INTRODUCTION:

A metabolic condition known as diabetes mellitus (DM) is typified by high blood glucose levels. 90% of individuals have type 2 diabetes, and one in eleven people worldwide are thought to have DM (T2DM). Experts estimate that by the end of 2040, there would be around 642 million instances of DM worldwide, up from the current 422 million cases^1,2.

Remogliflozin etabonate (RGF), with the chemical term [5-methyl-4-(4-(1-methylethoxy) benzyl)- 1-(1-methylethyl)-1H-pyrazol-3-yl 6-O-(ethoxy carbonyl)-b-D glucopyranoside] is classed as SGLT2 inhibitor.

The transporter known as SGLT2 plays a major role in the kidneys' capacity to reabsorb glucose. Vildagliptin (VGT) also recognized as (S)-1-[N-(3-hydroxy1-adamantyl) glycyl] pyrrolidine-2-carbonitrile. Metformin hydrochloride (MTH) is also known by its chemical name, 1,1-dimethyl biguanide hydrochloride.^3,6

The newer combination of RGF, VGT, and MTH was launched in October 2021 by Glenmark Pharmaceuticals Ltd., India. It works through three synergistic mechanisms to help control glycemic levels in people with diabetes (type 2) peoples may benefit from consuming RGF and VGT together with MTH.

RGF, VGT, and MTH alone and in combination with other drugs was determined using ultraviolet (UV) spectrophotometry^7,8,17,18, HPLC^4,5,6, LCMS⁹,HPTLC^12,15,19,20UPLC^13,14andRP-HPLC^10,11,16. However, only two research articles reported simultaneous estimation of all three drugs under study.

Based on the available literature it can be confirm that no UPLC method was available for the separation and simultaneous quantification of vildagliptin and remogliflozin etabonate. Hence the present work aimed to develop simple and precise analytical UPLC method for the separation and simultaneous estimation of vildagliptin remogliflozin etabonate and metformin in bulk drug as well as in pharmaceutical formulations.

MATERIALS AND METHODS:

Chemicals and Reagents:

Remogliflozin etabonate (REM), Vildagliptin (VLD), Metformin hydrochloride (MEH) Water, NaHPO₄, sodium hydroxide (NaOH), Acetonitrile and Hydrochloric acid (HCl) chemicals are made use in the research.

Instruments:

UPLC system from “Waters Alliance” corporation.

Detector - Photodiode array from “Waters Alliance” corporation.

Empower 2^nd software from “Waters Alliance” corporation.

Inertsil ODS 3V (250x4.6 mm, 5µm)

Preparation Mobile phase:

In a 1000 ml beaker HPLC water, then adjust the pH with NaHSO₄ to 3.8.

500 mL of acetonitrile and 500 mL of NaHSO₄ was used as mobile phase. They are mixed and sonicate for 20min.

Preparation Diluents:

With a 50: 50 volume/volume proportionality, NaHSO₄ and Acetonitrile are combined.

Preparation of stock solution:

To make a stock REM, VLD, and MEH solution, 100 mg REM, 50 mg VLD, and 500 mg MEH were carefully balanced in a flask (100 ml), then diluted appropriately in diluting fluid (NaHPO4 and Acetonitrile are mixed in a 50:50 volume/ volume proportionality). The concentrations are as follows: 100 microg/ml for REM, 50 microg/ml for VLD, and 500 microg/ml for MEH.

Preparation of working solution:

5 ml stock REM (100 µg/ml), VLD (50 µg/ml), and MEH (500 µg/ml) combined with 45 ml dissolving solvent NaHPO₄and Acetonitrile (pure) merged in a 50:50 v/v fraction. REM - 100 µg/ml, VLD - 50 µg/ml, and MEH – 500 µg/ml.

Optimization of chromatographic conditions:

After a series of trials, the final chromatographic conditions were determined as follows. The mobile phase was a buffer with pH 4.5 and acetonitrile: NaHSO₄ (50:50% v/v), and the stationary phase was a inertsil ODS 3V column with dimensions 5 µm, 4.6 mm x 250 mm to obtain the best peak shape. The separation of remogliflozin, vildagliptin and metformin hydrochloride was good at 260 nm, a flow rate of 0.4 mL/min, and Run time is 5 min as shown in graph 1.

Figure 1: Chromatograms - REM, VLD and MEH

Validation of the Analytical Method:

Linearity:

The method linearity was verified with 5 dilutions of the 100% concentration: 50µg/ml, 75µg/ml, 100µg/ml, 125µg/ml and 150 µg/ml for remogliflozin and 250µg/ml, 375µg/ml, 500µg/ml, 625 µg/ml and 750µg/ml for metformin hydrochloride. And 25 µg/ml,37 µg/ml,50 µg/ml,62 µg/ml and 75 µg/ml for vildagliptin. The acceptance criterion of the regression coefficient (R²) was NLT 0.99 as shown in table 1.

Table 1: Linearity of REM, VLD and MEH

REM		VLD		MEH
µg/ml	Area	µg/ml	Area	µg/ml	Area
50	1428645	25	939075	250	1870170
75.00	2151622	37.5	1393759	375.00	2820502
100.00	2871012	50	1867469	500.00	3768618
125	3590232	62.5	2334467	625	4710338
150	4317678	75.00	2808166	750	5669957

Selectivity:

Metformin hydrochloride 500 ng/mL and remogliflozin 100 ng/mL were injected in triplicate preparations of 100% concentration to confirm the method validation's exactness and selectivity. To check for carryover, another blank was injected as shown in graph 2.

