ArticlePDF Available

DEVELOPMENT AND VALIDATION OF HIGH-PERFORMANCE LIQUID CHROMATOGRAPHIC METHOD FOR DETERMINATION OF DAPAGLIFLOZIN AND ITS IMPURITIES IN TABLET DOSAGE FORM

Authors:

Abstract and Figures

Objective: The aim of the present work is the development of new, sensitive, specific, and accurate high-performance liquid chromatographic method for the separation and determination of dapagliflozin and its impurities in tablet dosage form. Methods: The chromatographic separation of drug and its impurities was achieved using Hypersil BDS C18 column (250 mm × 4.6 mm, 5 μ) with mobile phase consisted of mobile phase-A (Buffer pH 6.5) and mobile phase-B (acetonitrile:water 90:10) by gradient program at a flow rate of 1 mL/min with ultraviolet detection at 245 nm. Results: Dapagliflozin and its impurities A, B, C, D, E, and impurity-F were successfully eluted at the retention time of 16.95, 2.72, 7.82, 10.58, 21.11, 30.37, and 34.36 min, respectively, with good resolution. The method was validated according to the international conference on harmonization guidelines. The validation results showed good precision, accuracy, linearity, specificity, sensitivity, and robustness. Conclusion: Successful separation and determination of dapagliflozin and its six impurities were achieved by the proposed method. The developed method can be applied for the routine analysis of dapagliflozin and its impurities in pharmaceutical formulations.
Content may be subject to copyright.
Vol 12, Issue 3, 2019
Online - 2455-3891
Print - 0974-2441

TABLET



Received: 03 November 2018, Revised and Accepted: 09 January 2019

 The aim of the present work is the development of new, sensitive, specific, and accurate high-performance liquid chromatographic method
for the separation and determination of dapagliflozin and its impurities in tablet dosage form.
 The chromatographic separation of drug and its impurities was achieved using Hypersil BDS C18 column (250 mm × 4.6 mm, 5 µ) with
mobile phase consisted of mobile phase-A (Buffer pH 6.5) and mobile phase-B (acetonitrile:water 90:10) by gradient program at a flow rate of
1 mL/min with ultraviolet detection at 245 nm.
 Dapagliflozin and its impurities A, B, C, D, E, and impurity-F were successfully eluted at the retention time of 16.95, 2.72, 7.82, 10.58, 21.11,
30.37, and 34.36 min, respectively, with good resolution. The method was validated according to the international conference on harmonization
guidelines. The validation results showed good precision, accuracy, linearity, specificity, sensitivity, and robustness.
 Successful separation and determination of dapagliflozin and its six impurities were achieved by the proposed method. The developed
method can be applied for the routine analysis of dapagliflozin and its impurities in pharmaceutical formulations.
 Dapagliflozin, High-performance liquid chromatography method, Impurity.

Dapagliflozin is an antidiabetic drug used for the management of type 2
diabetes mellitus and belongs to a novel class called sodium glucose
cotransporter 2 (SGLT2) inhibitor. It is a potent, competitive, reversible,
highly selective, and orally active inhibitor of SGLT2, the major
transporter protein responsible for the renal glucose reabsorption.
By suppressing the SGLT2, dapagliflozin reduces plasma glucose
concentration by elevating the renal glucose excretion. Hence, it is used
to treat patients with type 2 diabetes. Dapagliflozin’s mechanism of
action is different from the mechanisms of other antidiabetic drugs as it
involves the direct and insulin-dependent elimination of glucose by the
kidney. It is a synthetic aryl glycoside contains multiple chiral centers,
but the drug is a single enantiomer and it is chemically described as (2S,
3R, 4R, 5S, 6R)-2-(4-chloro-3-[4-ethoxybenzy] phenyl)-6-(hydroxyl
methyl) tetrahydro-2H-pyran-3,4,5-triol [1-3].
Impurities are unwanted chemicals present within the formulation
and active pharmaceutical ingredient which affects the quality, safety,
and efficacy of the medicinal products. A significant aspect of ensuring
the safety of drug products is the qualification of impurities [4].
Identification of impurities is done by a variety of chromatographic and
spectroscopic techniques, either alone or in combination with other
techniques. The objective of the study was to identify and quantify the
dapagliflozin and its related impurities A, B, C, D, E, and F [5] (Table 1) in
the marketed pharmaceutical formulation. Literature survey revealed
that few analytical methods have been reported for the estimation of
dapagliflozin alone or in combination with other drugs by ultraviolet
(UV) spectrometry [6-9], high-performance liquid chromatography
(HPLC) [10-19], and LC-mass spectrometry [20]. However, there
is no reported method about the separation and determination of
dapagliflozin impurities. Hence, an attempt was made to develop
simple, accurate, precise, and sensitive HPLC method for estimation of
dapagliflozin in the presence of its above-mentioned impurities.


