Development and validation of UV spectrophotometric method for Simultaneous estimation of Empagliflozin and Metformin hydrochloride

in bulk drugs

 

  Sushil D. Patil*, Sayali K. Chaure, Sanjay Kshirsagar

MET’s Institute of Pharmacy, MET League of Colleges, Bhujbal Knowledge City, Adgaon, Nashik, MS 422003 *Corresponding Author E-mail: sushilpharma@rediffmail.com

ABSTRACT:

A new, simple, accurate and sensitive UV-spectrophotometric absorption correction method has been developed for simultaneous determination of Metformin HCl and Empagliflozin in bulk utilizing concept of standard addition. The method is based upon determination of Empagliflozin at 224 nm & Metformin HCl at 230 nm methanol as a solvent. Overlay spectra of both drugs shows absorbance at 227 nm. Linearity was observed in range of 10-50 µg/ml and 1-3 µg/ml for Metformin HCl and Empagliflozin respectively. The correlation coefficient value was found to near to 1.All methods were statistically validated as per ICH guidelines.

 

 

 

INTRODUCTION:

Empagliflozin:

Empagliflozin is a sodium glucose co-transporter-2 (SGLT-2) inhibitor and to improve the glycemic control in adult patient with type 2 diabetes. Chemically known as (2S, 3R, 4R, 5S, 6R)-2-[4-chloro-3-({4-[(3S)-oxolan-3-yloxy] phenyl}methyl) phenyl]-6-(hydroxymethyl)oxane-3,4,5-triol. The chemical structure of Empagliflozin shown in Fig.1.[1]

 

Fig 1: Structure of Empagliflozin

 

As per literature review, several method were there for the determination of its pharmacologic action. Empagliflozin was estimated by only few method UV spectroscopy, HPLC and UPLC. Only one stability indicating RP-HPLC method. The aim of present work was to develop and validate a accurate, cost effective and precise stability indicating RP-HPLC method for determination Empagliflozin.

 

Metformin hydrochloride:

Metformin (MET) is chemically named as 4 1-carbamimidamido-N, N-dimethylmethanimidamide and it is as shown in Fig. 1. It is used as a biguanide antihyperglycemic agent used for treating non-insulin-dependent diabetes mellitus (NIDDM) [2,3]. It improves glycemic control by decreasing hepatic glucose production, decreasing glucose absorption, and increasing insulin-mediated glucose uptake. MET may induce weight loss and is the drug of choice for obese NIDDM patients. Use of MET is associated with modest weight loss.

 

Fig 2: Structure of Metformin Hydrochloride

 

Literature survey revealed that few analytical methods are reported for analysis of both the drugs alone as well as in combination using ultraviolet (UV) spectrophotometry [4], high-pressure liquid chromatography (HPLC) [4-9], and ultra-pressure liquid chromatography [10].

 

MATERIALS AND METHODS:

Apparatus and Equipments:

A Shimadzu UV–visible spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) was used for all absorbance measurements with 1 cm paired quartz cell.

 

Reagents and Chemicals:

Pharmaceutical grade Empagliflozin and Metformin hydrochloride was supplied as a gift sample from Macleod Pharmaceutical Pvt. Ltd. Gujrat, India. Methanol used in analysis was of Analytical grade and all other chemicals and reagents were of analytical grade and were purchased from Thomas Baker (Chemicals), Mumbai, India.

 

Preparation of standard stock solution : (100µg/mL)

An accurately weighed quantity of 10 mg empagliflozin was transferred to 100 mL volumetric flask, dissolved with 10 mL of methanol and sonicated for 15 min, volume was then  made up to  the mark with water.

 

An accurately weighed quantity of 10 mg Metformin hydrochoride was transferred to 100 mL volumetric flask, dissolved with 10 mL of methanol and sonicated for 15 min, volume was then  made up to  the mark with water.

 

Selection of wavelength for analysis:

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 Methanol. (figure 3) and MET shows λmax at  230 nm (figure 4).

 

Fig 3. UV Spectra of Empagliflozin

	230 nm

 

Figure 4 : UV spectra of Metformin Hydrochloride

	224 nm
		227 nm

 

Figure 5 : Overlay UV spectra of EMPA and MET

 

Method validation:

The UV Spectrophotometric method was validated as per ICH guidelines for method validation. The performance parameters like linearity, precision and accuracy were evaluated. [12]

 

Limit of Detection (LOD):

LOD is the lowest level of concentration of analyte in the sample that can be detected, though not necessarily quantitated. It was calculated by using the formula [11, 12].

LOD= 3.3σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.

 

Limit of Quantitation (LOQ):

LOQ is the lowest concentration of analyte in a sample that may be determined with acceptable accuracy and precision when the required procedure is applied. It was calculated by using the formula, [12]  

LOQ=10σ/S

Where,

σ = Standard deviation of the response,

S = Slope of calibration curve.           

