INTRODUCTION:

Remogliflozin Etabonate is a medication of the gliflozin class used to treat type 2 diabetes and non-alcoholic steatohepatitis ("NASH"). IUPAC name was Ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-{[5-methyl-1-(propan-2-yl)-4-{[4-(propan-2yloxy)phenyl]methyl}-1H-pyrazol-3-yl]oxy}oxan-2-yl]methyl carbonate.

Remogliflozin is being developed by BHV Pharma, a fully owned subsidiary of Avolynt in North Carolina, USA, and Glenmark Pharmaceuticals through a partnership with BHV. It was initially discovered by the Japanese company Kissei Pharmaceutical. In May 2019, Glenmark launched Remogliflozin commercially in India for the first time^1-9.

Metformin is a member of the biguanide class of anti-diabetics with antihyperglycemic action. Lactic acidosis is an infrequent side effect of Metformin. IUPAC's name was N, N-Dimethylimidodicarbonimidic diamide. In 1922, Metformin was discovered. The research on people was started in 1950 by French physician Jean Sterne. It was first made available as a medicine in France in 1957 and the US in 1995^10-²².

According to a survey of the literature, there are two methods available for determining Remogliflozin Etabonate and Metformin HCl using UV spectroscopy²³^,²⁴ and two RP-HPLC techniques^25,26 has been published for the estimation of Remogliflozin Etabonate and Metformin HCl in their pharmaceutical dosage form or API for stability, suggesting an analytical method and validation. The goal of the current work was to develop a stability-indicating RP-HPLC method for the estimation of Remogliflozin Etabonate and Metformin HCl in their dosage forms. The proposed RP-HPLC detection method was validated in accordance with International Conference on Harmonisation (ICH) guidelines²⁷^-2⁹ because analytical methods must be approved before use by the pharmaceutical industry. This method's selectivity, linearity, accuracy, and precision, as well as its limit of detection and limit of quantification, were evaluated.

Figure 1: (a) Chemical structure of Remogliflozin Etabonate (b) Chemical structure of Metformin

MATERIALS AND METHODS:

Chemicals and reagents:

Glenmark Pharmaceuticals made the tablet formulation [REMO-ZEN M 500], and each film-coated tablet contained Remogliflozin Etabonte (100mg) and Metformin HCL (500mg), respectively. Analytically pure samples of Remogliflozin and Metformin were acquired from Actis Generics Pvt. Ltd., Vishakhapatnam, and Yarrow Chem Products, respectively. Analytical-grade orthophosphoric acid was used. Thermo Fisher Scientific Pvt. Ltd. was used to obtain sodium hydroxide (AR grade), hydrochloric acid, and methanol (HPLC grade). Water (HPLC quality) from Sisco Research Laboratories Pvt. Ltd. was used throughout the investigation. Molychem provided the hydrogen peroxide (30% w/v).

Instrumentation:

A Shimadzu (Kyoto, Japan) chromatographic system with an SPD M20A prominent photodiode array detector and LC 20 AD binary pump was used for the chromatography. The manual Rheodyne injector had a 20μL fixed loop. Chromatographic separation was accomplished on a Phenomenex Luna (250mm X 4.6mm, 5μm) C8 column maintained at ambient conditions. The LC Solution programme was used to acquire the data. On an electronic balance made by Shimadzu, the samples were weighed.

Preparation of combined stock solution:

A 100mL volumetric flask was filled with 100mg of Remogliflozin and 500mg of Metformin after they had been precisely weighed. Remogliflozin and Metformin were added to 50mL of methanol and sonicated to create a clear solution, and then the volume was made up with methanol to achieve concentrations of 1000μg/mL and 5000μg/mL, respectively.

Preparation of combined working standard solution:

10mL of the mixed stock solution was poured into a 100 mL volumetric flask and diluted with methanol to the desired strength. A working standard solution of remogliflozin (100μg/mL) and metformin (500μg/mL) was made.

