Development and Validation of a Stability-Indicating RP-HPLC Method for Simultaneous Estimation of Gimeracil, Oteracil, and Tegafur in Bulk and Dosage forms in the presence of Stress Degradation Impurities

Gandhi Santosh Kumar, Badal Kumar Mandal*

Department of Chemistry, School of Advance Sciences, Vellore Institute of Technology (VIT),

Vellore, Tamil Nadu – 632014, India.

*Corresponding Author E-mail: badalmandal@vit.ac.in, santoshkumar.gandhi@gmail.com

ABSTRACT:

In this study, an efficient, reproducible, and reliable stability indicating reverse-phase high-performance liquid chromatography (HPLC) method has been developed for the simultaneous quantification of Tegafur (TGFR), Gimeracil (GMRL), and Oteracil (ORCL) in bulk and dose formulation with a shorter run time (6min).TFGR, GMRL, and ORCL were separated using an isocratic method on a Waters C18 (250 mm x 4.6 mm × 5 µm) column with a mobile phase containing methanol and phosphate buffer (0.1M KH₂PO₄, pH 4.0) in a 50:50 (v/v) ratio. The detection wavelength was 275 nm and the flow rate was 1 mL/min. The new method demonstrated excellent linearity for TFGR (10 to 30µg/ml), GMRL (2.9 to 8.7µg/ml), and ORCL (7.9 to 23.70µg/ml). The method was found to be precise and accurate, with an RSD of 0.057% to 0.183% and a recovery rate of 98.66% to 100.52%. Stress degradation experiments have demonstrated that this method can be utilized to estimate TFGR, GMRL, and ORCL in the presence of their degradants. The method is suitable for determining TFGR, GMRL, and ORCL in bulk and capsule formulation (Tegonat).

KEYWORDS: Stomach carcinoma, S-1 Therapy, Quality control, Stress Degradation, RP-HPLC.

INTRODUCTION:

Stomach carcinoma remains one of the most common and deadly neoplasms worldwide.^1-5 The stomach has a mucus membrane that is made up of glands and columnar epithelial cells. These cells are susceptible to gastritis, an inflammatory condition that may cause peptic ulcers and, in the long run, stomach cancer.^6,7 In 2018, stomach cancer was estimated to have caused 783,000 fatalities, making it the third deadliest malignancy and the fifth most common neoplasm. Teysuno (S1) is an oral anticancer medication that contains potassium Oteracil, a therapy that prevents fluorouracil from being phosphorylated in the digestive system, Gimeracil, a dihydropyrimidine dehydrogenase inhibitor, and Tegafur, a prodrug of fluorouracil. The first-line treatments that are most effective for advanced, unresectable gastric cancer are S-1-based chemotherapy and S-1 and cisplatin together.

S-1 is additionally used to treat several cancers, most notably colorectal, gastric, breast, and pancreatic cancers.^8-12 It minimizes the development and proliferation of tumour cells, boosts the effectiveness of the treatment and lessens the likelihood of adverse effects. Pharmaceutical drug products must be marketed as being consistent, predictable, and therapeutically effective formulations. A crucial process in the pharmaceutical industry is quality control. Quality control is the sum of all the steps taken to ensure the identity and purity of a certain drug.^13,14 Therefore, it is essential to develop a reliable test method for quality control. Haritha et al. have only ever published a method for performing a quality control test on a combination of TFGR, GMRL, and ORCL.¹⁵ Analytical methods continue to be improved to develop ones that are simple to use, reliable, inexpensive, consistent, and, most importantly, have a high level of sensitivity, accuracy, and precision. The Haritha et al. technique, on the other hand, has a lengthy run duration of 12 minutes. An increase in execution time includes downsides such as consuming more solvent and requiring longer to complete each analysis. The combination of all of the aforementioned factors makes a single analysis costlier. Therefore, the goal of this investigation is to establish an efficient, reproducible, and reliable RP-HPLC technique to assay the mix of TFGR, GMRL, and ORCL with a shorter run time.

