charmee final binder

A Dissertation ON
DEVELOPMENT AND VALIDATION OF ANALYTICAL
METHODS FOR ESTIMATION OF ASPIRIN AND
ESOMEPRAZOLE IN BULK AND COMBINATION
Submitted By
Ms. Charmee B. Gandhi
B. PHARM.
To Savitribai Phule Pune University
In partial fulfillment of the requirements for the
degree of
MASTER OF PHARMACY
IN
QUALITY ASSURANCE TECHNIQUES
OF
THE FACULTY OF PHARMACY
Under the Guidance of
DR. GIRISH K. JANI
Ph.D., M.Pharm., B.Pharm.
Principal
DEPARTMENT OF QUALITY ASSURANCE TECHNIQUES
SSR COLLEGE OF PHARMACY, SAYLI-SILVASSA ROAD
SAYLI, SILVASSA – 396 230, U.T. OF D. & N.H., INDIA
JULY 2014

SSR COLLEGE OF PHARMACY
Sayli, Silvassa – 396 230, U. T. of Dadra and Nagar Haveli
CERTIFICATE OF GUIDE
This is to certify that a seminar on dissertation entitled “Development
and validation of analytical methods for estimation of Aspirin and
Esomeprazole in bulk and combination” submitted by Ms. CHARMEE
B. GANDHI of M.Pharm. Semester-IV (QUALITY ASSURANCE
TECHNIQUES) as a part of curriculum requirement for the syllabus of
M.Pharm. in the faculty of Quality assurance techniques of Savitribai Phule
Pune University under the guidance of Dr. Girish K. Jani, Principal, SSR
College of Pharmacy, Silvassa.
Dr. Girish K. Jani
Principal
Date:
Place: Silvassa

SSR COLLEGE OF PHARMACY
Sayli, Silvassa, Dadra and Nagar Haveli-396230 INDIA
INSTITUTE CERTIFICATE
This is to certify that a dissertation report entitled “Development and
validation of analytical methods for estimation of Aspirin and
Esomeprazole in bulk and combination” submitted by MS. CHARMEE
B. GANDHI of M.Pharm. (Quality Assurance Techniques) as a part of
curriculum requirement for the degree ‘Master of Pharmacy in ‘Quality
Assurance Techniques Specialization’ in the faculty of Pharmacy under
Savitribai Phule Pune university represents her own bonafide investigation
work and contains the result of her own studies and tends to the general
advancement of knowledge.
I further certified that this research work was carried out in the research
laboratory of the Department of Quality Assurance Techniques, SSR College
of Pharmacy, Silvassa during 2012-2014 under the guidance and
supervision of Dr. GIRISH K. JANI, Principal, SSR College of Pharmacy,
Silvassa-396230, UT of Dadra & Nagar Haveli, India. For a dissertation
that she is submitting, she has not been conferred any
diploma/degree/doctorate degree by either this university or other
University to the best of my knowledge.
Date:
Place: Silvassa
Dr. Girish K. Jani
PRINCIPAL

DECLARATION
I hereby declare that the research work embodied in this dissertation is a
bonafide and genuine research work carried out by me, under the
supervision and guidance of Dr. Girish K. Jani, Principal, S.S.R. College of
Pharmacy.
I further declare that the reported results and discussion of the dissertation
shows the novelty of research work as a part of curriculum work of Master
of Pharmacy in Quality Assurance Techniques Specialization of academic
year 2012-2014 for the degree of Master of Pharmacy in Quality
Assurance Techniques under the faculty of Pharmacy, Savitribai Phule
Pune University.
MS. CHARMEE B. GANDHI
Roll No:
18 Exam Seat No: 985
M.Pharm Semester IV
SSR College Of Pharmacy,
Sayli, Silvassa-396230, UT of D.& N.H.,
India.
Date:
Place: Silvassa

COPYRIGHT
DECLARATION BY THE STUDENT
I hereby declare that the Department of Quality Assurance Techniques,
SSR College of Pharmacy, Silvassa, UT of Dadra and Nagar Haveli, India
and the faculty of Pharmacy , Savitribai Phule Pune University, Shall have
right to preserve, use and disseminate this dissertation in the part or
electronic format for academic/ research purpose through prior
permission of research guide.
MS. CHARMEE B. GANDHI
Roll No: 18 Exam Seat No: 985
M.Pharm Semester IV
SSR College Of Pharmacy,
Sayli, Silvassa-396230, UT of D.& N.H.,
India.
Date:
Place: Silvassa

Dedicated
to
my beloved parents

Acknowledgment
“As one flower makes no garland, this presentation would not have taken shape without
wholehearted encouragement and live involvement of some generous souls”.
It is great pleasure for me to acknowledge all those who have contributed towards the
conception; origin and nurturing of this project.
I would like to express my salutation to SARASWATI MAA for giving me the strength,
confidence and moral boost to successful completion of this project.
I am heartily thankful to my guide respected principal sir, Dr. Girish K. Jani,
(M.Pharm., Ph.D) S.S.R. College of Pharmacy, University of Pune, for his help and
valuable guidance, scholarly inputs and consistent encouragement I received throughout
the research work. I shall forever be indebted to them for providing me the care, support,
encouragement and confidence in me.
I am extremely thankful to my source of inspiration, Dr. Keyur B. Ahir, formal
Assistant professor, Department of Quality assurance Techniques, S.S.R. College of
Pharmacy, for his valuable guidance throughout the research work. The feat was
possible only because of his unconditional support, he always made himself available to
clarify my doubts despite his busy schedules.
Thank you sir, for all your help and support.
I wish to convey my special thanks to Mr. Parixit Prajapati, Assistant Professor, Mr.
Vishal Modi, Assistant Professor and all the other staff members, Lab technicians,
librarian S.S.R College of Pharmacy for helping me in finishing my project work
successfully.
A special thanks to Sovereign Pharma, Daman and West coast Pharma, Ahemdabad,
Gujarat for gift samples of drugs.
It was pleasure working with my colleagues cum good friends Harsh, Dharti, Panthini,
Kimpy, Akshay, Neel and Hemraj because of their selfless support that made the

working environment full of joy and enthusiasm. I want to thank my class mates
Tejash, Jaydip, Hetal, Khushboo, Sushma, Chetan and Aakanksha.
I express my heartiest gratitude to My Parents for their love and blessing to complete
the project successfully and who led me from darkness to light, ignorance to enlighten
and confusion to clarity throughout my life.
I also extent my heart fell thanks to my fiancé Vishal, my sister Ami for all their love,
care and affection. They have always been my strengths and always encouraged me to
give my best.
Last but not the least, I pay my reverence to this institute, S.S.R. College of Pharmacy,
Silvassa, Savitribai Phule Pune University. I am undeniably proud to be associated with
this college.
- Charmee B. Gandhi

Table of content
Chapter Title Page
no.
List of abbreviations i
List of tables iii
List of figures iv
Abstract vi
1. Introduction 1
1.1 Classification of analytical method 2
1.2 UV-Visible spectroscopic method 3
1.3 High performance liquid chromatography 4
1.4 Analytical method development 5
1.5 Analytical method validation 10
1.6 Justification for combination of aspirin and esomeprazole 12
2. Review of literature 14
3. Aim and objective 23
4. Drug profiles 27
5. Experimental work 33
5.1 Equipments and materials 33
5.2 Identification of drugs 36
5.3 Development and validation of UV-visible spectroscopic method 40
5.4 Development and validation of High performance liquid
chromatographic method
42
6. Result and discussion 46
6.1 UV-Visible spectroscopic method 46
6.2 High performance liquid chromatographic method 51
7. Conclusion 62
8. Publication related to present investigation 63
9. References 65

i
List of abbreviations
ASP : Aspirin
ATOR : Atorvastatin
CAF : Caffeine
CAS : Chemical abstract service
DIC : Diclofenac
DL : Detection limit
DOM : Domperidone
DSC : Differential scanning calorimetry
DTA : Differential thermal analysis
ESO : Esomeprazole
FDA : Food and drug administration
FTIR : Fourier transform infra red
HPLC : High performance liquid chromatography
HPTLC: High performance thin layer chromatography
ICH : International conference of harmonization
IR : Infrared spectroscopy
IUPAC: International union of pure and applied chemistry
LEVO : Levosulpiride
LC : Liquid chromatography
LOD : Limit of detection
LOQ : Limit of quantitation
NAP : Naproxen

ii
NaOH : Sodium hydroxide
NF : National formulary
NSAID: Non-steroidal anti-inflammatory drug
PCM : Paracetamol
PDA : Photo-diode array detector
QL : Quantitation limit
RAM : Ramipril
ROS : Rosuvastatin calcium
RP : Reverse phase
RSD : Relative standard deviation
SD : Standard deviation
THF : Tetrahydro furan
UPLC : Ultra performance liquid chromatography
UV : Ultra violate spectroscopy
USP : United states pharmacopoeia
VIS : Visible

iii
List of tables
Table
no.
Title Page no.
1 The electromagnetic spectrum 3
2 The ranges of spectroscopic interest 3
3 Important information concerning sample composition and
properties
8
4 Identification of drugs by melting point 36
5 IR correlation of Aspirin 39
6 IR correlation of Esomeprazole 40
7 Wavelength maxima of Aspirin 46
8 Wavelength maxima of Esomeprazole 47
9 Statistical data for ASP and ESO by UV method 48
10 Intraday precision for ASP and ESO 49
11 Interday precision for ASP and ESO 49
12 Accuracy study of ASP & ESO by the proposed UV method 50
13 Analysis of synthetic mixture 50
14 Parameters and results 51
15 Optimized chromatographic conditions for HPLC 52
16 Statistical data for ASP and ESO by HPLC method 52
17 Repeatability of sample application data for ASP and ESO 53
18 Precision data for ASP 53
19 Precision data for ESO 54
20 Accuracy study of ASP & ESO by the proposed HPLC method 54
21 Robustness results of ASP and ESO 55
22 Analysis of synthetic mixture 55
23 Result of calibration readings for ASP by HPLC method 59
24 Result of calibration readings for ESO by HPLC method 60
25 Summary of validation parameters of HPLC 61

