Anticoagulant

ACKNOWLEDGEMENT
I take this privilege and pleasure to acknowledgethe contributions of many individuals

who have beeninspirational and supportive throughout my work andendowed me with the
most precious knowledge to see success in my endeavor.
With great pleasure and profound sense of gratitude, I express my most cordial and
humble thanks to my eminentguide respected Dr. Pawar A, AssistantProfessor,Department of
Pharmaceutics, for their constructive and meticulous guidance which exonerated me to
consummate the small work assigned to me. Their criticism, inspirations, profound knowledge
rectitude isinexplicable in few words. With small interaction with themduring the course of
dissertation work made me adventand prudent about research in pharmaceutical field.
I am grateful to Dr.Umesh Bhosale, Head of Analytical Development,Bharat Serums and

Vaccines,Ltd. for their valuable guidance during project work.
I am thankful toDr. Deshmukh Sir, Principal, Mula Education SocietiesCollege of Pharmacy,

Sonai,for providing necessary facilities to carry out this work.
Our head of department Dr.Sunil Pande, I am grateful to his for holding me to high
research standard and enforcing strict validations for each research result.
A novel and good dissertation is never the work of just one person. Thanking all of
them individually would be difficult but I would specially thank some of them.
I express my immense sense of gratitude to all the other staff members, librarian. I
express my deepest thanks to Mr. Nilesh Bawaskar sir and Prasenjeet Chahande sir and
Mr.Amar Mhase, Swapnil Munde, Shailendra Dhole, Vishal More, Rajendra Gadhave sir (ADL
department) for taking part in useful decision and giving guidance and advice s.Store in
chargeMR. Anil, Mangesh, Shridhar, Lab Technicians and all other lab technicians for
contributing to the successful completion of my project.
I would like to express my profound feelings of gratitude and affection for my father
Mr. Ambadas Date and my mother Mrs. Shanta Date and Brother Mr.Kiran Date for their
everlasting blessing, love, patience and support they gave me. Their trust has always
Analytical Method Development &Validation Of Anticoagulant Drug & its Quantitative Estimation
In marketed Preparation Page 1
inspired me to do my best. I owe much to their initiative and ethusionm without which I
could not have ventured into this field of study.
I am grateful to my bathmats Ganesh, Rahul, Shubham, Salman, AK, Surendra,

AnilAnd all of my juniors for their help, encouragement and constant support.
I am also extremely thankful to Bharat Serums and Vaccines.Ltd .for giving me opportunity
for doing my dissertation work at your esteemed organization as a Project Trainee.
List of Abbreviations
Abbreviations Remark
µ Micron
µg Microgram
M Molar
Mol Mole
PPM Parts Per Million
NaOH Sodium Hydroxide
HCl Hydrochloric Acid
RH Relative Humidity
Λmax Maximum Wavelength
°C Degree Celsius
WS Working Standard
SST System Suitability Test
Sr. No. Serial Number
RPM Round Per Minute
CAN Acetonitrile
HETP Height Equivalent to Theoretical Plate
Avg Average
Wt. Weight
RT Room Temperature
NMT Not More Than
NLT Not Less Than
Std Standard
RT Retention Time
µ Micron
µg Microgram
M Molar
Mol Mole
PPM Parts Per Million
NaOH Sodium Hydroxide
HCL Hydrochloric Acid
H2O2 Hydrogen Peroxide
RH Relative Humidity
Λmax Maximum Wavelength
°C Degree Celsius
WS Working Standard
SST System Suitability Test
Sr. No. Serial Number
RPM Round Per Minute
CAN Acetonitrile
HETP Height Equivalent to Theoretical Plate
Avg Average
Wt Weight
RT Room Temperature
NMT Not More Than
NLT Not Less Than
Std Standard
TABLE OF CONTENTS
Chapter
Title Page No.
No.
1. Introduction 1
2. Drug profile 50
3. Literature Review 51
4. Aim And Objective 55
5. Plan of work 57
6. Experimental Work 58
7. Result And Discussion 70
8. Conclusion 102
9. Bibliography 103
LIST OF TABLES
Table Page
Description
no. No.
1. Types of HPLC columns 29
2. Detectors Employed in HPLC Analysis 35
3. Validation Parameter & Acceptance criteria 49
4. Drug Solubility 50
5. Drug Sample with its Percent Assay 59
6. Materials and Reagent 59
7. Instrument and Equipment’s 59
8. Trial 1 62
9. Trial 2 62
10. Trial 3 63
11. Trial 4 63
12. Trial 5 64
13. Trial 6 64
14. Trial 7 65
15. Trial 8 65
16. Trial 9 66
17. Trial 10 66
18. Trial 11 67
19. Observation for trial 1 69
30. Optimized Chromatographic conditions 80
31. Injection sequence for specificity 82
32. Specificity results of Anticoagulant Drug 82
33. Linearity solutions for Anticoagulant Drug 83
34. Injection sequence for Anticoagulant Drug 84
35. Linearity of Anticoagulant Drug 85
36. Linearity regression data for calibration curve 85
37. Injection sequence for precision 86
38. Repeatability studies for Anticoagulant Drug 87
39. Injection sequence for intermediate precision 88
40. Intermediate precision studies for Anticoagulant Drug 89
41. Sequence of Accuracy level 91
42. Accuracy data for Anticoagulant Drug 92
43. Injection sequence for Robustness 94
44. Robustness (Change in temperature-23°C) 95
45. Robustness (Change in temperature-23°C) 96
46. Robustness(Change in Flow rate-0.95ml/min) 97
47. Robustness(Change in Flow rate-0.95ml/min) 98
48. Solution stability data for Anticoagulant Drug 99
49. Acceptance Criteria of validation 100
LIST OF FIGURES
Figure
Description Page No.
No.
1. Type of Classical Method 19
2. History of column chromatography 23
3. Separation of Column in HPLC 24
4. HPLC Instrument 31
5. Validation Parameter 43
6. Selection of analytical wavelength 70
Trial 1-Mobile phase- Methanol: Water (50:50 v/v), Flow Rate –0.8 ml/min),
7. 71
BDS Hypersil C18 250X4.6mm,5µm(40°C)
Trial 2-Mobile phase- Methanol: Water (70:30 v/v),Thermo scientific

8. 72
250X4.6mm, 5µm, column (40°C)
Trial 3-Mobile phase- Buffer: Acetonitrile: Methanol (50:30:20 v/v),Supeiro

9. 73
LC-CN (cyno), 250X4.6mm, 5µm column(40°C)
Trial 4 -Mobile phase- Buffer: Acetonitrile: Methanol (50:40:10 v/v),Zorbax

10. 74
NH2 ,250X4.6mm, 5µm, column(40°C)
Trial 5- Buffer: Methanol (50:50 v/v),Inersil 100A° 300X3.9mm, 5µm,

11. 75
column(40°C)
Trial 6- Buffer: Acetonitrile (70:30 v/v),Water 300X3.9mm,10µm, column

12. 75
(40°C)
Trial 7- Buffer: Acetonitrile (70:30 v/v),Waters, 300X3.9mm,5µm, column

13. 76
(40°C)
Trial-8 Buffer: Acetonitrile (60:40 v/v),Inersil ODS 250X4.6mm,5µm,

14. 77
column (40°C)
Trial-9 Buffer: Acetonitrile (55:45 v/v),Luna Phenomenax,100A° C18

15. 78
250X4.6mm,5µm, column(40°C)
Trial-10 Buffer: Acetonitrile (55:45 v/v),Luna Phenomenax,100A° C18

16. 79
250X4.6mm,5µm, column(30°C)
Trial-11 Mobile phase- Buffer: Acetonitrile (55:45 v/v),Flow Rate – (1.0
17. 80
ml/min), Luna Phenomenax,100A°, C18 250X4.6mm,5µm column (25°C)
18. Linarity Calibration curve 88
ABSTARCT
The simple, specific, precise and accurate method has been developed for RP-HPLC for
simultaneous estimation of Anticoagulant Drug in capsule dosage form. Spectra scan was
achieved by SHIMADZU UV spectrophotometer, quartz duvets were used for sample analysis.
The detection wavelength of Anticoagulant Drug by taking overly was found to be 226 nm .In
RP-HPLC method separation was achieved by Luna Phenomenax 100A° C18(250×4.6mm,5µm)
column with the mobile phase Triethyl amine buffer : Acetonitrile (55:45v/v).The pH of Triethyl
amine Buffer was adjusted by orthophosphoric acid (pH 3.0). The flow rate is 1.0 ml/min. The
retention time Anticoagulant Drug was found to be 6.15min and 4.96min for Anticoagulant Drug
respectively. A good linear response was obtained in the range of 5-10µg/ml of each
respectively. The LOD (Limit of detection) of Anticoagulant Drug was found to be
0.04262µg/ml and 0.004065µg/ml respectively and LOQ (Limit of Quantitation) of
Anticoagulant Drug was found to be 0.0371µg/ml and 0.0246µg/ml .The recovery of
Anticoagulant Drug was found to be. The proposed method were validated as per ICH guidelines
by means of different parameters like Linearity, Precision, Accuracy, LOD and LOQ, range of
selectivity, Robustness and Raggedness as per ICH guidelines and successfully applied to the
estimation of Anticoagulant Drug in tablet dosage form.
Keywords:- Dapagliflozin, Saxagliptin, RP-HPLC, Simultaneous estimation, UV, Validation
a. INTRODUCTION
The quality is important in every product or service but it is more vital in drug or
medicine as it involves in life. Analytical chemistry concerned with determining the qualitative
and quantitative composition of material under study. Analytical monitoring of pharmaceutical
product or of specific ingredients within the product is necessary to ensure its safety and efficacy
throughout its all the phases including storage, distribution and use.
Pharmaceutical analysis is a branch or Analytical chemistry that involves a series of

process for identification, determination, Quantification and purification of a substance and also
separation of the components of the solution or a mixture, or determination of structure of
chemical compounds. The substance may be a single compound or a mixture of compounds and
it may be in any of the dosage form. The drug substance used as pharmaceuticals are form any of
the sources like animals, plants, microorganisms, minerals and various synthetic products1 .
1.1 ANALYTICAL CHEMISTRY

Analytical chemistry is a scientific discipline that develops methods, instruments and
strategies to obtain information on the composition and nature of matter. Unlike other major sub
disciplines of chemistry such as inorganic and organic chemistry, analytical chemistry is not
restricted to any particular type of chemical compound or reaction. Analytical chemistry is
concerned with the chemical characterization of matter and thus pharmaceutical analysis covers
matter having pharmaceutical applications.
Analytical method development and validation play important roles in the discovery,
development, and manufacture of pharmaceuticals. Pharmaceutical products formulated with
more than one drug, typically referred to as combination products, are intended to meet
previously unmet patients need by combining the therapeutic effects of two or more drugs in one
product. These combination products can present daunting challenges to the analytical chemist
responsible for the development and validation of analytical methods2.
Market is flooded with combination of drugs in various dosage forms. The multi-
components formulations have gained a lot of importance nowadays due to greater patient
acceptability, increased potency, multiple action, fewer side effects and quicker relief. There is a
plethora of analysis of such formulations without priorseparation. For the estimation of multi-
component formulation, the instrumentaltechniques, which are commonly employed, are
spectrophotometry, GLC, high performance thin layer chromatography (HPTLC), HPLC etc.
These methods are based upon the measurement of specific and nonspecific physical properties
of thesubstances.
Knowledge of chemical composition of many substances is important in our daily life.

Analytical chemistry plays an important role in nearly all aspects of chemistry vise agricultural,
clinical, environmental, forensic, Manufacturing, metallurgical, and pharmaceutical chemistry3.
1.1.1 Importance of Analytical Chemistry
The importance of analytical chemistry in related scientific areas is illustrated by

considering its impact on clinical analysis, pharmaceutical research and quality control4.
 Sensitive chemical and instrumental tests are advised in order to detect abnormal and
normal components of body fluids.
 Blood and urine samples are determined for quantitation of glucose, urea, nitrogen,
protein, sodium potassium, calcium and uric acid etc.
 Similarly, the quality of manufactured drug in tablets, solution and emulsion form must
be carefully controlled in pharmaceutical industry otherwise the drug can itself affect the
therapeutic value.
 In other pharmaceutical studies, it is important to establish the properties and therapeutic
value of a drug before the drug is approved and made available to the patients.
1.1.2 Types of Chemical analysis
Pharmaceutical analysis, deals with qualitative and quantitative analysis of drug in bulk,
dosage forms and in biological samples.
There are main two types of chemical analysis.
1. Qualitative (identification)
2. Quantitative (estimation)
1.1.2.1 Qualitative analysis
Qualitative analysis deals with the identification of elements, ions or compounds present
in a sample. It gives information about atomic or molecular species or functional groups that are
present in the sample2.
1.1.2.2 Quantitative analysis
Quantitative analysis deals with the determinations of how much of one or more
constituent is present in a sample. The sample may be solid, liquid or gas5. 6.
Methods of detecting Analyte
There are mainly three methods of analyte which are as follows7,
Physical mean
 Mass
 Colour
 Refractive index
 Thermal conductivity
With electromagnetic radiation
 Absorption
 Emission
 Scattering
By an electric charge
 Electro Chemistry
 Mass Spectroscopy
Anticoagulant Drug
Anticoagulants, commonly referred to as blood thinners, are chemical substances that

prevent or reduce coagulation of blood, prolonging the clotting time. Some of them occur
naturally in blood-eating animals such as leeches and mosquitoes, where they help keep the bite
area unclotted long enough for the animal to obtain some blood. As a class of medications,
anticoagulants are used in therapy for thrombotic disorders. Oral anticoagulants (OACs) are
taken by many people in pill or tablet form, and various intravenous anticoagulant dosage forms
are used in hospitals. Some anticoagulants are used in medical equipment, such as test tubes,
blood transfusion bags, and dialysis equipment.
Anticoagulants are closely related to antiplatelet drugs and thrombolytic drugs by

manipulating the various pathways of blood coagulation. Specifically, antiplatelet drugs inhibit
platelet aggregation (clumping together), whereas anticoagulants inhibit the coagulation cascade
by clotting factors that happens after the initial platelet aggregation.
Use of Anticoagulant Drug
An anticoagulant medicine is used in patients to prevent blood clots from forming in

veins, arteries, the heart, and the brain of a patient. For example, if the clot travels to the patient's
heart it can cause a heart attack or if one forms in the brain it may cause a stroke or TIA (mini-
stroke, transient ischemic attack).
Examples of diseases and health conditions that require treatment with anticoagulants to
reduce the risk of clots forming, or are used to prevent life-threatening problems include:
 Deep vein thrombosis (DVT)

