INTRODUCTION:

About 463 million adults are living with diabetes globally. The proportion of people with type 2 diabetes is increasing in most countries¹. Diabetes mellitus type 1 is a disease caused by the lack of insulin secretion, and type 2 diabetes mellitus (T2DM) is a disease caused by insulin resistance by cells. Anti-diabetic drugs are used to treat diabetes mellitus by reducing the glucose level in the blood. Type 2 diabetes mellitus, which is characterized by polyphagia, polyuria, and polydipsia and needs a lifetime treatment with anti diabetic drugs². Metformin hydrochloride (MTF), [3-(diaminomethylidene)-1,1-dimethylguanidine] is used for the management of T2DM as it improves glucose tolerance, and decreases the postprandial and basal plasma glucose.

It improves insulin sensitivity by decreasing glucose intestinal absorption and hepatic production, and it is the most important therapy which is used in combination with other orally administered hypoglycemic^3,4,5. Empaglifozin and Metformin structure is shown in the Fig 1.

Fig 1: (a) Empaglifozin

Fig 1: (b) Metformin

Literature review shows stability-indicating micellar electrokinetic chromatography method⁶, Metformin and Empaglifozin combination drugs by spectrophotometry^7,8 Liquid chromatography method for drug⁹, HPTLC^10,11,12,13 RP-HPLC for single empaglifozin¹⁴ and LC-MS/MS¹⁶. Comparative evaluation of metformin¹⁷ and metformin with other drugs are shows for HPLC and UV-Visible spectrophotometric methods^{15,18,19,20,21,22,23,24,25}. However, good stability-indicating chromatographic method for determining Empaglifozin and Metformin HCl in the dosage form is unavailable. The present study is to develop a faster, best separatated analytes and validated stability-indicating RP-HPLC method for the simultaneous estimation of Empaglifozin and Metformin in bulk and tablet.

MATERIALS AND METHOD:

Chemicals and Reagents:

Reference sample of Empaglifozin and Metformin HCl, Acetonitrile, Water (HPLC grade) Methanol (HPLC Grade), Orthophosphoric acid were used in the analysis.

Instruments and Software:

A Schmidazu LC 2010 CHT with quaternary constant flow system and equipped with auto sample injector was utilized in the study. Schimadzu LC Solution software was employed to monitor and integrate the output signals. Other instrument and apparatus such Contech Analytical balance, Eutech pH meter, Leela Sonic Ultra-sonicator were used in this study.

Optimized Chromatographic conditions:

Mobile Phase:

It was prepared in the ratio of Buffer and Aetonitrile in the ratio of 50:50.

Buffer Solution:

1ml of Orthophosphoric acid was diluter up to 1000ml with HPLC grade water. The pH of the solution was found to be 1.8.

Solvent Mixture:

It was prepared by mixing Acetonitrile and water in the ratio of 50:50.

The separation of Empaglifozin and Metformin was achieved by using Column: Sunniest ECO C₁₈, 250mm X 4.6mm, 5µm at a Column temperature of 35°C with Buffer: Aetonitrile (50:50) as mobile Phase, at a Flow rate of 1.2ml/minute. Elution effected in isocratic mode with Injection volume of the analyte sample of 10µl and detection at 225nm with Run Time 6.0minutes

Standard Stock Solution of Empaglifozin:

50mg of empaglifozin was weighed and transferred into a 100ml volumetric flask containing 40ml of solvent mixture. Then the flask was sonicated with the help of ultrasonicator to dissolve the drug. Then the flask was diluted up to the mark with the solvent mixture. Again transfer accurately 5ml of this solution into another 25ml volumetric flask and then it was diluted up to the mark with the solvent mixture to obtain the concentration of 100µg/ml.

Standard Stock Solution of Metformin:

500mg of Metformin HCL was weighed and transferred into a 100ml volumetric flask containing 40ml of solvent mixture. Then the flask was sonicated with the help of ultra sonicator to dissolve the drug. Then the flask was diluted up to the mark with the solvent mixture to get the concentration of 5000µg/ml.

