INTRODUCTION:

Heart failure occurs when the heart is unable to pump an adequate amount of oxygenated blood to meet the metabolic demands of the body. This impairment can lead to systemic venous congestion and respiratory complications.

It may arise from dysfunction in systolic or diastolic processes, or both, and is often associated with structural changes such as altered atrial function and extracellular matrix remodeling, including abnormal collagen turnover, which may represent a fundamental pathological mechanism¹. To address these clinical challenges, a fixed-dose combination of Bisoprolol Fumarate and Dapagliflozin Propanediol Monohydrate was approved by the Central Drugs Standard Control Organization (CDSCO) on August 11, 2023, for patients with heart failure with reduced ejection fraction². Bisoprolol is a selective β1-adrenergic receptor blocker that reduces cardiac workload and improves hemodynamic stability. Dapagliflozin, a highly selective inhibitor of the Sodium-Glucose Co-Transporter 2 (SGLT2), reduces renal glucose reabsorption and promotes glycosuria, thereby improving glycemic control in individuals with Type 2 Diabetes Mellitus. Its unique insulin-independent mechanism also offers additional cardiovascular and renal benefits. Dapagliflozin acts through an insulin-independent mechanism by promoting the excretion of glucose via the kidneys, thereby distinguishing its mode of action from that of traditional antidiabetic agents³. It selectively inhibits Sodium-Glucose Co-Transporter 2 (SGLT2) over SGLT1, leading to reduced renal glucose reabsorption and improved glycemic control. Chemically, dapagliflozin propanediol monohydrate is described as (2S,3R,4R,5S,6R)-2-{4-chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6-(hydroxymethyl)oxane-3,4,5-triol, with a molecular formula of C₂₄H₃₅ClO₉ and a molecular weight of 502.99 g/mol. It appears as a white to off-white crystalline powder and is soluble in solvents such as ethanol, methanol, dimethyl sulfoxide (DMSO), and dimethylformamide (DMF)⁴. Bisoprolol fumarate (BISO), on the other hand, is a highly selective β1-adrenergic receptor blocker that reduces cardiac output and heart rate by inhibiting sympathetic stimulation of the heart⁵. This pharmacological activity contributes to decreased cardiovascular risk in patients with hypertension and heart failure. Its chemical name is bis(1-[(propan-2-yl)amino]-3-[4-({[2-(propan-2-yloxy)ethoxy]methyl}phenoxy)]propan-2-ol) (2E)-but-2-enedioate, with a molecular formula of C₄₀H₆₆N₂O₁₂ and a molecular weight of 766.97 g/mol. It is off-white to white crystalline powder, readily soluble in water and methanol, and moderately soluble in alcohol, glacial acetic acid, and chloroform, but only slightly soluble in acetone and ethyl acetate⁶.

Forced degradation studies play a critical role in understanding the stability profile of pharmaceutical compounds and their formulations. These studies provide insight into degradation pathways and by-products, aiding in the development of robust analytical methods. A significant challenge in developing a stability-indicating method (SIM) is generating adequately degraded samples that reflect both the typical degradation seen under storage conditions and potential degradation under stress. Such samples are essential to ensure the method can reliably distinguish the active pharmaceutical ingredient (API) from its degradation products. Stability studies are designed to assess how a compound's chemical integrity changes over time when exposed to stress conditions such as elevated temperature, light, humidity, and oxidative or hydrolytic environments. These evaluations help establish product expiry dates, recommended storage conditions, and reanalysis intervals⁷. Several RP-HPLC methods have been reported for the quantification of Bisoprolol Fumarate (BISO), either in its pure form, in dosage formulations, or in the presence of its degradation products^8–18. Similarly, validated HPLC methods have also been documented for the analysis of Dapagliflozin Propanediol Monohydrate (DAPA) in various matrices^19–30.

