1.1. ANALYTICAL CHEMISTRY

Analytical chemistry 1-4 was the science of making quantitative measurement. In

practice, quantifying analytes in a complex becomes an excise in problem solving. To be

effective and efficient, analyzing samples requires expertise in:

1. The chemistry that can occur in sample

2. Analysis and sample handling methods for a wide variety of problems (the tools – of – the

trade)

3. Proper data analysis and record keeping.

Traditionally, analytical chemistry had been split into two main types, qualitative and

quantitative.

1.1.1. Types:

1.1.1.1. Qualitative

Qualitative seeks to establish the presence of a given element or inorganic compound

in a sample.

Qualitative organic analysis seeks to establish the amount of a given element or

compound in sample.

1.1.1.2. Quantitative

Quantitative analysis seeks to establish the amount of a given elements or compound

in a sample.

Most modern analytical chemistry was categorized by two different approaches such as

analytical targets or analytical methods.

1.1.1.3. By analytical targets

Bioanalytical chemistry

Material analysis

Chemistry analysis

Environment analysis

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Forensics

By analytical methods

Spectroscopy

Mass Spectroscopy

Chromatography and electrophoresis

Crystallography

Microscopy

Electrochemistry

1.1.1.4 Techniques

There were many techniques available for the analysis of materials, however; they

were all based on the material’s interaction with energy.

This interaction permits the creation of a signal that was subsequently detected and

processed for its information content.

The types of analysis techniques confirm with the various types of energy.

1.1.1.4.1 Spectroscopic analysis

Spectroscopy measures the interaction of the material with the electromagnetic radiation.

Spectroscopy consists of many different merits such as

Atomic absorption spectroscopy

Atomic emission spectroscopy

Ultraviolet-visible spectroscopy

Infrared spectroscopy

Raman spectroscopy

Nuclear magnetic resonance spectroscopy

Photoemission spectroscopy

1.1.1.4.2. Electrochemical analysis

Electrochemistry measure the interaction of the material with an electric field.

1.1.1.4.3. Mass analysis

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Mass spectrometry measures mass-to-charge ratio of molecules using electric

magnetic fields.

There are several ionization methods: electron impact, chemical ionization, electrospray,

matrix assisted laser desorption ionization, others.

Also, mass spectrometry was categorized by approaches of mass analyzers: magnetic-

sector, quadrupole mass analyzer, quadrupole ion trap, time-of-flight, Fourier transform

ion cyclotron resonance.

1.1.1.4.4. Thermal analysis

Calorimetry and thermogravimetric analysis measures the interaction of a material and

heat.

1.1.1.4.5. Separation science

Separation processes were used to decrease the complexity of the material mixtures.

The most utilized separation method was chromatography.

After the material was sufficiently isolated and a signal was generated, the signal must

be detected and interpreted.

1.1.1.4.6. Data acquisition and analysis

Specific data acquisition and data analysis technique were required to obtain the

information produced by the various techniques for the material analysis named above.

Research and development in this area of analytical chemistry involves interdisciplinary

efforts in physics, electronics, optics, statistics and computer science.

1.1.1.5. Hybrid techniques

Combinations of the above techniques produce “hybrid” or “hyphenated” techniques.

Several examples were in popular use today and new hybrid techniques were under

development.

1.1.1.5.1. Methods

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Analytical methods rely on scrupulous attention to cleanliness, sample preparation,

accuracy and precision.

A standard method for analysis of concentration involves the creation of a calibration

curve.

If the concentration of elements or compound in a sample is too high for the detection

range of a technique, it can simply be diluted in a pure solvent.

If the amount in sample was below an instruments grange of measurement, the method

of addition can be used.

In this method a known quantity of the elements or compound under study was added and

the concentration observed in the amount actually in the sample.

1.1.1.5.2. Trends

Analytical chemistry research was largely driven by performance (Sensitivity,

selectivity, robustness, linear range, accuracy, precision and speed) and cost (purchase,

operation, training, time and space).Effort was also put into analyzing biological system.

Examples of rapidly expanding fields in this area were

Genomics

DNA sequencing and its related research. Genetic Finger printing and DNA microarray

are very popular tools and research filed.

Proteomics

The analysis of protein concentrations and modifications especially in response to

carious stressors, at various development stages or in various parts of the body.

Metabolomics

Similar to proteomics, but dealing with metabolites.

Transcriptomics

mRNA and its associated field.

Lipidomics

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Lipids and its associated field.

Peptidomics

Peptides and its associated field.

Metabolics

Similar to proteomics and metabolomics, but dealing with metal concentrations and

especially with their binding to proteins and other molecules.

1.2. ANALYTICAL METHOD DEVELOPMENT[5,6]

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8. Validate method for release to routine laboratory

1. Information on sample, define separation goals

2. Need for special HPLC Proceedure, sample pretreatment edure, sample pre-treatment.

3. Choose detector and detector settingssettings.

4. Choose LC method; preliminary run; estimate best separation conditions

7a. Recover purified material

5. Optimize separation conditions

6. Check for problems or requirement for special procedure

7b. Quantitative calibration7c. Qualitative method

Every day many chromatographers face the need to develop a High Perfor-mance Liquid

Chromatography (HPLC) separation. A good method development strategy should require only

as many experimental runs as are necessary to achieve the desired final result. Finally method

development should be as simple, as possible, and it should allow the use of sophisticated tools

such as computer modeling.

Method development often follows the series of steps summarized below:

1.2.1. WHAT IS KNOWN BEFORE STARTING A METHOD DEVELOPMENT

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A. Nature of the sample:

Before beginning method development, there is a need to review what is known about the

sample in order to define the goals of separation.

Important information concerning sample composition and properties:

Number of compounds present

Chemical structures of compounds

Molecular weights of compounds

pKa values of compounds

UV spectra of compounds

Concentration range of compounds in samples of interest

Sample solubility.

The chemical composition of the sample can provide valuable clues for the best choice

of initial conditions for the HPLC separation.

