Stability –indicating RP-HPLC analytical method

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.

http://www.jdeafrikana.com

mailto:[email protected]



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.




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.




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




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:




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




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




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.




Photo degradation


of Metformin and 1mg of Empagliflozin


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




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




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




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




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




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




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




Figure 7. Typical chromatogram of base degradation pattern of Metformin and Empagliflozin

Figure 8. Typical chromatogram of oxidative degradation pattern of Metformin and Empagliflozin




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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Stability –indicating RP-HPLC analytical method