 Heart attack
 Stroke
 For the prevention or treatment of:
 Pulmonary embolism
 Blood clots within venous and arterial catheters
 Stent thrombosis
Side effects
The most common side effect of treatment with anticoagulant medicine is bleeding.
Treatment with these products may cause various degrees of bleeding, including fatal bleeds.
This list of adverse effects associated with anticoagulants are compiled from adverse
effects listed for various anticoagulants and may not apply to every medicine.
Common side effects include:
 Bleeding
 Abdominal pain
 Flatulence (intestinal gas)
 Headache
 Lethargy
 Dizziness
 Fever
 Local injection site reactions
1.2 ANALYTICAL METHOD DEVELOPMENT
Method development is done for
1. New products
2. Existing products
Methods are developed for new products when no official methods are available.
Alternate methods for existing (non-pharmacopoeia) products are developed to reduce the
cost and time for better precision and ruggedness. Trial runs are conducted, method is
optimized and validated. When alternate method proposed is intended to replace the
existing procedure comparative laboratory data including merit/demerits are made
available8.
1.2.1 Selection of analytical method

First stage in the selection or development of method is to establish what is to be
measured and how accurately it should be measured. The following analyticaltechniques
are usually employed for estimations of different components in formulations :-
 Titrimetric and gravimetric
 Ultraviolet and visible spectrophotometry
 Thin layer chromatography
 High performance liquid chromatography (HPLC)
 Gas Chromatography (GC)
 Atomic absorption spectrometry (AAS)
 Infra-Red absorption spectrophotometry
1.2.2 Steps of method development

Documentation starts at the very beginning of the development process, a system for
full documentation of the development studies must be established. Alldata relating to
these studies must be recorded in laboratory notebook or an electronicdatabase 9. The
steps involved in method development are,
1.2.2.1 Analyte standard characterization
 All known information about the analyte and its structure is collected such as physical
and chemical properties, toxicity, purity, hygroscopic nature, solubility and stability.
 The standard analyte (100% purity) is obtained. Necessary arrangement is made for the
proper storage (refrigerator, Desiccators and freezer).
 When multiple components are to be analyzed in the sample matrix, the number of
components is noted, data is assembled and the availability of standards for each one is
determined.
 Only those methods (spectroscopic, MS, GC, HPLC etc.,) that are compatible with
sample stability are considered.
1.2.2.2 Method Requirement
The goals or requirements of the analytical method that need to be developed are considered
and the analytical figures of merit are defined. The required detection limits, selectivity,
linearity, range, accuracy and precision are defined.
1.2.2.3 Literature search and prior methodology
The literatures related to the analyte are reviewed degrading synthesis, physical and chemical
proper ties, solubility and relevant analytical methods. Books,periodicals, chemical
manufacturers and regulator y agency compendia such as USP /NF, AOAC and ASTM
publications should be reviewed. Chemical Abstracts Service (CAS) automated computerized
literature searches are convenient.
1.2.2.4 Choosing a method

Using the information in the literatures and prints, methodology is adapted.The methods are
modified wherever necessary. Sometimes it is necessary to acquireadditional instrumentation to
reproduce, modify, improve or validate existingmethods for in-house analytes and samples.
If there is no prior method tor the analyte in the literature, from analogy, thecompounds that
are similar in structure and chemical properties are investigated andare worked out. There is
usually one compound for which analytical method alreadyexist that is similar to the analyte of
interest.
1.2.2.5 Instrumental set up and initial studies
 The required instrumentation is setup. Installation, operational and performance
qualification of instrumentation using laboratory standard operating procedures
(SOP's) are verified.
 Always new consumables (e. g. solvents, filters and gases) are used, for example,
method development is never started, on a HPLC column that has been used earlier.
 The analyte standard in a suitable injection/introduction solution and in known
concentrations and solvents are prepared. It is important to start with an authentic,
known standard rather than with a complex sample matrix. If the sample is extremely
close to the standard (e. g., bulk drug), then it is possible to start work with the actual
sample.
 Analysis is done using analytical conditions described in the existing literature.
1.2.2.6 Optimization
During optimization one parameter is changed at a time, and set of conditions are
isolated, rather than using a trial and Error approach. Work has been done from an organized
methodical plan, and every step is documented (in a lab notebook) in caseof dead ends.
1.2.2.7 Determination of percent recovery of actual sample and demonstration of

quantitative sample analysis
Percent recovery of spiked, authentic standard analyte into a sample matrixthat is shown
to contain no analyte is determined. Reproducibility of recovery (average ± standard deviation)
from sample to sample and whether recovery has beenoptimized has been shown. It is not
necessary to obtain 100% recovery as long as theresults are reproducible and known with a high
degree of certainty.
The validity of analytical method can be verified only by laboratory studies.Therefore
documentation of the successful completion of such studies is a basicrequirement for
determining whether a method is suitable for its intended applications9.
1.2.3 CLASSIFICATION OF ANALYTICAL METHODS
The analytical methods can be broadly classified into two categories
1.2.3.1 Classical methods
For qualitative analysis the separated compounds are treated with reagents that could be
recognized by either color, by their boiling or melting points, their solubility in a series of
solvents, their optical activities or their refractive indices. For quantitative analysis, the amount
of analyte was determined by gravimetric or titrimetric measurements10.
Advantages of Classical Methods

 Procedure is simple and accurate.
 The equipment needed is cheap.
 Methods are based on absolute measurements.
 Specialized training is not required.
Limitations of Classical Methods
 Chemical environment is critical.
 There is a lack of specificity and versatility.
 Accuracy decreases with decreasing amount.
 Procedure is time consuming.
Types of Classical Methods
Volumetric Methods Classical Methods Gravimetric Methods
Fig.1:Flow chart of classical method
 Volumetric Method
It is based on the determination of a solution of known strength requiredtocomplete a
chemical reaction with the substance being analyzed.
 Gravimetric Method
In this method of analysis, the assay results generally are obtained either bydetermining
the weight of a substance in the sample, or the weight of some othersubstance derived from the
sample, the equivalent weight of which serves as the basisfor calculating the result10, 11.
1.2.3.2 Instrumental Methods
These methods are based upon the measurement of some physical property ofsubstance
using instrument like conductivity, electrode potential, light absorption andemission, mass to
charge ratio and fluorescence. These methods are now being used for quantitative analysis of a
variety of inorganic, organic and biochemical analyte12.
Advantages of Instrumental Methods
 Small Samples can be used.
 High sensitivity is obtained.
 Measurements obtained are reliable.
 The determination is very fast.
 Complex samples can be handled.
Limitations of Instrumental Methods
 Skilled person is required.
 The sensitivity and accuracy depends on type of instrument.
 Cost of equipment is high.
 Sizable space is required.
There are many techniques available for the analysis of materials, however, they are all
based on the material's interaction with energy. This interaction permitsthecreationof a signal
that is subsequently detected and processed for its informationcontent. There are many
techniques available for the analysis of analytes,
A. Spectroscopic Analysis
Spectroscopy measures the interaction of the material with electromagnetic radiation 12. Different
types are,
 Ultraviolet and visible spectrophotometry-Excitation of valence electrons.
 Infra-red spectroscopy-Excitation of molecular vibrations.
 Raman spectroscopy-Excitation of molecular vibrations by light scattering.
 Atomic absorption spectroscopy-Absorption of atomic resonance line.
 Atomic emission spectroscopy-Light emission from excited electronic states of atoms.
 X-ray diffraction-Diffraction of X-rays from crystal planes.
 X-ray fluorescence-Re-emission of X-rays from excited atoms.
 Fluorimetry and phosphorimetry-Emission of light energy by electrons.
 Mass spectroscopy- lionization and conversion of molecule into fragment ions.
 NMR spectroscopy-Reorientation of magnetic nuclei in a magnetic field.
 Nephelometry and Turbidimetry-Intensity measurement of transmitted light as function of
concentration of the dispersed phase.
 Electron spins resonance spectroscopy-Reorientation of magnetic electrons in a magnetic field.
B. Chromatographic techniques
Separation processes are used to decrease the complexity of material mixtures .The most utilized
separation method is chromatography13,16. Following types of techniques are used,
 Gas chromatography (GC)
 High performance liquid chromatography (HPLC)
 Size-exclusion chromatography
 High-performance thin layer chromatography (HPTLC)
 Paper chromatography
 Thin layer chromatography (TLC)
 Affinity chromatography
 Ion- exchange chromatography
After the isolation of material signal is generated, the signal must be detected and
interpreted.
C. Hyphenated Techniques
Combinations of the above techniques are called as "hybrid"or"hyphenated” techniques.
Several examples are in popular use today and new hybrid techniques are under development.17-20
 GC-MS (gas chromatography-mass spectrometry)
 ICP-MS (inductively coupled plasma-mass spectrometry)
 GC-IR (gas chromatography-infrared spectroscopy)
 MS-MS (mass spectrometry-mass spectrometry)
Instrumental methods are sensitive and it needs small amount of sample.Complex
mixtures can be analyzed with or without their prior separation withsufficient reliability and
accuracy of results.
D. Electrochemical methods
Electrochemical methods are of following types,21
 Conductometry
 Voltametry
 Potentiometry
 Coulometer
 Atomic Absorption Spectroscopy
 Emission ( Plasma) Spectroscopy
E. Miscellaneous Techniques
Following types of miscellaneous techniques are used 22-26
1. Mass Analysis:
Mass spectrometry measures the interaction of charged materials and electric and
magnetic fields.
2. Thermal Analysis:
Calorimetric and thermo gravimetric analysis measure the inter action of material and
heat. In or der to utilize the techniques availablecurrently, complex material mixtures
must be separated into simpler samples forindividual analysis.
1.3 CHROMATOGRAPHY
1.3.1 Introduction
Chromatography is defined as a procedure by which solutes are separated by a dynamic
differential migration process in a system consisting of two or more phases, one of which moves
continuously in a given direction and in which the individual substances exhibit motilities by
reason of differences in adsorption, partition, solubility, vapors pressure, molecular size or ionic
charge density. The individual substances thus obtained can be identified or determined by
analytical methods24. Chromatography was invented nearly 100 years ago, but it is only in the
past few years that the development of the technique and associated instrumentation has reached
a level that might be called the steady state36.
1.3.2 History of Chromatography
Chromatography is a technique by which a mixture sample is separated into components.
Although originally intended to separate and recover (isolate and purify)the components of a
sample, today, complete chromatography systems are often used to both separate and quantify
sample components.
Fig. 2 Diagram showing

The term, "chromatography" was coined by the Russian botanist, Tswett, who
demonstrated that, when a plant extract was carried by petroleum ether through a column
consisting of a glass tube packed with calcium carbonate powder, a number of dyes were
separated, as shown in Fig. 8. He named this analysis method "Chromatography" after "chroma"
and "graphos", which are Greek words meaning "color" and to draw, respectively37.
1.3.3 Separation Process
The Chromatographic method of separation involves the following steps37
 Adsorption or retention of sub stances on the stationary phase.
 Separation of the adsorbed substances by the mobile phase.
 Recovery of the separated substances by the continuous flow of the mobile phase.
 Qualitative and quantitative analysis of the eluted substances.
Principle of Chromatography
The speed of a migrating sample component depends on whether the component has an
affinity for the stationary or mobile phase. This affinity appears via various actions: adsorption,
partition, ion exchange, etc. As shown in Fig. 9, components that have a higher affinity for the
mobile phase compared with the stationary phase migrate more rapidly, while components that
have a higher affinity for the stationary phase are eluted from the column later. The order and
resolution of the components emerging from the column depend on the type of selected
stationary and mobile phases 38, 39.
Fig. 3 Diagram shows separation in HPLC
1.3.4 Types of Chromatography6,11

The chromatography is divided into mainly four types based upon the nature of stationary
and mobile phase, principle of separation, mode of chromatography and miscellaneous which are
as follows 6,11
1.3.4.1 Based upon the nature of stationary and mobile phase
 Gas-solid chromatography
 Gas-liquid chromatography
 Solid-liquid chromatography
 Liquid-liquid chromatography
1.3.4.2 Based on the principle of separation
 Adsorption chromatography
 Partition chromatography
1.3.4.3 Based on the modes of chromatography
 Normal phase mode
 Reverse phase mode
1.3.4.4 Miscellaneous
 Ion exchange chromatography
 Gel permeation chromatography
 Chiral chromatography
1.4 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)

HPLC is an analysis method that yields high performance and high speed compared with
traditional column chromatography because of the forcibly pumped mobile phase. Recently,
ultrafast analysis using a high-pressure-resistant apparatus has been attracting attention. UPLC
(Ultra performance Liquid Chromatography) is becoming established as an abbreviation for this
ultrafast Liquid Chromatography method6.
This technique is based on the same method of separation as classical column
chromatography such as adsorption, partition, ion exchange and gel permeation but it differ from
column chromatography, in that mobile phase is pumped through the packed column under high
pressure38.
It is the most popular technique today among the different chromatographic procedure.
Due to significant evolution of Liquid Chromatography (LC) instruments providing the superior
qualitative and quantitative results. The development of HPLC has enabled Liquid
Chromatography to achieve great success in providing following features,
 Speed of separation
 High resolving power
 Monitoring the column effluent
 Repetitive and reproducible analysis
 Automation of analytical procedure
Principle of HPLC
In all types of chromatography the two phases are chosen so that the components of the
sample distribute themselves between the mobile and stationary phases to varying degrees.
Those components strongly retained by the stationary phase move slowly with the flow of
mobile phase and vice versa. As a consequence of these differences in migration rates, sample
components separate into discrete bands, or zones, that can be analyzed qualitatively and
quantitatively39.
1.4.1 Criteria for selecting proper HPLC method

It is based on nature of the sample like regular and special. Regular samples are typical
mixtures of small molecules (< 2000 Da) that can be separated, using more or less standardized
conditions. Regular samples can further classified into neutral or ionic: Ionic samples include
acids, bases, amphoteric compounds and organic salts (ionized strong acids or bases). In neutral
samples, generally buffers or additives are not required in mobile phase while acidic or basic
compounds need addition of buffer to mobile phase. For basic or cationic samples, reversed
phase columns are recommended and amine additives for mobile phase may be beneficial.
Alternatively, the sample may bestrongly retained with 100 % Acetonitrile as mobile phase,
suggesting the use of non-aqueous reversed phase (NARP) or normal phase HPLC methods25.
Review of International conference on harmonization (ICH) guidelines for impurities in
new drug substance and new products. And accompanying guidelines formethod validation
quickly underscore the usefulness of HPLC for pharmaceutical analysis6. It provides
 Reliable quantitative precision and accuracy
 Linear dynamic range
 Determination of Active Pharmaceutical Ingredient (API) and related substances in the
same run using variety of detectors
 Can be performed on fully automated instrumentation
 Excellent reproducibility
 Applicable to wide array of compounds by judicious choice of HPLC column chemistry
HPLC is in some respects is more versatile than gas chromatography since
 It is not limited to volatile and thermally stable sample

 The choice of mobile phase and stationary phase is wider
HPLC is the method of choice for the analysis of
 Non-volatile substances (for volatile substances GC is an alternative)