MATERIAL AND METHODS:

Chemicals and Reagents:

All analytical grade reagents, including hydrochloric acid, trifluoroacetic acid, sodium hydroxide, potassium dihydrogen phosphate, and hydrogen peroxide (30%, v/v), as well as HPLC grade acetonitrile, methanol were procured from Merck India. Milli-Q water was used throughout the analysis. Working standards of TFGR, GMRL, and ORCL and Tegonat capsules containing TGFR (20 mg labelled strength), GMRL (5.8 mg labelled strength), and ORCL (15.8 mg labelled strength) were procured from a local vendor.

Instruments and Method Conditions:

A "Waters" HPLC system (model 2995) with a PDA detector (model 2998) and Empower 2.0 software was used for the method development and validation. The separation was carried out on a Waters C18 reverse phase column (250 mm x 4.6 mm × 5 µm) using an isocratic method with a mobile phase containing methanol and phosphate buffer (0.1M KH₂PO₄, pH 4.0) in a 50:50 (v/v) ratio. The column temperature was kept at 25°C, the mobile phase flow rate was kept at 1.0 mL/min, and a UV detector with a 275 nm wavelength was used.

Preparation of standard solutions:

Stock TFGR, GMRL, and ORCL solution having concentrations of 200µg/ml (TGFR), 58µg/ml (GMRL) and 158µg/ml (ORCL) were prepared in diluent (0.1M KH₂PO₄ buffer: methanol, 1:1 v/v). Working TFGR, GMRL, and ORCL solution having concentrations of 20.0µg/ml (TGFR), 5.8µg/ml (GMRL) and 15.8µg/ml (ORCL) was made from stock TFGR, GMRL, and ORCL solution using diluent.

Preparation of calibration solutions:

TFGR, GMRL, and ORCL calibration solutions having concentrations ranging from 10 to 30µg/ml (TGFR), 2.9 to 8.7µg/ml (GMRL) and 7.9 to 23.70µg/ml (ORCL) were made from stock TFGR, GMRL, and ORCL solution using diluent. The calibration graphs for the TFGR, GMRL, and ORCL were generated by plotting the peak areas acquired by injecting and analyzing all calibration TFGR, GMRL, and ORCL solutions against the respective concentrations.

Preparation of sample solution (Tegonat):

To prepare a Tegonat sample solution with concentrations of 200µg/ml (TGFR), 58µg/ml (GMRL), and 158µg/ml (ORCL), one Tegonat capsule (equivalent to 20 mg TGFR, 5.8 mg GMRL, and 15.8 mg ORCL) was placed into a 100 mL standard flask and dissolved in the diluent. After twenty minutes of ultra-sonication, the sample was filtered using a 0.45µ nylon syringe filter. To obtain concentrations of 20µg/ml of TGFR, 5.80µg/ml of GMRL, and 15.8µg/ml of ORCL the sample was further diluted using diluent. These Tegonat samples were assessed under the conditions specified in the "Instruments and Method conditions" section. Based on the obtained peak areas, the amount of TFGR, GMRL, and ORCL in the capsule (Tegonat) was calculated.

Preparation of forced degradation samples:

The method's specificity and stability indicating properties were assessed by forced degradation studies.^16-2010mL of Tegonat sample solution (200µg/ml for TGFR, 58µg/ml for GMRL, and 158µg/ml for ORCL) were pipetted into three separate 100 mL standard flasks. Thereafter, the sample solution was treated with 0.1 N HCl (10 ml), 0.1 N NaOH (10 ml), and (30%, v/v) H₂O₂ at room temperature for half an hour to promote acidic, alkaline, and oxidative degradation. The mixture was then neutralized and further diluted to volume using the diluent. Similarly, 10 mL of the Tegonat sample solution was subjected to thermal degradation for 30 minutes at 105°C in an oven and photolytic degradation for 6 hours under the sunlight. After cooling to room temperature, these solutions were further diluted to 100 mL with diluent, resulting in solutions that contained 20µg/ml (TGFR), 5.8µg/ml (GMRL), and 15.8µg/ml (ORCL). The method conditions listed in the "Instruments and maintained conditions" section were used when evaluating these forced degradation samples. The amount of TFGR, GMRL, and ORCL that remained after the forced degradation was determined by performing the assay. Using a PDA detector, the HPLC peak purity of the TFGR, GMRL, and ORCL peaks from the stressed Tegonat samples was assessed.

