INTRODUCTION:

Empagliflozin chemically 1-chloro-4-[b-D-glucopyranos-1-yl]-2-[4-([S]-tetrahydrofuran -3- yl -oxy) benzyl]-benzene (fig.2) which belongs to gliflozin class used in the treatment of type-2 diabetes. Empagliflozin is an inhibitor of the sodium glucose co transporter -2 (SGLT-2) thus SGLT-2reduces blood glucose by blocking glucose reabsorption in the kidney and thereby excreting glucose (i.e., blood sugar) via the urine.[9,1]

Metformin is chemically known as 1, 1-dimethylbiguanide hydrochloride (fig 1) a biguanide derivative which is the most commonly prescribed drug to the patients with type-2 diabetes. it bring down the blood glucose levels by decreasing the hepatic glucose production, declining intestinal absorption of glucose and enhancing insulin sensitivity by elevating peripheral glucose utilization and uptake. [4,5]. The literature reveal that various analytical technique viz; uv spectrophotometry, high performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC). Our aim was to develop proper method which estimates both the analytes in a shorter time and to develop low cost method. [9]

Fig No. 1 Metformin

Fig No. 2 Empagliflozin

MATERIALS AND METHODS:

Apparatus and equipment:

A Shimadzu UV-visible spectrophotometry UV-1800, (Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurement with 1 CM paired quartz cell.

Reagent and chemical:

Pharmaceutical grade Empagliflozin and Metformin hydrochloride was supplied as a gift sample from Macleod Pharmaceutical Pvt. Ltd. Gujrat, India. tablet for analysis was purchased from local marked manufacture by Boehringer Ingelheim International GmbH

Binger Strasse 173 55216 Ingelheim Germany.

Preparation of standard stock solution:

Standard stock solution of pure drug containing of 1000 µg/ml of EMPA and MET were prepared in water. Standard stock solution were further diluted with water to get standard working solution of analysts in the concentration range of 2-10µg/ml and 4-20µg/ml for EMPA and MET, respectively and scanned in range of 200-400nm.

Preparation of sample solution and formulation analysis:

Twenty tablet were weight accurately and quantity of tablet equivalent to 500 mg of MET and 10mg of EMPA was weighted and dissolve in 50 ml of water and sonicated for 15 min in 100 ml volumetric flask, volume was make upto the mark with water.[9]

Selection of wavelength:

By appropriate dilution of standard stock solution, solution containing 10 μg/ml of MET and 10 μg/ml EMPA separately, these diluted solutions were scanned in range 200-400 nm separately. EMPA showed λmax at 224 nm in water and MET shows λmax at 230 nm[1,9].

Fig1: Isobestic for Empagliflozin and Metformin

Fig: 2 Metformin calibration Curve

Fig:3 Empagliflozin calibration Curve

Theoretical aspect:

Method A: Q-absorbance [12,13]

Q-Absorbance method uses the ratio of absorbance at two selected wavelengths, one at isoabsorptive point and other being the max of one of the two compounds. From the stock solutions, working standard solutions of Empagliflozin and Metformin were prepared by appropriate dilution and were scanned in the entire UV range to determine the maximum absorbance (λmax) and isoabsorptive point. Empagliflozin and Metformin have λmax at 224 nm and at 232 nm, respectively. Both the drugs were found to have same absorbance at 203 nm (isoabsorptive point). The wavelengths selected for analysis were 224 nm and 232 nm respectively. A series of standard solutions ranging from 2-10µg/mL for empagliflozin and 4-14-20µg/mL metformin were prepared to plot a calibration curve of absorbance versus concentration. The calibration curves were found to be linear in the concentration range under study. Absorptivity values of empagliflozin and metformin were determined at selected wavelength.[6]

The concentration of two drugs in mixture was calculated

By using following equations:

Qm – Qy A1

CDEXI = --------------- _ ------ (1)

Qx - Qy ax1

Qm – Qx A2

CPCM = --------------- _ ------ (2)

Qy – Qx ay1

Method B: Area under curve method:

For the simultaneous determination using the area under the curve method, suitable dilutions of the standard stock solutions (1000 μg/mL) of EMPA and MET were prepared separately in water. The solutions of drugs were scanned in the range of 200-400 nm. (13) For Area Under Curve method, the sampling wavelength ranges selected for estimation of EMPA and MET were 219-229 nm (λ1-λ2) and 227-238 nm (λ3-λ4). Mixed standards were prepared and their Area under the Curve were measured at the selected wavelength ranges.[7]

Concentration of two drugs in mixed standard and the sample solution were calculated using equation (1) and (2).

CMFA = A2 × aX2 - A1 × ay2 / aX2 × aY1 - aX1 × aY2 ……… (1)

CPCT = A2 - aX2 × CMFA/ aY2 ……………... (2)

Method C: Dual wavelength:

The mechanism of this method is that the difference between absorbance at two particular wavelengths where one component shows no difference in absorbance. (13) This difference between absorbance is directly proportional to the concentration of the components. Spectra of EMPA in the range of 2-10μg/ml and of MET in the range of 4-20μg/ml were collected in the range of 200nm to 400nm.At wavelengths of 224nm and 238nm, EMPA was shown same absorbance and MET was shown significant difference in absorbance, hence were selected for estimation of EMPA. At wavelengths of 232nm and 244nm, MET was shown same absorbance and EMPA was shown significant difference in absorbance, hence were selected for estimation of MET. To establish two separate curves for both the drugs, difference in absorbance at 224nm and 238nm were plotted against the concentration of EMPA and difference in absorbance at 232nm and 244nm were plotted against the concentration of MET.(8)

Method validation:

The method was validated with respect to linearity, precision, accuracy, specificity, and robustness and ruggedness, limit of detection, and limit of quantification accordingly to ICH guidelines.[2,3]

Linearity:

The linearity was determined by analyzing independed level of calibration range 2-20µg/ml for EMPA and 4-20µg/ml for MET. Absorbance of each solution was recorded against water with different method A, B, C at different wavelength. The calibration curve absorbance vs. concentration was plotted and correlation coefficient regression line equations were obtained for determination empagliflozin and metformin.