iv
List of figures
Figure no. Title Page no.
1 HPLC system block diagram 5
2 Steps in HPLC method development 7
3 Structure of Aspirin 27
4 Structure of Esomeprazole 30
5 UV-Visible instrument 34
6 Digital balance 34
7 IR instrument 35
8 HPLC system 35
9 DSC of ASP 37
10 DSC of ESO Na 37
11 IR spectra of Aspirin (API) 38
12 IR spectra of Aspirin (standard) 38
13 IR spectra of Esomeprazole (API) 39
14 UV spectra of 10 ppm solution of ASP in methanol 46
15 UV spectra of 10 ppm solution of ESO in methanol 47
16 Overlay of 10 ppm solution of ASP and ESO 47
17 Calibration Curve of ESO at 237 nm and at 275 nm 48
18 Calibration Curve of ASP at 237 nm and at 275 nm 49
19 Chromatogram of ASP and ESO using methanol : water (80 :
20)
56
20 Chromatogram of ASP and ESO using methanol : water (50 :
50)
56
21 Chromatogram of ASP and ESO using methanol : water : THF
(60 : 40 : 0.05)
57
22 Chromatogram of standard Asp and ESO ( 60 µg/ml and 15
µg/ml respectively ) using mobile phase methanol : water :
tetrahydrofuran (70 : 30 : 0.05 v/v)
57
23 Chromatogram of ASP and ESO of synthetic mixture ( 60 58

v
µg/ml and 15 µg/ml respectively ) using mobile phase
methanol : water : tetrahydrofuran (70 : 30 : 0.05 v/v)
24 Multicurve report 58
25 Calibration curve of ASP by HPLC method 59
26 Calibration curve of ESO by HPLC method 60

vi
ABSTRACT
Development of validated method of analysis for pharmaceutical drugs using simple and
sensitive analytical techniques like, UV and RP-HPLC.
UV method was developed using methanol as solvent. Absorbance readings were taken at
two wavelengths, 275 nm (λmax of ASP) and 237 nm (iso-absorptive point). UV method
was found to be linear in the range of 20-100 µg/ml for ASP and 5-25 µg/ml for ESO. The
limit of quantification was found to be 15.6 µg/ml for ASP and 0.26 µg/ml for ESO
respectively.
RP-HPLC method was developed using methanol : water : THF (70 : 30 : 0.05 v/v/v) as
mobile phase, C18 column as stationary phase. HPLC method was found to be linear in the
range of 20-120 µg/ml for ASP and 5-30 µg/ml for ESO. The limit of quantification was
found to be 5.93 µg/ml for ASP and 0.35 µg/ml for ESO respectively. The percentage
recovery was 99.27-100.19 % for ASP and 99.88-101.05 % for ESO.
UV and HPLC methods were developed for estimation of ASP and ESO from synthetic
mixture. Both the methods were validated and found to be accurate, precise and robust.

CHARMEE B. GANDHI; Roll no: 18; Exam seat no: 985 Chapter 1
SSR COLLEGE OF PHARMACY, SILVASSA. 1 M.PHARM. Dissertation; QAT, 2012-14
1. INTRODUCTION
Analytical chemistry is a scientific discipline used to study the chemical composition,
structure and behavior of matter. Analytical chemistry involves the application of range of
techniques and methodologies to obtain and assess qualitative, quantitative and structural
information on the nature of matter.
Qualitative Analysis is the identification of elements, species and/or compounds present
in the sample.
Quantitative analysis is the determination of the absolute or relative amounts of
elements, species or compound present in the sample.
Structural analysis is the determination of the spatial arrangement of atoms in an element
or molecule or identification of characteristics groups of atoms (functional groups).
The gathering and interpretation of qualitative, quantitative and structural information is
essential to many aspects of human endeavor, both terrestrial and extra-terrestrial. The
maintenance of, and improvement in, the quality of life throughout the world, and the
management of resources rely heavily on the information provided by chemical analysis.
Manufacturing industries use analytical data to monitor the quality of raw materials,
intermediates and finished products. Progress and research in many areas is dependent on
establishing the chemical composition of man-made or natural materials, and the
monitoring of toxic substances in the environment is of ever increasing importance.
Studies of biological and other complex systems are supported by the collection of large
amounts of analytical data.
Analytical data are required in a wide range of disciplines and situations that include not
just chemistry and most other sciences, from biology to zoology, but the arts, such as
painting and sculpture, and archaeology. Space exploration and clinical diagnosis are two
quite disparate areas in which analytical data is vital.[1]
A complete chemical analysis even for a single substance, involve a series of steps and
procedure. Each one of them has to be carefully considered and assessed in order to
minimize errors and maintain accuracy and reproducibility.[2]
The following steps are involved in the analysis of sample-
1. Sampling
2. Preparation of analytical sample

3. Dissolution of sample
4. Removal of interferences
5. Sample measurement and control of instrument factors
6. Result(s)
7. Presentation of data
1.1 Classification of analytical method
This classification includes the modern and latest method of analysis.
1. Chemical methods
A. Volumetric methods
a) Acidimetric and alkalimetric methods
b) Non-aqueous titration methods
c) Oxidation-reduction methods
d) Complexometric method
B. Gravimetric methods
2. Physical methods
a) Refractometry
b) Polarimetry
c) Optical rotator dispersion
3. Electrochemical methods
a) Electro analytical methods
b) Polarography
4. Spectroscopic methods
a) UV and visible spectroscopy
b) Fluorescence and phosphorescence spectroscopy
c) Atomic spectroscopy (emission and absorption)
d) Infra red spectroscopy (IR)
e) Nuclear magnetic resonance spectroscopy (NMR)
f) Mass spectroscopy (MS)
g) Electron spin resonance spectroscopy
5. Separation methods
a) Chromatographic methods
b) Electrophoresis
6. Radiochemical methods

a) Methods using isotopes
7. Miscellaneous type (instrumental)
a) Particle size analysis
b) Differential thermal analysis (DTA) and differential scanning calorimetry
(DSC)
1.2 UV- Visible spectroscopic method[3,4]
Most organic molecules and functional groups are transparent in the portions of the
electromagnetic spectrum that we call the ultraviolet (UV) and visible (VIS) regions-
that is, the regions where wavelengths range from 100 nm to 800 nm.
Table 1 : The electromagnetic spectrum
Approximate wavelengths range (cm)a
Region of spectrum
10-12
– 10-11
Cosmic rays
10-11
– 10-8
Gamma rays
10-8
– 10-6
X rays
10-6
– 10-5
Ultraviolet
10-5
– 10-4
Visible
10-4
– 10-2
Infrared
10-2
– 10 Microwave
10 - 108
Radio frequency
a
Rounded to orders of magnitude
Table 2: The ranges of spectroscopic interest
Region Wavelength range
Far ultraviolet 100 - 200 nm
Ultraviolet 200 - 400 nm
Visible 400 - 750 nm
Near infrared 0.75 - 4 µm
Infrared 4 - 25 µm
The fundamental law that governs the quantitative spectrophotometric analysis is
the
Beer- Lambert’s law:[5,6]
“When a beam of monochromatic light is passed through a transparent cell
containing a solution of an absorbing substance, reduction of intensity of the light

may occur; the rate of reduction in intensity with the thickness of the medium is
proportional to the intensity of the light and the concentration of the absorbing
substances.”
Mathematically Beer- Lambert’s law is expressed as:
A = a × b × c
Where,
A = absorbance or optical density
a = absorptivity or extinction coefficient
b = path length of radiation through sample (cm)
c = concentration of solute in solution
For the quantitative analysis of substances in single and multicomponent samples;
Following techniques are generally used;[6]
 Simultaneous equation method
 Multi-component mode method
 Method of least squares (use of calibration curve)
 Absorbance ratio method
 Geometric correction method
 Orthogonal polynomial method
 Difference spectrophotometric method
 Derivative spectrophotometric method
 Chemical derivatization method
1.3 High performance liquid chromatography[1]
High-performance liquid chromatography (HPLC) is a separation technique where
solutes migrate through a column containing a micro particulate stationary phase at
rates dependent on their distribution ratios. These are functions of the relative affinities
of the solutes for the mobile and stationary phases, the elution order depending on the
chemical nature of the solutes and the overall polarity of the two phases. Very small
particles of stationary phase are essential for satisfactory chromatographic efficiency
and resolution, and the mobile phase must consequently be pumped through the
column, resulting in the generation of a considerable back-pressure. The composition of
the mobile phase is adjusted to elute all the sample components reasonably quickly.
Solutes eluted from the end of the column pass through a detector that responds to each

one. There are a number of modes of HPLC enabling an extremely wide range of solute
mixtures to be separated. The modes are defined by the type of stationary phase and
associated sorption mechanism.
A schematic diagram of a high-performance liquid chromatography instrument is
shown
in Figure 1. It consists of five major components:
● solvent delivery system;
● sample injection valve;
● column;
● detection and recording system;
● microcomputer with control and data-processing software.
Figure 1: HPLC system block diagram
1.4 Analytical method development[7]
Analytical method development is the process by which a specific analytical method is
to be developed for drug products from the stage of in process to finished product and
mini validation to be done before starting the analysis of routine samples, investigation
samples and stability samples. Analytical method development and finalizing the
method consists of :
1. Standardizing the working standard from reference standard.
2. Optimizing the chromatographic condition, concentration of standard and sample
solution and extraction procedure of the sample.

