STABILITY-INDICATING RP-HPLC METHOD AND ITS ......Department of Pharmaceutical Analysis & Quality Assurance, JNTU Kakinada University, Vikas College of pharmacy, Andhra Pradesh, India

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Lathareddy et al. World Journal of Pharmacy and Pharmaceutical Sciences

STABILITY-INDICATING RP-HPLC METHOD AND ITS

VALIDATION FOR ANALYSIS OF METFORMIN & SITAGLIPTIN

IN BULK AND PHARMACEUTICAL DOSAGE FORM

Yaraveda Lathareddy*, Naidu Srinivasa Rao

Department of Pharmaceutical Analysis & Quality Assurance, JNTU Kakinada University,

Vikas College of pharmacy, Andhra Pradesh, India.

ABSTRACT

A simple, rapid, precise, sensitive and reproducible reverse phase high

performance liquid chromatography (RP-HPLC) method has been

developed for the quantitative analysis of Metformin & Sitagliptin

(JANUMET) in pharmaceutical dosage forms. Chromatographic

separation of MET & SITA was achieved on AGILENT CN , 250mm

x 4.6mm, 5µm and the mobile phase containing OPA buffer &

Acetonitrile in the ratio of 80:20 v/v. The flow rate was 1.0 ml/min,

detection was carried out by absorption at 205nm using a photodiode

array detector at ambient temperature. The number of theoretical plates

and tailing factor for MET & SITA were NLT 3000 and should not

more than 2 respectively. JANUMET was exposed to thermal,

photolytic, hydrolytic, acid, alkali, and oxidative stress, and the

stressed samples were analyzed by use of the proposed method &

chromatograms from the stressed samples, obtained by use of the photodiode-array detector.

The linearity of the method was excellent over the range 25-750µg/ml and 3-75 µg/ml for

MET & SITA respectively. The correlation coefficient was 0.999. Relative standard

deviations of peak areas of all measurements were always less than 2.0%. The proposed

method was validated according to ICH guidelines. And it was found to be suitable and

accurate method for quantitative analysis of JANUMET and study of its stability.

Keywords: High performance liquid chromatography, Metformin, Sitagliptin & JANUMET

tablet dosage form.

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Article Received on 05 August 2013, Revised on 25 August 2013, Accepted on 28 September 2013

*Correspondence for

Author:

* Yaraveda Lathareddy

Department of Pharmaceutical

Analysis & Quality Assurance,

JNTU Kakinada University,

Vikas College of pharmacy,

Andhra Pradesh, India.

[email protected]

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INTRODUCTION

SITAGLIPTINR)-4-oxo-4-[3-(trifluromethyl)-5,6-dihydro[1,2,4]triaz-olo[4,3-a]pyrazin-

7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine and Metformin is N,Ndimethyl

imidocarboni-midic diamide are used in the treatment of type 2 diabetes. Structures are

shown in fig 1 and 2 respectively. SITAGLIPTIN works to competitively inhibit the enzyme

dipeptidyl peptidase 4 (DPP-4). This enzyme breaks down the incretins GLP-1 and GIP

inactivation, they are able to potentiate the secretion of insulin and suppress the release the

glucagon by the pancreas. This drives blood glucose levels to normal. Metformin activates

AMP-activated proteinkinase (AMPK), a liver enzyme that plays an important role in insulin

signaling, whole body energy balance, and the metabolism of glucose and fats; activation of

AMPK is required for metformin inhibitory effect on the production of glucose by liver cells.

The literature reveals that there are some of the methods have been reported for Sitagliptin

UV, HPLC and spectrophotometry. For metformin, Development and Validation of RP-

HPLC method5Simultaneous determination of metformin and sitagliptin by reverse phase

HPLC in pharmaceutical dosage forms. As no HPLC method have been reported for the

determination of Sitagliptin and Metformin an attempt was made to report a simple, sensitive,

validated and economic method for the determination of Sitagliptin and Metformin

Sitagliptin Metformin

MATERIALS AND METHODS

Metformin and Sitagliptin were obtained as gift samples from Dr. Reddy’s laboratories,

Hyderabad, India. Distilled, 0.45 µm filtered water used for HPLC analysis and preparation

of buffer. Buffers, Acetonitrile and all other chemicals were analytical grade. The HPLC

system consisted of a Waters model LC-20AD dual pump, a Waters model DGU-20A

degasser, Waters model SPD-M20A photo diode array (PDA) detector and a Waters model

SIL-20A HT auto injector. It was operated by Empower2 software. The AGILENT CN,

250mm x 4.6mm, 5µm column was used and mixture of Acetonitrile and buffer in the

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proportion of 20:80 v/v was used as mobile phase at a flow rate of 1.0 mL/min. The column

was maintained at ambient temperature. Detector was programmed at 205 nm for detection of

MET and SITA.

