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Development and validation of RP-HPLC method for simultaneous estimation

of Dapagliflozin and Metformin in bulk and in synthetic mixture.

Article  in  WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES · January 2018

DOI: 10.20959/wjpps20177-9657

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 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
Urooj et al. World Journal of Pharmacy and Pharmaceutical Sciences
SJIF Impact Factor 6.647

Volume 6, Issue 7, 2139-2150 Research Article ISSN 2278 – 4357

DEVELOPMENT AND VALIDATION OF RP-HPLC METHOD FOR

SIMULTANEOUS ESTIMATION OF DAPAGLIFLOZIN AND
METFORMIN IN BULK AND IN SYNTHETIC MIXTURE

Afshan Urooj*1, P. Shyam Sundar1, R. Vasanthi1, M. Alagar Raja1, K. Rajeswar Dutt1,

K. N. V. Rao1 and H. Ramana2

1
Nalanda College of Pharmacy, Charlapally, Nalgonda, T.S.
2
Venkateshwara Institute of Pharmaceutical Sciences, Charlapally, Nalgonda, T.S.

Article Received on
ABSTRACT
20 May 2017, A simple, accurate and precise reverse phase high performance liquid
Revised on 10 June 2017,
Accepted on 01 July 2017, chromatography method has been developed for simultaneous
DOI: 10.20959/wjpps20177-9657 estimation of Metformin and Dapagliflozin in bulk and synthetic
mixture. The determination was carried out by using HPLC of waters
*Corresponding Author (Model: Alliance 2695) with Phenomenex Luna C18 (4.6mm I.D. ×
Afshan Urooj 250mm, 5µm) column was used for chromatographic separation. It
Nalanda College of contains waters injector and PDA detector (Deuterium). Mobile phase
Pharmacy, Charlapally,
consists of Acetonitrile: Water (75:25% v/v) and flow rate adjusted
Nalgonda, T.S.
was 1ml/min. Wavelength selected for detection was 285nm and
injection volume was 10 µl. By using the developed method, retention time of Metformin and
Dapagliflozin was found to be 3.2min and 5.4min respectively. The method has been
validated for linearity, accuracy and precision. Linearity of Metformin and Dapagliflozin
were in the range of 20–100μg/ml and 10–50μg/ml respectively. The percentage recoveries
obtained for Metformin and Dapagliflozin were found to be in range of 99.3 – 99.6%. LOD
and LOQ were found to be 5.0µg/ml and 15.2µg/ml for Metformin 3.7 and 11.4µg/ml for
Dapagliflozin. Results of validation parameters demonstrated that the analytical procedures is
suitable for its intended purpose and meets the criteria defined in ICH Q2R1.
KEYWORDS: Metformin, Dapagliflozin, Simultaneous Estimation, RP-HPLC.

INTRODUCTION
Dapagliflozin (DAPA) (Figure2) 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

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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 is a first generation, selective SGLT inhibitor
that blocks glucose transport with about 100-fold selective for SGLT2 over SGLT1.[1]
Metformin (MET) (Figure1) is a biguanide antihyperglycemic agent used for treating non-
insulin dependent diabetes mellitus (NIDDM). It improves glycemic control by decreasing
hepatic glucose production, decreasing glucose absorption and increasing insulin-mediated
glucose uptake. Metformin is the only oral antihyperglycemic agent that is not associated
with weight gain. Metformin may induce weight loss and is the drug of choice for obese
NIDDM patients. When used alone, metformin does not cause hypoglycemia; however, it
may potentiate the hypoglycemic effects of sulfonylureas and insulin. It is also used in the
treatment of polycystic ovary syndrome and has been investigated for other diseases where
insulin resistance may be an important factor.[2] The combination of Dapagliflozin and
metformin is used as a therapeutic option for the treatment of patients with type 2 diabetes
mellitus (T2DM). This unique combined mechanism of action and favorable efficacy and
safety profile of dapagliflozin and metformin support consideration of this fixed-dose
combination as a treatment option for patients with T2DM. Literature survey reveals that few
HPLC and LC-MS methods have been reported for determination of metformin. A successful
study is done for estimation of dapagliflozin by HPLC individually. In this present study a
successful attempt has been made to develop a rapid, precise, accurate and comparatively
economical RP-HPLC method. The developed method validated and recovery studies were
conducted and studied by using various statistical parameters according to ICH guidelines.[3]

MATERIALS AND METHODS

Chemicals and Reagents
Acetonitrile, Water and Methanol of HPLC grade were obtained from LICHROSOLV
(MERCK) Mumbai, India. Pharmaceutical grade Dapagliflozin and Metformin were obtained
from Hetero labs, Hyderabad, India.