Dapagliflozin reference standard and impurities were obtained from
Veeprho laboratories Pvt. Ltd., Pune. Dapagliflozin tablet, Forziga 10 mg,
was purchased from local market. Analytical grade orthophosphoric
acid, HPLC grade acetonitrile, methanol, and water were purchased
from Merck (Mumbai, India).
Instrumentation
The HPLC system used for the method development and validation
composed of Waters alliance system 2695 separation module with
autosampler and UV detector. Separation was carried out in a Hypersil
BDS column C18 column (250 mm × 4.5 mm, 5 µ). The data analysis was
processed with empower software.
Preparation of mobile phase
Mobile phase-A
Two milliliters of 88% orthophosphoric acid was measured and
transferred into 2000 ml standard flask, and the volume was made up
to the mark with HPLC grade water and adjusted the pH of the solution
to 6.50 using triethylamine.
Mobile phase-B
Acetonitrile and water mixture were prepared in the ratio of
75:25 % v/v and degassed by sonication.
Preparation of diluent
Mobile phase-B was used as diluent.
Preparation of standard solution
Accurately weighed and transferred about 50 mg of dapagliflozin
standard into a 50 ml volumetric flask. To this, 30 ml of diluent was
Research Article
© 2019 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.
org/licenses/by/4. 0/) DOI: http://dx.doi.org/10.22159/ajpcr.2019.v12i3.30853
448
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
Grace et al.
added and was sonicated with intermittent shaking to dissolve and
diluted to the volume with diluents (1000 µg/ml). 5 ml of this solution
was diluted to 50 ml with diluent (100 µg/ml). Further 5 ml of this
solution was diluted to 50 ml with diluents (10 µg/ml).
Preparation of sample solution
The commercial dapagliflozin 10 tablets (Forxiga tablet 10 mg) were
accurately weighed and powdered. From the tablet, powder transferred
about 50 mg equivalent dapagliflozin sample into a 50 ml volumetric flask.
To this, 30 ml of diluent was added and sonicated with intermittent shaking
to dissolve and diluted to the volume with diluent. The solution was filtered
through a 0.45-µ-membrane filter. The above solution was appropriately
diluted to get the final concentration of 10 µg/ml sample solution.

About 10 mg of all the six impurities were accurately weighed and
transferred into six different 10 ml volumetric flask, and the volume
was made up to the mark with diluent (1 mg/ml). 1 ml each of impurity
stock solution was taken separately in six different 100 ml volumetric
flask; and the volume was made up to the mark with mobile phase to
get the concentration of 10 µg/ml for each; and all the solutions were
studied individually on the HPLC system.
Preparation of impurities mixture
Accurately weighed and transferred about 2 mg each of impurity A, B,
C, D, E, and F in 10 ml of volumetric flask. To this, 5 ml of diluent was
added and was sonicated with intermittent shaking to dissolve and
diluted to volume with diluent (200 µg/ml). The solution was filtered
through a 0.45-µ-membrane filter.

Transferred 1ml of the stock solution of the known impurities in 200 ml
of volumetric flask made up to volume with Dapagliflozin sample
solution (Dapagliflozin 10 µg/ml and impurities 1.0 µg/ml).
Preparation of resolution solution
Accurately weighed and transferred about 5 mg of impurity-D and 50 mg
of Dapagliflozin into a 50 ml volumetric flask. To this, 30 ml of diluent
was added and was sonicated with intermittent shaking to dissolve and
made up to the volume with diluent. From the above solution, 5 ml was
diluted to 100 ml with diluents to get the concentration of dapagliflozin,
50 µg/ml and impurity-D, 5 µg/ml.
ic conditions
The separation and analysis of all compounds were carried out on
Hypersil BDS C18 column (250 mm × 4.5 mm, 5 µ) at 50°C, and the
analytes were monitored with UV detection at 245 nm. A gradient
mixture of mobile phase-A and mobile phase-B was used at a flow
rate of 1 ml/min. The LC gradient program was set as follows,
Time (min)/mobile phase-A: mobile phase-B percentage (%). It was
programmed as 0–8/75:25, 8–12/55:45, 12–25/55:45, 25–35/40:60,
35–65/30:70, 65–66/30:70, and 66–75/75:25.
Method validation
The method validation was performed as per the international
conference on harmonization (ICH) guidelines [21]. The parameters such
as specificity, linearity and range, precision, accuracy, limit of detection
(LOD), limit of quantitation (LOQ), and robustness were evaluated.