B. Separation goals

The goals of HPLC separation need to be specified clearly, which include:

The use of HPLC to isolate purified sample components for spectral identifica-

tion or quantitative analysis

It may be necessary to separate all degradants or impurities from a product for

reliable content assay or not

In quantitative analysis, the required levels of accuracy and precision should known (a

precision of ± 1 to 2% is usually achievable)

Whether a single HPLC procedure is sufficient for raw material or one or more different

procedures are desired for formulations.

When the number of samples for analysis at one times is greater than 10, a run

time of less than 20 minutes often will be important.

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Knowledge on the desired HPLC equipment, HPLC experience and academic training do

the operators have?

1.2.2. SAMPLE PRETREATMENT AND DETECTION

Samples come in various forms:

Solutions ready for injections

Solutions that require dilution, buffering, addition of an internal standard or other

volumetric manipulation

Solids that must first be dissolved or extracted

Samples that require sample pretreatment to remove interferences and or protect the

column or equipment from damage.

Direct injection of the samples is preferred for its convenience and greater precision

however most samples for HPLC analysis require weighing and/or volumetric dilution before

injection. Best results are often obtained when the composition of the sample solvent is close to

that of the mobile phase, since this minimizes base line upset and other problems.

Some samples require a partial separation (pretreatment) prior to HPLC, because it is

necessary to remove interferences, concentrate sample analyte or eliminate “columnkillers”

In many cases the development of an adequate sample pretreatment procedure can be

more challenging than achieving a good HPLC separation. The samples may be of two types,

regular or special. The regular samples are typical mixture of small molecules (< 2000 Da) that

can be separated by normal starting conditions. Where as special samples are better separated

under customized conditions given in the following table.

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Table 1.1. Type of samples and their requirements

Type of Sample Requirements

Inorganic ions Detection is primary problem; use ion chromatography

Isomers Some isomers can be separated by reversed phase HPLC and are

then classified as detection is primary regulations of isomers are

obtainable using either normal phase normal phase or reversed

phase HPLC separations with

cyclodextrin-silica columns.

Enantiomers These compounds require chiral conditions for their separation.

Biological

compounds

Several factors make samples of this kind “special” mole

molecular conformation, polar functionally, and a wide range of

hydrophobicity.

Macromolecules “Big” molecules require column packings with large pores;

(>> 10-nm diameters); in addition, biological molecule require

special conditions

1.2.3. DEVELOPING THE SEPARATION

A. Selecting an HPLC Method and Initial Conditions:

If the HPLC is chosen for the separation, the next step is to classify the sample, as regular

or special. Regular samples are typical mixtures of small molecules that can be separated using

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more or less standardized starting conditions. Special samples are usually better separated with a

different column and customized conditions.

Choice of the Column

The selection of the column in HPLC is somewhat similar to the selection of columns in

GC, in the sense that, in the adsorption and partition modes, the separation mechanism is based

on inductive forces, dipole-dipole interactions and hydrogen bond formation. In case of ion-

exchange chromatography, the separation is based on the differences in charge, size of the ions

generated by the sample molecules and the nature of ionisable group on the stationary phase. In

the case of size-exclusion chromatography the selection of the column is based on the molecular

weight and size of the sample components. Selection of columns based on the method is briefly

summarized in the following table.

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Table 1.2. Different methods of HPLC

Method /Description /Columns Preferred Method

Reversed–phase HPLC

Uses water-organic mobile

phase Columns: C18 (ODS), C8,

Phenyl, trimethylsilys (TMS),

cyano.

First choice for most samples, especially

neutral or non-ioniged compounds that

dissolve in water-organic mixtures

Ion pair HjPLC

Uses water-organic mobile

phase, A buffer to control pH,

and an Ion-pair reagent.

Columns: C18, C8, Cyano

Acceptable choice for ionic or ionisable com-

pounds, especially bases or cations.

Normal-phase HPLC

Uses mixture of organic solvents

as mobile phase Columns:

cyano, diol, amino, silica.

Good second choice when reversed phase or

ion-pair HPLC is ineffective; first choice for

lipophilic samples that do not dissolve well in

water-organic mixtures.

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Nature of Sample

TLCSFCGCHPLC CE

Regular

Neutral Ionic

Exploratory run (RP)

peptides

Carbohydrates

nucleotides

Special

Inorganic ions

Biological Samples

isomers

enantiomers

Normal Phase

isocratic

Gradient

NARP

Ion-pair

Synthetic polymers

macromolecules

proteins

Carbohydrates

nucleic acids

The following chart show the strategy recommended for choosing the experimental conditions

for the first separations.

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Regular samples can be further classified as neutral or ionic. Samples classified as ionic

include acids, bases, amphoteric compounds, and organic salts (ionized strong acids or bases).

Table 1.3. Experimental conditions for the initial separation of regular sample

Separation Variable Preferred Initial Choice

Column:

Dimensions (length, ID)

Stationary phase

Particle size

15 x 0.46 cm

C8 or C18

5 µm

Mobile Phase: Solvents A and B

%B

Buffer

Additives

Buffer-ACN

80-100%

25 mM potassium phosphate

Do not use initially

Flow rate:

Temperature:

Sample Size:

Volume

Weight

1.5-2 ml/min

35-45o C

< 25 µl

< 100 µg

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If the sample is neutral, buffers or additives are not required in the mobile phase. Acids or

bases usually require the addition of a buffer to the mobile phase. For basic or cationic samples,

less acidic reversed phase columns are recommended, and amine additives for the mobile phase

may be beneficial using these conditions, the first exploratory run is carried out and then

improved systematically.

On the basis of the initial exploratory run, isocratic or gradient elution can be selected as

most suitable. At this point it may also be apparent that typical reversed phase conditions provide

insufficient sample retention, suggesting the use of either ion–pair or normal phase HPLC

alternatively, the sample may be strongly retained with 100% ACN as mobile phase, suggesting

the use of non aqueous reversed phase chromatography or normal phase HPLC.