Instruments
The analysis was performed by using the weighing machine, pH meter (Lab India). The
HPLC used is of WATERS Alliance 2695seperation module with 996 PDA detector. The
output signal was monitored and integrated using Empower 2 software. A Symmetry C18, X-

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bridge column, Xterra. Phenomenex Luna C18 (4.6 x 150mm, 5 µm) was found to be ideal as
it gave good peak shape and resolution at 1ml/min flow.

REAGENTS AND SOLUTIONS

Preparation of Mobile Phase
Accurately measured 750ml (75%) of HPLC Acetonitrile and 250ml of Water (25%) were
mixed and degassed in a digital ultrasonicator for 10 minutes and then filtered through 0.45µ
filter under vaccum filtration. The mobile phase tried was Methanol: Water, Acetonitrile:
Water with varying proportions. Finally, the mobile phase was optimized to Acetonitrile and
Water in proportions 75:25v/v respectively.

Preparation of Standard solution

Accurately weigh and transfer 10 mg of Metformin and Dapagliflozin working standard into
a 10ml of clean dry volumetric flasks add about 7ml of Methanol and sonicate to dissolve and
removal of air completely and make volume up to the mark with the same Methanol.

Further pipette 0.6 ml of the above Metformin and 0.3ml of the Dapagliflozin stock solutions
into a 10ml volumetric flask and dilute up to the mark with diluent.

Preparation of Sample solution

Take average weight and crush in a mortar by using pestle and weight powder 10 mg
equivalent weight of Metformin and Dapagliflozin sample into a 10mL clean dry volumetric
flask and add about 7mL of diluent and sonicate to dissolve it completely and make volume
up to the mark with the same solvent.

Further pipette 0.6 ml of the above stock solutions into a 10ml volumetric flask and dilute up
to the mark with diluent.

Assay
10 µL of the standard solution was injected five times into the chromatographic system,
chromatograms were recorded and peak areas were measured. 10 µL of the sample solution
was injected in five times into the chromatographic system, chromatograms were recorded
and peak areas were measured.

METHODS DEVELOPMENT[4-12]
The developed method was fully validated for the parameters as per ICH guidelines.

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System Suitability
System suitability is done by replicate analysis injected for five times and measured the area
for all five injections in HPLC. The %RSD for the area of five replicate injections was found
to be within the specified limits.

Linearity
Linearity is determined by a series of three to five injections of five or more standards. Plot a
graph of peak area (or heights) of the calibration standards are usually plotted in the Y-axis
against the nominal standard concentration, and the linearity of the plotted curve is evaluated
through the value of the co-relation coefficient (r2). The methods were linear in the range of
10-50ppm Dapagliflozin 20-100ppm for metformin and inject each level into the
chromatographic system and measure the peak area.

Accuracy
Accuracy of the method was determined by inject the three replicate injections of individual
concentrations (50%, 100%, 150%) were made under the optimized conditions. Recorded the
chromatograms and measured the peak responses. Calculate the amount found and amount
added for Metformin and Dapagliflozin and calculate the individual recovery and mean
recovery values.

Precision
To determine the precision, intra-day and inter-day analysis was performed. The standard
solution was injected for five times and measured the area for all five injections in HPLC.
The %RSD for the area of five replicate injections was found to be within the specified
limits. Solutions corresponding to each concentration level were injected in duplicate. The
precision of an analytical method is a measure of the random error and is defined as the
agreement between replicate measurements of the same sample.

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.

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Robustness
Robustness of the method was performed in different conditions to find the variability of test
results. The sample was analyzed at 0.9 mL/min and 1.1 mL/min instead of 1mL/min,
remaining conditions are same. 10µl of the above sample was injected and chromatograms
were recorded. Their effects on the retention time (TR), tailing factor (T), theoretical plate
numbers (N) and repeatability of peak areas (n = 6) were studied.

Limit of detection and Limit of quantification

The determination of the limit of detection of instrumental procedures is carried out by
determining the signal-to-noise ratio by comparing test results from the samples with known
concentration of analyte with those of blank samples and establishing the minimum level at
which the analyte can be reliably detected. A signal-to-noise ratio of 2:1 or 3:1 is generally
accepted. The determination of the limit of quantification is carried by minimum
concentration at which the analyte can reliably be quantified is established.

