A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 mm column in isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow rate was 1.0 mL min-1 and effluent was monitored at 245 nm using PDA detector. The injection volume was 10 µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc. in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively. The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No chromatographic interference from the tablet excipients was found. The proposed method was successfully used for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.

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Available online at www.icjpir.com ISSN: 2349-5448
Intercontinental journal of pharmaceutical
Investigations and Research
ICJPIR |Volume 2 | Issue 4 | Oct – Dec- 2015 Research Article
Stability indicating RP-HPLC method for estimation of dapagliflozin in
bulk and tablet dosage form
Jeyabaskaran M,1*
Prof. Rambabu C,2
and Lakshmi Maneka S
1
Research scholar, Department of Chemistry, Acharya Nagarjuna University, Nagarjuna
Nagar,Guntur (Dist), Andhra Pradesh, India.
2
Professor and Head, Department of Chemistry, Acharya Nagarjuna University, Nagarjuna
Nagar,Guntur (Dist), Andhra Pradesh, India.
Corresponding Author: Jeyabaskaran M
ABSTRACT
A simple, specific, accurate, precise and stability-indicating reverse phase high performance liquid
chromatography (RP-HPLC) method is developed for estimation of Dapagliflozin (DGF) in bulk and
Pharmaceutical dosage form. The method employed, Hypersil BDS C18 250 mm x 4.6 mm, 5 m column in
isocratic mode with mobile phase of 0.1% Ortho phosphoric acid buffer and acetonitrile 50:50% v/v. The flow
rate was 1.0 mL min-1
and effluent was monitored at 245 nm using PDA detector. The injection volume was 10
µl and the total runtime was set as 5min. The retention time for DGF was found to be 2.226min.The method was
validated in terms of Linearity, accuracy, precision, limit of detection (LOD), limit of quantification (LOQ) etc.
in accordance with ICH guidelines. Linear regression analysis data for the calibration plot showed that there
was a good linear relationship between response and concentration in the range of 25 - 150 µg/ml respectively.
The LOD and LOQ values for HPLC method were found to be 0.04 and 0.121 µg/ml respectively. No
chromatographic interference from the tablet excipients was found. The proposed method was successfully used
for estimation of Dapagliflozin (DGF) in Bulk and Pharmaceutical dosage form.
Keywords: Dapagliflozin, RP-HPLC, Stability indicates, Stress conditions
INTRODUCTION
Dapagliflozin (DGF) is a potent and selective
SGLT-2(SLC5A2) inhibitor with EC50 of 1.1nM.
Chemically, DGZ is (2S,3R,4R,5S,6R)-2-{4-
chloro-3-[(4-ethoxyphenyl)methyl]phenyl}-6-
(hydroxymethyl)oxane-3,4,5-triol with empirical
formula of C21H25ClO6 and Molecular weight is
408.87 g/mol.2
It has good permeability across
Caco-2 cell membranes and is a substrate for P-
glycoprotein (P-gp). Dapagliflozin is not a
significant P-gp inhibitor.1
Dapagliflozin is
indicated for the management of diabetes mellitus
type 2, and functions to improve glycemic control
in adults when combined with diet and exercise.

