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METHOD DEVELOPMENT AND VALIDATION FOR THE

SIMULTANEOUS ESTIMATION OF SITAGLIPTIN AND

METFORMIN BY HPLC AND UV-SPECTROSCOPY METHODS

Under the guidance of

GUIDE CO-GUIDE

Dr. M.LAXMI SUREKHA Mr. NITIN.S. JADAV

M.Pharm, Ph.D. M.Pharm.

Presented by

SRILATHA. K , B.Pharm.(H.No: 06712885020)

Department of pharmaceutical Analysis,

Trinity College of Pharmaceutical analysis.

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2

CHAPTER TOPIC

1 INTRODUCTION

2 DRUGS PROFILE

3 LITERATURE REVIEW

4 OBJECTIVES AND PLAN OF WORK

5 EXPERIMENTAL WORK

6 RESULTS AND DISCUSSION

7 CONCLUSIONS

8

REFERENCES

CONTENTS

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3

Chapter 1

Introduction

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4

Introduction

Pharmaceutical analysis is the branch of analytical chemistry involved in

the separation, identification and determination of the substances present in bulk and

pharmaceutical preparations. Main uses of analysis include :

Qualitative analysis (Identification)

Quantitative analysis (estimation).Qualitative analysisis performed to indicate whether the substance or compound is

present in the sample or not. Qualitative tests identify the specific analyte by

detection of evolved gas, formation of precipitates, colour change reactions, melting

point and boiling point test etc.

Quantitative analytical techniques are mainly used to quantify any compound or

substance in the sample.

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Chapter 2

Drug Profile

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DRUG PROFILE OF SITAGLIPTIN

STRUCTURE OF ZOLPIDEM TARTRATE

6

Category

Hypoglycemic Agents, Dipeptidyl-Peptidase IV Inhibitors

IUPAC name

7 - [(3R) - 3 - amino - 1 - oxo - 4 - (2,4,5 - trifluorophenyl) butyl] -

5,6,7,8 - tetrahydro - 3 - (trifluoromethyl) - 1,2,4 - triazolo[4,3 - a]

pyrazine phosphate monohydrate

Molecular formulaC16H15F6N5OH3O4PH2O

Molecular mass Average: 407.314g/mol.

Pka 6.16

Description white to off-white crystalline powder

Chemical Structure of Sitagliptin

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Mechanism of Action:

Sitagliptin works to competitively inhibit the enzyme dipeptidyl peptidase 4 (DPP-4). This

enzyme breaks down the incretins GLP-1 and GIP, gastrointestinal hormones released in

response to a meal. By preventing GLP-1 and GIP inactivation, they are able to increase thesecretion of insulin and suppress the release of glucagon by the pancreas. This drives blood

glucose levels towards normal. As the blood glucose level approaches normal, the amounts of

insulin released and glucagon suppressed diminishes, thus tending to prevent an "overshoot"

and subsequent low blood sugar (hypoglycemia) which is seen with some other oral

hypoglycemic agents.

Pharmacodynamics :

Sitagliptin is an orally-active member of the new dipeptidyl peptidase-4 (DPP-4) inhibitor

class of drugs. The benefit of this medicine is expected to be its lower side-effects of

hypoglycemia in the control of blood glucose values. The drug works to diminish the effects of a

protein/enzyme (by the inhibition of this protein/enzyme) on the pancreas at the level of release

of glucagon (diminishes its release) and at the level of insulin (increases its synthesis and

release) until blood glucose levels are restored toward normal, in which case the

protein/enzyme-enzyme inhibitor becomes less effective and the amounts of insulin released

diminishes thus diminishing the "overshoot" of hypoglycemia seen in other oral hypoglycemic

agents.

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Pharmacokinetics:

Absorption: oral administration, Bioavailability 87%

Volume of distribution:198 L PH:3.5-4.5

Plasma Protein Binding:The fraction of Sitagliptin reversibly bound to plasma

proteins is low 38%

Metabolism:Metabolized to a limited extent by CYP isoenzymes 3A4 and 2C8 to

inactive metabolites.

Route of Elimination:Eliminated principally by kidneys via active tubular secretion.Excreted in urine (87%) mainly as unchanged drug and in feces (13%).

Clearance :renal clearance =350 mL/minfor healthy one receiving 100 mg oral

dose

Half life (t1/2):12.4 hours.

Shelf-Life:36 months

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DRUG PROFILE OF METFORMIN

9

Category Hypoglycemic Agents

IUPAC name 1-carbamimidamido-N,N-dimethylmethanimidamide

Molecular formula C4H11N5

Molecular mass 165.62g.mol-1

Pka 12.4

Description white to off-white crystalline powder

Class Biguanides

Fig No. 2.2 Chemical Structure of Metformin

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Mechanism of Action:

Metformin's mechanisms of action differ from other classes of oral antihyperglycemic

agents. Metformin decreases blood glucose levels by decreasing hepatic glucose production,

decreasing intestinal absorption of glucose, and improving insulin sensitivity by increasingperipheral glucose uptake and utilization. These effects are mediated by the initial activation by

metformin of AMP-activated protein kinase (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's inhibitory effect on the production of glucose by

liver cells. Increased peripheral utilization of glucose may be due to improved insulin binding to

insulin receptors.

.

Pharmacodynamics:

Metformin is an oral antihyperglycemic agent that improves glucose tolerance in

patients with NIDDM, lowering both basal and postprandial plasma glucose. Metformin is

not chemically or pharmacologically related to any other class of oral antihyperglycemic

agents. Unlike sulfonylureas, metformin does not produce hypoglycemia in either patients

with NIDDM or healthy subjects and does not cause hyperinsulinemia. Metformin does

not affect insulin secretion.

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Pharmacokinetics:

Absorption : Absorbed over 6 hours, bioavailability is 50 to 60% under fasting

conditions. Administration with food decreases and delays absorption.. Peak action

occurs 3 hours after oral administration.

Volume of distribution : 654 L for metformin 850 mg administered as a single

dose. The volume of distribution following administration is 63-276 L.

Protein binding : Metformin is negligibly bound to plasma proteins.

Metabolism : Metformin is not metabolized.

Route of elimination : Metformin is excreted unchanged in the urine and does not

undergo hepatic metabolism nor biliary excretion. Approximately 90% of the drug is

eliminated in 24 hours in those with healthy renal function.

Clearance : 718-1552 mL/minute following single oral dose of 0.5-1.5 g. Metformin

is removed by hemodialysis at a rate of approximately 170 ml/min under good

hemodynamic conditions.

Half life: 6.2 hours. Duration of action is 8-12 hours.

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Chapter 3

Literature Review

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REVIEW OF LITERATURE

T. Raja and A. Lakshmana Rao et al.26A new simple high performance thin layer

chromatographic method for simultaneous determination of antidiabeticdrugs, metformin

hydrochloride and sitagliptin phosphate in bulk and tablet dosage form were investigated.