The fixed-dose combination of Bisoprolol Fumarate and Dapagliflozin Propanediol Monohydrate was recently approved by the Central Drugs Standard Control Organization (CDSCO) in August 2023 and is currently undergoing Phase III clinical trials. However, to date, no validated RP-HPLC method has been reported for the simultaneous quantification of these two drugs in a synthetic mixture. Furthermore, no stability-indicating method (SIM) using RP-HPLC has been established for their concurrent analysis. Therefore, the objective of the present study is to develop and validate a precise, accurate, and linear RP-HPLC method capable of simultaneously estimating Bisoprolol and Dapagliflozin in a synthetic blend. The method was validated in accordance with ICH Q2 (R1) guidelines. To evaluate the stability-indicating capability, forced degradation studies were conducted under various stress conditions, including acidic, basic, oxidative, thermal, and photolytic environments, following ICH Q1A (R2) recommendations. The proposed method successfully achieved separation of the active pharmaceutical ingredients from their degradation products, demonstrating its suitability as a stability-indicating analytical tool ^31–32.

Figure 1: Structure of A) Bisoprolol fumarate B) Dapagliflozin propanediol Monohydrate

MATERIALS AND METHODS:

Chemicals:

Dapagliflozin propanediol monohydrate (DAPA) was obtained as a gift sample from Precise Chemipharma Pvt. Ltd., Pawne, Navi Mumbai, India. Bisoprolol fumarate (BISO) was generously provided by Mehta Pharmaceutical Industries, Virar, Thane, India. All reagents and solvents used were of analytical or HPLC grade. These included Milli-Q purified water, methanol (HPLC grade), acetonitrile (HPLC grade), sodium hydroxide (NaOH, AR grade), hydrochloric acid (HCl, AR grade), hydrogen peroxide (H₂O₂, AR grade), and buffer components (AR grade), which were procured from standard commercial suppliers.

Chromatographic Conditions:

Chromatographic separation was performed on an Agilent Zorbax ODS C18 column (150 mm × 4.6 mm, 5 μm) maintained at a temperature of 25 °C. The mobile phase consisted of 0.1% triethylamine and acetonitrile in a 66:33 (v/v) ratio, with the pH adjusted to 3.0 using orthophosphoric acid. The mobile phase was delivered at a flow rate of 1.5 mL/min in isocratic mode. The injection volume was set to 100 µL, and the detection wavelength was fixed at 225 nm using a UV detector. The total run time for the chromatographic analysis was 15 minutes. The retention times for Bisoprolol Fumarate and Dapagliflozin Propanediol Monohydrate were found to be 2.99 minutes and 9.348 minutes, respectively.

Preparation of Solutions:

Preparation of 0.1% Triethylamine (TEA) Solution:

To prepare the 0.1% TEA solution, 1 mL of triethylamine was added to 1000 mL of distilled water. The pH was adjusted to 3.0 using diluted orthophosphoric acid.

Preparation of Mobile Phase:

The mobile phase was prepared by mixing 0.1% triethylamine solution and acetonitrile in a 66:33 (v/v) ratio. The pH was adjusted to 3.0 using orthophosphoric acid. The solution was sonicated to degas and then filtered through a 0.45 μm membrane filter. This mobile phase was also used as the diluent for all sample and standard preparations.

Preparation of Standard Solutions:

Bisoprolol Fumarate (BISO) Stock Solution:

Accurately weighed 10 mg of bisoprolol fumarate was transferred into a 100 mL volumetric flask. Approximately 50 mL of methanol was added, and the solution was sonicated to ensure complete dissolution. The volume was then made up to the mark with methanol. This served as the stock solution for BISO.

Dapagliflozin Propanediol Monohydrate (DAPA) Stock Solution:

Similarly, 10 mg of dapagliflozin propanediol monohydrate was accurately weighed and transferred into a separate 100 mL volumetric flask. About 50 mL of methanol was added, followed by sonication until fully dissolved. The volume was adjusted to 100 mL with methanol to obtain the stock solution of DAPA.

Preparation of Sample Solution:

A quantity equivalent to 200 mg of the synthetic mixture (approximately equal to one tablet) was transferred into a 100 mL volumetric flask. About 50 mL of methanol was added, and the mixture was sonicated for 15 minutes to ensure complete extraction. The solution was then filtered using a 0.45 μm syringe filter. From the filtered solution, 1 mL was transferred to a 10 mL volumetric flask and diluted with mobile phase to achieve a final concentration of 10 μg/mL each for BISO and DAPA.

