INTRODUCTION:

Remogliflozin Etabonate chemically ethyl [(2R,3S,4S,5R,6S)-3,4,5-trihydroxy-6-[5-methyl-1-propan-2-yl-4-[(4-propan-2-yloxyphenyl)methyl]pyrazol-3-yl]oxyoxan-2-yl]methyl carbonate, is an oral selective Sodium-Glucose co-transporter 2 (SGLT2) inhibitor used for the management of type 2 Diabetes Mellitus. Vildagliptin chemically (S)-1-{2-(3-Hydroxyadamantan 1 ylamino) acetyl] pyrrolidine-2-carbonitrile;

1-[(3-hydroxyadamant- 1 ylamino)- acetyl}-pyrrolidine-2-(s)-carbonitrile) belongs to class of medicines called ‘islet enhancers’. Vildagliptin inhibits the inactivation of GLP-1 and GIP by DPP-4, allowing GLP-1 and GIP to potentiate the secretion of insulin in the beta cells and suppress glucagons release by the islets of Langerhans in the pancreas. Vildagliptin has been shown to reduce hyper glycaemia in type 2 diabetes mellitus^1-2

Various UV methods are reported for estimation of both drus in individually and combined with other drugs.^4-7.^.various UV methods are reported for individualdrugs but there is no method reported for simultaneous estimation of Remogliflozin Etabonate and Vildagliptin in bulk and Pharmaceutical Dosage form^8-17. Simultaneous equation (SE) or Vierordt’s method is typically applied to estimate drug combinations that contain two drugs or more than two drugs in combined dosage form. Technical hitches involved in this method is very less when compared to other UV methods. Hence an attempt has been made to develop a simple and a reproducible Simultaneous equation method to ensure the safety and efficacy of this selected combination. This developed method was fully validated and applied successfully for the simultaneous estimation of Remogliflozin Etabonate and Vildagliptin in pure and pharmaceutical dosage form.

MATERIALS AND METHODS:

Apparatus:

Shimadzu 1800 UV-VIS double beam spectrophotometer with UV probe software was used. Absorbance measurements were recorded with a pair of 1 cm matched quartz cells.

Chemicals and Reagents:

Reference standard of Remogliflozin Etabonate and Vildagliptin was purchased from Benzchem Enterprise Vadodara and TriveniInterchem Pvt. Ltd., Vapi respectively and they were used as such without further purification. The marketed tablet (Remo V) used contains 100mg Remogliflozin Etabonate and 50mg Vildagliptin and was manufactured by Glenmark Pharmaceuticals Ltd.

Preparation of Standard Stock Solutions:

Standard stock solution of Remogliflozin Etabonate (100µg/mL) was prepared by dissolving 10mg of Remogliflozin Etabonate in 60mL of Methanol. Resulting solution was sonicated for 10 minutes and the final volume was adjusted to 100mL with Methanol. From this standard stock solution, 1mL was withdrawn and diluted using same solvent to get working standard solution of 10µg/mL. Similarly standard stock solution of Vildagliptin (20µg/mL) was prepared by dissolving 2 mg of Vildagliptin in 60mL of Methanol. Resulting solution was sonicated for 10 minutes and the final volume was adjusted to 100mL with Methanol. This solution was further diluted to get working standard solution.

Simultaneous Equation Method Development:

Working solutions of both drugs were scanned in the UV range 200– 400nm. The overlay spectra of both drugs were recorded (Figure 1). From overlain spectra, wavelengths 236nm (𝜆max of Remogliflozin Etabonate) and 215nm (𝜆max of Vildagliptin) were selected for analysis of both drugs using simultaneous equation method (𝜆1- 236nm for Remogliflozin Etabonate and 𝜆2-215nm for Vildagliptin). Consequently, it may be possible to determine both drugs by the technique of from method or simultaneous equation method.