RESULTS AND DISCUSSION:

HPLC method development:

The isocratic method was used for the present study due to its many advantages over gradient elution, including greater simplicity, lower cost, and simpler instrumentation. To develop a reliable and robust method many different parameters were evaluated during method development, such as different column chemistries, type of buffer, pH of the mobile phase, the organic solvent used for the mobile phase, and the composition of the mobile phase^21-22. The resolution, peak symmetry, theoretical plates, and repeatability of the retention time for the TFGR, GMRL, and ORCL peaks were all investigated during the method optimization studies. The final method condition was optimized on Waters C18 reverse phase column (250 mm x 4.6 mm × 5 µm) using isocratic elution with a mobile phase containing methanol and phosphate buffer (0.1M KH₂PO₄, pH 4.0) in a 50:50 (v/v) ratio. The column temperature was kept at 25 °C, the mobile phase flow rate was kept at 1.0 mL/min, and a UV detector with a 275 nm wavelength was used. Under those particular experimental conditions, all of the TFGR, GMRL, and ORCL peaks were precisely separated and devoid of tailing (Figure 1).

Method Validation:

The validation research included an examination of the following components as per ICH guideline.²³

System Suitability:

System suitability (SS) is a commonly performed test to ensure that a chromatographic system is appropriate for a given analysis. It measures the system's repeatability, resolution, and column efficiency. The SS solution containing 20µg/ml (TGFR), 5.8µg/ml (GMRL), and 15.8µg/ml (ORCL) was prepared in diluent (KH₂PO₄ buffer: methanol, 50:50 v/v) and analysed six times according to the method conditions. The SS results, which met the criteria for the factors taken into consideration for TFGR, GMRL, and ORCL, are shown in Table 1.

Figure 1: TFGR, GMRL, and ORCL peaks with optimized HPLC method conditions

Table 1: TFGR, GMRL, and ORCL analysis: system suitability

Statistical value	Retention period	Peak area	Plate count	Symmetry	Resolution
GMRL
Average (n=5)	2.275	411252	4286.000	1.330	-
SD (n=5)	0.0078	1376.3529	80.8455	0.0100	-
RSD	0.3438	0.3347	1.8863	0.7519	-
ORCL
Average (n=5)	2.917	612948	5264	1.258	4.204
SD (n=5)	0.0116	1053.236	101.7384	0.0084	0.0439
RSD	0.3986	0.1718	1.9328	0.6651	1.0450
TGFR
Average (n=5)	4.029	949474	4782	1.188	5.526
SD (n=5)	0.0257	1118.090	59.7093	0.0084	0.0623
RSD	0.6382	0.1178	1.2486	0.7043	1.1272

Specificity:

By injecting mobile phase/diluent (KH2PO4 buffer: methanol, 5:5 v/v), working TFGR, GMRL, and ORCL solution (20µg/ml – TGFR; 5.8µg/ml – GMRL; 15.8µg/ml – ORCL) and Tegonat sample solution (20µg/ml – TGFR; 5.8µg/ml – GMRL; 15.8µg/ml - ORCL), specificity evaluation was performed (Figure 2). Due to the excipients and blank, no significant interference was seen at the retention times of TFGR, GMRL, and ORCL.

Linearity:

The method's linearity was assessed by constructing calibration curves for TFGR (10 to 30µg/ml), GMRL (2.9 to 8.7µg/ml), and ORCL (7.9 to 23.70µg/ml) at five concentration levels. Plotting the peak area and corresponding concentration for each level resulted in a calibration plot. Regression analysis was used to determine the linearity.