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Dapagliflozin is a sodium-glucose cotransporter 2
inhibitor, which prevents glucose reabsorption in
the kidney. Using dapagliflozin leads to heavy
glycosuria (glucose excretion in the urine), which
can lead to weight loss and tiredness.3
The
chemical structure of DGZ was shown in Fig.01.
The drug product in a stability test sample needs to
be determined using a stability indicating method,
as recommended by the International Conference
on Harmonization (ICH) guidelines4
and U.S.
Pharmacopoeia (USP) 265
. Although stability
indicating methods have been reported for assay of
various drugs in drug products, most of them
describe assay procedures for drug products
containing only one active drug substance. Only
few stability indicating methods are reported for
assay of drug products containing two or more
active drug substances. The objective of this work
was to develop a simple, precise, and rapid column
liquid chromatography (LC) procedure that would
serve as stability indicating assay method for drug
product of DGF.
An extreme literature survey revealed that very
few analytical methods have been reported such as
HPLC6-8
for DGF in individual and combination
with other drugs. In order to minimize the batch –
to- batch variation, it is very important to develop
suitable analytical methods for day –to- day
analysis of drugs. It was found that one attempt has
been made to develop stability indicating studies
and estimation of DGF by RP-HPLC at the starting
of my work. Therefore, it was thought of interest in
development and validating an advanced new
sensitive, specific, precise, accurate stability
indicating RP-HPLC method for estimation of SOF
in bulk drug and in pharmaceutical dosage form.
We here in report a simple, rapid and reliable
HPLC for the estimation of DGF in bulk and
pharmaceutical dosage forms as per ICH
guidelines.9-13
Fig 01: Structure of Dapagliflozin.
MATERIALS AND METHODS
Chemicals and reagents
Pure standard of DGF was obtained as gift
sample from Dr,Reddy’s Laboratory in Hyderabad.
HPLC grade acetonitrile, HPLC grade water, Ortho
phosphoric acid HPLC (Merck. Mumbai, India),
Potassium dihydrogen phosphate and Triethylamine
(RANKEM, Mumbai, India.) and All solvents used
in this work are HPLC grade. Forxiga tablets (Astra
Zeneca Ab) Containing Dapagliflozin marketed
formulation was purchased from local market, high
precision weighing balance (wensar instruments,
hyderabad), micro pipette (in labs 10-100 µl) were
employed in the study. All the glassware employed
in the work cleaned with hot water followed acetic
anhydride then acetone and dried in hot air oven
whenever required. Working environment was
maintained in between 25o
C.
HPLC and chromatographic conditions
The analysis was performed on Waters 2695
RP-HPLC separation module (Waters Corporation,
Milford, USA) equipped with PDA detector having
back pressure 5000psi, automatic injector and
Hypersil BDS C18 (250 mm × 4.6 mm, 5 µm) were
used as stationary phase. Single pan Balance
(Shimadzu, model AY-120), Control Dynamics pH
meter (Mettler Toledo), Sonicator (Labindia
Instruments). The chromatographic separation was
achieved by using 0.1 % Ortho phosphoric acid
buffer and acetonitrile 50:50 %v/v as mobile phase
at a flow rate of 1ml/min. The mobile phase was
filtered through 0.45µm nylon membrane filter and

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degassed before use. The injection volume was 10
µl and the total runtime was set as 5min. The
determination of analytes was carried out at 245 nm
using PDA detector.
HPLC METHOD
Preparation of stock solution
Accurately Weighed and transferred 10mg
Dapagliflozin working Standard into a 10 ml clean
dry volumetric flask, add 5 ml of diluents (Water
and acetonitrile 50:50), sonicated for 30 minutes
and make up to the final volume with diluents.
Preparation of standard solution
From the above stock solution, 1 ml was pipette
out into a 10ml volumetric flask and then make up
to the final volume with diluent. HPLC spectrum of
DGF in optimized conditions is shown in Fig.2.
Fig.02; A typical chromatogram of DGF
Method validation
The accuracy of the proposed method was
determined by standard addition method. It is the
closeness of the analytical results obtained by the
analysis to the true value. A known amount of
standard drug was added to the fixed amount of
injection solution. Accuracy was expressed as
percentage recovery. Recovery test was performed
with three different concentrations i.e. 50 µg/ml,
100µg/ml and 150 µg/ml for Dapagliflozin. The %
recovery results were calculated and given in
Table.1.
Table.1: % Recovery results of Dapagliflozin
Conc. Dapagliflozin
Amount added (µg/ml) Amount recovered
(µg/ml)
% Recovery
50% 50 50.07 100.14
50 50.30 100.59
50 49.94 99.87
100% 100 99.77 99.77
100 100.23 100.23
100 101.72 101.72
150% 150 148.43 98.95
150 148.57 99.04
150 151.12 100.75

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Precision
Repeatability or Precision of the method was
determined by injecting six replicates of standard
solution at 100µg/ml of dapagliflozin HPLC
system. From the results obtained it was found that
the proposed method was precise shown in Table.2.
Table.2: Precision data
Injection Dapagliflozin concentration Area
1
100µg/ml
1539364
2 1541556
3 1545028
4 1548226
5 1523848
6 1545504
Mean 1540588
STDV 8773.6
%RSD 0.6
Linearity
A series of six concentrations in the range of 25
to 150µg/ml of Dapagliflozin has been prepared
and peak areas were recorded at 245 nm. A
calibration curve was plotted between peak area
versus concentration of respective Dapagliflozin
and the response of the drug was found to be linear.
The linear regression equation (y=mx+c) was found
to be y = 15079x + 4232.3 (Fig.3) for
Dapagliflozin. The linearity results were given in
Table.3.
Fig.3: Calibration curve of Dapagliflozin
Table.3: Linearity results of Dapagliflozin
Concentration
(µg/ml)
Area Average area % RSD
25
385637
381984
0.82
380158
380158
50
757872
761708
0.44
764259
762992
75
1120567
1124143
0.55
1120527
1131335
y = 15079x + 4232.3
R² = 0.9998
0
500000
1000000
1500000
2000000
2500000
0 50 100 150 200