3. Analytical method verification or mini validation to be done before analyzing
(routine samples) tests like assay, dissolution and related substance in development
levels or stages etc.
4. Prior starting the validation the satisfactory result should be found in mini
validation and formulation should be finalized.
1.4.1 Basic criteria for new method development of drug analysis:
 The drug or drug combination may not be official in any pharmacopoeias,
 A proper analytical procedure for the drug may not be available in the literature due
to patent regulations,
 Analytical methods may not be available for the drug in the form of a formulation
due to the interference caused by the formulation excipients,
 Analytical methods for the quantification of the drug in biological fluids may not be
available,
 Analytical methods for a drug in combination with other drugs may not be available,
 The existing analytical procedures may require expensive reagents and solvents. It
may also involve cumbersome extraction and separation procedures and these may
not be reliable.
1.4.2 Method development in HPLC[7]
There exists a today a good practical understanding of chromatographic separation and
how it varies with the sample and with experimental conditions. A good method
development strategy should require only as many experimental runs as are necessary to
achieve the desired final result. Ideally, every experiment will contribute to the end result
so that there are no wasted runs. Usually, this requires that the results of each
chromatographic run be assessed before proceeding with the next experiment. Sometimes
the chemical structures of the sample components are known, other times this is not the
case. Finally, method development should be as simple as possible, yet it should allow the
use of sophisticated tools such as computer modeling if these are available.

Figure 2 : Steps in HPLC method development
 Prerequisites
Nature of the sample
Before beginning method development, we need to review what is known about the
sample. The chemical composition of the sample can provide valuable clues for the best
1. Information on sample, define
separation goals.
2. Need for special HPLC procedure,
sample pretreatment, etc. ?
3. choose detector and detector settings
4. preliminary run; estimate best
separation conditions
5. optimize separation techniques
6. check for problems or requirements
for special procedure
7b. Quantitative
calibration
7a. Recover purified
material
7c. Qualitative method
8. Validate method for release to routine
laboratory

choice of initial conditions for HPLC separation. Depending on the sample information,
different approaches to HPLC method development are possible. Some analysts try to
match the “chemistry” of the sample to a best choice of initial HPLC conditions. To do
this, they rely heavily on their own past experience and/or they supplement this
information with data from the literature.
Table 3: Important information concerning sample composition and properties
Number of compounds present
Chemical structures of compounds
Molecular weights of compounds
pKa values of different compounds
UV spectra of compounds
Concentration range of compounds in samples of interest
Sample solubility
 Sample pretreatment and detection
Samples come in various forms:
 Solutions ready for injection
 Solutions that require dilution, buffering, addition of an internal standard or
other volumetric manipulation
 Solids that must first be dissolved or extracted
 Samples that require pretreatment to remove interferences and or protect the
column or equipment from damage
Direct injection of the sample is preferred for its convenience and greater precision.
However, most samples for samples for HPLC analysis require weighing and/or
volumetric dilution before injection. Best results are often obtained when the
composition of the sample solvent is close to that of the mobile phase, since this
minimizes baseline upset and other problems.
Before the first sample is injected during HPLC method development, we must be
reasonably sure that the detector selected will sense all the sample components of
interest. Variable wavelength ultraviolet (UV) detectors normally are the first choice,
because of their convenience and applicability for most samples. For this reason,

information on the UV spectra can be an important aid for method development. UV
spectra can be found in the literature, estimated from the chemical structures of sample
components of interest, measured directly (if the pure compounds are available), or
obtained during HPLC separation by means of a photodiode-array (PDA) detector.
When the UV response of sample is inadequate, other detectors are available or the
sample can be derivatized for enhanced detection.
 Developing the separation
If HPLC is chosen for separation, the next step is to classify the sample as regular or
special. Regular samples are typical mixtures of small molecules (< 2000 Da) that can
be separated using more or less standardized starting conditions. Exceptions or special
samples are usually better separated with a different column and customized
conditions.
Regular samples can be further classified as neutral or ionic. Samples classified as
ionic include acids, bases, amphoteric compounds and organic salts (ionized strong
acids or bases). If the sample is neutral, buffers or additives are generally not required
in the mobile phase. Acids or bases usually require the addition of a buffer to the
mobile phase. For basic or cationic samples less acidic reverse phase columns are
recommended, and amines additives for the mobile phase is beneficial. Using these
initial conditions, first exploratory run is carried out. On the basis of initial exploratory
run, isocratic or gradient elution can be selected as most suitable.
 Improving the separation
The separation achieved in the first one or two runs usually will be less than adequate.
After a few additional trials, it may be tempting to accept a marginal separation,
especially if no further improvement is observed.
Separation or resolution is primary requirement in quantitative HPLC analysis.
Resolution depends on the life of the column and can vary from day to day with minor
fluctuation in separation conditions.
The time required for separation should be as short as possible. This assumes that the
goals have been achieved and the total time spent on method development is
reasonable.

 Completing the HPLC method
The final procedure should meet all the goals that were defined at the beginning of
method development. The method should also be robust in routine operation and usable
by all laboratories and personnel for which it is intended.
1.5 Analytical method validation[8-14]
Validation is defined as ‘finding or testing the truth of something’. When the analytical
methods are used to generate results about the characteristics of drug related samples it
is vital that the results are trustworthy: they may be used as the basis for decisions
relating to administering the drug to patients. A validation study is performed on an
analytical method to ensure that reliable results are always obtained.
 Method validation is defined as the process of defining and proving an analytical
method acceptable for its intended use.
 The objective of any analytical measurement is to obtain consistent, reliable and
accurate data. Validated analytical methods play a major role in achieving this
goal. The results from method validation can be used to judge the quality,
reliability and consistency of analytical results, which is an integral part of any
good analytical practice. Validation of analytical methods is also required by most
regulations and quality standards.
 Method validation is the process used to confirm that the analytical procedure
employed for a specific test is suitable for its intended use.
 Analytical methods need to be validated, verified, or revalidated in the following
instances:
1. Before initial use in routine testing
2. When transferred to another laboratory
3. Whenever the conditions or method parameters for which the method has
been validated change (for example, an instrument with different
characteristics or samples with a different matrix) and the change is outside
the original scope of the method.
4. In order to demonstrate equivalence between two methods ( e.g. a new
method and a standard ).
5. When quality control indicates an established method is changing with time.

Typical validation characteristics which should be considered are given below:-[9-14]
Accuracy:
Accuracy is the measure of how close the experimental value is to true value. It is
measured as the percent of analyte recovered by assay or by spiking samples in a blind
study. For the drug product, this is performed by analyzing synthetic mixtures
(placebos) spiked with known quantities of active drug.
The FDA and ICH guidelines Q2 (R1) recommends that recovery should be performed
at the 80, 100 and 120 % or 50, 100 and 150 % of label claim.
Precision:
Precision is the measure of how close the data values are to each other for a number of
measurements under the same analytical conditions. In USP 23/NF 18, general chapter
<1225>, precision is defined as the degree of agreement among individual test results
obtained by repeatedly applying the analytical method to multiple samplings of a
homogenous sample. Precision is usually expressed as % RSD.
Repeatability:
Repeatability expresses the results of the method operating over a short time interval
under the same conditions.
Intermediate precision:
Intermediate precision expresses within laboratory variations. The attribute evaluates
the reliability of the method in an environment different from that used during the
method development phase such as the method can be evaluated on different days,
with different analysts and equipments, etc.
Reproducibility:
Reproducibility is assessed by performing collaborative studies between laboratories.
Specificity/Selectivity:
The USP defines specificity as the ability to measure accurately and specifically the
analyte of interest in the presence of other components as excipients, impurities and
degradation products which are present in the sample matrix. According to the ICH,
the validation procedure should be able to demonstrate the ability of the method to
assess unequivocally the analyte in the presence of impurities, matrix components and
degradation products.
Limit of detection (LOD)/ Detection limit (DL):
The LOD is the lowest concentration of the analyte in the sample that can be detected,
but not necessarily quantitated as an exact value.

Limit of quantitation (LOQ)/ Quantitation limit (QL):
The LOQ is the lowest concentration of the analyte in a sample which can be
quantitatively determined with suitable precision and accuracy. This is a parameter of
the quantitative assays for low concentration of compounds which are present in
sample matrices with other components as excipients, impurities and degradation
products.
Linearity:
The linearity of an analytical procedure is its ability to obtain test results that are
directly proportional to the concentration of analyte in the sample within a given
range. Linearity is generally reported as the variance of the slope of the regression line
calculated according to an established mathematical relationship from test results
obtained by the analysis of samples with varying concentration of analyte.
Range:
The range of an analytical method is the interval between the upper and the lower
concentration levels of analyte (including concentration) for which the method as
written has been shown to precise, accurate and linear.
Robustness :
The robustness of an analytical procedure is a measure of its capacity to remain
unaffected by small but deliberate variations in some parameters and provide an
assurance of its reliability during normal usage.
1.6 Justification for combination of aspirin and esomeprazole[15-16]
Low dose aspirin therapy plays a fundamental role in both the primary and
secondary prevention of cardiovascular events. Although the evidence using low
dose aspirin for secondary prevention is well-established, the decision to use aspirin
for primary prevention is based on an evaluation of the patient’s risk of
cardiovascular events compared to their risk of adverse events, such as bleeding. In
addition to the risk of bleeding associated with long term aspirin administration,
upper gastrointestinal side effects, such as dyspepsia often lead to discontinuation of
therapy, which places patients at an increased risk for cardiovascular events. One
option to mitigate adverse events and increase adherence is the addition of
esomeprazole to the medication regimen. The addition of esomeprazole to low dose

aspirin therapy in patients at high risk of developing gastric ulcers for the prevention
of cardiovascular disease significantly reduced their risk of ulcer development.
Therefore, for those patients who are at a high risk of developing a gastrointestinal
ulcer, the benefit of adding esomeprazole likely outweighs the risks of longer term
proton pump inhibitor use, and the combination can be recommended.