Method Validation:

System Suitability:

System suitability parameters including theoretical plates (USP), tailing factor, retention time, and peak area were assessed by injecting six replicate samples of standard solutions containing Bisoprolol Fumarate and Dapagliflozin Propanediol Monohydrate. The results, presented in Table 1, confirmed the reproducibility and efficiency of the chromatographic system.

Specificity:

Specificity was evaluated by comparing the chromatograms of blank, standard, and sample solutions. The retention times of Bisoprolol and Dapagliflozin (Figures 4–6) showed no interference from excipients or impurities, confirming that the method is specific to the analytes of interest.

Linearity:

Linearity was established by preparing a series of standard solutions at concentrations of 0.5, 0.75, 1.0, 1.25, and 1.5 times the working concentration for both analytes. This corresponded to a concentration range of 5–15 µg/mL for Bisoprolol and 5–15 µg/mL for Dapagliflozin. Calibration curves were plotted using the least squares regression method. The correlation coefficients (r²) were found to be 0.9995 for Bisoprolol and 0.9994 for Dapagliflozin, indicating excellent linearity.

Accuracy:

Accuracy was determined through recovery studies at three concentration levels: 50%, 100%, and 150%. Known quantities of Bisoprolol and Dapagliflozin standards were spiked into the sample solution containing 10 µg/mL of each drug. The percent recovery values at all three levels demonstrated that the method is accurate and reliable.

Precision:

Precision was assessed in terms of repeatability and intermediate precision. Repeatability was determined by analyzing six replicates of the sample solution, and the % relative standard deviation (%RSD) for peak areas was found to be below 2%. Intermediate precision was evaluated by performing intraday and interday analyses over three consecutive days. The concentrations tested were 50, 100, and 150 µg/mL for both Bisoprolol and Dapagliflozin. All %RSD values were within acceptable limits, confirming the method's precision.

Limit of Detection (LOD) and Limit of Quantification (LOQ):

LOD and LOQ were calculated based on the standard deviation of the response and the slope of the calibration curve. The method demonstrated good sensitivity, as indicated by low LOD and LOQ values for both analytes.

Robustness:

Robustness was evaluated by deliberately varying method parameters, including flow rate (±0.1 mL/min), organic phase composition (±2%), and column temperature (±5 °C). Standard solutions of 10 µg/mL for both drugs were used for the analysis. The %RSD of peak areas remained below 2%, indicating that the method is robust and capable of withstanding small, deliberate changes in analytical conditions.

Forced degradation study:

Determining the ideal storage conditions is aided by these studies, which describe the drug material's stability under varied stress conditions. Degradation investigations were conducted using the sample stock solution. The sample solution was refluxed with 1 N HCl, 2 N HCl and 1 N NaOH, 2N NaOH for two hours at 60°C to degrade the acid and base. A sample was refluxed with 3% hydrogen peroxide at 60°C for 6 hours in order to undergo oxidative degradation. The samples were subjected to photolytic degradation Exposure (sunlight, 6 h) and thermal degradation for 4 hours at 65°C & 70℃ for 4 hours in a hot air oven. All of the deteriorated samples were diluted using diluent to reach 10 μg/ml BISO and 10 μg/ml DAPA.

RESULTS:

Method Development:

The objective of the present study was to develop a robust and stability-indicating RP-HPLC method for the simultaneous estimation of Dapagliflozin Propanediol Monohydrate (DAPA) and Bisoprolol Fumarate (BISO) in a synthetic mixture. Solubility testing was initially conducted for both analytes in various solvents, including water, methanol, and acetonitrile, as well as their combinations. Since both drugs exhibited good solubility in methanol, it was selected as the diluent for standard and sample preparations.

During method optimization, various chromatographic parameters were systematically adjusted to enhance separation efficiency and resolution. These included mobile phase composition, flow rate, detection wavelength, stationary phase (column type), and column temperature. The detection wavelength of 225 nm was chosen based on the overlain UV absorption spectra of BISO and DAPA, which exhibited suitable absorbance at this wavelength (Figure 1).

