Quantitative Estimation and Validation of Dapagliflozin and Metformin Hydrochloride in Pharmaceutical Dosage form by RP-HPLC

 

Nachiket S. Dighe¹*, Priyanka R. Varade¹, Ganesh S. Shinde¹, Priya S. Rao²

¹Department of Pharmaceutical Chemistry, Pravara Rural College of Pharmacy, Pravaranagar, Tal-Rahata,

Dist.-Ahmednagar.

²Department of Pharmacognosy, Pravara Rural College of Pharmacy, Pravaranagar, Tal- Rahata,

Dist.- Ahmednagar.

*Corresponding Author E-mail: nachiket1111@rediffmail.com

A simple and more economic RP-HPLC method was developed and subsequently validated for the simultaneous determination of Metformin and Dapagliflozin in bulk and pharmaceutical dosage form. The chromatographic conditions were standardized using a Cosmosil C18 column with 250mm in length and internal diameter of 4.6mm with size 5μm. The analyte detection was carried out by using a UV detector set at a wavelength of 228 nm. The mobile phase consisted of Methanol: Potassium dihydrogen phosphate buffer with pH 3.0 (80:20%v/v) and retention time of Metformin and Dapagliflozin was found to be 3.6 min and 5.2 min respectively. The calibration curves of two drugs were linear with correlation coefficients of 0.999 and 0.998 over a concentration range of 100-500μg/ml for Metformin and 1-5μg/ml for Dapagliflozin. This method has been validated and shown to be accurate, precise, specific, sensitive, linear, robust and fast. Metformin and Dapagliflozin were subjected to different degradation stress conditions. The degradation products were well resolved from that of pure standard drugs (Metformin and Dapagliflozin) with significant different retention time values. The current method has been statistically validated according to the ICH guidelines and this method has been subsequently developed and applied successfully to determine the levels of Metformin and Dapagliflozin in a combined formulation and in the routine quality control analysis with good accuracy and sensitivity.

 

 

INTRODUCTION:

Dapagliflozin (DAPA) belongs to a new class of oral anti-diabetic drugs, called Sodium Glucose Co-Transporter 2 (SLGT2) inhibitor. It is indicated for the management of Diabetes Mellitus type 2, and functions to improve glycemic control in adults when combined with diet and exercise. It is a Sodium- glucose co-transporter 2 inhibitor which prevents glucose reabsorbption in kidney.

 

 

 

 

Dapagliflozin could be a initial first generation, selective SGLT inhibitor that blocks glucose transport with about 100-fold selective for SGLT2 over SGLT^[1]. The chemical name of dapagliflozin is (2S, 3R, 4R, 5S, 6R)-2-[-4-Chloro-3-(4-ethoxybenzyl) phenyl]-6-(hydroxymethyl) tetrahydro-2H-pyran-3,4,5-triol and has the structure (Figure 1), molecular formula C₂₁H₂₅CLO₆ with molecular weight 408.875 g/mol^[2]. Dapagliflozin is a white to off-white powder, non-hygroscopic and soluble in many polar organic solvents eg. DMSO, water, ethanol, dimethyl formaide, and sparingly soluble in aqueous buffer^[3].

 

Fig. 1: Structure of Dapagliflozin

Metformin (MET) is a biguanide antihyperglycemic agent used for treating non-insulin dependent diabetes mellitus (NIDDM)^[4]. It improves glycemic management by decreasing hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated glucose uptake^[5]. Metformin is the only oral antihyperglycemic agent that’s not related to weight gain. Metformin may induce weight loss and is that the drug of selection for obese NIDDM patients. When used alone, antidiabetic doesn’t cause hypoglycemia, but, it should enhance the hypoglycemic effect of sulfonylurea and internal secretion. It is conjointly employed in the treatment of polycystic ovary syndrome and has been investigated for different diseases wherever hypoglycemic agent resistance could also be a vital issue. The chemical name of metformin hydrochloride (metformin HCL) is 1,1-Diamethylbiaguanidehyrochloride and has the structure (Figure 2), molecular formula C₄H₁₁N₅.HCL with molecular weight 165.63 g/mol^[6]. Metformin freely soluble in water, slightly soluble in alcohol and acetonitrile; practically (much) insoluble in acetone, ether and chloroform.^[7]

 

Fig. 2: Structure of Metformin

 

The combination of Dapagliflozin and metformin is used as a therapeutic choice for the treatment of patients with type 2 diabetes mellitus (T2DM)^[8,9]. This distinctive combined mechanism of action and favorable effectiveness and safety profile of dapagliflozin and metformin (antidiabetic) support consideration of this fixed-dose combination as a treatment selection for patients with T2DM^[10,11].