B. Getting Started on Method Development:

One approach is to use an isocratic mobile phase of some average organic solvent

strength (50%). A better alternative is to use a very strong mobile phase first (80-100%) then

reduce % B as necessary. The initial separation with 100% B results in rapid elution of the entire

sample, but few groups will separate. Decreasing the solvent strength shows the rapid separation

of all components with a much longer run time, with a broadening of latter bands and reduced

retention sensitivity.

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Table 1.4.Goals that are to be achieved in method development

Goal Comment

Resolution Precise and rugged quantitative analysis requires that

RS be greater than 1.5.

Separation time < 5-10 min is desirable for routine procedures.

Quantitation < 2% (ISD) for assays; < 5 % for less-demanding

analysis; < 15% for trace analysis

Pressure < 150 bar is desirable, < 200 bar is usually essential

(new column assumed)

Peak height Narrow peaks are desirable for large signal/noise ratios.

Solvent consumption Minimum mobile-phase use per run is desirable.

The separation achieved in the first one or two runs usually will be less than adequate.

After a few additional tries, it may be tempting to accept a marginal separation, especially if no

further improvement is observed.

Separation or resolution is a primary requirement in quantitative HPLC analysis. Usually,

for samples containing five or fewer components, baseline resolution (RS >1.5) can be obtained

easily for the bands of interest. This level of resolution favors maximum precision in reported

results. Resolution usually degrades during the life of the column and can vary from day to day

with minor fluctuations in separation conditions.

Therefore, values of RS = 2 or greater should be the goal during method development for

simple mixtures. Such resolution will favor both improved assay precision and greater method

ruggedness. Some HPLC assays do not require base line separation of the compounds of interest.

In such cases only enough separation of individual components is required to provide

characteristic retention times for peak identification.

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The time required for a separation should be as short as possible. This assumes that the

other goals of previous table have been achieved, and the total time spent on method

development is reasonable. The run time goal should be compared with the 2-h setup time

typically required for an HPLC procedure. Thus if only two or three samples are to be assayed at

one time, a run time of 20-30 min is not excessive. When lots of 10 or more samples are to be

assayed, run times of 5 to 10 min are desirable.

Conditions for the final HPLC method should be selected so that the operating pressure

with a new column does not exceed 170 bar (2500 psi, 17 MPa), and an upper pressure limit

below 2000 psi desirable. There are two reasons for this pressure limit, despite the fact that most

HPLC equipment can be operated at much higher pressures. First, during the life of a column,

the back pressure may rise by a factor of as much as 2, due to the gradual plugging of the column

by particulate matter. Second, at lower pressures (< 170 bar), pumps, sample valves, and

especially auto samplers operate much better, seals last longer, columns tend to plug less, and

system reliability is significantly improved. For these reasons, a target reassure of less than 50%

of the maximum capability of the pump is desirable. When dealing with more challenging

samples or if the goals of separation stringent, a large number of method developments run may

be required to achieve acceptable separation.

C. Repeatable Separation

As the experimental runs described above are being carried out, it is important to confirm

that each chromatogram can be repeated. When changing conditions (mobile phase, column,

temperature) between method developments, experiments, enough time must elapse for the

column to come into equilibrium with the new mobile phase and temperature.

Usually, column equilibrium is achieved after passage of 10-20 column volumes of the

new mobile phase through the column. However, this should be confirmed by carrying out a

repeat experiment under the same conditions.

When constant retention times are observed in two such back to back repeat experiments

it can be assumed that the column is equilibrated and the experiments are repeatable. For

reversed-phase separations, longer equilibration times can result when one of the two mobile

phases being interchanged contains <10% organic.

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1.2.4. COMPLETING THE METHOD DEVELOPMENT

The final procedure should meet all the goals of the method development, the method

should also robust in routine operation and usable by all laboratories and personnel for which it

is intended

Completing the Method

1. Preliminary data to show required method performance.

2. Written assay procedure developed for use by other operators.

3. Systematic validation of method performance for more than one system or operator, using

samples that cover the expected range in composition and analyte concentration; data

obtained for day to day and inter laboratory operation.

4. Data obtained on expected life of column and column-to-column reproducibility.

5. Deviant results studied for possible correction of hidden problems.

6. All variables (temperature, mobile phase composition, etc.) studied for effect on separation;

limits defined for these variables; remedies suggested for possible problems (poor

resolution of key band pair, increased retention for last band with longer run times, etc.).

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1.3. INTRODUCTION TO HPLC [7-10]

Chromatography:

Chromatography is a technique by which the components in a sample, carried by the

liquid or gaseous phase, are resolved by sorption-desorption steps on the stationary phase.

1.1.1 High Performance Liquid Chromatography

High Performance Liquid Chromatography (HPLC) is one mode of chromato -graphy;

the most widely used analytical technique

HPLC utilizes a liquid mobile phase to separate the components of a mixture. These

components (or analytes) are first dissolved in a solvent, and then forced to flow through a

chromatographic column under a high pressure. In the column, the mixture is resolved into its

components.

The interaction of the solute with mobile and stationary phases can be manipulated

through different choices of both solvents and stationary phases. As a result, HPLC acquires a

high degree of versatility not found in other chromatographic systems and it has the ability to

easily separate a wide variety of chemical mixtures.

HPLC as compared with the classical technique is characterized by

Small diameter(2-5 mm), reusable stainless steel columns

Column packing with very small (3, 5 and 10 µm) particles

Relatively high inlet pressures and controlled flow of the mobile phase

Precise sample introduction without the need for large samples

Special continuous flow detectors capable of handling small flow rates and

detecting very small amounts

Automated standardized instruments

Rapid analysis

High resolution

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High performance is the result of many factors:

Very small particles of narrow distribution range and uniform pore size and

distribution

High pressure column slurry packing techniques

Accurate low volume sample injectors

Sensitive low volume detectors

Good pumping systems

Retention mechanism

In general, HPLC is a dynamic adsorption process. Analyte molecules, while moving

through the porous packing bead, tend to interact with the surface adsorption sites. Depending on

the HPLC mode, the different types of the adsorption forces may be included in the retention

process:

Hydrophobic (non-specific) interactions are the main ones in reversed-

phase separations.