The specificity was demonstrated by injecting blank solution,
dapagliflozin standard solution, and sample spiked with impurity
solution, and the chromatograms were checked for interferences [22].
Linearity
The linearity was tested in the concentration range of 0.20–13.00 µg/ml
for dapagliflozin, 0.26–2.00 µg/ml for impurity-A, 0.21–2.00 µg/ml
for impurity-B, 0.52–2.00 µg/ml for impurity-C, 0.26–2.00 µg/ml for
impurity-D, 0.37–2.00 µg/ml for impurity-E, and 0.37–2.00 µg/ml for
impurity-F.
Precision
The precision was studied by repeatability and intermediate precision
(ruggedness). The repeatability was checked by injecting the sample
solution spiked with impurities in six replicates, and the intermediate
precision was evaluated by different analyst using different columns
on different days. The percentage relative standard deviation (%RSD)
of % total impurity was calculated.
Name of compound  
Dapagliflozin (2S,3R,4R,5S,6R)-2-[4-chloro-3-(4-ethoxybenzy) phenyl]-6-(hydroxymethyl)
tetrahydro-2H-pyran-3,4,5-triol
Impurity-A (2S,3R,4R,5S,6R)-2-(4-bromo-3-(4-ethoxybenzyl) phenyl)-6-(hydroxymethyl)
tetrahydro-2H-pyran-3,4,5-triol
Impurity-B (3R,4S,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl) phenyl)-6-(hydroxymethyl)
tetrahydro-2H-pyran-2,3,4,5-tetraol
Impurity-C 4-bromo-1-chloro-2-(2-ethoxybenzyl) benzene
Impurity-D 1,4-dibromo-2-(4-ethoxybenzyl) benzene
Impurity-E (2S,3R,4R)-2-(4-chloro-3-(4-ethoxybenzyl) phenyl)-5-(1,2-dihydroxyethyl)
tetrahydrofuran-3,4-diol
[5]
Impurity-F (2R,3R,4R,5S,6R)-2-(4-chloro-3-(4-ethoxybenzyl) phenyl)-6-(hydroxymethyl)
tetrahydro-2H-pyran-3,4,5-triol
449
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
Grace et al.
Accuracy
The accuracy of the method was verified by injecting the sample
solution spiked with impurities at different levels ranging from 70% to
130% in three replicates, and % mean recovery of each impurity was
calculated.
Limit of detection and limit of quantitation
LOD is defined as the lowest concentration of an analyte that an
analytical method differentiates from background levels. The LOQ
is defined as the lowest concentration that can be measured with
acceptable accuracy, precision, and variability. The LOD and LOQ were
calculated from the linearity curve by using the formulae:
LOD= 3.3
S
σ
LOQ=10
S
σ
Where σ is the standard deviation of the y-intercept and S is the slope
of the calibration plot.

Robustness of the method was verified by deliberately varying the
instrumental conditions by flow rate (±10%), organic phase ratio
(±2%), pH of buffer (solution-A) (±0.2), and column oven temperature
(±5°C). The %RSD of % total impurity was calculated [21-25].

Method development
Analytical detection wavelength of 245 nm was selected for the
proposed HPLC method based on the UV absorption of dapagliflozin
and its impurities. Several trials were done to separate dapagliflozin
from its impurities using various columns and by changing mobile
phase composition and pH. Finally, the separation of all the impurities
from the drug peak was achieved with Hypersil BDS column C18
column (250 mm × 4.5 mm, 5 µ) and with a gradient mixture of the
mobile phase-A (Buffer pH 6.5) and mobile phase-B (acetonitrile:water
75:25 % v/v), and the peak shape of the drug and the impurities was
good. The retention time (RT) of the drug and the impurities were
identified by analyzing the chromatograms (Fig. 1) of individual
standard solutions of dapagliflozin and the impurities in five replicates
under the optimized method conditions.