 

MATERIAL AND METHOD:

List of Instruments:

Table 1: List of apparatus/ instruments used.

Sr. No.	Name	Model	Manufacturer/Supplier
1.	Weighing balance	PGB 100 Max: 100gm Min: 0.001gm	Wenser High Precision Balance
2.	Digital PH Meter	PICO+	Lab India pvt ltd
3.	Sonicator	WUC- 4L Capacity - 4 liter	Wenser Ultra Sonicator
4.	Magnetic stirrer		Remi Equipment
5.	HPLC	HPLC 3000 Series	Analytical Technologies Ltd.

 

Table 2: HPLC Instrument Information.

Parts of Instruments	Information
System	HPLC Binary Gradient System
Model no.	HPLC 3000 Series
Company	Analytical Technologies Ltd.
Pump	P-3000-M Reciprocating (40 MPa)
Column	Cosmosil C18 (250mm×4.6ID, particle size- 5 micron)
Detector	UV-3000-M
Software	HPLC Workstation

 

List of Chemicals

Table 3: List of chemical used.

Sr. No.	Name	Specification	Manufacturer/Supplier
1.	Methanol	HPLC grade	Merck
2.	Potassium dihydrogen phosphate buffer	HPLC grade	Merck

 

Table 4: List of API used.

Sr. No.	Name	Specification	Manufacturer/ Supplier
1.	Dapagliflozin	Working standard	Aurobindo pharma Ltd.
2.	Metformin Hydrochloride	Working standard	Aurobindo pharma Ltd.

 

Preparation of mobile phase:

Mixed a HPLC grade Methanol: Potassium dihydrogen phosphate buffer with pH 3.0 (80:20) in volumetric flask. Filter through 0.45μ filter under vacuum filtration.

 

Diluent preparation:

Use mobile phase as diluent.

 

Preparation of standard solutions:

Accurately weigh and transfer 0.01gm (10mg) of pure Metformin hydrochloride and Dapagliflozin working standard separately into 10ml clean and dry volumetric flask. Add diluent and sonicated to dissolve it completely and made volume upto the mark with same solvent (mobile phase). From this, five working standard solution of concentration covering the range 100-500 ppm and 1-5 ppm for Metformin HCL and Dapagliflozin respectively, were prepared by transferring and diluting different aliquots into series of 10ml volumetric flask with same diluent.

 

Preparation of sample solutions:

Weighed and transfer 20 tablets of OXRAMET^TM XR 1280mg [containing Dapagliflozin 10mg and Metformin HCL 1000 mg label claim] into mortar and pestle. Crush the above tablets into fine powder. Weigh and transfer sample powder quantity equivalent to 10 mg of Dapagliflozin and Metformin in 10 ml volumetric flask containing mobile phase and shaken vigorously, sonicated for 15 min and made up volume up to the mark with diluent. Aliquots of the above solution was pipetted and transferred into a series of clean and dry 10 ml volumetric flask and diluent was added up to the mark to get final concentration of Dapagliflozin and Metformin HCL. 20μL volume each of these standard and sample solution were injected five times and the peak areas were recorded.

 

Selection of wavelength:

UV spectrum of 10μg/ml Dapagliflozin and Metformin HCL diluents (mobile phase composition) was recorded by scanning in the range of 200nm to 400nm. From the spectrum wavelength selected as 228nm. At this wavelength, both drugs show good absorbance.

 

Fig. 3: Wavelength of Dapagliflozin and Metformin HCL.

 

 

 

 

HPLC instrumentation and chromatographic conditions:

The HPLC system was Binary Gradient system consisting of pump P-3000-M Reciprocating (40MPa), detector UV-3000-M, column Cosmosil C18 (250mm×4.6ID, Particle size- 5micron), thermo scientific injector rheodyne injector (20μl capacity) and syringe Hamilton (25μl). Data were processed using HPLC workstation software. A freshly prepared mixture of methanol: Potassium dihydrogen phosphate buffer (80:20 v/v) (PH) used as the mobile phase. Mobile phase was sonicated and filtered through 0.45μm membrane filter before used. The flow rate of mobile phase was maintained at 0.9 ml/min. The eluents were monitored at 228nm. The injection volume of both sample and standard were 20μl. Total run time is 10 min.