Two C18 reverse-phase columns were evaluated during method development: Inertsil ODS-3 C18 (250 mm × 4.6 mm, 5 μm) and Agilent Zorbax ODS C18 (150 mm × 4.6 mm, 5 μm). System suitability parameters such as retention time, theoretical plates, tailing factor, and resolution were compared between columns. Based on superior performance in these criteria, the Agilent Zorbax ODS C18 column was selected for further analysis.

Several mobile phase combinations consisting of water, methanol, acetonitrile, and buffers were tested under isocratic conditions. The optimized mobile phase consisted of 0.1% triethylamine and acetonitrile in a 66:33 (v/v) ratio, with the pH adjusted to 3.0 using orthophosphoric acid. Chromatographic separation was achieved under isocratic elution at a flow rate of 1.5 mL/min and detection at 225 nm. This final method demonstrated suitable retention times and peak shapes for both analytes with acceptable system suitability parameters.

Method Validation:

System suitability:

A system suitability study was conducted for both Bisoprolol Fumarate (BISO) and Dapagliflozin Propanediol Monohydrate (DAPA) to evaluate the performance of the chromatographic system. The assessed parameters included resolution, theoretical plates, and tailing factor. The results confirmed that all values were within the acceptable limits as per ICH guidelines—resolution greater than 2, theoretical plates above 2000, and tailing factor less than 2. The detailed results are presented in Table 1.

Table 1: System Suitability Study – Observation Details

Parameters	BISO (Mean ± SD)	% RSD	DAPA (Mean ± SD)	%RSD
Retention time (min)	2.9646 ± 0.021	0.70	9.3436 ± 0.0792	0.84
Theoretical plates	2193.4 ± 7.92	0.36	2383.2 ± 9.88	0.41
Tailing factor	1.26 ± 0.01	0.79	1.18 ± 0.01	0.84
Resolution	0.0	0.00	11.14 ± 0.04	0.35

Specificity:

It was verified that neither drug's retention time showed any additional peaks and the current approach was specific for the concurrent quantitation of both drugs in laboratory prepared mixture.

Linearity:

Throughout the concentration range of 5–15 μg/ml, the technique performed consistent with an r² of 0.9995 for BISO and 0.9994 for DAPA. Summary of table given below in the table 2.

Table 2: Linearity and Precision Summary for BISO and DAPA

Sr No	Parameters	BISO	DAPA
1	Linearity range (µg/ml)	5-15	5-15
2	_R2	0.9995	0.9994
3	Slope	183355	292795
4	Intercept	101553	50904

Figure 2: Calibration curve of BISO

Figure 3: Calibration curve of DAPA

Figure 4: Chromatogram of Diluent

Accuracy:

Evaluating the method’s validity involves assessing its percentage recovery performance. To illustrate this, three different known concentrations of the drug (50%, 100%, and 150%) were added to the placebo to test the procedure's accuracy. For BISO and DAPA, the average recovery percentages were 99.92% and 100.24%, respectively. The results are summarized in Table 3.

Figure 5: Chromatogram of BISO and DAPA Sample

Figure 6: Chromatogram of BISO and DAPA standard

Precision:

The interday and intraday precision results for BISO and DAPA were found to be 0.28 and 0.40, respectively. The system precision, technique precision, and intermediate precision studies showed %RSD values all within ±2.0%, confirming the method's reliability and consistency.

LOD and LOQ:

The limits of detection (LOD) and quantitation (LOQ) were established as 0.99 µg/ml and 3.02 µg/ml for BISO, and 1.06 µg/ml and 3.23 µg/ml for DAPA, respectively.

