Development and Validation of Novel Analytical Method for Empagliflozin and Metformin Hydrochloride in Bulk and Pharmaceutical Dosage Form by Four Different Simultaneous Estimation Approaches using UV Spectroscopy

Manojkumar K. Munde ^1,2*, Nilesh S. Kulkarni², Rahul H. Khiste³, Dhanya B. Sen¹

¹Department of Pharmacy, Sumandeep Vidyapeeth Deemed to be  University, Piparia,

Vadodara-391760, Gujarat, India.

²PES Modern College of Pharmacy (for Ladies), Moshi, Maharashtra, Pune, India.

Affiliated to Savitribai Phule Pune University, Pune.

³Marathwada Mitra Mandal’s College of Pharmacy, Thergaon, Pune-411033, Maharashtra. India.

Affiliated to Savitribai Phule Pune University, Pune.

*Corresponding Author E-mail: manojpcist@gmail.com

ABSTRACT:

Four new UV spectrophotometric methods namely simultaneous equation, absorbance ratio, area under curve and first derivative (zero crossing) spectroscopic methods were developed and validated for simultaneous estimation Empagliflozin and Metformin hydrochloride in bulk and tablet formulation. In simultaneous equation method, absorbance was measured at 224 and 232 nm for both the drugs. Empagliflozin and Metformin hydrochloride was estimated using 224 and 232 nm in absorbance ratio method. In Area under curve method both drugs were estimated at 224 and 232 nm respectively. First derivative (zero crossing) method was based on the transformation of UV spectra in to first derivative spectra followed by measurement of first derivative signal at 224 and 232 nm for Empagliflozin and Metformin hydrochloride, respectively using 2 nm as wavelength interval (Δλ) and 1 as scaling factor. Methods were found to be simple, fast, highly sensitive, cost effective and hence can be useful for simultaneous estimation of Empagliflozin and Metformin hydrochloride in commercial tablet formulation for routine quality control analysis.

KEYWORDS: Simultaneous equation, absorbance ratio, area under curve method, first derivative (zero crossing) spectroscopic methods, tablet formulation.

INTRODUCTION:

Empagliflozin (EN) chemically,(1-chloro-4-[b-D-glucopyranos-1-yl]-2-[4-([S]-tetrahydrofuran-3-yl-oxy) benzyl]-benzene is an orally administered selective sodium glucose cotransporter-2 (SGLT-2) inhibitor, which lowers blood glucose in people with type 2 diabetes by blocking the reabsorption of glucose in the kidneys and promoting excretion of excess glucose in the urine. Empagliflozine have the potential to reduce cardiovascular risk in patients with type 2 diabetes^1,2.

In patients with type 2 diabetes and hyperglycaemia a higher amount of glucose is filtered and reabsorbed. Empagliflozin improves glycaemic control in patients with type 2 diabetes by reducing renal glucose reabsorption. The content of glucose moiety removed by renal excretion, through this glucuretic mechanism is dependent on blood glucose concentration and GFR. Inhibition of SGLT2 in patients with type 2 diabetes and hyperglycaemia leads to excess glucose excretion in the urine^3-5. Metformin hydrochloride (MET) is given orally in the treatment of type 2 diabetes mellitus and is the drug of choice in overweight patients. They do not stimulate insulin release but require that some insulin be present in order to exert their antidiabetic effect. Possible mechanism of action includes the delay in the absorption of glucose from the GIT, decrease hepatic glucose production and increase in insulin sensitivity and glucose uptake in to cells and inhibition of hepatic gluconeogenesis^6-8. Handful clinical trials were reported in the literature for Empagliflozin and Metformin. Literature survey shows that there are many methods for the estimation of Empagliflozin and Metformin separately and in combination with other drugs. To our knowledge, spectrophotometric determination of Empagliflozin and Metformin in bulk and combined dosage form has not been developed and reported so far. So, an attempt was made to develop and validate an economic and spectrophotometric determination of Empagliflozin and Metformin in bulk and combined dosage form. The method was validated as per ICH guidelines.