 

Method based on Standard Deviation of blank:

Analysis were carried out using water as a blank or diluents. The absorbance of water was measured 10 times at 227 nm.

 

Method based on Calibration Curve:

Prepare 0.001ppm to 1.0 ppm of empagliflozin and Metformin HCL solution from stock solution respectively. The absorbance of resulting solution was measured 10 times at 227 nm.

Cut off range for empagliflozin was found to be 0.001µg/mL

Cut off range for Metformin HCL was found to be 0.002µg/mL

 

 

Table 1. Determination LOD and LOQ based on calibration curve calculations

Sr. No.	Conc. µg/mL	Absorbance at 227nm EMPA	Conc. µg/mL	Absorbance at 227nm MET
1	0.001	0.002	0.002	0.00195
2	0.002	0.002	0.004	0.0034
3	0.003	0.003	0.006	0.00575
4	0.004	0.003	0.008	0.00895
5	0.005	0.005	0.01	0.0091
6	0.006	0.007	0.02	0.0124
7	0.007	0.009	0.03	0.0295
8	0.008	0.01	0.04	0.0345
9	0.009	0.011	0.05	0.0485
10	0.01	0.009	0.06	0.06
11	0.02	0.008	0.07	0.07
12	0.03	0.011	0.08	0.078
13	0.04	0.011	0.09	0.09
14	0.05	0.013	0.1	0.0995
15	0.06	0.014	0.2	0.195
16	0.07	0.017	0.3	0.29
17	0.08	0.01	0.4	0.395
18	0.09	0.015	0.5	0.48
19	0.1	0.019	0.6	0.585
20	0.2	0.027	0.7	0.676
21	0.3	0.037	0.8	0.7805
22	0.4	0.048	0.9	0.9085
23	0.5	0.057	1	0.979
24	0.6	0.066
25	0.7	0.071
26	0.8	0.081
27	0.9	0.089
28	1	0.098

 

 

Fig 6: Calibration curve of empagliflozin for LOD and LOQ (Conc 0.001-1 µg/mL)

 

Fig 7: Calibration curve of Metformin HCL for LOD and LOQ (Conc 0.002-1 µg/mL)

 

Linearity:

Linearity was studied by diluting standard stock solution of Empagliflozin to 1-3 μg/ml and Metformin 2-12μg/ml concentrations (n=3). Calibration curves with concentration verses absorbance were plotted at their respective wavelengths and the obtained data was subjected to regression analysis using the least square method. The standard curves for Empagliflozin and Metformin are shown in (Fig. 6, 7) respectively and data is presented in Table 2.[11]

 

Table 2: Calibration study data EMPA

Sr. No.	Concentration (µg/mL)	Absorbance at 227 nm
1	1.0	0.134
2	1.5	0.158
3	2.0	0.188
4	2.5	0.216
5	3.0	0.239

 

Table 3: Calibration study data MET

Sr. No.	Concentration (µg/mL)	Absorbance at 227 nm
1	1.0	0.134
2	1.5	0.158
3	2.0	0.188
4	2.5	0.216
5	3.0	0.239

 

Fig 8: Calibration curve of empagliflozin (1.0-3.0 µg/mL)

 

Fig 9: Calibration curve of MET (2.0-10.0 µg/mL)

 

Specificity:

Specificity is the ability to measure unequivocally the desired analyte in the presence of components such as excipients and impurities.[11,12]

 

Here, Magnesium stearate was used as excipient and which is prepare a stock solution (B) (100µg/mL). Spiking Magnesium stearate in 3 different levels 80,100,120% respectively from stock solution B and stock solution (A) which is spike 100% and mix with 3 different level of Magnesium stearate solution to  determine the amount of ℅recovery at 227nm. 

 

Table 4: % Recovery studies for specificity of EMPA

Sr. No.	Conc. of API(µg/mL)	Add % level	Spike stock sol.(µg/mL)	Abs. at 227nm	Drug found (µg/mL)	% Recovery
1	2	80	1.6	0.181	1.924	96.226
2	2	100	2.0	0.181	1.924	96.226
3	2	120	2.4	0.182	1.943	97.169

 

Table 5: % Recovery studies for specificity of MET

Sr. No.	Conc. of API(µg/mL)	Add % level	Spike stock sol.(µg/mL)	Abs. at 227nm	Drug found (µg/mL)	% Recovery
1	4	80	3.2	0.358	3.632653	90.81632653
2	4	100	4.0	0.354	3.604021	90.05102041
3	4	120	3.8	0.361	3.673469	91.83673469

 

Accuracy:

To check the accuracy of the developed methods and to study interference of formulation additives, analytical recovery experiments were carried out by using standard addition method. Reference standard solution of each drug was added to tablet samples at three different

concentrations level (80, 100 and 120%). At each level, samples were prepared in triplicate and the mean percentage recoveries and % RSD value were calculated. Table .6 shows the result for accuracy of the method.[12,13]

 

Table 6: Accuracy study data (Plane drug + Product) of EMPA

 

Table 7: Accuracy study data (Plane drug + Product) of MET

 

Precision:

It is the degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings. It was determined by studying repeatability, intra-day and inter-day precision of method. The average recovery of the analyte of 80%, 100% and 120% solution.