Chromatographic separation of the drugs were performed The method was validated forlinearity,accuracy, robustness and application for assay as per ICH guidelines. The study

shows that the developed method issimple and accurate and it would be suitable for the

simultaneous determination of metformin hydrochloride and sitagliptin phosphate in bulk drug

and pharmaceutical formulations.

Sani A. Ali, et al.25

have reported High Performance Liquid Chromatography MethodDevelopment and Validation Indicating Assay for metformin . A new simple, rapid, selective,

precise and accurate isocratic reverse phase high performance liquid chromatography assay

has been developed for the estimation of metformin Hydrochloride in tablet formulation. The

separation was achieved by using C-18 column (LichroCART 125x4mm, 5m) coupled with a

guard column of silica in mobile phase methanol : buffer (0.025M Orthophosphoric acid with

the pH adjusted to 3.0 0.1 with triethylamine) (40:60 v/v). The flow rate was 2.0ml/min and

the drug was detected using UV detector at the wavelength of 278nm. The retention time waswithin 1.7531.757 minutes. The method was validated as per ICH guidelines. The proposed

method was found to be accurate, repeatability and consistent and the method was used for

the routine analysis of formulation containing the drug without any alteration in the

chromatography conditions.

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M. S Bhatia, et al.27have developed a High Performance Liquid Chromatographic Estimation of

sitagliptin and glibenclimide from Tablets. This chromatographic method utilises a 12.5 cm

Nucleosil C18 bonded phase column with a mobile phase consisting of acetonitrile : methanol :

0.002 M phosphoric acid (20:30:50) at a flow rate of 1.2 ml/min. Results of analysis gave

standard deviation values below 1.5 and recovery study values between 98 to 103 per cent.

Thus the method is suitable for routine analysis of multicomponent formulations of these two

drugs. Ranjit Singha, et al.28stated that have developed Simultaneous Estimation of metformin and

glibenclamide by Reverse Phase - High Performance Liquid Chromatography. A new, simple,

rapid, selective, precise and accurate isocratic reverse phase high performance liquid

chromatography assay has been developed for simultaneous estimation of metformin and

glibenclamide in tablet formulations. The separation was achieved by using C-18 column

(Phenomenax, 250 x 4.6mm i.d.) coupled with a guard column of same material, in mobilephase Acetonitrile: Water: Triethylamine (25:75:1). The flow rate was 1.0 ml/min and the

separated drugs were detected using UV detector at the wavelength of 300 nm. The method

was validated as per ICH guidelines.

Jain Pritam, Chaudhari Amar 29et al.stated that Development and validation of first order

derivative UV-Spectrophotometric method for determination of Sitagliptin in bulk and in

Formulation, A simple, rapid, accurate and economical Firs torder UV-derivative

spectrophotometric method has been developed for estimation of sitagliptin from bulk and

pharmaceutical formulation. Materials and methods: The maxof sitagliptin in methanol and

water was found to be 267 nm. The same spectrum was derivatised in to first order derivative;

showed maximum amplitude of the trough at 275 nm. The drug follows linearity in the

concentration range 10-60 g/ml with correlation coefficient value 0.998. Results: The

proposed method was applied to pharmaceutical formulation and % amount of drug estimated99.19 % was found in good agreement with the label claim. The accuracy of the method was

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Sumithra M et al.30stated that A simple, sensitive and rapid reverse phase high performance

liquid chromatographic method was developed for simultaneous estimation of Sitagliptin and

Metformin. A BDS hypersil C18 column (250x4.0mm,5)was used with a mobile phase

containing a mixture of phosphate buffer (Ph-4) and Acetonitrile and in the ratio of 60:40.

The flow rate was 1.0ml/min and effluents were monitored at 260nm and eluted at 2.8min

and 2.0min respectively. Calibration curve was plotted with a range from 2-12g/mlfor

Sitagliptin and 20-120g/mlfor Metformin. The assay was validated for parameters likeaccuracy, precision, robustness and system suitability parameters. The proposed method

can be useful in the routine analysis for determination on sitagliptin and metformin.

Sumithra M et al.30stated that A simple, sensitive and rapid reverse phase high performance liquid

chromatographic method was developed for simultaneous estimation of Sitagliptin and Metformin.

A BDS hypersil C18 column (250x4.0mm,5)was used with a mobile phase containing a mixture of

phosphate buffer (Ph-4) and Acetonitrile and in the ratio of 60:40. The flow rate was 1.0ml/min and

effluents were monitored at 260nm and eluted at 2.8min and 2.0min respectively. Calibration curve was

plotted with a range from 2-12g/mlfor Sitagliptin and 20-120g/mlfor Metformin. The assay was

validated for parameters like accuracy, precision, robustness and system suitability parameters.

The proposed method can be useful in the routine analysis for determination on sitagliptin and metformin.

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Chapter 4

Objective and Plan of Work

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AIM AND SCOPE OF THE WORK

It is necessary to find the content of each drug either in pure or single, combined dosage

forms for purity testing. It is also essential to know the concentration of the drug and its

metabolites in biological fluids after taking the dosage form for treatment.

The scope of developing and validating analytical methods is to ensure suitable methods for

a particular analyte of more specific, accurate, precise and robust. The main objective for

this is to improve the conditions and parameters, which should be followed in the

development and validation.

A survey of literature reveals that good analytical methods are not available for the drugs

like ZolpidemTratarate and very few simultaneous estimation methods for Metformin

Hydrochloride and Repaglinide. Even though very few methods of estimation of above drugs

are available, many of them suffer from one disadvantage or the other, such as low

sensitivity, lack of selectivity and simplicity etc.

The existing physicochemical methods are inadequate to meet the requirements; hence it is

proposed to improve the existing methods and to develop new methods for the assay of

ZolpidemTratarate, Metformin Hydrochloride and Repaglinide in pharmaceutical dosage

forms adapting different available analytical techniques like HPLC.

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OBJECTIVE AND PLAN OF WORK

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OBJECTIVE AND PLAN OF WORK

Objective

The objective of the proposed method is to develop simple and accurate methods for the

determination ZolpidemTratarate, Metformin Hydrochloride and Repaglinide by RP-HPLC

methods in pharmaceutical dosage forms.

To develop simple, greater sensitive and faster elution method by RP-HPLC to reduce time

of analysis based on the literature survey made. Main objective is to reduce the retention time of

Zolpidem Tartrate, Metformin Hydrochloride and Repaglinide.

Plan of Work

The plan of the proposed work includes the following steps:

To undertake solubility studies and analytical studies of ZolpidemTratarate, Metformin

Hydrochloride and Repaglinide to develop initial UV and chromatographic conditions.

