Final

ANALYTICAL METHOD DEVELOPMENT AND VALIDATION
FOR SIMULTANEOUS ESTIMATION OF ANTIHYPERTENSIVE

DRUGS
By
Ms. BINDHYASHREE K M (19P4561)
Ms. SAHANA K M (19P4560)
Dissertation submitted to
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCESBENGALURU,
KARNATAKA
In partial fulfillment of the requirements for the degree of
BACHELOR OF PHARMACY
Under the guidance of
Mr. SUBHAJITH GHOSH

Department of Pharmaceutical Chemistry
Sarada Vilas College of pharmacy Mysore
SARADA VILAS COLLEGE OF PHARMACY

KRISHNAMURTHYPURAMMYSURU –570004
2021-2022
RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES
BENGALURU, KARNATAKA
This is to certify that this dissertation entitled Analytical method development and
validation for simultaneous estimation of antihypertensive drugs has been
submitted by Ms. Bindhyashree K M, Ms. Sahana K M, to the university.
Date: Registrar
(Evaluation)
EVALUATION CERTIFICATE
This is to certify that this dissertation has been evaluated.
Internal Examiner External Examiner
Signature
Name
College
Date
I
DECLARATION BY THE CANDIDATES
We hereby declare that the dissertation entitled Analytical method development and
validation for simultaneous estimation of antihypertensive drugs has been
submitted by Ms. Bindhyashree K M, Ms. Sahana K M, is a bonafide and genuine
research work carried out in partial fulfillment of the requirement for the degree of
Bachelor of Pharmacy under the guidance and supervision of Mr. Subhajith Ghosh,
Associate Professor, Sarada Vilas College of Pharmacy, Mysuru. The contents of this
dissertation, in full or in parts, have not been submitted to any other institute of
university for the award of any degree or diploma.
Signature of Candidates
Date:
(Ms. Bindhyashree K M)
Place: Mysuru
(Ms. Sahana K M)
II
CERTIFICATE BY THE GUIDE
This is to certify that the dissertation entitled Analytical method development and
validation for simultaneous estimation of antihypertensive drugs submitted by
Ms. Bindhyashree K M, Ms. Sahana K M, is a bonafide research work carried out
in partial fulfillment of the requirements for the degree of Bachelor of Pharmacy
under my supervision and guidance. The contents of this dissertation, is in full or in
parts, have not been submitted to any other institute or university for the award of any
degree or diploma.
Date: Signature of the Guide
Place: Mysuru
Mr. Subhajith Ghosh

Associate Professor,Department of
Pharmaceutical chemistry
Sarada Vilas College Pharmacy
Mysuru
III
CERTIFICATE BY THE CO-GUIDE
degree or diploma.
Date: Signature of the Co-Guide
Place: Mysuru
Mr. Harshith Kumar
Assistant Professor Department of

Pharmaceutical chemistry
Sarada Vilas College of Pharmacy

Mysuru
IV
ENDORSEMENT BY THE PRINCIPAL

degree or diploma.
Date: Signature of the Principal
Place: Mysuru
Dr. Hanumanthachar Joshi

Principal
Mysuru
V
ENDORSEMENT BY THE HOD

degree or diploma.
Date: Signature of the HOD
Place: Mysuru
Dr. Jinesh B Nagavi
Head of the Department of
pharmaceutical chemistry

Mysuru
VI
DECLARATION BY THE CANDIDATES
COPYRIGHT
We hereby declare that Rajiv Gandhi University of Health Sciences, Bengaluru, Karnataka,
shall have the rights to preserve, use and disseminate the dissertation / thesis in print or
electronic format for academic/ research purpose.
Signature of Candidates
Date:
Place: Mysuru
(Ms. Bindhyashree K M)
(Ms. Sahana K M)
© Rajiv Gandhi University of Health Sciences, Karnataka
VII
ACKNOWLEDGEMENT
First and foremost, we would like to thank God Almighty for giving us the strength, ability and
opportunity to undertake this study and to preserve and complete itsatisfactorily.
We thank our parents and family for all the love and support they have provided us throughout
our project. Their encouragement has helped us to achieve our goal.
It is genuine pleasure to express our sincere thanks and gratitude to our guide, Mr. Subhajith
Ghosh, Professor of Pharmaceutical chemistry, Sarada Vilas College of Pharmacy. His timely
advice, meticulous scrutiny, scholarly advice and scientific approach have helped us to a very
great extentto accomplish our task.
We whole heartedly remember the help and support provided by our beloved lecturer and
Institutional Co-guide, Mr. Harshith Kumar R, Assistant professor in the Department of
Pharmaceutical chemistry Sarada Vilas College of Pharmacy, and without whose cooperation
and suggestions our work wouldnot have been completed successfully.
We respect and thank Dr. Hanumanthachar Joshi, Principal, Sarada Vilas College of
Pharmacy, for giving us an opportunity to do our project and providing us all guidance and
support to complete this project in time.
It’s our privilege to thank all teaching and non-teaching staff of our institution for their support
and encouragement.
We wish to acknowledge the support and guidance of Dr. Jinesh B Nagavi professor and
HOD of department of pharmaceutical chemistry, Mr. G Gokul Nanda Assistant
professor, Mrs. Chaitra Shree Assistant professor, Mr. Chethan M B Assistant professor,
Dr. Prabitha P Assistant professor, Department of pharmaceutical chemistry, Sarada Vilas
College of Pharmacy, for their valuable help to carry out the studies and completion of my
project.
Our special thanks to Mr. Shanmukha Priya P, Vijnana Bhavana, Mysore University for their
valuable help to carry out the studies and completion of our project.
ACKNOWLEDGEMENT
We are very grateful to our family for their constant support throughout our dissertation work
emotionally and financially.
We would also like to express our gratitude to all who directly or indirectly have helped us in the
completion of our dissertation work.
This work is a team work, it is more a combination of ideas, suggestions, reviews,
contributions and efforts of many. We wish to express our appreciation to all those with whom
we have worked interacted and whose thoughts and insights have helped us in furthering our
knowledge and understanding of the subject.
Ms. Bindhyashree K M
Ms. Sahana K M
ACKNOWLEDGEMENT
VIII
ABBREVATIONS
LIST OF ABBREVIATIONS
Abbreviations Expansions
RP-UFLC Reverse Phase- Ultra Fast liquid Chromatography
℃ Degree Centigrade
abs Absorbance
conc Concentration
Cm Centimeter
hr Hour
M Molar
Max Maximum
µg Microgram
Mg Milligram
Min Minute
ml Milliliter
mM Millimolar
RPM Revolution Per Minute
UV Ultraviolet
ICH International Conference Harmonization
FDA Food and Drug Administration
USFDA United States Food and Drug Administration
LLOQ Lowest Limit of Quantification
HQC High Quality Control
LQC Lower Quality Control
MQC Medium Quality Control

ABBREVATIONS
% Percentage
SD Standard Deviation
IP Indian Pharmacopoeia
CC Calibration Curve
CV Coefficient of Variation
TLM Telmisartan
AZL Azelnidipine
r2 Correlation Coefficient
RSD Relative Standard Deviation
RT Retention Time
ng Nanogram
AR Analytical Reagent
AUC Area Under Curve
Cmax Maximum Concentration
ER Extraction Recovery
(RdRp) RNA Dependent RNA Polymerase
Rt Retention Time
XI
TABLES
LIST OF TABLES
Sl. No Table Page no.

