INTRODUCTION:

Diabetes mellitus type-2 is asscociated withhyperglycaemia in which resistance of insulin reduction and pancreatic β cell function disruption was noticed. Bexagliflozin is a novel category of gliflozin that meant for type-II diabetes mellitus treatment by inhibiting SGLT2 cotransporter located at proximal renal tubules of S1 and S2 segments^1-3. Bexagliflozin (BEX) is known chemically as (2S,3R,4R,5S,6R).6-(hydroxymethyl) oxane-3,4,5-triol or -2-(4-chloro-3-{[4-(2-cyclopropoxyethoxy)phenyl]methyl}phenyl). It has been noted that bexagliflozin is utilised to lower systolic blood pressure, albuminuria, and excess body weight [4-6]. Analytical techniques like UV, HPLC, HPTLC, UPLC, and LC-MS/MS were discovered in the literature review for the estimation of various gliflozins both alone and in conjunction with other anti-diabetic medications^7-25. It was predicted by literature that no HPLC, Spectroscopy method was developed for bexagliflozin^26-34. Hence an effort was undertaken to design a straightforward, sensitiveRP-HPLC technique for bexagliflozin and validated the developed method based on ICH guidelines.

Fig. 1: Bexagliflozin structural formula

MATERIALS AND METHODS:

Instrumentation of HPLC:

Waters HPLC Analyser 2695 with integration of software Empower 2 and it is equipped with Auto sampler, quartenary pumps and PDA detector.

Chemical solutions and reagents:

Bexagliflozin was procured from BMR labs. Ortho phosphoric acid, HPLC grade water and acetonitrile bought from Merck chemicals private limited in Mumbai, India.

Procedure for standard and working standard solution preparation:

Bexagliflozin 20mg was accurately measured and put into a volumetric flask that holds 100ml. Then by adding 70 millilitres of methanol as a diluent and then sonicating the mixture for around 25minutes. The volume was then diluted with diluent till it reached 100 millilitres. One millilitre of the stock solution described above was taken out and the volume was increased to ten millilitres using diluent to obtain the standard bexagliflozin concentration 20µg/ml.

Sample preparation:

10 Bexagliflozin tablets were weighed precisely and grounded into fine powder.Bexagliflozin powder which is equivalent to one tablet wasadded in 100ml volumetric flask along with 50ml diluent and sonicated for 25minto dissolve the gases. Above solution was filtered to obtain clear solution and make the final volume upto the mark with diluent. Suitable dilutions were done for above sample stock solution to acquire concentration of 20 µg/ml bexagliflozin and can be applied for routine analysis.

Mobile phase:

pH-4 Dihydrogen potassium ortho phosphate (0.01N): methanol [60:40 v/v] was chosen because of peak symmetry, low tailing factor of bexagliflozin peak.

Method development:

The development of the method involved the utilisation of various columns and solvents. Ultimately, the proposed method was established using a mobile phase consisting of Potassium dihydrogen ortho phosphate 0.01N (pH-4) and Methanol in a ratio of 60:40 v/v, with a flow rate of 1.2 ml and employing the AscentisC18 column as the stationary phase.The chromatogram of Bexagliflozin was observed using a PDA detector at a wavelength peak of 225nm.

Method validation:

ICH Q2R1 guidelines were used to validate the present chromatographic technique.Linearity,Robustness, LOD, LOQ, accuracy,system suitability, precision were validation parameters³⁵.

System suitability:

In order to determine the best chromatographic parameters for bexagliflozin evaluation, system suitability experiments were carried out. The chromatogram was evaluated for system appropriateness characteristics after six replicate injections of bexagliflozin onto the column. These criteria include USB theoritical plate count, peak area % RSD, and tailing factor of peak.

Linearity and calibration curve:

The current technique's linearity was assessed using the least squares method. The calibration curve for bexagliflozin was plotted at concentrations ranging from 5 to 30µg/ml.

Precision:

The evaluation of precision involved the administration of six replicate injections at a concentration level of 100% for bexagliflozin, specifically at 20µg/ml. The terms intra and inter day precision are employed to illustrate precision, which is assessed by the %RSD of peak areas, ideally remaining below 2.