Setting up of initial chromatographic conditions for the assay of ZolpidemTratarate, Metformin

Hydrochloride and Repaglinide in pure and pharmaceutical dosage forms.

Optimization of initial chromatographic conditions.

Finally validation of developed method in terms of accuracy, precision, linearity, robustness and

system suitability results will be validated statistically according to ICH guidelines.

To check the specificity and stability of the method forced degradation studies has to beconducted.

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19

Chapter 5

Experimental

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CHEMICALS AND REAGENTS USEDSL NO: CHEMICALS/STANDARDS ANDREAGENTS GRADE COMPANY

1 Ammonium Acetate AR Thomas Baker2 Glacial Acetic Acid AR Merck3 Potassium di-hydrogen Ortho phosphate AR Finar4 Ortho phosphoric acid AR Finar5 Methanol HPLC Merck6 Acetonitrile HPLC Merck7 Water HPLC LobaChemi8 Conc. Hydrochloric acid NA Merck9 Sodium hydroxide pellets NA SD fine chemicals10 Hydrogen Peroxide NA Merck

11 Zolpidem Tartrate (API) NA12 Metformin Hydrochloride (API) NA13 Repaglinide (API) NA

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INSTRUMENTS AND EQUIPMENTS USED

SL NO INSTRUMENTS AND EQUIPMENTS1 HPLC (Company: WATERS, Model: Aliance 2695, Detector 2487 with Empower 2

software),

2 UV Spectrophotometer (Company: Labindia, Model: UV-3000+ with UV win 5software).

3 Electroinc Balance (Company: Ascoset, Model:ER200A)4 Sonicator (Company: Enertech, Model:SE60US)5 pH Meter (Company: ADWA, Model:AD102U)6 Heating Mantle (Company: Bio Technics India, Model: BTI)7 Thermal oven (Company: Nargang)

8 Filter Paper 0.45 microns (Company: Milli Pore)21

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DEVELOPMENT AND VALIDATION OFNEW RP-HPLC METHOD FOR THE

DETERMINATION OF ZOLPIDEMTARTRATE IN PURE ANDPHARMACEUTICAL FORMULATIONS

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METHODDEVELOPMENT

Optimisation of UV conditions:

Initially method development work was started by taking UV-visible spectra from 400-200 nm

of standard solutions (20ppm). By observing the spectra of standard solution max 305, 300

and 295 nm were taken for trials to develop UV method.

Different mobile phases in different compositions, with different columns the Trials were

made

Mobile phases used:

Phosphate buffer (pH 5.5) : Methanol-70:30,60:40

Acetate buffer (pH4.5) : Methanol-50:50,40:60

Acetate buffer (pH4.5) : Acetonitrile-50:50

Methanol : Water-50:50

Columns used: Waters C18 (754.5mm,3.5)

Waters C18 (1504.5mm,5) Xterra

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UV Absorption spectrum of Zolpidem Tartrate (20ppm) in

Ammonium Acetate Buffer : Methanol (40:60)

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Based on pka of the drug that is 6 16

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Based on pka of the drug that is 6.16

for Zolpidem Tartrate initially the

buffer pH was adjusted to 5.5 using the

potassiumhydroxide

TRIAL NO 1:Buffer preparation:Potassium di hydrogen ortho

phosphate pH adjusted to 5.5

using potassium hydroxide

Mobile phase:Buffer : Methanol

(70:30) were mixed and sonicated

to degas.

Diluent:

Buffer :Methanol (70:30)

Chromatographic conditions:

Flow rate : 0.8 ml/min

Column : WatersC18 (754.5mm,3.5)

Detector wavelength : 305nm

Column temp : Ambient

I nj ection volume : 20l

Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry is not good due to this the peak

area is more. And the RT is about 5.3 .CONCLUSION:

Due to the lack of improper ionization the peak is

asymmetric.

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With out changing the pH and by

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changing the mobile phase composition

by increasing the organic phase the

second trial was conducted. To reduce

the RT. TRIAL NO 2:Buffer preparation:

Potassium di hydrogen ortho

phosphate pH adjusted to 5.5 using

potassium hydroxide

Mobile phase: Buffer : Methanol

(60:40) were mixed and sonicatedto degas.

Diluent:




Column : Waters C18

(754.5mm,3.5)




Run time : 10minSample conc : 20ppm

RESULT:

Here the peak symmetry is better than the last trial, but

the plate count has been drastically reduced withincreased tailing factor. And the RT is about 1.47, but

the voide volume is 1, so to increase the RT above 2

min the third trial is done.

CONCLUSION:

Due to increase in organic phase the RT has been

reduced. 26

Based on pka of the drug that is

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Based on pka of the drug that is

6.16 for Zolpidem Tartrate the

buffer pH was adjusted to 4.6 using

the Glacial Acetic Acid

TRIAL NO 3:Buffer preparation:Ammonium Acetate buffer pH

adjusted to 4.6 using Glacial Acetic

Acid



to degas.

Diluent:




Column : Waters C18

(1504.5mm,5) Xterra





RESULT:

Change in the Buffer with pH and Column with length

and particle size gives a very good Symmetrical Peakwith good Plate Count.

CONCLUSION:

Due to proper ionization good peak is obtained. But to

reduce the RT the fourth trial has been done.

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changing the mobile phase composition

by increasing the organic phase the

fourth trial was conducted. To reduce

the RT. TRIAL NO 4:Buffer preparation:

Ammonium Acetate buffer pH


Acid



Diluent:




Column : Waters C18

(1504.5mm,5) Xterra





RESULT:

The RT has been reduced to 3.268 with good symmetry.

CONCLUSION:As to increase the plate count slightly the fifth trial has

been done.

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B h i th i h

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By changing the organic phase

from methanol to acetonitrile the

fifth trial has been carried out.

TRIAL NO 5:Buffer preparation:



Acid

Mobile phase: Buffer : Acetonitrile


to degas.Diluent:

Buffer : Methanol (50:50)



Column : Waters C18(1504.5mm,5) Xterra




Run time : 10min

Sample conc : 20ppm

RESULT:

The RT has been reduced to 3.053 with good symmetry

and reduced plate count.CONCLUSION:

As to see the method also to economical the acetonitrile

is replaced with methanol again.

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By changing the Buffer with

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By changing the Buffer with

water and acetonitrile with

methanol the sixth trial has

been carried out.

TRIAL NO 6:Mobile phase: Water : Methanol


to degas.

Diluent:

Buffer : Methanol (50:50)Chromatographic conditions:


Column : Waters C18

(1504.5mm,5) Xterra




Run time : 10min

Sample conc : 20ppm

RESULT:

The RT has been increased to 4.170 with good symmetry

and reduced plate count.CONCLUSION:

As to decrease the RT seventh trial has been done with

the conditions of Trial 3 with slight changes.