1 Drug Information for Telmisartan 14
2 Drug Information for Azelnidipine 15
3 List of Chemicals, Reagents and Solvents 17
4 List of Pure Drugs and Their Formulations 17
5 List of Instruments and Equipment’s 17-18
6 Chromatographic Conditions of TLM & AZL 22
7 Intra-Day and Inter-Day Precision Data of TLM 29-33
8 Intra-Day and Inter-Day Precision Data of AZL 34-38
9 Accuracy Data of TLM 39

10 Accuracy Data of AZL 40
11 Stability Study of TLM 41
12 Stability Study of AZL 42
XII
FIGURES
LIST OF FIGURES
Sl. No Figures Page no.
1 Mechanism of Telmisartan 3
2 Mechanism of Azelnidipine 4
3 Chemical Structure of TLM 14
4 Chemical Structure of AZL 15
5 Chromatograms of Blank 23
6 Chromatogram of TLM and AZL (5/10) µg/ml) 24
10 Chromatogram of TLM and AZL with Plasma (80/160) 26
µg/ml)
11 Standard Calibration Graph of Telmisartan 27
12 Standard Calibration Graph of Azelnidipine 27
13 Selectivity Chromatogram of Blank 28
14 Selectivity Chromatogram of TLM and AZL 28
XIII
CONTENTS
TABLE OF CONTENTS
CHAPTER CONTENTS PAGE NO.

1-4
1 Introduction
5-6
2 Objectives
7-15
3 Review of Literature
16-20
4 Materials and Methods
21-42
5 Results and Discussion
43-44
6 Summary
45-46
7 Conclusion
47-49
8 Bibliography
ANNEXURE
International conference certificate
admin
ABSTRACT
ABSTRACT
Aim: Aim of the study was to be establish the analytical method development and
validation for simultaneous estimation of Telmisartan and Azelnidipine according to ICH
guidelines.
Methods: The chromatographic separation was achieved by using waters C 18 column
(250mm x 4.6mm, 5μm) column by applying gradient elution using the mobile A
consists of 0.1% formic acid in water as an aqueous phase and acetonitrile as an organic
modifier the following gradient in the ratio 80: 20 (v/v) detection was carried at the 260
nm by employing 0.8ml/min flow rate.
Results: The retention time was found to be 5.950min and 7.293 min for Telmisartan
and Azelnidipine respectively. The developed method has the linearity range was
obtained for RP-UPLC method was 10-160 µg/ml and 5-80 µg/ml for Telmisartan and
Azelnidipine respectively and the regression co-efficient (r 2) is 0.999 for the both drugs.
Accuracy & precision is evaluated for the method and found be within the limit and the
results were reproducible.
This method has been adopted for simultaneous estimation of telmisartan and azelnidipine
formulation available in the market.
Conclusion: Based on these results, conclude that the developed method was precise and
accurate for the simultaneous estimation of Telmisartan and Azelnidipine. This method can be
adopted for simultaneous estimation of drugs will be helpful for in-vivo studies in future days
& pharmacokinetics studies.
Keywords: Telmisartan, Azelnidipine, Validation, UPLC.

INTRODUCTION
CHAPTER :1
INTRODUCTION
Department of Pharmaceutical Chemistry Page 1

INTRODUCTION
INTRODUCTION
High blood pressure, also known as hypertension, is a common condition that is generally
characterized by a higher amount of pressure in blood vessels than the normal value.[1]
Hypertension is one of the main gamble factor for both coronary supply route illness and
cardiovascular infection.[2] High blood pressure (BP), otherwise called hypertension, as a clinical
blood pressure of 140/90mmHg or higher affirmed by a subsequent ambulatory blood pressure
(or then again home blood pressure checking normal) of 135/85 mmHg or higher. Hypertension
is a critical risk factor for cardiovascular infection.[3]
UPLC is a modern technique which gives a new direction for liquid chromatography. UPLC
refers to ultra-performance liquid chromatography, which enhance mainly in three areas: “speed,
resolution and sensitivity. In twenty first centenary pharmaceutical industries are focusing for
new ways to in economy and shorten time for development of drugs In UPLC main advantage is
better efficiency with speedy analysis and this achieved by only smaller particle size.
UPLC analysis improves in three areas of
1. Produced Chromatogram with resolved peak.
2. Fast analysis
3. Sensitive analysis It uses fine particles and saves time and reduces solvent consumption.
The new method make a very big difference by retains the same analytical separation method as
HPLC while other things which drastically changes are speedy analysis, sensitivity and high
resolution. Today’s pharmaceutical industries are focusing for new path to reduce cost and less
time for development of drugs and in mean time the quality of their products are not suffer
analytical laboratories maintain the whole things.[4]
Telmisartan is a 4-((2-n-propyl-4-methyl-6-(1-methylbenzimidazol-2-yl)-benzimidazol-1-yl)
methyl)-biphenyl-2-carboxylic acid, is an angiotensin II receptor antagonist(ARAII) widely used
in the treatment of high blood pressure.[5]

INTRODUCTION
Mechanism of telmisartan;
Angiotensin II AT1-receptor by binding reversibly and specifically to the receptors in vascular
smooth muscle and the adrenal gland. As Angiotensin II is a vasoconstrictor, which additionally
stimulates the synthesis and release of an aldosterone blockage of its actions leads to decreases in
systemic vascular resistance. It is used for the treatment of hypertension, decreases high blood
pressure helps prevent strokes, heart attacks, and kidney issues. Telmisartan is official in Indian
pharmacopoeia and Japanese Pharmacopoeia [6] [shown in figure 1].
Figure 1 : Mechanism of Telmisartan

INTRODUCTION
Mechanism of Azelnidipine;
Azelnidipine is chemically known as (3) -(1-diphenylmethylazetidin-3-yl)-5-isopropyl-2-amino-
1,4-dihydro-6-methyl-4-(3-nitrophenyl) 3,5pyridinedicarboxylate.[7] Mechanism of azelnidipine;
Azelnidipine is third generation calcium channel antagonist can hold Ca+ ions outside the
cardiac muscle and vascular smooth muscle. They enter in the cells through cell membrane it
expands blood vessel, lower peripheral vascular resistance and arterial pressure. In clinic, it is
utilized for treatment of hypertension and angina pectoris.[8] [shown in figure 2]
Figure 2:- Mechanism of Azelnidipine.