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100
1522931
1529862
0.79
1543878
1522777
125
1870059
1869125
0.15
1871352
1865965
150
2272551
2275299
0.20
2272709
2280636
Robustness
Influence of small changes in chromatographic
conditions such as change in flow rate (
0.1ml/min), Temperature ( 50
C), mobile phase (
5%) studied to determine the robustness of the
method are also in favour of (Table.4, % RSD <
2%) the developed RP-HPLC method for the
analysis of dapagliflozin ( API).
Table. 4: Robustness of method
Parameter Variation Average Area %RSD
Standard - 1540588 0.60
Flow rate 0.9 ml 1571462 0.81
1.1 ml 1457151 0.59
Mobile phase 55:45 1612324 0.38
65:35 1572705 0.66
Temperature -5o
C 1556328 1.27
+5o
C 1372587 1.90
Limit of Detection (LOD)
Limit of detection is the known concentration of
Dapagliflozin and establishing minimum
concentration at which the Dapagliflozin can be
reliably detected. It was calculated based on the
standard deviation of the response and the slope of
the standard calibration curve. The LOD was found
to be 0.040 µg/ml of Dapagliflozin.
Limit of Quantification (LOQ)
Limit of quantification is the known
concentration of Dapagliflozin and establishing
minimum level at which the Dapagliflozin can be
quantified with acceptable accuracy and precision.
The LOQ was found to be 0.121 µg/ml of
Dapagliflozin. The LOD and LOQ results were
given Table.5.
Table.5: LOD and LOQ results of Dapagliflozin
Sample LOD LOQ
Dapagliflozin 0.040 µg/ml 0.121 µg/ml
Specificity and stability in analytical solution
The results of specificity indicated that the peak
was pure in presence of degraded sample. It is
important to mention here that the dapagliflozin
was stable in solution form up to 24 hrs at 250
C.
The results of linearity, precision, inter & intraday
assays, method robustness, LOD, LOQ, specificity
and stability in analytical solution established the
validation of the developed RP-HPLC method for
analysis of dapagliflozin.
Assay of DGF in dosage form
10 tablets were weighed and average weight
was calculated. Then from the transferred the
equivalent to one tablet to 100ml volumetric flask,
70ml of diluent was added and sonicated for 15
min, further the volume was made up with diluent.
From the filtered solution, 1ml was pipette out into

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10ml volumetric flask and made up to 10ml with
diluent. From the solution, 10µl was injected into
HPLC system and peak area was recorded with
detector at 245nm. The % assay was calculated
with obtained peak area of detector response. This
indicates that developed method can be used for
routine analysis. (Fig.4)
Fig.4: Chromatogram showing the assay of DGF marketed dosage form.
Optimization of chromatographic conditions
The chromatographic conditions were optimized
by different means. (Using different column,
different mobile phase, different flow rate, different
detection wavelength & different diluents for
sample preparation etc.(Table.6)
Table.6: Optimized chromatographic conditions
Parameter Condition
RP-HPLC Water 2695 separation module with PDA detector
Mobile phase 0.1% OPA(Ortho phosphoric acid) :ACN 50:50 v/v
Column Hypersil BDS 250 mm x 4.6 mm, 5 µm.
Column
Temperature
250
C
Wavelength 245nm
Diluent First dissolved in methanol and make up quantity with Acetonitrile: Water in the
ratio of (50:50)
Injector volume 10µl
Flowrate 1ml min-1
Runtime 5min
Retention time 2.226min
Stability indicating studies
The API (Dapagliflozin) was subjected to stress
conditions in various ways to observe the rate and
extent of degradation that is likely to occur in the
course of storage and/or after administration to
body. This is one type of accelerated stability
studies that helps us determining the fate of the
drug that is likely to happen after along time
storage, within a very short time as compare to the
real time or long term stability testing. The various
degradation pathways studied are acid hydrolysis,
alkali hydrolysis, oxidative degradation, Photolytic
degradation and dry heat degradation.

