Figure 3.2: Specimen chromatogram of blank...

Figure 3.2: Specimen chromatogram of blank solution

Figure 3.3: Specimen chromatogram of placebo solution

Figure 3.4: Specimen chromatogram of standard solution

Figure 3.5: Specimen chromatogram of sample solution

Figure 3.6: Specimen chromatogram of spiked impurities solution

Figure 3.7: Specimen chromatogram of spiked impurities and standard

Figure 3.8: Purity plot of unstressed sample solution

Figure 3.9: Purity plot of heat stressed sample solution

Figure 3.10: Purity plot of humidity stressed sample solution

Figure 3.11: Purity plot of sun light stressed sample solution

Figure 3.12: Purity plot of UV light stressed sample solution

Figure 3.13: Purity plot of acid stressed sample solution

Figure 3.14: Purity plot of base stressed sample solution

Figure 3.15: Purity plot of peroxide stressed sample solution

Figure 3.16: Purity plot of water stressed sample solution

Figure 4.2: The trial method chromatogram using mobile phase composition of buffer:

methanol: acetonitrile (89:8:3 v/v/v) respectively.

Fgure 4.3: The trial method system suitability chromatogram using increased flow rate of

0.5mL/ minute and decreased injection volume of 2µL.

Figure 4.4: The trial method system suitability chromatogram using sodium-1-heptane

sulphonate buffer and increased injection volume of 5µL.

Figure 4.5: The trial method system suitability chromatogram using sodium

buffer.

Figure 4.6: The trial method system suitability chromatogram using 0.8 mL / minute flow rate.

The trial method system suitability chromatogram using sodium-1-octane sulphonate

l method system suitability chromatogram using 0.8 mL / minute flow rate.

Figure 4.7: Chromatogram of blank

octane sulphonate

l method system suitability chromatogram using 0.8 mL / minute flow rate.

Figure 4.8: Chromatogram of placebo

Figure 4.9: Chromatogram of mesalamine standard solution

Figure 4.10: Chromatogram of mesalamine sample solution

Figure 4.11: Chromatogram of mesalamine system suitability solution

Figure 4.12: Chromatogram of mesalamine impurity mix solution

Figure 4.13: Chromatogram of mesalamine and impurity mix solution

Figure 4.14: Chromatogram of stressed mesalamine under visible light

Figure 4.15: Chromatogram of stressed mesalamine under UV light

Figure 4.16: Chromatogram of stressed mesalamine under heat

Figure 4.17: Chromatogram of stressed mesalamine under humidity

Figure 4.18(a): Chromatogram of stressed mesalamine under acid hydrolysis

Figure 4.18(b): Purity plot of stressed mesalamine under acid hydrolysis

Figure 4.19(a): Chromatogram of stressed mesalamine under base hydrolysis

Figure 4.19(b): Purity plot of stressed mesalamine under base hydrolysis

Figure 4.20: Chromatogram of stressed mesalamine under water

Figure 4.21: Chromatogram of stressed mesalamine under peroxide oxidation

Figure 5.3: Chromatograms of (a) blank solution and (b) placebo solution

Figure 5.4: Chromatogram of standard solution

Figure 5.5: Chromatogram of sample solution

Figure 5.6: The linearity graph of metformin

Figure 5.7: The linearity graph of vildagliptin

R² = 0.9999

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

1200000

2000 3000 4000 5000 6000 7000 8000Concentration (µg mL-1)

Met

form

in p

eak a

rea

(AU

) Metformin linearity graph

R² = 0.9999

50000

100000

150000

200000

250000

300000

350000

200 300 400 500 600 700 800

Vildagliptin linearity graph

Concentration (µg mL-1)

Vil

dag

lipti

n p

eak a

rea

(AU

)

Figure 6.2: Specimen chromatogram of blank solution

Figure 6.3: Specimen chromatogram of placebo solution

Figure 6.4: Specimen chromatograms of (a) standard and (b) sample solutions

Figure 7.2: Chromatogram of blank (diluent)

Figure 7.3: Chromatogram of placebo solution

Figure 7.4: Chromatogram of ciprofloxacin system suitability solution

Figure 7.5: Chromatogram of ciprofloxacin standard solution

Figure 7.6: Chromatogram of ciprofloxacin sample solution

ANALYTICAL CHROMATOGRAPHY STUDIES TO DEVELOP NOVEL METHODS FOR

EVALUATION OF

SRI KRISHNADEVARAYA UNIVERSITY, ANANTAPUR

In partial fulfillment for the Award of the degree of

ANATAPUR

ANALYTICAL CHROMATOGRAPHY STUDIES TO DEVELOP NOVEL METHODS FOR

EVALUATION OF PHARMACEUTICAL DRUG PRODUCTS

Thesis submitted to

SRI KRISHNADEVARAYA UNIVERSITY, ANANTAPURAMU

In partial fulfillment for the Award of the degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

By

NANDIPATI SUBHAKAR

Under the Supervision of

Prof. V. KRISHNA REDDY

Department of Chemistry

SRI KRISHNADEVARAYA UNIVERSITY

ANATAPURAMU-515 003, ANDHRA PRADESH, INDIA

MARCH – 2014

ANALYTICAL CHROMATOGRAPHY STUDIES TO DEVELOP NOVEL METHODS FOR

PHARMACEUTICAL DRUG PRODUCTS

In partial fulfillment for the Award of the degree of

Prof. V. KRISHNA REDDY,

Department of Chemistry.