Dipole-dipole (polar) interactions are dominated in normal phase mode

Ionic interactions are responsible for the retention in ion-exchange

chromatography.

All these interactions are competitive. Analyte molecules compete with the molecule at

adsorption sites. So the stronger analyte molecules interacts with the surface and the weaker the

eluent interaction, the longer analyte will be retained on the surface.

SEC (size-exclusion chromatography) is a special case. It is the separation of the mixture

by the molecular size of its components. In this mode any positive surface interactions should be

avoided. Basic principle of SEC separation is that the bigger the molecule, the less possibility for

her to penetrate into the adsorbent pore space, so, the bigger the molecule the less it will be

retained.

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1.1.2. TYPES OF HPLC TECHNIQUES

A. Based on modes of chromatography:

Reverse phase chromatography

Normal phase chromatography

B. Based on principle of separation:

Adsorption chromatography

Ion exchange chromatography

Size exclusion chromatography

Affinity chromatography

Chiral phase chromatography

C. Based on elution technique:

Isocratic separation

Gradient separation

D. Based on the scale of operation:

Analytical HPLC

Preparative HPLC

A. Based on modes of chromatography:

1. Reverse Phase Chromatography

The stationary bed is non polar (hydrophobic) in nature, while the mobile phase is a polar

liquid, such as mixtures of water and methanol or acetonitrile. Here the more non polar the

material is, the longer it will be retained.

The object was to make silica less polar or non-polar so that polar solvents can be used to

separate water-soluble polar compounds. Since the ionic nature of the chemically modified silica

in now reversed i.e., it is non-polar or the nature of the phase is reversed, the chromatographic

separation carried out with such silica is referred to as reversed phase chromatography.

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A large number of chemically bonded silica based stationary phases are available

commercially. Silica based stationary phases are still more popular in reversed phase

chromatography; however other adsorbents based on polymer (styrene divinyl benzene

copolymer) are slowly gaining ground.

The less water–soluble compounds are better retained by the reversed phase surface. The

retention time decreases in the following order: Aliphatic > induced dipoles (E.g. CCl4) >

permanent dipoles (E.g. CHCl3) > weak Lewis bases (Ethers, aldehydes, ketones) > strong Lewis

bases (amines ) > weak Lewis acids (alcohols, phenols) > strong Lewis acids (carboxylic acids ).

Also the retention increases as the number of carbon atoms increases.

As general rule the retention increases with an increase in the contact area between

sample molecule and stationary phase i.e., with an increase in the number of water molecules,

which are released during the adsorption of a compound. Branched chain compounds are eluted

more rapidly than their corresponding normal isomers.

In reversed phase system the strong attractive forces between water molecules arising

from the 3-dimensional intermolecular hydrogen bonded network present in the structure of

water must be distorted or disrupted when a solute is dissolved.

Only higher polar or ionic solutes can interact with the water structure. Now polar solutes

are squeezed out of the mobile phase and are relatively insoluble in it but with the hydrogen

carbon moieties of the stationary phase.

Chemically bonded octadecyl silane (ODS) and alkane with 18 carbon atoms is the most

popular stationary phase used in pharmaceutical industry. Since most pharmaceutical compounds

are polar and water soluble, the majority of HPLC methods used for quality assurance,

decomposition studies, quantitative analysis of both bulk drugs and their formulations use ODS

HPLC columns. The solvent strength in reverse phase chromatography is reversed from that of

adsorption chromatography (silica gel) as stated earlier. Water interacts strongly and highly with

silanol groups, so that, adsorption of sample molecules become highly restricted and they are

rapidly eluted as a result. Exactly opposite applies in reversed phase system; water cannot wet

the non-polar (hydrophobic) alkyl group such as C18 of ODS phase and therefore does not

interact with the bonded moiety. Hence water is the weakest solvent of all and gives slowest

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elution rare. The elution time (retention time) in reversed phase chromatography increases with

increasing amount of water in the mobile phase.

2. Normal phase Chromatography

In normal phase chromatography the stationary phase is polar adsorbent (like silica gel or

any other silica based packing) and the mobile phase is generally a mixture of non-aqueous

solvents (such as n-hexane or tetra hydro furan) the separation is based on repeated adsorption

desorption steps polar samples are thus retained on the polar surface of the column packing

longer than less polar materials.

B. Based on principle of separation:

1. Adsorption Chromatography

The principle of separation is adsorption. Separation of components takes place because

of the difference in affinity of compounds towards stationary phase. This principle is seen in

normal phase as well as reverse phase mode, where adsorption takes place.

2. Ion Exchange Chromatography

The stationary bed has an ionically charged surface of opposite charge to the sample ions.

This technique is used almost exclusively with ionic or ionizable samples. The stronger the

charge on the sample, the stronger it will be attracted to the ionic surface and thus, the longer it

will take to elute. The mobile phase is an aqueous buffer, where both pH and ionic strength are

used to control elution time.

3. Size Exclusion Chromatography

The column is filled with material having precisely controlled pore sizes, and the sample

is simply screened or filtered according its solvated molecules. Large molecules are rapidly

washed through the column smaller molecules penetrate inside the porous of the packing

particles and elute later.

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4. Affinity / Ion- Pair Chromatography

Separation is based on a chemical interaction specific to the target species. The more

popular reversed phase mode uses a buffer and an added counter-ion of opposite charge to the

sample with separation being influenced by pH, ionic strength, temperature, concentration and

type of organic co-solvent(s). Affinity chromato-graphy, common for macromolecules, employs

a ligand (biologically active molecule bonded covalently to the solid matrix).Which interacts

with its homologous antigen (analyte) as a reversible complex that can be eluted by changing

buffer conditions.

5. Chiral Chromatography

Separation of the enantiomers can be achieved on chiral stationary phases by formation

of diastereomers via derivatizing agents or mobile phase additives on a chiral stationary phase.

