Limit of Detection (LOD): 3.364 ug/ml

Limit of Quantitation (LOQ): 10.19 ug/ml

4.4.7 System suitability:

The system suitability test was an integral chromatographic techniques development that was carried out in accordance with ICH (Q2) guidelines. The % RSD of system suitability parameters such as peak area, retention duration, theoretical plates, and tailing factor of standard solution is within limit and mentioned in Table 7.

Table 7: system suitability parameters of deferiprone

System suitability parameters	Mean	SD	% RSD (NMT 2%)
Retention time	4.75	0.001	0.03
Peak area	280187.39	1308.32	0.47
Tailing factor	1.37	0.02	1.40
Theoretical plates	7731.2	118.37	1.53

4.4.7 Assay:

The obtained test results of assay exhibited good percentage recoveries and low SD values, confirming that the method is appropriate for routine analysis of deferiprone in tablet dosage form and is tabulated in Table 8.

Table 8: Assay results of deferiprone

Sr. No.	Weight of standard	Weight of sample	Average area of standard	Area of sample	% Assay
1	10.52 mg	104.52 mg	278676.56	267070.91	99.60
2				268850.94	100.26
3				267952.22	99.93
4				267520.70	99.77
5				268157.63	100.008
Mean					99.91
SD					0.2504
%RSD (NMT 2%)					0.2506

4.5 Force degradation studies:

Peak area differences indicate degradation when compared to pre-analyzed deferiprone solutions. Chromatograms of acidic, alkaline, and oxidative degradation, as well as thermal, photolytic, and water degradation, revealed additional peaks indicating mild degradation were shown in Figure 5.

The most severe deterioration, 93.75%, was seen under oxidative conditions. Deferiprone showed degradation slightly by acidic hydrolysis and less slightly by basic hydrolysis. Deferiprone was found to be more stable in hydrolytic, thermal, and photolytic stress conditions, resulting in no degradations as described in Table 9. Under established conditions, the degradation products generated were well resolved. Peak purity of the primary peak was found to be 100% under each circumstance because the peak of degradation products was successfully isolated and resolved from the main peak of deferiprone without any interference.

5. DISCUSSION:

The simple, and precise stability-indicating RP-HPLC method of deferiprone was developed. Deferiprone was eluted at a retention time of 4.7 min with a good peak shape. All validation parameter results were found to be within the specified limits.

Deferiprone shows minor degradation in oxidative circumstances, but it is stable in acidic, alkaline, thermal, water, and photolytic stress conditions, according to the stress study. During forced degradation tests, the degradation products were isolated from the active pharmaceutical component which indicates 100 % purity of deferiprone. The results of assay showed that, the suggested RP-HPLC method can be employed for routine deferiprone analysis in capsule dosage form.

6. CONCLUSION:

The unique, simple, accurate, precise, sensitive, and stability-indicating RP-HPLC method of deferiprone was discovered sucessfully. All validation parameters were found to be within the acceptable limits according to ICH guidelines. The current method also indicates the stability study of deferiprone. It shows that this method is also applicable for routine analysis of deferiprone in pharmaceutical capsule dosage form.

7. ACKNOWLEDGEMENT:

The authors are thankful to the Management of Gahlot Institute of Pharmacy College, Koparkhairane, and Navi Mumbai for the encouragement to carry out this project work. The authors are extremely thankful for Cipla Ltd, India for the gift sample of deferiprone capsule formulation.

Authors are extremely thankful for Central Drug Testing Laboratory, Mumbai Ltd for providing facilities of sophisticated Instrumentations for carrying out this analytical research work. Special thanks to Dr. Vijay Kumar, Mr. Ashok Kumar and Pankaj Thakur, Hemant Magar, and others for their valuable guidance and support. Special thanks to Dr. V. H. Bhaskar (Principle), Dr. Bharti Fegade (Assist. Prof.), of Gahlot institute of Pharmacy College, for providing technical guidance and for checking the manuscript.

8. REFERENCE:

1. Brancaleoni V, Di Pierro E, Motta I, Cappellini MD. Laboratory diagnosis of thalassemia. International Journal of Laboratory Hematology. 2016; May; 38: 32-40. doi:10.1111/ijlh.12527

2. Entezari S, Haghi SM, Norouzkhani N, Sahebnazar B, Vosoughian F, Akbarzadeh D, Islampanah M, Naghsh N, Abbasalizadeh M, Deravi N. Iron chelators in treatment of iron overload. Journal of Toxicology. 2022 May 5; 2022. doi:10.1155/2022/4911205

3. Wijaya AB, Marhaeni W, Devi WR, Saputra M, Rahman G. Oxidative Stress and Renal Function in Pediatric Patients with Beta Thalassemia Major (β-TM) Receiving Deferiprone and Deferasirox: A Cross-Sectional, Single Center Study. Research Journal of Pharmacy and Technology. 2023 Mar 1; 16(3): 1225-30. doi: 10.52711/0974-360X.2023.00203

4. Chaudhary JL, Rathi SG, Shah S, Patel JT, Vaghela S. Deferiprone (Ferriprox) as newer iron Chelator in Thalassaemia major patients. Research Journal of Pharmacy and Technology. 2021; 14(2): 1041-4. doi: 10.5958/0974-360X.2021.00186.4

5. Marchande DD, Jain A. Deferiprone: A review of Analytical methods. International Journal of Pharmaceutical Research and Applications. 2022; Dec 22; 1724-30 doi: 10.35629/7781-070617241730