Preparation of sample solution from tablets:

A pool of tablets [REMO-ZEN M 500] (each tablet contains 100mg of Remogliflozin and 500mg of Metformin) were weighed, taken into a mortar, crushed to fine powder, and mixed uniformly. Weight (896.4 mg), which is equal to 100mg of Remogliflozin Etabonate, was dissolved in 100mL of methanol, the solution was then sonicated for 50 minutes, and the volume was then diluted to 100mL with methanol. A 0.22-micron syringe filter was used to filter the clear supernatant after centrifuging the final solution at 3000 rpm. Further dilutions were prepared to obtain the solutions containing Remogliflozin Etabonate and Metformin Hydrochloride in the ratio 1:5 respectively, as in formulation.

Standardized chromatographic conditions:

Standard solutions of 100μg/mL of Remogliflozin and 500μg/mL of Metformin hydrochloride were injected in column with 20μl micro syringe. The chromatogram was run for appropriate minutes with mobile phase 0.1% Orthophosphoric Acid: Acetonitrile (30:70). The detection was carried out at wavelength 230 nm.

Method validation:

According to ICH criteria, the devised technique was validated for robustness, linearity, precision, accuracy, limit of detection, and system appropriateness.

System suitability:

To make sure the instrument is appropriate for its intended use, system suitability parameters were examined. The standard solution was administered fresh from the working standard solution. The peak area, theoretical plates (N), resolution (R), and tailing factors of each solution were examined.

Linearity:

By diluting an adequate volume of the working standard solution with diluent, the linearity of this approach was examined using concentrations in the range of 0.1–20 μg/mL for Remogliflozin and 0.5–100μg/mL for Metformin. Triplicate injections of 20μL of each solution were made in accordance with the ideal chromatographic conditions. Remogliflozin and metformin concentrations were plotted against peak regions to create calibration curves, and then regression equations were computed.

Precision:

By analysing the samples in accordance with ICH rules, repeatability and intermediate precision were defined as precision. Decisions were made both that day (with intraday precision) and the day after (with interday precision). Three sequential duplicates injections of three sample solutions of remogliflozin and Metformin (3:15, 4:20, and 5:25μg/mL) were used to examine each phase of precision. The relative standard deviation (RSD) of the mean assay value was used to express precision.

Limit of detection and limit of quantitation:

The response's standard deviation and the calibration curve's slope were used to determine the limit of detection (LOD) and limit of quantitation (LOQ). Remogliflozin and Metformin's LOD and LOQ were used to determine the method's sensitivity, which was then computed using the slope method.

LOD = 3.3 × σ /S,

LOQ = 10 × σ /S

where σ is the standard deviation of the response and S is the slope of the standard curve.

Robustness:

Variations in the flow rate, pH, and mobile phase ratio were evaluated to examine the method's resilience. The system appropriateness parameters were examined, and the relative standard deviation and percentage recovery were documented.

Force degradation studies:

Acid degradation:

Transferring 0.4mL of the combined working standard solution of remogliflozin and Metformin (100 µg/mL and 500µg/mL), 1mL of 0.1 N HCl was added, and the mixture was refluxed at 60℃ for 30 minutes. After being cooled to room temperature, the solution was neutralized with 0.1 N NaOH. The solution was diluted with diluent until it met the required level. This solution was injected in triplicate and examined in a volume of 20µL. However, there is no degradation.

Alkaline degradation:

0.4mL of combined working standard solution (100 µg/mL and 500µg/mL) of Remogliflozin and Metformin was transferred to a 10mL volumetric flask, 1mL of 0.1 N NaOH was added and refluxed at 60ºC for 30 minutes. The solution was cooled to room temperature and neutralized with 0.1 N HCl. The solution was further diluted up to the mark with diluent. A 20µL of this solution was injected in triplicate and analysed. However, there is no degradation.

Peroxide degradation:

Remogliflozin and Metformin working standard solutions (100µg/mL and 500µg/mL) totaling 0.4 mL were transferred to a 10mL volumetric flask together with 1mL of 3% hydrogen peroxide, and the mixture was refluxed at 60°C for 30 minutes. The solution was brought to room temperature and diluted with diluent to the proper concentration. This solution was injected in triplicate and examined in a volume of 20µL. Degradation occurred, and extra peaks were acquired.