Preparation of Working Standard Stock solution:

14, 12, 10, 8, 6, 4 and 7, 6, 5, 4, 3 and 2 of standard stock solution of Empaglifozin and metformin were transferred into seven different 10ml volumetric flasks and then the flasks were diluted up to the mark with the help of solvent mixture to obtain the concentration of both drugs mention in the Table 1. Then this solutions were injected into the HPLC system and the areas were recorded and it reflects in the below mentioned Table 1. The linear regression equation of empaglifozine is Y = 29132.9524 X – 625.6667 and Correlation coefficient r 2 = 0.999. The linear regression equation of Metformin is Y = 26991.1493 X + 703795.4211 and Correlation coefficient r 2 = 0.9953. The linearity curve of Empaglofozin is shown in the Fig 3. The linearity curve of the Metformin is shown in the Fig 2. The representative chromatogram of the empa and metformin is shown in the Fig 4.

Table 1: Linearity data of Metform and Empaglifozin

Sl. No.	Metformin		Empaglifozine
Sl. No.	Conc. (µg/ml)	Area	Conc. (µg/ml)	Area
1	0	0	0	0
2	200	6963477	2	60527
2	300	9164547	3	83429
3	400	11532600	4	113628
4	500	14106638	5	147898.5
5	600	16351299	6	171664
6	700	19485408	7	203250.5
7	800	22495417	8	234251

Fig: 2: Linearity curve of Metformin

Fig: 3: Linearity curve of Empaglifozin

Preparation of Sample Solution from Marketed Tablets:

Commercial tablets available in local market procured and an average weight of 20 tablets was determined and powdered finely in a mortar. Powdered tablet equivalent to 50 mg of Empaglifozin and 500 mg of Metformin HCl was accurately weighed and transferred into a 100 ml clean dry volumetric flask which was dissolved with diluents (or solvent mixture) and sonicated and made volume up to volume with the diluents and filtered. Further transferred 3 ml of filtrate into another 50ml volumetric flask and made up to the mark with the diluent. The results of the tablet analysis is shown in the below Table 2. The representative chromatogram of tablet sample is shown in the Fig 5.

Fig 4: Representative chromatogram of standard (5 empa and 500 met µg/ml)

Fig 5: The representative chromatogram of tablet sample.

Method Validation:^10,11

The HPLC method was validated in terms of System linearity, specificity, sensitivity, precision, and accuracy, robustness in accordance with ICH Q2 (R1) guideline and system suitability test as per USP.

Precision:

The intraday precision/repeatability can be determined by injecting three working standard solutions and test sample injections. The areas of all the injections were taken and % Relative standard deviations were calculated. The % RSD was found to be less than 1.5 in. The inter day precision can be determined by utilizing three different concentration working standard solutions and three different test sample solutions were injected on three different days. The areas of all the injections were taken and % Relative standard deviation was calculated. The results obtained were found to be less than 1.5% in standard and test sample solutions.

Accuracy:

A spiked recovery studies using the conventional addition method was performed to test the accuracy of the proposed method. It was performed at 50 % and 100 % level. The results of accuracy studies are shown in the below Table 3. ¹¹

Table No: 2: The results of tablet analysis

Analyte	Label claim (mg/Tablet)	Amount Found (mg/Tablet)	C.I.	% RSD	SE	t
Empaglifozin	5	5.026	100.520±1.355	1.084	0.488	1.065
Metformin	500	500.342	100.068±0.260	0.208	0.093	0.728

Table 3: The results of recovery study of the drug

% Level of recovery	Analyte	Formulation (µg/ml)	Amount of drug added (µg/ml)	Amount of drug found (µg/ml)	CI	% RSD	SE	t
50	Empa	3	1.5	4.520	100.448±1.30	1.045	0.470	0.955
50	Met	300	150	450.948	100.210±0.340	0.272	0.122	1.72
100	Empa	3	3	6.034	100.566±2.293	1.835	0.827	0.686
100	Met	300	300	599.918	99.98±0.290	0.233	0.104	0.13

Peak purity:

The peak purity of Empaglifozin and Metformin were assessed by comparing the retention time (R_t) of samples of standard mixture and tablet samples of Empaglifozin and Metformin. Overlay chromatogram in Fig 5 depicts good correlations between the retention time of standard and dosage form drugs.

Fig 6: Overlay Chromatogram of Empaglifozin and Metformin

Robustness:

The robustness of the proposed assay method was determined by deliberately small changes in the chromatographic condition which included wavelength (222 nm 228 nm), flow rate (1.8 and 2.2 mL/ min) and organic phase (+5% to -5%). The % RSD shows with in the 1.5. So it indicates that the method is robust.