Five standard solutions having concentrations of 5, 10, 15, 20, and 25µg/mL for Remogliflozin Etabonate and 1, 2, 3, 4, and 5µg/mL for Vildagliptin were prepared in Methanol and their corresponding absorbance was measured at 236nm and 215nm. The concentration of drugs x (Remogliflozin Etabonate) and y (Vildagliptin) in sample solutions were determined by the SE method using the following formula:³

𝐶𝑥 = 𝐴2𝑎𝑦1 − 𝐴1𝑎𝑦2 / 𝑎𝑥2𝑎𝑦1 − 𝑎𝑥1𝑎𝑦2,

𝐶𝑦 = 𝐴1𝑎𝑥2 − 𝐴2𝑎𝑥1 / 𝑎𝑥2𝑎𝑦1 − 𝑎𝑥1𝑎𝑦2,

where 𝐶𝑥 and 𝐶𝑦 are the concentration of Remogliflozin Etabonate and Vildagliptin, 𝐴1 and 𝐴2 are the absorbance of sample solution at 236nm and 215nm, respectively, 𝑎𝑥1 and 𝑎𝑥2 are absorptivity of Remogliflozin Etabonate at 236nm and 215nm, and 𝑎𝑦1 and 𝑎𝑦2 are absorptivity of Vildagliptin at 236nm and 215nm, respectively.

Determination of Absorptivity Value:

The absorptivity value of Remogliflozin Etabonate and Vildagliptin from each solution was calculated using following formula and the results were presented in Tables 1 and 2:

Absorptivity = Absorbance/conc. (gm/100mL)

Developed method was validated as per ICH guidelines.

RESULTS AND DISCUSSION:

Specificity:

Specificity of the method was dogged by measuring the absorbance of Remogliflozin Etabonate and Vildagliptin individually at 236 nm and 215 nm against the blank and synthetic excipients and their absorbance was compared with the blank and synthetic excipients. No interference was observed at 236 nm and 215 nm indicating that the method is specific.

Linearity:

The calibration curves (Figures 4 and 5) were constructed by plotting the absorbance versus the concentration ranges from 5, 10, 15, 20, to 25µg/mL and 1, 2, 3, 4, to 5µg/mL for Remogliflozin Etabonate and Vildagliptin. It was found that, the calibration curves were linear in these concentration ranges with their correlation coefficient values (𝑅²) 0.9993 for Remogliflozin Etabonate and 0.9998 for Vildagliptin. Results revealed that good correlation exists between the concentration of the sample and their absorbance.

Precision:

Precision study was established by measuring the absorbance of sample solution without changing the assay procedure and the results were presented in Table 4. The low % RSD (<2%) for Remogliflozin Etabonate and Vildagliptin indiczte that the method is precise.

Accuracy:

Accuracy of the method was determined by applying this described method to synthetic excipients containing known amount of each drug corresponding to 80%, 100%, 120%, of Remogliflozin Etabonate and Vildagliptin. The amount of Remogliflozin Etabonate and Vildagliptin recovered in each level was calculated and results were presented in Table 3.

The percentage recovery varied from 99.25 to 101.06% for Remogliflozin Etabonate and 99.58 to 100.41% for Vildagliptin, indicating good accuracy of the method.

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

LOD and LOQ were calculated based on the standard deviation of the response (50% concentration solution and the slope of calibration graph. LOD and LOQ for Remogliflozin Etabonate and Vildagliptin were found to be 0.0246µg/mL and 0.0745µg/mL for Remogliflozin Etabonate and 0.0278µg/mL and 0.0842µg/mL for Vildagliptin.

Table 1: Linearity Value for Remogliflozin Etabonate and Vildagliptin

Parameters	Remogliflozin Etabonate	Vildagliptin
Regression Equation	Y=0.2683x – 0.0441	Y=0.1865x + 0.0928
Linearity (µg/ml)	5-25	1-5
Correlation Coefficient (r²)	0.9993	0.9998

Table 2: Precision results for Remogliflozin Etabonate and Vildagliptin.