LOD and LOQ:

The standard deviation of intercepts and mean slopes from bexagliflozin's calibration curve was used to interpret its LOD and LOQ.

LOD (Detection limit) = 3.3 σ/ s

LOQ (Quantification limit) = 10 σ/ s

σ symbolises standard deviation of responses and s symbolises mean of slopes. The calibration curve was used to figure out σ and s values.

Accuracy:

The accuracy of the suggested analytical method was assessed using the standard addition method, which involved spiking the standard with samples at 50%, 100%, 150%, and recovery and RSD percentages for the corresponding solutions.

Specificity:

Specificity primarily defines the examination of bexagliflozin in the presence of other compounds without interference. The determination was made by administering successive injections of blank, standard solution, and placebo augmented with the standard solution. Observations have been conducted to ascertain the no interference from the placebo and blank at bexagliflozin retention time.

Robustness:

Robustness implies to the method's capacity to yield consistent replies despite purposeful alterations to the method's conditions to a certain degree. To assess the robustness of the current approach, minor alterations were intentionally implemented in the temperature, flow rate, and mobile phase ratio. %RSD of bexagliflozin peak regions were evaluated to determine the method's robustness.

Stability indicating studies:

Stability-indicating investigations were conducted following the ICH Q1A, Q1B, and Q2B criteria. Intrinsic stability of drug substance was demonstrated by performing stability indicating studies which aids to develop ideal storage conditions. The drug substance was stressed under acid/photo/ base/thermal/neutral/ peroxide degradation conditions and stability of drug under respective degradation conditions was measured³⁶.

Acid hydrolysis:

1ml of bexagliflozin (200 µg/ml) standard stock solution and 1ml of 2N HCl were combined and put into a 10ml volumetric flask, then refluxed at 60°C for approximately 30 minutes. 1N NaOH was used to neutralize above solution and dilute the volume to 10ml with diluent and 10μl of above solution was injected on to column and recorded the respective chromatogram

Base hydrolysis:

A volumetric flask of 10ml was filled with 1ml of bexagliflozin (200µg/ml) standard stock solution and 1 ml of 2N NaOH. The mixture was refluxed for approximately 30 minutes at 60°C. 1N HCl was used to neutralize above solution and dilute the volume to 10ml with diluent and 10μl of above solution was injected on to column and recorded the respective chromatogram

Peroxide hydrolysis:

1ml of bexagliflozin (200µg/ml) standard stock solution and 1ml of H₂O₂ were combined and put into a 10ml volumetric flask. The mixture was refluxed at 60°C for approximately 30minutes. Dilute the volume to 10ml with diluent and 10μl of above solution was injected on to column and recorded the respective chromatogram

Thermal degradation:

1ml of bexagliflozin (200µg/ml) standard stock solution was placed in volumetric flask of 10ml and the volume was adjusted to the mark using diluent, then the resulting solution was incubated in an oven at 105°C for 24hours. After specified period the solution was taken from oven and dilute the volume to 10ml with diluent and 10μl of above solution was injected on to column and recorded the respective chromatogram

Photo degradation:

1ml from standard stock solution ofbexagliflozin (200 µg/ml) was tranferred in a beaker and it is placed in U.V. chamber at 254nm wavelength. Dilute the resultant solution to achieve a concentration of bexagliflozin at 20 µg/ml, then inject it into the HPLC system and record the chromatogram.

Neurtral degradation:

1ml of bexagliflozin (200µg/ml) standard stock solution and 1ml water was placed in 10ml volumetric flask, refluxed at 60^oC for 30min. Dilute the obtained solution in a volumetric flask to a final volume of 10mL with diluent, then inject 10μL of the solution onto the column, thereafter recording the corresponding chromatogram.

Application of method to commercialized formulations:

The current method was employed to determine the assay of bexagliflozin marketted tablets (Brenzavvy), by injecting both standard and sample solutions sequentially. The bexagliflozin %purity was calculated by taking into account bexagliflozin peak areas in both solutions.