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Preparation of Acid induced degradation
Product
10ml (10mg) of Dapagliflozin stock solution
was taken into 100ml volumetric flask and refluxed
with 10ml 2N Hydrochloric acid at 800
C for 30
min. The resultant solution was collected, diluted
with diluent to get the concentration of 100µg/ml
and 10µl solution was injected into HPLC system
and chromatograms were recorded. (Fig.5)
Preparation of Alkali induced Degradation
Product
10ml of Dapagliflozin stock solution was taken
into 100ml volumetric flask and refluxed with
0.1N sodium hydroxide at 80 0
C. After 30 min the
resultant solutions was diluted with diluent to get
the concentration of 100µg/ml and 10µl solution
was injected into HPLC system and chromatograms
were recorded.(Fig.6)
Preparation of hydrogen peroxide induced
degradation product
10ml of Dapagliflozin stock solution was taken
into 10ml volumetric flask. 10 ml of freshly
prepared 10% H2O2 solution was added into
volumetric flask and solution was kept at 80 0
C for
30min. Then the resultant solution was injected into
HPLC system to get the chromatograms were
recorded. (Fig.7)
Preparation of water induced degradation
product
10ml of Dapagliflozin stock solution was taken
into 10ml volumetric flask. 10ml of water was
added into volumetric flask and solution was kept
at 80ºC for 8 hrs. the resultant solution was diluted
and 10µl solution was injected into the HPLC
system to get the chromatogram were recorded.
(Fig.8)
Photochemical Stability induced Product
The photochemical stability study of the drug
was studied by exposing the sample concentration
of 100µg/ml to UV light in UV chamber for 7 days
or 200 Watt hours/m2.
Then the resultant solution
was diluted and 10µl solution was injected into the
HPLC system to get the chromatograms were
recorded. (Fig.9)
Dry heat induced degradation product
To study the dry heat degradation studies, the
standard drug solution of Dapagliflozin was placed
in oven for 6hrs at 1050
C. The resultant solutions
were diluted to get the concentration of 100µg/ml
and 10µl solution was injected into HPLC system
and chromatograms were recorded to assess the
stability studies. (Fig.10)
Results of degradation studies
The specificity was successfully performed
under various stress conditions like acid, base,
oxidative, dry heat and photolytic and all the
degraded products were separated from sample
peaks. It was found that no interference of the
degraded products was seen with the drug products.
All Dapagliflozin peaks were tested for purity test
by comparing purity of angle and purity of
threshold. Purity of angle and purity of threshold
was estimated by chromatographic software, where
in all the conditions purity of angle was always less
than purity of threshold. This indicates that the
proposed method was specific. The results of
forced degradation shown in Table.7.
Table.7: Forced degradation studies of Dapagliflozin
Stress condition Dapagliflozin
Purity of
angle
Purity of
Threshold
Acid degradation 1.656 1.925
Base degradation 0.766 1.442
Peroxide degradation
Water degradation
0.476
0.434
0.530
0.608
Dry heat degradation 0.367 0.548
Photolytic degradation 0.419 0.583

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RESULTS AND DISCUSSION
To develop a precise, linear, specific & suitable
stability indicating RP-HPLC method for analysis
of dapagliflozin, different chromatographic
conditions were applied & the results observed are
presented. Isocratic elution is simple, requires only
one pump & flat baseline separation for easy and
reproducible results. So, it was preferred for the
current study over gradient elution.
In case of RP-HPLC various columns are
available, but here Hypersil BDS C18 (250mm × 4.6
mm, 5µm) column was preferred because using this
column peak shape, resolution and absorbance were
good. Mobile phase & diluent for preparation of
various samples were finalized after studying the
solubility of API in different solvents of our
disposal (methanol, acetonitrile, dichloromethane,
water, 0.1M NaOH, 0.1M HCl). The drug was
found to be highly soluble in acetonitrile & ethanol.
Drug was insoluble in water. Using these solvents
with appropriate composition newer methods can
be developed and validated.
The result shows the developed method is yet
another suitable method for assay and stability
studies which can help in the analysis of
dapagliflozin in different formulations.
Fig.5: Chromatogram of Acid degradation
Fig.6: Chromatogram of base degradation

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Fig.7: Chromatogram of oxidative degradation
Fig.8: Chromatogram of water degradation
Fig.9: Chromatogram of dry heat degradation

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Fig.10: Chromatogram of photolytic degradation
CONCLUSION
A sensitive & selective RP-HPLC method has
been developed & validated for the analysis of
Dapagliflozin (API). Further the proposed RP-
HPLC method has excellent sensitivity, precision
and reproducibility. The result shows the developed
method is yet another suitable method for assay,
impurity studies which can help in the analysis of
Dapagliflozin in different formulations.
Acknowledgement
I am thankful to Dr.Reddy’s laboratory for
providing the drug sample for this project.
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