OBJECTIVES
CHAPTER:2
OBJECTIVES

OBJECTIVES
2. OBJECTIVES
1.The aim of study is to establish a newer method for the simultaneous estimation of
hypertensive drugs by analytical method development and validation.
2.To develop analytical method for the simultaneous estimation of selected hypertensive
drugs by chromatographic method.
3. To validate the method according to the ICH guidelines.

REVIEW OF LITERATURE
CHAPTER:3

3. REVIEW OF LITERATURE
1. Bangaruthalli J et al., (2019): This method has been established for the simultaneous
estimation of Telmisartan and Atorvastatin calcium by RP-HPLC method. The
chromatographic conditions were successfully developed for the separation of Telmisartan and
Atorvastatin calcium by using boston ODS C18 column, flow rate was 1.0ml/min, mobile
Phase consists of methanol: Acetonitrile: buffer in ratio of 35:25:40. Detection wave length was
235nm.The instrument used was SHIMADZU HPLC auto sampler. The retention time of
Atorvastatin calcium and Telmisartan was found to be 2.350 and 3.490 minutes respectively.
The analytical method was validated according to ICH guidelines (ICH Q2b). The correlation
coefficient (r2) was found to be 0.997 and 0.999 for Telmisartan and Atorvastatin calcium
respectively. % mean recovery was found to be 100.943% and 100.576% for Telmisartan and
Atorvastatin calcium respectively. %RSD for precision on replicate injection was 0.46 and 0.70
for Telmisartan and Atorvastatin calcium respectively. The validation study was found to be
precise, robust, and repeatable.[9]
2. K.N. Jayaveera et al., (2018): The method was develop a rapid, precise, accurate and sensitive
reverse phase liquid chromatographic method for the estimation of azelnidipine in the plasma
of rat animal model studies for transdermal drug delivery. The chromatographic method was
standardized for azelnidipine using Shimadzu HPLC model reverse phase analytical inspire
C18 column (250 mm x 4.5 mm, 5 μm particle size) with LC10AD pump and SPD-10A UV
Detector, the mobile phase consists of75:25 methanol: water and 0.1%glacial acetic acid, wave
length at 254nm, with flow rate of 1ml/min. The retention REVIEW OF LITERATURE Dept
of Pharmaceutical Analysis. Analysis 8 time of azelnidipine found to be 6.130 min. The method
was statistically validated and %RSD was found to be less than 2 indicating high degree of
accuracy and precision. Hence this is proposed method can be successfully applied for the
estimation of azelnidipine in various dosage forms and animal model in –vivo studies.[10]
3. Megha G. GorE et al., (2018): A simple and precise method was developed for the assay of
azelnidipine from tablet formulation. The solvent system and wavelength were optimized in
order to maximize the sensitivity of the proposed method, azelnidipine shows the maximum

absorbance at 257 nm. The separation was achieved on HPLC binary gradient system equipped
with HPLC 3000 series. The mobile phase was prepared with Methanol: Water (80: 20%v/v) o-
phosphoric acid used for the pH adjustment (pH-3). The method was validated for accuracy,
precision, linearity, LOD & LOQ of sample solution. Linearity was observed in the
concentration range of 20-100 µg/ml & gave mean correlation coefficient 0.998. The developed
RP-HPLC method was found to be accurate, precise and was successful applied to a
pharmaceutical tablet formulation for qualitative estimation of Azelnidipine. In this work
HPLC method for assay in tablet formulation was developed and validated. Methanol as diluent
was selected which give sharp spectra[11]
4. P Harshalatha et al., (2017): The aim of present study is to develop a validated high
performance liquid chromatographic method for the simultaneous analysis of two
antihypertensive drugs, Telmisartan and Hydrochlorothiazide in their tablet dosage form.
Chromatographic process was carried on a Hypersil gold C18, 25× 4.6 mm, 5 µm SS column
employing degassed and filtered mobile phase of buffer: methanol in their 10:90 ratio. Flow
rate was maintained at 0.7ml/min and the eluent were monitored at 327nm. Developed method
was validated for the various validation parameters such as precision, specificity, accuracy,
linearity, robustness, ruggedness and stability. A good linearity was observed for the drugs
Telmisartan and Hydrochlorothiazide over the concentration range of 48-72 µg/ml and 32- 48
µg/ml respectively [12]
5. Sharma Shaina et al., (2016): This work was designed to develop and validate an accurate,
precise and rapid method for the estimation of Telmisartan as Active Pharmaceutical Ingredient
(API) as well as in tablet dosage form and prepared spherical agglomerates by RPHPLC. The
developed method was found to be simple, accurate, precise and sensitive. The separation was
achieved on an Isocratic High Pressure Liquid Chromatography (HPLC) (Thermo Scientific)
using pumps Jasco PU 2080 Plus, UV detector, column oven (Jasco), and a Reverse Phase C-18
(phenyl) Column (25 cm x 4.6 mm) i.d., particle size 5 µm. The HPLC system was run with
flow rate: 0.8 ml/min Injection Volume: 10µl and run time: 10 min, Detector temp: 40 o C. The
method was validated for specificity, precision, linearity, and accuracy, robustness, LOD and