Table 3: % Recovery data of accuracy

Drug	Level (%)	Amount added (µg/ml)	Amount recovered (µg/ml)	Mean % recovery ± SD	%RSD
BISO	50%	5	5.02	98.3 ± 0.188	0.19
	100%	10	10.01	99.9 ± 0.094	0.09
	150%	15	15.00	101.2 ± 0.249	0.24
DAPA	50%	5	5.04	101.5 ± 1.517	1.19
	100%	10	10.03	100.7 ± 0.124	0.12
	150%	15	15.01	100.4 ± 0.188	0.18

Table 4: Robustness summary

Sr No	Parameter	Variations	Mean peak area ± SD (n=3)		%RSD
Sr No	Parameter	Variations	BISO	DAPA	BISO	DAPA
1	Temperature (±5℃)	20 ℃	1416014 ± 3412.11	2765343 ± 3399.21	0.24	0.12
		25 ℃	1690315 ± 5091.01	2818357 ± 10465.79	0.30	0.37
		30 ℃	1556055 ± 2232.29	2785457 ± 7831.8	0.25	0.28
2	Flow rate (± 0.1 ml/min)	1.4	1886299 ± 9549.30	2563814 ± 5010.80	0.50	0.21
		1.5	1690315 ± 5091.01	2818357 ± 10465.79	0.30	0.37
		1.6	1559492 ± 3292.55	2635673 ± 14027	0.53	0.14
3	Organic phase (±2)	-31	1617960 ± 2491.69	2734982 ± 6567.03	0.15	0.24
		33	1690315 ± 5091.01	2818357 ± 10465.79	0.30	0.37
		+35	1573961 ± 3333.84	2774597 ± 6337.09	0.19	0.36

A B

Figure 7: Acid degradation Chromatogram of BISO and DAPA sample A) For 1N HCl B) 2N HCl

C D

Figure 8: Alkali degradation Chromatogram of BISO and DAPA sample A) For 1N NaOH B) 2N NaOH

Figure 9: Oxidative degradation Chromatogram of BISO and DAPA sample (3% H2O2)

Figure 10: Photolytic degradation Chromatogram of BISO and DAPA sample (sunlight Exposure)

Figure 11: Thermal degradation Chromatogram of BISO and DAPA sample E) For 65 ℃ F) For 70 ℃

Table 5: Forced degradation study results

	Stress conditions
	Acidic condition		Alkali condition		Oxidative condition	Photolytic condition	Thermal condition
Drug	1N HCl, 60 ℃, 2 h	2N HCl, 60 ℃, 2 h	1N NaOH, 60 ℃, 2 h	2N NaOH, 60 ℃, 2 h	3% H₂O_2, 60 ℃, 2 h	Exposure (sunlight, 60 ℃, 6 h)	65 ℃, 4 h	70 ℃, 4 h
BISO (API)	3.1	15.7	2.3	7.9	6.1	0.2	1.7	2.4
DAPA (API)	9.2	19.5	3.7	9.3	7.7	7.8	3.8	12.8
BISO (Sample)	1.4	18.5	3.0	5.8	8.3	2.7	2.2	3.1
DAPA (Sample)	7.5	17.7	4.2	10.5	10.1	7.5	5.8	13.1

Table 6: Method validation summary

Parameters	BISO	DAPA
Linearity	5-15 µg/ml	5-15 µg/ml
Regression Equation	y =183355x - 101553	y = 292795x - 50904
Correlation Co-efficient (r²)	0.9995	0.9994
LOD (µg/ml)	0.99	1.06
LOQ (µg/ml)	3.02	3.23
Repeatability (% RSD, n=6)	0.28	0.40
Intraday Precision (% RSD, n=3)	0.13-0.75	0.30-0.86
Interday Precision (% RSD, n=3)	0.83 -1.20	0.56 -0.90
Accuracy (% recovery, n=3)	98.3 -101.2%	100.4 - 101.5 %
Assay ± SD (% RSD)	0.4	0.3
Robustness (% RSD)	< 2.0 % in each parameter

CONCLUSION:

A novel RP-HPLC approach has been developed for determining Bisoprolol Fumarate and Dapagliflozin Propanediol Monohydrate in a synthetic mixture, offering precise and accurate stability indication. This method improves resolution and separation of medicines and degradants, revealing that additives or degradation products impact on analytical outcomes. The method's adaptability and use are attributed to its regression results, % RSD, and standard deviations, making it suitable for regular quantification in labs.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENT:

The authors are grateful to Himatnagar Kelavani Mandal, Himatnagar, Gujarat, India, for providing all the amenities needed to perform the study.