Chapter 2
Review Of
Literature

2. REVIEW OF LITERATURE[17-20]
 Aspirin was synthesized by the German chemist Felix Hoffman (1868-1946) in the
laboratories of Farbenfabriken Bayer, Elberfeld, Germany in 1897. The compound
was tested pharmacologically by Wohlgemuth and Witthauer who documented the
antirheumatic, antipyretic and analgesic properties free of the undesirable side
effects of salicylic acid.
 Aspirin is official in Indian, British, United states and European Pharmacopoeia.
 Esomeprazole is the S- isomer of omeprazole. Omeprazole showed an inter-
individual variability and therefore a significant number of patients with acid-
related disorders required higher or multiple doses to achieve symptom relief and
healing. Astra started a new research program in 1987 to identify a new analogue
to omeprazole with less interpatient variability. Only one compound proved
superior to omeprazole and that was the S-isomer, esomeprazole, which was
developed as the magnesium salt. Esomeprazole magnesium (Nexium) received its
first approval in 2000.
 Esomeprazole is official in Indian, British, United States and European
Pharmacopoeia.
 There are number of Spectroscopic and Chromatographic methods have been
reported for the estimation of Esomeprazole magnesium and Aspirin individually
and as well as in combination with others drugs. There are few methods reported
for determination of aspirin and esomeprazole in combination with each other.
Review of literature for Aspirin[21-32]
:-
UV methods reported for estimation of Aspirin in combination with other drugs:
Sr.
No.
Drug Sample
matrix
Experimental condition Ref.
1. Aspirin +
Rosuvastatin
calcium
Tablet A. Solvent:- methanol
B. Absorption maximum:-
ASP: 207 nm
ROS: 243 nm
C. Concentration range:-
ASP: 2-12 µg/ml
21

ROS: 3-18 µg/ml
2. Aspirin +
Rosuvastatin
calcium
Capsule A. Solvent:- 0.1 N NaOH
ASP: 241 nm
ROS: 296 nm
ASP: 10-50 µg/ml
ROS: 2-10 µg/ml
22
3. Aspirin +
Paracetamol
Tablet A. Solvent:- 0.1 N HCl : methanol (1:1)
ASP: 265 nm
PCM: 257 nm
C. Concentration range:- 2-64 µg/ml
(for both)
23
4. Aspirin +
Ramipril +
Atorvastatin
Capsule A. Absorption maximum: 210 -320 nm
B. Concentration range:
Asp: 10-50 µg/ml
RAM: 1-5 µg/ml
ATOR: 2-10 µg/ml
24
5. Aspirin +
Caffeine
Tablet A. Solvent: 0.1 N NaOH
B. Absorption maximum:
ASP: 297 nm
CAF: 272 nm
ASP: 0-40 µg/ml
CAF: 0-25 µg/ml
25
6. Aspirin +
Rosuvastatin
calcium
Tablet A. Solvent:- 0.1 N NaOH
B. Absorption maximum:
ASP: 297 nm
ROS: 242 nm
Isoabsorptive point: 287.5 nm
ASP: 5-25 µg/ml
26

ROS: 2-26 µg/ml
7. Aspirin +
Atorvastatin
calcium
Capsule A. Solvent:- methanol
ASP: 222 nm
ATOR: 242 nm
Isoabsorptive point: 232 nm
ASP: 5-30 µg/ml
ATOR: 5-40 µg/ml
27
8. Asprin +
Rosuvastatin
calcium
ASP: 225 nm
ROS: 243 nm
Isobestic point: 237.5 nm
ASP: 15-50 µg/ml
ROS: 3-21 µg/ml
28
Chromatographic methods reported for estimation of Aspirin
Sr.
No.
Drug Sample
matrix
1. Aspirin Bulk drug A. Column:- hypersil BDS C 18
B. Mobile phase:- sodium perchlorate
buffer (pH 2.5) :acetonitrile :
isopropyl alcohol (85:14:1 % v/v)
C. Flow rate:- 1.5 ml/min
D. Injection volume:- 20 µl
E. Detection wavelength:- 275 nm
F. Column temperature:- 250
C
G. Retention time:- 3.8 min
29
2. Aspirin Bulk and
tablet
A. Column: C 18
B. Mobile phase:- water : acetonitrile
(50:50)
30

ASP: 6-14 µg/ml
ROS: 45-105 µg/ml
D. Flow rate:- 1.0 ml/min
F. Retention time:-
ASP: 3.44 min
ROS: 4.30 min
Chromatographic methods reported for estimation of Aspirin in combination with
other drugs
Sr.
No.
Drug Sample
matrix
1. Aspirin +
Rosuvastatin
calcium
Capsule A. Column:- C 18
B. Mobile phase:- Acetonitrile in ratio
50:50 (%v/v) pH adjusted to 4.0 with
orthophosphoric acid
C. Flow rate:- 1.0 ml/min
E. Run time:- 6 min
F. Detection wavelength:- 243 nm
G. Retention time:-
ASP: 3.44 min
ROS: 4.30 min
31
2. Aspirin +
Paracetamol
Tablet A. Column:- Phenomenex-luna C8
B. Mobile phase:- Acetonitrile :
phosphate buffer pH 7.0 (60:40)
%v/v
E. Injection volume:- 20 µl
32

G. Retention time:-
ASP: 4.85
PCM: 3.05
Review of literature for Esomeprazole:-[33-45]
UV methods reported for estimation of Esomeprazole
Sr.
No.
Drug Sample
matrix
1. Esomeprazole Tablet A. Solvent:- methanol
B. Concentration range:- 5-40 µg/ml
C. Absorption maxima:- 303 nm
(method A)
D. Zero crossing point:- 303 nm with
sharp peak at 292 nm (method B)
294nm -310 nm for area under curve
(method C)
33
UV methods reported for estimation of Esomeprazole in combination with other
drugs:
Sr.
No.
Drug Sample
matrix
1. Esomeprazole
+
Levosulpiride
ESO: 300 nm
LEVO:- 234 nm
34
2. Esomeprazole
magnesium
trihydrate +
Diclofenac
sodium
Bulk
drug &
synthetic
mixture
A. Solvent:- methanol : water
B. Diluents:- methanol : water
C. Absorption maxima:-
Simultaneous equation method
ESO: 301 nm
DIC: 280 nm
Q- method
35

ESO: 302.80 nm
Dic: 280 nm
D. Concentration range:-
ESO: 2-10 µg/ml
DIC: 5-25 µg/ml
3. Esomeprazole
magnesium +
Domperidone
Capsule A. Solvent:- methanol and various
analytical grade reagents for
degradation studies
B. Absorption maxima:-
ESO: 299 nm
DOM: 287 nm
ESO: 1-6 µg/ml
DOM: 5-30 µg/ml
36
4. Esomeprazole
magnesium
trihydrate +
Naproxen
Tablet A. Solvent: methanol
B. Absorption maxima:
ESO: 302 nm
NAP: 276 nm
ESO: 1-11 µg/ml
NAP: 10-35 µg/ml
37
5. Esomeprazole
+
Domperidone
Pure drug
and
capsule
A. Solvent:- methanol, 0.1 NaOH
B. Absorption maxima:-
ESO: 301 nm
DOM: 285 nm
ESO: 5-20 µg/ml
DOM: 8-30 µg/ml
38
Chromatographic methods reported for estimation of Esomeprazole
Sr.
No.
Drug Sample
matrix
1. Esomeprazole Tablet A. Column:- Zodiac C18 39

B. Mobile phase:- acetonitrile :
methanol (20:80)
C. Detection wavelength:- 274 nm
D. Retention time:- 3.30 min
2. Esomeprazole Bulk drug A. Column:- prevail C8
phosphate buffer (35 : 65)
E. Injection volume:- 20 µl
F. Retention time:- 5.957 min
40
3. Esomeprazole Bulk drug
and
capsule
A. Column:- C18
phosphate buffer (50 : 50)
E. Run time:- 10 min
F. Retention time:- 6.5 min
41
Chromatographic methods reported for estimation of Esomeprazole in combination
with other drugs
Sr.
No
.
Drug Sample
matrix
1. Esomeprazole
+ Naproxen
Tablet A. Column:- L 1 (X Terro RP 18)
B. Mobile phase:-
A:- Buffer pH 8.7 : acetonitrile :
methanol (70:20:10)
B:- Buffer pH 8.7 : acetonitrile
(20:80)
C. Diluent 1:- 800 ml methanol: 200 ml
milli-Q water: 4 ml triethylamine
Diluent 2:- 0.25 N NaOH
42