 Substances with high polarity or ionic samples
 Substances with high molecular weight
 Thermally unstable and decomposable substances
1.4.2 Types of HPLC method39-43
1.4.2.1 Based on modes of separation
 Normal Phase Chromatography
 Reverse Phase Chromatography
1.4.2.2 Based on principle of separation
 Adsorption chromatography
 Ion exchange chromatography
 Size exclusion chromatography
 Affinity chromatography
 Chiral phase chromatography
1.4.2.3 Based on elution technique
 Isocratic separation
 Gradient separation
 Normal phase chromatography
In normal phase mode, the stationary phase (e. g. silica gel) is polar in natureand the
mobile phase is non-polar (isopropane with hexane) in nature, non-polar compounds travel faster
and are eluted first. This is because less affinity between solute and stationary phase. Polar
compounds are retained for longer time in thecolumn because more affinity towards stationary
phase and takes more time to be eluted from the column. This is not advantageous in
pharmaceutical applications since most of the drug molecules polar in nature and takes longer
time to be elutedand detected. Hence this technique is not widely used in pharmacy.
 Reverse Phase Chromatography
In reversed phase mode, the stationary phase is non-polar in nature and the mobile phase is
aqueous, moderately polar in nature. One common stationary phase issilica which has been
treated with RMe2SiCl, where R is a straight chain alkyl group such as C I8H37 or C8H17 and most
commonly used solvents for mobile phase are water, methanol and acetonitrile. With these
stationary phases, retention time is longer for molecules which are more non-polar, while polar
molecules elute more readily. An investigator can increase retention time by adding more water
to themobile phase ; there by making the affinity of the hydrophobic analyte for thehydrophobic
stationary phase stronger relative to the now more hydrophilic mobilePhase. Reverse phase
chromatography is so commonly used that it is often incorrectly referred to as "HPLC" without
further specification. The Pharmaceutical industryregularly employs Reverse phase
chromatography to quality drugs before theirrelease.
Structural properties of the analyte molecule play an important role in itsretention
characteristics. In general, an analyte with a larger hydrophobic surface area(C-H, C-C, and
generally non-polar atomic bonds, such as S-S and others) results in a longer retention time
because it increases the molecule's non-polar surface area, which is non-interacting with the
water structure. On the other hand, polar groups, such as-OH,-NH2, COO-or-NH3+ reduce
retention as they are well integrated into water, Very large molecules. However, can result in an
incomplete interaction between the large analyte surface and the ligand's alkyl chains and can
have problems entering the pores of the stationary phase.
Retention time increases with hydrophobic (non-polar) surface area. Branchedchain
compounds elute more rapidly than their corresponding linear isomer s because the overall
surface area is decreased. Similarly organic compounds with single C-C-bonds elute later than
those with a C=C or C-C triple bond, as the double or triplebond is shorter than a single C-C-
bond.
Table 1: Normal vs. Reversed Phase Chromatography
Parameters Normal Reverse
Packing Polarity High Low
Solvent Polarity Low High
Elution Order Non polar first, then Polar Polar First, then Non polar
Effect of increasing solvent polarity Decrease Retention time Increase retention time
1.4.2.2 Based on principle of separation

 Adsorption Chromatography
The principle of separation in normal phase mode and reverse phase mode is adsorption;
separation of components takes place because of difference in affinity of compounds towards
stationary phase. The component which has more affinity towards stationary phase travels slower
and eluted later, the component which has less affinity towards stationary phase travels faster
and eluted first. Since no two components have the same affinity towards stationary phase, the
components are separated.
 Size Exclusion Chromatography
Size Exclusion Chromatography (SEC), also known as gel permeation chromatography
or gel filtration chromatography, separates particles on the basis of size. It is generally a low
resolution chromatography and thus it is often reserved for the final, "polishing" step of
purification. It is also useful for determining the tertiary structure and quaternary structure of
purified proteins. It is used primarily for the analysis of large molecules such as proteins or
polysaccharides. SEC works by trapping these smaller molecules in the pores of a particle. The
larger molecules simply pass by the pores as they are too large to enter the pores. Larger
molecules therefor e flow through the column quicker than smaller molecules, that is, the smaller
the molecule, the longer the retention time.
 Ion Exchange Chromatography
In Ion Exchange Chromatography, retention is based on the attraction between solute
ions and charged sites bound to the stationary phase. Ions of the same charge are excluded.
Types of ion exchangers include
 Polystyrene resins: These allow cross linkage which increases the stability of the chain.
Higher cross linkage reduces swerving, which increases the equilibration time and ultimately
improves selectivity.
 Cellulose and dextran ion exchangers (gels) : These possess larger pore sizes and low
charge densities making them suitable for protein separation.
 Controlled-pore glass or porous silica: In general, ion exchangers favour the binding of
ions of higher charge and smaller radius.
An increase in counter ion (with respect to the functional groups in resins) concentration
reduces the retention time. An increase in pH reduces the retention time in cation exchange while
a decrease in pH reduces the retention time in anion exchange.
 Affinity Chromatography
This is the most selective type of chromatography employed. It utilizes the specific
interaction between one kind of solute molecule and a second molecule that is immobilized on a
stationary phase. For example, the immobilized molecule may be an antibody to some specific
protein. When solute containing a mixture of proteins is passed by this molecule, only the
specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later
extracted by changing the ionic strengthor pH.
 Chiral phase chromatography
Separation of optical isomers can be done by using chiral stationary phases. Different
principles operate for different types of stationary phases and for different samples. The
stationary phases used for this type of chromatography are mostly chemically bonded silica gel.
 Isocratic separation
In this technique, the same mobile phase combination is used throughout the process of
separation. The same polarity or elution strength is maintained throughout the process.
 Gradient separation
In this technique a mobile phase combination of lower polarity or elution strength is used
followed by gradually increasing the polarity or elution strength39-43.
1.4.3 Instrumentation of HPLC
Fig. no 4 - Instrumentation of HPLC system
 HPLC instrumentation containing following parts 6. 11

 Mobile Phase Reservoir and solvent System treatment
 pumps (Displacement Pump, Reciprocating Pump, Pneumatic Pump)
 Sample Injectors
 Pre columns
 Liquid chromatographic column (Analytical Column)
 Detectors
 Data systems
COMPONENTS OF HPLC
HPLC is a technique for separation, identification and quantification of components in a

mixture. It is especially suitable for compounds which are not easily volatilized, thermally
unstable and have high molecular weights.
1.4.3.1 Mobile Phase Reservoir and Solvent System Treatment
Modern HPLC apparatus is equipped with one of more glass or stainless steel reservoirs. It
is alien equipped with a means of removing dissolve gases usually 02and N2 that interfere by
forming bubbles in column and detector system. Degasser may consist of
 vacuum pumping system

 A distillation system
 Devices for heating and stirring the solvent
 Devices for sparging (in which the, dissolved gases are swept out of the solution by fine
bubbles of inert gas of low solubility).
 Isocratic System: A separation that employ s a single solvent of constant composition is
called as isocratic system.
 Gradient System: In this technique the proportion of the tow or more solvent is varied in
a programmed way.
1.4.3.2 Pump
Pumps are required to deliver a constant flow of mobile phase at pressuresranging from
1-550 bars. Pumps capable or pressure up to 8000 psig provide a wide range of flow rates of
mobile phase, typically from 0. 1 to 10 ml/min. Low now rates (10-100µl/min) are used with
microbore columns, intermediate now rates (0. 5-2ml/min) are used with conventional analytical
HPLC columns, and first flow rates are used for preparative or semi preparative columns and for
slurry packingtechniques. Mechanical pumps of the reciprocating piston type view a pulsating
supply of mobile phase.
The various types of the pump used are
 Constant displacement pumps or syringe pump

 Dual piston reciprocating pump
 Pneumatic pump or constant pressure pump
1.4.3.3 Injection System
Commercial chromatographs use valves for sample injection. With these devices sample
is first transferred at atmospheric pressure from a syringe into asample loop. Turning the valve
from load to inject position connects the sample loopin to the high-pressure mobile Phase stream.
Where by the contents of the sample loop are transferred on to the column. In Rheodyne 7125
valve, sample from a micro litter syringe is loaded into the needle port. Filling the sample
loop.which is a small piece of stainless steel tube connected between ports. Any excess goes to
waste from another port. On turning to 'inject', the loop contents are flushed on to the column. A
variety of loop volumes is available, commonly 10-50µL.
Injection pores are of basically of two types
 Those in which the sample is injected directly into the column.

 Those in which the sample is deposited into the column inlet and then swept by a valving
action into the column by the mobile phase.
1.4.3.4 Precolumn
Some HPLC instruments are equipped with a precolumn, which contains a packing
chemically identical to that in the analytical column. Particle size is largehence the pressure drop
across the pre column is negligible with respect to the analytical column. The pre column is
mainly used to remove the impurities from the solvent, and thus prevent contamination of the
analytical column.
1.4.3.5 Liquid Chromatographic Column (Analytical Column)
HPLC column are made up of high quality stainless steel, polished internally to a mirror
finish. Standard analytical columns are 4-5 mm internal diameter and 1000cm in length. Shorter
columns 3-6 cm in length containing a small particle size Packing material (3-5µm) produce
similar or better efficiencies, in terms of the no oftheoretical plates (about 7000), that those of 20
cm columns containing 10µmirregular particles and are used when short analysis times and high
through put of samples are required. Micro bore columns of 1-2 mm internal diameter and 10-
25cmin length have certain advantages of lower detection limit, and lower consumption
solvent37.
Column Packing Materials
Two basic types of packing have been used in HPLC.
1. Based on Size
 Pellicular
It consists of non-porous, spherical glass or polymer beads with a diameter of 30 to 40 µm.
 Porous particles
These particles have sizes ranging from 3-10 µm and are composed of silica alumina or ion
exchange resin. These particles are often wetted with thin organic films which are physically of
chemically bounded to the surface.42
2. Based on Chemical Composition
a) Normal Phase partition Chromatography
In this type of chromatography the stationary phase is polar and mobile phase is
comparatively non polar.
b) Reverse phase partition Chromatography
The stationary phase is less polar than the mobile phase. The stationary phase is silica,
chemically bounded through a siloxane (Si-O-Si-C) linkage to a low polar functional group.
These phases are prepared by treating the surface silanol groups of silica with an
organochlorosilane reagent43.
1.4.3.6 Detectors
A detector is required to sense the presence and the amount of sample in thesample
component h the column current. The output of the detector is an electricalsignal that is
proportional to some property of the mobile phase and/or the solute. A detector that measures
property which is possessed by both mobile phase and solute iscalled bulk property detector. e. g.
Refractive Index detector, alternatively if the Property is possessed essentially by the solute e. g.
absorption of UV/visible light ofelectrochemical property, the detector is called a solute properly
detector. The mostcommonly used detectors in the HPLC analysis of pharmaceutical substances
aredescribe below39.40
Table 2: Most common HPLC detectors
Sr.No. Detectors Advantages Limitations

Non-specific; complex
1. UV-Visible Works with all molecules samples, absorption
wavelength
2. Works with nearly all Temperature sensitive,
Refractive Index (RI)
molecules high LOD
3. Infrared Radiation (IR) Works with all molecules Many solvents IR active
4. Photo Diode Array (PDA) Works for all wavelengths High LOD
5. Fluorescence Very specific, low LOD Not everything fluoresces
6. Electrochemical Commercially available Non-specific; high LOD
7. Uniform response; Non-specific; interference

Scattering
5ng/25ml LOD from solvent
8. Low LOD, analyte

Mass Spectrometer (MS) Ability to ionize analyte
identification
1.4.3.7 Data systems
Chromatographic data systems serve many functions in HPLC, of which themost notable
functions include experimental aids for method development systemcontrol for all operational
settings data collection and processing report generationand regulatory functions44-46
1.4.4 Quantitative Analysis in HPLC
The sample or solute is analyzed quantitatively in HPLC by either peak heightor peak
area measurements. Peak area are proportional to the amount of the materialeluting from the
column as long as the solvent flows at constant rate. Peak heights areproportional to the amount
of the material only when peak widths are constant and arestrongly affected by the sample
injection techniques. They are five principlesevaluation methods for quantifying the solute47
1.4.4.1 Calibration by Standards
Calibration curves for each component are prepared from pure standards, using identical
injection volumes of operating conditions for standards and samples.The concentration of solute
is read from its curve if the curve is linear
X = K × Area
Where,
X = concentration.
K=proportionality constant (slope of the curve).
In this evaluation method, only the area of the peaks of interest is measured. Relative
response factors must be considered when converting areas to volume and when the response of
the given detector differs for each molecular type ofcompounds.
1.4.4.2 Internal Standard Method
In this technique, a non-quantity of internal standard is chromatographic andarea versus

concentration is ascertained. Then a quantity of internal standard is addedto the raw sample prior
to any sample pretreatment or separation operations. Thepeak area of the standard in the sample
run is compared with the peak are when the standard is run separately. This ratio serves as
correction factor for variation insample size, for losses in any preliminary pre-treatment
operations, or for incomplete elution of the sample. The material selected for the internal
standard must becompletely resolved from adjacent sample components and should not interfere
withthe sample components and never be present in samples.
Area ratio− Area oF sample

Area ratio=
Area of internal standar d
Area of sample
sample conc= Xconc . of standard
Area of internal standard
1.4.4.3 Area Normalization
This technique is often used for the sample having identical components. It issued to
evaluate the absolute purity of the sample. The procedure is to total up the areas under all peaks
and then calculates the percentage of the total area that iscontributed by the compound of
interest. For this method the entire sample must beeluted, all components must be separated and
each peak must be completely resolved.
1.4.4.4 Standard Addition methods
If only few samples are to be chromatographic, it is possible to employ the method of

standard edition (s). The chromatogram of the unknown is recorded, then a known amount of
analyte (s) is added and the chromatogram is repeated using same reagents, instruments and
other conditions. From the increase in the peak area (orpeak height), the original concentration
can be computed by interpolation.
If all instrumental reading (area/height)'Rx' is obtained, from a sample of unknown'x'and

a reading'Rt' is obtained from the sample to which a known concentration ‘ a'of analyte has been
added, then ‘ x' can be calculated from
X Rx
= −−−−(5)
x+ a Rt
A correction for dilution must be made if the amount of standard added, changes the total
sample volume significantly. It is always advisable to check the result by adding at least one
other standard47.
1.4.4.5 External standard method
The external standard method involves the use of a single standard or up tothree
solutions. The peak area or the height of the sample and the standard use arecompared directly.
One can also use the slope of the calibration curve based onstandard that contain known
concentrations of the compound of interest 48.
Steps Involved In Development of HPLC Method
Collect detailed account of all analytical methods developed tor the drug to avoid duplication
of the next method development. Details about the structure of the drugs and their
physicochemical properties are also collected and detailed drug profile collected.
 Selection of chromatographic method.