REFERENCES:

1. Tanai E, Frantz S. Pathophysiology of heart failure. Compr Physiol. 2016; 6: 187–214.

2. Central Drugs Standard Control Organization. FDC approved drug combination of Dapagliflozin and bisoprolol fumarate [Internet]. 2023 Aug 11 [cited 2025 May 21]. Available from: https://cdsco.gov.in/opencms/opencms/en/Approval_new/CT-Approvals/

3. Ravinandan AP, Vishwas HN, Hosur SS, Mustafa GM, Kavya HB. Dapagliflozin: a molecule for type-2 diabetes mellitus. Int J Pharm Chem Anal. 2022; 9(4):155–6.

4. CDSCO. Dapagliflozin Propanediol Monohydrate [Internet]. 2024 [cited 2025 May 21]. Available from: https://cdsco.gov.in/opencms/opencms/en/Approval_new/CT-Approvals/

5. Gorre F, Vandekerckhove H. Beta-blockers: focus on mechanism of action. Acta Cardiol. 2010; 65(5):565–70.

6. PubChem [Internet]. Bethesda (MD): National Center for Biotechnology Information; 2004– [cited 2025 Mar 22]. Available from: https://pubchem.ncbi.nlm.nih.gov/compound/Bisoprolol-Fumarate

7. Patel JR, Bedi HS, Middha A, Prajapati LM, Parmar VK. RP-HPLC method for estimation of nitazoxanide in oral suspension formulation. Der Pharma Chemica. 2012; 4(3):1140-1144.

8. Manimala M, Reddy RK, Rani HD. Development and validation of UV spectrophotometric and RP-HPLC methods for the determination of bisoprolol fumarate in pure and pharmaceutical dosage form. World J Pharm Res. 2013; 2:3165–71.

9. RB Kakde, VH Kotak, DL Kale. Estimation of Bisoprolol Fumarate in Pharmaceutical Preparations by HPTLC. Asian J. Research Chem. 2008; 1(2): 70-73.

10. Laxman M. Prajapati, Anjali Patel, Jimish R. Patel, Amit K. Joshi, Mohammadali Kharodiya. High Performance Thin Layer Chromatography Method for Simultaneous Estimation of Cefepime Hydrochloride and Sulbactam Sodium. Asian J. Pharm. Ana. 2016; 6(4): 207-212.

11. Tripathy SN, Swain S, Jena BR, Sahu A. New validated UV spectrophotometric method for the quantification of bisoprolol fumarate in its pharmaceutical dosage form. Acta Sci Pharm Sci. 2023; 7(2): 4–9.

12. Todevska EL, Piponski M, Stefova M. Development and validation of an analytical method for the determination of related substances in bisoprolol fumarate in dosage forms by HPLC-UV-DAD. Maced J Chem Chem Eng. 2021; 40:263–76.

13. Bhavyasri K, Goud JS, Sewthasri R, Sumakanth M. Development and validation of stability indicating RP-HPLC method for the estimation of bisoprolol fumarate in bulk and pharmaceutical dosage form. Int J Pharm Phytopharmacol Res. 2020; 10(4): 49–70.

14. Sandip A Telavane, Seema Kothari, Manohar V. Lokhande. Method of Validation for Residual Solvents in Bisoprolol Fumarate by GC Technique. Asian Journal of Pharmaceutical Research. 2021; 11(3):147-5.

15. Kakde RB, Kotak VH, Kale DL. Estimation of bisoprolol fumarate in pharmaceutical preparations by HPTLC. Asian J Res Chem. 2008; 1(2):70–3.

16. Mahu SC, Space AF, Ciobanu C, Hancianu M, Agoroaei L, Butnar E. Quantitative determination of bisoprolol fumarate by HPLC. Rev Chim (Bucharest). 2016; 67(3): 414–7.

