stability –indicating rp-hplc analytical method
TRANSCRIPT
G Veerabhadram et al journal de Afrikana, 2016, 3(5); 314-328
© journal de afrikana www.jdeafrikana.com 314
Research Article ISSN; 2411-1376
Title: Stability –indicating RP-HPLC analytical method development and validation for the Metformin and Empagliflozin in pharmaceutical dosage
form
N. Padmaja1, Mulagiri Sharath Babu2, G.Veerabhadram2* 1 Department of pharmacy, University College of Technology, Osmania University, Hyderabad-
500007, India. 2 Department of Chemistry, University College of Science, Osmania University, Hyderabad-500007,
India. _________________________________________________________________________
Corresponding Author:
Dr. G Veerabhadram
Contact: [email protected] Office: 040-27682337 Ext.313 Mobile: + 91- 9885179305 Article Statistics Received: 26th Oct 2016 Revised: 18th Nov 2016 Accepted: 26th Nov 2016 ISSN; 2411-1376
Abstract: A simple and accurate stability-indicating RP-HPLC method was developed for the simultaneous determination of Metformin and Empagliflozin in bulk and pharmaceutical dosage form. The chromatographic conditions were standardised using a Thermosil C18 (4.6mmx250mm, 5µ particle size) with PDAdetection at 225nm. The mobile phase consisted of methanol: acetonitrile: 0.025M potassium hydrogen phosphate buffer (adjusted to pH: 3 by ortho phosphoric acid) (45:30:25, v/v/v).The retention times of Metformin and Empagliflozin were 2.383 and 3.119min respectively. The calibration curves were linear with correlation coefficients of 0.999 and 0.999 over a concentration range of 4-20 µg/ml for Metformin and 15-75 µg/ml for Empagliflozin. This method has been validated and shown to be specific, precise, sensitive, linear, accurate,robust and fast. Metformin andEmpagliflozin were subjected to different degradation stress conditions. The degradation products were well resolved from the purestandard drug with significantly different retention time values.The proposed method has been validated according to the ICH guidelines and this method was successfully applied to estimate the levels of two drugs in a combined formulation with good accuracy and precision.
Key words: Metformin, Empagliflozin, RP-HPLC, Method
development, Validation
Site this Article:
N. Padmaja, Mulagiri Sharath Babu, G.Veerabhadram, Stability –indicating RP-HPLC analytical
method development and validation for the Metformin and Empagliflozin in pharmaceutical dosage
form, journal de afrikana, 2016, 3(5); 314-328.
G Veerabhadram et al journal de Afrikana, 2016, 3(5); 314-328
© journal de afrikana www.jdeafrikana.com 315
1. Introduction
Diabetes and its most abnormalities
constitute a major health problem in the
modern society.Metformin is an oral anti-
diabetic drug and belongs to biguanide
class.It is chemically N, N-
dimethylimidodicarbonimidicdiamide
hydrochloride (1,1-dimethyl biguanide
hydrochloride). It lowers blood glucose
concentrations in type 2 diabetes without
causing overt hypoglycemia.Metformin
inhibits hepatic gluconeogenesis in mice
independently of the LKB1/AMPK pathway
via reduction in hepatic energy state.(1-3).
Empagliflozin is an orally carry on
exacting sodium glucose co-transporter-2
(SGLT-2) inhibitor. Chemical formula of
Empagliflozin is (1-chloro-4-[b-d-
glucopyranos-1-yl]-[4-([s]-tetrahydrofuran-
3-yl-oxy)benzyl]-benzene(figure.1).The
empirical formula is C17H19ClO2Si and
molecular weight is 455.91.This is a new
antidiabetic drug from the gliflozin
class,recently approved for the treatment of
type 2 diabetes.
Empagliflozin(SGLT2 inhibitor) is a
white to yellowish, non-hygroscopic
crystalline solid. It lowers blood glucose in
kinfolk amongst kind 2 diabetes by
surpassing the reabsorption of glucose in the
kidneys and promoting excretion of excess
glucose in the urine (4-8 ).