d
c
b
f
a
e
450
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
Grace et al.
The chromatographic system performance was checked by injecting
the resolution solution prepared with dapagliflozin standard and the
closely eluted impurity-D, and the chromatogram was recorded (Fig. 2).
The resolution was found to be 9.80. From the chromatograms of
resolution, and individual standard solutions of dapagliflozin, and the
impurities, the system suitability parameters such as the number of
theoretical plates, tailing factor, and the resolution were calculated. The
results are listed in Table 2. The chromatographic system is considered
suitable when it meets the following criteria. The resolution between
the peaks must be >2, the number of theoretical plates should be more
than 2000, and tailing factor must be lower than 2.
Method validation
Specificity
The specificity of the method was tested by comparing the
chromatograms of blank (Fig. 3), dapagliflozin standard solution
(Fig. 4), and sample spiked with impurity solution (Fig. 5). No
interference peaks were observed at the RT of dapagliflozin due to the
blank, impurities, and placebo.
Linearity
The linearity graph was plotted by taking the concentration in the
x-axis and peak area in y-axis over the calibration ranges tested
for dapagliflozin and impurities. The data were subjected to linear
regression analysis, and the regression statistics such as concentration
range, slope, y-intercept, and correlation coefficient are shown in
Table 3.
Precision
Precision of the method by repeatability and intermediate precision was
assessed by injecting sample solution spiked with impurities six times,
and the results were expressed in terms of %RSD of the percentage of
total impurities. The results are presented in Table 4.
Accuracy
The accuracy of the proposed method was determined by analyzing
the dapagliflozin sample solution spiked with impurities at three
concentration levels of 70%, 100%, and 130% of each impurity in
triplicate. The mean percentage recovery was calculated and reported
in Table 5.



impurities



       
Retention time 2.72 7.82 10.58 16.95 21.11 30.37 34.36
Theoretical plates 6168 43,350 64,182 18,298 64,685 82,991 87,950
Symmetry factor 1.12 1.03 1.11 0.64 1.11 1.05 1.04
Peak area 128,579 119,169.6 66,577.8 7,906,417 254,283.6 143,306 73,306.6
Percentage RSD of peak area 0.21 0.15 0.13 0.026 0.12 0.21 0.11
Resolution 34.68 17.27 18.75 9.80 24.15 8.81
Imp: Impurity, RSD: Relative standard deviation

 µ  Intercept 
Impurity-A 0.26–2.00 11978 9.2664 0.9981
Impurity-B 0.21–2.00 15070 82.95 0.9996
Impurity-C 0.52–2.00 6497.5 140.47 0.9979
Dapagliflozin 0.20–13.00 8897.7 490.18 0.9999
Impurity-D 0.26–2.00 25152 25.71 0.9988
Impurity-E 0.37–2.00 15795 980.3 0.9952
Impurity-F 0.37–2.00 8742.6 264.97 0.999
451
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
Grace et al.
Limit of detection and limit of quantitation
LOD and LOQ were predicted from the linearity curve using the slope
and standard deviation of y-intercepts of regression lines, as per the
ICH guideline. The predicted LOD and LOQ values were verified by
checking the precision of six replicate injections at that concentration
for impurity-A, impurity-B, impurity-C, Dapagliflozin, impurity-D,
impurity-E, and impurity-F, and the results are shown in Table 6.

To test the robustness small but deliberate changes were made in the
method conditions, the samples were analyzed in triplicate and the
%RSD of percentage of total impurities were calculated for each altered
condition. In the varied method conditions, all the analytes are well
resolved, and the elution order of analytes was unchanged. The results
were summarized in Table 7.

  
 
1 0.146 0.146 0.142
2 0.148 0.148 0.142
3 0.145 0.145 0.161
4 0.146 0.146 0.135
5 0.148 0.148 0.159
6 0.146 0.146 0.145
Mean 0.146 0.146 0.147
SD 0.0012 0.0012 0.0104
Percentage RSD 0.82 0.82 7.07
Overall mean 0.147
Overall SD 0.0071
Overall percentage RSD 4.83
RSD: Relative standard deviation, SD: Standard deviation
 µ µ Precision at LOD level 
   
Impurity-A 0.084 0.253 944±125.81 13.33 3078±212.25 6.90
Impurity-B 0.042 0.127 749±113.12 15.10 2027±198.75 9.81
Impurity-C 0.217 0.434 1286±99.40 7.73 2676±127.59 4.77
Dapagliflozin 0.065 0.196 949±57.54 6.06 2092±153.63 7.34
Impurity-D 0.071 0.216 1688±213.6 12.66 5151±212.38 4.12
Impurity-E 0.114 0.345 1478±271.3 18.36 5027±243.61 4.85
Impurity-F 0.176 0.352 2046±156.9 7.67 3495±140.28 4.01
Overall mean percentage RSD 11.55 5.97
*n=6. LOD: Limit of detection, LOQ: Limit of quantitation, RSD: Relative standard deviation, SD: Standard deviation