Parameters	Remogliflozin Etabonate			Vildagliptin
Parameters	Concentration (µg/ml)	Absorbance ± SD (n=3)	%RSD	Concentration (µg/ml)	Absorbance ± SD (n=3)	%RSD
Intraday	10	0.497±0.002	0.402	2	0.496±0.001	0.325
	15	0.775±0.001	0.129	3	0.656±0.001	0.152
	20	1.025±0.002	0.202	4	0.835±0.003	0.453
Interday	10	0.496±0.001	0.307	2	0.470±0.001	0.324
	15	0.774±0.001	0.149	3	0.642±0.008	1.305
	20	1.021±0.001	0.149	4	0.833±0.003	0.454
Repeatability	15	0.773±0.001 0.776±0.001 0.775±0.001 0.774±0.001 0.776±0.001 0.775±0.001	0.150	3	0.659±0.001 0.655±0.001 0.657±0.001 0.658±0.001 0.605±0.001 0.654±0.001	0.299

Table 3: Absorptivity value for Remogliflozin Etabonate and Vildagliptin

Absorptivity value for Remogliflozin Etabonate
Concentration (µg/mL)	Absorbance 𝜆1 − 236 nm	Absorptivity 𝜆1 − 236 nm	Absorbance 𝜆2 − 215 nm	Absorptivity 𝜆2 – 215 nm
5	0.215	0.043	0.296	0.0086
10	0.496	0.049	0.376	0.0376
15	0.776	0.051	0.635	0.042
20	1.025	0.051	0.784	0.0392
25	1.285	0.051	0.962	0.0384
	Absorptivity for 𝜆1	0.049	Absorptivity for 𝜆2	0.03316

Absorptivity value for Vildagliptin.
Concentration (µg/mL)	Absorbance 𝜆1 − 215 nm	Absorptivity 𝜆1 − 215 nm	Absorbance 𝜆2 − 236 nm	Absorptivity 𝜆1 − 236 nm
1	0.278	0.278	0.319	0.319
2	0.482	0.241	0.465	0.232
3	0.661	0.22	0.532	0.177
4	0.829	0.207	0.752	0.188
5	1.025	0.205	0.973	0.194
	Absorptivity for 𝜆1	0.2302	Absorptivity for 𝜆2	0.222

Table 5: Recovery results for Remogliflozin Etabonate and Vildagliptin

Drug	Level of Recovery in (%)	Amount added (µg/ml)	Total Amount Recovered (µg/ml)	%RSD	% Drug Recovery
Remogliflozin Etabonate	80	4.5	11.9±0.001	0.249	99.25
	100	7.5	15.2±0.001	0.197	100.01
	120	9.5	17.4±0.001	0.163	101.06
Vildagliptin	80	0.9	2.3±0.002	0.363	99.58
	100	1.5	3.2±0.001	0.232	99.92
	120	2.1	3.7±0.001	0.198	100.41

Figure 3: Calibration graph for Vildagliptin

Table 6: LOD and LOQ

Validation Parameters	Remogliflozin Etabonate	Vildagliptin
LOD (µg/ml)	0.0246	0.0278
LOQ (µg/ml)	0.0745	0.0842

Application of Developed Method to Marketed Dosage Forms:

20 tablets were weighed and flattened into powder. Powder weight equivalent to 10mg of Remogliflozin Etabonate and 2mg of Vildagliptin were transferred into a 100mL volumetric flask. 50mL of solvent was added and sonicated for 20 minutes. Then the final volume was diluted up to the mark with the solvent (Methanol) and filtered. 2mL of the above filtrate was transferred into a 25mL volumetric flask, and the final volume was adjusted up to mark with the same solvent to get sample solution with the concentration of 100µg/mL of Remogliflozin Etabonate and 20µg/mL of Vildagliptin. The absorbance of the resulting solution was measured at 236nm and 215nm. The amount of Remogliflozin Etabonate and Vildagliptin was found to be 100.62 and 99.74 of label claim respectively.

Finally the developed new simple simultaneous equation method was applied successfully to the marketed tablet dosage form and the assay results were indicating that this method can be effectively used for the estimation of both drugs in combined dosage form.

CONCLUSION:

The developed simultaneous equation method is simple, precise, specific, and accurate. Statistical analysis proved that the method was repeatable and selective for the simultaneous estimation of Remogliflozin Etabonate and Vildagliptin in pure and pharmaceutical dosage forms without any interference from the excipients. This new simple method can be used routinely for the estimation of these drugs.

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Received on 19.05.2022 Modified on 10.09.2022

Asian J. Pharm. Ana. 2023; 13(2):69-73.

DOI: 10.52711/2231-5675.2023.00012