Thermal degradation:

Remogliflozin and Metformin working standard solutions (100µg/mL and 500µg/mL) totaling 0.4 mL was transferred to a 10mL volumetric flask and refluxed at 80°C for 30 minutes. The solution was cooled to room temperature and diluted with diluent until it reached the desired strength. This solution was injected in triplicate and examined in a volume of 20 µL. However, there is no degradation.

Photolytic degradation:

Remogliflozin and Metformin (100µg/mL and 500 µg/mL) were combined in 0.4mL of working standard solution, and the solution was then placed in a 10mL volumetric flask and subjected to UV radiation at a shorter wavelength of 254nm for 30 minutes. With diluent, the solution was thinned to the proper consistency. This solution was injected in triplicate and examined in a volume of 20µL. However, there is no degradation.

RESULTS AND DISCUSSION:

Method optimization:

The proposed method was optimized by varying the chromatographic parameters to satisfy the system suitability. 0.1% Orthophosphoric acid and Acetonitrile in the ratio of 30:70 % v/v was used as the mobile phase at flow rate 0.9 ml/min was chosen as an optimized condition for determination of Remogliflozin (REM) and Metformin (MET). A good symmetrical peak was achieved with PDA detection at 230nm. The optimized chromatogram are as shown below.

Figure 2: Chromatogram of standard consisting of Metformin and Remogliflozin

Linearity:

A good linear relationship between concentration and peak areas was seen over concentration ranges of 0.1–20 µg/ml (Remogliflozin) and 0.5–100µg/ml (Metformin). The regression equations for Remogliflozin and Metformin were y = 35482x + 16491 and y = 89523x + 214323, respectively, with correlation coefficients of 0.9944 and 0.9955.

Precision:

The sample solutions were used in the intra-day and inter-day precision investigations at three different concentrations, and the %RSD varied from 0.01 to 0.08% for intra-day and 0.06 to 0.87% for inter-day studies.

Accuracy:

The traditional addition technique was used to conduct the accuracy experiments. Remogliflozin and Metformin had recovery rates ranging from 99.87 to 100.23% and 99.62 to 100.74%. (Table-1).

Robustness:

Three parameters from the optimal chromatographic settings, including the mobile phase composition (± 5 parts) and flow rate (± 0.1 mL/min), were changed to maintain robustness. The peak tailings, the theoretical plates, or the percent assays of remogliflozin and Metformin were not significantly impacted by deliberate modifications to the procedure, such as adjustments to the mobile phase's composition, flow rate, or wavelength. (Table-2).

(a) (b)

Figure 3: Calibration curve (a) Remogliflozin (b) Metformin

Table 1: Accuracy results of Remogliflozin (REM) and Metformin (MET)

Level (%)	Tablet Conc. (µg/mL)		Pure drug Conc. (µg/mL)		Area		Conc. found (µg/mL)		Recovery (%)		*% Recovery ± SD, % RSD
	Tablet Conc. (µg/mL)		Pure drug Conc. (µg/mL)		230 nm		Conc. found (µg/mL)		Recovery (%)		*% Recovery ± SD, % RSD
	MET	REM	MET	REM	MET	REM	MET	REM	MET	REM	MET	REM
50	15	3	7.5	1.5	2228591	176160	22.50	1.5	100	100	99.94± 0.07, 0.07	99.75 ± 0.33, 0.33
	15	3	7.5	1.5	2227912	175489	22.49	1.5	99.95	99.37
	15	3	7.5	1.5	2226845	176025	22.48	1.5	99.87	99.87
100	15	3	15	3	2910013	229383	30.11	3	100.74	100	100.20 ± 0.47, 0.47	99.62 ± 0.38, 0.38
	15	3	15	3	2898756	228987	29.99	3	99.91	99.63
	15	3	15	3	2899486	228578	29.99	3	99.96	99.24
150	15	3	22.5	4.5	3571436	282849	37.48	4.5	100	100.23	99.91± 0.08, 0.08	99.71 ± 0.45, 0.45
	15	3	22.5	4.5	3569458	282048	37.48	4.5	99.85	99.48
	15	3	22.5	4.5	3569875	281987	37.48	4.5	99.88	99.42