6. Vedantika Das, Bhushan Bhairav, R. B. Saudagar. Quality by Design approaches to Analytical Method Development. Research Journal of Pharmacy and Technology 2017; 10(9): 3188-3194. doi: 10.5958/0974-360X.2017.00567.4

7. Malik A, Firke S, Patil R, Shirkhedkar A, Kalaskar M. Development and validation of zero and first-order derivative area under curve spectrophotometric methods for the determination of deferiprone in bulk material and capsules. Asian Journal of Pharmaceutical Analysis. 2019; 9(2): 49-54. doi: 10.5958/2231-5675.2019.00011.5

8. Sachin kothawade N, vishal pande V. Development and validation of an rp-hplc method for deferiprone estimation in pharmaceutical dosage form. Asian Journal of Pharmaceutical and Clinical Research. 2023; April 7; 16(4): 100-03.doi: 10.22159/ajpcr.2023.v16i4.47000

9. Mounika A, Kiran Kumar V. Method development and validation for deferiprone by RP-HPLC method. Indo American Journal of Pharmaceutical Sciences. 2022; 09(12). doi: 10.5281/zenodo.7548262

10. Sireesha KS, Sai Kiran BS, Sekhar KB, Muneer S. A new RP-HPLC method development and validation of deferiprone in bulk and its pharmaceutical dosage form. International Journal of Advanced Research. 2016; 4(8): 2174-9. doi: 10.21474/IJAR01/1435

11. Abbas M, Nawaz R, Iqbal T, Alim M, Asi MR. Quantitative determination of deferiprone in human plasma by reverse phase high performance liquid chromatography and its application to pharmacokinetic study. Pakistan Journal of Pharmaceutical Sciences. 2012; Apr 1; 25(2).

12. Fares MY, Hegazy MA, El-Sayed GM, Abdelrahman MM, Abdelwahab NS. Quality by design approach for green HPLC method development for simultaneous analysis of two thalassemia drugs in biological fluid with pharmacokinetic study. RSC Advances. 2022; 12(22): 13896-916. doi: 10.1039/D2RA00966H

13. Sutar S, Patil P, Marchande D, Patil S, Bandgar S, Kumbhar R. RP-HPLC Method Development for Simultaneous Estimation of Oral Iron Chelator Deferiprone and its related Impurity. Research Journal of Pharmacy and Technology. 2023; Apr 29; 16(4): 1890-4. doi: 10.52711/0974-360X.2023.00310.

14. Asmari M, Abdel-Megied AM, Michalcová L, Glatz Z, El Deeb S. Analytical approaches for the determination of deferiprone and its iron (III) complex: Investigation of binding affinity based on liquid chromatography-mass spectrometry (LC-ESI/MS) and capillary electrophoresis-frontal analysis (CE/FA). Microchemical Journal. 2020; May 1; 154: 104556. doi: 10.1016/j.microc.2019.104556

15. Guideline ICH. Validation of analytical procedures: text and methodology. Q2 (R1). 2005; Nov; 1(20): 05.

16. Raul SK, Padhy GK, Mahapatra AK, Charan SA. An overview of concept of pharmaceutical validation. Research Journal of Pharmacy and Technology. 2014; 7(9): 1081-90.

17. Sowmya HG, Priya M, Murugan V. Implementation of quality by design approach for method development and validation: A review. Research Journal of Pharmacy and Technology. 2022; 15(1): 436-40. doi: 10.52711/0974-360X.2022.00072

18. Dara SK, Tiwari RN. Pharmaceutical process validation: An industrial perspective. Research Journal of Pharmacy and Technology. 2014; 7(7): 810-4.

19. Jetani JM, Pai KG. A comparative review of the USFDA guidelines on process validation focusing on the importance of quality by design (QbD). Research Journal of Pharmacy and Technology. 2017; 10(4): 1257-60. doi: 10.5958/0974-360X.2017.00223.2

20. Sutar SV, Yeligar VC, Patil SS. Stability Indicating Forced Degradation Studies. Research Journal of Pharmacy and Technology. 2019; 12(1): 429-33. doi: 10.5958/0974-360X.2019.00078.7

21. Kushwaha P. Significance of Stability Studies on Degradation Product. Research Journal of Pharmacy and Technology. 2009; 2(4): 621-7.

Received on 12.09.2023 Modified on 18.12.2023

Research J. Pharm. and Tech 2024; 17(6):2725-2731.

DOI: 10.52711/0974-360X.2024.00427

Degradation Type	Stress Conditions	Retention time of degradation products (min)	% Residual drug	Peak Purity (%)
Control standard and sample solution	Not exposed to any stress conditions	-	100	100
Acid degradation	3ml 5N HCl at 60 ⁰C for 3 hr	3.675, 4.102, 7.757	97.23	100
Alkali degradation	3ml 5N NaOH at 60⁰C for 3 hr	3.894, 4.085	98.90	100
Peroxide degradation	1ml 3% H₂O₂ for 1 hr	3.995 , 7.567	93.75	100
Thermal degradation	Under dry heat 60⁰C for 1 hr	3.824,4.133	99.83	100
Water degradation	Under water bath at 60⁰C for 3 hr	1.605, 1.710 3.912	99.61	100
Photolytic degradation	Under UV light at 254 nm for 48 hr	3.935, 4.251	99.41	100