 
     
70 103.02 96.08 92.14 96.37 104.27 103.02
100 104.44 101.47 99.99 101.37 98.50 102.22
130 104.42 104.36 98.17 102.25 99.38 102.76
Overall mean 103.96 100.63 96.76 99.99 100.71 102.67
Overall SD 1.940 3.647 4.883 2.788 3.528 1.209
Overall percentage RSD 1.87 3.66 4.96 2.80 3.48 1.18
*n=3. RSD: Relative standard deviation, SD: Standard deviation

Limit of detection and limit of quantitation data
According to the literature review, there is no HPLC method development
was reported for simultaneous estimation of dapagliflozin and its
impurities. In the proposed method under optimized chromatographic
conditions, the dapagliflozin and its impurities were well separated with
good peak shape and proper RT. System suitability test results confirm
that the developed method is suitable for the analysis of impurities in the
drug product. The results of method validation parameters are
within the acceptance criteria as per the ICH guidelines [21]. The
specificity study meets the acceptance criteria [21], as that no
interference was observed from the blank at the RT of known
impurities. It is evident that from the obtained data that all the
peaks were well resolved, and the method is said to be specific.
The result of linearity study shows excellent correlation existed
between the peak area and concentration of the drug dapagliflozin and
impurities, and the correlation coefficient of the drug and
impurities complied with the acceptance limit (not ≥0.95) [22].
The obtained precision data represent no significant variation in
the measured response and demonstrate that the method is
repeatable and rugged with the %RSD value below 4.83 which meets
the acceptance limit (<10%) [22]. The mean % recovery of
recovery study was in the range of 96.76–103.96. This range
complies with the acceptance criteria [22] 85%–115%, thus
confirming the accuracy of the method. The mean %RSD of
precision at LOD and LOQ level was below 11.55 (acceptance limit –
<15%) [22], and the very low LOD and LOQ values when compared to
the reported method [25] indicate the sensitivity of the developed
method for determination of dapagliflozin impurities. The
satisfactory mean %RSD value of robustness study exhibits the
proposed method which is robust enough to withstand the small
variations of method conditions.
452
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
Grace et al.

The quality and safety of the drug product not only depends on the
adopted manufacturing procedure and toxicological properties of an
active substance but also depends on the impurities that it contains.
Hence, a thorough examination of related impurities plays a significant
role in controlling the quality of a drug product. In the current research
work, a simple, sensitive, specific, and accurate HPLC method was
developed for separation and determination of dapagliflozin in the
presence of its related impurities. The developed method was validated
as per the ICH guidelines and it was found to be precise, linear, rugged,
and robust. Hence, the present method can be adapted to separate,
identify, and quantify the related impurities of dapagliflozin in its
pharmaceutical formulations. Application of this method in quality
control shall improve the safe use of medicinal products which contain
dapagliflozin as an active substance.

The research work, manuscript preparation, and grammar check using
the software Grammarly were done by Mrs. A. CAROLINE GRACE, the
research work was guided by Dr. T. PRABHA, and critical revision and
final proofreading of the manuscript were done by Dr. T. SIVAKUMAR.

The authors declare that there are no conflicts of interest.