LOQ parameters. The recovery range was within the range of 99.0–102.0% and the method
could be successfully applied for the routine analysis of the drug substance as well as the
spherical agglomerates prepared by crystallin coagglomeration technique.[13]
6. Gaikwad R B, et al., (2016) A simple, rapid and selective HPLC method has been developed
quantitation of Bisoprolol fumarate and Telmisartan from bulk drug and pharmaceutical
formulations using a mobile phase consisting mixture of methanol and water (75:25 v/v) at the
flow rate of 1ml/min. A Waters X Bridge RP C18 (4.6 x 250 mm) column was used as
stationary phase. The retention time of Bisoprolol fumarate and Telmisartan were 5.7 min. and
7.6 min. respectively Linearity was observed in the concentration range of 5-25 μg/ml for
Bisoprolol fumarate and 40- 200 μg/ml for Telmisartan. Percent recoveries obtained for
Bisoprolol fumarate and Telmisartan were 99-101 % and 99-100 % respectively. The proposed
method is precise, accurate, selective and rapid for the simultaneous determination of
Bisoprolol fumarate and Telmisartan.[14]
7. Reema H et al., (2013): A simple, precise, and accurate RP-HPLC method has been developed
and validated for the simultaneous assay of Telmisartan and Cilnidipine in tablets. Isocratic RP-
HPLC method was developed on Waters C18 250 × 4.6 mm, 5 𝜇m column using mobile phase
as acetonitrile (ACN): buffer pH 3.0 with orthophosphoric acid (68: 32) at a flow rate of 1.0
mL/min and the detection was carried out at 245 nm using photodiode array detector. Forced
degradation study was carried out by oxidation, hydrolysis, photolysis, and heating the drug.
The method was validated for specificity, linearity, precision, accuracy, robustness, and
solution stability. The method was found to be linear in the concentration range of 40–160
𝜇g/mL with correlation coefficient of 0.9990 for Telmisartan and 10–40 𝜇g/mL with correlation
coefficient of 0.9989 for Cilnidipine.[15]
8. Sunil Jawla et al., (2010): This method has been developed and subsequently validated for
simultaneous determination of Telmisartan and Ramipril in pharmaceutical dosage forms. The
method employs an Inertsil ODS C18 column, 5 μ, 250mm x 4.60mm id with flow rate of 1.5
ml/min using PDA detection at 210nm. The separation was carried out using a mobile phase
consisting of potassium di hydrogen phosphate buffer having pH 2.8 and Acetonitrile in the

ratio of 60:40 respectively. The retention time for Telmisartan and Ramipril was found to be
5.7min and 10.8min respectively. A linear response was observed over the concentration range
of 2.5-25.5ppm and 3.0-7.25ppm for the assay of Telmisartan and Ramipril respectively. the
proposed method was found to be accurate, precise, reproducible and specific and can be used
for simultaneous analysis of these drugs in tablet formulation [21] SUJANAK. et al (2011) A
simple, selective, precise and stability indicating RP High Performance Liquid
Chromatographic (HPLC) method of analysis of Telmisartan in pure and pharmaceutical
dosage form was developed and validated. The chromatographic conditions comprised of a
reversed phase C8 column (4.6 x 150mm, 3.5 μm, make: XTerra), with a mobile phase
composed of Buffer and Methanol (40:60v/v, Adjusted the pH to 3.0 with ortho Phosphoric
acid). Flow rate was 0.5 mL / min. Detection was carried out at 230 nm. The retention time of
Telmisartan was 2.6 min. The linear regression analysis data for the calibration plots showed
good linear relationship in the concentration range 20‐100 μg/ml. The values of correlation
coefficient, slope and intercept were, 0.9998, 2.326and6.708, respectively the method was
successfully validated in accordance to ICH guidelines acceptance criteria for specificity,
linearity, precision, recovery, ruggedness and robustness. All the peaks of degraded product
were resolved from the active pharmaceutical ingredient with significantly different retention
time6 the chromatographic conditions comprised of a reversed phase C8 column (4.6 x 150mm,
3.5 μm, make: XTerra), with a mobile phase composed of Buffer and Methanol (40:60v/v,
Adjusted the pH to 3.0 with ortho Phosphoric acid). Flow rate was 0.5 mL / min. Detection was
carried out at 230 nm. The retention time of Telmisartan was 2.6 min. The linear regression
analysis data for the calibration plots showed good linear relationship in the concentration
range 20100 μg/ml. The values of correlation coefficient, slope and intercept were, 0.9998,
2.326and6.708, respectively. The method was successfully validated in accordance to ICH
guidelines acceptance criteria for specificity, linearity, precision, recovery, ruggedness and
robustness. The drug undergoes degradation under acidic, basic, Peroxide and thermal
degradation conditions. All the peaks of degraded product were resolved from the active
pharmaceutical ingredient with significantly different retention time.[16]
9. Kaliappan Ilango et al., (2013): The present study describes development and subsequent
validation of stability indicating HPLC methods for simultaneous estimation of Telmisartan

(TLM) and Atorvastatin (ATV) in their combined formulation. The proposed RP-HPLC
method utilizes a Phenomenex Luna C18 column using acetonitrile: 0.025 M ammonium
acetate (38: 52%, v/v) as mobile phase (pH 3.8), flow rate of 1.0 mL/min. Quantification was
achieved with UV detection at 281 nm over concentration range of 12 to 72 g/mL for TLM and
3 to 18 g/mL for ATV respectively. In HPTLC, separations were performed on silica gel 60
F254 using toluene methanol-ethyl acetate-acetic acid (5: 1: 1: 0.3, v/v) as mobile phase. The
compact bands of TLM and ATV at 0.37 ± 0.02 and 0.63 ± 0.01 respectively were scanned at
279 nm.[17]
10. Swathi S. Gaikwad et al., (2019): Azelnidipine, dihydropyridine based calcium channel
blocker has been used for treating ischemic heart disease and cardiac remodeling after
myocardial infarction but it is having a low bioavailability due to its poor solubility. The
present study is to investigate the formulation and evaluation of floating tablets of Azelnidipine
for controlled release and to increase bioavailability by increasing the gastrointestinal transit
time and muco adhesion of drug. The gastro retentive tablets were prepared by direct
compression method using different concentrations of combination of Polyoxyethylene oxide
WSR 303 as hydrophilic polymer and Potassium bicarbonate as gas generating agent. Main
effects of the formulation variables were evaluated quantitatively using design approach
showing that both independent variables have significant effects on floating lag time, % drug
release at 1 h (D1 h) and time required to release 90% of the drug (t90). The statistically
optimized formulation(F3) released 95.11 ± 1.43% drug for 12 h followed K-Peppas drug
release kinetics indicating release of drug by diffusion and erosion mechanism. Evaluation of
the optimized formulation in vivo in human volunteers showed that the GFT was buoyant in
gastric fluid and that its gastric residence time was enhanced. Pharmacokinetics studies carried
out revealed significant (P < 0.05) equivalent Cmax, longer Tmax and higher AUC values for
the optimized formula compared to the marketed oral product. From the results obtained it can
be concluded that Azelnidipine Gastro retentive tablets with enhanced bioavailability and better
release pattern is suitable for more effective treatment compared to marketed conventional
tablets.[18]