 First reversed phase should be tried.
 If not successful nor mal phase should be taken into consideration.
 For ion exchange or ion pair chromatography, first ion suppression by Ph control and
reversed phase chromatography should be tried.
 Selection of column stationary phase.
 Matching the polarity of sample and stationary phase and using a mobile phase of
different polarity achieve a successful separation.
1.4.5 Selection of mobile phase
Reversed phase bonded packing, when used in conjunction with highly polar solvents;
approach is ideal and is a universal system tor liquid chromatography. Mobile phase may be
either single liquid or combination of liquids, which arecompatible with sample, column and
instrument.
Selection of suitable detector
Detector is the eye of HPLC system and measures the compounds alter theirseparation on
the column. There are basically two types of detectors the bulk property detectors and solute
property detectors. Detectors, in order of their popularity are UV,fluorescent, conductivity, Polari
meter and refractive index detectors. UV detector is the first choice because of its convenience
and applicability in case of most of theSamples 49, 50
1.4.6 System Suitability
A system suitability test is an integral part of gas and liquid chromatographic method.
They are used to verify that the resolution and reproducibility of thechromatographic system are
adequate for the analysis to be done. The test is based onconcept that the equipment electronic,
analytical operation and sample to be analyzedconstitute an integral system that can be evaluated
as such.
It is the verification of the system to ensure system performance before or during the
analysis. Parameter such as plate count, tailing factor, reproducibility andresolution are
determined and compared against the specification set for the method. The area under curve
(AUC) of five replicate injections should not be more than 2%of relative standard deviation
(RSD) 51. 52
The parameters that are affected by the changes in chromatographic conditions are.
1.4.6.1 Retention Time (Rt)
Retention Time is the time of elution of maximum peak after injection of compound. It is
the time in between the sample is injected and chromatographic peakis recorded. The total
retention time (tR1 or tR2) is the time, which is needed by sample component to migrate from
column inlet (sample injection) to the column end(detector).
Recommendations
The resolution between two or more drugs should be more than 2.
1.4.6.2 Theoretical Plates (N)
It is also called as column efficiency. A column can be considered as being made of large
number of theoretical plates where distribution of sample betweenliquid-liquid or solid-solid
phase occurs.
The number of theoretical plates in column is even by the relationship
N=16(t/w)2----------(6)
Where, t is the retention time and w is the width at the base of the peak.
HETP = L/N----------(7)
Where, L is the length of column and N is the number or theoretical plates.
Recommendations
Theoretical plates should be more than 2000.
1.4.6.3 Resolution (R)
It is a function of Column efficiency and is specified to ensure that closely eluting

compounds are resolved from each other to establish the general resolving power of the system.
For the separation of two components in mixture the resolutionis determined by equation
2(t 2−t 1)
R=
w 1+w 2
Where, t2 and t1 is the retention time of second and first compounds respectively and
W2 and W1 are the corresponding widths at the bases of peak obtained byextrapolating straight
sides of the peaks to baselines.
Recommendations
R should be more than 2 between peak of-interest and the closest eluted potential interferences
(impurities, excipients, degradation products or internal standard).
1.4.6.4 Tailing factor (T)
It is the measure of peak symmetry, is unity for perfectly symmetrical peaksand its value
increases as tailing become more pronounced.
w 0.05
T= −−−−(9)
2F
Where, W0.05 is the width of peak at 5% height and F is the distance from the peakmaximum to
the leading edge of the peak height from the baseline. Tailing factorshould be less than 2.
Recommendations
T should be less than 2.
1.4.6.5 Capacity Factor (k')
The capacity factor is a measure of the degree of retention of an analyte relative to an

unrestrained peak, where tR is the retention time for the sample peak andto is the retention time
for an unrestrained peak.
It is calculated by the formula
' t
K= −1−−−−−(10)
t2
Where, t is the retention time of the drug [hat is the retention time of non retarded component, air
with thermal conductivity detection.
Recommendations
The peak should be well resolved from other peaks and the void volume. Generally the value of
kis > 2.
1.4.6.6 Selectivity (α)
These tests are used to verity that the resolution and reproducibility of the system are
adequate for the analysis to be performed. Parameters such as plate count, tailing factor,
resolution and reproducibility (%, RSD retention time and area for six repetitions) are
determined and compared against the specifications set for the methodon this basis of
measurement of above parameters decided that the performed method is suitable or selective for
development or not.
K' 2
α= −−−−−(11)
K'1
1.5 VALIDATION OF ANALYTICAL METHOD
Validation is defined as follows by different agencies
Food and Drug administration (FDA): Establishing documentation evidence, which provides a
high degree of assurance that specific process will consistently produce a product meeting its
predetermined specification and quality attributes45.
World Health Organization (WHO): Action of providing that any procedure process,
equipment, material, activity, or system actually leads to the expected results47.
European Committee (EC): Action of providing in accordance with the principles of good
manufacturing practice, that any procedure, process, equipment material,activity or system
actually lead to the expected results. In brief validation is a key process for effective Quality
Assurance52.
 Objective of Validation
The primary objective of validation is to form a basis for written procedures for production
and process control which are designed to assure that the drugproducts have the identity,
strength, quality and purity they purport or are representedto process. Quality, safety and efficacy
must be designed and built into the products. Each step of the manufacturing process must be
controlled to maximize theprobability that the finished product meets all quality and design
specifications 52.
1.5.1 Analytical method validation
Method validation can be defined as (ICH)"Establishing documented evidence, which

provides high a degree of assurance that specific activity will consistently produce a desired
result or product meeting its predetermined specifications and quality characteristics".
Method needs to be validated or revalidated
 Before their introduction into routine use

 Whenever the conditions changes for which the method has been validated, e. g.
Instrument with different characteristics
 Whenever the method is changed, and the change is outside the original scope of the
method.
 Purpose of Validation
 Enable the scientists to communicate scientifically and effectively or technical matter.
 Setting the standards of evaluation procedures for checking compliance and taking
remedial action.
 Economic: Reduction in cost associated with process sampling and testing.
 As quality of the product cannot always be assured by routine quality control because of
testing of statistically insignificant number of samples.
 Retrospective validation is useful for trend comparison of results compliance to
cGMP/cGLP.
 Closure interaction with Pharmacopoeial forum to address analytical problems.
 International pharmacopoeial harmonization particularly in respect of impurities
determination and their limits.
 Steps involved in validation procedures
 Proposed protocols or parameters for validations are established.
 Experimental studies are conducted.
 Analytical results are evaluated.
 Statistical evaluation is carried out.
 Report is prepared documenting all the results53
1.5.2 Types of Validation
The validation are mainly divided into two types which are,
1.5.2.1 Prospective Validation
This is performed for all new equipment, products and processes. It is aProactive
approach of documenting the design, specifications and performance beforethe system is
operational. This is most defendable type of validation.
1.5.2.2 Concurrent Validation
This is performed in two instances that are for existing equipment andverification of
proper installation along with specific operational tests is done. In caseof an existing,
infrequently made product, date is gathered from at least threesuccessful trials.
1.5.2.3 Retrospective Validation
This is establishing documented evidence that the Process is performed satisfactorily and
consistently over time, based on review and analysis of historicaldata. The source of such data is
production and QA/Qc records. The issues to be addressed here are changes to equipment,
process, specifications and other relevant changes in the past48.
1.5.3 Validation Parameters
The validation parameters as per ICH guideline are categorized mainly of following types53
Fig. 5: Systematic diagram for Validation Parameters
1.5.3.1 Precision
The precision of analytical method is the degree of agreement amongindividual test

results when the method is applied repeatedly to multiple sampling of homogenous sample. The
precision of an analytical method is usually expressed asthe standard deviation or relative
standard deviation (coefficient of variation) of a series of measurement. Reproducibility
expresses the precision between laboratories (collaborative studies usually applied to
standardization of methodology).
The ICH requires repeatability to be tested from at least six replicationsmeasured at 100% of
the test target concentration or from at least nine replications covering the complete specified
range. For example, the results can be obtained atthree concentrations with three injections at
each concentration.
 The repeatability of a test procedure is assessed by carrying out complete separate

determinations on the separate samples of the same homogeneous batch of the material
and this will provide a measure of the precision of the procedure under normal laboratory
operating conditions
 Intermediate precision is a term that has been defined by ICH (4) as the long term
variability of the measurement process. It is determined by comparing the results of a
method run within a single laboratory over a number of weeks. A methods inter mediate
precision may reflect discrepancies in results obtained
 From different operators.
 From inconsistent working practice (thoroughness) of the same operator.
 From different Instruments.
 With standards and reagents from different suppliers,
 With columns from different batches or
 A combination of these.
The objective of intermediate precision validation is to verify that in the same laboratory the
method will provide the same results once the development phase is over.
 Reproducibility, as defined by the ICH, represents the precision obtained between

different laboratories. The objective is to verify that the method will provide the same
results in different laboratories. The reproducibility of an analytical method is
determined by analyzing aliquots from homogeneous lots in different laboratories
with different analysts, and by using operational and environmental conditions that
may differ from, but are still within, the specified parameters of the method (inter
Laboratory tests).
Validation of reproducibility is important if the method is to be used in different laboratories.
 Differences in room temperature and humidity

 Operators with different experience and thoroughness
 Equipment with different characteristics, e. g. Delay volume of an HPLC system
 Variations in material and instrument conditions, e. g. In HPLC, mobile phases
composition, pH, flow rate of mobile phase
 Variation in experimental details not specified by the method
 Equipment and consumables of different ages
 Columns from different suppliers or different batches
 Solvents, reagents and other material with varying quality
1.5.3.2 Accuracy
The accuracy of an analytical method is the closeness of test results, obtainedby that method
to the true value. The accuracy of an analytical method should beestablished across its range. In
the case of the assay or a drug in the formulated product, accuracy may be determined by
application of the analytical method tosynthetic mixtures of drug product components to which
known ai71ount of analytehave been added within the range of the method. Average recovery
should be 99 to101 % of drug at each level.
The ICH documents recommended that accuracy should be assessed using aMinimum of nine
determinations over a minimum of three concentrations levels,Covering the specified range
(three concentrations and three replicates of each Concentration).
1.5.3.3 Limit of Detection
The lowest conc. of the analyte in the sample that the method can detect butnot necessarily
quantify under the stated experimental conditions simply indicatesthat the sample is below or
above certain level. Limit test prescribed as percentage or as parts per million. The limit of
detection will not only depend on the procedure ofanalysis but also on type of instrument.
1.5.3.4 Limit of Quantitation
The limit of Quantitation (LOQ) is the lowest amount of analyte in a sample that can be
determined with acceptable precision and accuracy under the stated experimental conditions. It is
expressed as the conc. of analyte (e. g., percentage, parts per billion) in the sample. The S/N ratio
should not less than 10 and RSD >2%.
1.5.3.5 Specificity
The specificity is the ability to assess unequivocally the analyte of interest in the
presence of component that may be expected to be present, such as impurities,degradation
products, and matrix components. In case of assay, demonstration ofspecificity requires that the
procedure is unaffected by presence of impurities orexcipients. In practice, this can be done by
spiking the drug substances or productwith appropriate levels or impurities or excipients, and
demonstrating that the assay result is unaffected by the presence of these extraneous materials. If
impurities of degradation product standards are unavailable, specificity may be demonstrated by
comparing the test result ofsamples containing impurities of degradation products to second well
characterized procedure. These comparisons should include samples stored under relevant stress
condition (e.g. light, heat, humidity, acid or base hydrolysis and oxidation)
1.5.3.6 Linearity and range
The linearity of an analytical method is its ability to elicit test results that are directly, or by a
well-defined mathematical transformation, proportional to the conc. of analyte in sample within
the given range. It should be established across the rangeof the analytical procedure. Linearity is
generally reported as the correlationcoefficients, the slope of regression line should be r2> 0. 999.
The range of analytical method is the interval between the upper and lowerlevel of analyte
that have been demonstrated to be determined with suitable level of conditions (e. g., light, Heat,
Humidity, acid or base, precision, accuracy, and linearity using method written. The range is
normallyexpressed in the same unit as test results (e. g., percent, part per billion).
1.5.3.7 Ruggedness
The ruggedness of analytical method is the degree or reproducibility of test results obtained
by the analysis of the same samples under a variety of conditions such as different laboratories.
different instruments. Different lots of reagents, different temperatures, Different days, different
analysts, etc. It is normally expressed as the lack of influence on test results of proportional and
environmental variables ofthe analytical method. For ruggedness study the conc. of analyte is
measured using different parameters such as.
 Different operator in same laboratory

 Different equipment in same laboratory
 Different source of segment and solution
 Different laboratory
1.5.3.8 Robustness
The robustness of analytical method is the measure of its capacity to remain unaffected by
small but deliberate variations in method parameters and provides an indication of its reliability
during normal usage. Experiments are per formed by changing conditions such as temperature
(+50cc), butter pH (±0. 5), and ionic strength of buffers, Level of additives to mobile phase. The
method must be robust enough to withstand slight changes and allow routine analysis of sample.
Table 3 : Acceptance Criteria of validation Parameter (ICH Guideline)
Characteristics Acceptance Criteria
Accuracy/Trueness Recovery 98-102%
Precision RSD < 2%
Repeatability RSD < 2%
Intermediate Precision RSD < 2%
Detection Limit S/N> 2 or 3
Quantitation Limit S/N> 10
Linearity Correlation coefficient r > 0.999
Range 80-120 %
2. DRUG PROFILE
Physico-Chemical Information of Molecule:
Molecular weight-627.75 g/mol

Pharmacological Category-Anticoagulant
BCS Class- Class II (Low solubility/High permeability)
Route of administration-Oral
Protein Binding- Relatively low binding (34-35%) to plasma proteins.
Physical Appearance: Yellow-white powder.
Melting Point: 155.08°C and 184.49 °C.
Dissociation Constants-pKa1 = 4.0 ± 0.1
pKa2 = 6.7 ± 0.1
Apparent Partition Coefficient: The partition coefficient of the neutral form (free base) is log P
= 3.8
Drug Solubility
Table 4: Drug Solubility:
Sr.No Solvent Solubility
1. Methanol Freely soluble
2. Ethanol Slightly soluble
3. Isopropanol Sparingly soluble
4. Acetone very slightly soluble
5. Ethyl Acetate Practically insoluble
Mechanism of Action
Anticoagulant Drug is an inactive pro-drug that is converted to Anticoagulant Drug, the active
form, by esterase-catalyzed hydrolysis in the plasma and liver. Anticoagulant Drug, the main
active principle in plasma, is a rapid-acting competitive and reversible direct inhibitor of
thrombin. Thrombin, a serine protease, is responsible for the conversion of fibrinogen to fibrin in
the coagulation cascade. Inhibition of thrombin consequently prevents thrombus development.
Anticoagulant Drug inhibits free thrombin, fibrin-bound thrombin and thrombin-induced platelet
aggregation.
3. Literature Review
The extensive literature survey have been carried out on various analytical method for
estimation of Anticoagulant Drug in pharmaceutical formulation, which describes of
qualification of Anticoagulant Drug by various methods like UV,HPLC etc, in pharmaceutical
dosage as well as in biological matrix, which provides the importance reference and the initiative
information about the molecule behavior under various chromatographic conditions.
Bhavani G. et al (2016) : have reported the estimation of Anticoagulant Drug in capsule

dosage form by Dissolution method development of RP-HPLC .The detection wavelength of
341 nm using UV detector. The chromatographic study was performed on a Phenomenex Luna
C18 (250 X 4.6mm;5µ,column at flow rate of 1.0ml/min. The solvent system of
triethylammonium phosphate buffer (pH-3.0) and acetonitrile in the proportion of 40:60 v/v,
diluent as 0.01 N HCL. The mentioned method was developed and validated as per ICH
guidelines.
Mrinalini C.Damle et al (2014): A simple, selective, proven, high performance, high