System Suitability:

The system suitability test was carried out on 5µg/ml and 500µg/ml of Empaglifozin and Metformin respectively by using five replicate injections. The system suitability is conformity of chromatographic parameters that ensures the performance of the analytical system. All the parameters of system suitability are shown in the below Table no 4.

Table 4: Results of system suitability parameter of Empaglifozin and Metformin

Sl. No.	Parameter	Specification	Empaglifozin	Metformin
1	Retention Time	----	4.0822	2.104
2	Tailing factor	< 2.0	1.298	1.5016
3	Theoretical Plates	> 2000	7110.0748	3862.2094
4	Area (% RSD)	< 2	0.52	0.42
5	Resolution (Rs)	> 2.0	10.1784	----

Forced degradation study:

Force degradation studies have done to develop a stability-indicating assay by involving the proposed optimized RP-HPLC conditions by utilizing acidic, basic and oxidative, conditions at 90 minutes. The results of forced degradation studies are shown in the Table 5.

Table 5: The results of forced degradation studies

Stress Condit ion	Degra dation times	Metformin					Empaglifozin
Stress Condit ion	Degra dation times	taken conc. (µg/ml)	Found conc. (µg/ml)	Area	% Assay	Degra Dation	Taken Conc. (µg/ ml)	Area	Found conc. (µg/ml)	% Assay	Degrad ation
0.1 N HCL	90	500	441.845	12629707	88.369	11.63	5	134532.50	4.639	92.8	7.2
0.1 N Na OH	90	500	485.164	13798942.50	97.033	2.967	5	147555	5.086	101.72	0
% 3 H₂O₂	90	500	495.42	14075922.5	99.084	0.91	5	151348	5.21	104	0

Fig 7: Blank chromatogram of 0.1 N HCl

Fig 8: Representative chromatogram for degradation of drugs in 0.1 N HCl

Fig 9: Blank Chromatogram of 0.1 N NaOH

Fig 10: Representative chromatogram of drug in 0.1 N NaOH

Fig 11: Blank Chromatogram of 3% H₂O₂

Fig 12: Representative chromatogram of drug in % 3 Hydrogen Peroxide

Sensitivity:

The sensitivity of the method was estimated by calculating LOD and LOQ. The LOD and LOQ were separately determined based on the standard calibration curve. The Lower limit of detection and limit of quantization were found to be 0.296, and 0.896µg/ml respectively.

RESULTS AND DISCUSSION:

To optimize the proposed method several trials were performed systematically on chromatographic conditions like column type, and mobile phase conditions. Reverse phase solvents like mixtures of water: organic solvent and buffer: organic solvent were studied at different ratios on HPLC columns to optimize this research article. Validation results, indicates that all the parameters were within the acceptable limits of ICH guidelines. The system suitability test results proved that more efficient separation (N>2000), with well-resolved (R>2) and symmetric peaks (T<2) were obtained consistently. Moreover, the analytical output was reproducible since the %RSD for the retention time of six system suitability determinations was less than 2%. The method was found to be selective because formulation excipients didn't interfere with blank and placebo determinations. The r² obtained from the linear regression analysis for both drugs was closer to 1 which indicates the better linearity of the proposed method. Recovery results within 98% - 102% reveals the accuracy of the method and, the %RSD values for repeatability and intermediate precision determinations never exceeded 1.5% showing the better precision of the proposed HPLC method. Analytical results from a deliberate slight variation on the optimized method condition indicated that variations have no significant influence on the analytical output and thus the method was robust

CONCLUSION:

A new simple and faster liquid chromatographic method was successfully developed and validated for simultaneous quantification of Empaglifozin and Metformin in bulk and Tablet formulation. The method was demonstrated to be stability-indicating, fast, sensitive, accurate, precise, and robust. Thus, the developed method can be easily adopted for the routine quality control of bulk and in the combination tablet dosage form.

Acknowledgement:

The authors thanks to the Director of Fine Cure Pharmaceuticals Limited for providing necessary facilities to develop and validate this method

AUTHORS CONTRIBUTION:

All authors have contributed equally.

CONFLICT OF INTERESTS:

Author declares that there have been no conflicts of interest.

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Received on 28.01.2023 Modified on 17.04.2023

Research J. Pharm. and Tech 2024; 17(3):1135-1140.

DOI: 10.52711/0974-360X.2024.00177