RESULTS AND DISCUSSION:

Optimisation of chromatographic conditions:

The current approach is to design the fine method and optimizing the chromatographic conditions with variety of trails at various conditions. Column was chosen based on theotitical plates, tailing factor and symmetry of peak shape and finally Ascentis C18 column [150mm × 5.0mm, 2.8µm particle size] was selected. Multiple trials were conducted utilising various solvent ratios, ultimately resulting in [pH-4] Potassium dihydrogen ortho phosphate 0.01N: Methanol in a 60:40 v/v ratio was chosen as mobile phase, with a flow rate of 1.2ml/min and an injection volume of 20 µl. The analyte, Bexagliflozin, was identified using a PDA detector at a wavelength of 225 nm (λmax), with an elution occurring at a retention time of 2.857 minutes. The optimised chromatographic conditions are presented in Table 1. The standard peak, sample peak, and blank for Bexagliflozin are summarised in Figures 2, 3, and 4.

Table 1: Optimized chromatographic conditions

Parameter	Condition
Column:Stationary phase	Ascentis [150mm×5.0mm,2.8µm ] C18
Rate of flow	1.2 ml per minute
Mobile phase	0.01N Dihydrogen potassium ortho phosphate[pH-4]: Methanol [60:40 v/v]
Run time	6min
Retention time	2.857 minute
Injection volume	20µl
λmax	225nm
Temperature at column	Ambient

Fig.2: Blank chromatogram

Fig. 3: Standard Bexaglifozin chromatogram

Fig. 4: Sample Bexagliflozin chromatogram

System suitability:

The new approach is confirmed to be suitable for the analysis of bexagliflozin, as it exhibits over 2000 theoretical plates and a %RSD of peak area, tailing factor, and retention durations of less than 2. The developed method demonstrated over 2000 theoretical plates, with a %RSD of peak area, tailing factor, and retention times all below 2. The recorded chromatograms indicated a tailing factor of less than 2, confirming the method's suitability for the analysis of bexagliflozin. The findings are presented in Table 2.

Table 2: System suitability of bexagliflozin

Parameter	Bexagliflozin	Acceptable standards
Theoretical plates	8644.5	>2000
Tailing factor	1.145	<2
Retention time	2.873min	…….

Linearity:

The proposed method linearity was evaluated over 5-30 µg/ml concentration of bexagliflozin. The linear regression equation and correlation coefficient that was obtained from calibration curve of bexagliflozin was found to beY=136616x+18149 and 0.9997 respectively. Bexagliflozin calibration curve represents a strong linear relationship around 5-30µg/ml range of concentration and results were provided in table-3 and 4 and data was displayed in fig. 5. The linearity of the proposed approach was assessed across a concentration range of 5-30 µg/ml for bexagliflozin. The linear regression equation derived from the bexagliflozin calibration curve is Y = 136616x + 18149, with a correlation coefficient of 0.9997. The calibration curve for Bexagliflozin demonstrates a robust linear correlation within the concentration range of 5-30 µg/ml, with results presented in Tables 3 and 4 and data illustrated in Figure 5.

Fig. 5: Standard bexagliflozin calibration curve

Table 3: Bexagliflozin linearity and range

S. No.	Concentration of Bexagliflozin (µg/ml)	Peak area
1	0	0
2	5	692522
3	10	1388924
4	15	2087528
5	20	2788839
6	25	3430378
7	30	4083541
Y-intercept	18149
Slope	136616
Correlation coefficient	0.9997

Precision:

The current method precision was approached by Intra and inter-day precision measures were used to approach the precision of the current method, and the corresponding measures' percentage RSDs were analysed as 0.7 and 0.8, respectively, ensuring the precision of the current method.

Limit of detection (LOD) and limit of quantification (LOQ):

The estimated levels of detection and quantification for bexagliflozin at lower concentrations were 0.08µg/ml and 0.23µg/ml, respectively. These values demonstrate the sensitivity of the current approach.

Accuracy:

The proposed chromatographic method accuracy was approached by spiking standard solution to sample solution at 50%, 100% and 150% level and % recovery was found to be 99.9%, 100.1% and 99.1% respectively and %RSD of mean recoveries was estimated at 0.7, indicated the reliability of current method accuracy. The outcomes were displayed in table 5.