Table 2: Robustness data for Metformin (MET)

Parameter	Condition	Std. Area	Sample Area	Mean Sample Area	Theoretical plates	Tailing Factor	Assay (% w/w) ± SD, % RSD*
		MET	MET	MET	MET	MET	MET
Flow rate (± 0.1mL/min.)	1.0	1792782	1792888	1792943.5	3788.978	1.520	100.01 ± 0, 0
			1792999
	0.8	2238122	2237897	2237947	4474.472	1.442	99.99 ± 0.003, 0.003
			2237997
Mobile phase composition (± 5 parts)	35:65	1775648	1775999	1775832.5	4258.744	1.731	100.01 ± 0.01, 0.01
			1775666
	75:25	1904625	1904599	1904855	4119.43	1.759	100.01 ± 0.019, 0.019
			1905111

Table 3: Robustness data for Remogliflozin (REM)

Parameter	Condition	Std. Area	Sample Area	Mean Sample Area	Theoretical plates	Tailing Factor	*Assay (% w/w) ± SD, RSD
		REM	REM	REM	REM	REM	REM
Flow rate (±0.1ml/min)	1.0	135787	135689	135777	8153.836	1.397	99.99 ± 0.09, 0.09
			135865
	0.8	144887	144812	144911.5	10228.107	1.388	100.02 ± 0.09, 0.09
			145011
Mobile phase composition (± 7.7 %)	35:65	133758	133611	133755	8443.815	1.355	100 ± 0.15, 0.15
			133899
	75:25	145009	144999	145049	8744.996	1.453	100.03 ± 0.048, 0.048
			145099

CONCLUSION:

According to ICH guidelines, the RP-HPLC and UV spectrophotometric techniques mentioned above were developed and validated for determining remogliflozin and Metformin. Comparing the procedures to those already set, they were straightforward, precise, accurate, and affordable. As seen from the LOD and LOQ values, one advantage of these newly created methods is that they are far more sensitive than those in use. The RP-HPLC method might also report the Remogliflozin and Metformin stability characteristics under various stress situations. As a result, it can be used to determine the stability of medications in tablet form. Due to the absence of excipients or degradants, both techniques were precise.

ACKNOWLEDGEMENT:

Metformin HCl and Remogliflozin Etabonate were kindly provided by Yarrow Chem Products and Actis Generics Pvt Ltd., and the authors are grateful for their support. I'm also thankful to Dr. G. Sowjanya for offering crucial advice for conducting research.

REFERENCES:

1. Napoleon A.A. Pharmaceutical Titrimetric Analysis theory and practical, Kalaimani Publishers and Distributors, 1.1-1.4.

2. S. Ashutosh Kar. Pharmaceutical Drug Analysis 2nd Edition, New Age International Private Limited Publishers, 2005; 452-474.

3. Beckett AH and Stenlake JB. Text Book of Practical Pharmaceutical Chemistry, Part II, C.B.S., Publishers and Distributors, 157-162.

4. Waller DG, Sampson AP, Renwick AG, Hiller K. Medical pharmacology and therapeutics; 4th edn, 457.

5. Anthony Markham. Remogliflozin etabonate: first global approval. 2019; 79(10): 1157-1161.doi:10.1007/s40265-019-01150-9.

6. Dutta D, Jindal R, Mehta D, Khandelwal D, Sharma M. Efficacy and Safety of Novel Sodium Glucose Cotransporter-2 inhibitor Remogliflozin in The Management of Type 2 Diabetes Mellitus: A Systematic Review and Meta-Analysis. Diabetes Metab Syndr. 2011; 15(6). https://doi.org/10.1111/j.1463-1326.2011.01462.x

7. Sykes AP, O'Connor-Semmes R, Dobbins R, Dorey DJ, Lorimer JD, Walker S. Randomized trial showing efficacy and safety of twice-daily remogliflozin etabonate for the treatment of type 2 diabetes. Diabetes, Obesity and Metabolism. 2014; 17(1): 94-97. https://doi.org/10.1111/dom.12391