H3CN
HN NH2
CH3
NH NH
. HCl
Figure .1: Structure of Metformin
Cl O
O
OH
HO OH
OH O
Figure .2: Structure of Empagliflozin
According to the literature review,
only one spectrophotometric method(9) and
two chromatographic methods (10-11 ) were
developed for simultaneous determination
of Metformin and Empagliflozin. The aim of
the new methods is to present Stability –
indicating RP-HPLC method development
and validation for the Metformin and
Empagliflozin in pharmaceutical dosage
form. A new combination dosage form of
Metformin and Empagliflozin is indicated
for the treatment and management of
diabetis. The focus of present study was to
develop and validate simple precise method
for the same purpose.
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2. Materials and methods
2.1 Chemical and reagents
Working standards, Metformin and
Empagliflozin were kindly provided by
Boehringer Ingelheim India ltd,
Maharashtra, India. Potassium hydrogen
phosphate, Orthophosphoric acids are of AR
grade purchased from Merck. (Darmstadit,
Germany). HPLC solvents like, Acetonitrile,
Methanol from Rankem, Mumbai, India.
Synjardy® tablets nominally containing
12.5mg of Empagliflozin and 500 mg of
Metformin per tablet were supplied from the
Boehringer Ingelheim pharmaceutical
company (Germany).
2.2 Instrumentation
The waters 2695 binary pump, auto sampler
and a 2996 photo diode array detector were
used for method development, stability and
validation studies. The output signal was
monitored and processed using Empower
software on Pentium computer (Digital
Equipment Co).
2.3 Preparation of mobile phase
The mobile phaseprepared, contain
methanol: acetonitrile: 0.025M potassium
hydrogen phosphate buffer (adjusted to pH:
3 by ortho phosphoric acid) (45:30:25,
v/v/v) filtered through 0.45 µm and
degassed before use.Mobile phase was used
as the diluent.
2.4 Preparation of standard working
solution
Stock solutions of Empagliflozin and
Metformin(1 mg/ml) were prepared
separately in the diluent sonicated for 10
min and then working solutions (10µg/ml)
were prepared by transferring 1ml of each
stock solution to a 100 ml volumetric flask
and completed to volume with diluent.
2.5 Preparation of working sample
solution
Twenty tablets of Synjardy® were weighed,
powdered and mixed in a mortar. An
accurately weighed amount of the finely
powdered tablets equivalent to 2.5 mg of
Empagliflozin and 100 mg of Metformin
was made up to 100 mL with diluent,
sonicated to dissolve, filtered and then 1 mL
of the extract was transferred to a 100 mL
volumetric flask, spiked with 10 mL of
Empagliflozin working solution and finally
completed to volume with diluent.The final
concentration of the diluted tablet extract
was 2.25 μg /mLEmpagliflozin (0.25 μg
from tablet and 2 μg from spiking) and 10
μg /mLMetformin.
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3. Experimental
3.1 Method development
The RP-HPLC separation and quantitation
were achieved on Thermosil C18
(4.6mmx250mm),5µ column using mobile
phase composition of methanol: acetonitrile:
0.025M potassium hydrogen phosphate
buffer (adjusted to pH: 3 by ortho
phosphoric acid) (45:30:25, v/v/v).at a flow
rate of 1.2 ml /min. The detector was set at
225 nm.
3.2 Method validation
The method was validated for its accuracy,
linearity, precision, sensitity and specificity.
Method validation was carried out as per
ICH guidelines.
3.2.1 Linearity
Calibration curve was constructed by
plotting area vs. concentration of Metformin
and Empagliflozin solutions, the regression
equation was calculated and calibration
curve was plotted over the concentration
range of 4-20µg/ml for Metformin and 15-
75µg/ml for Empagliflozin.