11. Sree Janardhan et al., (2012): Developed and optimized a validated isocratic reverse phase
HPLC separation of Rosuvastatin, Telmisartan, Ezetimibe and Atorvastatin in pharmaceutical
preparation using response surface methodology. The separation was carried out by using
phenomenex C18 column (15 cm · 4.6 mm id, 5 lm particle size) and UV detection at 239 nm.
The ranges of the independent variables used for the optimization were MeCN: 33–38%, buffer
conc.: 10–20 mM and flow rate: 1–2 ml/min. The influence of these independent variables on
the output responses: capacity factor of the first peak (k1), resolutions of the 2nd and 3rd peak
(Rs2,3), and capacity factor of the fifth peak (k5) were evaluated. Using this strategy, a
mathematical model was defined and a response surface was derived for the separation. The
three responses were simultaneously optimized by using Derringer’s desirability functions.
Optimum conditions chosen for the assay were MeCN, MeOH, 20 mM K2HPO4 (pH 3.0 ± 0.2)
solution (34.27:20:45.73 v/v/v) and flow rate 2 ml/min. Total chromatographic analysis time
per sample was approximately 10 min. The optimized assay condition was validated as per the
ICH guidelines and applied for the quantitative analysis of Rosavel EZ, Avas-EZ and Lipisar
20 tablet. The developed method was simple, accurate and precise. Hence, it can be employed
for the routine analysis in quality control laboratories.[19]

DRUG PROFILE:
a) Telmisartan
Table no:1
Structure Fig3: Structure of Telmisartan
Chemical name 4'-[(1,7'-dimethyl-2'-propyl-1H,3'H-2,5'- bibenzimidazol-

3' yl)methyl]bipheny l- 2-
carboxylic acid
Category Anti-hypertensive drug
Molecular Weight 514.617 g/mol
Molecular Formula C33H30N4O2
Melting Point 261 to 263°C
Solubility Very soluble in n dimethyl acetamide; sparingly soluble

in methanol; freely soluble in a acetonitrile
, practically insoluble in water.
pKa (Strong Acid) 3.65
pKa (Strong Basic) 4.1

b) Azelnidipine:
Table no:2
Fig4: Structure of Azelnidipine

Structure
Chemical name 3-(1-Benzhydrylazetidin-3-yl) 5-isopropyl 2- amino-6-methyl-

4-(3-nitrophenyl)-1,4-
dihydropyridine-3,5-dicarboxylate
Category Anti-hypertensive drug
Molecular weight 582.6g/mol
Molecular formula C33 H34N4O6
Melting point 108℃-113°C
Solubility Very Soluble in N Dimethyl acetamide; sparingly

soluble in methanol; freely soluble in
acetone, practically insoluble in water
pKa (Strong Acid) 7.16
pKa (Strong Base) 5.18

METHODOLOGY
CHAPTER:4
METHODOLOGY

METHODOLOGY
4. METHODOLOGY
A. Chemical, reagents and solvents
The chemicals, reagents and solvents used in the analytical method development, validation
were listed in Table 3.
Table 3: List of chemicals, reagents and solvents
Sl.No Name of the chemicals/Reagents Manufacturer
1. Methanol (HPLC grade) S D Fine Chem Pvt.Ltd, Mumbai
2. Formic Acid (A R grade) Rankem Chemicals, Mumbai
3. Acetonitrile (HPLC grade) S D Fine Chem Pvt.Ltd, Mumbai
4. Millipore Water (HPLC grade) In house
B. Pure drugs and their formulations

The list of drugs and their manufacturers are listed in Table 4.
Table-4 List of pure drugs and their formulations
Sl.No Drugs Manufacturer
1. Yarrow Chemicals
Telmisartan (99% pure)
2. Azelnidipine (99% pure) Yarrow Chemicals
C.Instruments and equipment

The instruments and equipment used in the analytical method development, validation were
listed in Table 5.
Table 5: List of instruments and equipment
Sl.no Instruments/equipment Mode/maker

1. Acquity H class UPLC, PDA detector Waters Corporation, USA
2. Automatic sample manager (Serial # Waters Corporation, Singapore
C10UPA554M)
3. Centrifuge (CAT – 1010) Tarsons, India
4. C18 column (5µm 4.6×250nm) Waters

METHODOLOGY
5. Electronic microbalance Mettler Toledo, India

6. Micropipettes Eppendorf AG, Germany.
7. Vortex mixer Grant Bio, UK.
8. Sonicater Oscar Ultrasonic, India.
D. Preparation of mobile phase:

The Mobile Phase is made up of 0.1% Formic acid in Water as an aqueous phase (A) and
Acetonitrile as an Organic modifier (B). It was then filtered through 0.45 μm membrane
filter. Finally, the mobile phase was sonicated for 20 min to degas it.
E. Preparation of standard solution

a) Telmisartan.
By dissolving 0.1g of pure telmisartan drug in 10ml of methanol solution (100mg/ml), the
stock solution (A) was prepared and sonicated for 10 mins. The standard solution (B) were
prepared by adding 1ml sol(A) and diluting with 10ml of methanol (1000μg/ml) and series
of dilution were made to obtain concentration of 10 - 160 µg/ml with the required dilution of
the stock standard solution of Telmisartan with diluents.
b) Azelnidipine.
By dissolving 0.1g of pure azelnidipine drug in 10ml of methanol solution (100mg/ml), the
stock solution (A) was prepared and sonicated for 10 mins. The standard solution (B) were
prepared by adding 1ml sol(A) and diluting with 10ml of methanol (1000μg/ml) and series
of dilution were made to obtain concentration of 5-80 µg/ml with the required dilution of the
stock standard solution of azelnidipine with diluents.
Validation of developed RP UPLC method according to ICH guidelines

Q2(R2)
A. Linearity and calibration curve:
Linearity was evaluated by carrying out the chromatographic analysis of several telmisartan
& azelnidipine standard solutions with increasing concentrations and by establishing the

METHODOLOGY
Calibration plot of the response vs. concentration, which visually approximates a straight
line. Additionally, linear regression analysis was performed to assess the linearity of the
calibration curve by employing the least square linear regression method to obtain the slope,
intercept and correlation coefficient. To determine the linearity of the evaluated method,
standard solutions of telmisartan & azelnidipine stock solution was diluted in solvent,
yielding standard solutions with concentrations of 10,20,40,80, &160 μg/ml & 5,10,20,40,80
μg/ml. Subsequently, the solution was filtered using Whatmann’s filter paper, which was
later sonicated for 5 minutes before injecting into the UPLC system to obtain the
chromatograms.
B. Limit of detection (LOD):
The limit of detection (LOD) or detection limit (DL) can be defined as the lowest
concentration of an analyte which can be reliably detected in the test sample. The LOD was
calculated by taking 3.3 times of standard deviation of the response(σ) and the slope of the
calibration curve (S).
LOD=3.3𝜎/𝑆
Where, σ = the standard deviation of the response S = the slope of the calibration curve
C. Limit of quantification (LOQ):
The lowest concentration level at which a measurement is quantitatively meaningful is

called the limit of quantitation (LOQ) or quantification limit (QL). This is most often
defined as 10 times the signal-to-noise ratio. Thus, the LOQ is 10 times the standard
deviation (σ) of the response and slope (S) of the calibration curve.
LOQ=10𝜎/𝑆
Where, σ = the standard deviation of the response S = the slope of the calibration curve
D. Accuracy:
The accuracy of the analytical method is determined as the nearness of the obtained value to the
true value. In the present study, accuracy was determined by spiking the reference standards of