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Changes to the Trial 3 by

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Changes to the Trial 3 by

adjusting the pH to 4.5 and and

max to 300 nm the seventh

trial has been carried out.TRIAL NO 7:Buffer preparation:



Acid



Diluent:




Column : Waters C18

(1504.5mm,5) Xterra

Detector wavelength : 300 nm



Run time : 6 minSample conc : 20ppm

RESULT:

The RT has got to 3.1 with good symmetry of peak and

good plate count.CONCLUSION:

As all the system suitability parameters are well with in

the limits. This parameters are fixed to be final for the

method development of Zolpidem Tartrate.

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OPTAMIZEDMETHODPARAMETERS

PARAMETERS CONDITIONSColumn(Stationary Phase) Symmetry C18 (4.6 x 150mm, 5 m, Make:

XTerra) or equivalent

Mobile PhaseAmmonium Acetate Buffer(4.5pH):Methanol

(60:40) pH adjusted with glacial acetic acid

Flow rate (ml/min) 0.8

Run time (min) 6

Column temperature(C) Ambient

Volume of injection loop (l) 20

Detection wavelength (nm) 300

Drug RT (min) 3.14 32

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METHODPREPARATION OF MOBILE PHASE

Weigh 7.0 grams of Ammonium Acetate in to a 1000ml

beaker, dissolve and diluted to 1000ml with HPLC water

Adjusted the pH to 4.5 with Glacial Acetic acid

Mix a mixture of above buffer 400ml (40%) and 600ml of Methanol HPLC (60%) and degas in ultrasonic

water bath for 5 minutes. Filter through 0.45 filter

under vacuum filtration.

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PREPARATION OF STOCK SOLUTION & DILUTIONS

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PREPARATION OF STOCK SOLUTION & DILUTIONSOF DIFFERENT CONCENTRATIONS

10mg of Zolpidem Tartrate pure drug was weighed and

dissolved in the mobile phase with 7 ml and sonicated

for 15 min

And the volume was made up to 10 ml in 10ml

volumetric flask to get the stock solution (1000g/ml).

From this further aliquots of 0.2-0.6 ml from the

stock solution were taken and diluted with the mobile

phase to get solutions in the concentration range of

20g to 60g/ml.34

PREPARATION OF SAMPLE SOLUTION

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PREPARATION OF SAMPLE SOLUTIONFive tablets containing the drug were taken and powdered. The powder

equivalent to 10mg of the active ingredient was accurately weighed and

taken in a 10ml volumetric flask and mobile phase was added to make up to

volume.

The volumetric flask was sonicated for 30 minutes to effect complete

dissolution of drug and the solution was made up to volume with mobile

phase and filtered through Whatman filter paper (0.45 m) made up of

cellulose nitrate.

Aliquots solutions were prepared by taking 0.3ml of the filtered solution into

10ml volumetric flasks, separately and made up to volume with mobile phase to

yield concentrations of drug in range of linearity previously described.

The amount of drug present in each

pharmaceutical formulation was calculated by

using the standard calibration curves

(concentration in g/ml was taken on X-axis and

peak area on Y-axis) 35

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36

VALIDATION OF ZOLPIDEM TARTRATE BY RP-HPLC

The following experimental design is drawn in order to prove the test method is

capable to yield consistent, reliable and reproducible results within the pre-determinedacceptance limits.

Acceptance criteria for above validation parameters are specified in individual experimental

design.

Observations and results are recorded in individual method validation data sheets.

Summarize the findings of the method validation and draw interference.

Based on the interpretation of the results in method validation, draw the conclusion.

The following parameters have been validated.

Linearity

Precision

Accuracy

Robustness

System Suitability

Specificity

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37

METHOD VALIDATION

LINEARITY

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38

LINEARITY

A Series of solutions were prepared using Zolpidem tartrate working standard at

concentration levels from 20ppm to 60ppm of target concentration. Measure the peak area

response of the solution.

ACCEPTANCE CRITERIA

Correlation Coefficient should be not less than 0.990.

OBSERVATION

The correlation coefficient was found to be 0.999, slope is 47336and intercept is 13206

respectively. The results were shown in table no: 7 and 6

CONCLUSION

Response of Zolpidem Tartrate were linear over the concentration range of about 20 ppm

to 60 ppm.

PROCEDURE FOR CALIBRATION CURVE

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Prior to injection of the drug solutions, the column was equilibrated

with the mobile phase flowing through the systems.

The chromatographic separation was achieved using a mobile phase

consisting of Ammonium Acetate Buffer:Methanol (40:60 v/v) at a

flow rate of 0.8ml/min. The eluent was monitored using UV

detection at a wavelength of 300 nm.

an injection volume of 20l of each of standard and sample solutions were injectedinto the HPLC system to get the chromatograms

A graph was plotted by taking concentration of the drug on x-axis and peak area

on y-axis 39

PRECISION

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40

PRECISION

a) System precision: Standard solution was prepared as per the test method and injected five times as per the

test procedure.

ACCEPTANCE CRITERIA

The % Relative standard deviation of Peak area of Zolpidem tartrate from the five replicate injections

should be not more than 2.0

OBSERVATION

Test results of Zolpidem Tratrate were shown that the %RSD of peak areas, and retention times are within

limits. The results were shown in table no: 10

b)Method precision

Prepared six sample preparations of Zolpidem Tratrate as per test method and injected each solution.

ACCEPTANCE CRITERIA

The % relative standard deviation of %Assay from six test preparations of Zolpidem Tratrate should

be not more than 2.0%.

OBSERVATION

Test results for Zolpidem Tratrate are showing that the %RSD of Assay results are within limits. The

results were shown in table no: 11

CONCLUSION Hence, test method was precise.

ACCURACY

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41

ACCURACY

Prepared solutions in triplicate by spiking Zolpidem Tartrate drug substance on

Formulation at of 40 ppm of target concentration as per the test method and analyzed.

ACCEPTANCE CRITERIA

The mean % recovery of the Zolpidem Tartrate at each level should be not less than

98.0 and not more than 102.0

OBSERVATION

The Mean %Recovery results were within limits. The results were shown in table no: 12

and 13

CONCLUSION

It is concluded that the test method has an acceptable level of accuracy from target

concentration.

ROBUSTNESS

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42

ROBUSTNESS

Chromatographic conditions variation

To demonstrate the robustness of the method, prepared standard solution as per test

method and injected at different variable conditions like using different flow rates and mobile

phase compositions. Peak areas and Retention times were compared with that of method

precision.

ACCEPTANCE CRITERIA

The Peak areas and Retention times should pass as per the test method at variable

conditions.

OBSERVATION

From the observation it was found that the Peak areas and Retention times were within

limit at all variable conditions. The results were shown in table no: 14

CONCLUSION

Hence, it was concluded that the test method was Robust.