3.2.2Accuracy
The accuracy of proposed method was
determined by calculating % recoveries
ofMetformin and Empagliflozin. It was
carried out by adding quantities of each
analyte corresponding to three concentration
levels(80,100,120%) of the labelled claim to
the excipients.
3.2.3 Precision
Precision of the method was of intra-day and
inter-day variations and also as
repeatability.Intra-day precision was
determined by analysing, the three different
concentrations4µg/mL, 6µg/mL and
8µg/mL of Metformin ; 24µg/mL,32µg/mL
and 40µg/mL of Empagliflozin
respectively, for three times in the same day.
Day to day variability was assessed using
above mentioned three concentrations
analysed on three different days, over a
period of one week. This result shows
reproducibility of the assay.
3.2.4 Robustness
The robustness of proposed method was
assessed by varying two parameters, which
includes variation of flow rate, change in
mobile phase composition using 4µg/mL of
Metforminand 24 µg/mL of Empagliflozin
solutions respectively.
3.2.5 Sensitivity
Sensitivity of the proposed method was
estimated in terms of Limit of detection
(LOQ) and Limit of quantitation (LOQ).
The stock standard solutions were injected
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in concentration range of 6 µg/mL
(Metformin) and 32 µg/mL (Empagliflozin).
3.2.6 System suitability test
According to ICH, system suitability is used
to verify the suitability for the resolution and
reproducibility of the chromatographic
system. Solutions of Metformin 4µg/mL and
Empagliflozin 24µg/mL for
chromatographic conditions were tested for
system suitability testing.
3.2.7 Forced degradation of Empagliflozin
and Metformin
Acid degradation:
Accurately weighed and transferred 100 mg
of Metformin and 2.5mg of Empagliflozin
working standard into a 100ml volumetric
flask and was added about 2 ml of 0.1N Hcl
and sonicated for 10minutes and kept it in
darkness for 48 hours then refluxed under
heat at 600Cin a heating mantle for 1 hour.
These solutions were diluted with the diluent
to reach a final concentration of 10µg/mL
of Metformin and 2.25 µg/mL of
Empagliflozin.
Base Degradation:
Accurately weighed and transferred 100 mg
of Metformin and 2.5mg of Empagliflozin
working standard into a 100ml volumetric
flask and added about 2 ml of 0.1N NaOH
and sonicated for 10minutes and kept it in
darkness for 12 hours then refluxed under
heat at 450Con a heating mantle for 1 hour.
These solutions were diluted with the diluent
to reach a final concentration of 10µg/mL
of Metformin and 2.25 µg/mL of
Empagliflozin.
Thermal Degradation
Accurately weighed and transferred 100 mg
of Metformin and 2.5mg of Empagliflozin
working standard into a 100ml volumetric
flask and kept in oven under heat at
105oCfor 12 hours. These solutions were
diluted with the diluent to reach a final
concentration of 10µg/mL of Metformin
and 2.25 µg/mL of Empagliflozin.
Peroxide Degradation
Accurately weighed and transferred 100 mg
of Metformin and 2.5mg of Empagliflozin
working standard into a 100ml volumetric
flask and was added about 3ml of 3%
Hydrogen Peroxide (H2O2) and sonicated for
10 minutes and kept in darkness for 12 hours
and refluxed under heat at 650C in a heating
mantle for 1 hour. These solutions were
diluted with the diluent to reach a final
concentration of 10µg/mL of Metformin
and 2.25 µg/mL of Empagliflozin.
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Photo degradation
Accurately weighed and transferred 100 mg
of Metformin and 1mg of Empagliflozin
working standard into a 100ml volumetric
flaskThe solutions were kept in the sun light
for 12 h. These solutions were diluted with
the diluent to reach a final concentration of
10µg/mL of Metformin and 2.25 µg/mL of
Empagliflozin.