17. RB Kakde, VH Kotak, AG Barsagade, NK Chaudhary, DL Kale. Spectrophotometric Method for Simultaneous Estimation of Amlodipine Besylate and Bisoprolol Fumarate in Pharmaceutical Preparations. Research J. Pharm. and Tech. 2008; 1(4): 513-515.

18. Bhusnure OG, Dongare RB, Gholve SB, Rajmane TM, Munde AB, Giram PS. Development and validation of UV spectroscopic method for the determination of bisoprolol fumarate tablets. Int J Pharm Biol Sci. 2018; 8(2): 338–44.

19. United States Pharmacopeia-National Formulary. Dapagliflozin Propanediol. Rockville (MD): USP; 2023 Dec. doi:10.31003/USPNF_M9215_02_01

20. Sarkar S, Patel VP. Method development and validation of dapagliflozin drug in bulk and tablet dosage form by RP-HPLC. Int J Pharm Res Health Sci. 2017; 5(4): 1755–9.

21. Sumaa BV, Deveswaran RB, Shenoy P. A new high-performance thin-layer chromatographic method development and validation of dapagliflozin in bulk and tablet dosage form. Int J Pharm Pharm Sci. 2019; 11(8): 58–63.

22. Manoharan G, Ismaiel AM, Ahmed ZM. Estimation of dapagliflozin in raw and tablet formulation. Chem Res J. 2018; 3(2): 159–64.

23. Vinutha Kommineni, K.P.R. Chowdary, S.V.U.M. Prasad. Formulation of Dapagliflozin and Saxagliptin Tablets and In vitro Evaluation by RP-HPLC Method. Asian J. Pharm. Ana. 2019; 9(2): 93-98. doi: 10.5958/2231-5675.2019.00018.8

24. Sayali S. More, Sandeep S. Sonawane, Santosh S. Chhajed, Sanjay J. Kshirsagar. Development and Validation of RP-HPLC Method for Simultaneous Estimation of Saxagliptin and Dapagliflozin in Tablets. Asian J. Pharm. Tech. 2018; 8 (3):145-148.

25. Chavhan PG, Patil SA, Pawar SP. Analytical method development and validation of dapagliflozin in bulk drug and pharmaceutical dosage form by U-HPLC method. Int J Pharm Res Technol. 2024: 661–9.

26. Navya Sree V, Bhavyasri K, Sumakanth M, Swethasri R. Estimation of dapagliflozin in pure and marketed formulation by validated reverse phase-high performance liquid chromatographic method. Int J Life Sci Pharm Res. 2020; 10(1):70–84.

27. Bhagwat JB. Quantitative estimation of dapagliflozin in blood plasma by using UV spectroscopy. Pharm Anal Acta. 2010; 10(2): 608.

28. Sanagapati M, Dhanalakshmi K, Reddy NG, Kavitha B. Method development and validation of dapagliflozin API by UV spectroscopy. Int J Pharm Sci Rev Res. 2014; 26(1): 270–7.

29. Debata J, Kumar S, Jha SK, Khan A. A new RP-HPLC method development and validation of dapagliflozin in bulk and tablet dosage form. Int J Drug Dev Res. 2017; 9(2):48–51.

30. U.S. Food and Drug Administration. Guidance for industry Q1A(R2): stability testing of new drug substances and products [Internet]. 2003 [cited 2025 May 21]. Available from: https://www.fda.gov/media/71707/download

31. Iram F, Iqbal MA, Husain A. Forced degradation studies. J Anal Pharm Res. 2016; 3(9).

32. Ueyama E, Takahashi F, Ohashi J, Konse T, Kishi N, Kano K. Mechanistic study on degradation of azelnidipine solution under radical initiator-based oxidative conditions. J Pharm Biomed Anal. 2012; 61: 277–83.

Received on 28.04.2025 Revised on 31.05.2025

Accepted on 30.06.2025 Published on 06.10.2025

Available online from October 13, 2025

Asian J. Pharm. Res. 2025; 15(4):355-363.

DOI: 10.52711/2231-5691.2025.00055

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.