LIMIT OF DETECTION

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LIMITOFDETECTIONPreparation of 40g/ml solution:

Accurately weigh and transfer 10mg of Zolipidem tartarate Working standard into a 10 mL Volumetric

flasks add about 7 mL of Diluent and sonicate to dissolve it completely and make volume up to the

mark with the same solvent. (Stock solution)Further pipette 0.4 ml of the above stock solution into a 10ml volumetric flask and dilute up to the

mark with diluent. Mix well and filter through 0.45m filter.

Preparation of 0.09% solution At Specification level (0.036g/ml solution):

Pipette 1mL of 10g/ml solution into a 10 ml of volumetric flask and dilute up to the mark with

diluent.

Further pipette 0.09mL of above diluted solution into a 10 ml of volumetric flask and dilute up to the

mark with diluent.

Calculation of S/N Ratio:

Average Baseline Noise obtained from Blank : 43V

Signal Obtained from LOD solution (0.09% of target assay concentration): 128 V

S/N = 128/43 = 3.0943

LIMIT OF QUANTIFICATION

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LIMIT OF QUANTIFICATION

Preparation of 40g/ml solution:





Preparation of 0.3% solution At Specification level (0.12g/ml solution):

Pipette 1mL of 10g/ml solution into a 10 ml of volumetric flask and dilute up to the mark with

diluent.


mark with diluent.




S/N = 419/43 = 9.7444

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Chapter 6

Results and Discussion

45

TYPICAL CHROMATOGRAM OF MOBILE PHASE

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G

YPICAL CHROMATOGRAM OF ZOLPIDEM TARTRATE STANDARD (40PPM)

46

LINEARITY RANGE OF ZOLPIDEM TARTRATE

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S.No Linearity Level Concentration Area

1 I 20g/ml 926213

2 II 30g/ml1402091

3 III 40g/ml1862724

4 IV 50g/ml2352834

5 V 60g/ml2844035

Correlation Coefficient 0.999

Slope (a) 47336

Intercept (b)13206

LINEARITY RANGE OF ZOLPIDEM TARTRATE

47

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y = 47336x - 13206

R = 0.999

0

500000

1000000

1500000

2000000

2500000

3000000

0 10 20 30 40 50 60

PeakArea

Cocentration (g/ml)

CALIBRATION CURVE OF ZOLPIDEM

TARTRATE

48

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ASSAY

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30 ppm 50

TYPICAL CHROMATOGRAMS OF MARKETEDFORMULATION

(30ppm)

SYSTEM PRECISION

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SYSTEM PRECISIONINTRA DAY INTER DAYInjection Area

Injection-11858787

Injection-21851176

Injection-3

1851848

Injection-41851874

Injection-51851123

Average1852962

Standard Deviation3275.6

%RSD0.18

Injection Area

Injection-11892556

Injection-21898440

Injection-31896326

Injection-41896644

Injection-51899562

Average1896705


%RSD 0.14 51

METHOD PRECISION

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INTRA DAYInjection Area

Injection-1 1910172

Injection-21912204

Injection-31918943

Injection-41920794

Injection-51928479

Injection-61932712

Average1920551


%RSD 0.46

INTER DAYInjection Area

Injection-1 1882144

Injection-21881694

Injection-31880161

Injection-41882126

Injection-51886945

Injection-61879498

Average1882095


%RSD 0.14 52

ACCURACY STUDIES

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Sample ID

Concentration (g/ml)%Recovery

Statistical

AnalysisPure drug Formulation

S1: 50 % 20 40 100.84 Mean = 100.87

SD = 0.1868

%RSD = 0.185

S2: 50 % 20 40 100.70

S3: 50 % 20 40 101.07

S4 : 100 % 40 40 98.47 Mean = 99.65

SD = 0.3175

%RSD = 0.318

S5: 100 % 40 40 99.02

S6: 100 % 40 40 98.47

S7 : 150 % 60 40 99.09 Mean = 98.90

SD = 0.2753

%RSD = 0.278

S8: 150 % 60 40 98.59

S9: 150 %

60

40

99.04 53

ROBUSTNESS

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Robustness of the method reflects the reliability of an analysis with respect to deliberate

variations in the method parameters. The results obtained with changes in the parameters on a

40g/ml solution are as shown

Sl.

No.

Parameter Condition Peak area

Statistical

analysis

Retention

time

Statistical

analysis

1Flow rate

(ml/min)

0.7 1942204

Mean= 1921616

SD= 18550

%RSD= 0.965

3.156

Mean= 3.128

SD= 0.032

%RSD= 1.0230.8 1916444 3.137

0.9 1906202 3.092

2Mobile phase

ratio

45:55 1975874

Mean= 1950589

SD= 30689

%RSD= 1.573

3.159Mean= 3.130

SD= 0.031

%RSD=

0.990

40:60 1916444 3.137

35:65 1959450 3.09654

SYSTEM SUITABILITY PARAMETERS

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System suitability parameters can be defined as tests to ensure that the method can

generate results of acceptable accuracy and precision.

Parameters Obtained Values

Theoretical plates (N) 2405.28

Asymmetry (As) 1.65

LOD (g/ml) 0.036

LOQ (g/ml) 0.12

55

A t C it i

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Acceptance Criteria

S/N Ratio value shall be 3 for LOD solution.

56

AcceptanceCriteria

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p

S/N Ratio value shall be 10 for LOD solution.

57

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SPECIFICITY OF ZOLPIDEM TARTRATE

The specificity of the method was demonstrated through forced degradation studies

conducted on the sample using acid, alkaline, oxidative and thermal degradations to find

whether the degradation products could be clearly separated from the pure drug peak.

Typical chromatogram of blank58

TYPICAL CHROMATOGRAM OF UNTREATED

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GSAMPLE (40g/ml)

S.NORetentio

n TimeArea Area % Height

Height

%

1 3.203 1959609 100.000 174974 100.00

Totals

1959609 100.000 174974 100.00059

ACID DEGRADATION

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About 10mg of the pure drug was accurately weighed and transferred to 10ml

volumetric flask. 3ml of 0.1N HCl was added and kept for 24 hour and 12 hours

of heating in water bath at 70C.

It was then cooled, neutralized using 0.1N NaOH and the volume was made up to 10mlwith mobile phase.

Then from this 40g/ml solution was prepared and 20l of it

was injected into the HPLC system to obtain the

chromatogram.

60

TYPICAL CHROMATOGRAM OF A 40g/ml

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SOLUTION (ACIDIC DEGRADATION)

S.NORetention

TimeArea Area % Height Height %

1 2.616 102738 5.25 9046 5.17

2 3.205 1856871 94.75 165928 94.83

Totals

1959609 100.000 174974 100.00061

ALKALINE DEGRADATION

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About 10mg of the pure drug was accurately weighed and transferred to 10ml volumetric

flask. 3ml of 0.1N NaOH was added and kept for 24 hour and 12 hours of heating in

water bath at 70C.