Fig:1 Chemical structures of  Metformin hydrochloride (a) and Empagliflozin (b)

MATERIALS AND METHODS:

Chemicals and reagents:

Empaliflozin and Metformin reference standards used throughout the experiment were received as gift sample from Lupin Ltd. Pune, Maharashtra, India. The pharmaceutical formulation, Jardiance MetTM (Boehringer Ingelheim India Private Limited Bandra (East) Mumbai, India) 5 mg of EN along with 500 mg of MET was purchased from commercial sources. AR grade methanol was used as solvent and procured from Loba Chemie Pvt. Ltd., Mumbai, India.

Apparatus:

Shimadzu double beam UV visible spectrophotometer (UV-1800, UV Probe, Shimadzu Corporation) with matched quartz cell of 1 cm path length was used throughout the experiment.

Preparation of Standard Solution:

Stock solution of EN and MET were prepared individually by weighing accurately 10 mg of standard drugs and transferred to a 10 ml volumetric flask separately. Standard drugs were diluted to 10 ml with methanol to get the concentration of the drugs 1000 μg/ml. Further dilutions were made to get required concentration with methanol.

Procedure:

1. Simultaneous Equation Method:

Standard stock solutions containing 1000 μg/ml of EN and MET were suitably diluted separately with methanol to obtain the drug solutions containing 10 μg/ml. Both the solutions were scanned in the UV region (200 - 400 nm) and spectra were recorded. Based on the spectral pattern, SE (simultaneous equation) methods [Beckett and Stenlake] were chosen for the estimation of both the drugs. From the overlain spectra, 232 nm (λmax of MET) and 224 nm (λmax of EN) were selected for SE method.

Varying concentrations ranging from 5-30 μg/ml of EN and MET were prepared by diluting respective stock solutions. All the solutions were scanned in the UV region and absorbances were noted at 224 and 232 nm for SE. Absorptivity values were calculated for EN and MET at their relevant wavelengths by applying following formula:

Absorptivity = absorbance / concentration (gm/100 ml)

2. Absorbance Ratio Method:

Based on the spectral pattern, AR (absorbance ratio) methods [Beckett and Stenlake] were chosen for the estimation of both the drugs. In Absorbance ratio method 214 (isobestic point) and 232 nm (λmax of MET) was selected, which showed excellent linearity and therefore used for simultaneous determination. All the solutions were scanned in the UV region and absorbances were noted at 232 and 214 nm for AR method. Absorptivity value of individual solution at their respective wavelength was calculated and average absorptivity value (Table 1) at specific wavelength of particular drug was used for calculating concentration of drugs.

Table1: Absorptivity values for SE and AR methods

SE				AR
Avg. Absorptivity				Avg. Absorptivity
EN		MET		EN		MET
224	232	224	232	214	232	214	232
450.4	296.6	421.9	425.6	532.4	289.8	938.5	605.7

3. First Derivative (zero crossing) Method:

The normal UV spectra of EN and MET were transformed into first and second derivative spectra. Based on the spectral pattern and zero crossing points, 1^st DR (derivative spectroscopic) method was chosen for the study. First derivative spectra showed typical zero-crossing points at 277 nm for EN and 237 nm for MET applying 2 nm as wavelength interval (Δλ) and 1 as scaling factor. After assessing overlain spectra, 237 nm and 277 nm were selected for further studies (Figure 3). Calibration curve was plotted for both EN and MET in the concentration range of 5 to 30 μg/ml. Results were subjected to regression analysis by least square method to determine the values of slope, intercept and correlation coefficient.

Fig. 2: Overlain UV spectra of EN and MET (10 μg/ml).

Fig. 3: Overlain 1st derivative (zero crossing) UV spectra of EN and MET (10 μg/ml).