When used as an impurity test method, the sensitivity is enhanced if the enantiomeric impurity

elutes before the enantiomeric drug.

C. Based on elution technique:

1. Isocratic Separation

In this technique the constant eluent composition is pumped through the column during

the whole analysis.

2. Gradient Separation

In this technique the eluent composition (and strength) is steadily changed during the

whole analysis.

D. Based on the scale of operation:

1. Analytical HPLC

In this only analysis of the samples are done. Recovery of the samples for reusing is

normally not done, since the samples used are very low.

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2. Preparative HPLC

Where analysis of the individual fractions of pure compounds can be collected using

fraction collector. The collected samples are reused.

1.1.3. INSTRUMENTATION

Fig-1: HPLC Instrument

In order to realize eluent flow rates with packing in the 2 to 10 µm particle sizes, which

are common in modern liquid chromatography, pumping pressures of up to several thousand

pounds per square inch are required. As a consequence of these high pressures, the equipment

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required for HPLC tends to be more elaborate and expensive than that encountered in other types

of chromatography.

A. Stationary Phases (Adsorbents)

HPLC separations are based on the surface interactions, and depend on the types of the

adsorption sites (surface chemistry). Modern HPLC adsorbents are the small rigid porous

particles with high surface area

Main adsorbent parameters are:

Particle size: 3 to 10 µm

Particle size distribution: As narrow as possible, usually within 10% of the mean

Pore size: 70 to 300 Å

Depending on the type of the ligand attached to the surface, the adsorbent could be normal

phase (-OH-NH2), or reversed-phase (C8, C18, Phenyl), and even anion (NH4+), or cation (-

COO-) exchangers.

B. Mobile phase (eluents)

In HPLC type and composition of the mobile phase (eluent) is one of the variables

influencing the separation. Despite of the large variety of solvents used in HPLC, there are

several common properties:

Purity

Detector compatibility

Solubility of the sample

Low viscosity

Chemical inertness

Reasonable price

Each mode of HPLC has its own requirements. For normal phase mode solvents are

mainly non polar, for reversed-phase eluents are usually a mixture of water with some polar

organic solvent such as acetonitrile. Size-exclusion HPLC has special requirements, SEC eluents

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has to dissolve polymers, but the most important is that SEC eluent has to suppress all possible

interactions of the sample molecule with the surface of the packing material.

C. Mobile phase reservoir, filtering

The most common type of solvent reservoir is a glass bottle. Most of the manufacturers

supply these bottles with the special caps, Teflon tubing and filters to connect to the pump inlet

and to the purge gas (helium) used to remove dissolved air. Helium purging and storage of the

solvent under helium was found not to be sufficient for degassing of aqueous solvents. It is

useful to apply a vacuum for 5-10 min. and then keep the solvent under a helium atmosphere.

D. Pumps

The HPLC pump is considered to be one of the most important components in a liquid

chromatography system which has to provide a continuous constant flow of the eluent through

the HPLC injector, column, and detector.

High pressure pumps are needed to force solvents through packed stationary phase beds.

However, many separation problems can be resolved with larger particle packing that requires

less pressure. Flow rate stability is another important pump feature that distinguishes pumps. For

most types of separation stable flow rate is not very important. However, for size exclusion

chromatography the flow rate has to be extremely stable.

Modern pumps have the following parameters:

Flow rate range: 0.01 to 10 ml/min

Flow rate stability: Not more than 1% (short term)

For SEC flow rate stability should be less than 0.2%

Maximum pressure: Up to 5000 psi (345 bar, 340 atm).

It is desirable to have an integrated degassing system, either helium purging, or better

vacuum degassing. The two basic classifications are the constant-pressure and the constant-flow

pump.

The constant-pressure pump is used only for column packing. The constant-flow pump is

the most widely used in all common HPLC applications.

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E. Injectors

Sample introduction can be accomplished in various ways. The simplest method is to use

an injection valve. In more sophisticated LC systems, automatic sampling devices are

incorporated where sample introduction is done with the help of auto samplers and

microprocessors.

In liquid chromatography, liquid samples may be injected directly and solid samples need

only be dissolved in an appropriate solvent. The solvent need not be the mobile phase, but

frequently it is judiciously chosen to avoid detector interference, column/component

interference, and loss in efficiency or all of these. It is always best to remove particles from the

sample by filtering, or centrifuging since continuous injections of particulate material will

eventually cause blockage of injection devices or columns.

Injectors should provide the possibility of injecting the liquid sample within the range of

0.1 to100 ml of volume with high reproducibility and under high pressure (up to the 400

psi).They should also produce minimum band broadening and minimize possible flow

disturbances. The most useful and widely used sampling device for modern LC is the micro

sampling injector valve.

F. Columns

The heart of the system is the column. Typical analytical columns are 10, 15 and 25 cm

in length and are fitted with extremely small diameter (3, 5 or 10 µm) particles. The internal

diameter of the columns is usually 4 or 4.6 mm; this is considered the best compromise among

sample capacity, mobile phase consumption, speed and resolution. Preparative columns are of

larger diameter.

Packing of the column tubing with the small diameter particles requires high skill and

specialized equipment. For this reason, it is generally recommended that all but the most

experienced chromatographers purchase pre-packed columns, since it is difficult to match the

high performance of professionally packed LC columns without a large investment in time and

equipment.

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In general, LC columns are fairly durable and one can expect a long service life unless

they are used in some manner which is intrinsically destructive, as for example, with highly

acidic or basic eluents, or with continual injections of 'dirty' biological or crude samples. It is

wise to inject some test mixture (under fixed conditions) into a column when new, and to retain

the chromatogram. If questionable results are obtained later the test mixture can be injected again

under specified conditions. The two chromatograms may be compared to establish whether or

not the column is still used.