It was then cooled, neutralized using 0.1N Hcl and the volume was made up to 10mlwith mobile phase.



chromatogram.

62


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gSOLUTION (ALKALINE DEGRADATION)

S.NORetention


1 2.694 118551 6.05 12265 7.01

2 3.265 1841058 93.95 162709 92.99

Totals

1959609 100.000 174974 100.00063

OXIDATIVE DEGRADATION

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About 10mg of the drug was accurately weighed and transferred to a 10ml volumetric

flask and 3 ml of 3%w/v of hydrogen peroxide solution was added and kept for 24 hour

and 12 hours of heating in water bath at 70C.

It was then cooled and made up to 10ml with mobile phase.



chromatogram.

64

TYPICAL CHROMATOGRAM OF PURE 3% W/V

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TYPICAL CHROMATOGRAM OF PURE 3% W/VHYDROGEN PEROXIDE SOLUTION

65


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SOLUTION (OXIDATIVE DEGRADATION)

S.NORetention

Time

Area Area % Height Height %

1 2.143 56721 0 5058 0

2 3.273 1764441 90.04 16132 90.78

3 10.270 195168 9.96 158842 9.22

Totals

1959609 100.000 174974 100.00066

THERMAL DEGRADATION

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About 10mg of the pure drug was accurately weighed and transferred to a 10ml

volumetric flask and kept in the hot air oven for 12 hours at a temperature of 70C and

diluted with mobile phase to 10ml.

From this 0.4 ml was diluted to 10 ml so as to obtain a concentration of 40g/ml.

It was then allowed to cool and then 20L of this was injected

into the HPLC system.

67

TYPICAL CHROMATOGRAM OF A 40G/MLSOLUTION (THERMAL DEGRADATION)

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SOLUTION (THERMAL DEGRADATION)

S.NORetention


1 3.202 1946063 99.03 173925 99.40

2 11.01 13546 0.97 1049 0.60

Totals

1959609 100.000 174974 100.00068

The % degradation was calculated on the basis of the response obtained in case of theuntreated pure drug sample under similar experimental conditions

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untreated pure drug sample, under similar experimental conditions.

RESULT OF THE FORCED DEGRADATION STUDY.

Conditions

appliedPeak area

% drug

recovered

RT of the

analyte (min)

RT of major

degradants (min)

Untreated 1959609 100 3.203 -

Acid degradation 1856871 94.75 3.205 2.616

Alkaline

degradation1841058 93.95 3.265 2.694

Oxidative

degradation1764441 90.04 3.273 10.270

Thermal

degradation

1946063 99.03 3.202 11.01 69

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DEVELOPMENT AND VALIDATION OFNEW RP-HPLC METHOD FOR THE

SIMULTANEOUS ESTIMATION OFMETFORMIN HYDROCHLORIDEANDREPAGINIDE IN PURE AND

PHARMACEUTICAL FORMULATIONS

71

METHODDEVELOPMENT

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M H PM

Optimisation of UV conditions:

Initially method development work was started by taking UV-Visible spectra from 400-200 nm

of standard solutions (20ppm). By observing the spectra of standard solution max of

Metformin Hydrochloride and Repaglinide were found to be 240 and 246 nm. Initially 240 and

255 nm were taken for trials to develop UV method.

Different mobile phases in different compositions, trials were made.

Mobile phases used:

Potassium di-hydrogen ortho phosphate pH 3.0: Methanol (50:50 v/v)

Potassium di-hydrogen ortho phosphate pH 2.5: Acetonitrile (35:65, 50:50 v/v)

Potassium di-hydrogen ortho phosphate pH 2.3: Acetonitrile (35:65, 60:40 v/v)

Potassium di-hydrogen ortho phosphate pH 2.2: Acetonitrile (60:40v/v)

Columns used: Waters XBridge C18 (4.6 x 150mm, 3.5 m

72

UV spectrum of Metformin Hydrochloride and Repaglinide (20 ppm) in

the mobile phase ( = 240nm and 246nm) (20ppm) in Potassium di

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the mobile phase (max= 240nm and 246nm) (20ppm) in Potassium di-

hydrogen ortho phosphate pH 2.2 and acetonitrile (60:40v/v)

73

MetforminHydrochloride20ppm Repaglinide 20ppm

Metformin Hydrochloride and Repaglinide (20ppm)

Metformin Hydrochloride

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TRIAL NO 1:Buffer preparation:Potassium di hydrogen ortho

phosphate pH adjusted to 3.0using ortho phosphoric acid



to degas.

Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry is not good due to this the plate

count is very less.

CONCLUSION:


asymmetric.

74

Metformin hydrochloride and

Repaglinide

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TRIAL NO 2:Buffer preparation:

Potassium di hydrogen orthophosphate pH adjusted to 3.0

using ortho phosphoric acid

Mobile phase: Buffer :

Acetonitrile (40:60) were mixed

and sonicated to degas.

Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)Detector wavelength : 255nm



Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry for Repaglinide is not good. due

to this the plate count is very less for both Metformin

hydrochloride and Repaglinide.

CONCLUSION:


asymmetric for Repaglinide.

75

Repaglinide

Metformin Hydrochloride and

R i id

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TRIAL NO 3Buffer preparation:

Potassium di hydrogen ortho

phosphate pH adjusted to 3.0




and sonicated to degas.Diluent:




Column :C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry for Repaglinide is not good. due

to this the plate count is very less for both Metformin

hydrochloride and Repaglinide.

CONCLUSION:

After changing the max from 255-240 nm also there is

no change.

76

Repaginide

Repaglinide

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Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry for Repaglinide is good. Tailing

factor and Plate count are also with in the limits.

CONCLUSION:

This can be used as final method.

77


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Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry for Metformin Hydrochloride

but the Tailing factor is more here

CONCLUSION:

But the plate count is not with in the limits so this

composition can not be used.

78


R li id

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Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here after changing the mobile phase ration the

Repaglinide peak symmetry is not good and the

metformin Hydrochloride plate count is not good

CONCLUSION:

Due to less plate count of Metformin Hydrochloride and

assymetric peak of Repaglinide this composition can not

be used. 79

Repaglinide


Repaglinide

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Diluent:




Column : C18

(1504.5mm,3.5) (Xbridge)




Run time : 10min

Sample conc : 20ppm

RESULT:

Here the peak symmetry for Repaglinide is not good and

the retention time has been increased to 7.3 min.

CONCLUSION:

Due to more retention time of Repaglinide it is not

selected.