Diluent 3:- mobile phase A
D. Retention time:-
ESO: 6.112 min
NAP: 3.352 min
2. Esomeprazole
+
Domperidone
(HPTLC)
Tablet A. Stationary phase:- Alum.foil
precoated with silica gel 60F254
B. Mobile phase:- Toluene : ethyl
acetate : methanol (2:7:0.5)
C. Concentration:-
ESO: 200-600 ng/spot
DOM: 400-1200 ng/spot
43
3. Esomeprazole
+ Naproxen
Bulk drug A. Column:- C18
B. Mobile phase:- phosphate buffer :
acetonitrile (60 :40)
D. Retention time:-
ESO: 2.105 min
NAP: 3.555 min
E. Flow rate:- 1.0 ml/min
F. Run time:- 7 min
G. Injection volume:- 20 µl
44
4. Esomeprazole
+ Naproxen
(RP-UPLC
PDA method)
Tablet A. Column:- C18
B. Mobile phase:- potassium dihydrogen
phosphate buffer :acetonitrile (pH of
buffer adjusted to 2.8 with
orthphosphoric acid) (40 : 60)
E. Run time:- 4 min
F. Injection volume:- 20 µl
G. Retention time:-
ESO: 0.713 min
NAP: 1.264 min
45

Review of literature for Aspirin and Esomeprazole in combination:-[49,47]
Chromatographic methods reported for estimation of Aspirin and esomeprazole in
combination
Sr.
No.
Drug Sample
matrix
1. Aspirin +
Esomeprazole
magnesium
Tablet A. Column:- hyperchrom ODS-BP C18
methanol : 0.05 M phosphate buffer
at pH 3 adjusted with
orthophosphoric acid (25:25:50 v/v)
C. Flow rate:- 1 ml/min
E. Run time:- 10 min
G. Retention time:-
ASP: 4.286 +/- 0.03983 min
ESO: 6.088 +/- 0.10740 min
46
2. Aspirin +
Esomeprazole
magnesium
Binary
mixture
A. Column:- hypersil C 18
B. Mobile phase:- methanol :
acetonitrile (90:10 v/v)
C. Flow rate:- 1 ml/min
D. Temperature:- 250
C
F. Retention time:-
ASP: 1.92 +/- 0.3 min
ESO: 3.4 +/- 0.05 min
47

3. AIM AND OBJECTIVE:-
With the reference of few articles it can be determined that aspirin and esomeprazole in
combined dosage form can be used for the prevention of cardiovascular events. The
addition of esomeprazole to low dose aspirin therapy in patients at high risk of developing
gastric ulcers for the prevention of cardiovascular disease, significantly reduced their risk
of ulcer development. At present there are few methods reported for analysis of aspirin
and esomeprazole in combined form.
Aim of work:-
Development and validation of analytical method for estimation of Aspirin and
Esomeprazole in bulk and combination by spectroscopic and chromatographic techniques.
Objective of work:-
To develop and validate simple, sensitive, rapid, accurate, precise and cost effective
spectrophotometric and chromatographic method using Aspirin and Esomeprazole.
The objective of an above method is to develop an analytical method, which can be used
to measure accurately and specifically the analyte in the presence of components that may
be expected to be present in sample matrix.
The developed method was validated as per ICH guidelines.
It was carried out in 3 steps.
1. Method development
2. Method validation
3. Method application

1. Method development
Wavelength selection
Selection of detectable wavelength, which will sense the analyte
of interest in sample.
Literature survey
To collect information related to drug substance. Its physical,
chemical and pharmacological properties.
Procurement of drug sample
To procure working standard of drug having high degree of
purity.
Selection of solvent
To check the solubility and determine the polarity of the drug in
particular solvent for complete separation and resolution.
Best column Best mobile
phase
Best condition Best system
Chromatographic condition
To achieve separation goal

2. Method validation
Linearity
To check the ability to obtain test results which are directly proportional
to the concentration of analyte in sample.
Range
To determine the interval between the upper and lower concentration of
the analyte in the sample.
Accuracy
To determine the closeness of agreement between the values which is
accepted either as a conventional true value or an accepted reference
value and the value found.
Robustness
To study the effect of deliberate variations by altering the concentration
of solvent to check method capacity to remain unchanged.
Precision
To determine the closeness of agreement between the series of
measurement from multiple sampling of homogenous sample.
Statistical validation
To know that to what extent it is reliable.

3. Method application
To check the utility of developed and validated analytical method by applying in
analysis of Aspirin and Esomeprazole.

4. DRUG PROFILES[48-53]
1. ASPIRIN:-
Structure:
Figure 3 : Structure of aspirin
Generic name Aspirin
Molecular formula C9H8O4
IUPAC name 2-(acetyloxy)benzoic acid
Molecular weight 180.157 g/mol
CAS number 50-78-2
Category Anti inflammatory, Analgesic, Antipyretic,
antithrombotic
Description White color granules
Solubility Slightly soluble in water, Freely soluble in methanol
Pka 3.49 (at 250
C)
Log p 1.19
Melting point 1350
C
Metabolism Aspirin is rapidly hydrolyzed primarily in the liver to
salicylic acid, which is conjugated with glycine (forming
salicyluric acid) and glucuronic acid and excreted largely
in the urine.
Bioavailability 68 %
Protein binding High (99.5 %) to albumin. Concentration increases, with
reduced plasma albumin concentration or renal
dysfunction and during pregnancy.

Half life 0.25 hour
Storage Store oral forms at room temperature in tightly closed
containers.
Administration Take this medicine after meals or with food to lessen
stomach irritation (except for enteric coated capsules or
tablets and aspirin suppositories).
Indication For use in the temporary relief of various forms of pain,
inflammation associated with various conditions
(including rheumatoid arthritis, juvenile rheumatoid
arthritis, systemic lupus erythematosus, osteoarthritis and
ankylosing spondylitis), and is also used to reduce the
risk of death and/or nonfatal myocardial infarction in
patients with a previous infarction or unstable angina
pectoris.
Contraindication Hypersensitivity to salicylates or NSAIDs; hemophilia,
bleeding ulcers, or hemorrhagic states.
Special precaution Pregnancy- category D.
Lactation- excreted in breast milk.
Children- Reye syndrome has been associated with
aspirin administration in children (including teenagers)
with acute febrile illness.
Hypersensitivity- reaction may include bronchospasm
and generalized urticaria or angioedema; patients with
asthma have greatest risk.
Renal function- may cause renal dysfunction or
aggravate kidney diseases.
Hepatic function- may cause hepatotoxicity in patients
with impaired liver function.
GI disorders- can cause gastric irritation and bleeding.
Surgical patients- aspirin may increase risk of
postoperative bleeding. If possible, avoid use 1 week
before surgery.
Adverse drug reaction EENT- dizziness, tinnitus.

GI- bleeding, dyspepsia, heartburn, nausea.
Hematologic- anemia, decreased iron concentration,
increased bleeding times.
Miscellaneous- hypersensitivity reactions may include
urticaria, hives, rashes, angioedema and anaphylactic
shock.
Drug interaction Alcohol- may increase the risk of GI ulceration and
prolong bleeding time.
Antacids, corticosteroids, urinary alkalinizers- may
decrease aspirin levels.
Carbonic anhydrase inhibitors (e.g. acetohexamide),
methotrexate, valproic acid- may increase level of these
drugs.
Heparin, oral anticoagulants- may increase risk of
bleeding.
Insulin, sulfonylureas- aspirin (more than 2 g/day) may
potentiate glucose lowering.
Probenecid, sulfinpyrazone- may decrease uricosuric
effect.
Food interaction Alcohol appears to cause 50 to 100 % increase in aspirin
serum levels.
Mechanism of action:-
The analgesic, antipyretic, and anti-inflammatory effects of acetylsalicylic acid are due to
actions by both the acetyl and the salicylate portions of the intact molecule as well as by
the active salicylate metabolite. Acetylsalicylic acid directly and irreversibly inhibits the
activity of both types of cyclooxygenase (COX-1 and COX-2) to decrease the formation of
precursors of prostaglandins and thromboxanes from arachidonic acid. The platelet
aggregation-inhibiting effect of acetylsalicylic acid specifically involves the compound's
ability to act as an acetyl donor to cyclooxygenase; the nonacetylated salicylates have no
clinically significant effect on platelet aggregation. Irreversible acetylation renders

cyclooxygenase inactive, thereby preventing the formation of the aggregating agent
thromboxane A2 in platelets. Since platelets lack the ability to synthesize new proteins, the
effects persist for the life of the exposed platelets (7-10 days). Acetylsalicylic acid may
also inhibit production of the platelet aggregation inhibitor, prostacyclin (prostaglandin
I2), by blood vessel endothelial cells.
2. ESOMEPRAZOLE:-
Structure:
Figure 4 : Structure of Esomeprazole
Generic name Esomeprazole
Molecular formula C17H19N3O3S
IUPAC name (S)-5-methoxy-2-[(4-methoxy-3,5-dimethylpyridin-2-
yl)methylsulfinyl]-3H-benzoimidazole
Molecular weight 345.417 g/mol
CAS number 161796-78-7
Category Proton pump inhibitor, antiulcerative, antihistamines
Description Pale yellow powder
Solubility Freely soluble in ethanol, methanol; slightly soluble in
acetone, isopropanol, very slightly soluble in water.
Pka 3.97
Log p 1.66
Melting point 1560
C
Metabolism Mainly hepatic. Esomeprazole is completely metabolized
N
H3C
OCH3
CH3
S
O
N
H
N
OCH3