 

Optimized chromatographic condition:

In the present study the separation of Dapagliflozin and Metformin HCL was achieved by using C18 column Cosmosil C18 (250mm×4.6ID, Particle size- 5micron) with mobile phase consisting of mixture of methanol and Potassium dihydrogenphosphate buffer (pH 3.0) in the ratio of 80:20 at a flow rate 0.9 ml/min with uv detection wavelength of 228nm at ambient temperature. The retention time for Dapagliflozin and Metformin HCL were found to be 4.180min and 6.274min respectively.

 

 

Time	Conc.	Area	Resolution	T. Plate num	Asymmetry
4.180 min	100 ppm	1394652	0.00	8363	1.27

Fig. 4: Retention time of Metformin.

 

 

Time	Conc.	Area	Resolution	T. Plate num	Asymmetry
6.274 min	100 ppm	9834338	0.00	6639	1.22

Fig. 5: Retention time of Dapagliflozin.

 

 

Time	Conc.	Area	Resolution	T. Plate num	Asymmetry
3.624 min	100 ppm	980326	6.80	8707	1.28
4.585 min	1 ppm	16019	0.00	13487	1.00

Fig. 6: Chromatogram of standard solution for Metformin and Dapagliflozin.

 

RESULT AND DISCUSSION:

Method Validation:

The developed method was validated as per ICH guidelines for its specificity, system suitability, linearity, accuracy, precision, robustness, ruggedness, limit of detection, limit of quantification.

 

System suitability:

System suitability and chromatographic parameters were validated such as resolution, theoretical plates, and the tailing factor was calculated. The results are given in table 5.

 

 

 

 

 

 

Table 5: System suitability parameters for Dapagliflozin and Metformin HCL.

System suitability parameters	Dapagliflozin	Metformin HCL
Retention time	4.585 min	3.624 min
Theoretical plate no.	13487	8707
Tailing factor	1.00	1.28
Resolution	0.00	6.80

 

Linearity

The linearity of this method was evaluated by linear regression analysis and calculated by the least square method and studied by preparing standard solutions of Metformin and Dapagliflozin at different concentration levels. The calibration curve showed (Fig. 7 and 8) good linearity in the range of 100-500 μg/ml, for Metformin with a correlation coefficient (r²) of 0.999 and 1-5 μg/ml for Dapagliflozin with a correlation coefficient (r²) of 0.998. Results are given in table 6.

Table 6: Linearity data for Metformin and Dapagliflozin.

Drug	Concentration (ppm)	Area
Metformin	100	980326
	200	2080503
	300	3115256
	400	4382832
	500	5491934
Dapagliflozin	1	16019
	2	79785
	3	153156
	4	218389
	5	297570

 

Fig. 7: Linearity graph of Metformin.

 

Fig. 8: Linearity graph of Dapagliflozin.

 

% Recovery (Accuracy):

Recovery studies were administered by addition of the standard or quality drug to the sample at 3 completely different concentration levels (50%, 100% and 150%) taking into through proportional purity of added bulk drug samples. At every concentration, the sample was injected thrice to visualize repeatability and from the % RSD values it was analyzed that the method was accurate as % recovery values found to be in the range of 99.57-100.15% for the Metformin and 99.36-99.89% for Dapagliflozin at three different concentrations 50%, 100%, 150%. The results are given in respectively table 7 and 8.

 

 

Table 7: %Recovery data for Metformin.

Conc (%)	Sample amount (ppm)	Amount added (ppm)	Amount recovered (ppm)	% recovery	% mean recovery
50%	200	100	300.33	100.11	100.15
	200	100	300.57	100.19
	200	100	300.48	100.16
100%	200	200	398.97	99.74	99.89
	200	200	400.00	100
	200	200	399.81	99.95
150%	200	300	499.05	98.81	99.57
	200	300	500.05	100.01
	200	300	499.56	99.91

 

Table 8: %Recovery data for Dapagliflozin.

Conc (%)	Sample amount (ppm)	Amount added (ppm)	Amount recovered (ppm)	% recovery	% mean recovery
50%	2	1	3.03	99.66	99.36
	2	1	3.02	99.30
	2	1	3.02	99.13
100%	2	2	3.99	99.79	99.89
	2	2	4.00	100
	2	2	3.99	99.89
150%	2	3	4.97	99.49	99.83
	2	3	5.03	100.79
	2	3	4.96	99.20

 

Precision:

Intraday precision:

A standard solution containing Metformin and Dapagliflozin were analyzed three times on the same day and % RSD was calculated. The results are given in table 9.