80

Repaglinide

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OPTAMIZEDMETHODPARAMETERS

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82

PARAMETERS CONDITIONS

Column(Stationary Phase)

XBridge C18 (4.6 x 150mm, 3.5 m, Make: Waters) or

equivalent

Mobile PhasePotassium dihyrogenortho phosphate (2.2 pH) : Acetonitrile

(35:65%v/v) pH adjusted with ortho phosphoric acid.

Flow rate (ml/min) 0.6

Run time (min) 7

Column temperature(C) Ambient

Volume of injection loop (l) 10

Detection wavelength (nm) 240

Drug RT (min)Metformin Hydrochloride Repaginide

2.517 3.825 82

METHOD

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PREPARATION OF MOBILE PHASEWeigh 7.0 grams of Potassium di hyrogen ortho

phosphatein to a 1000ml beaker, dissolve and diluted to

1000ml with HPLC water

Adjusted the pH to 2.2 with Ortho phosphoric acid

Mix a mixture of above buffer 350ml (35%) and 750ml of Acetonitril HPLC (75%) and degas in

ultrasonic water bath for 5 minutes. Filter through

0.45 filter under vacuum filtration.

83

PREPARATION OF STOCK SOLUTION & DILUTIONSOF DIFFERENT CONCENTRATIONS

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OF FF R N CONC N RA ONS

10mg of Metformin Hydrochloride and Repaglinide

pure drug was weighed and dissolved in the mobile

phase with 7 ml and sonicated for 15 min

And the volume was made up to 10 ml in 10ml volumetric

flask to get the stock solution (1000 g/ml) of bothMetformin Hydrochloride and Repaglinide..

From this further aliquots of 0.05-0.5 ml from

the stock solution were taken and diluted with the

mobile phase to get solutions in the concentration

range of 5g to 50g/ml.84

PREPARATION OF SAMPLE SOLUTIONT t t bl t t i i th d t k d d d Th d

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Twenty tablets containing the drug were taken and powdered. The powder

equivalent to 2500mg and 10mg of the active ingredient of Metformin

Hydrochloride and Repaglinide was accurately weighed and taken in a

100ml volumetric flask and mobile phase was added to make up to volume.

The volumetric flask was sonicated for 45 minutes to effect complete

dissolution of drug and the solution was made up to volume with mobile

phase and filtered through Whatman filter paper (0.45 m) made up ofcellulose nitrate.

Aliquots solutions were prepared by taking 2.0 ml of the filtered solution into10ml volumetric flasks, separately and made up to volume with mobile phase to

yield concentrations of drug in range of linearity previously described for

Repaglinide.

85

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86

The amount of drug present in each

pharmaceutical formulation was calculated by

using the standard calibration curves

(concentration in g/ml was taken on X-axis andpeak area on Y-axis)

Aliquots solutions were prepared by taking 2.0 ml of the filtered solution

into 10ml volumetric flasks, separately and made up to volume with mobile

phase. From this take 0.2 ml in to 10 ml volumetric flask and made up thevolume with mobile phase to yield concentrations of drug in range of linearity

previously described for Metformin Hydrochoride.

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LINEARITY

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89

A Series of solutions were prepared using Metformin Hydrochloride and Repaglinide

working standard at concentration levels from 5ppm to 50ppm of target concentration.

Measure the peak area response of the solution.

ACCEPTANCE CRITERIA

Correlation Coefficient should be not less than 0.990.

OBSERVATION

The correlation coefficient was found to be 0.9999 for both drugs Metformin

Hydrochloride and Repaglinide , slope is 73266 and 24865, intercept is 1485.3 and 1922.3

for Metformin Hydrochloride and Repaglinide respectively. The results were shown in table

no: 7 and 6

CONCLUSION

Response of Metformin Hydrochloride and Repaglinide were linear over the

concentration range of about 5 ppm to 50 ppm.


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Prior to injection of the drug solutions, the column was equilibrated

with the mobile phase flowing through the systems.

The chromatographic separation was achieved using a mobile phase

consisting of Potassium dihyrogenortho phosphate (2.2 pH) :

Acetonitrile (35:65%v/v) at a flow rate of 0.6ml/min. The eluent

was monitored using UV detection at a wavelength of 240 nm.

an injection volume of 20l of each of standard and sample solutions were injected

into the HPLC system to get the chromatograms

A graph was plotted by taking concentration of the drug on x-axis and peak area

on y-axis 90

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ACCURACY

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92

Prepared solutions in triplicate by spiking Metformin Hydrochloride and Reapaglinide

drug substance on Formulation at of 20 ppm of target concentration as per the test method

and analyzed.

ACCEPTANCE CRITERIA

The mean % recovery of the Metformin Hydrochloride and Reapaglinide drug at each

level should be not less than 98.0 and not more than 102.0

OBSERVATION

The Mean %Recovery results were within limits. The results were shown in table no: 12

and 13

CONCLUSION

It is concluded that the test method has an acceptable level of accuracy from target

concentration.

ROBUSTNESS

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93

Chromatographic conditions variation

To demonstrate the robustness of the method, prepared standard solution as per test

method and injected at different variable conditions like using different flow rates and mobile

phase compositions. Peak areas and Retention times were compared with that of method

precision.

ACCEPTANCE CRITERIA

The Peak areas and Retention times should pass as per the test method at variable

conditions.

OBSERVATION

From the observation it was found that the Peak areas and Retention times were within

limit at all variable conditions. The results were shown in table no: 14

CONCLUSION

Hence, it was concluded that the test method was Robust.

LIMIT OF DETECTION OF METFORMIN HYDROCHLORIDE

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mark with the same solvent. (Stock solution)

Further pipette 0.2 ml of the above stock solution into a 10ml volumetric flask and dilute up to the


Preparation of 0.09% solution At Specification level (0.018g/ml solution):Pipette 1mL of 10g/ml solution into a 10 ml of volumetric flask and dilute up to the mark with

diluent.


mark with diluent.




S/N = 124/42 = 2.98594

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LIMIT OF DETECTION OF REPAGLINIDE

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mark with the same solvent. (Stock solution)

Further pipette 0.2 ml of the above stock solution into a 10ml volumetric flask and dilute up to the


Preparation of 0.25% solution At Specification level (0.05 g/ml solution):Pipette 1mL of 10g/ml solution into a 10 ml of volumetric flask and dilute up to the mark with

diluent.


mark with diluent.




S/N = 128/42 = 3.0496

LIMIT OF QUANTIFICATION OF REPAGLINIDE

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Preparation of 0.95% solution At Specification level (0.19 g/ml solution):Pipette 1mL of 10g/ml solution into a 10 ml of volumetric flask and dilute up to the mark with

diluent.


mark with diluent.