4. Results and discussion
4.1 Optimization of HPLC method
Different mobile phases were tried using
various proportions of different phases and
organic modifiers. Methanol and acetonitrile
were tried as organic modifiers and different
potassium hydrogen phosphate buffer
strengths were tried (0.025 and 0.0075M).
Metformin and Emphagliflozin exhibit UV
absorption mainly at 225 nm. A typical
absorption spectra of Metformin and
Empagliflozinare shown in figure.3. The
resolution of standard Empagliflozin and
Metformin in the presence of degradation
products was satisfactory. Ultimately, a
mobile phase consisting of methanol:
acetonitrile: 0.025M potassium hydrogen
phosphate buffer (adjusted to pH: 3 by ortho
phosphoric acid) (45:30:25, v/v/v) was
selected for validation purposes and stability
studies. Optimized standard chromatogram
for Metformin and Empagliflozinis shown
in figure 4.
Fig.3: Overlay spectra of Metforminand Empagliflozin
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Figure 4: Optimized standard chromatogram of Metformin and Empagliflozin
4 .2 Validation of the method
4.2.1 Linearity
The linear equations for Metformin and
Empagliflozin were y = 2410.4x – 238.1 and
y = 12807x – 6432.9 with correlation
coefficient(r) being 0.999 and 0.999 for
Metformin and Empagliflozin respectively
and results are given in Table 1 and 2 ,figure
5 and 6.
Table 1: Linearity data
Serial
no.
Metformin Empagliflozin
Concentration
(µg/ml) Peak area
Concentration
(µg/ml) Peak area
1 4 9613 15 201932
2 8 18226 30 388071
3 12 28839 45 597859
4 16 38452 60 770854
5 20 48065 75 961396
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Table 2: Calibration data
Parameters Metformin Empagliflozin
Linearity range (µg/ml) 4-20 15-75
Correlation coefficient 0.999 0.999
Slope 2410.4 12807
Y-intercept 238.1 6432.9
Figure 5: Linearity curve of Metformin
Figure 6: Linearity curve of Empagliflozin
y = 12807x + 6432.R² = 0.999
0
200000
400000
600000
800000
1000000
1200000
0 20 40 60 80
Peak
are
a
Concentration
y = 2410.x - 238.1R² = 0.999
-10000
0
10000
20000
30000
40000
50000
60000
0 5 10 15 20 25
Peak
are
a
Concentration
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4.2.2 Accuracy
To the pre analysed sample, a known
amount of a standard solution of pure
drug(Metformin and Empagliflozin ) was
applied at three different levels.These
showed acceptable % recoveries in the range
of 98.5-100.51 for Metformin and 98.7-
100.3 for Empagliflozin.The results are
tabulated in Table 3.
Table 3: Percentage recovery of Metformin and Empagliflozin
Name of drug Spiked level (%) % Recovery % RSD
Metformin 80 98.5 0.81
100 99.7 0.22
120 100.51 0.35
Empagliflozin 80 99.2 0.42
100 98.7 0.65
120 100.3 0.12
4.2.3 Precision
Precision of the method was determined in
terms of intraday and inter-day
variation(%RSD). The low %RSD value
was indicated that the method was precise
and reproducible.The results are shown in
Table 4.
Table 4: Precision
Name of drug
Amount applied(µg/ml)
Intraday precision (Mean peak area±S.D)
%RSD Interday precision
(Mean peak area±S.D)
%RSD
Metformin 4 35653±690.96 1.9 35175±696.05 1.97
6 36512±700.96 1.9 35300±639 1.8
8 35123±525 1.49 35721±717 0.47
Empagliflozin 24 466344±4302 0.92 464123±7859.4 1.6
32 473081±7534 1.59 472843±6414 1.3
40 476632±7642 1.6 474286±5498 1.15
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4.2.4 Robustness
The standard deviation and % R.S.D of
peak areas were calculated for each
parameter and % R.S.D .was found to be
less than 2% and values are shown in Table
5.