4. Area under curve method:

The absorption spectrum (from 200 to 400 nm) of these solutions were recorded using distilled water as a blank. The AUC (area under the curve) values for each component were recorded over the wavelength ranges of (227-237) nm and (217-227) nm and the calibration graphs were constructed. The area absorptivity values were calculated at each wavelength range for the two components then the concentration of EN and MET was calculated from the equations⁹:

Preparation of Sample Solution:

Analysis of Sample Solution:

1. Simultaneous Equation Method:

After scanning the sample solution (formulation) between 200 to 400 nm, responses were noted at 224 and 232 nm. The unknown concentration of drugs present in the sample solution was estimated by solving following formula,

Where Cx and Cy are the concentrations of EN and MET, a_x1 and a_x2 are absorptivities of EN at 232 and 224 nm, respectively. a_y1 and a_y2 are absorptivities of MET at 232 and 224nm, respectively. A1 and A2 are the absorbances of sample solution at 232 and 224nm.

2. Absorbance Ratio Method:

The unknown concentration of drugs in the sample solution was estimated by AR method applying following formula:

Where, a_x1 and a_x2 are absorptivities of EN at 232 and 214 nm, respectively. a_y1 and a_y2 are absorptivities of MET at 232 and 214 nm.

A1 and A2 are the absorbances of sample solution at 232 and 214 nm. Cx and Cy are the concentrations of EN and MET, respectively in sample solution.

3. First Derivative (zero crossing) Method:

Sample solution was scanned in the UV region (200-400nm) and spectrum was recorded and transformed into their 1^st derivative spectra and amplitude was measured at 224 or 232 nm. The unknown concentration of drugs present in the sample solution was estimated by using regression equation¹⁰.

Validation of Spectroscopic Methods:

Specificity:

To check the interference between tablet excipients used in the formulation and drug substance, specificity study was carried out. All the tablet excipients (as per marketed formulation) were mixed in proportion and diluted using methanol and filtered using Whatman filter paper no 41. All the solutions (Placebo and standard) were scanned in the UV region and compared to assess the interference among excipients and drugs.

Linearity and Range:

Linearity and range of all the four  methods were checked by analysing all the standard solutions separately ,containing EN and MET (5, 10, 15, 20, 25 and 30 μg/ml) in methanol and absorbance’s were noted at 224 and 237 nm for SE method; 232 and 214nm for AR method; 224 and 232 nm for 1^st DR method. Calibration graphs were constructed using absorbance of standard drug solutions versus concentration in SE and AR method; 1st derivative signal of standard drug solutions versus concentration in DR method. Regression analysis was performed by least squares method to determine the values of slope, intercept and correlation coefficient.

Precision:

Precision of the methods were evaluated by performing repeatability, intra-day and inter-day precision studies. Repeatability of the methods were evaluated by analyzing sample solutions (EN and MET:10 μg/ml) six times by measuring the absorbances of both the drugs solution at 224 and 232 nm in SE method; 232 and 214 nm for AR method; 224 and 232 nm for 1st DR method, respectively and % RSD was calculated. Intra-day precision was performed by analyzing sample solutions (EN and MET: 10 μg/ml) in triplicate for three times on the same day within the linearity range and % RSD was calculated. Inter-day precision was evaluated by repeated analysis of sample solutions (EN and MET: 10 μg/ml) in triplicate within the linearity range on three different days and percentage RSD was calculated.

Accuracy:

In order to ensure the suitability and reliability of the projected methods, recovery studies were performed by standard addition method. To an equivalent quantity of pre-analyzed sample solution (EN and MET: 4, 8 and 12 μg/ml), a known concentration of standard EN and MET were added at 50, 100 and 150% level and the resulting solutions were reanalyzed by projected methods and % recoveries were calculated. The outcomes of accuracy studies were assessed based on the percentage of standard EN and MET recovered from the formulation by applying following formula:-

LOD and LOQ:

Sensitivity of the proposed methods were determined in terms of LOD and LOQ. The limit of detection and limit of quantification of EN and MET were calculated applying following equation as per ICH guidelines.

Where, S = The slope of the calibration curve and σ = Standard deviation of the response

Fig. 4: Overlain UV spectra of standard drugs for SE and AR methods.

Fig. 5: Overlain first derivative (zero crossing) UV spectra of standard drugs for 1^st  DR method.