 

Table 9: Intraday data for Metformin and Dapagliflozin.

Metformin HCL		Dapagliflozin
Morning	Area	Morning	Area
	3115256		153156
	3121250		152094
	3120296		151813
Evening	3112917	Evening	151589
	3124732		152347
	3118868		151497
Mean	318886.5	Mean	152082.7
% RSD	0.14	%RSD	0.40

 

Inter day precision:

A standard solution containing Metformin and Dapagliflozin were analyzed three times on a different day and % RSD was calculated. The results are given in table 10.

 

Table 10: Interday data for Metformin and Dapagliflozin.

Metformin		Dapagliflozin
Day 1	Area	Day 1	Area
	3115256		153156
	3121250		152094
	3120296		151813
Day 2	3121611	Day 2	153216
	3120054		154820
	3122253		151277
Mean	3122253	Mean	151277
%RSD	0.08	%RSD	0.84

Robustness:

Small deliberate changes in chromatographic conditions such as a change in wavelength (±2 units) and flow rate (±2 units) were studied to determine the robustness of the method. The results were within the factor of (% RSD<2%) the developed RP-HPLC method for the analysis of metformin and dapagliflozin. The results are given in table 11 & 12.

 

 

Table 11: Robustness data for Metformin and Dapagliflozin at different flow rate.

Name of drug	Flow rate (ml/ min)	Area	Mean	S. D	% S. D
Metformin	0.8	2075330	2080675	5433.55	0.261
	0.9	2080503
	1.0	2086193
Dapagliflozin	0.8	80068	79593.7	593.59	0.745
	0.9	79785
	1.0	78928

 

Table 12: Robustness data for Metformin and Dapagliflozin at different Wavelength.

Name of drug	Wavelength (nm)	Area	Mean	S. D	% S. D
Metformin	226	2089054	2082840	5436.29	0.261
	228	2080503
	230	2078963
Dapagliflozin	226	79935	79745	212.83	0.266
	228	79785
	230	79515

Ruggedness:

To evaluate the ruggedness of the method, precision was performed on different days by maintaining same conditions. The testing of ruggedness is normally suggested when the method is to be used in more than one laboratory. Ruggedness is normally expressed as the lack of the influence on the test results of operational and environmental variables of the analytical method. %RSD between the test obtained should not be more than 2%. The results are given in table 13.

 

Table 13: Ruggedness data for Metformin and Dapagliflozin.

Drug	Concentration (ppm)	Area
Metformin	100	984588
	200	20788963
	300	3118952
	400	4380782
	500	5491109
Dapagliflozin	1	15808
	2	79515
	3	150825
	4	218151
	5	296279

 

 

Fig. 9: Ruggedness graph of Metformin.

 

Fig. 10: Ruggedness graph of Dapagliflozin.

 

Limit of Detection and Limit of Quantification:

Limit of detection result for Dapagliflozin and Metformin was found to be 0.052 and 0.837 respectively and also limit of quantification result for Dapagliflozin and Metformin was found to be 0.158 and 2.538 respectively and were within the limits. Results are summarized in table no.14, 15.

 

Table No.14: Limit of Detection for Dapagliflozin and Metformin.

Sr. No	Name	Standard Deviation	Slope	LOD
1	Dapagliflozin	1115.52	70171	0.052
2	Metformin	2874.81	11326	0.837

 

Table No.15: Limit of Quantification for Dapagliflozin and Metformin.

Sr. No	Name	Standard deviation	Slope	LOQ
1	Dapagliflozin	1115.52	70171	2.538
2	Metformin	2874.81	11326	0.158

 

 

Assay:

300 ppm and 30ppm of the standard solution metformin and dapagliflozin was injected into the chromatographic system, chromatograms were recorded and peak areas were measured. The sample solution metformin and dapagliflozin was injected into the chromatographic system, chromatograms were recorded and peak areas were measured. The Results are shown in table 16.

 

Table 16: Assay data for Metformin and Dapagliflozin.