Reagents required

1. Ortho Phosphoric Acid (HPLC grade)

2. Acetonitrile (HPLC grade)

3. HPLC grade Water (Milli Q or equivalent)

Preparation of Buffer

Pipette out 1ml Ortho Phosphoric acid is dissolved into 1lt Water.

Mobile phase:

Prepare a mixture of Buffer and Acetonitrile in the ratio of (80:20). Filter and degas.

Chromatographic condition

Use suitable High Performance Liquid Chromatography equipped with UV-visible detector.

Column : AGILENT CN 250mm x 4.6mm, 5µm.

Wavelength : 205 nm

Injection Volume : 10µL

Column Temperature : Ambient

Flow rate : 1.0 mL/min

Retention time of Metformin is about 3.0 min and Sitagliptin is about 6min.

Preparation of Diluent

Used mobile phase as diluents

Preparation of standard solution of Metformin

Weigh accurately about 250 mg of Metformin working standard and 50mg of Sitagliptin

working standard are taken into 50ml volumetric flask. Add 70mL of diluent, sonicate to

dissolve and dilute to volume diluent. Further dilute 5mL to 50mL with the diluent.

Preparation of standard solution of Sitagliptin

Weigh accurately about 50mg of Sitagliptin working standard are taken into 100ml

volumetric flask. Add 70 mL of diluent, sonicate to dissolve and dilute to volume diluent.

Further dilute 5mL to 50mL with the diluent.

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Preparation of Sample solution

Weigh 10tablets and crush the tablets weigh powder then take 5 tablets equivalent of sample

into a 100mL volumetric flask. Add 70mL of diluent, sonicate to dissolve and dilute to

volume with diluent. Further dilute 1mL to 50mL with the diluent. Filter through 0.45µ

Nylon syringe filter.

Procedure

Inject 10µL of Standard preparation five times and Sample preparation in the

Chromatograph. Record the chromatograms and measure the peak responses for Metformin

& Sitagliptin. The System suitability parameters should be met. From the peak responses,

calculate the content of Metformin & Sitagliptin in the sample.

Evaluation of system suitability

1. Relative Standard Deviation of five replicate injections of Standard preparation for

Metformin & Sitagliptin peaks should not be more than 2.0%.

2. The tailing factor for Metformin & Sitagliptin peaks should be more than 2.0 and plate

count will be not less than 3000.

Forced Degradation studies

Forced degradation study was carried out by treating the sample under the following

conditions. Sample Stock solution is from Method Precision sample flask.

a) Acid degradation

5 ml of the above stock solution was transferred into 100ml volumetric flask and added 60ml

of diluent, treated with 5.0ml of 5N hydrochloric acid and heated at 60°c for 10 minutes, and

cooled, neutralized with 5ml of 5N sodium hydroxide and diluted to volume with diluent and

was analyzed as per the test method.

b) Alkali degradation

5 ml of the above stock solution was transferred into 100ml volumetric flask and added 60ml

of diluent, treated with 5.0ml of 5N sodium hydroxide and heated at 60°c for 10 minutes, and

cooled, neutralized with 5ml of 5N hydrochloric acid and diluted to volume with diluent and

was analyzed as per the test method.

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c) Peroxide degradation

5 ml of the above stock solution was transferred into 100ml volumetric flask and added with

60ml diluent was treated with 5 ml of 30% v/v solution of hydrogen peroxide and heated at

60°c for 10 minutes, cooled and diluted to volume with diluent and was analyzed as per the

test method.

d) Reduction

5 ml of the above stock solution was transferred into 100ml volumetric flask and added with

60ml diluent was treated with 5 ml of 1N solution of sodium bicarbonate and heated at 60°c

for 10 minutes, and cooled, made to volume with diluent and was analyzed as per the test

method.

e) Photolytic degradation

Sample was exposed to 1.2 Million lux hours of light and analyzed the exposed sample as per

test procedure.

f) Thermal degradation

Sample was kept in hot air oven at 60°C for 1 hour. Treated sample was analyzed as per the

test method.

RESULT AND DISCUSSION

A reversed-phase column procedure was proposed as a suitable method for the simultaneous

determination of Sitagliptin and metformin in combined dosage forms. The chromatographic

conditions were optimized by changing the mobile phase composition, pH, and buffers used in

the mobile phase. Different ratios were experimented to optimize the mobile phase. Finally a

mixture of OPA buffer & Acetonitrile in the ratio of 80:20 v/v was used. A typical

chromatogram obtained by using the above mentioned mobile phase from 20µl of the assay

preparation is illustrated below. The retention times of Sitagliptin and Metformin were 2.7

and 6.0 min, respectively. The linearity of the method was tested from 3-75µg/ml for

Sitagliptin and 25-750µg/ml for metformin. Correlation coefficients for the regression line

were 0.9982 and 0,9991 for Sitagliptin and metformin respectively. The accuracy of the

method was studied by recovery experiments. The recovery was determined at three levels,

viz. 50%, 100%, and 150% of the selected concentrations. Three samples were prepared for

each recovery level. The recovery values for Sitagliptin and metformin ranged from 99.8-

100.4%, respectively. The precision of the method was determined from one lot of combined

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dosage form. To determine the robustness of the developed method experimental conditions

were purposely altered and RSD of the peak areas of Sitagliptin and metformin were found

not greater than 2.0 illustrates the robustness of the method.