A short guard column is introduced before the analytical column to increase the life of

the analytical column by removing not only particulate matter and contaminants from the solvent

but also sample components that bind irreversibly to the stationary phase. In addition, in liquid-

liquid chromatography, the guard column serves to saturate the mobile phase with the stationary

phase so that losses of this solvent from the analytical column are minimized. The composition

of the guard column packing should be closely similar to that of the analytical column; the

particle size is usually larger, however, to minimize pressure drop. When the guard column has

become contaminated, it is repacked or discarded and replaced with a new one of the same type.

Thus, the guard column is a sacrificed to protect the more expensive analytical column.

For many applications, close control of column temperature is not necessary, and

columns are operated at ambient temperature. Often, however, better chromato- grams are

obtained by maintaining column temperatures constant to a few tenths degree centigrade. Most

modern commercial instruments are now equipped with column heaters that control column

temperatures to a few tenths of a degree from near ambient to 100oC to 150oC. Columns may

also be fitted with water jackets fed from a constant temperature bath to give precise temperature

control.

G. Detectors

The function of the detector in HPLC is to monitor the mobile phase as it emerges from

the column.

28

1. Basic detector requirements:

High sensitivity

Fast response

Wide linear dynamic range (this simplifies quantitation)

Low dead volume (minimal peak broadening)

Cell design which eliminates remixing of the separated bands

Insensitivity to changes in type of solvent, flow rate, and temperature

Operational simplicity and reliability

It should be tune able so that detection can be optimized for different compounds

It should be non-destructive.

2. Choosing a Detector:

Table 1.5. Types of Detectors

RI UV/VIS Fluor MS

Response Universal Selective Selective Selective

Sensitivity 4 microgram 5 nanogram 3 picogram 1 picogram

Flow sensitive Yes No No Yes

Temp. sensitive Yes No No No

3. Detector sensitivity

Detector sensitivity is one of the most important properties of a LC detector. Sensitivity

can be associated with the slope of the calibration curve. It is also dependent on the standard

deviation of the measurements. The higher the slope of your calibration curve the higher the

sensitivity of your detector for that particular component, but high fluctuations of your

measurements will decrease the sensitivity.

29

4. Response

For mass-sensitive detectors, the response R (mV/mass/unit time) is:

R = hW / sM

For the concentration sensitive detector, the response R (mV/mass/unit volume) is:

Where:

h = peak height (mV)

W = peak width at 0.607 of the peak height (cm)

F = flow rate (ml/min)

M = mass of solute injected

s = chart speed (cm/min)

5. Types of Detectors

Generally, there are two types of HPLC detectors, bulk property detectors and solute

property detectors.

Bulk property detectors:

These detectors are responding to a mobile phase bulk property, such as refractive

index, and dielectric constant detectors.

Solute property detectors:

Solute property detectors respond to some property of the solutes, which is not exhibited

by the pure mobile phase. Such as UV absorbance, fluorescence.

Optical detectors are most frequently used. These detectors pass a beam of light through

the flowing column effluent as it passes through a low volume (~ 10 ml) flow cell.

30

The most commonly used detector in LC is the ultraviolet absorption detector. A variable

wavelength detector of this type, capable of monitoring from 190 to 460-600nm, will be found

suitable for the detection of the majority samples.

Other types of detectors

RI – Refractive Index-Universal analyte detector. Solvent must remain the same

throughout separation. Very temperature sensitive. Sometimes difficult to stabi-lize baseline.

FD – Fluorescence- Excitation wavelength generates fluorescence emission at a higher

wavelength. Analytes must have fluorophore group. Can react analyte with fluorophore reagent.

Very sensitive and selective. More difficult methods transfer. Results very dependent upon

separation conditions.

MS – Mass Spec- Mass to charge ratio (m/z). Allows specific compound ID. Several

types of ionization techniques: electrospray, atmospheric pressure chemical ionization, electron

impact. The detector usually contains low volume cell through which the mobile phase passes

carrying the sample components.

H. Data systems

The main goal in using electronic data systems is to increase analysis accuracy and

precision, while reducing operator attention. In routine analysis, where no automation (in terms

of data management or process control) is needed, a pre-programmed computing integrator may

be sufficient.

1.1.4. PARAMETERS USED IN HPLC [ 5,8]

A. Retention time (tR)

The time it takes after sample injection for the analyte peak to reach the detector is called

the retention time and is given the symbol tR

OR

Retention time is the difference in time between the point of injection and appearance of

peak maxima.

31

α = (t2 - ta) / (t1 - ta)

Where,

α = Relative retention

t2 = Retention time of the second peak measured from point of injection.

t1 = Retention time of the first peak measured from point of injection.

ta = Retention time of an inert peak not retained by the column measured

from point of injection

B. Retention volume

Retention volume is the volume of mobile phase required to elute 50% of the component

from the column. It is the product of retention time and flow rate.

C. Resolution (Rs)

Resolution is measure of the extent of separation of two components and the base line

separation achieved.

Rs = 2(t2 - t1) / (w1 + w2)

Where,

t1 and t2 are the retention times of the first and second adjacent bands.

w1 and w2 are their baseline band widths.

An alternative approach gives more reliable values of Rs band widths at half height (w1/2)

are measured for bands 1 and 2, W0.5.1 and W0.5.2 then calculations of Rs using above equation

(or) below equation may not be reliable when Rs is less than 1.

Rs = 1.18(t2 - t1) / (W0.5.1 + W0.5.2)

32

Resolution can be estimated or measured in 3 different ways:

1. Calculations based on below e.q.

Rs = 2(t2 - t1) / (w1 + w2)

2. Comparison with standard resolution curves.

3. Calculations based on the valley between the 2 bands.

Resolution can be expressed in terms of three parameters (k, α, and N) which are directly related

to experimental conditions.

Rs = 1/4(α - 1) N1/2 K / (1 + k)

Where,

K = The average retention factor for the two bands

N = is the column plate number

α = is the separation factor

α = k2 / k1: k1 and k2 are values of k for adjacent bands 1and 2.

The above equation is useful in method development because it classifies the dozen (or)

so many experimental variables into 3 categories: retention (k), column (N) and selectivity (α).