8. Sykes AP, Kemp GL, Dobbins R, O'Connor-Semmes R, Almond SR, Wilkison WO. Randomized efficacy and safety trial of once-daily remogliflozin etabonate for the treatment of type 2 diabetes. Diabetes, Obesity and Metabolism. 2014; 17(1): 98-101. https://doi.org/10.1111/dom.12393

9. Avolynt Announces Completion of Phase 2b BRID Study of SGLT2 inhibitor Remogliflozin Etabonate. Avolynt, Inc. July 24, 2018.

10. George Fantus, R. Brossea. Mechanism of action of Metformin: insulin receptor and postreceptor effects in vitro and in vivo. The Journal of Clinical Endocrinology and Metabolism. 1986; 63(4): 898–905. doi: 10.1210/jcem-63-4-898.

11. S.M. Charjan, S.K. Bais, R.R. Shiradkar, R.L. Bakal, V.M. Durge, A.V. Chandewar. Comparative Evaluation of Quality Control Parameters of Marketed Antidiabetic Tablet. Research J. Pharm. and Tech. May 2011; 4(5): 801-805.

12. Nachiket S. Dighe, Ganesh S. Shinde, Jyoti. J. Vikhe. Simultaneous Estimation, Validation and Force Degradation Study of Metformin Hydrochloride and Empagliflozin by RP-HPLC Method. Research J. Science and Tech. 2019; 11(2): 135-147. DOI: 10.5958/2349-2988.2019.00021.4

13. Jayshree Pawar, Sandeep Sonawane, Santosh Chhajed, Sanjay Kshirsagar. Development and Validation of RP-HPLC method for simultaneous Estimation of Metformin HCl and Gliclazide. Asian J. Pharm. Ana. 2016; 6(3): 151-154. DOI: 10.5958/2231-5675.2016.00024.7

14. Khushbu Patel, Ujashkumar A. Shah, Hirak V. Joshi, Jayvadan K. Patel, Chhaganbhai N. Patel. QbD Stressed Development and Validation of Stability-Indicating RP- HPLC Method for the Simultaneous Estimation of Linagliptin and Metformin HCl in Pharmaceutical Dosage Form. Research Journal of Pharmacy and Technology. 2022; 15(5): 1917-3. DOI: 10.52711/0974-360X.2022.00319

15. S. J. Daharwal, S Prakash Rao, Vijay Kumar Singh, Chandraprakash Dwivedi, Veena D. Singh. Comparative evaluation of marketed formulations of Metformin HCl. available in India. Asian J. Research Chem. 2015; 8(7): 441-444. DOI: 10.5958/0974-4150.2015.00070.X

16. Ravi Pratap Pulla, B.S. Sastry, Y. Rajendra Prasad, N. Appala Raju. Simultaneous Estimation of Metformin HCL and Sitagliptin Phosphate in Tablet Dosage Forms by RP-HPLC. Research J. Pharm. and Tech., 2011; 4(4): 646-649.

17. Ashok Kumar P, Suresh V Kulkarni, Basavaraj, Nikunj Patel, Someshwara Rao B, Ramesh B. Formulation and Evaluation of Locust Bean Gum Based Matrix Tablets for Oral Controlled Delivery of Metformin Hydrochloride and Its Comparison with Marketed Product. Research J. Pharm. and Tech. 2010; 3(4): 1082-1087.

18. Vitarani Dwi Ananda Ningrum, Ari Wibowo, Isma Fuaida, Zullies Ikawati, Ahmad Hamim Sadewa, Mohammad Robikhul Ikhsan. Validation of an HPLC-UV Method for the Determination of Metformin Hydrochloride in Spiked-human Plasma for the Application of Therapeutic Drug Monitoring. Research J. Pharm. and Tech., 2018; 11(6): 2197-2202. DOI: 10.5958/0974-360X.2018.00406.7

19. Inzucchi SE, Lipska KJ, Mayo H, Bailey CJ, McGuire DK. Metformin in patients with type 2 diabetes and kidney disease: a systematic review. 312(24): 2668–75. doi: 10.1001/ jama.2014.15298.