Drug name	Composition (ppm)	Area of standard	Area of sample	% Assay
MET	300	3115256	3124700	100.30
DAPA	3	153156	153455	100.19

 

CONCLUSION:

The developed RP-HPLC method offers several advantages such as rapidity, usage of simple mobile phase and easy sample preparation steps. From the present study, it can be concluded that the proposed method is simple, specific, sensitive, precise, accurate and reproducible. Results of validation parameters demonstrated that the analytical procedure is suitable or appropriate for its intended purpose. Further, improved sensitivity makes it and reliable specific for its intended use. Hence, this method can be applied for the analysis of pharmaceutical dosage forms and pure drug.

 

ACKNOWLEDGEMENT:

The authors express their gratitude to the Pravara Rural College of Pharmacy, Loni for providing all the facilities and Aurobindo Pharmaceuticals Ltd, for providing me the gift samples of Metformin HCL and Dapagliflozin.

 

REFERENCES:

1.        Afshan Urooj, P. Shyam Sundar1, R. Vasanthi, M. Alagar Raja, K. Rajeswar Dutt, K. N. V. Rao and H. Ramana, Development and Validation of RP-HPLC method for simultaneous estimation of dapagliflozin and metformin in bulk and in synthetic mixture. World Journal of Pharmacy and Pharmaceutical Sciences, 2017, 6(7): 2139-2150.

2.        Mohammad Yunoos, Gowri sankar D., A validated stability indicating high-performance liquid chromatographic method for simultaneous determination of metformin hcl and dapagliflozin in bulk drug and tablet dasage form. Asian J Pharm Clin Res, 2015; 8(3): 320-326.

3.        N. Padmaja and G. Veerabhadram, Stability–indicating RP-HPLC method for the determination of Metformin and Dapagliflozin in bulk and pharmaceutical dosage form. journal de Afrikana, 2017, 4(2): 409-425.

4.        K. Lakshmi Prameela, P. Rama Krishna Veni P. V.V. Satyanarayana, B. Hari Babu, Development and validation of stability indicating reverse phase high performance liquid chromatography method with photodiode array detection for the simultaneous estimation of hypoglycemic agents, dapagliflozin and Metformin. Int J Pharma Bio Sci, 2017 July, 8(3), 328-336.

5.        Khyati J. Patel, Ankit B. Chaudhary, Shweta M. Bhadani and Ruchi J. Raval, Stability indicating RP-HPLC method development and validation for estimation of dapagliflozin and metformin HCL. World Journal of Pharmacy & Pharmaceutical Sciences, 2017, 6(9): 796-809.

6.        T. Deepan, M.V. Basaveswara Rao and M.D., Dhanaraju, Development of Validated Stability Indicating Assay Method for Simultaneous Estimation of Metformin and Dapagliflozin by RP- HPLC. European Journal of Applied Sciences, 2017, 9 (4): 189-199.

7.        Syeda Kulsum, G. Vidyasagar, Tasneem Mohammed, A Simple and Validated RP-HPLC Method for the Simultaneous Estimation of Metformin and Dapagliflozin in Bulk and Pharmaceutical Dosage Forms. Journal of Pharmaceutical and Medicinal Chemistry, 2015; 1(2): 99-104.

8.        Sherif A. Abdel-Gawad, Abdul Malik S. Al-Tamimi, Elsadig H.K. Adam, Chromatographic Simultaneous Quantification of Dapagliflozin And Metformin Hydrochloride In Presence of Their Degradation Products. IJBPAS, 2017; 6(10): 2007-2021.

9.        Syed Abdul Hadi and Bharath Rathna Kumar P., Simultaneous estimation of metformin and dapagliflozin tablets by RP-HPLC. World Journal of Pharmacy and Pharmaceutical Sciences, 2017; 6 (11) :1188-1199.

10.      Shyamala, Nidhi B, Kavitha M, Pooja and JVC Sharma, Validated RP-HPLC method for simultaneous estimation of Metformin Hydrochloride and Dapagliflozin in tablet dosage form. American Journal of Biological and Pharmaceutical Research, 2015; 2(2):109-113.

11.      Ghadir A Khalil, Ismail Salama, Mohammed S Gomaa1 and Mohammed A Helal, Validated RP-HPLC Method For Simultaneous determination Of Canagliflozin, Dapagliflozin, Empagliflozin And Metformin. IJBPAS ,2018; 8(1): 1-13.

 

 

 

 

 

Received on 26.02.2019                    Modified on 02.04.2019

Accepted on 10.05.2019                   ©AJRC All right reserved