performance liquid phase (RP-HPLC) liquid chromatography (RP-HPLC) was developed and
approved to evaluate Anticoagulant Drug. The analysis was carried out on a Neosphere C8
column (150 mm x 4.6 mm). The mobile phase contains methanol: phosphate buffer (0.01 M, pH
3) in the ratio of 60:40 and is processed for ultrasonic degassing. It is served at room temperature
at a flow rate of 1 ml / min and a residence time of about 4.4 ± 0.05 minutes. The study was
conducted on an HPLC system equipped with a 225 nm PDA detector. The active ingredients are
subject to strong hydrolysis conditions (acidic, alkaline, and neutral), oxidation, photolysis and
thermal decomposition. The accuracy of this method is based on recovery studies.
Asmaa A. El Zaher et al (2015): Simple, accurate, accurate and fast LC (LC) was
developed for Anticoagulant Drug powder and pharmaceutical preparations. Chromatographic
separation was performed by isocratic elution on Eclipse XDB C8 (4.6 × 250 mm, 5) using 0.01
M orthophosphate acid (pH 2.6): acetonitrile (60:40 v / v). The flow rate is 1.5 ml min -1, and
UV detection is performed at 225 nm. This method was successfully used to separate DEM from
the decomposition product (Anticoagulant Drug). This method is approved in accordance with
ICH recommendations.
Kumar Raja Jayavarapu et al (2016): Simple and sensitive UV spectroscopy is

described for measuring Anticoagulant Drug in bulk and in pharmaceutical preparations. These
solubilization showed the maximum absorption at 326 nm using 0.1 n. HCl as a solvent. The
acceptable beer content range is from 03 to 21 µg / ml. When analyzing the formulas available
on the market, the results obtained by the proposed method correspond to the specified
quantities. The developed method was statistically verified in accordance with the
recommendations of the ICH. The developed ultraviolet spectrometry is simple, sensitive and
unique and can be successfully used in laboratory studies, as well as in the routine analysis of
pharmaceutical preparations of Anticoagulant Drug in industry.
P. Manasa et al (2015): The purpose of this article is to develop fast, sensitive, accurate,
accurate linear stability, demonstrating reverse-phase HPLC, and test it in accordance with ICH
guidelines for assessing Anticoagulant Drug inside the capsule. The optimized method was
Phenomenex Kinetex EVO C18 (250 × 4.6 mm, 5 μm), triethylammonium phosphate buffer (pH
2.0) in a ratio of 30: 30: 40 vol. / About.: Methanol: acetonitrile phase, Min and detection
wavelength 254 nm. The optimized method separates all pollutants from the top of the
preparation by forced decomposition.
D. VIJAY KUMAR, et al (2015):To develop a rapid, sensitive, accurate, precise and

linear Reverse Phase High Performance Liquid Chromatographic (RP-HPLC) method. The
optimized method uses a reverse phase column, Waters Symmetry C18 (250 X 4.6 mm; 5μ), a
mobile phase of tri ethyl ammonium phosphate buffer (pH 2.5): acetonitrile in the proportion of
40:60 v/v, flow rate of 1.0 ml/min and a detection wavelength of 313 nm using a UV detector.
The developed method resulted in Anticoagulant Drug eluting at 2.44 min. A sensitive, rapid,
accurate, precise and linear RP-HPLC method was developed and validated for the quantitative
estimation of Anticoagulant Drug in capsules as per ICH guidelines and hence it can be used for
the routine analysis in various pharmaceutical industries.
P. SOWNDARYA, et al (2015): Development of a simple and cost-effective UV

spectrophotometric method for encapsulating and validating Anticoagulant Drug (150 mg) in
accordance with ICH guidelines Optimized procedure used 0.05 n. HCL as a solvent to evaluate
the analysis of Anticoagulant Drug in capsules with a wavelength of 325 nm. Thus, ICH
recommendations can be used for routine analysis in various pharmaceutical industries.
Shoumik Roy et al (2017): Accurate, sensitive, and rapid RP-HPLC methods have been
validated to evaluate Anticoagulant Drug from a capsule dosage form. The separation was
carried out on a Zorbax C18 column (100 × 4.6 mm, 3.5 μm) in the isocratic mode, and the
mobile phase contained acetonitrile: water in a ratio of 70:30 vol. / about. The flow rate of the
mobile phase is 1.0 ml / min, and detection is carried out at a wavelength of 225 nm. The
residence time of the Anticoagulant Drug is 3.0 minutes. This method is approved in accordance
with ICH recommendations. In addition, the proposed method can be used for routine analysis of
Anticoagulant Drug in quality control laboratories.
RAJESH NAWALE et al (2018): to develop and validate new, simple, and selective
reverse-phase–high-performance liquid chromatography (RP-HPLC) method for the quantitative
determination of Anticoagulant Drug and its impurities in pharmaceutical dosage form as per the
International Conference on Harmonization guidelines. Chromatographic analysis was
performed on Princeton SPHER-l00 C18 (250 × 4.6 mm, 5 μm) HPLC column, maintained at
50°C column temperatures, 6°C sample tray temperature, and detection monitored at 225 nm.
The mobile phase consisted of acetonitrile:phosphate buffer (pH 2.5) (33:67 V/V). The flow rate
was maintained at 1.0 ml/min.
N.Madhavi Latha et al (2016):The RP- HPLC method was developed and validated for
the determination of Anticoagulant Drug in bulk form. The chromatography was carried out on
Unisol C18 (4.6 × 150 mm, 3μm) using a mobile phase of methanol and ammonium acetate
buffer in the ratio of 90:10, at a flow rate of 1 ml/minute. The analytes were monitored at 226 nm
using a PDA detector. The retention time of the DEM was 2.52 min. The method was linear in
the concentration range of 20-100 µg/ml with a correlation coefficient of 0.999. The method was
validated as per ICH guidelines.
K.Sridhar Reddy et al (2016):A simple, precise, accurate, economical and reproducible

RP-HPLC method for estimation of Anticoagulant Drug in capsule dosage form has been
developed. Quantitative HPLC was performed with shimadzu LC 20atwith Spin chrome
Software with UV-Visible Detector (SPD-20A), Phenomenex Luna C18, 5µm, 250 x 4.6mm
(size) column was used in the study. The mobile phase of Methanol: Water (70:30) used in this
study. The conditions optimized were: flow rate (1.2 ml/minute), wavelength (230 nm) and run
time was 10 min, column temperature was maintained at 500C. Retention time was found to be
4.60 min. The developed method was evaluated in the assay of commercially available capsules
Paradaxa containing Anticoagulant Drug. The amount of drug in capsule was found to be 75mg.
By using method, stability of the drug has been studied.
Dare M et al (2015):Method Validation for Stability Indicating Method of Related

Substance in Active Pharmaceutical Ingredients Anticoagulant Drugby Reverse Phase
Chromatography the stability indicating, highly accurate precise and linear method for related
substance of the Anticoagulant Drug through the reverse phase high performance liquid
chromatography and it is validated as per the current ICH guideline. The optimize method uses a
reverse phase column, Poroshell 120 EC -18 (150 mm × 4.6 mm, 2.7µ), Mobile Phase of hexane-
1 Sulfonic acid sodium salt monohydrate (6.5 ± 0.05) and Methanol through gradient flow rate of
0.6ml/min. Keeping the column at temperature 30°C and using the sample amount 10 µL at 5°C
and detected all the impurities at 230 nm by the UV detector. In the developed method, elution of
Anticoagulant Drug was at 26.9 min and all the eluted Impurities were well separated and met
the system suitability criteria.
SYED SHAHED HUSSAIN, et al (2015): To develop a simple and a cheap UV

spectrophotometric method for the quantitative estimation of Anticoagulant Drug in capsules and
validate as per ICH guidelines.The optimized method uses triethyl ammonium phosphate
aqueous solution (pH 2.5) as a solvent for the estimation of assay of Anticoagulant Drug in
capsules at a wavelength of 325 nm. The developed method resulted in Anticoagulant
Drugexhibiting linearity in the range 5-15μg/ml. Method was found to be robust with respect to
wavelength and pH of the solvent. A simple and a cheap UV spectrophotometric method was
developed and validated for the quantitative estimation of Anticoagulant Drug in capsules as per
ICH guidelines and hence it can be used for routine analysis in various pharmaceutical
industries.
4. AIM AND OBJECTIVES
Market survey revealed that, day by day new drugs and their combinations with other
drugs are being introduced, as these are more effective than single drug. So, the analytical
chemists are facing challenges for the development and validation of analytical methods for
these combinations. Now a day's instrumental methods of analysis are widely accepted over
classical methods, as these extremely sensitive, provide precise, accurate results and detailed
information from small samples of material. They are rapidly executed and in general are readily
amenable to automation. Examples: Spectrophotometric method and Chromatographic method
(HPLC, HPTLC and GC.) After a thorough literature survey it was evident that, there is no any
method reported tor simultaneous estimation of Anticoagulant Drugby RP-HPLC method.
Present work is an attempt to develop a novel, rapid, sensitive and reproducible UV

Spectrophotometric and Reverse Phase High Performance Liquid Chromatographic (RP-HPLC)
method for quantitative determination of Anticoagulant Drug in pharmaceutical dosage forms.
The performance of the method will be validated according to the present ICH guidelines for
specificity, limit of detection, limit of quantification, linearity, accuracy, precision, robustness
and ruggedness.
The main aim and objectives behind this project work is

1. To establish accurate, simple, sensitive precise and cost effective RP-HPLC method for
simultaneous estimation capsule dosage form.
2. To describe the system suitability parameters or proposed method for estimation of
Anticoagulant Drug
3. To validate the proposed method as per ICH guidelines And parameters,
3.2] PROPOSED PLAN OF WORK:
Literature search
Method development
 Solubility studies
 Buffer selection
 Mobile phase composition
 Selection of column
 Chromatographic conditions
 Sample preparation
Method validation
 Specificity
 Linearity
 Accuracy
 Precision
 Robustness
 Solution stability
 Intermediate precision
 Filter interference
5. PLAN OF WORK:
An outline of proposed work is given below-
 Selection of marketed formulation.

 Study of physicochemical properties of marketed capsule formulation.
 Literature Survey
 Method development
Trial of the instrumental methods on pure drug samples which includes the following steps-
1) Spectrophotometry:
 Determination of scanning wavelength
 Study of additivity of absorbance at selected wavelength
2) High Performance Liquid Chromatography

 Selection of mobile phase.
 Selection of column.
 Selection of chromatographic conditions.
 Application of developed method for working standards.
 Application of developed method for estimation in capsule formulation.
 System suitability parameters
3) Validation of the proposed methods

 Specificity
 Accuracy
 Precision
 System precision
 Method precision
 Intermediate precision or Ruggedness
 Linearity and range Robustness
 Filter paper study Solution stability study
 System suitability Testing Assay
6. Experimental Work
UV Spectrophotometric studies on Anticoagulant drug
1. Materials and Reagent

2. Instrument and Equipment
3. Selection of analytical wavelength
RP-HPLC Method Development for Anticoagulant drug
1. Material and Reagents

2. Instruments and Equipment
3. Preparation of Solutions
4. Optimization of Chromatographic Condition
Method Validation
1. Specificity
2. Linearity and Range
3. Precision
4. Intermediate Precision
5. Recovery (accuracy) studies
6. Robustness studies
7. Solution Stability
System Suitability Test Parameters:
 Drug Sample
Table no.5: Drug Sample with its Percent Assay

Sr.No. Name of Drug % Assay
1 Anticoagulant drug 100.2%
 Materials and Reagent used in studies of Anticoagulant drug.
Table no 6: Materials and Reagent

Procured from/supplied
Sr.No. Name of Chemical Grade
by
1. Orthophosphoric acid Excela R Thermo Fisher Scientific
2. Triethyl amine HPLC Thermo Fisher Scientific
3. Methanol Qualigens (HPLC) Thermo Fisher Scientific
4. Acetonitrile Qualigens (HPLC) Thermo Fisher Scientific
 Instruments used for estimation of Anticoagulant drug

Table no 7: Instrument and Equipment’s
Sr.No Instrument Name Make Model Software

1. UV- Visible double beam Shimadzu, Japan UV-1800 UV Probe
spectrophotometer
2. Electrical Balance Mettler Toledo AB204-S/FACT -
3. HPLC System DGU-20A.3 LC solution

Shimadzu, Japan
4. Ultra Sonication 08895-34 -
Cole-parmer
5. pH meter Agilent 3200P -
Technologies
Software’s Used
 M-power for waters Aquity HPLC.
 Spectra manager for UV Spectrophotometer.
Methodology
6.1 UV SPECTROPHOTOMETRIC METHOD

6.1.1 Preparation of standard stock solution
Accurately weighed quantity of Anticoagulant drug 100 mg was transferred to the 100 ml
volumetric flask and dissolve in methanol. The volume made up to the mark with methanol to
make (1000 µg/mL) concentration of Anticoagulant drug.
Standard Solution preparation :
Further 1 mL of standard Stock solution Diluted to 100mLwith MeOH (10µg/mL). again

4.0mLof this solution diluted to 10.0mL with MeOH (4.0µg/mL).
The standard solution of Anticoagulant drugwas scanned at different concentrations in

the range of 200-400 nm and λmax was found to be 226 nm against reagent blank.
6.2 RP HPLC Method Development for Anticoagulant drug

6.2.1 Selection Of Mobile Phase
For selection of optimum mobile phase for development of HPLC method of Anticoagulant
drug estimation of various experiment were performed finally the mobile phase with composition
of Buffer and Acetonitrile was selected
Buffer Prepration: Take 14 ml Tri ethyl amine in 1000 ml of water and add mix sonicate to
dissolve adjust the pH 3.0 with Ortho phosphoric acid.
Mix above buffer and Acetonitrile in the ratio of (550 :450), filter with 0.45µm Nylon filter.
Sonicate and degassed the mobile phase. Use this mobile phase for the analysis and for
chromatographic study.
6.2.2 Selection of flow rate
Different mobile phase flow rate (0.8,-1.0 ml/min) was tried. The optimum flow rate for
which theoretical plate number was maximum, with best resolution of all peaks and with
reasonable run time (10 min) was selected.
Preparation of solution
6.2.3 Preparation of Diluent
Water: Methanol (300: 700)
6.2.4 Preparation of Standard stock solution and Calibration Curves:

Accurately weigh and transfer about 11 mg of Anticoagulant drug working standard into a
10 ml volumetric flask,add about 7.0 ml diluent . Dissolve by sonication, cool and make up the
volume with diluent and mix well, and further 5 ml of above standard solution transfer in to 50
ml volumetric flask and make up with diluent. Filter through 0.45µ nylon membrane filter.
6.2.5 Preparation of Sample solution