Table 5: Accuracy of bexagliflozin (n=3)

Drug name	Bexagliflozin % Level of addition	Added amount (mg)	% Recovery	Drug found (mg/ml)
Drug name	Bexagliflozin % Level of addition	Added amount (mg)	Mean±SD	Mean±SD
Bexagliflozin	50	10	99.9±0.7	19.9±0.13
	100	20	100.1±0.6	40.02±0.3
	150	30	99.1±0.7	59.5±0.4

n is number of bexagliflozin determinations, SD is bexagliflozin standard deviation

Robustness:

Small changes in the mobile phase composition, temperature, and flow rate were used to test robustness of the suggested chromatographic method. The method validation parameter did not change in a way that could be predicted. Table 6 shows the findings.

Table 6: Bexagliflozin robustness bexagliflozin (n=6)

Parameter	Peak area (Mean±SD)	% RSD
Mobile phase minus	1871554±10089.1	0.5
Mobile phase plus	1878284±13561.5	0.7
Thermal minus	1838356±6691.2	0.4
Thermal plus	1820742±10969.2	0.6
Flow rate minus	1836776±15225.0	0.8
Flow rate plus	1877596±14647.7	0.8
Mobile phase minus	1871554±10089.1	0.5
Mobile phase plus	1878284±13561.5	0.7

n is number of bexagliflozin determinations, SD is bexagliflozin standard deviation

Specificity:

The specificity of the current chromatographic procedure was evaluated by analysing chromatograms generated from injections of a blank, standard, and sample. The standard and sample chromatograms exhibited strong correlation, while the blank and placebo chromatograms demonstrated no interference from excipients, with no peaks observable during the retention times of the standard and sample chromatograms. The findings were represented in fig. 2, 3 and 4.

Stability-indicating studies:

Stability of drug substance was predicted by performing stability indicating studies and are performed by exposure of BEX under specified stress conditions like acid, base, neutral, photo, thermal, oxidation degradation conditions. Under each stressed condition reduction in percentage assay of bexagliflozin was observed and degradation chromatograms were observed under acid and alkaline degradation conditions. Bexagliflozin degradation chromatograms were shown in fig. 6. The results were disclosed in table 7.

Fig. 6: Bexagliflozin degradation chromatograms

Assay:

Bexagliflozin % assay in commercial tablets was ascertained as 100.92% (table 8), The calculated data was in compliance with the ICH regulations.

Table 8. Assay of bexagliflozin

Drug	Peak name	Peak Area	Label claim	% Assay
Bexagliflozin	Standard	1850841	20mg	100.92
Bexagliflozin	Test	1869779	20mg

CONCLUSION:

The proposed trouble shooting RP-HPLC method is pulse free and adopted high accuracy and sensitivity for quantification of bexagliflozin in film coated tablet dosage forms and in bulk. The present methodology for method development and validation incorporates the approval criteria outlined in ICH standards. The present method has acceptable degradation (< 20%) as per ICH guidelines. The current method effectively resolute the analyte and potential degradants effectively. Hence, this method has an incredible application in pharmaceutical industry.

ACKNOWLEDGMENT:

The authors were grateful to Mallareddy Pharmacy College, Maisammaguda, Hyderabad, for providing facilities and their encouragement to carryout research work.

AUTHOR CONTRIBUTION:

All authors contributed equally to carry out the research work.

CONFLICTS OF INTERESTS:

Declaration none.

REFERENCES:

1. Allegretti AS, Zhang W, Zhou W, Thurber TK, Rigby SP, Bowman-Stroud C, Trescoli C, Serusclat P, Freeman MW, Halvorsen YC. Safety and Effectiveness of Bexagliflozin in Patients with Type 2 Diabetes Mellitus and Stage 3a/3b CKD. American Journal of Kidney Diseases. 2019; 74(3): 328-37. doi: 10.1053/j.ajkd.2019.03.417.

2. Pasqualotto E, Figueiredo WJM, Gewehr DM, da Silva MR, van de SLS, de Araujo GN, Leal FS, Pinheiro CEA. Efficacy and safety of bexagliflozin in patients with type 2 diabetes mellitus: A systematic review and meta-analysis. Diabetes Obesity and Metabolism. 2023; 25(7): 1794-802. doi: 10.1111/dom.15051.