METHODOLOGY
the telmisartan & azelnidipine which were administered into the blank matrix, yielding solutions
containing analyte with concentration levels of 50%, 100%, 150% & 50%, 100%, 150%. At
every concentration level, about three samples were prepared and later analyzed. The recovery
studies were performed in triplicates (for each sample), and the percentages of recovery and
mean recovery were calculated for telmisartan & azelnidipine.
𝑏−𝑎
% Recovery = x 100
𝑐
where
a is the amount of drug found before addition of standard
b is the amount of drug found after addition of standard drug
c is the amount of standard drug added
The accuracy data is shown in table 9 and 10.
E. Precision:
Precision is the degree of agreement/closeness among the results of each test when the
method is subjected to multiple homogenous samples. It can be explained as the
reproducibility of measurement, and to determine the same, series of measurements (of
similar quantities) were carried out. Thus, the system and method precision were evaluated
using a standard solution of 20,40,80, & 10,20,40μg/mL of telmisartan & azelnidipine which
was administered three times and the chromatograms were recorded for the same. Further, to
determine the precision, statistical analysis such as Standard Deviation (SD) and the percent
Relative Standard Deviation (% RSD) of variables such as Retention Time (RT), peak area
etc.…were measured for the test results.
F. Robustness:
The robustness of the method determines the susceptibility of a method to small changes
such as pH values, temperature, mobile phase composition, etc. which might occur during
routine analysis at the laboratory. In the present study, robustness was investigated by
deliberately Changing the pH of the mobile phase.

RESULTS AND DISCUSSION
CHAPTER:5
RESULT AND DISCUSSION

5. RESULT AND DISCUSSION
Analytical method development validation
A. Development and optimization of chromatographic condition

The Telmisartan and Azelnidipine was eluted on a C18 (5µm×4.6mm×250mm) column using
Mobile phase A consist of (0.1% Formic acid in water + Acetonitrile) and Mobile Phase B
consist of and it is delivered at a flow rate of 0.8 ml/min in the following gradient elution. The
eluents were monitored at 260 nm, the retention time was found to be 5.950 min and 7.293 min
for telmisartan and azelnidipine respectively showed in the below Figure5,6,7,8,9,10,11, &12.
Table6: Standard chromatographic conditions for telmisartan and

azelnidipine
Instrument Ultra-Performance liquid chromatography (UPLC) Acquity H-class,

Waters
Column C18 Waters (4.6×250mm, 5µm)
Mobile phase 0.1% Formic acid in water and Acetonitrile
Wavelength 260 nm
Flow rate 0.8 ml/min
Retention time 5.950 min and 7.293 min for Telmisartan and Azelnidipine
Run Time 10 min

Figure 5: Chromatogram of Blank.
Method development
Figure 6: Chromatogram of Azelnidipine.

Figure 7: Chromatogram of Telmisartan
Figure 8: Chromatogram of Azelnidipine &Telmisartan (5/10)

Figure 9: Chromatogram of Azelnidipine & Telmisartan (10/20)
Figure 10: Chromatogram of Azelnidipine & Telmisartan (20/40)

Figure 11: Chromatogram of Azelnidipine &Telmisartan (40/80).
Figure 12: Chromatogram of Azelnidipine & Telmisartan (80/160).

Validation of the developed method as per ICH guideline.

5.1. Linearity and calibration curve
5.1.1 Azelnidipine.
The linearity was tested in the concentration range of 5-80 µg/ml and the calibration curve
constructed was evaluated by its correlation coefficient. The correlation coefficient (r2) for all the
calibration curves was consistently greater than 0.999 represented in Figure 13.
Figure:13 Standard calibration graph of azelnidipine

5.1.2 Telmisartan
The linearity was tested in the concentration range of 10-160 µg/ml and the calibration curve
constructed was evaluated by its correlation coefficient. The correlation coefficient (r2) for all the
calibration curves was consistently greater than 0.999 represented in Figure 14.
Figure 14: Standard calibration graph of telmisartan

5.2 Method Sensitivity and Specificity

Confirmation of azelnidipine and telmisartan could be readily achieved by comparing the
retention time obtained from the sample with standard drug. All the chromatograms were
analyzed and it has been noted that endogenous components did not interfere with azelnidipine
and telmisartan.
5.3 Selectivity
No significance interference from endogenous components was observed
Figure 15: Selectivity chromatogram of blank
Figure 16: Selectivity chromatogram of azelnidipine & telmisartan.

5.4Precision
5.4.1Intraday precision
Table 07: Intra-intraday precision data of telmisartan by ICH method
Morning:
Sl.NO Conc. In Peak area Conc. Average STD DEV % CV
ng/ml Obtained
in µg/ml
1 20 4486.647 20.10 20.08 0.075 0.37
4492.547 19.95
4493.574 20.02
2 40 10006.645 40.02 39.6 0.55 1.3
10008.756 39.99
10004.755 39.05
3 80 20511.768 80.1 80.58 0.005 0.54
20509.678 80.65
20510.647 80.99
SD- Standard Deviation, RSD- Relative Standard Deviation

Evening:
Sl.NO Conc. In Peak area Conc. Average STD DEV % CV
ng/ml Obtained
in µg/ml
1 20 4486.647 20.10 20.08 0.075 0.37
4492.547 19.95
4493.574 20.02
2 40 10006.645 40.02 39.6 0.55 1.3
10008.756 39.99
10004.755 39.05
3 80 20511.768 80.1 80.58 0.005 0.54
20509.678 80.65
20510.647 80.99

Interday precision
Day 1
SI.NO Conc. In Peak area Conc. Average STD DEV % CV

ng/ml
Obtained
in µg/ml
1 20 4485.258 20.08 20.05 0.036 0.17
4490.563 20.06
4497.238 20.01
2 40 10005.236 40.36 40.23 0.19 0.47
10009.154 40.32
10002.587 40.01
3 80 20512.65 79.59 80.08 0.17 0.58
20505.32 80.12
20511.85 80.53

Day 2
ng/ml Obtained
in µg/ml
1 20 4485.121 20.09 20.03 0.05 0.24
4483.453 20.03
4485.893 19.99
2 40 10006.321 40.11 40.20 0.28 0.69
10003.542 40.52
10006.586 39.98
3 80 20511.352 80.21 79.99 0.37 0.46
20509.745 79.56
20513.529 80.20