Weigh accurately 282.0mg of capsule fill equivalent to 110mg of anticoagulant
drug.Transfer this fill in to a 100 ml of volumetric flask . Add about 70 ml of diluent and
sonicate 20 min with occational shaking and dilute to volume with diluent and mix . Filter 10 ml
of this solution with 0.45 µ nylon membrane filter and further dilute 5 ml of this solution to 50
ml volumetric flask and make up with diluent and mix well . Filter the solution through 0.45µ
nylon filter
6.3 METHOD DEVELOPMENT
Trial 1:
Table No.8: Table showing chromatographic conditions for trial 1.
Parameters Conditions
Mobile Phase Methanol :Water(500:500)
Column Thermo scientific 250X4.6mm,5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Column oventemperature 40°C
Injectionvolume 5 µL
Runtime 15 min
Detector UV Detector
Trial 2:
Table No.09 : Table showing chromatographic conditions for trial 2.
Mobile Phase Methanol :Water (700:300)
Column Thermo scientific 250X4.6mm, 5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Injectionvolume 5 µl
Runtime 15 min
Trial 3:
Table No.10 : Table showing chromatographic conditions for trial 3.
Methanol: Acetonitrile:Buffer
Mobile Phase
(200:300:500)
Buffer pH 4.75
Column Supero LC-CN(Cyno) 250X4.6mm, 5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 4:
Table No. 11: Table showing chromatographic conditions for trial 4.
Methanol: Acetonitrile:Buffer
Mobile Phase
(100:400:500)
Buffer pH 4.75
Column Zorbax NH2250X4.6mm, 5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 5:
Mobile Phase Methanol:Buffer (500:500)
Buffer pH 4.75
Column Inersil 100 A° 300X3.9mm, 5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Injectionvolume 5 µL
Runtime 15 MIN
Trial 6:
Mobile Phase Acetonitrile:Buffer (700:300)
Buffer pH 4.75
Column Water 300X3.9mm,10µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Injectionvolume 5µl
Runtime 15 min
Trial 7:
Mobile Phase Acetonitrile:Buffer (700:300)
Buffer pH 3.0
Column Water 300X3.9mm,5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 8:
Table No. 15: Table showing chromatographic conditions for trial 8 .
Mobile Phase Acetonitrile: Buffer (600:400)
Buffer pH 3.0
Column Inertsil ODS 250X4.6mm,5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 9:
Buffer pH 3.0
Column Luna Phenomenax 100A° C18 250X4.6mm,5µm
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 10:
Buffer pH 3.0
Wavelength 226 nm
Flowrate 0.8 ml/min
Runtime 15 min
Trial 11: Final optimized method :
Buffer pH 3.0
Wavelength 226 nm
Flowrate 1.0 ml/min
Runtime 10 min
6.4 Validation:
The developed chromatographic method was validated for system suitability, linearity,
range, accuracy, precision, LOD-LOQ and robustness parameters as per Q2 (R1) ICH guidelines.
6.4.1 Specificity
Specificity of an analytical method is its ability to measure accurately and a specifically
the analyte of interest without interferences from blank and placebo.Blank and sample
preparation as per test procedure was injected and the interference of blank peaks with the
analyte peak was checked. The purity for the main peak in sample preparation and standard
preparation was also checked.
6.4.2 Linearity and range
Working standard solutions were injected in the range of 55.14- 165.42μg/ml under the
optimized chromatographic conditions and peak areas were calculated at 226 nm. The calibration
curve was plotted between areas against concentrations of the drug. Linear regression data as
well as calibration curve were shown in table no 18 under result and discussion section.
6.4.3 Precision
Repeatability study was carried out with nine replicates and intermediate (inter-day)
precision was carried out with three concentrations of Drug with three replicates. The values of
% relative standard deviation (% RSD) for both the parameters are shown in table no 39 and 40
under result and discussion.
6.4.4 Intermediate Precision
The intermediate precision of the method was checked by determining precision on a

different instrument, analysis being performed in on a different day. The % content of all DAB
impurities and total impurities was found to be below quantification level even when it is
performed on a different instrument. The method is said to be precise with respect to the criteria
of the intermediate precision.
6.4.5 Accuracy studies
The accuracy of the method was determined by calculating percentage recovery of

Anticoagulant drug from combined gel formulation. Recovery studies were carried out by
applying the method to gel sample containing Anticoagulant drug at 75, 100 and 125% levels. At
each level three determinations were carried out and the results obtained were compared.
6.4.6 Robustness studies
Robustness of the optimized method was studied by changing column temperature

(±2°C), wavelength (±2 nm) and mobile phase composition (±2%) during analysis. The sample
was injected in triplicate for every condition and % RSD was calculated for each condition is
shown in table no 33,34,35 and 36 under result and discussion section.
6.4.7 System suitability:
Standard solution of Anticoagulant Drug was injected six times into HPLC system as per
test procedure. The system suitability parameters were evaluated from standard chromatograms
and the % RSD of area, retention times, tailing factor, theoretical plates and resolution was
calculated from six replicate injections. Result for system suitability parameters are given in
table no 37 under result and discussion section
7. Result And Discussion
7.1 UV Spectrophotometric studies on Anticoagulant Drug
7.1.2 Selection of analytical wavelength:
Anticoagulant Drug was examined using Shimadzu UV-1800 in the range of 200-800
nm. Solution of 4.0 µg/mL concentration of drug samples was prepared in Methanol and
scanned in UV-visible range (200 to 800 nm) using methanol as blank.
093321 - Raw Data - 181121_STD 5ppm_083041.spc

0.51
2
0.40
1
Abs.
0.20
0.00
-0.05
200.00 300.00 400.00 500.00 600.00
nm.
Fig no.6:Selection of analytical wavelength
The standard solution of Anticoagulant drugwas scanned at different concentrations in

the range of 200-400 nm and λmax was found to be 226 nm against reagent blank
7.2 Optimization of RP-HPLC Method for Anticoagulant Drug
Different mobile phases composition were tried in order to find the best condition for
elution Anticoagulant Drugat same Chromatographic conditions such as Column – Luna
Phenomenax,100A° C18 (250X4.6mm,5µm) column , column temperature 25° C, detection by
using UV detector. Different flow rate were tried as follows:
Trial No 1:
1850.00
1800.00
psi
1750.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes
Fig. no 7 - Mobile phase- Methanol: Water (50:50 v/v),

Flow Rate –0.8 ml/min),BDS Hypersil C18 250X4.6mm,5µm(40°C)
Table no 19: Observation for trial 1
Observations Observations for Anticoagulant
Retention time -
Area -
Base line Good
Peak shape -
Conclusion Peak not observed, Need to be optimize
Trial No 2:
2750.00
2700.00
psi
2650.00
2600.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes
Fig. no 8 - Mobile phase- Methanol: Water (70:30 v/v),

Flow Rate –(0.8 ml/min),BDS Hypersil C18 250X4.6mm, 5µm, column (40°C)
Retention time -
Area -
Base line Noisy
Peak shape -
Trial No 3:
0.001
0.000
AU
-0.001
-0.002
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes
Fig. no 9 - Mobile phase- Buffer: Acetonitrile: Methanol (50:30:20 v/v),

Flow Rate –(0.8 ml/min), Supeiro LC-CN (cyno), 250X4.6mm, 5µm column(40°C)
Retention time -
Area -
Base line Noisy
Peak shape -
Trial No 4:
0.002
0.000
AU
-0.002
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes
Fig. no 10 - Mobile phase- Buffer: Acetonitrile: Methanol (50:40:10 v/v),

Flow Rate –(0.8 ml/min),Zorbax NH2,250X4.6mm, 5µm, column(40°C)
Retention time -
Area -
Base line Wavy
Peak shape -
Trial No 5:
0.02
0.00
AU
-0.02
-0.04
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes
Fig. no 11 - Mobile phase- Buffer: Methanol (50:50 v/v),

Flow Rate –(0.8 ml/min), Inersil 100A° 300X3.9mm, 5µm, column(40°C)
Retention time -
Area -
Base line Wavy
Peak shape -
Conclusion Low Peak Response, Need to be optimize
Trial No 6:
0.006
0.004
5.6
74
0.002
AU
0.000
-0.002
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes
Fig. no 12 - Mobile phase- Buffer: Acetonitrile (70:30 v/v),

Flow Rate –(0.8 ml/min),Water 300X3.9mm,10µm, column(40°C)
Retention time 5.674
Area 21321
Asymmetry 1.03
Theoretical Plates 7947
Base line Wavy
Peak shape Good
Trial No 7:
0.020
0.010
5.1
54
AU
0.000
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes

Flow Rate – (0.8 ml/min),Waters, 300X3.9mm,5µm, column(40°C)
Area 88333
Asymmetry 1.36
Base line Good
Peak shape Good
Trial No 8:
0.15
7.6
10
0.10
AU
0.05
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes

Flow Rate – (0.8 ml/min),Inertsil ODS250X4.6mm,5µm, column(40°C)
Area 7322165
Asymmetry 0.72
Base line Wavy
Peak shape Bad
Conclusion Need to be optimize
Trial No 9:
0.40
5.1
58
0.20
AU
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
Minutes
Fig. no.15 - Mobile phase- Buffer: Acetonitrile (55:45 v/v),

Flow Rate – (0.8 ml/min), Luna Phenomenax,100A° C18 250X4.6mm,5µm, column(40°C)
Area 5327737
Asymmetry 1.42
Base line Good
Peak shape Good
Due to high temperature Drug might be
Conclusion degrading so thermostat temperature needs to
be change (40°C25°C)
Trial No 10:
5.4
0.40
56
AU
0.20
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.0011.0012.0013.0014.0015.00
Minutes

Flow Rate – (0.8 ml/min), Luna Phenomenax,100A° C18 250X4.6mm,5µm, column(30°C)
Area 5868568
Asymmetry 1.14
Base line Good
Peak shape Good
Conclusion Flow needs to be Increased (0.81.0mL/min)
Trial No 11: Method Develop
0.60
0.50
4.13
5
0.40
0.30
AU
0.20
0.10
0.00
0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Minutes
Fig. no 17-Mobile phase- Buffer: Acetonitrile (55:45 v/v),

Flow Rate – (1.0 ml/min), Luna Phenomenax,100A°,C18 250X4.6mm,5µm column (25°C)
Area 5088114
Asymmetry 1.31
Base line Good
Peak shape Good
Conclusion Method Ready for the validation
7.2 System suitability test parameters:
Initially, various chromatographic conditions were tried in order to obtain better
separation characteristics by changing mobile phase composition, ACN: Buffer (55:45v/v) and
1.0min /ml flow was selected and detected by using UV detector. The retention time of
Anticoagulant drug was found to be and 4.0-5.0 min respectively. Optimized chromatographic
conditions are mentioned in table no.
Table no 30: Optimized Chromatographic conditions
Parameters Drug
Column Luna Phenomenax,100A° C18 (250X4.6mm,5µm)
Flow rate 1 ml/min
Injection volume 20 µl
Retention time 10 min
Detection UV Detector
Mobile Phase ACN: Buffer (55:45v/v)
Diluent Methanol:Water
Theoretical plate NLT 2000
Tailing factor NMT 2.0
7.3.Validation of the analytical method

The developed chromatographic method was validated for Specificity, linearity,
recovery, precision and robustness and solution stability parameters as per ICH guideline Q2
(R1).
7.3.1 Specificity
Specificity of an analytical method is its ability to measure accurately and a specifically
the analyte of interest without interferences from blank and placebo.
Solution preparations :
Mobile phase: Buffer Preparation: Take 14 ml Tri ethyl amine in 1000 ml of water and add
mix sonicate to dissolve adjust the pH 3.0 with Ortho phosphoric acid.
Mix above buffer and Acetonitrile in the ratio of (550:450), filter with 0.45µm Nylon
filter. Sonicate and degassed the mobile phase. Use this mobile phase for the analysis and for
chromatographic study.
Diluent- Methanol:Water (70:30 v/v) .
Placebo Preparation: 172.5 mg of excipient into 100 ml of diluent.
Preparation of standard solution:
Transfer an accurately weighed quantity of about 11.05 mg of Anticoagulant Drug

reference standard into a 10 ml volumetric flask, dissolve and dilute to volume with
Methanol:Water (70:30 v/v)and take 5 ml of std solution into dilute 50 ml diluents .
Preparation of Sample solution:
282.5 mg of average weight of capsule dissolved it to diluent and make up the volume
upto 100 ml. And take 5 ml of this sample dilute 50 ml diluents.
Placebo spiked with API at target concentration-
172 mg of placebo and 110 mg of Anticoagulant drug into 100 ml volumetricflask, Add
about 70 ml of diluent and sonicate 20 min with occational shaking and dilute to volume with
diluents and mix. Filter this solution with 0.45 µ nylon membrane filter.
Table no.31- Injection sequence for specificity
Sr. No. Name of solution No. of Injections
1. Mobile phase 1
2. Diluent 1
3. Placebo 1
4. Identification of Drug 1
5. Standard Solution 1
6. Sample solution 1
7. Placebo spiked with API 1
Table No.32
Injection Retention
Sample Name Area Response
No. Time
1. Mobile Phase - Nil
2. Diluent - Nil
3. Placebo Solution - Nil
4. Identification Standard 4.125 5088114
5. Identification Sample 4.126 5166624
6. Placebo Spiked with API 4.120 5147882
7.3.2 Linearity and range

The linearity of an analytical procedure is its ability (within a given range) to obtain test
results which are directly proportional to the concentration (amount) of analyte in the samples.
For Anticoagulant drug the linearity was performed at five levels viz, 50%, 75%, 100%, 125%
and 150% of limit concentration.
Range
The range of analytical procedure is the interval between the upper and lower
concentration of analyte in the sample for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and linearity.
The range derived from linearity studies.
Blank-Methanol: Water (70:30 v/v) .
Placebo Preparation-172.5 mg of excipient into 100 ml of diluent.
Linearity standard stock solution
Accurately weighed and transfer about 55.030 mg of Anticoagulant Drug into 50 ml of

diluent made further dilutions from above stock solution.
Table no.33-Preparation of linearity solution for anticoagulant Drug:
Dilutions from standard anticoagulant Drug(concentration in

Level
stock solution ppm)
50 1.0 ml → 20 ml 55.14
75 1.5 ml → 20 ml 82.71
100 2.0 ml → 20 ml 110.28
125 2.5 ml → 20 ml 137.85
150 3.0 ml →20 ml 165.42
Procedure -
Inject as per sequence for anticoagulant Drug and calculate regression coefficient, % y intercept
and response factor.
Injection sequence-
Table no.35– Injection sequence for anticoagulant Drug

Sr. No. Injection Name No. of Injections
1 Mobile phase 1
2 Diluent 1
3 Linearity level-1 1
1.5.1 Acceptance Criteria

1. The correlation coefficient value should not be less than 0.995 over the selected
range.
2. % y –intercept should within ± 2.0%
Results
Table no 36- Linearity of Anticoagulant Drug
Concentration Peak Areas for
Sr. No. Level
µg/ml Drug
1 50% 55.14 2560654
2 75% 82.71 3919121
3 100% 110.28 5113107
4 125% 137.85 6400159
5 150% 165.42 7721425
Fig no.18 – Calibration curve