Method validation

This method described above had been validated as per the ICH guidelines for the parameters

like accuracy, linearity, precision, detection limit, quantitation limit and robustness. And the

results were summarized below.

Linearity

The linearity responses in the concentration range of 25-750 µg/mL for MET and 3-75

µg/mL for SITA was determined. And the co-relation coefficient was NLT 0.99

Precision

Precision was measured in terms of repeatability of application and measurement. Study was

carried out by injecting six replicates of the standard at a concentration of 500µg/mL for

MET and 50µg/mL for SITA. And the RSD calculated from replicates of assay values NMT

2.0%

Accuracy

Accuracy (Recovery) of the method was determined by spiking 50, 100 and 150% of

working standard at a concentration of 500µg/mL for MET and 50µg/mL for SITA. Samples

were injected in triplicate across its range according to the assay procedure. The RSD

calculated from replicates of assay values NMT 2.0% and the percentage recovery was in

between 99% to 102%

Detection and quantitation limits

The LOD and LOQ values were determined by the formulae LOD = 3.3 s/ m and LOQ = 10

s/m (Where, s is the standard deviation of the responses and m is mean of the slopes of the

calibration curves).

System suitability

The system suitability was assessed using five replicate analyses of drugs at concentration of

500µg/mL for MET and 50µg/mL for TEL by increasing the injection volumes 10-50µL.

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Specificity

Specificity studies were carried for both pure drug and drug product by comparing the plots

with blank and placebo. Peak purity tests were also carried out to show that the analyte

chromatographic peak is not attributable to more than one component as the impurities are

not available by purity index data.

Robustness

Robustness of the method was determined by making slight changes in the chromatographic

conditions, such as flow rate (1± 0.1 mL/min), wavelength (±1nm),organic phase(± 10%) and

ph(±0.2)

Fig 1 Control peak

Fig 2 purity plot for Metformin

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Fig 3 purity plot for Sitagliptin

Chromatograms for Forced Degradation Studies

Fig 4 Blank peak

Fig 5 Acid degradation

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Fig 6 Alkali degradation

Fig 7 Hydrolysis degradation

Fig8 Peroxide degradation

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Fig 9 Photolytic degradation

Fig 10 Reduction degradation

Fig 11 Thermal degradation

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Table1 Data for forced degradation studies of Metformin

Table2 Data for forced degradation studies of Sitagliptin

Accuracy

Table 3 Data for Metformin

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Table 4 Data for Sitagliptin

LINEARITY

Table5 linearity data for Metformin

Fig12 calibration curve for Metformin

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Table6 linearity data for Sitagliptin:

Fig12 calibration curve for Sitagliptin

Method

Precision

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Table7 precision data for Metformin

Table8 precision data for Sitagliptin

Intermediate Precision

Table9 IP data for Metformin

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Table10 IP data for Sitagliptin

ASSAY

SAMPLE

Table11 Assay data for sample

STD

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Table12 Assay data for STD

Metformin comparison

Table13 comparative data for Metformin

Sitagliptin comparison

Table14 comparative data for Sitagliptin CONCLUSION

The proposed method was found to be simple, precise, accurate and rapid for simultaneous

determination of Sitagliptin and metformin. The mobile phase is simple to prepare and

economical. The sample recoveries in all formulations were in good agreement with their

respective label claims and they suggested non-interference of formulation excipients in the

estimation. The most striking feature of this method is its simplicity and rapidity also best

separation of the analyte against the method available. The recovery studies revealed

excellent accuracy and high precision of the method. The HPLC method was found to give

better results and can be employed for routine analysis in quality control analysis. The

described method gives accurate and precise results for determination of Sitagliptin and

metformin mixture in tablet dosage form.

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ACKNOWLEDGEMENTS

The authors are thankful to the Head of the pharmaceutical Analysis Department & my guide

for his moral support and encouragement during the work. And to the Cystron pharmaceutical

laboratories, Vijayawada, for providing the necessary facilities to carry out this research

work.

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Documents

STABILITY-INDICATING RP-HPLC METHOD AND ITS ......Department of Pharmaceutical Analysis & Quality Assurance, JNTU Kakinada University, Vikas College of pharmacy, Andhra Pradesh, India