Fig. 6: Overlain UV spectra of EN and MET (5-30 μg/ml) for SE and AR methods.

Fig. 7: Overlain 1st derivative (zero crossing) UV spectra of  EN and MET (5-30μg/ml) for 1st DR method.

Table 2: Summary of linear regression and method validation data for the proposed methods

Parameters	SE				AR
	EN		MET		EN		MET
Wavelength (nm)	224	232	224	232	232	214	232	214
Linearity range (µg/ml)	5-30
Correlation coefficient	0.9993	0.9947	0.9986	0.9997	0.996	0.9991	0.9976	0.9975
Regression equation Slope Intercept	0.0471 0.0271	0.0299 0.0061	0.0426 0.0055	0.0427 0.003	0.0036 0.0024	0.0038 0.0081	0.0066 0.0077	0.0094 0.0005
LOD (µg/ml)	0.15	0.27	0.06	0.29	0.16	0.06	0.14	0.17
LOQ (µg/ml)	0.45	0.39	0.21	0.35	0.19	0.24	0.43	0.29
Specificity	No Interference
Precision (%RSD) repeatability of measurement (n=6) Intra-day(n=3) Inter-day (n=3)	0.49 0.51  0.77	0.71 0.45 1.05	0.83 0.78 0.19	0.48 0.74 1.04	0.92 0.87 0.61	0.84 0.65 0.93	0.52 0.84 0.71	0.56 0.38 0.72

Table 2: continued

Parameters	AUC				DR
	EN		MET		EN		MET
Wavelength (nm)	217-227	227-237	217-227	227-237	224	232	224	232
Linearity range (µg/ml)	5-30
Correlation coefficient	0.998	0.9954	0.9993	0.9969	0.995	0.9996	0.9978	0.9981
Regression equation Slope Intercept	0.0019 0.001	0.0002 0.0033	0.0026 0.0024	0.0058 0.0065	0.0001 0.001	0.00008 0.00007	0.00002 0.00002	0.0004 0.0003
LOD (µg/ml)	0.25	0.13	0.04	0.28	0.21	0.22	0.07	0.12
LOQ (µg/ml)	0.49	0.51	0.25	0.36	0.41	0.46	0.25	0.27
Specificity	No Interference
Precision (%RSD) repeatability of measurement (n=6) Intra-day(n=3) Inter-day (n=3)	1.01 0.79 0.53	1.14 0.43 0.64	0.52 0.61 0.87	0.91 0.75 1.05	0.93 0.58 0.19	0.28 0.62 1.12	0.82 0.67 0.51	0.84 0.65 0.93

Table 3:  Recovery data of the proposed methods.

Drugs	Level of recovery	% recovery				%RSD
		SE	AR	AUC	DR	SE	AR	AUC	DR
Empagliflozin	50	98.14 ± 0.51	98.25 ± 0.44	97.23 ± 0.14	98.23 ± 0.28	0.87	0.56	0.89	0.54
	100	101.01 ± 0.18	99.19 ± 0.41	99.29 ± 0.25	97.25 ± 0.23	0.56	0.87	0.75	0.39
	150	98.19 ± 0.82	98.71 ± 0.44	97.14 ± 0.23	98.36 ± 0.29	0.38	0.85	0.26	0.47
Metformin	50	98.49 ± 0.43	97.93 ± 0.73	98.26 ± 018	99.37 ± 0.27	0.45	0.54	0.49	0.98
	100	97.81 ± 0.26	98.86 ± 1.03	99.16 ± 0.14	97.46 ± 0.23	0.39	0.24	0.53	0.37
	150	98.28 ± 0.45	98.28 ± 0.25	98.35 ± 0.46	99.23 ± 28	0.89	0.38	0.32	0.54

Table 4: Results of formulation analysis using different methods.