1. Committee for Medicinal Products for Human Use, European Medicine
Agency. Forxiga-Assessment Report. Available from: http://www.ema.
europa.eu/docs/en_GB/document_library/EPAR_-Public_assessment_
report/human/002322/WC500136024.pdf. [Last cited on 2016 Nov 16].
2. Therapeutic Goods Administration, Department of Health and Ageing,
Australian Government. Australian Public Assessment Report for
Dapagliflozin Propanediol Monohydrate. Available from: https://
www.tga.gov.au/sites/default/files/auspar-dapagliflozin-propanediol-
monohydrate-130114.pdf. [Last cited on 2016 Nov 16].
3. Drug Bank. Dapagliflozin. Available from: https://www.drugbank.ca/
drugs/DB06292. [Last cited on 2016 Nov 14].
4. Federal Register International Conferences on Harmonization. Draft
Revised Guidance on Impurities in New Drug Substances, Q3A(R);
2000. p. 45085-90.
5. Veeprho Pharmaceuticals. Dapagliflozin Impurity. Available from:
https://www.veeprhopharma.com/dapagliflozin-impurity-manufacturer-
supplier.php. [Last cited on 2016 Nov 14].
6. Mante GV, Gupta KR, Hemke AT. Estimation of dapagliflozin from its
tablet formulation by UV spectrometry. Pharm Methods 2017;8:102-7.
7. Sanagapati M, Dhanalakshmi K, Nagarjunareddy G, Kavitha. B.
Method development and validation of dapagliflozin API by UV
spectroscopy. Int J Pharm Sci Rev Res 2014;27:270-2.
8. Jani BR, Shah KV, Kapupara PP. Development and validation of UV
spectroscopic first derivative method for simultaneous estimation
of dapagliflozin and metformin hydrochloride in synthetic mixture.
J Bioequiv Stud 2015;1:1-8.
9. Chitra KP, Eswaraiah MC, Rao MV. Unique UV spectrophotometric
method for reckoning of dapagliflozin in bulk and pharmaceutical
dosage forms. J Chem Pharm Res 2015;7:45-9.
10. Jeyabaskaran M, Rambabu C, Dhanalakshmi B. RP-HPLC method
development and validation of dapagliflozin in bulk and tablet
formulation. Int J Pharm Anal Res 2013;2:221-6.
11. Sanagapati M, Dhanalakshmi K, Nagarjunareddy G, Sreenivasa S.
Development and validation of a RP-HPLC method for the estimation
of dapagliflozin in API. Int J Pharm Sci Res 2014;5:5394-7.
12. Yunoos M, Sankar DG. A validated stability indicating high performance
liquid chromatographic method for simultaneous determination of
metformin HCL and dapagliflozin in bulk drug and tablet dosage form.
Asian J Pharm Clin Res 2015;8:320-6.
14. Debata J, Kumar S, Jha SK, Khan A. A new RP-HPLC method
development and validation of dapagliflozin in bulk and tablet dosage
form. Int J Drug Dev Res 2017;9:48-51.
15. Patel KJ, Chaudhary AB, Bhadani SM, Raval RJ. Stability indicating
RP-HPLC method development and validation for estimation of
dapagliflozin and metformin HCl. World J Pharm Pharm Sci 2017;
6:796-809.
16. Verma MV, Patel CJ, Patel MM. Development and stability indicating
HPLC method for dapagliflozin in API and pharmaceutical dosage
form. Int J Appl Pharm 2017;9:1618-32.
17. Prameela KL, Veni PR, Narayana PV, Haribabu B. Development
and validation of stability indicating high performance liquid
chromatography method with photodiode array detection for the
simultaneous estimation hypoglycemic agents, dapagliflozin and
metformin. Int J Pharm Bio Sci 2017;8:328-36.
18. Patel A, Maheshwari D. Development and validation of UV
spectrophotometric method and RP-HPLC method for simultaneous
estimation of dapagliflozin propanediol and glimepiride in synthetic
mixture. Eur J Pharm Med Res 2017;4:416-34.
19. Phani RS, Prasad KR, Mallu UR. A study of new method development,
validation and forced degradation for simultaneous analysis of
dapagliflozin and saxagliptin in pharmaceutical dosage form by HPLC
method. Pharm Chem 2017;9:96-103.
20. Aubry AF, Gu H, Magnier R, Morgan L, Xu X, Tirmenstein M, et al.
Validated LC-MS/MS methods for the determination of dapagliflozin,
a sodium-glucose co-transporter 2 inhibitor in normal and ZDF rat
plasma. Bioanalysis 2010;2:2001-9.
21. International Conference on Harmonization of Technical Requirements
for Registration of Pharmaceuticals for Human Use. Validation of
Analytical Procedures: Text and Methodology ICH Q2 (R1); 2005.
22. Daksh S, Goyal A, Pandiya CK. Validation of analytical methods-
strategies and significance. Int J Res Dev Pharm Life Sci 2015;
4:1489-97.
23. Tsvetkova B, Pencheva I, Zlatkov A, Peikov P. High performance liquid
chromatographic assay of indomethacin and its related substances in
tablet dosage forms. Int J Pharm Pharm Sci 2012;4:549-52.
24. Oruganti SS. Development and validation of analytical methods for
the determination of impurities in some selected drug substances using
high performance liquid chromatographic technique. Int Curr Pharm
Res 2016;8:49-53.