33

D. Capacity factor (K I )

Retention factor is related to the retention time and is a reflection of the proportion of

time particular solute resides in the stationary phase as opposed to the mobile phase.

Long retention times results in large values of K. The capacity factor is not the same as

the available binding capacity, which refers to the mass of the solute that a specified amount of

medium is capable of binding under defined conditions.

The capacity factor K1can be calculated for every peak defined in a chromatogram, using

the following equations.

K1 = tR - t0/t0

Where,

tR = Retention time of a solute peak.

t0 = Column dead time or Column void time solvent peak

E. Column efficiency

Two related terms are widely used as quantitative measures of chromatographic column

efficiency.

1. Plate height

2. Plate count N

The two are related by the equation: N = L/H

The efficiency of chromatographic columns increases as the plate count becomes greater

and as the plate height becomes smaller. A theoretical plate is an imaginary or hypothetical unit

of a column where equilibrium has been established between stationary phase and mobile phase.

N is dimensionless number and reflects the kinetics of the chromatographic retention

mechanism. Efficiency depends primarily on the physical properties of the chromatographic

medium together with the chromatographic column and system dimensions.

34

Theoretical plate:

A theoretical plate is an imaginary or hypothetical unit of a column where distribution of

solute between stationary phase and mobile phase has attained equilibrium. A theoretical plate

can also be called as a functional unit of the column.

The column plate number increases with several factors:

1. Well-packed column (column quality)

2. Longer columns

3. Lower flow rates

4. Smaller column-packing particles

5. Lower mobile phase viscosity and higher temperature

6. Smaller sample molecules

7. Minimum extra column effects.

Column performance can be defined in terms of values of N and band asymmetry (band

shape) for a test substance run under “favorable” conditions. The column plate number N is

defined by

N = 16(tR / W)2

Manual measurement of the base line band width W may be subject to error. Therefore a more

practical equation for N is

N = 5.54(tR / W1/2)2

Here,

tR = is band retention time

W1/2 = is the band width at half height.

35

1.4. VALIDATION

Analytical method validation is the process of demonstrating that analytical

procedures are suitable for their intended use and provide accurate test results that evaluate a

product against its defined specification and quality attributes .

The U.S. Federal Register states “Validation data must be available to establish that

the analytical procedures used in testing meets proper standards of accuracy and reliability

[15]” any analytical test methods are expected to be used in a Quality Control environment

they require an additional degree of refinement compared to research methods [12].

The following observation will explain the relationship between validation and method

development.

When methods are properly developed, they readily validate.

Validation is not a method development tool and it does not make a method good or

efficient.

Validation acceptance criteria should be based on method development experience.

36

VALIDATION OF ANALYTICAL PROCEDURES [13-17]

Different Types of Validation characteristics [18]

Generalized validation process for an HPLC assay method:

Validation is the process of collecting documented evidence that the method performs

according to its intended purpose.

1. 4.1. Precision:

The closeness of agreement between a series of measurements multiple samplings of

the same homogeneous sample under prescribed condition.

The precision of test method is usually expressed as the standard deviation or relative

standard deviation of a series of measurements.

Precision may be considered at three levels: Repeatability, Intermediate Precision and

Reproducibility.

37

Acceptance Criteria:

Percentage Relative standard deviation (%RSD) NMT 1 % (Instrument precision)

(%RSD) NMT -2% (Intra- assay precision)

1.4.2. Accuracy [18]:

The ICH guideline recommends that accuracy should be determined using a

minimum of nine determinations over a minimum of three concentration levels covering the

specified range (ICH, 1996). Spiked samples are prepared in triplicate at three levels over a

range that covers 80 -120% of the target concentration for assay methods and over a range that

covers the expected impurity content of a sample for impurity methods (Shabir, 2003).

There are several methods that can be used for determining accuracy. The most common

include:

Analyze a sample of known concentration and compare the measurement to the true value.

In this case, method accuracy is the agreement between the difference in the measured analyte

concentration and the known amount of analyte added. That is the accuracy or % recovered is

calculated as:

Cm × 100

Where Cm is the measured concentration and Ct is the theoretical concentration.

Accuracy has also been reported as a sample is analyzed and the measured value should ideally

be identical to the true value. Accuracy is represented and determined by recovery experiments.

The usual range is being 10% above or below the expected range of claim. The % recovery was

calculated using the formula,

%Recov ery=(a+b )−a

bX 100

Where,

a – Amount of drug present in sample

b – Amount of standard added to the sample.

38

Ct

Acceptance Criteria:

For an assay method, mean recovery will be 100%± 2% at each concentration over

the range of 80-120% of the target concentration.

For an impurity method, mean recovery will be 0.1% absolute of the theoretical

concentration or 10% relative, whichever is greater for impurities in the range of

0.1-2.5 % (V/W).

1.4.3. Detection Limit:

It is lowest amount of analyte in a sample that can be detected but not necessarily

quantitated under the stated experimental conditions.

Following are different approaches:

Visual Evaluation Method:

Prepare the sample solutions with known lowest possible concentrations of analyte and

establish the minimum concentration at which the analyte can be reliably detected by analyzing

as per test method.

Based on Signal to Noise Ratio Method:

The LOD can be expressed as a concentration at specified signal-to-noise ratio obtained

from samples spiked with analyte. A signal-to-noise ratio between 3:1 and 2:1 is generally

considered acceptable.

Based on the standard Deviation of the Response and the Slope:

Prepare the blank solution as per test method and inject six times into the

chromatographic system.

Similarly prepare the linearity solution staring from lowest possible concentration of

analyte to 150 % (or as per protocol) of target concentration and establish the linearity

curve.

39

The detection limit (DL) may be expressed as:

3.3 X Standard deviation of the response of the blank (σ)

LOD =

Slope

The slope shall be estimated from the calibration curve of the analyte.

1.4.4. Quantitation Limit:

It is lowest amount of analyte in a sample, which can be quantitatively determined with

acceptable accuracy and precision.