Figure 2: TFGR, GMRL, and ORCL: Specificity

Regression validation for TFGR: y = 46427.42 x + 11396.2; R² = 0.99970

Regression validation for GMRL: y = 70887.72414x - 2137.4; R² = 0.99999

Regression validation for ORCL: y = 38802.02532x - 2070.2; R² = 0.99999

The proposed method's excellent linearity is apparent from the obtained R² values.

Sensitivity:

The limit of detection (LOQ) and limit of quantitation (LOD) were calculated using the below formulas as per ICH guideline: LOD =3.3 × SD/s and LOQ = 10 × SD/s, where ‘SD’ is the standard deviation of the y-intercept of the regression lines and ‘s’ is the slope of the TFGR, GMRL, and ORCL calibration graphs. The LOD and LOQ values were found to be 0.0337μg/mL and 0.101μg/mL for TFGR, 0.040μg/mL and 0.133μg/mL for GMRL and 0.133μg/mL and 0.418μg/mL for ORCL indicating good sensitivity for the proposed method.

Precision:

Six replicate working standard injections containing GMRL (5.80µg/ml), ORCL (15.8µg/ml), and TFGR (20µg/ml) were analyzed in order to determine system precision. Furthermore, the six Tegonat sample preparations' assay was analyzed to determine the method precision. To illustrate system and method precision, respectively, the SD and RSD for peak areas (Table 2) and assay percentages (Table 2) of TFGR, GMRL, and ORCL were calculated.

Accuracy:

The accuracy was determined at three levels of 50%, 100%, and 150% of the sample concentration by using the standard addition method. In order to determine the recovery (%), a pre-quantified sample solution was added with a known amount of standard, and each level was analyzed in triplicate. (Table 3). The results demonstrate the accuracy of the method being used. Between 98.66% and 100.52% was the mean recovered percentages for GMRL, ORCL, and TFGR.

Robustness:

The robustness is a measurable indicator of the current method's capacity to resist being impacted by purposeful changes in parameters, including the flow rate, column’s thermal condition, solvent proportion in the mobile phase and its pH, and analysis wavelength. The standard solution comprising 20µg/ml (TGFR), 5.8µg/ml (GMRL), and 15.8µg/ml (ORCL) was analysed by purposeful small controlled changes in method parameters. The mean areas of GMRL, ORCL, and TFGR, including their corresponding SD and RSD (Table 4) were computed (Table 4). Minor adjustments in flow rate, column temperature, mobile phase composition, mobile phase pH and wavelength were shown to have no effect on the assessment of the drugs.

Table 2: TFGR, GMRL, and ORCL: Precision

Precision →	System	Method	System	Method	System	Method
Statistical value ↓	Area of GMRL	% GMRL Assay	Area of ORCL	% ORCL Assay	Area of TFGR	% TFGR Assay
Average (n=6)	409883	99.26	611033	99.38	948193	99.66
SD (n=6)	749.468	0.182	588.222	0.096	548.822	0.058
RSD	0.182	0.183	0.096	0.096	0.057	0.058

Table 3: TFGR, GMRL, and ORCL : Accuracy

Level →	50% included level		100% included level		150% included level
Statistical value ↓	Added (µg/ml)	Recovered (%)	Added (µg/ml)	Recovered (%)	Added (µg/ml)	Recovered (%)
GMRL
Average (n=3)	2.871	99.38	5.742	100.25	8.613	100.18
SD (n=3)	-	0.222	-	0.204	-	0.162
RSD	-	0.224	-	0.204	-	0.162
ORCL
Average (n=3)	7.821	99.91	15.642	100.37	23.463	100.52
SD (n=3)	-	0.287	-	0.126	-	0.053
RSD	-	0.287	-	0.125	-	0.053
TFGR
Average (n=3)	10.00	99.24	20.00	99.63	30.00	98.66
SD (n=3)	-	0.111	-	0.046	-	0.369
RSD	-	0.112	-	0.046	-	0.374