20. Diamanti-Kandarakis E, Economou F, Palimeri S, Christakou C. Metformin in polycystic ovary syndrome. Annals of the New York Academy of Sciences. 2010; 1205(1): 192–8. doi: 10.1111/j.1749-6632.2010.05679.x.

21. Diamanti-Kandarakis E, Christakou CD, Kandaraki E, Economou FN. Metformin: an old medication of new fashion: evolving new molecular mechanisms and clinical implications in polycystic ovary syndrome. European Journal of Endocrinology. 2010; 162(2): 193–212. doi: 10.1530/EJE-09-0733.

22. Nathan DM, Buse JB, Davidson MB, Ferrannini E, Holman RR, Sherwin R, Zinman B. Medical management of hyperglycaemia in type 2 diabetes mellitus: a consensus algorithm for the initiation and adjustment of therapy: a consensus statement from the American Diabetes Association and the European Association for the Study of Diabetes. Diabetologia. 2009; 52(1): 17–30. DOI: 10.1007/s00125-008-1157-y.

23. Nathan DM, Buse JB, Davidson MB, Ferrannini E, Holman RR, Sherwin R, Zinman B. Medical Management of Hyperglycaemia In Type 2 Diabetes Mellitus: A Consensus Algorithm For The Initiation and Adjustment Of Therapy: A Consensus Statement From The American Diabetes Association and The European Association For The Study Of Diabetes. Diabetologia. 2009; 52(1): 17–30. DOI: 10.1007/s00125-008-1157-y.

24. Mahesh Attimarad, Anroop Balachandran Nair, Sreeharsha Nagaraja, Bandar Essa Aldhubiab, Katharigatta Narayanaswamy Venugopala, Shinu Pottathil. Smart UV derivative spectrophotometric methods for simultaneous determination of Metformin and Remogliflozin: development, validation and application to the formulation. Indian Journal of Pharmaceutical Education and Research. 2021; 55(1): 293-302. DOI: 10.5530/ijper.55.1s.62.

25. Shivani V. Trivedi. Stabilty indicating RP-HPLC method development and validation for simultaneous estimation of Remogliflozin Etabonate and Metformin Hcl in synthetic mixture and tablet dosage form. World Journal of Pharmaceutical Research. 2021; 10(1).

26. Ruchi Vasa, Nimit Vasa, Neha Tiwari, Pragnesh Patani, Bansi Solanki. Development and validation of stability indicating RP- HPLC method for estimation of Metformin Hcl and Remogliflozin Etabonate in pharmaceutical dosage form. International Journal of All Research Education and Scientific Methods. 2021; 9(1).

27. ICH Harmonized Tripartite Guideline; Stability Testing of New Drug Substances and New Drug Products Q1A (R2); International conference on Harmonization, IFPMA, Geneva, Switzerland; 2003.

28. ICH Harmonized Tripartite Guideline; validation of analytical procedures of New Drug Substances and New Drug Products Q2 (R1); International conference on Harmonization, IFPMA, Geneva, Switzerland; 2003.

Received on 01.10.2023 Revised on 21.03.2024

Accepted on 23.06.2024 Published on 10.12.2024

Available online on December 30, 2024

Asian Journal of Pharmaceutical Analysis. 2024; 14(4):234-240.

DOI: 10.52711/2231-5675.2024.00042

Parameters	Metformin	Remogliflozin
Retention time	2.091 ± 0.014	4.635 ± 0.012
Theoretical plates	3257.191 ±508.1	8292.240 ± 478.7
Tailing factor	1.570 ± 0.045	1.443 ± 0.026
Resolution	----------	14.354 ± 0.044

Brand name	Label claim (mg)		Amount obtained (mg)		*Assay (% w/w) ± SD
REMO-ZEN M 500	REM	MET	REM	MET	REM	MET
REMO-ZEN M 500	100	500	98.25	497.66	98.24 ± 1.31, 1.33	99.47 ± 1.29