Table no 37- Linearity regression data for calibration curve
Parameter Anticoagulant Drug
Linearity Range 55.14-165.42
r² ± SD* 0.9997
Y= mx + c Y=46,436,5835x + 21,861.2000
7.3.3 Precision studies
The precision of an analytical method is the closeness of test results between series
ofmeasurements obtained from multiple sampling of the same homogenous sample underthe
prescribed conditions.
Transfer an accurately weighed quantity of about 11.05 mg of Anticoagulant Drug reference

standard into a 10 ml volumetric flask, dissolve and dilute to volume with Methanol:Water
(70:30 v/v)and take 5 ml of std solution into dilute 50 ml diluents .
Sample Preparation: (Prepare the Six sample preparation )

Weighed accurately 285.5 mg of Anticoagulant drug capsule content and dilute with
Methanol:Water (70:30 v/v) diluent and make up the volume up to 100 ml.Prepared in duplicate.
Procedure
Separately inject equal volume of solutions as per sequence of injection into the
chromatograph and record the peak area responses for the major peaks and check for the system
suitability requirements.
Injection sequence
Table no- 38 Injection sequence for precision
1. Mobile phase 1
2. Diluent 1
3. Standard Solution 6
4. Precision set – 1 1
Acceptance criteria
% RSD of results for assay from six replicate test solution preparations should be not more than
2.0%.
Procedure-
Repeatability was studied by six replicates of the working standard (0.11 mg/ml)
solutions for Anticoagulant drug. Intermediate (Inter day) precision studies were performed by
repeated injections of standard drug solutions at (0.11mg/ml) on differentHPLC system. The
method is precise as the % RSD values (Table 24 and 25) were found within an acceptable
limit.
Table no.39 - Repeatability studies
Sample Sample Drug Drug Spl Dil Dil Dil

Name Area (mg/Cap) (%) (mg) (mL) (mL) (mL)
Precision Set I 5151970 110.30 100.28 285.2 100 5 50
Precision Set II 5197058 110.30 100.27 287.7 100 5 50
Precision Set III 5110513 109.30 99.36 285.5 100 5 50
Precision Set IV 5093701 109.79 99.81 283.3 100 5 50
Precision Set V 5198308 110.95 100.86 286.1 100 5 50
Precision Set VI 5145734 110.17 100.15 285.2 100 5 50
Average 100.12
SD 0.50
%RSD 0.50
7.3.4 Intermediate precision-
To demonstrate the reproducibility (Ruggedness) of the test results obtained by an
analytical method for the variability of instrument, column, analyst and day. Analyze six test
preparation as per analytical method representing a single batch and determine the assay for the
same. Evaluate the intermediate precision by computing the results and relative standard
deviation of the results of assay.
Transfer an accurately weighed quantity of about 11.048 mg of Anticoagulant Drug

reference standard into a 10 ml volumetric flask, dissolve and dilute to volume with
Methanol:Water (70:30 v/v)and take 5 ml of std solution into dilute 50 ml diluents.
Sample Preparation: (Six sample preparations)
Methanol:Water (70:30 v/v) diluent and make up the volume up to 100 ml. Prepared in duplicate.
Procedure
Separately inject equal volume of solutions as per sequence of injection into the
chromatograph and record the peak area responses for the major peaks and check for the system
suitability requirements.
Table no.39–Injection sequence for intermediate precision
1 Mobile phase 1
2 Diluent 1
3 Standard Solution 6
4 Intermediate Precision set – 1 1
Table no 40- Intermediate precision studies
Sample Sample Drug Drug Spl Dil Dil Dil

Name Area (mg/tab) (%) (mg) (mL) (mL) (mL)
Precision Set 1 5151970 110.30 100.28 285.2 100 5 50
Precision Set 2 5197058 110.30 100.27 287.7 100 5 50
Precision Set 3 5110513 109.30 99.36 285.5 100 5 50
Precision Set 4 5093701 109.79 99.81 283.3 100 5 50
Precision Set 5 5198308 110.95 100.86 286.1 100 5 50
Precision Set 6 5145734 110.17 100.15 285.2 100 5 50
Int Precision set – 1 5101979 109.87 99.88 281.6 100 5 50
Average 100.9
SD 0.58
%RSD 0.58
7.3.5 Accuracy Studies for Anticoagulant Drug
The accuracy of an analytical procedure expresses the closeness of agreement between

thevalue which is accepted either as a conventional true value or an accepted reference valueand
the value found, Accuracy may often be expressed as present recovery byassay of known, added
amounts of analyte. Accuracy is a measure of the exactness of theanalytical method that is true
for all practical purpose.Perform accuracy at three levels and each level in triplicate preparation,
50%, 100% and150%.
Blank and Standard solution.
Preparation of stock solution for Accuracy
Standard Solution: Accurately weighed about 11.026 mg reference standard into10 ml diluent
and take 5ml std stock solution dilute 50 ml diluents.
a) 50% Accuracy Level preparation :
Accurately weighed about 55.0 mg anticoagulant Drug reference standard into a 100 ml
volumetric flask, dissolve and dilute to volume with Methanol:Water (70:30 v/v)
b) 100% Accuracy Level preparation :

c) 150% Accuracy Level preparation :

Table no 41: Sequence of Accuracy level
1 Diluent 1
2 Standard Solution 6
3 Recovery (50%) - Set I 1
4 Recovery (50%) - Set II 1
5 Recovery (50%) - Set III 1
Result-
Table no. 42 - Accuracy data
Actual
Wt. of API Theoretical
Sample Dil. conc. %
Sample Name Placebo added conc.
Area (mL) (mg/mL Recovery
in (mg) (mg) (mg/mL)
)
Recovery (50%) - I 2534258 177.1 55.0 100.0 0.055 0.055 99.54
Recovery (50%) - II 2562481 178.0 55.0 100.0 0.055 0.055 100.65
Recovery (50%) - III 2516874 177.8 55.0 100.0 0.055 0.054 98.86
Recovery (100%) - I 5116204 176.9 110.0 100.0 0.110 0.111 100.48
Recovery (100%) - II 5080509 176.8 110.0 100.0 0.110 0.110 99.78
Recovery (100%) - III 5057898 177.0 110.0 100.0 0.110 0.109 99.33
Recovery (150%) - I 7652497 176.8 165.0 100.0 0.165 0.165 100.19
Recovery (150%) - II 7609847 178.0 165.0 100.0 0.165 0.164 99.64
Recovery (150%) - III 7612541 177.5 165.0 100.0 0.165 0.164 99.67
Level Area SD %RSD

50% 99.68 0.90 0.91
100% 99.86 0.58 0.58
150% 99.83 0.31 0.31
The recovery studies were performed on standard solution at 55,110,165μg/ml

concentrations of Anticoagulant Drug of 50%, 100%, 150 % accuracy level. The mean %
recovery ± S.D. values corresponding to 3 levels were found to be 99.68± 0.90, 99.86 ± 0.58 and
99.83 ± 0.31 for Anticoagulant drug. The results are shown in Table no .42
7.3.6 Robustness of the method:

one factor was change at one time to estimate the effect. Robustness of method was evaluated at
a concentration level 10 μg/ml for Anticoagulant Drug. Insignificant difference in peak area and
less variability in retention time were observed.
The robustness of an analytical procedure is a measure of its capacity to remain unaffected
by small, but deliberate variation in method parameters and provides indication of its reliability
during normal usage.
Blank, placebo, standard and sample solution preparation
Procedure
Change following parameters one by one and observe their effect.
1. Change in flow rate of mobile phase ± 0.15ml (i.e.0.95 ml/min & 1.05ml/min).
2. Change in column oven temperature ± 2ºC (i.e. 23ºC and 27ºC).
Calculate individual % assay value and % RSD
Table no.43– Injection sequence for Robustness

1 Diluent(Change in temperature-23°C) 1
2 Standard Solution (Change in temperature-23°C) 6
3 Precision set-1(Change in temperature-23°C) 2
4 Diluent(Change in temperature-27°C) 1
5 Standard Solution (Change in temperature-27°C) 6
6 Precision set-1(Change in temperature-27°C) 2
7 Diluent(Change in Flow rate -0.95 ml/min) 2
8 Standard Solution(Change in Flow rate -0.95 ml/min) 6
9 Precision set-1(Change in Flow rate -0.95 ml/min) 2
10 Diluent(Change in Flow rate -1.05 ml/min) 2
11 Standard Solution(Change in Flow rate -1.05 ml/min) 6
12 Precision set-1(Change in Flow rate -1.05 ml/min) 2
Results-
Table no.33 –Robustness (Change in temperature-23°C)
Drug
Sample Sample Drug Spl Dil Dil Dil
(mg/tab
Name Area (%) (mg) (mL) (mL) (mL)
)
Robustness 23°C
5109970 110.74 100.68 282.2 100 5 50
Prep-1
Robustness 23°C
5196058 112.01 101.83 283.7 100 5 50
Prep-1
Anticoagulant Drug
Precision 1 100.28
Precision 2 100.27
Precision 3 99.36
Precision 4 99.81
Precision 5 100.86
Precision 6 100.15
Robustness 0.95 mL/min 100.68
Avg. 100.406
SD 0.742
RSD % 0.74
Table no.34 –Robustness (Change in temperature-27°C)
Drug
(mg/tab
)
Robustness 23°C
5152970 111.83 101.66 282.2 100 5 50
Prep-1
Robustness 23°C
5097058 110.03 100.02 283.7 100 5 50
Prep-1
Anticoagulant Drug
Precision 1 100.28
Precision 2 100.27
Precision 3 99.36
Precision 4 99.81
Precision 5 100.86
Precision 6 100.15
Robustness 1.05mL/min 101.66
Robustness 1.05mL/min 100.02
Avg. 100.303
SD 0.695
RSD % 0.69
Table no.35 –
Robustness (Change in Flow rate-0.95ml/min)
Drug
(mg/tab
)
Robustness 23°C
5131970 111.22 101.11 282.2 100 5 50
Prep-1
Robustness 23°C
5157058 111.17 101.06 283.7 100 5 50
Prep-1
Anticoagulant Drug
Precision 1 100.28
Precision 2 100.27
Precision 3 99.36
Precision 4 99.81
Precision 5 100.86
Precision 6 100.15
Avg. 100.364
SD 0.616
RSD % 0.61
Table no.36 – Robustness (Change in Flow rate-1.05 ml/min)
Drug
(mg/tab
)
Robustness 23°C
5150970 111.72 101.57 282.2 100 5 50
Prep-1
Robustness 23°C
5177058 111.70 101.54 283.7 100 5 50
Prep-1
Anticoagulant Drug
Precision 1 100.28
Precision 2 100.27
Precision 3 99.36
Precision 4 99.81
Precision 5 100.86
Precision 6 100.15
Avg. 100.481
SD 0.787
RSD % 0.78
7.3.7 Solution Stability
Prepared blank, (as diluents) system suitability solution as per the method and spiked all the
impurities which are mentioned in Table 1 as per the specification label. Sample solution was
preparing freshly and injected. This study was repeated at the interval of 6 hours till 48 hours to
measure the solution stability at 5°C temperature
Transfer an accurately weighed quantity of about 11.036 mg of Anticoagulant Drug reference

standard into a 10 ml volumetric flask, dissolve and dilute to volume with Methanol:Water
(70:30 v/v) and take 5 ml of std solution into dilute 50 ml diluents .
Sample preparation:
Methanol:Water (70:30 v/v) diluent and make up the volume up to 100 ml. Prepared in duplicate.
Sample
Time(Hrs.) RT % Variation
Area
Initial 4.102 5154210 100.00
After 6.0 Hrs 4.102 5121548 0.63
After 24.0 Hrs 4.104 5130587 0.46
After 30.0 Hrs 4.103 5127254 0.52
After 48.0 Hrs 4.102 5109897 0.86
Table no.37
Sr. No. Validation parameters Desired limits