Table 4: TFGR, GMRL, and ORCL : Robustness

Statistical value ↓	GMRL area	ORCL area	TFGR area
Methanol concentration (45 ± 5% volume)
Average (n=3)	413966.3	604323.0	952794.0
SD (n=3)	7516.1	8103.4	17669.7
RSD	1.8	1.3	1.9
Flow rate (1.0 ± 0.1 mL/min)
Average (n=3)	414588.3	611293.7	949716.0
SD (n=3)	6672.2	11053.9	14166.9
RSD	1.6	1.8	1.5
Column temperature (27 ± 2°C)
Average (n=3)	415108.3	612483.7	952320.7
SD (n=3)	6433.0	8626.3	11955.5
RSD	1.5	1.4	1.3
pH (3.6 ± 0.2 value)
Average (n=3)	411816.4	613531.3	949754.2
SD (n=3)	1851.0	296.2	1555.0
RSD	0.4	0.1	0.2
Wavelength (275 ± 3nm)
Average (n=3)	413693.7	612290.0	952817.0
SD (n=3)	7177.7	6756.0	17129.9
RSD	1.7	1.1	1.8

Degradation studies:

The method specificity was also demonstrated by performing the stress degradation studies on TFGR, GMRL, and ORCL (Fig. 3). The fraction of TFGR, GMRL, and ORCL degraded for every stress condition was found by performing the assay of the samples (Table 5). The effective resolution of the degradants’ peaks from TFGR, GMRL, and ORCL peaks served as evidence of the method specificity which was further confirmed by the peak purity assessment of TFGR, GMRL, and ORCL peaks in the degraded samples. The purity of the TFGR, GMRL, and ORCL peaks was evaluated using Empower 2 software. The purity angle values of TFGR, GMRL, and ORCL peaks were found less than purity threshold values confirming the purity of TFGR, GMRL, and ORCL peaks and demonstrating no elution with degradants (Table 5).

Figure 3: TFGR, GMRL, and ORCL analysis: degradation/specificity results

Comparison with the reported method:

Haritha et al. have only ever reported a single method for the quality control test of a mixture of TFGR, GMRL, and ORCL.¹¹ Unlike Haritha et al.'s method, which used acetonitrile as the organic solvent, our mobile phase used methanol. Acetonitrile is significantly more expensive than methanol, which is also less toxic. However, the new method takes less than 6 minutes to execute, as opposed to the lengthy 12 minutes for the Haritha et al. method. As a result, the proposed method has a high throughput and is cost-effective. The R² values of 0.99970 for TFGR, 0.99999 for GMRL, and ORCL make evident the developed method's strong linearity compared to Haritha et al.'s method (R² = 0.9996 for TFGR, 0.9995 for GMRL and 0.9994 for ORCL). In comparison, the LOD (TFGR - 0.101μg/ml; GMRL - 0.040μg/ml; ORCL - 0.133μg/ml) and LOQ (TFGR - 0.337μg/ml; GMRL - 0.133μg/ml; ORCL - 0.418μg/ml) in current method was low than Haritha et al.'s method (LOD: TFGR - 0.2μg/ml; GMRL - 0.058μg/ml; ORCL - 0.158μg/ml; and LOQ: TFGR - 0.606μg/ml; GMRL - 0.175μg/ml; ORCL - 0.478μg/ml). Therefore, it showed superior sensitivity compared to the method established by Haritha et al. The precision range was 0.243% to 1.894% RSD in Haritha et al. while the current method has a precision range from 0.058% to 0.183%. In the Haritha et al. method, the effect of changes in the column’s temperature, mobile phase pH, and analysis wavelength were not assessed during the robustness test.