A typical acceptable signal- to- noise ratio is 10:1. Other approaches depend on the
determination of the slope of the calibration curve and the standard deviation of
responses.

RESULTS AND DISCUSSION

The goal of this study was to develop a new RP-HPLC method, several mobile phase
compositions were tried for separation and quantification of Dapagliflozin and Metformin in
bulk and pharmaceutical dosage forms. To develop an effective method for the analysis of the
drugs preliminary tests were performed in order to select adequate and optimum conditions.
Parameters such as detection wavelength, mobile phase composition and pH, mobile phase
comprising of Acetonitrile: Water in 75: 25v/v at a flow rate 0.5 mL/min to get a better
reproducibility and repeatability. Quantification was achieved with UV detection at 240 nm
and the retention time for Dapagliflozin and Metformin were found to be 3.202 and
5.463mins respectively. A typical chromatograms of Dapagliflozin and Metformin is shown
in Figure No.3, 4. The optimized method was validated as per ICH guidelines.

System suitability
System suitability tests were carried out on freshly prepared standard solutions and the
parameters are summarized in Table.No.1.

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Linearity
The correlation coefficient for linear curve obtained between concentration vs. Area for
standard preparations of Dapagliflozin and Metformin is 0.999and 0.999 respectively. It
shows that the good correlation exist between the drug and response. The results are
summarized in the Table No.2, 3.

Accuracy
The % Recovery for each level obtained for Dapagliflozin was found to be within the limits
(98-102.0 %).The results obtained for recovery at 50%, 100%, 150% are within the limits.
Hence method is accurate. The %Recovery for each level obtained for Metformin was found
to be within the limits (98-102%) as per the ICH guidelines the results were within the limit.
The results are shown in Table No.4, 5.

Precision
The % RSD of 6 determinations of Dapagliflozin and Metformin for System precision
intraday and inter day was found to be within the acceptance criteria of not more than 2.0%.
The results are tabulated in Table No. 6, 7, 8, 9.

Limit of Detection and Limit of Quantification

Limit of detection result for Dapagliflozin and Metformin was found to be 2.5 and 3.3
respectively and were within the limits. S/N ratio for Dapagliflozin and Metformin were
found to be within the limits. Results are summarized in Table No.10, 11.

Robustness
The analysis was performed in different conditions to fine the variability of test results. The
conditions are checked for variation of results. Results are summarized in Table No. 12, 13.

Table No.1: System Suitability of Proposed Method

S. No Parameters Dapagliflozin Metformin
1. Theoretical plates 7946 2385635
2. Resolution 7.52 ---
3. Tailing Factor 1.1 1.2
4. Retention Time(min) 3.297 5.409

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Table No.2: Linearity data of Dapagliflozin

S.No Linearity level Concentration Area
1. I 10ppm 61953
2. II 20ppm 130213
3. III 30ppm 198697
4. IV 40ppm 267002
5. V 50ppm 321658
Correlation Coefficient 0.99

Table No: 3 Linearity data of Metformin

S.No Linearity level Concentration Area
1. I 20ppm 909889
2. II 40ppm 1583641
3. III 60ppm 2395378
4. IV 80ppm 3185089
5. V 100ppm 3943725
Correlation Coefficient 0.99

Table No.4: Accuracy results for Metformin

%Concentration Amount Amount
% Mean
(at specification Area Added Found
Recovery Recovery
Level) (ppm) (ppm)
50% 1217218 30 29.4 99.1
100% 2397141 60 59.5 99.6 99.5
150% 3514547 90 89.7 99.8

Table No.5: Accuracy results for Dapagliflozin

%Concentration Amount Amount
Mean
(at specification Area Added Found % Recovery
Recovery
Level) (ppm) (ppm)
50% 98598.67 15 14.6 99.9
100% 198359.7 30 30.0 100 99.6
150% 291512.3 45 44.7 99

Table No.6: Results of Intra-day precision for Metformin

S.No Injection Area
1. Injection- 1 2397164
2. Injection-2 2391741
3. Injection-3 2371846
4. Injection-4 2361748
5. Injection-5 2371649
Mean 2378830
Std.dev 14958
%RSD 0.628

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Table No.7: Results of Intra-day precision for Dapagliflozin

S.No Injection Area
1. Injection- 1 198464
2. Injection-2 193643
3. Injection-3 196462
4. Injection-4 194644
5. Injection-5 198464
Mean 196335.4
Std.dev 2190.191
%RSD 1.115536