Precision:

Method repeatability was determined by three times repetitions of assay procedure. For intra-day precision method was repeated 3 times in a day and the average % RSD was determined. Similarly the method was repeated on 3 different day’s foe inter-day precision and average % RSD was determined. Precision of analyst was determined by repeating study by another analyst working in the laboratory. [6]

Accuracy:

The accuracy studies were carried out at different concentration by spiking a known concentration of standard drug to the placebo at three levels (80%, 100%, 120%) on three different preparations and analyzed by the developed methods.[9]

Specificity:

Specificity is a procedure to detect quantitatively the analyst in the presence of component that may be expected to be present in the sample matrix commonly used excipient in tablet preparation were spiked in a pre-working quantity of drugs and then absorbance was measured and calculation done to determined the quantity of the drugs.[7,8]

Robustness:

The robustness of proposed method was tested by changing parameters such as wavelength range, slit width, temperature, and shaking time. None of the variables significantly affected the absorbance of the drugs indicating that the method could be considered as robust.[6]

Ruggedness:

The ruggedness of proposed method was determined by analyzing aliquots from homogeneous lot in different under graduate laboratories using similar operation and environmental condition, data is presented in table no 1.

Table 1: optical characteristics, precision study and result of formulation analysis

Parameter		Empagliflozin			Metformin
Parameter		Method A	Method B	Method C	Method A	Method B	Method C
Wavelength (nm)		203	224	238	203	232	244
Beer’s law limit (µg/mL)		2-4µg/ml	2-4µg/ml	2-4µg/ml	4-20µg/ml	4-20µg/ml	4-20µg/ml
Regression Equation*	Slope (m)	0.1943	0.0517	0.0204	0.0643	0.0807	0.0406
Regression Equation*	Intercept(c)) (c)	0.0285	0.0181	0.001	0.0181	0.0224	0.021
Correlation coefficient (r)		0.9989	0.9967	0.9995	0.9993	0.9991	0.9993
Precision (%RSD)	Repeatability (n=3)	0.966	1.086	1.52	1.526	1.516	1.602
	Intra-day	1.999	1.972	0.873	1.84	1.957	1.902
	Inter-day	1.738	1.685	.966	1.689	1.764	1.816
Formulation Analysis (% Assay, %RSD), n=3		98.92, 0.863	99.5, 0.828	104.5, 1.470	97.21, 0.8843	100, 1.103	96.85, 1.326
LOD (µg/mL)		0.008	0.02	0.04	0.373	0.0249	0.0379
LOQ (µg/mL)		0.02	0.4	0.12	0.610	0.0082	0.169
Ruggedness (%RSD)	Analyst I	0.613	0.589	0.381	1.75	0.38	1.06
Ruggedness (%RSD)	Analyst II	0.606	0.586	0.385	0.97	0.21	0.59

Table 2: Accuracy Studies EMPA

Recovery level	Analyte name	Amount spiked (µg/ml)	Mean recovery, % RSD
Recovery level	Analyte name	Amount spiked (µg/ml)	Method A	Method B	Method C
80%	EMPA	3.2	70.28, 1.997	88.75,1.875	89, 1.865
100%	EMPA	4	66.58, 1.844	103.1, 1.301	80.9, 2.435
120%	EMPA	4.8	78.58,1.978	97.36, 1.873	94.30, 1.704

Table 3: Accuracy Studies MET

Recovery level	Analyte name	Amount spiked (µg/ml)	Mean recovery, % RSD
Recovery level	Analyte name	Amount spiked (µg/ml)	Method A	Method B	method C
80%	EMPA	9.6	99.8, 1.618	88.5, 1.58	88.39, 1.318
100%	EMPA	12	101.16, 1.343	70.75, 1.345	91.38, 1.491
120%	EMPA	14.4	100.8, 1.685	123.5, 1.061	96.84, 1.213

DISCUSSION:

The proposed method was found to be simple, accurate and rapid for the routine determination empagliflozin and metformin in tablet formulation. To study validity and reproducibility of proposed method, recovery studies were carried out. The method was validated in terms of linearity, accuracy, precision, specificity and reproducibility. The three methods can be successfully used for simultaneous estimation of empagliflozin and metformin in combined dosage form.

ACKNOWLEDGMENT:

The authors are thankful to the management and trustees of Mumbai Educational Trust’s Bhujbal Knowledge City, Nashik, for providing necessary chemicals and analytical facilities and to Macleods Pharmaceutical Pvt. Ltd. Gujarat, India, for providing pharmaceutical grade Empagliflozin and Metformin Hydrochloride as gift sample.

REFERENCES:

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2. ICH Harmonized Triplicate Guidelines, “Validation of analytical procedures: text and methodology, Q2 (R1),” in International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use, 2005.

3. International Conference of Harmonization (ICH) of Technical Requirements for the Registration of Pharmaceuticals for Human Use, Validation of Analytical Procedures: Methodology, Adopted in Geneva (1996).

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Received on 14.02.2019 Modified on 05.03.2019

Asian J. Research Chem. 2019; 12(2):94-98.

DOI: 10.5958/0974-4150.2019.00021.X