Day 3
ng/ml Obtained
in µg/ml
1 20 4485.452 20.45 19.99 0.41 2.09
4484.253 19.63
4482.236 19.89
2 40 10008.123 39.79 40.17 0.58 1.44
10007.582 40.85
1000.335 39.89
3 80 20512.593 80.09 80.15 0.80 0.99
20513.236 80.99
20315.547 79.39

Table 08: Intra-interday precision data of azelnidipine by ICH method:
5.4.2 Intraday precision

Morning:
Conc. In Peak area Conc. Average STD DEV % CV
SI.NO
ng/ml
Obtained in
µg/ml
1 10 2372.423 10.01 10.01 0.01 0.09
2371.667 10.02
2373.546 10.00
2 20 5171.147 20.02 20.01 0.01 0.04
5172.124 20.01
5170.214 20.03
3 40 10517.913 40.01 40.01 0.02 0.05
10516.254 40.03
10513.125 39.99
SD- Standard Deviation, RSD- Relative Standard Deviation,

Evening

SI.NO
ng/ml
Obtained in
µg/ml
1 10 2375.421 10.01 10.03 0.05 0.4
2374.541 9.9
2376.852 10.01
2 20 5172.253 20.01 20 0.02 0.1
5173.586 20.02
5170.214 19.9
3 40 10513.124 40.03 40.05 0.05 0.09
10516.265 40.1
10510.125 40.02

Interday precision
Day 1

SI.NO
ng/ml
Obtained in
µg/ml
1 10 2372.978 10.4 10.19 0.2 1.9
2371.972 10.2
2370.854 9.9
2 20 5170.523 20.4 20.19 0.2 0.9
5172.586 20.2
5171.214 19.99
3 40 10516.241 40.4 40.19 0.2 0.4
10515.247 40.2
10513.586 39.9

Day 2

SI. NO
ng/ml Obtained
in µg/ml
1 10 2372.124 10.02 10.04 0.05 0.4
2374.143 10.1
2375.365 10.0
2 20 5171.245 20.03 20.07 0.1 0.5
5172.547 20.2
5175.214 19.99
3 40 10517.365 40.1 40.05 0.04 0.09
10519.325 40.03
10518.386 40.2

Day 3

SI.NO
ng/ml Obtained
in µg/ml
1 10 2373.257 10.3 1014. 0.1 0.9
2372.354 10.02
2375.526 10.1
2 20 5172.258 20.01 20.03 0.05 0.2
5176.149 20.1
5176.257 19.99
3 40 10517.214 40.3 40.11 0.1 0.2
10516.254 40.02
10515.325 40.01
5.5 Accuracy:
A known quantity of standard solution has been added to the sample solutions previously
analyzed at three different levels (50%, 100% and 150%). The amount recovered for telmisartan
and azelnidipine has been calculated for three concentrations. Percent RSD values for all
analyses were less than 2% indicating that no impurities interfere and analytical method is very
accurate. (Table 9 and Table 10)

Table 09: Accuracy data for telmisartan by ICH method (% recovery data)
Sl.No % of Amount of Amount of Total Total % Mean %SD %RSD

drug drug drug amount of amount Recovery
added taken added drug(n=3) of drug
(ng/ml) (ng/ml) found
(STD) (sample)
1 50% 20 10 30 30 100 99.7 0.125 0.115
29.9 99.2
30 100
2 100% 20 20 40 40 100 99.4 0.67 0.77
40.2 99.8
38.8 98.5
3 150% 20 40 60 60 99.1 99 0.57 0.57
59.9 98
58.9 100
STD- Standard, SD- Standard Deviation, RSD- Relative Standard Deviation

Table 10: Accuracy data for azelnidipine by RP UPLC method (% recovery

data
Sl. No % of Amount of Amount of Total Total % Mean %SD %RSD

drug drug drug amount of0 amount Recovery
added taken added drug(n=3) of drug
(ng/ml) (ng/ml) found
(STD) (sample)
1 50% 40 20 60 59.9 99.8 99.9 0.94 0.94
60.0 100
60.0 100
2 100% 40 40 80 78.8 98.5 99.5 0.77 0.77
80.2 100
80.0 100
3 150% 40 80 120 100 100 99.6 0.47 0.47
99.9 99
100 100
STD- Standard, SD- Standard Deviation, RSD- Relative Standard Deviation

5.6 Stability
The stability study was investigated for the sample under various stability periods and storage
conditions. They were carried out at three concentration levels (low, medium and high) at six
replicates. The percentage stability was estimated by comparing the mean of back calculated
concentrations of all analytes from the stored stability samples with that of freshly sample
concentration. The results indicated that each analyte had an acceptable stability under those
conditions, as shown in Table 11 and Table 12
Table 11: Stability of telmisartan during Storage and Sample Handling
Stability sample Mean ± SD (µg/ml) Recovery RSD (%)

Conc.(µg/ml) (%)
Long-term 20 20.8±11.2 103.19 1.86
(30 days) 40 40.8±12.3 102.40 2.15
80 81.1±13.3 104.3 3.86
Short-term 20 20.5±14.6 104.1 1.95
(8 hours) 40 40.8±23.1 105.5 4.9
80 81.6±21.5 109.1 4.3
Auto-sampler 20 21.5±14.2 105.0 3.0
(24 hours) 40 41.6±25.4 109.9 5.48
80 81.3±30.2 108.9 6.2
Freeze-Thaw 20 21.3±29.2 108.9 6.3

40 41.6±17.5 107.4 3.7
80 81.9±29.7 109.7 6.1

Table 12: Stability of Azelnidipine during Storage and Sample Handling
sample Conc.
Stability (µg/ml) Mean ± SD (µg/ml) Recovery (%) RSD (%)
Long-term 10 10.8±12.2 104.19 2.67
(30 days) 20 21.8±15.3 106.40 3.26
40 40.1±13.3 105.3 2.86
Short-term 10 10.5±14.6 105.1 1.9
(8 hours) 20 21.8±23.1 107.5 4.9
40 40.1±21.5 109.1 4.3
Auto-sampler 10 10.5±14.2 105.0 3.0
(24 hours) 20 20.6±25.4 109.9 5.48
40 40.3±30.2 108.3 6.2
10 10.3±29.2 108.9 6.3
Freeze-Thaw 20 20.6±17.5 107.4 3.7
40 80±29.7 109.7 6.1

SUMMARY
CHAPTER:6
SUMMARY

SUMMARY
6. Summary
For routine analysis purpose it is necessary to establish methods capable of analyzing huge
number of samples in short period of time with good accuracy and precision.
Chromatographic technique can generate a large amount of quality data which serves as a highly
powerful and convenient tool. In the present work UPLC methods were developed and validated
for simultaneous estimation of telmisartan and azelnidipine by using analytical method.
Literature survey revealed that only few HPLC method was available to quantify the telmisartan
and azelnidipine &other combination drug and individual drug.
A very quick, cost-effective, precise and accurate UPLC method for the simultaneous estimation
for telmisartan and azelnidipine has been developed and validated in compliance with ICH.
Besides the short run time (10 min), retention time of telmisartan and azelnidipine was found to
be 5.950 and 7.293 minutes respectively and flow rate of mobile phase (0.8 mlmin-1) made the
method attractive because these features save analysis time and cost. This method is sensitive,
selective, and rapid for telmisartan and azelnidipine in active pharmaceutical ingredient (API).
The accuracy and precision are within reasonable limits and finally analytical method is reliable,
precise and accurate.
After developing the method, it was validated by using different parameters. Based on the
obtained results the proposed UPLC method is proven to be suitable as well as found to be
simple, precise and economical for the determination and can be routinely adopted technique for
the quantification of telmisartan and azelnidipine.