by the cytochrome P450 system via CYP2C19 and
CYP3A4. Metabolism produces inactive hydroxyl and
desmethyl metabolites, which have no effect on gastric
acid secretion. Less than 1 % of parent drug is excreted
in urine.
Bioavailability 50 to 90 %
Protein binding 97 %
Half life 1-1.5 hours
Storage Store at room temperature in tightly closed container.
Administration Orally
Indication For the treatment of acid-reflux disorders (GERD),
peptic ulcer diseases, H.pylori eradication and
prevention of gastrointestinal bleeds with NSAID use.
Contraindication ESO is contraindicated in patients with known
hypersensitivity to proton pump inhibitors.
Special precaution Do not use esomeprazole if patient is allergic to it, if
patient is taking atazanavir, clopidogrel, dasatinib,
nelfinavir, rifampin, rilpivirine, or St. John's wort
Adverse drug reaction Common side effects include headache, diarrhea, nausea,
flatulence, decreased appetite, constipation, dry mouth,
and abdominal pain. More severe side effects are severe
allergic reactions, chest pain, dark urine, fast heartbeat,
fever, paresthesia, persistent sore throat, severe stomach
pain, unusual bruising or bleeding, unusual tiredness,
and yellowing of the eyes or skin.
Drug interaction Diazepam- esomeprazole can potentially increase the
concentration in blood of diazepam by decreasing the
elimination of diazepam in the liver.
Ketoconazole- esomeprazole reduce the absorption and
concentration of ketoconazole in blood and lead to
reduce effectiveness of ketoconazole.
Digoxin- esomeprazole increase the absorption and
concentration of digoxin in blood and leads to increase

digoxin toxicity.
Saquinavir, nelfinavir and atazanavir- esomeprazole
increase the blood level of sequinavir and reduce the
blood levels of nelfinavir and atazanavir.
Clopidogrel- ESO reduces the activity of clopidogrel.
Cilostazol- ESO increases the concentration of cilostazol
and its metabolites.
Food interaction Food may interfere with the absorption of esomeprazole.
Esomeprazole should be taken at least one hour before
meals and at the same time every day. This will make it
easier for your body to absorb the medication.
Mechanism of action:-
Esomeprazole is a proton pump inhibitor that suppresses gastric acid secretion by specific
inhibition of H+/K+-ATPase in the gastric parietal cell. By acting specifically on the
proton pump, esomeprazole blocks the final step in acid production, thus reducing gastric
acidity.

5. EXPERIMARNTAL WORK
5.1 EQUIPMETNS & MATERIALS:
5.1.1 INSTRUMENTS:
 Ultraviolet-visible spectrophotometer
 Model:- Agilent cary 60
 Make:- Agilent technologies
 Software:- Cary win UV
 High performance liquid chromatography (HPLC)
 Model:- LC 100
 Make:- Cyberlab
 Column:- C18 column
 Particle size:- 5 µm
 Pump:- LC-P-100 binary system
 Injector:- Mannual rheodyne injector 7725i six-port sample injection valve
with 25 µL fixed loop
 Detector:- UV-Visible detector
 Wavelength:- 237, 275, 300 nm
 Software:- HPLC
 Infra red spectrophotometer:
 Model:- Alpha spectrophotometer
 Make:- Bruker
 Scan range:- 600 cm-1
to 4000 cm-1
 Software:- OPUS/Mentor
 Analytical balance:- Precisa, XB 220A
 Sonicator:- Equitron, digital ultrasonic cleaner

Figure 5: UV-visible instrument
Figure 6: Digital balance

Figure 7: IR instrument
Figure 8: HPLC system

5.1.2 MATERIALS:
 Aspirin working standard- procured as gift sample from West coast pharma,
Ahemdabad, Gujarat.
 Esomeprazole sodium working standard- procured as a gift sample from
Sovereign pharma, Daman.
5.1.3 REAGENTS:
 Methanol (laboratory reagent) – was obtained from Astron chemicals,
Ahemdabad, India.
 Methanol, THF, water (HPLC grade) – RFCL limited, Ankleshwar, Gujarat.
5.2 Identification of drugs
These drugs were identified by following ways:
5.2.1 By melting point:
Table 4: Identification of drug by melting point
NAME OF DRUG STANDARD MELTING
POINT RANGE
OBSERVED MELTING
POINT RANGE
Aspirin 133 -1360
C 135 – 1370
C
Esomeprazole 154 -1570
C 154 – 1560
C

5.2.2 Identification by DSC:
Figure 9: DSC of ASP
Figure 10 : DSC of ESO Na

5.2.3 Identification of drug by IR spectroscopy:
IR spectroscopy study was performed by preparing pellets of drug and KBr using
hydraulic press and FT-IR was scanned from 4000 cm-1
to 600 cm-1
. IR spectra was
compared with the reference standard.
ASPIRIN
Figure 11: IR spectra of Aspirin (API)
Figure 12: IR spectra of Aspirin (standard)

Table 5: IR correlation of Aspirin
Sr.
No.
Observed Frequency (cm-1
) Types of Vibration
1. 1681.75 cm-1
C = O in acid
2. Broad peak between 3273.27
to 2402.16 cm-1
O – H in acid
3. 1749.58 cm-1
C = O in ester
4. 1178.62 cm-1
1295.40 cm-1
C – O in ester
5. Sharp peak between 900-600
cm-1
Aromatic ring
6. 2919.51 cm-1
C – H stretching
7. 1455.04 cm-1
C = C in aromatic ring
ESOMEPRAZOLE
Figure 13: IR spectra of Esomeprazole (API)

Table 6: IR correlation of Esomeprazole
Sr. No. Observed Frequency (cm-1
) Types of Vibration
1. 3353.39 N - H
2. 1151.90 C – N stretching
3. 1568.31 C = C in aromatic ring
4. 1075.85 S = O
5. 2938.84 C – H stretching
5.3 DEVELOPMENT AND VALIDATION OF UV SPECTROPHOTOMETRIC
METHOD
5.3.1 EXPERIMENTAL
Preparation of standard stock solution
ASP (100 mg) and ESO Na(107 mg) were accurately weighed and transferred to two
separate 100 ml volumetric flasks and dissolve them in few ml of methanol. Volumes were
made up to mark with methanol to yield solution containing 1000 µg/ml of ASP and ESO.
Appropriate aliquots from stock solution of ASP and ESO were taken and diluted with
mobile phase to obtain final concentration of 100 µg/ml of ASP and ESO.
Calibration curve for ASP and ESO
Appropriate aliquots of ASP stock solution (2, 4, 6, 8, 10 ml) were taken in different 10 ml
volumetric flasks. To the another flask aliquots of ESO stock solution (0.5, 1.0, 1.5, 2.0,
2.5 ml) were taken in different 10 ml volumetric flask. Volume was made up to the mark
with solvent and final concentration of 20, 40, 60, 80, 100 µg/ml of ASP and 5, 10, 15, 20,
25 µg/ml of ESO.

Determination of A (1%, 1 cm) values of drugs at selected wavelengths:
A (1%, 1 cm) values of drugs were calculated using the following formula:
(1)
A set of equations for absorbance ratio method were framed using these A (1%, 1cm)
values which are given below:
QX = ax2/ax1, QY = ay2/ay1, QM = A2/A1 (2)
Where A1 and A2 are absorbance of mixture at 237 nm and 275 nm; ax1 and ax2, A (1%,
1cm) of ESO at 237 nm and 275 nm; ay1 and ay2, A (1%, 1cm) of ASP at 237 nm and 275
nm respectively. CESO and CASP are concentration of ESO and ASP in mixture.
Concentration CESO and CASP can be calculated by following formula-
X
= X (3)
Validation of UV method
Linearity of calibration curves
Linearity of method was evaluated by constructing calibration curves at five concentration
levels over a range of 20 – 100 µg/ml for ASP and 5 – 25 µg/ml for ESO. The calibration
curves were developed by plotting peak area versus concentration (n = 5).
Accuracy
The accuracy of method was determined by calculating recoveries of ASP and ESO
method of standard additions. Known amount of ASP (50 %, 100 %, 150 %) were added
to a prequalified sample solution and the amount of ASP and ESO were estimated by
measuring the peak area and by fitting these values to the straight line equation of
calibration curve.
Precision
The intra-day and inter-day precision studies were carried out by estimating the
corresponding responses 3 times on the same day and on next day for 3 different

concentration for ASP (20,60,100 µg/ml) and ESO (5,15,25 µg/ml) and the results were
reported in terms of relative standard deviation.
LOD and LOQ
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can
reliably be differentiated from background levels.
The limit of quantification (LOQ) of an individual analytical procedure is the lowest
amount of analyte that can be quantitatively determined with suitable precision and
accuracy. LOD and LOQ were calculated using following equation as per ICH guidelines.
LOD = 3.3 × σ / S
LOQ = 10 × σ / S
Where σ is the standard deviation of Y- intercepts of regression lines and S is the slope of
calibration curve.
Analysis of synthetic mixture
Withdraw 5 ml solution from 100 µg/ml solution of ESO and dilute up to 10 ml (50
µg/ml). Withdraw appropriate aliquot from 50 µg/ml solution of ESO and from 100 µg/ml
solution of ASP and transfer it to 10 ml volumetric flask and make up the volume with
solvent.
5.4 DEVELOPMENT AND VALIDATION OF HPLC METHOD
5.4.1 EXPERIMENTAL
Preparation of mobile phase
Mobile phase was prepared by mixing methanol, water and tetrahydrofuran in the ratio of
70 : 30 : 0.05 %. Solution was filtered, degassed and sonicated for 10 minutes and used as
a mobile phase.
Preparation of standard stock solution
ASP (100 mg) and ESO Na(107 mg) were accurately weighed and transferred to two
separate 100 ml volumetric flasks and dissolve them in few ml of methanol. Volumes were
made up to mark with methanol to yield solution containing 1000 µg/ml of ASP and ESO.