Table No.8: Results of Intermediate precision for Metformin

S. No Injection Area
1. Injection- 1 2389562
2. Injection-2 2381654
3. Injection-3 2381946
4. Injection-4 2391741
5. Injection-5 2386452
6. Injection-6 2374763
Mean 2384353
Std.dev 6183.339
%RSD 0.25933

Table No.9: Results of Intermediate precision for Dapagliflozin

S. No Injection Area
1. Injection- 1 197486
2. Injection-2 197486
3. Injection-3 196746
4. Injection-4 195862
5. Injection-5 196582
6. Injection-6 198463
Mean 197104.2
Std.dev 903.542
%RSD 0.458408

Table No.10: Limit of Detection Chromatogram of Dapagliflozin and Metformin

S.No Name Retention time Area s/n
1. Dapagliflozin 5.463 194627 2.5
2. Metformin 3.202 2391746 3.3

Table No.11: Limit of Quantification Chromatogram of Dapagliflozin and Metformin

S.No Name Retention time Area s/n
1. Dapagliflozin 6.250 194627 7.6
2. Metformin 3.639 2391746 10.1

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Table No. 12: Results for Robustness Metformin

Peak Retention Theoretical Tailing
Parameter used for sample analysis
Area Time plates factor
Actual Flow rate of 1.0mL/min 2391746 3.202 9028 1.2
Less Flow rate of 0.9mL/min 2371831 3.639 7381 1.2
More Flow rate of 1.1mL/min 2218319 2.859 9311 1.1
Less organic phase
2294821 3.460 7462 1.2
(about 5 % decrease in organic phase)
More organic phase
2394811 3.022 6817 1.1
(about 5 % Increase in organic phase)

Table No. 13: Results for Robustness Dapagliflozin

Peak Retention Theoretical Tailing
Parameter used for sample analysis
Area Time plates factor
Actual Flow rate of 1.1mL/min 194627 5.463 7398 1.1
Less Flow rate of 0.9mL/min 183738 6.250 6883 1.1
More Flow rate of 0.8mL/min 198373 4.863 9917 1.2
Less organic phase
178471 6.196 8372 1.1
(about 5 % decrease in organic phase)
More organic phase
189462 5.010 7716 1.2
(about 5 % Increase in organic phase)

Table No.14: Peak results for Assay standard

Metformin
S.No Name RT Area Height USP Tailing USP Plate Count
1 Metformin 3.211 2397162 397161 1.2 9472
2 Metformin 3.222 2394721 389173 1.2 9745
3 Metformin 3.254 2389461 391723 1.2 8917

Dapagliflozin
S.No Name RT Area Height USPTailing USP PlateCount Resolution
1 Dapagliflozin 5.414 198462 7811 1.1 8492 7.49
2 Dapagliflozin 5.453 198472 8193 1.1 8916 7.52
3 Dapagliflozin 5.424 198735 7972 1.1 9372 7.44

Table No. 15: Peak results for Assay sample

Metformin
S. No Name Rt Area Height USP Tailing USP Plate Count
1. Metformin 3.297 2391741 381612 1.2 9472
2. Metformin 3.294 2389166 391746 1.2 8927
3. Metformin 3.295 2361731 381634 1.2 9017

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Dapagliflozin
USP Plate
S. No Name RT Area Height USP Tailing Resolution
Count
1. Dapagliflozin 5.435 198641 8174 1.1 9284 7.18
2. Dapagliflozin 5.417 196547 8942 1.1 8974 7.44
3. Dapagliflozin 5.434 194027 7294 1.1 9017 7.38

Figure No.1: Metformin

Figure No.2: Dapagliflozin

Figure No.3: Standard Chromatograms of Dapagliflozin and Metformin

Figure No.4: Sample Chromatograms of Dapagliflozin and Metformin

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Figure No.5: Linearity study of Metformin

Figure No.6: Linearity study of Dapagliflozin

CONCLUSION
The developed 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, sensitive, precise, specific, accurate and reproducible.
Results of validation parameters demonstrated that the analytical procedure is suitable for its
intended purpose. Further, improved sensitivity makes it specific and reliable for its intended
use. Hence, this method can be applied for the analysis of pure drug and pharmaceutical
dosage forms.

ACKNOWLEDGEMENT
The authors are grateful to the Management of Nalanda College of Pharmacy, Nalgonda for
providing the facilities carry out the present research our work.

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