Residence : (08554) 273252

Mobile : 9490121379

E-Mail : krvanka@gmail.com

This is to certify that the thesis entitled “

studies to develop novel methods for evaluation of pharmaceutical drug

products” is a bonafide research work done by

under my supervision in the department of chemistry, Sri Krishnadevaraya

University, Anantapuramu

Philosophy. The work embodied in this thesis is original

submitted in part or in full for the award of any degree or diploma by any

other University.

Date:

Place: Anantapuramu

: krvanka@gmail.com

CERTIFICATE

certify that the thesis entitled “Analytical chromatography

studies to develop novel methods for evaluation of pharmaceutical drug

is a bonafide research work done by Mr.Nandipati Subhakar,

under my supervision in the department of chemistry, Sri Krishnadevaraya

amu, for the award of the degree of Doctor of

Philosophy. The work embodied in this thesis is original and has not been

submitted in part or in full for the award of any degree or diploma by any

(Prof. V. KRISHNA REDDY)

Research Supervisor

SRI KRISHNADEVARAYA UNIVERSITY

ANANTAPURAMU – 515 003, ANDHRA PRADESH, INDIA

Analytical chromatography

studies to develop novel methods for evaluation of pharmaceutical drug

Nandipati Subhakar,

under my supervision in the department of chemistry, Sri Krishnadevaraya

, for the award of the degree of Doctor of

and has not been

submitted in part or in full for the award of any degree or diploma by any

(Prof. V. KRISHNA REDDY)

515 003, ANDHRA PRADESH, INDIA

DECLARATION

The work incorporated in this thesis entitled “Analytical chromatography

studies to develop novel methods for evaluation of pharmaceutical drug

products” has been carried out by me in the Department of Chemistry, Sri

Krishnadevaraya University, Anantapuramu, Andhra Pradesh, India. The

work embodied in this thesis is original and I declare that it has not been

submitted in part or in full for the award of any degree or diploma of any

other university.

Date: (NANDIPATI SUBHAKAR)

Place: Anantapuramu

ACKNOWLEDGMENTS

This thesis would have remained a distant dream had it not been for several wonderful

people. I would like to express my sincere gratitude to all of them.

First of all, I am extremely grateful to my adored research supervisor, Prof. V Krishna Reddy,

the retired Professor of Department of Chemistry, Sri Krishnadevaraya University, Anantapuramu,

for his valuable guidance, patience and consistent encouragement I received throughout the

research work. He is a person with an amicable and positive disposition; I consider it as a great

opportunity to do my doctoral program under his guidance.

I am sincerely thankful to Dr Reddy`s Laboratories Limited, Hyderabad, for providing moral

support in carrying out this research work along with professional services and I am highly thankful

to Sri Krishnadevaraya University, Anantapuramu, for giving an opportunity as a scholar, of the

university.

I am grateful to Prof. P Raveendra Reddy and Dr. P Venkata Ramana, for their advice during

the course of my thesis work.

I am thankful to Dr. Y Ramachandra Reddy, Mr.U Sreenivas and E SasikiranGoud for their

support and encouragement.Many friends have encouraged and helped me to achieve my goals. I

greatly value their friendship and I deeply appreciate their belief in me.

I owe my special gratitude to my beloved parents Sri N Chinna Rao and Smt. N Subhadra.

I have no suitable words that can fully describe their everlasting love for me. I am also thankful to

my brother Mr N Lakshminarayana and each individual of all my family members for their

affectionate encouragement extended to me throughout my career.

I owe my deepest gratitude towards my better half MrsN Bhanu Bhagyavathi, daughter N

Sahasra and son N Anunay, for their affection and support which enabled me to pursue my goal

whole heartedly.

CONTENTS

S. No Content Page No

Chapter - 1 Introduction to analytical techniques and drugs

Section –(i) : Introduction to analytical techniques 1 - 12

Section -(ii) : Introduction to drugs 13 - 33

Chapter - 2 Introduction to analytical method development and validation

Section -(i) : Introduction to analytical method development 34 - 60

Section –(ii) : Introduction to analytical method validation 60 - 80

Chapter - 3 Determination of omeprazole assay by UPLC 81 - 111

Chapter - 4 Determination of mesalamine assay by UPLC 112 - 141

Chapter - 5 Simultaneous estimation of vildagliptin and metformin by RP

– HPLC 142 - 167

Chapter - 6 Estimation of telmisartan by RP - HPLC 168 - 194

Chapter - 7 Determination of ciprofloxacin by UPLC 195 - 218