CONCLUSION:

A stability-indicating reverse phase HPLC method was developed for the simultaneous quantification of TFGR, GMRL, and ORCL in both bulk material and tablet formulation. The results demonstrate the method's accuracy, sensitivity, and specificity. This method can be utilized to quantify TFGR, GMRL, and ORCL simultaneously in bulk material and tablet formulation (Tegonat) for routine quality control analysis. Stress degradation studies indicate that the method is suitable for evaluating TFGR, GMRL, and ORCL in the presence of their degradants.

Table 5: TFGR, GMRL, and ORCL: Forced Degradation

Condition	Drug	Non-degraded (%)	Degraded (%)	Purity angle	Purity threshold	Purity flag: pass/fail
Acid Degradation	GMRL	89.51	10.49	0.353	0.768	Pass
	ORCL	88.82	11.18	0.238	0.772	Pass
	TFGR	89.52	10.48	0.237	0.671	Pass
Base Degradation	GMRL	92.75	7.25	0.268	0.967	Pass
	ORCL	92.00	8.00	0.26	0.67	Pass
	TFGR	91.35	8.65	0.45	0.77	Pass
Peroxide Degradation	GMRL	94.35	5.65	0.371	0.769	Pass
	ORCL	95.51	4.49	0.26	0.67	Pass
	TFGR	93.50	6.50	0.26	0.67	Pass
Thermal Degradation	GMRL	90.13	9.87	0.257	0.668	Pass
	ORCL	88.10	11.90	0.25	0.57	Pass
	TFGR	88.99	11.01	0.35	0.67	Pass
Photo Degradation	GMRL	94.63	5.37	0.279	0.767	Pass
	ORCL	93.15	6.85	0.27	0.67	Pass
	TFGR	94.01	5.99	0.36	0.67	Pass

CONFLICTS OF INTEREST:

None declared by authors.

REFERENCE:

1. Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians. 2018; 68(6): 394-424. https://doi.org/10.3322/caac.21492

2. Yadav AR, Mohite SK. Cancer- A Silent Killer: An Overview. Asian Journal Pharmaceutical Research. 2020; 10(3): 213-216. https://doi.org/10.5958/2231-5691.2020.00036.2

3. Saha D, Maity T, Jana M, Mandal S. Cancer Treatment Strategy-An Overview. Asian Journal of Pharmaceutical Technology. 2011; 1(2): 28-33.

4. Tiwari KJ, Jadhav K. Cancer is a Life-Threatening Disease: A Review. Research Journal of Pharmaceutical Dosage Forms and Technology. 2020; 12(2): 111-114. https://doi.org/10.5958/0975-4377.2020.00020.8

5. Kaur C, Kumar S, Singh S, Kaur H. Cancer: A Black Spot to Human Race!. Research Journal of Science and Technology. 2020; 12(1): 01-12. https://doi.org/10.5958/2349-2988.2020.00001.7

6. Rawla P, Barsouk A. Epidemiology of gastric cancer: global trends, risk factors and prevention. Gastroenterology Review. 2019;14(1):26-38. https://doi.org/10.5114/pg.2018.80001

7. Shin WS, Xie F, Chen B, Yu P, Yu J, To KF, Kang W. Updated Epidemiology of Gastric Cancer in Asia: Decreased Incidence but Still a Big Challenge. Cancers (Basel). 2023; 15(9): 2639. https://doi.org/10.3390/cancers15092639

8. Kobayakawa M, Kojima Y. Tegafur/gimeracil/oteracil (S-1) approved for the treatment of advanced gastric cancer in adults when given in combination with cisplatin: a review comparing it with other fluoropyrimidine-based therapies. Onco Targets and Therapy. 2011; 4: 193-201. https://doi.org/10.2147/OTT.S19059

9. Liu H, Wang Y, Li G, Song W, Wang R. Clinical study of tegafur-gimeracil-oteracil potassium capsule (s-1) and oxaliplatin combination chemotherapy in advanced colorectal cancer. Journal of Cancer Research and Therapy. 2015; 11(2): 331-335. https://doi.org/10.4103/0973-1482.157339

10. Ishizuna K, Ninomiya J, Ogawa T, Kojima M, Tsuji E, Kawashima M, Nozaki M, Yamagishi H, Ueda Y. Effectiveness and safety of tegafur-gimeracil-oteracil potassium (TS-1) for metastatic breast cancer: a single-center retrospective study. Gan To Kagaku Ryoho. 2014; 41(13): 2577-2582.