CONCLUSION
CHAPTER 7
CONCLUSION

CONCLUSION
7.CONCLUSION
• A simple, precise and accurate method for the simultaneous estimation of telmisartan and
azelnidipine has been developed and validated in compliance with ICH guidelines.
• Sensitive and specific analytical method was developed & validated.
• This method has been adopted for simultaneous estimation of telmisartan and azelnidipine
formulation available in the market.
Future perspectives
➢ The experimental studies are needed to investigate the pharmacokinetic parameters in animal
model.
➢ Toxicity study.

BIBILOGRAPHY
CHAPTER :8
BIBILOGRAPHY

BIBILOGRAPHY
8. BIBILOGRAPHY
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estimation of azelnidipine and telmisartan. Asian journal of pharmaceutical research and
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2. Mandale D, Mistry R, Chauhan N. An Analytical Approach of Azelnidipine: a Review. World J.
Pharm. Pharm. Sci. 2021 Jan 2; 10:682-92.
3. Kitt J, Fox R, Tucker KL, McManus RJ. New approaches in hypertension management: a review
of current and developing technologies and their potential impact on hypertension care. Current
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4. Taleuzzaman M, Ali S, Gilani SJ, Imam SS, Hafeez A. Ultra-performance liquid
chromatography (UPLC)-a review. Austin J Anal Pharm Chem. 2015;2(6):1056
5. Torrealday N, Gonzalez L, Alonso RM, Jimenez RM, Lastra EO. Experimental design approach
for the optimisation of a HPLC-fluorimetric method for the quantitation of the angiotensin II
receptor antagonist telmisartan in urine. Journal of pharmaceutical and biomedical analysis.
2003 Aug 8;32(4-5):847-57
6. Eswarudu MM, Roja P, Sankar PR, Babu PS. An Updated Review On Analytical Methods For
Estimation Of Azelnidipine And Telmisartan. Asian Journal of Pharmaceutical Research and
Development. 2022 Apr 15;10(2):59-76.
7. Kawabata K, Samata N, Urasaki Y, Fukazawa I, Uchida N, Uchida E, Yasuhara H.
Enantioselective determination of azelnidipine in human plasma using liquid chromatography–
tandem mass spectrometry. Journal of Chromatography B. 2007 Jun 1;852(1-2):389-97.
8. Rane AS, Mahajan SK. Validation and forced stability-indicating HPTLC method for
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9. Jagirapu B, Harini U, Divya M, Sushma P. Simultaneous estimation of telmisartan and
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BIBILOGRAPHY
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estimation of telmisartan as active pharmaceutical ingredient in tablet dosage form and prepared
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HPLC Method
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Cilnidipine with Forced Degradation Behavior Study by RP-HPLC in Tablet Dosage Form.
International Scholarly Research Notices. 2013;2013
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ANALYTICAL METHOD DEVELOPMENT AND VALIDATION

FOR SIMULTANEOUS ESTIMATION OF ANTIHYPERTENSIVE

In partial fulfillment of the requirements for the degree of

Mr. SUBHAJITH GHOSH

Sarada Vilas College of pharmacy Mysore

SARADA VILAS COLLEGE OF PHARMACY

This is to certify that this dissertation has been evaluated.

Internal Examiner External Examiner

DECLARATION BY THE CANDIDATES

CERTIFICATE BY THE GUIDE

Date: Signature of the Guide

Mr. Subhajith Ghosh

CERTIFICATE BY THE CO-GUIDE

Date: Signature of the Co-Guide

Assistant Professor Department of

Sarada Vilas College of Pharmacy

ENDORSEMENT BY THE PRINCIPAL

Date: Signature of the Principal

Dr. Hanumanthachar Joshi

ENDORSEMENT BY THE HOD

Date: Signature of the HOD

Sarada Vilas College of Pharmacy

DECLARATION BY THE CANDIDATES

© Rajiv Gandhi University of Health Sciences, Karnataka

RPM Revolution Per Minute

ICH International Conference Harmonization

FDA Food and Drug Administration

USFDA United States Food and Drug Administration

LLOQ Lowest Limit of Quantification

HQC High Quality Control

LQC Lower Quality Control

MQC Medium Quality Control

RSD Relative Standard Deviation

AUC Area Under Curve

Cmax Maximum Concentration

(RdRp) RNA Dependent RNA Polymerase

Sl. No Table Page no.

7 Intra-Day and Inter-Day Precision Data of TLM 29-33

8 Intra-Day and Inter-Day Precision Data of AZL 34-38

9 Accuracy Data of TLM 39

Sl. No Figures Page no.

CHAPTER CONTENTS PAGE NO.

International conference certificate

Keywords: Telmisartan, Azelnidipine, Validation, UPLC.

Department of Pharmaceutical Chemistry Page 1

Department of Pharmaceutical Chemistry Page 2

Figure 1 : Mechanism of Telmisartan

Department of Pharmaceutical Chemistry Page 3

Figure 2:- Mechanism of Azelnidipine.

Department of Pharmaceutical Chemistry Page 4

Department of Pharmaceutical Chemistry Page 5

3. To validate the method according to the ICH guidelines.

Department of Pharmaceutical Chemistry Page 6

Department of Pharmaceutical Chemistry Page 7

Department of Pharmaceutical Chemistry Page 8

Department of Pharmaceutical Chemistry Page 9

Department of Pharmaceutical Chemistry Page 10

Department of Pharmaceutical Chemistry Page 11

Department of Pharmaceutical Chemistry Page 12

Department of Pharmaceutical Chemistry Page 13

Structure Fig3: Structure of Telmisartan

Chemical name 4'-[(1,7'-dimethyl-2'-propyl-1H,3'H-2,5'- bibenzimidazol-

Category Anti-hypertensive drug