Figure 2: Chromatograms - REM, VLD and MEH selectivity

Precision:

After passing the specificity and system suitability criteria, the method was verified for system precision and method precision with the limit of % RSD for the RT and area NMT 2%. The intermediate precision was verified on the next day with another column by setting the limit as % RSD for the RT and NMT 2% for the area as shown in the table 2 and graph 3.

Table 2: Precision of REM, VLD and MEH

REM		VLD		MEH
Area	Data	Area	Data	Area	Data
2877948	Mean 2879061	1861503	Mean 1868493	3760348	Mean 3766333
2870091		1879535		3771365
2888678	SD 7671.2	1868574	SD 6219.3	3765778	SD 4213.3
2873609		1863788		3765331
2875986	RSD 0.3	1868384	RSD 0.3	3764186	RSD 0.1
2888054		1869171		3770991

Figure 3: Chromatograms - REM, VLD and MEH selectivity

Accuracy and recovery:

To verify the method accuracy, triplicate preparations were prepared at 50%, 100%, and 150% of the 100% concentrations. (100 µg/ml for remogliflozin, 50 µg/ml for vildagliptin and 500µg/ml for metformin hydrochloride) The percent recovery was calculated with acceptance criteria of 95%-105% as shown in the table 3.

Table 3: Accuracy and recovery of REM, VLD and MET

S. No.	Drug Name	% Of Concentrations	Add in (µg/ml)	Area
1	Remogliflozin	50	49.5	1422423
			49.5	1431257
			49.5	1436080
2		100	99	2882003
			99	2879440
			99	2877690
3		150	148.5	4315520
			148.5	4313699
			148.5	4322146
1	Vildagliptin	50	24.75	939618
			24.75	939319
			24.75	940555
2		100	49.5	1872092
			49.5	1869064
			49.5	1862787
3		150	74.25	2814481
			74.25	2807805
			74.25	2803777
1	Metformin	50	247.5	1870330
			247.5	1875722
			247.5	1882941
2		100	495	3769731
			495	3767081
			495	3775878
3		150	742	5665164
3		150	742	5676346

LOD and LOQ:

By considering the 10% concentration of the target concentration, the sample was injected into the system with the acceptance criteria S/N ratio NLT 10. From the LOQ and LOD, preparations of different concentrations were injected to identify the detectability with the acceptance criteria 3:1 as shown in graph 4.

Figure 4: Chromatograms - REM, VLD and MEH sensitivities

Robustness:

To verify the method efficiency when minor changes occurred in optimized method parameters such as mobile-phase composition, column temperature and flow, and buffer PH. These parameters were tested with the criteria that they should pass the system suitability criteria as shown in graph 5.

Figure 5: Chromatograms - REM, VLD and MEH robustness

Degradation studies:

To test the developed method for stability indicating method the formulation sample was subjected to acid and base, and thermal, photo, and peroxide degradation were carried with the aim of detection of degradants in the chromatogram. Acid degradation was carried out by adding 10 mL of 0.1N HCL to the stock solution, and from that 1 mL was removed and added to a 1000 mL volumetric flask and the volume adjusted to the mark. In the same way, 6 mL 1N NaOH was added to test for base degradation. To test for thermal degradation, the sample was subjected to heat at 105°C for 3 hours and the sample prepared as per the assay procedure. For photo degradation, the sample was exposed to ultraviolet light with an intensity NLT 2000 lux power for 6 hours and the sample prepared as per the assay procedure. For peroxide degradation, 8mL H₂O₂ were added to the stock 1000 mL volumetric flask, 1 mL was removed and added to a 1000 mL flask, the volume adjusted to the mark with the diluent, and the sample was injected as shown in the table 4 and graph 6,7,8,9 and 10.

Table 4: Degradation outcomes of REM, VLD and MEH

S. No.	Drug Name	Condition	Area	% Recovery	% Degradation
1	Remogliflozin	Acid	2590632	89.37	10.63
		Base	2712950	93.59	6.41
		Peroxide	2655568	91.61	8.39
		Heat	2615862	90.24	9.76
		Sun light	2751275	94.91	5.09
2	Vildagliptin	Acid	1710901	90.66	9.34
		Base	1791933	94.95	5.05
		Peroxide	1764278	93.49	6.51
		Heat	1658657	87.89	12.11
		Sun light	1730808	91.71	8.29
3	Metformin	Acid	3411175	89.48	10.14
		Base	3499230	92.18	7.82
		Peroxide	3615080	95.24	4.76
		Heat	3320727	87.48	12.52
		Sun light	3539251	93.24	6.76


Figure 6:	Figure 7:

Figure 8:	Figure 9:

Figure 10:

Chromatograms (Acid, Base, Peroxide, Thermal and photo stability) REM, VLD and MEH degradation

CONCLUTION:

Based on the results obtained in the current study, the developed method was very sensitive, accurate, linear, and economical. Due to the short duration of the chromatographic program, more samples can be analysed within a short period, which will be helpful in the industry at a time when multiple products are manufactured continuously. The method met all the predefined acceptance criteria. With this method, the sample of bulk and formulation samples and surface cleaning samples can be analysed. As the method is capable of detecting degradant formulations, bulk shelf-life samples can also be analysed by using this method.

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Received on 10.09.2024 Revised on 14.11.2024

Accepted on 12.12.2024 Published on 28.02.2025

Available online from March 04, 2025

Asian Journal of Pharmaceutical Analysis. 2025;15(1):40-44.

DOI: 10.52711/2231-5675.2025.00007

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