Chromatographic solution
A Kromasil C18 (250 × 4.6 mm i.d.) column equilibrated with mobile phase methanol :
water : THF (70 :30: 0.05 v/v/v) was used. Mobile phase rate was maintained at 1 ml/min.
and effluents were monitored at 237 nm. The sample was injected using a 20 µl fixed loop
and the total run time was 7 minutes.
Calibration curve for ASP and ESO
Appropriate aliquot of ASP stock solution was taken in different 10 ml volumetric flask.
To the same flask different aliquot of ESO stock solution was added , volume was made
up to the mark with the mobile phase to obtain final concentration of 20,40,60,80,100,120
µg/ml of ASP and 5,10,15,20,25,30 µg/ml of ESO.
Validation of HPLC method
Linearity of calibration curves
Linearity of the method was evaluated by constructing calibration curves at six
concentration levels over a range of 20 -120 µg/ml and 5 – 30 µg/ml for ASP and ESO
respectively. The calibration curves were developed by plotting peak area versus
concentration.
Accuracy
The accuracy of method was determined by calculating recoveries of ASP and ESO by
method of standard additions. Known amount of ASP (50 %, 100 %, 150 %) and ESO (50
%, 100 %, 150 %) were added to a prequalified sample and the amount of ASP and ESO
were estimated by measuring the peak area and fitting these equation to the straight line
equation of calibration curve.
Precision
The precision of an analytical method is the degree of agreement among individual test
results when the method is applied repeatedly to multiple samplings of homogenous
samples. It provides an indication of random error results and was expressed as % RSD.
Precision is evaluated in terms of intra-day and inter-day precision. Intra-day precision
was determined by analyzing sample solutions of ASP (20, 60, 120 µg/ml) and ESO (5,

15, 30 µg/ml) at three levels covering low, medium and high concentrations of the
calibration curve three times on the same day (n = 3). Inter-day precision was determined
by analyzing sample solutions of ASP (20, 60, 120 µg/ml) and ESO (5, 15, 30 µg/ml) at
three levels covering low, medium and high concentrations over a period of time. The
peak areas obtained were used to calculate mean and % RSD values.
LOD and LOQ
The limit of detection (LOD) is defined as the lowest concentration of an analyte that can
reliably be differentiated from background levels. The limit of quantification (LOQ) of an
individual analytical procedure is the lowest amount of analyte that can be quantitatively
determined with suitable precision and accuracy. LOD and LOQ were calculated using
following equations as per ICH guidelines.
LOD = 3.3 × σ / S
LOQ = 10 × σ / S, where σ is the standard deviation of the response of regression lines and
S is the slope of calibration curve.
Robustness
Small changes in the flow rate and the ratio of mobile phase were carried out and effects
on the results were examined. Robustness of the method was determined in triplicate at a
concentration level of 60 µg/ml and 15 µg/ml of ASP and ESO respectively. The mean
and the % RSD of peak areas were calculated.
Weigh accurately 100 mg of ASP, transfer it to the 100 ml volumetric flask and dissolve it
in few ml of methanol (A). Now, weigh accurately 107 mg of ESO Na which is equivalent
to 100 mg of ESO, transfer it to the 100 ml volumetric flask and dissolve it in few ml of
methanol (B). Sonicate both the solutions and filter through the membrane filter paper.
Make up the final volume with methanol.
Withdraw 40 ml from the solution A and 10 ml from the solution B and transfer both the
solutions to the same 100 ml volumetric flask and make up the final volume with
methanol. Withdraw 1.5ml from the above solution and transfer it to separate 10 ml

volumetric flasks and make up the volume with methanol to make the final concentration.
Concentration ranges obtained are 60µg/ ml and 15µg/ml for ASP and ESO respectively.

Chapter 6
Results And
Discussion

6. RESULTS AND DISCUSSION
6.1 UV method
6.1.1 Selection of wavelength
10 µg/ml solutions of ASP and ESO were used to detect the wavelength.
A. 10 µg/ml solution of Aspirin in methanol:-
Figure 14 : UV spectra of 10 µg/ml solution of ASP in methanol
Table 7 : Wavelength maxima of Aspirin
Sr. No. Wavelength Absorbance
1 275 0.623
2 205 0.391

B. 10 µg/ml solution of ESO in methanol:-
Figure 15 : UV spectra of 10 µg/ml solution of ESO in methanol
Table 8 : Wavelength maxima of Esomeprazole
Sr. No. Wavelength Absorbance
1 300 0.372
2 210 0.817
Figure 16 : Overlain of 10 ppm solution of ASP and ESO

6.1.2 Validation
Linearity
The six point calibration curve that were constructed were linear over the selected
concentration range for both ESO and ASP that were 5-25 µg/ml for ESO and 20-100
µg/ml for ASP. Each concentration was repeated for 5 times. The linearity of the
calibration graphs and adherence of the system to Beer’s law were validated by the high
value of the correlation coefficient and the intercept value.
Table 9 : Statistical data for ASP and ESO by UV method
Parameter ASP ESO
Linearity 20-100 µg/ml 5-25 µg/ml
Correlation coefficient 0.9998 0.9976
Standard deviation of slope 0.025882 0.00016432
Standard deviation of
intercept
0.013307 0.00086487
Figure 17 : Calibration Curve of ESO at 237 nm and at 275 nm
y = 0.0322x - 0.1165
R² = 0.9976
0
0.2
0.4
0.6
0.8
0 10 20 30
Absorbance(nm)
Concentration (µg/ml)
Calibration curve
at 237 nm
y = 0.0243x - 0.0513
R² = 0.9989
0
0.1
0.2
0.3
0.4
0.5
0.6
0 10 20 30
Absorbance(nm)
Calibration curve
at 275 nm

Figure 18 : Calibration Curve of ASP at 237 nm and at 275 nm
Precision
The % RSD for intraday precision was found to be range of 0.325685 for ESO and
0.085951 for ASP. The % RSD for interday was found to be range of 0.413003 for ESO
and 1.202587 for ASP. The RSD values within the limit indicate that the method is
precise.
Table 10 : Intraday precision for ASP and ESO
Name of Drug Avg. Abs. (n=6) % RSD
ASP (60 µg/ml) 1.540717 0.085951
ESO (15 µg/ml) 0.3549 0.325685
Table 11 : Interday precision for ASP and ESO
Name of Drug Avg. Abs. (n=6) % RSD
ASP (60 µg/ml) 1.526183 1.202587
ESO (15 µg/ml) 0.350683 0.413003
y = 0.0268x - 0.1137
R² = 0.9959
0
0.5
1
1.5
2
2.5
3
0 50 100 150
Absorbance(nm)
Calibration curve
at 237 nm
y = 0.0066x - 0.0518
R² = 0.9998
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 50 100 150
Absorbance(nm)
Calibration curve at
275 nm

Accuracy
Accuracy of the method was checked by the recovery studies at 3 different levels, that is
50%, 100%, 150% and amount recover for ESO were 100.34%, 100.26%, 100.51% and for
ASP were 99.79%, 99.87% and 99.72%.
Table 12 : Accuracy study of ASP & ESO by the proposed UV method
Recovery
level
Initial amount
(µg/ml)
Concentration of
standard drug
added(µg/ml)
% Recovery
ESO ASP ESO ASP ESO ASP
0% 10 40 0 0 100.23 99.52
50% 10 40 5 20 100.34 99.79
100% 10 40 10 40 100.26 99.87
150% 10 40 15 60 100.51 99.72
Limit of detection and limit of quantification
The detection limits for ASP and ESO were 5.150829 µg/ml and 0.088307 µg/ml,
respectively at 237 nm, while quantitation limits were 15.60857 µg/ml and 0.267596 µg/ml
respectively at 237 nm. The above data shows that a microgram quantity of both the drugs
can be accurately and precisely determined.
The proposed was applied for the synthetic mixture and % label claim of ESO and ASP
was found to be 99.93% and 100.14% respectively.
Table 13: Analysis of synthetic mixture
Name of drug Amount taken
(µg/ml)
Amount found
(µg/ml)
% amount recovered
(n=3)
ESO 15 14.93757451 99.58383
ASP 60 60.0295091 100.0492

Table 14: Parameters and results
Sr.
No.
Parameters Esomeprazole Aspirin
237 nm 275 nm 237 nm 275 nm
1 Beer’s Law (µg/ml) 5-15 5-15 20-100 20-100
2 Slope 0.03232 0.02824 0.02624 0.0066
3 Intercept 0.11686 0.04736 0.09418 0.0458
4 Correlation Cofficient
(r2
)
0.997 0.998 0.995 0.998
5 Limit of Detection 0.088307 1.069359 5.150829 2.178252
6 Limit of Quantitation 0.267596 3.240482 15.60857 6.600764
6.2 RP-HPLC METHOD
6.2.1 Optimization of mobile phase
The objective of the method development was to resolve chromatographic peaks for active
ingredients.
Various mixtures containing methanol and water were tried as mobile phase in the initial
stage of method development. Mixture of methanol : water (80 : 20 v/v), methanol : water
(50 : 50 v/v), methanol : water : tetrahydrofuran (60:40:0.05 v/v/v) were tried as mobile
phase but satisfactory resolution of drugs and peaks were not achieved.
The mobile phase Methanol : Water : Tetrahydrofuran (70 : 30 : 0.05 v/v/v) was found to
be satisfactory and gave two symmetric and well-resolved peaks for ASP and ESO. The
retention time for ASP and ESO were 1.81 min and 4.83 min, respectively. It indicates
good separation of both the compounds. The mobile phase flow rate was maintained at 1
ml min-1
.
6.2.2 Selection of detection wavelength
The sensitivity of HPLC method that uses UV detection depends upon proper selection of
detection wavelength. An ideal wavelength is the one that gives good response for the
drugs that are to be detected. Overlay UV spectra of both the drugs showed that ASP and
ESO absorbed appreciably at 237 nm, so detection was carried out at 237 nm.