Flow rate (±10%) 0.9 ml/min 0.143±0.0104 2.17
1.0 ml/min 0.147±0.0031 7.07
1.1 ml/min 0.141±0.0021 1.49
Mobile phase-B composition-organic phase ratio±2% Acetonitrile 73% 0.144±0.0111 7.71
Acetonitrile 75% 0.147±0.0031 7.07
Acetonitrile 77% 0.145±0.0040 2.76
Column oven temperature±5°C 45°C 0.147±0.0020 1.36
50°C 0.147±0.0031 7.07
55°C 0.132±0.0015 1.14
pH of mobile phase-A±0.2 6.3 0.164±0.0025 1.52
6.5 0.147±0.0031 7.07
6.7 0.148±0.0010 0.68
*n=3. RSD: Relative standard deviation, SD: Standard deviation
Altered method conditions  
13. Shyamala, Nidhi B, Kavitha M, Pooja, Sharma JV. Validated RP-HPLC
method for simultaneous estimation of metformin hydrochloride and
dapagliflozin in tablet dosage form. Am J Biol Pharm Res 2015;2:109-3.
25. Ravichandran V, Shalini S, Sundram KM,Rajak H.Validation of
analytical methods-strategies and importance. Int J Pharm Pharm Sci
453
Grace et al.
Asian J Pharm Clin Res, Vol 12, Issue 3, 2019, 447-453
2010;2:18-22.
... The LOD and LOQ were separately determined and calculated based on the calibration curve of standard solution. 17 The LOD and LOQ were calculated and the results were within limits i.e., less than 2. ...
Article
Full-text available
Objective: To develop precise, accurate and reproducible stability assay method by RP-HPLC for estimation of dapagliflozin in API and pharmaceutical dosage form.Methods: The adequate separation was carried using agilent C18 (4.6 ml (millimeter)*150,5 µm (micromiter), mixture of acetonitrile: di-potassium hydrogen phosphate with pH-6.5 adjusted with OPA (40:60 %v/v) as a mobile phase with the flow rate of 1 ml/min (milliliter/minute) and the effluent was monitored at 222 nm (nanometer) using photo diode array detector. The retention time of dapagliflozin API and dapagliflozin tablet were 3.160 min (minute) and 3.067 min (minute) respectively.Results: Linearity for dapagliflozin was found in the range of 50-150µg/ml (microgram/milliliter) (R2 = 0.99) respectively. The accuracy of the present method was evaluated at 50 %, 100% and 150%. The % recoveries of dapagliflozin API and tablet were found to be in the range of 99.00–99.99 % and 98.50–99.99 % respectively. Precision studies were carried out and the relative standard deviation values were less than two. The method was found to be robust.Conclusion: The proposed method was found to be specific, accurate, precise and robust can be used for estimation of dapagliflozin in API and Pharmaceutical dosage form.
Article
Full-text available
Dapagliflozin (DAP) is indicated for the management of diabetes mellitus Type 2, and functions to improve glycemic control in adults when combined with diet and exercise1. DAP is an inhibitor of sodium-glucose cotransporter 2 (SGLT2) responsible for the majority of the reabsorption of filtered glucose from the tubular lumen. By inhibiting SGLT2, DAP reduces reabsorption of filtered glucose and lowers the renal threshold for glucose, and thereby increases urinary glucose excretion2. In present work, a selective, specific, sensitive and economical UV spectroscopic method has been developed for the estimation of Dapagliflozin in Bulk and its pharmaceutical dosage forms. An absorption maximum was found to be at 233.65 nm. Dapagliflozin obeyed Beer’s law in the concentration range from 10-35 μg / ml. Proposed method was validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values with correlation coefficient of 0.9998. The percentage recovery of Dapagliflozin ranged from 99.7 in pharmaceutical dosage form. Results of the analysis for accuracy, precision, LOD, LOQ and were found to be satisfactory. The proposed method is simple, rapid and suitable for the routine quality control analysis. © 2015, Journal of Chemical and Pharmaceutical Research. All rights Reserved.
Article
Full-text available
The Novel, simple, sensitive, rapid, accurate and economical and reliable First derivative spectroscopic method has been developed for synthetic mixture of Dapagliflozin (DAPA) and Metformin hydrochloride (MET). This method involve solving of first derivative method based on measurement of absorbance at two wavelengths 235 nm and 272 nm using UV visible spectrophotometer with 1cm matched quartz cells and methanol solvent were employed in this method. The Developed method obeyed Beer’s-Lambert’s law in the concentration range of 0.5-2.5 μg/ml, having correlation coefficient for Dapagliflozin was 0.984 and 25-125 μg/ml, having correlation coefficient for Metformin hydrochloride was 0.982. A derivative spectrum shows better resolution of overlapping bands than the fundamental spectrum. Different validation parameters like, precision (intra-day and inter-day studies), limit of detection, limit of quantitation were studied and were found to be within the limit. Results of the methods were validated statistically.The validation results showed that the proposed method was sensitive, economical and simple and it could be successfully applied for evaluation and to estimate the amount of synthetic mixture containing Dapagliflozin and Metformin hydrochloride.