3. Omar A, Revathy C, Leslie MLG, Janis N, Vance B, Matthews, Markus PS. Bexagliflozin for type 2 diabetes: an overview of the data. Expert Opinion on Pharmacotherapy. 2021: 22(16): 2095-103. doi:10.1080/14656566.2021.1959915

4. Shubham B, Prabhjeet KB, Manjusha C. Sodium-glucose transporter (SGLT2) inhibition: A potential target for treatment of type-2 Diabetes Mellitus with Natural and Synthetic compounds. Egyptian Journal of Basic and Applied Sciences. 2023; 10(1): 69-82. doi:10.1080/2314808X.2022.2145734.

5. Jonali R, Harshil S, Vivek KV, Manmohan S. A review on the medicinal chemistry of sodium glucose co-transporter 2 inhibitors (SGLT2-I). European Journal of Medicinal Chemistry Reports. 2022; 6: 1-6. doi:10.1016/j.ejmcr.2022.100074

6. Andra F, Alana AH, ManuelOS, Laura AV, Sandra MF, Estefanía T, Esther RL, Manuel P, Oreste G, José Ramón GJ, Francisca L. Role of Sodium-Glucose Co-Transporter 2 Inhibitors in the Regulation of Inflammatory Processes in Animal Models. International Journal of Molecular Sciences. 2022; 23(10): 5634-44. doi:10.3390/ijms23105634

7. Kaur I, Wakode S, Pal Singh H. Development and validation of stability indicating UV spectroscopic method for determination of canagliflozin in bulk and pharmaceutical dosage form. Pharm Methods. 2016; 7(1): 63–9. doi:10.5530/phm.2016.7.10

8. Haritha G, Aanandhi VM, Shanmugasundaram P. Novel Method Development for Metformin, Ivabradine, Metoprolol and Ertugliflozin and its Validation in API and Pharmaceutical Dosage Form by RPHPLC Method. Research Journal of Pharmacy and Technology. 2021; 14(4): 2055-1. doi: 10.52711/0974-360X.2021.00365

9. Deepthi R, Gowri Sankar D. A Novel UPLC Method for Simultaneous Estimation of Ertugliflozin and Sitagliptin in Bulk and Tablet Dosage Form. Research Journal of Pharmacy and Technology. 2021; 14(9): 4859-2. doi: 10.52711/0974-360X.2021.00844

10. Ghante MR, Tangade RB, Sawant SD, Kulkarni PD, Bhusari VK. Development and Validation of stability indicating RP-HPLC method for the estimation of Ertugliflozin by forced degradation studies. Research Journal of Pharmacy and Technology. 2022; 15(11): 4945-9. doi: 10.52711/0974-360X.2022.00831

11. Manojkumar KM, Kulkarni NS, Khiste RH, Dhanya BS. Development and Validation of Novel Analytical Method for Empagliflozin and Metformin Hydrochloride in Bulk and Pharmaceutical Dosage Form by Four Different Simultaneous Estimation Approaches using UV Spectroscopy. Research Journal of Pharmacy and Technology. 2020; 13(3): 1236-1242. doi: 10.5958/0974-360X.2020.00228.0

12. Manasa M, Vijey AM. Stability Indicating Simultaneous Method development and Validation of Dapagliflozin and Saxagliptin by RP-HPLC. Research Journal of Pharmacy and Technology. 2021; 14(2): 1045-1049. doi: 10.5958/0974-360X.2021.00187.6

13. Patel SB, Vaghasiya EH, Amit RD, Amitkumar JV, Ajay IP, Nilesh KP, Hina MJ. Stability Indicating Photodiode Array Detector Based Estimation of Dapagliflozin Propanediol Monohydrate in API and Tablet Dosage Form by RP-UPLC. Research Journal of Pharmacy and Technology. 2021; 14(9): 4635-9. doi: 10.52711/0974-360X.2021.00805