11. Otsuka H, Fujii T, Toh U, Iwakuma N, Takahashi R, Mishima M, Takenaka M, Kakuma T, Tanaka M, Shirouzu K. Phase II clinical trial of metronomic chemotherapy with combined irinotecan and tegafur-gimeracil-oteracil potassium in metastatic and recurrent breast cancer. Breast Cancer. 2013; 22(4): 335-342. https://doi.org/10.1007/s12282-013-0483-1

12. Zhang X, Tian X, Wei Y, Deng H, Ma L, Chen Z. Activity and Safety of Tegafur, Gimeracil, and Oteracil Potassium for Nasopharyngeal Carcinoma: A Systematic Review and Meta-Analysis. Journal of Oncology. 2021; 2021: 6690275. https://doi.org/10.1155/2021/6690275

13. Khar Kilam RK. Evolution of quality control in pharmaceutical technology. Journal of Advanced Pharmaceutical Technology & Research. 2013; 4(4): 172. https://doi.org/10.4103/2231-4040.121409

14. Haleem RM, Salem MY, Fatahallah FA, Abdelfattah LE. Quality in the pharmaceutical industry - A literature review. Saudi Pharm J. 2015; Oct; 23(5): 463-9. https://doi.org/10.1016/j.jsps.2013.11.004

15. Haritha KM, Khandapu BMK., Raju RR. Liquid chromatography dependent stability indicating methodology: development and authentication for formulations of capsule type containing tegafur, gimeracil, and oteracil. International Journal of Applied Pharmaceutics, 2023; 15(4): 71–81. https://doi.org/10.22159/ijap.2023v15i4.47831.

16. Vyas AJ, Jadav CD, Patel AI, Patel AB, Shah SR, Sheth D, Dholakia S. Review on Stability Indicating Assay Method or Forced Degradation Study: Strategy and Regulatory Consideration. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 131-9. https://doi.org/10.52711/2231-5675.2023.00022

17. Roge AB, Tarte PS, Kumare MM, Shendarkar GR, Vadvalkar SM. Forced Degradation Study: An Important Tool in Drug Development. Asian Journal of Pharmaceutical Research. 2013; 3(4): 198-201.

18. Shinde NG, Bangar BN, Deshmukh SM, Sulake SP, Sherekar DP. Pharmaceutical Forced Degradation Studies with Regulatory Consideration. Asian Journal of Research in Pharmaceutical Sciences. 2013; 3(4): 178-188.

19. Sutar SV, Yeligar VC, Patil SS. A Review: Stability Indicating Forced Degradation Studies. Research Journal of Pharmacy and Technology. 2019; 12(2): 885-890. https://doi.org/10.5958/0974360X.2019.00152.5.

20. International conference on the harmonization. ICH Stability testing of new drug substances and products Q1A (R2), 2003.

21. Hamid K. Analytical Method Development in Pharmaceutical Research: Steps involved in HPLC Method Development. Asian Journal of Pharmaceutical Research. 2017; 7(3): 203-207. https://doi.org/10.5958/2231-5691.2017.00031.4

22. Rajmane AD, Shinde KP. A Review of HPLC Method Development and Validation as per ICH Guidelines. Asian Journal of Pharmaceutical Analysis. 2023; 13(2): 143-1. https://doi.org/10.52711/2231-5675.2023.00024.

23. International conference on the harmonization. ICH harmonized tripartite guideline. Validation of analytical procedures: Text and methodology Q2(R1), 2005.

Received on 30.11.2023 Modified on 20.03.2024

Research J. Pharm. and Tech 2024; 17(9):4569-4574.

DOI: 10.52711/0974-360X.2024.00705