Article
Full-text available
A reversed-phase high performance liquid chromatographic (RP-HPLC) method with UV detection was proposed for separation of indomethacin and its impurities from tablet dosage forms. The best separation was achieved on a LiChrosorb C18, 250 mm x 4.6 mm, 5 μm column at a detector wavelength of 240 nm. The utilization of mixture of 40 volumes 0.5 % v/v orthophosphoric acid, 20 volumes of methanol and 40 volumes of acetonitrile as mobile phase with a flow rate of 2ml/min enabled acceptable resolution of indomethacin, in large excess, from possible impurities, in a short elution time (9 min). Analytical parameters linearity, accuracy, precision and specificity were determined by validation procedure and found to be satisfactory. Overall, the proposed method was found to be simple, rapid, precise and accurate for quality control of indomethacin and its impurities in dosage forms and in raw materials. In this work the kinetic investigation of the alkaline hydrolysis of indomethacin was also carried out. The degradation reaction was monitored by means of HPLC method developed and was found to follow first-order kinetics. The rate constant and half-life of the hydrolytic decomposition were estimated.
Article
In the present study, a simple, novel, safe, sensitive and economic UV- Spectrophotometric method for the estimation of a Type II anti diabetic drug, Dapagliflozin was developed and assessed. The developed method was validated as per ICH guidelines. The drug showed tow different wavelengths of maximum absorption, at 203nm and 237nm. This method can be successfully applied for the estimation of Dapagliflozin in bulk for routine analysis with UV detection at 237nm. A Labindia UV-Visible spectrophotometer with 1cm matched quartz cells and ethanol solvent were employed in this method. The Developed method obeyed Beer's-Lambert's law in the concentration range of 0.5-0.9μg/ml, having correlation coefficient of 0.994. Different validation parameters like, precision (intra-day and inter-day studies), limit of detection, limit of quantitation, ruggedness and robustness were studied and were found to be within the limits.
Article
Objective: A simple and precise stability indicating reversed-phase high-performance liquid chromatography method was developed and validated for the simultaneous determination of metformin (MET) hydrochloride and dapagliflozin (DAP) in bulk and pharmaceutical dosage form. Methods: Chromatography was carried out on hypersil BDS C18(250 mm × 4.6 mm, 5 μ particle size) column containing mobile phase of buffer (0.1% orthophosphoric acid) adjusted to pH 6.8 with triethylamine:acetonitrile in the ratio of 50:50%/v/v at a flow rate of 1 ml/minutes. The analyte was monitored using photodiode array detector at 240 nm. Results: The retention time was found to be 2.791 minutes and 3.789 minutes for MET hydrochloride and DAP respectively. The proposed method was found to be having linearity in the concentration range of 85-510 μg/ml for MET (r2=0.99995) and 0.5-3.0 μg/ml for DAP (r2=0.99978), respectively. The mean % recoveries obtained were found to be 99.66-100.23% for MET and 99.61-100.38% for DAP respectively. Stress testing which covered acid, base, peroxide, photolytic and thermal degradation was performed on under test to prove the specificity of the method and the degradation was achieved. The developed method has been statistically validated according to ICH guidelines. Conclusion: Thus, the proposed method can be successfully applied for the stability indicating the simultaneous determination of MET hydrochlorideand DAP in bulk and combined tablet dosage form and in the routine quality control analysis. © 2015, Asian Journal of Pharmaceutical and Clinical Research. All rights reserved.
Article
Dapagliflozin is an inhibitor of sodium-glucose co-transporter 2 (SGLT-2) in development for the treatment of Type 2 diabetes. To support toxicology studies, LC-MS/MS methods were developed and validated for the quantitation of dapagliflozin in rat plasma. The assay uses solid phase extraction and LC-MS/MS analysis in negative ion electrospray ionization mode. Because dapagliflozin readily forms adducts in the presence of formic acid, the mobile phases were simple mixtures of water and acetonitrile. The assay was validated in the concentration range of 5-2000 ng/ml with good intra- and inter-day precisions and acceptable sample stability. The validated assay was successfully applied to the quantitation of dapagliflozin in plasma in support of preclinical studies in both normal and diabetic rats.