14. Nachiket SD, Ganesh SS, Vikas BS. Simultaneous Estimation and Validation of Canagliflozin and Metformin Hydrochloride in Bulk and Pharmaceutical Dosage Form by using RP-HPLC. Research J. Pharm. and Tech. 2019; 12(10): 4953-4957. doi: 10.5958/0974-360X.2019.00859.X

15. Vijaya SV, Choudhari VP, Venkata Reddy M. Development of new Validated HPTLC Method for simultaneous estimation of Canagliflozin and Metformin in Tablet Formulation. Research Journal of Pharmacy and Technology. 2022; 15(6): 2599-4. doi: 10.52711/0974-360X.2022.00434

16. Vidhi SD, Paresh UP. Development and Validation of Simultaneous Estimation of Canagliflozin and Metformin by Q Absorbance Ratio Method in its API and Tablet Formulation. Research Journal of Pharmacy and Technology. 2022; 15(10): 4637-0. doi: 10.52711/0974-360X.2022.00778

17. Rama KK, Sundararajan R. Stability Indicating and Simultaneous Determination of Saxagliptin, Dapagliflozin and Metformin in Tablet dosage form using RP-HPLC. Research Journal of Pharmacy and Technology. 2021; 14(11): 5813-8. doi: 10.52711/0974-360X.2021.01011

18. Ismail A, Mohammad H, Youssef Al. Estimation of Nine Organic Volatile Impurities in Bulk Dapagliflozin propandiol hydrate and Dosage forms from different sources using a new developed HS-GC-FID method. Research Journal of Pharmacy and Technology. 2022; 15(7): 3226-32. doi: 10.52711/0974-360X.2022.00541

19. Nihad KH, Abdulhakim N. A Comparison of Efficacy among Syrian diabetic patients treated with Empagliflozin versus Dapagliflozin, a Randomized, Triple-blind, Two-period crossover study. Research Journal of Pharmacy and Technology. 2023; 16(10): 4642-8. doi: 10.52711/0974-360X.2023.00755

20. Patel A. Method development for simultaneous estimation of dapagliflozin and saxagliptin in fixed-dose combination and validation on UV spectroscopic. Journal of Medical Pharmaceuticals Allied Sciences. 2020; 9(3): 2536–43. dooi: 10.22270/jmpas.v9i3.952

21. Siridevi MP, Kumar HT, Rao SY, Rao VPK. RP-HPLC method for quantification of empagliflozin in pharmaceutical formulation. Asian Journal Pharmacy Technology. 2019; 9(3): 208. doi:10.5958/2231-5713.2019.00035.7

22. Pathak S, Mishra P. Stability-indicating HPLC-DAD method for the determination of empagliflozin. Future Journal Pharmaceutical Sciences. 2021; 7(1). doi: 10.1186/s43094-021-00329-w

23. Vankalapati KR, Alegete P, Boodida S. Stability-indicating HPLC method development and validation for simultaneous estimation of metformin, dapagliflozin, and saxagliptin in bulk drug and pharmaceutical dosage form. Biomedical Chromatography. 2022; 36(7): e5384. doi:10.1002/bmc.5384

24. Nandre DS, Ahmed A. Stability indicating hplc method development and validation for simultaneous estimation of dapagliflozin and metformin tablet dosage form. Asian Journal Pharmaceutical and Clinical Research. 2022; 109–14. doi: 10.22159/ajpcr.2022.v15i10.45616

25. Gaikwad AV, Khulbe P. HPLC method development for the estimation of Empagliflozin in bulk and pharmaceutical formulation. Journal of Pharmaceutical Research International. 2022; 22–31. doi:10.9734/jpri/2022/v34i23b35926

26. M Hanif, Bushra R, Ismail NE, Bano R, Abedin S, Alam S. Empagliflozin: HPLC based analytical method development and application to pharmaceutical raw material and dosage form. Pakistan Journal Pharmaceutical Sciences. 2021; 34(3): 1081-7. doi:10.36721/PJPS.2021.34.3

27. Dayyih WA, Ani IA, Al-Shdefat RI, Zakareia Z, Hamid SA, Shakya AK. Development and validation of a stability indicating HPLC method for empagliflozin and linagliptin in tablet dosage form. Asian Journal of Chemistry. 2021; 33(2): 484–8. doi:10.14233/ajchem.2021.23019