 

For intraday, the analysis was carried out at different intervals on the same day and for inter day, the analysis was carried on different days. Table 8 and 9 give the results for intraday and inter-day studies respectively.[11,12,13]

 

Table 8: Precision study data EMPA

 

Table 9: Precision study data MET

 

Ruggedness:

Ruggedness of the proposed method is determined by analysis of aliquots from homogenous slots by different analysts using similar operational and environmental conditions. The results are shown in Table.10 and Table.11.[11,12,13]

 

Table 10: Robustness study data at different wavelength of EMPA

Sample No.	Wavelength (nm)
	222	224	226
1	0.1869	0.1861	0.1839
2	0.1862	0.1852	0.1842
3	0.1856	0.1849	0.1834
Avg. %Recovery	101.163	100.377	98.899
SD	0.613	0.589	0.381
%RSD	0.606	0.586	0.385

 

Table 11: Robustness study data at different wavelength of MET

Sample No.	Wavelength (nm)
	220	230	240
1	0.689	0.721	0.697
2	0.702	0.718	0.699
3	0.699	0.720	0.705
Avg. %Recovery	92.26	94.64	91.07
SD	1.73	0.38	1.06
%RSD	0.97	0.21	0.59

 

RESULTS AND DISCUSSION:

Development and optimization of the spectrophotometric method:

Proper wavelength selection of the methods depends upon the nature of the sample and its solubility. To develop a rugged and suitable spectrophotometric method for the quantitative determination of empagliflozin, the analytical condition were selected after testing the different parameters such as diluents, buffer, buffer concentration, and other chromatographic conditions.

 

Our preliminary trials were by using different compositions of diluents consisting of methanol best result was obtained and degassed in an ultrasonic bath. Below figure represent the spectrum (Figure 1).

 

Selection of wavelength:

Scan standard stock solution in UV spectrophotometer between 200 nm to 400 nm on spectrum mode, using Methanol as a blank. Empagliflozin shows λmax at 224nm and Metformin Hydrochloride shows λmax at 230nm.The combine overlay of both drugs shows λmax at 227nm. The proposed analytical method is simple, accurate and reproducible (Figure 3,4,5).

 

Method validation:

The set parameter the method is validated, LOD and LOQ were found to be 0.036,0.111 and 0.04,0.1402 for EMPA and MET respectively.(Table 1, and Fig. 3), 0.0068 and 0.0206 (Table 1, and Fig.6,7) by standard deviation of blank and calibration curve method respectively. Among the entire range Linearity range was found to be 1 to 3 μg/ml for EMPA(Table 2, and Fig. 8)  and 2 to 10 μg/ml for MET.(Table 3, and Fig. 9).Method was found to be specific for the EMPA(Table 3) and MET (Table 4). For accuracy data supported by ANNOVA test with at P ≤ 0.05 and other data indicates that the results lie between 97.79-99.71% for both the drugs (Table 5 and 6).The precision confidence interval of 99% was considered precise and the %RSD values for the repeatability and intermediate precision studies were <0.666%,<0.663% and <0.708%,<0.706%,for EMPA and MET respectively shown in (Table 7 and 8). The evaluation of robustness at different wavelength (Table 9 and 10), the value of the test preparation solution of robustness was not affected and it was in accordance with that of actual. Hence the analytical method would be concluded as robust.

 

Table 9: Result summary of various validation parameter

Parameter	Values for EMPA	Values for MET
LOD (µg/mL)	0.036 µg/mL	0.04 µg/mL
LOQ (µg/mL)	0.111 µg/mL	0.1402 µg/mL
Linearity and Range (µg/mL)	1-3 µg/mL	2-10 µg/mL
Accuracy (%Recovery)	99.44%	93.27%
ANOVA	P≤ 0.05	P≤ 0.05
Precision (%RSD)	RSD< 2%	RSD< 2%

 

CONCLUSION:

The developed UV methods were found to be more accurate, precise and reproducible. These statistical parameter of these methods showed good results. The recovery studies revealed excellent accuracy and high precision of the method. The methods were found to be simple & time saving. All three proposed methods could be applied for routine analysis in quality control laboratories.

 

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 Macleod Pharmaceutical Pvt. Ltd. Gujrat, India, for providing pharmaceutical grade Empagliflozin and Metformin Hydrochloride as gift sample.

 

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Received on 06.04.2017       Accepted on 18.05.2017     

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