Drug

Labelled  Amount (mg/ml)

Labelled  Found (mg/ml)

Amount found (%)

RSD %

SR

AR

AUC

DR

SR

AR

AUC

DR

SR

AR

AUC

DR

EN

5 mg

4.85

4.94

4.75

4.58

98.25 ± 0.50

97.26 ±0.25

99.03 ±0.25

98.32 ±0.56

0.59

0.69

0.42

0.34

MET

500 mg

490.03

492.56

490.25

496.25

97.25  ± 0.02

98.45 ±0.23

99.63 ±0.45

98.23 ±0.56

0.62

0.29

0.69

0.58

Stability of the Solution:

Stability of the solutions were checked by observing any changes in terms of absorbance and spectral pattern which was compared to freshly prepared solutions by keeping the solutions at room temperature and analysing at frequent intervals.

RESULTS AND DISCUSSION:

Four UV spectroscopic methods namely SE, AR, AUC  and 1st DR spectroscopic methods were developed and validated for simultaneous estimation of EN and MET in tablet dosage form which are simple, sensitive, precise and accurate. In SE method, absorbance was measured at 224 and 232 nm for both the drugs. In AR method 232 and 214 nm was used for the detection and quantification of EN and MET. AUC absorbance was measured at 227-237  and 217-227nm for both drugs. 1st DR method was based on the transformation of UV-spectra in to first derivative spectra and followed by measurement of first derivative signal at 224 and 232 nm for EN and MET, respectively using 2 nm as wavelength interval (Δλ) and 1 as scaling factor. Linear relation was established for EN and MET in the concentration range of 5-30 μg/ml for all the methods. Overlain spectra of EN and MET are shown in Figure 4 and 5. Calibration graphs were plotted using absorbance of standard drug solution versus concentration for SE, AR and AUC method. 1st derivative signal of standard drug solution versus concentration was used to plot calibration curve for 1st DR method. Regression analysis was performed by applying least square method for calculating values of slope, intercept and correlation coefficient for EN and MET at their relative wavelengths. Outcome of precision studies were evaluated in terms of % RSD, follows ICH guideline acceptable limits (˂2), which shows good repeatability, low intra and inter-day variability, indicating an excellent precision of the developed methods (Table 2). Regression analysis was performed by applying least square method for calculating values of slope, intercept and correlation coefficient for EN and MET at their relative wavelengths. Outcome of precision studies were evaluated in terms of % RSD, follows ICH guideline acceptable limits (˂2), which shows good repeatability, low intra and inter-day variability, indicating an excellent precision of the developed methods (Table 2). The outcome of recovery studies ranged from 97-102% for both the drug suggests suitability of the proposed methods (Table 3). Moreover, low LOD and LOQ values prove the sensitivity of the proposed methods (Table 2). Solution stability was checked at room temperature and it was found to be stable up to two days. The projected methods were successfully applied for the quantitative determination of EN and MET in tablet formulation (Jardiance Met^™:5 mg of EN and 500 mg of MET). Sample solutions were analyzed six times and experimental values were found to be within 96 and 100 % for both the drugs and hence the developed methods can be used for the simultaneous determination of both the drugs in combined tablet formulation (Table 4).

CONCLUSION:

Four different methods namely SE, AR, AUC and 1^st DR spectroscopic methods were developed for simultaneous estimation of EN and MET in combined tablet dosage form. Developed methods were validated according to ICH guidelines. Projected methods were found to be simple, sensitive, precise, accurate and cost effective. Moreover, all the developed UV-spectrophotometric methods require little sample preparation procedure and have wide concentration range with high sensitivity. Statistical data reveals that there is no statistical significant dissimilarity among all the three methods. Therefore, all the developed methods can be used successfully for routine quality control analysis of EN and MET in combined tablet dosage form.

ACKNOWLEDGEMENT:

Authors are thankful to the Department of Pharmacy, Sumandeep Vidyapeeth University, Piparia, Waghodia, Vadodara, Gujarat, India for providing all the facilities throughout the work.

CONFLICT OF INTERESTS:

There are no conflicts of interest.

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Received on 23.07.2019            Modified on 26.11.2019

Accepted on 08.12.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2020; 13(3):1236-1242.

DOI: 10.5958/0974-360X.2020.00228.0