Table 5: Robustness study of Metformin and Empagliflozin
4.2.5 Sensitivity
LOD was found to be 0.21 and 0.42 for
Metformin and Empagliflozin
respectively.LOQ was found to be 0.56 and
0.20 for Metformin and Empagliflozin
respectively.
4.2.6 System suitability test
The summary of System suitability validation parameters were listed in Table 6.
Chromatographic condition Metformin
[Peak area]
Empagliflozin
[Peak area]
Flow rate
0.80 36512 464501
1.00 35145 471215
1.20 35652 478621
Mean±S.D 35769±691.05 471445±67042
%RSD 1.931 0.01
Mobile phase composition
45:30:25 36557 468970
40:35:25 35128 456121
40:35:30 35982 467125
Mean±S.D 35889±719.02 464072±6947.2
%RSD 2.00 1.49
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Table 6: System suitability parameters
Parameters Metformin Empagliflozin
Retention time 3.118 2.836
Theoretical plates 4693 2766
Tailing factor 0.76 1.48
Resolution 4
4.2.7 Analysis of marketed tablet formulation:
The validated LC method was applied for
the quantitation of tablet formulation that
was obtained by injected 3 replicates of the
sample solutions.The amount of
Empagliflozin and Metformin estimated
were found to be 98.61 and
99.56,respectively in Table 7.A typical
chromatogram of Metformin and
Empagliflozin in tablet dosage formis shown
in figure 7.
Table 7:% Assay of marketed formulation
Brand
name
Name of
drug Dosage
Amount prepared
(μg /ml)
Amount found
(μg/ml) %Assay
Synjardy® Metformin 500mg 10 9.84 98.61
Empagliflozin 12.5 mg 2.25 2.21 99.56
Figure 7:A typical chromatogram of Metformin and Empagliflozin in tablet dosage form
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4.2.8 Stability indicating property
The chromatogram of sample
degraded by acid, base, hydrogen peroxide
and light showed well separated spots of
pure Empagliflozin and Metformin as well
as some additional peaks at different
retention times.
From the acidic degradation study of
Meformin and Empagliflozin, it was found
that no degradation took place over 48 h in
acidic conditions. The results of basic
hydrolysis showed degradation of
Metformin and Empagliflozin peak at 2.386
and 3.125 min respectively. In the oxidative
degradation study it was found that
Metformin and Empagliflozin were
degraded when kept at 600C for 12 h. The
degradant were retained at 2.385 and 3.126.
Degradation was not observed in Metformin
and Empagliflozin subjected to themal and
photo hydrolysis.The results of forced
degradation studies are given in table 8 and
chromatograms in figure 8 and 9.
Table 8: Stress degradation results for Metformin and Empagliflozin
Condition Degrad
-ation
time
Metformin Empagliflozin
RT
(Min)
%
Degradation
%
Recovery
RT
(Min)
%
Degradation
%
Recovery
Acid 48Hrs 2.383 7.13 92.87 3.119 5.71 94.29
Base 12 Hrs 2.385 9.63 90.37 3.126 9.49 90.51
Peroxide 12 Hrs 2.384 8.23 91.77 3.125 11.42 88.55
Thermal 12 Hrs 2.386 7.27 92.73 3.125 8.42 91.58
Photo 12 Hrs 2.386 4.49 95.73 3.118 6.44 93.56
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Figure 7. Typical chromatogram of base degradation pattern of Metformin and Empagliflozin
Figure 8. Typical chromatogram of oxidative degradation pattern of Metformin and Empagliflozin
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5. Conclusion
The developed stability indicating RP-HPLC
method provides simple, economical,
accurate, precise quantitative analysis for
simultaneous determination of Metformin
and Empagliflozin in bulk and
pharmaceutical dosage form.
Acknowledgement
One of the authors (Padmaja N) is thankful
to RGNF for awarding Senior Research
Fellowshipand Head, Department of
chemistry for providing the necessary
facilities.
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