Table 15: Optimized chromatographic conditions for HPLC
Parameters Conditions
Mobile phase Methanol : water : THF (70 : 30 : 0.05 v/v/v)
Column Kromasil C18 column, 250 × 4.6 mm i.d.(5 µm particle size)
Detection
wavelength
237 nm
Flow rate 1ml/ min
Temperature 250
C
Retention time 1.81 min and 4.83 min
Run time 7 min
Syringe size 20 µl
6.2.3 Validation
Linearity
The calibration curve for ASP was found to be linear in the range of 20-120 µg/ml with a
correlation coefficient of 0.999. the calibration curve for ESO was found to be linear in
range of 5-30 µg/ml with a correlation coefficient of 0.999. the regression analysis of
calibration curves are reported in table
Table 16 : Statistical data for ASP and ESO by HPLC method
Parameter ASP ESO
Linearity 20-120 µg/ml 5-30 µg/ml
Correlation coefficient 0.999 0.999
Standard deviation of slope 14.84251 18.20165
Standard deviation of
intercept
995.0426 94.21383
Precision
Instrument precision was determined by performing injection repeatability test and the %
RSD values for the ASP and ESO were found to be 0.294 and 0.925 respectively. The

intra-day and inter-day precision studies were carried out and the results are reported in
table 17 and 18.
Table 17 :Repeatability of sample application data for ASP and ESO
Concentration ASP
60 µg/ml
ESO
15 µg/ml
Area
(mAU)
104952.1 39813.6
105671 39892.4
105473.6 39854.1
104982.6 39814.3
105267.8 40513.4
105746.5 39239.8
Mean 105348.9 39854.6
Std. Dev 309.7277 368.9468
% RSD 0.294002 0.925732
Table 18 : Precision data for ASP
Conc.
µg/ml
Intraday Area
(mAU)
(n=3)
% RSD Interday Area
(mAU)
(n=3)
% RSD
20 37228.03 0.78605 37621.5 0.638201
60 103477.6 0.116146 104545.3 0.654173
120 206884.1 0.58986 207232.4 0.417697

Table 19 : Precision data for ESO
Conc.
µg/ml
Intraday Area
(mAU)
(n=3)
% RSD Interday Area
(mAU)
(n=3)
% RSD
5 13255.87 0.65365 13292.27 0.976388
15 39977.93 0.334746 39890.33 0.871002
30 78983.13 0.213733 78756.97 0.602816
Accuracy
The accuracy of method was determined by calculating recoveries of ASP and ESO by
method of addition. The recoveries found to be 99.27 – 100.197 % and 98.884 – 101.057
% for ASP and ESO respectively. The high values indicate that the method is accurate.
Table 20: Accuracy study of ASP & ESO by the proposed HPLC method
Amount of
sample taken
(µg/ml)
Amount of
standard drug
added
(µg/ml)
Amount of drug
recovered
(µg/ml)
% recovery ± % RSD (n=3)
ASP ESO ASP ESO ASP ESO ASP ESO
40 10 0.0 0.0 40.038 9.888 100.097 ± 0.974 98.884 ± 0.454
40 10 20 5 60.515 14.937 100.197 ± 0.962 99.585 ± 0.737
40 10 40 10 80.071 20.119 100.089 ± 0.902 100.597 ± 0.42
40 10 60 15 99.273 25.264 99.273 ± 0.372 101.057 ± 0.202
Limit of detection and limit of quantification
The detection limits for ASP and ESO were 1.958512 µg/ml and 0.118107 µg/ml,
respectively, while quantification limits were 5.934884 µg/ml and 0.357901 µg/ml
respectively. The above data shows that a microgram quantity of both the drugs can be
accurately and precisely determined.

Robustness
Robustness of method was studied by changing the flow rate of the mobile phase from 1
ml min-1
to 0.9 ml min-1
and 1.1 ml min-1
. Using 1.1 ml min-1
flow rate, retention time for
the ASP and ESO were observed to be 1.63 and 4.56 min respectively and with 0.9 ml
min-1
, the retention time for ASP and ESO were found to be 1.99 and 5.49 min
respectively without affecting resolution of the drug. When a mobile phase composition
was changed to methanol : water : THF (65 : 35 : 0.05 v/v/v) by increasing the percentage
of water the retention time for ASP and ESO were observed to be 1.78 and 6.20 min
respectively. When a mobile phase composition was changed to methanol : water : THF
(75 : 25 : 0.05 v/v/v) by decreasing percentage of water the retention time for ASP and
ESO were observed to be 1.74 and 4.28 min respectively. The assay result of both drugs
were found to be more than 98 %.
Table 21: Robustness results of ASP and ESO
Parameter Method
condition
Rt % RSD of peak area
ASP ESO ASP ESO
Flow rate 0.9 ml/min 1.99 5.49 0.367241 1.351209
1.1 ml/min 1.63 4.56 0.150274 0.32851
Mobile phase ratio
Methanol : water :
THF
65 : 35 : 0.05 1.78 6.20 0.193731 0.370898
75 : 25 : 0.05 1.74 4.28 0.377069 0.969278
6.2.4 Analysis of synthetic mixture
Synthetic mixture was analyzed by proposed method which gave percentage recovery for
ASP and ESO were more than 98 % respectively.
Table 22: Analysis of synthetic mixture
Formulation Amount taken
(µg/ml)
Amount found
(µg/ml)
% of drug found ± RSD
ASP ESO ASP ESO ASP ESO
Synthetic
mixture
61.4458 15.06572 102.0097
±
0.146591
101.102 ±
0.749904

Figure 19 : Chromatogram of ASP and ESO using methanol : water (80 : 20)
Figure 20 : Chromatogram of ASP and ESO using methanol : water (50 : 50)

Figure 21 : Chromatogram of ASP and ESO using methanol : water : THF (60 : 40 : 0.05)
Figure 22 : Chromatogram of standard Asp and ESO ( 60 µg/ml and 15 µg/ml
respectively) using mobile phase methanol : water : tetrahydrofuran (70 : 30 : 0.05 v/v/v)

Figure 23 : Chromatogram of ASP and ESO of synthetic mixture ( 60 µg/ml and 15 µg/ml
respectively ) using mobile phase methanol : water : tetrahydrofuran (70 : 30 : 0.05 v/v/v)
Figure 24 : Multicurve report

Table 23: Result of calibration readings for ASP by HPLC method
Concentration
(µg/ml)
Area (mAU)
Mean ± S.D. (n=5)
% RSD
20 37577.46 ± 185.3348 0.493207
40 70124.4 ± 530.745 0.756862
60 103742.1 ± 797.5031 0.768736
80 135276.2 ± 437.986 0.323772
100 168454.1 ± 1292.66 0.767366
120 207014.6 ± 1633.551 0.789099
slope 1676.7
Intercept 29993.9
r2
0.9991
Graph 25 : calibration curve of ASP by HPLC method
y = 1676.7x + 2993.9
R² = 0.9991
0
50000
100000
150000
200000
250000
0 20 40 60 80 100 120 140
Area
Concentration (ppm)
ASP Average

Table 24: Result of calibration readings for ESO by HPLC method
Concentration
(µg/ml)
Area (mAU)
Mean ± S.D. (n=5)
% RSD
5 13118.36 ± 99.75762 0.760443
10 26044.1 ± 125.6543 0.482467
15 39452.8 ± 387.4851 0.982149
20 52952.3 ± 128.3218 0.242335
25 66337.1 ± 536.0273 0.808035
30 78393.06 ± 563.3303 0.718597
Slope 2632.9
Intercept 25.567
r2
0.9997
Graph 26 : Calibration curve of ESO by HPLC method
y = 2632.9x - 25.567
R² = 0.9997
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
0 5 10 15 20 25 30 35
Area
Concentration (ppm)
ESO Average

Table 25: Summary of validation parameters of HPLC
Parameters ASP ESO
Range 20 -120 µg/ml 5 – 30 µg/ml
Retention time (min) 1.81 4.83
Detection limit (µg/ml) 1.958512 0.118107
Quantification limit
(µg/ml)
5.934884 0.357901
Accuracy (%) 99.27 – 100.197 % 98.884 – 101.057 %
Precision (% RSD)
Intra-day (n=3) 0.116146 0.334746
Inter-day (n=3) 0.654173 0.871002
Instrument precision (%
RSD)
0.294002 0.925732

7. CONCLUSION
The UV and HPLC method has been developed for estimation of Aspirin and
Esomeprazole in synthetic mixture.
UV method was developed using methanol as solvent. UV method was found to be linear
in the range of 20-100 µg/ml for ASP and 5-25 µg/ml for ESO. The limit of quantitation
was found to be 15.6 µg/ml for ASP and 0.26 µg/ml for ESO respectively.
HPLC method was found to be linear in the range of 20-120 µg/ml for ASP and 5-30
µg/ml for ESO. The limit of quantification was found to be 5.93 µg/ml for ASP and 0.35
µg/ml for ESO respectively. The percentage recovery was 99.27-100.19 % for ASP and
99.88-101.05 % for ESO.
Both the methods were validated and found to be accurate, precise and robust.

Chapter 8
Publication Related To
Present Investigation

8. PUBLICATION RELATED TO PRESENT INVESTIGATION
Sr. No. Journal name Date of
submission
Current status Type of article
1. Development and
validation of
analytical method for
estimation of aspirin
and esomeprazole by
RP-HPLC method
29th
March,
2013
Published on 9th
April, 2014
Research

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