1. Specificity No interference from other peaks
Correlation coefficient NMT 0.99.
2. Linearity
Report slope and intercept.
3. Method precision %RSD- NMT 2.0 for six preparations of samples
%RSD- NMT 2.0 for six preparations of samples with
4. Intermediate precision
respect to six preparation of precision.
% recovery should be between 98% to 102%.
5. Accuracy
% RSD of each level should NMT 2.0
Area response of Standard and Sample should be NMT
6. Solution Stability
2.0% for standard and sample
% RSD between the area response of filtered and
7. Filter paper interference
centrifuged samples NMT 2.0.
8. SUMMARY AND CONCLUSION
 A Simple, specific and reproducible RP-HPLC method was developed for simultaneous
estimation of Anticoagulant Drug dosage form.
 The RP-HPLC method was developed using Luna Phenomenax,100A° C18 column, 5μm
(4.6×250 mm) column, at 25˚ C using a mobile phase consisting of ACN: Buffer (55:45
v/v) at 1.0 ml flow rate. Detection was carried out by using UV detector.
 Retention time of Anticoagulant Drug was found about 4.0 to 5.0 min respectively.
 Assay results for anticoagulant drug using proposed method showed 100.12%.
 The developed method was validated for Linearity and Range, Precision, Intermediate
Precision, Recovery (accuracy) studies, Robustness Studies, Solution Stability.
 There were no interference at the retention time of main peak thus method was found to
be specific.
 The linearityRegression coefficient found 0.9997.
 %RSD value of repeatability, intra-day and inter-day precision studies were found
0.58%which is less than 2.0% and it showed that method was precise.
 As the method showed adequate recovery of the drugs it was concluded as accurate.
 There was not much change in retention times and peak areas of Anticoagulant drug by
change in flow rate, column oven temperature. Thus, method was found to be robust.
 There was not much % variation in the peak areas of Anticoagulant drug by injecting it
an after the specific time intervals. Thus,a solution preparation in the method was found
to be stable after an 48 hours.
9. Bibliography
1. Thomas L. L., Devid A. W., Victoria F. R. and William Z. Foye's principles of medicinal
Chemistry. 6thEdi., Lippincott Williams and wilkils, USA, 2008.
2. Watson D. G. Pharmaceutical Analysis. Harrcourt Publishers Ltd,. UK, 2000.
3. Anjeneyulu Y., Chandrasekhar K. and Manikam v. Analytical chemistry, Pharma Book
Syndicate. New Delhi. 2005.
4. christian G. Analytical chemistry.5th Ed., John Wiley And Sons ,New York,2001.
5. Kar A. Pharmaceutical analysis. 1stEd,. Vol-1, C. B. S. Publishers and Distributer,New
Delhi. 2007.
6. Sethi P. D. Quantitative Analysis of Drugs in Pharmaceutical Formulation. 3 rdEd.C. B.
S_publishers and Distributor s, New Delhi, 1997.
7. Christian G. D. Analytical Chemistry. 6th Ed., John wiley and Sons, New York,2003.
8. https://www.medicinenet.com/atrial_fibrillation_pictures_slideshow/article.htm
9. Adamovies J. Chromatographic Analysis of Pharmaceutical. 2nd Ed., Marcel Dekker Inc.,
New York, 2002.
10. Michael E., Schartz I . And Krull S. Analytical Method Development and Validation. John
wiley and Sons , New York, 2004.
11. Khopkar S. M. Basic concepts of analytical chemistry. New Age International Ltd,
Publisher, New Delhi, 1998.
12. Kasture A., Wadodkarar S., Mahadik S. and More H. Pharmaceutical Analysis. 1 0 th Ed.
Vol-2, Nirali prakashan, Pune. 2004.
13. Skoog D., Holler F. and crouch S. Principle of Instrumental Analysis. 'Thomson
Publication, India, 2007.
14. Gearien J., Bernard F. and Grabowski D. Methods of Drug Analysis. j. Lea and Febiger,
USA, 1969.
15. Kitson F. G., Larsen B. S. and McEwen c. N. Gas chromatography and Mass Spectroscopy-
A Practical Guide. Acadamic Press, London,1996.
16. Scott P. w. and Raymond. Gas Chromatography. Library science, UK. 2003.
17. Kennedy J.H. Analytical Chemistry Principles. Harcourt brace Jovanovic, London,1994
18. Stout T.H. and Dorsey J.G. High Performance Liquid Chromatography. Eurand America.
Inc.Vandalia,Ohia,2002
19. Kealey D. and Haines P. J. Analytical Chemistry. B.I. O. S. Scientific Publishers
Ltd.,UK,2002.
20. Connors K. A. Pharmaceutical Analysis. 3rd Ed. John wiley, and Sons, NewDelhi, 1999.
21. Kamboj P. C. Pharmaceutical Analysis. 1st Ed. Vol-1. Vallabh Publication, New Delhi,
2003.
22. Kennedy J. H. Analytical Chemistry Principles. Harcourt Brace Jovanovic,London. 1984.
23. Marvin C. HPLC-A Practical User’s Guide.2 nd Ed., John Wiley and Sons. Inc. Hoboken.
New Jersey. 2007.
24. Harvey D. Modern Analytical Chemistry. McGraw-Hill Companies, USA, 2000.
25. Skoog D. A., Holler J. E. and Crouch R. S. Instrumental Analysis. 5 th Ed.,Cengage
Learning India Pvt. Ltd., New Delhi, 2010.
26. Christian G. D. and Reilly J. E. Instrumental Analysis. Allyn and Bacon, USA,1945.
27. Higuchi T., Brochmann F. and Hanssen H. Pharmaceutical Analysis. C.B.S. Publishers and
Distributers, New Delhi, 2005.
28. Pernarowski M., KnevelA And Christian. j. J. Pharm sci., 1960.
29. Scott R.P.W Technique and Practice of chromatography. vol-7, Marcel Dekker. New York,
2007.
30. Willard H. H., Merritt L. L., Dean J. A. and Settle F. A. Instrumental Methods of 7 th Ed.,
CBS Publisher and Distributors, New Delhi, 2002.
31. Sharma B.M. Instrumental Methods of Chemical Analysis. 26th Ed., GOEL publishing
House,New Delhi,2007
32. Brochmann F. Basic Education in Analytical Chemistry. 17th Ed., C. B. S. Publisher and
Distributer, New Delhi. 2001.
33. Mendham J., Denney R. C., Barnes.J. D. and Thomas M. Vogel's Textbook of Quantitative
Analysis. 6th Ed., Pearson Education., Singapore, 2005.
34. Anjaneyulu Y. and Marayyah. Quality Assurance and Quality Management in
pharmaceutical Industry. Pharma book publishers, Hyderabad, 2005.
35. Stenlake J. and Beckett A. Practical Pharmaceutical Chemistry, 4th Ed., C. B. S,Publishers
and Distributors, New Delhi, 2007.
36. Weston A. and Brown P. R. High Performance Liquid Chromatography principles and
Practice. Academic Press, USA, 1997.
37. Synder K., Krikland J. and GlajchI J. Practical HPLC Method Development. 2 nd Ed.,
Wiley-Interscience Publication, USA, 1983.
38. International Conference on Harmonization (ICH), Harmonized Tripartite guideline on
validation of Analytical Procedure, Methodology, Q2 (R2) ; 1994.
39. http:// wnw.swgfast.org/... /validation/010808 validation 1.0UpdatedFormatarchived. pdf
40. http : //www.Chemistry.nmsu.edu
41. Skoog D., Holler J. and Nieman T. Principle of instrumental Analysis. 5 th Ed.,Saunders
College publishing, New Delhi, 1998.
42. Bruce P., Minkkinen P. and Riekkola M. Mikrochimica; 1998.
43. William K. Organic Spectroscopy. Palgrave, New York,2005.
44. product monograph , Anticoagulant Drug Capsules 75 mg, 110 mg and 150 mg ,APOTEX
INC 150 Signet Drive, Toronto, Ontario,M9L1T9, October 2017, Page 1 of 78
45. Mrinalini C.Damle, Rupesh A. Bagwe development and validation of stability-indicating
RP-HPLC method for estimation of Anticoagulant Drug, ISSN 0976-9595 Research
Article, Damle et al, J Adv Sci Res, 2014, 5(3): 39-44
46. Mehta HIREN R, Patel Paresh Anticoagulant Drug new Direct thrombin inhibitors
Anticoagulant international journal of pharmacy, ISSN 2230-8407 Mehta Hiren et al IRJP
2 (4) 2011 50-55
47. Kumar Raja Jayavarapu, Dr. T. Satyanarayana, B. Baby, Ch.Kesavarao, K.Sujatha,
K.Ramya, and M.S.N.Varaprasad ,validated uv-spectroscopic method for the estimation of
Anticoagulant Drug in formulation and tablet dosage form, Indo American Journal of
Pharmaceutical Research, 2016, ISSN NO: 2231-6876.
48. Asmaa A. El Zaher, Ehab F. El Kady, Ola M. El Houssini and Hind E. El Ghwas
Development and Validation of Stability-Indicating RP-LC Method for the Determination
of Anticoagulant Drug in Bulk and Pharmaceutical Formulations, Current Science
International ISSN 2077-4435, Volume : 04 ,2015 Pages: 402-408
49. P. Manasa, P. Sowndarya, K. Monika, A. Ashok Kumar a rapid stability indicating RP-
HPLC assay method development and validation for the quantitative estimation of
Anticoagulant Drug in capsules indo American journal of Pharmaceutical Science,
CODEN (USA): IAJPBB ,ISSN: 2349-7750, IAJPS 2015, 2 (10), 1382-1392
50. Brent E. Burbridge, MD, FRCPC Anticoagulant Drug: Management Before Invasive
Radiology Procedures ,Jr. of Radiology Nursing,Elsevier, JUNE 2013, VOLUME 32
ISSUE 2, 66-69
51. d. vijaykumar, k. balaraju, a. ashokkumar, a rapid RP-HPLC method development and
validation for the quantitative estimation of Anticoagulant Drug in capsules , International
Journal of Pharmacy and Pharmaceutical Sciences, ISSN- 0975-1491 Vol 7, Issue 10,
2015
52. p. sowndarya, k. mounika, s. laxmiprasanna, k. sandya, a. ashokkumar, assay method
development and validation of Anticoagulant Drug in capsules by uv spectroscopy,
International Journal of Pharmacy and Pharmaceutical Sciences, ISSN- 0975-1491
Vol 7, Issue 8, 2015
53. J. Nagadeep, P kamaraj ,Gradient RP-HPLC method for the determination of potential
impurities in DEM in bulk drug and capsule formulations, King Saud University,Arabian
Journal of Chemistry,2015,1-13
54. Bhavna Patel1,Shoumik Roy1,Hardik Ghelani2 , Shraddha Parmar, Development &
Validation of RP-HPLC Method for Estimation of Anticoagulant Drugfrom Capsule
Dosage Form, Bhavna Patel et al. / International Journal of Pharma Sciences and Research
(IJPSR), ISSN : 0975-9492 Vol. 8 No. 06 Jun 2017,143-151
55. Rajeshnawale, Shankar pol, prashantpuranik , anwardaud, vishalrajkondawar, analytical
method development and validation of Anticoagulant Drug related substance in
pharmaceutical dosage form by reverse-phase high-performance liquid chromatography ,
Asian J Pharm Clin Res, Vol 11, Issue 10, 2018, 357-364
56. G. Bhavani1, Syed Shahed Hussain1, Ch. Ranjith1, A. Ashok Kumar, Dissolution Method
Development and Validation of Anticoagulant Drug Capsules by RP-HPLC , RESEARCH
ARTICLE Am. J. PharmTech Res. 2016; 6(4) ISSN: 2249-3387, Journal home page:
http://www.ajptr.com
57. Pintu B. Prajapati, Arti J. Rakholiya, Bhavin P. Marolia, Stability Indicating HPTLC
Method for Estimation of Anticoagulant Drug in its Pharmaceutical Dosage Form, Eurasian
Journal of Analytical Chemistry ISSN: 1306-3057 2017 12(2):75-86
58. N.Madhavi Latha, P.Supriya, G.V.Ramana, K.B.V.Rohith, G.Chaitanya, A.K.M Pawar,
Development and validation of RP- HPLC method for the estimation of Anticoagulant
Drug (DEM) in bulk form, IJPAR Vol.5 Issue 1 , Jan- Mar -2016, ISSN:2320-2831,
Journal Home page: www.ijpar.com
59. A.Srinivas , K.Sridhar Reddy, G.Sai, Method Development and Validation of DEM by RP-
HPLC Method and its Degradation study , International Journal of Trends in Pharmacy and
Life Sciences Vol. 2, Issue: 1, 2016:769-777, e-ISSN: 2454-7867, Available online at
www.ijtpls.com
60. Dare M, Jain R, and Pandey A, Method Validation for Stability Indicating Method of
Related Substance in Active Pharmaceutical Ingredients Anticoagulant Drug by Reverse
Phase, Dare et al., J Chromatography Sep Tech 2015, Volume 6 , Issue 2 ,1000263, 6:2
http://dx.doi.org/10.4172/2157-7064.1000263
61. Syed Shahed Hussain, G. Bhavani, A. Ashok Kumar, uv Spectrophotometric assay method
development and validation of Anticoagulant Drug in capsules, International Journal of
Pharmacy and Pharmaceutical Sciences ,ISSN- 0975-1491 Vol 7, Issue 8, 2015
62. www.en.m.wikipedia.org/wiki/Dabigatran.com (accessed on 15/5/2014).
63. Validation of Analytical Procedures: Text and Methodology Q1A (R2), ICH Harmonized
Tripartite Guideline, Geneva Switzerland, 2003.
64. Validation of Analytical Procedures: Text and Methodology Q2(R1), ICH Harmonized
Tripartite Guideline, Geneva Switzerland, 2003
65. International Conference on Harmonization of Technical Requirements for Registration of
Pharmaceuticals for Human use. Validation of Analytical Procedures: Text and
Methodology ICH Q2 (R1);2005
66. A Rapid RP-HPLC Method Development And Validation For The Quantitative Estimation
of Anticoagulant Drug in capsules, D.Vijay kumar, K.Balaraju, A.Ashok kumar,
International Journal of Pharmacy and Pharmaceutical science.,Vol 7,issue:10,2015.Fgh
67. Leko, M., 2014.DEM, a thrombin inhibitor for the prevention of venous thromboembolism
and stroke. Curr. Opin.Invest. Drugs. 8(9): 758-68.
68. Sekhar reddy BRC, Vijaya bhaskar rao N. A stability indicating RP-HPLC method for
estimation of Dabigatran in pure and pharmaceutical dosage forms. South pacific journal of
pharma and bioscience 2014;2(1):80-92
69. Health Canada. Summary basis of decision Pradax. November 6, 2008. Retrieved from
http://www.hc-sc.gc.ca/dhp-mps/prodpharma/sbd-smd/drug
med/sbd_smd_2008_pradax_114887eng.php. January 5, 2013
70. Ankit P, Sharad K, Ashim KS, Aarti Z, Seth AK. Spectrophotometric method for
estimation of Anticoagulant Drug in bulk and its pharmaceutical dosage form. Pharma Sci
Monit 2014;5(2):31-9.
71. Zhe-Yi Hu, Robert BP, Vanessa LH, Casey L. Conventional liquid chromatography/triple
quadrupole mass spectrometry based metabolite identification and semi-quantitative
estimation approach in the investigation of in vitro Anticoagulant Drug metabolism. Anal
Bioanal Chem 2013;405(5):1695-704
72. http://pubchem.ncbi.nlm.nih.gov/compound/11434065
73. https://en.wikipedia.org/wiki/Dabigatran.
74. Drug bank database is a detailed drug data with comprehensive drug target
information.www.drugbank.ca/drugs/DB06695.

Anticoagulant

Uploaded by

Document Information

Original Description:

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Anticoagulant

Uploaded by

Copyright:

Available Formats

ACKNOWLEDGEMENT

I take this privilege and pleasure to acknowledgethe contributions of many individuals

I am grateful to Dr.Umesh Bhosale, Head of Analytical Development,Bharat Serums and

I am thankful toDr. Deshmukh Sir, Principal, Mula Education SocietiesCollege of Pharmacy,

I am grateful to my bathmats Ganesh, Rahul, Shubham, Salman, AK, Surendra,

PPM Parts Per Million

NaOH Sodium Hydroxide

HCl Hydrochloric Acid

Λmax Maximum Wavelength

SST System Suitability Test

Sr. No. Serial Number

RPM Round Per Minute

HETP Height Equivalent to Theoretical Plate

NMT Not More Than

NLT Not Less Than

PPM Parts Per Million

NaOH Sodium Hydroxide

HCL Hydrochloric Acid

H2O2 Hydrogen Peroxide

Λmax Maximum Wavelength

SST System Suitability Test

Sr. No. Serial Number

RPM Round Per Minute

HETP Height Equivalent to Theoretical Plate

NMT Not More Than

NLT Not Less Than

4. Aim And Objective 55

7. Result And Discussion 70

1. Types of HPLC columns 29

2. Detectors Employed in HPLC Analysis 35

3. Validation Parameter & Acceptance criteria 49

5. Drug Sample with its Percent Assay 59

6. Materials and Reagent 59

7. Instrument and Equipment’s 59

20. Observation for trial 2 70

21. Observation for trial 3 71

22. Observation for trial 4 72

23. Observation for trial 5 73

24. Observation for trial 6 74

25. Observation for trial 7 75

26. Observation for trial 8 76

27. Observation for trial 9 77

28. Observation for trial 10 78

29. Observation for trial 11 79

30. Optimized Chromatographic conditions 80

31. Injection sequence for specificity 82

32. Specificity results of Anticoagulant Drug 82

33. Linearity solutions for Anticoagulant Drug 83

34. Injection sequence for Anticoagulant Drug 84

35. Linearity of Anticoagulant Drug 85

36. Linearity regression data for calibration curve 85

37. Injection sequence for precision 86

38. Repeatability studies for Anticoagulant Drug 87

40. Intermediate precision studies for Anticoagulant Drug 89

41. Sequence of Accuracy level 91

42. Accuracy data for Anticoagulant Drug 92

43. Injection sequence for Robustness 94

44. Robustness (Change in temperature-23°C) 95

45. Robustness (Change in temperature-23°C) 96

46. Robustness(Change in Flow rate-0.95ml/min) 97

47. Robustness(Change in Flow rate-0.95ml/min) 98

48. Solution stability data for Anticoagulant Drug 99

49. Acceptance Criteria of validation 100