S/N = 419/42 = 10.0497

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Results and Discussion

98

TYPICAL CHROMATOGRAM OF MOBILE PHASE

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TYPICAL CHROMATOGRAM OF METFORMIN HYDROCHLORIDE AND REPAGLINIDE STANDARD (20PPM)

99

Area of Metformin

LINEARITY RANGE OF METFORMIN HYDROCHLORIDE ANDREPAGLINIDE

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S.No Linearity Level ConcentrationArea of Metformin

HydrochlorideArea of Repaglinide

1 I 5g/ml362120 120239

2 II 10g/ml726426 242570

3 III 20g/ml1492439 498282

4 IV 30g/ml2183380 749949

5 V 40g/ml2951238 988812

6 VI 50g/ml

3650958 1240721

Correlation Coefficient 0.9999 0.9999

Slope (a) 73266 24865

Intercept (b) 1485.3 1922.3 100

3500000

4000000

CALIBRATION CURVE OF METFORMIN HYDROCHLORIDE AT240 nm

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101

y = 73266x + 1485.3

R = 0.9999

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

0 10 20 30 40 50 60

Peak

Area

Concentration (g/ml)

y = 24865x - 1922.3

R = 0.9999

0

200000

400000

600000

800000

1000000

1200000

1400000

0 10 20 30 40 50 60

PeakA

rea

Concentartion (g/ml)

CALIBRATION CURVE OF REPAGLINIDE AT 240 nm

ANALYSIS OF COMMERCIAL FORMULATION

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ANALYSIS OF COMMERCIAL FORMULATION

Formulation Labeled Amount (mg)% Recovery by

proposed method%RSD

PRANDIMET

(NOVO

NORDISK)

Metformin

HclRepaglinide

Metformin

HclRepaglinide

Metformin

HclRepaglinide

500 2.0 98.44 0.0 98.010.04 0.01 0.02

102

ASSAYTYPICAL CHROMATOGRAMS OF MARKETED FORMULATION(20ppm)

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103

( pp )


Repaglinide

SYSTEM PRECISIONINTRA DAY INTER DAY

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Injection

Area of

Metformin

Hcl

Area of

Repaglinide

Injection-11497177 498308

Injection-21501852 499086

Injection-3

1496466 497665

Injection-41494920 496151

Injection-51501919 499217

Average1498466 498084

Standard

Deviation

3225.8 1249.7

%RSD

0.21 0.25

Injection

Area of

Metformin

Hcl

Area of

Repaglinide

Injection-11489301 494736

Injection-21492080 497000

Injection-3

1489889 496238

Injection-41493862 497617

Injection-51484376 494658

Average1489901 496949

Standard

Deviation

3582.5 1328.3

%RSD

0.24 0.26 104

METHOD PRECISIONNTRA DAYArea of

Area of

INTER DAYArea of

Area of

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Injection Metformin

Hcl

Area of

Repaglinide

Injection-1

1466304 486236

Injection-21472460 486345

Injection-31468940 486289

Injection-4 1470674 486446

Injection-51476102 486498

Injection-61480496 486309

Average1472496 486353

Standard

Deviation

5124.6 99.45

%RSD

0.34 0.02

Injection Metformin

Hcl

Area of

Repaglinide

Injection-1

1475716 486513

Injection-21477408 486329

Injection-31480323 486390

Injection-4 1486710 486358

Injection-51477798 486574

Injection-61477554 486299

Average1479251 486410

Standard

Deviation

3941.3 109.0

%RSD

0.26 0.02105

ACCURACY STUDIES FOR METFORMIN HYDROCHLORIDE

Concentration (g/ml) Statistical

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Sample ID

Co ce t at o ( g/ )

%RecoveryStatistical


S1: 50 % 10 20 98.96 Mean = 99.10

SD = 0.135

%RSD = 0.13

S2: 50 % 10 20 99.23

3S3: 50 % 10 20 99.11

S4 : 100 % 20 20 97.57 Mean = 97.58

SD = 0.032

%RSD = 0.03

S5: 100 % 20 20 97.63

S6: 100 % 20 20 97.58

S7 : 150 % 30 20 98.81 Mean = 98.80

SD = 0.005

%RSD = 0.005

S8: 150 % 30 20 98.80

S9: 150 % 30 20 98.81106

ACCURACY STUDIES FOR REPAGLINIDE

Concentration (g/ml) Statistical

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Sample ID

( g )

%RecoveryStatistical


S1: 50 % 10 20 98.10 Mean = 98.05

SD = 0.058

%RSD = 0.05

S2: 50 % 10 20 97.99

3S3: 50 % 10 20 98.08

S4 : 100 % 20 20 98.04 Mean = 98.03

SD = 0.045

%RSD = 0.04

S5: 100 % 20 20 97.98

S6: 100 % 20 20 98.07

S7 : 150 % 30 20 98.02 Mean = 98.03

SD = 0.015

%RSD = 0.01

S8: 150 % 30 20 98.05

S9: 150 % 30 20 98.04107

ROBUSTNESS FOR METFORMIN HYDROCHLORIDE Robustness of the method reflects the reliability of an analysis with respect to deliberate

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variations in the method parameters. The results obtained with changes in the parameters on a

40g/ml solution are as shown

Sl.

No.


Statistical

analysis

Retention

time

Statistical

analysis

1Flow rate

(ml/min)

0.5 1521762

Mean= 1493710SD= 26354

%RSD= 1.76

2.58

Mean= 2.53SD= 0.045

%RSD= 1.770.6 1489901 2.52

0.7 1469468 2.49

2Mobile phase

ratio

37:63

1448116

Mean= 1476634

SD= 24717

%RSD= 1.673

2.52

Mean= 2.523

SD= 0.005

%RSD=

0.198

35:65 1489901 2.52

33:67 1491885 2.53108

ROBUSTNESS FOR REPAGLINIDE

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Sl.

No.


Statistical

analysis

Retention

time

Statistical

analysis

1Flow rate

(ml/min)

0.5 512276

Mean= 50051SD= 10125

%RSD= 2.02

3.91

Mean= 3.83SD= 0.075

%RSD= 1.9580.6 496049 3.82

0.7 493457 3.76

2Mobile phase

ratio

37:63

509006

Mean= 505633

SD= 8421

%RSD= 1.665

3.93

Mean= 3.84

SD= 0.073

%RSD=

1.901

35:65 496049 3.82

33:67 511846 3.79109



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generate results of acceptable accuracy and precision.

Parameters

Obtained Values

Metformin

Hydrochloride

Repaglinide

Theoretical plates (N) 2133.6 2694.7

Asymmetry (As) 1.7 1.4

LOD (g/ml) 0.018 0.05

LOQ (g/ml) 0.06 0.19110

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AcceptanceCriteriaS/N Ratio value shall be 10 for LOD solution.

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112

Metformin Hydrochloride LOQ

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AcceptanceCriteriaS/N Ratio value shall be 10 for LOD solution.

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114

Repaglinide LOQ

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THANKYOUALL

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