28. L.B. Borse, M.S. Wagh, S.L. Borse, S.P. Ahire, I.S. Vaishnav, V.D. Naphade, et al. RP- HPLC method development and validation for estimation of dapagliflozin in tablet formulation. Journal of Pharmaceutical Negative Results. 2022; 364–72. doi:10.47750/pnr.2022.13.s05.53

29. Dhale CR, Rao. Stability indicating hplc method development and validation for the simultaneous estimation of metformin hydrochloride and dapagliflozin in api and pharmaceutical dosage form. International Research Journal Pharmacy. 2021; 12(8): 52–7. doi:10.7897/2230-8407.1208157

30. Verma MV, Patel CJ, Patel MM. Development and stability indicating hplc method for dapagliflozin in api and pharmaceutical dosage form. International Journal of Applied Pharmaceutics. 2017; 9(5): 33. doi:10.22159/ijap.2017v9i5.19185

31. Bhatt D, Thatavarthi P, Rajkamal B. Analytical method development and validation for the estimation of Canagliflozin in bulk and formulation by RP- HPLC. Intternational Journal of Pharmaceutical Research. 2018; 10(03). doi:10.25004/ijpsdr.2018.100306

32. Al-Shdefat R, Al-Ani I, Tamimi L, Awad R, Rayyan WA, Dayyih WA. Development and validation of a stability-indicating HPLC-DAD method for the determination of canagliflozin and metformin simultaneously in combination dosage form. Pharmaceutical Chemistry Journal. 2021; 55(4): 402–9. doi: 10.1007/s11094-021-02436-7

33. Moktar MM, Sujan MS, Heba MEA, Fotouh RM. A UPLC/DAD method for simultaneous determination of empagliflozin and three related substances in spiked human plasma. BMC Chemistry. July 2019; 13(1): 83-95. doi: 10.1186/s13065-019-0604-9

34. Asmaa A, El-Zaher, Hanaa AH, Ehab FE, Marwa AA. A validated LC-MS/MS bioanalytical method for the simultaneous determination of dapagliflozin or saxagliptin with metformin in human plasma. Microchemical Journal. 2019; 149: 1-10. doi: 10.1016/j.microc.2019.104017.

35. Ramya KB, Swetha A. Novel stability indicating RP-UPLC method for silmultaneous estimation of sitagliptin and ertugliflozin in bulk and pharmaceutical formulations. Future Journal of Pharmaceutical Sciences. 2021; 7: 86. doi: https://doi.org/10.1186/s43094-021-00231-5

36. ICH Harmonized Tripartite (2005), Validation of analytical procedures: text and methodology Q2 (R1). International Conference on Harmonization, Geneva Available from: http://www.ich.org/fileadmin/public_web_site/ICH_Products/Guidelines/Quality/Q2_R1/Step4/Q2_R1_Guideline.pdf. (Accessed 12 Mar 2020)

37. ICH Harmonized Tripartite, Stability testing of new drug substance and products Q1A(R2), 2003. International Conference on Harmonization, Geneva Available from: http://www.ich.org/fileadmin/public_web_site/ICH_Products/Guidelines/Quality/Q1A_R2_Guideline.pdf. (Accessed12 Mar 2020)

Received on 29.10.2023 Revised on 23.09.2024

Accepted on 06.05.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5942-5948.

DOI: 10.52711/0974-360X.2025.00859

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

Degradation parameter	Peak area of sample	Peak area of standard	% Assay	% Degradation
Alkali degradation	1725909	1850841	93.16	6.84
Acid degradation	1727004	1850841	93.22	6.78
Dry heat degradation	1802718	1850841	97.30	2.70
Photo degradation	1817454	1850841	98.10	1.90
Neutral degradation	1838440	1850841	99.23	0.77
Oxidative degradation	1798729	1850841	97.09	2.91

Drug	Concentration of bexagliflozin (µg/ml)	Intra-day precision		Inter-day precision
Drug	Concentration of bexagliflozin (µg/ml)	Mean±SD	%RSD	Mean±SD	%RSD
Bexagliflozin	20	1857959±12407.2	0.7	1842344±14966.4	0.8