Following are different approaches:

Visual Evaluation Method:

Prepare the sample solutions with known lowest possible concentrations of analyte and

establish the minimum concentration at which the analyte can be reliably quantified by analyzing

as per test method.

Based on signal to noise ratio method :

The LOQ can be expressed as a concentration at specified signal-to-noise ratio

obtained from samples spiked with analyte. A signal-to-noise ratio of 10:1 is generally

considered acceptable. The ratio recognized by the ICH (1996) is a general rule. It has been

stated that “The determination of LOQ is a compromise between the concentration and the

required precision and accuracy. That is, as the LOQ concentration level decreases, the precision

increases”.

Based on the standard Deviation of the Response and the Slope:

Prepare the blank solution as per test method and inject six times into the

chromatographic system.

Similarly prepare the linearity solution staring from lowest possible concentration of

analyte to 150% (or as per protocol) of target concentration and establish the linearity

curve.

40

The Quantification limit ( QL ) may be expressed as :

10 X Standard deviation of the response of the blank(σ)

LOQ =

Slope

The slope shall be estimated from the calibration curve of the analyte.

Perform the Precision and accuracy at the level of limit of quantification by spiking LOQ

concentration on placebo / Drug product / Drug substance.

For detail methodology and acceptance criteria refer Precision and accuracy of       test

method.

Acceptance Criteria:

In Pharmaceutical application, the LOQ is typically set at minimum 0.05% for active

pharmaceutical ingredients.

LOQ defined as the lowest concentration providing a RSD of 5%.

LOQ should be at least 10% of the minimum effective concentration for clinical applications

1.4.5. Specificity:

The ability to assess unequivocally the analyte in the presence of components that may be

expected to present, such as impurities, degradation products and matrix components, etc.

Specificity shall be demonstrated by performing Placebo / blank interference and forced

degradation studies.

Blank interference:

Prepare blank solution as per test method and analyse as per test method.

Placebo interference (In case of Drug products):

Prepare the placebo solution equivalent to the test concentration (Subtract the weight of

active ingredient) and analyse as per test method.

Force Degradation studies:

Degrade the sample forcefully under the various stress conditions like Light, heat, humidity,

acid / base / water hydrolysis and oxidation and ensure the degradation from 1 % to 20 %.

41

Light: Expose the Drug product, drug substance and placebo to UV light for about 200

watt hours / square meter and the overall illumination not less than 1.2 million Lu hours [17] for visible light. Prepare the sample and placebo solution as per test method and

analyse.

Heat: Expose the Drug product, drug substance and placebo at 105 °C for about 12

hours ( For substance having low melting point below 10°C of its melting point ). Prepare

the sample and placebo solution as per test method and analyse.

Humidity: Expose the Drug product, drug substance and placebo for about 80 % RH at

about 25°C for about one week. Prepare the sample and placebo solution as per test

method and analyse.

Acid / Base: Prepare the acid or base solution of 0.1N and reflux the sample and placebo

with 50 ml of acid / base solution for about 1 hour at 60°C. Neutralize the solution and

dissolve the contents in diluents as per test method. Change the strength of acid and base

or reflux time to ensure the desired degradation.

Water: Reflux the sample / placebo with 100 ml of purified water for 12 hour at 60°C.

Dissolve the contents in diluents as per test method. Change the reflux time so as to

ensure the desired degradation.

Oxidation: Reflux for 12 hour at 60°C with 1 % H2O2 or suitable oxidant. Dissolve the

contents in diluents as per test method. Change the reflux time so as to ensure the desired

degradation.

Note: Based on the physicochemical properties and literature stress conditions can be

decided.

Acceptance Criteria:

Placebo / Blank should not elute at the retention time of analyte peak and known impurity

peak.

Peak purity of analyte peak should be confirmed.

Degradation of active analyte peak should be from 1% to 20%.

42

1.4.6. 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 analyte concentration in

samples within a given range. The linear range of detectability that obeys Beer’s law is

dependent on the compound analyzed and the detector used. The working sample concentration

and samples tested for accuracy should be in the linear range. The claim that the method is linear

is to be justified with additional mention of zero intercept by processing data by linear least

square regression. Data is processed by linear least square regression declaring the regression co-

efficient and b of the linear equation

Y= aX + b

together with the correlation coefficient of determination r. For the method to be linear the r

value should be close to1. Where Y is the measured output signal, X is the concentration of

sample, a is the slop, b is the intercept.

The range of an analytical method is the interval between the upper and lower levels of

the analyte (including these levels) that have been demonstrated to be determined with precision,

accuracy and linearity using the method as written.

If linearity is not meeting the acceptance criteria, establish the range of concentration in

which it is linear.

Acceptance criteria:

Coefficient of correlation should be NLT 0.99.

1.4.7. Robustness:

It is a measure of method's ability to remain unaffected by small but deliberate

variations in method parameters and provides an indication of its reliability during normal usage.

For example a chromatographic method, the typical method parameters need to

change deliberately and verify during method validation:

Flow rate : (+/- 0.2ml/minutes).

Mobile phase composition : (+/- 10% of organic phase).

Column oven temperature : (+/- 5°C).

pH of buffer in mobile phase : (+/- 0.2 units).

Filter suitability : (At least two filters).

43

For Variations:

1. System suitability should meet the acceptance criteria as per test method.

2. If system suitability doesn’t meet, narrow the variation range and carryout the experiment

again to meet system suitability.

1.4.8. Ruggedness:

The United States of Pharmacopeia (USP) defines Ruggedness as “the degree of

reproducibility of test results obtained by the analysis of the same samples under a variety of

normal test conditions, such as different labs, different analysts, and different lots of reagents.

Ruggedness is a measure of Reproducibility of test results under normal, expected operational

conditions from laboratory to laboratory and from analyst to analyst”.

The following are the typical method parameters need to be tested during method validation:

Analyst-to-Analyst variability.

Column-to-Column variability.

System-to-System variability.

Different days.

Different Laboratories.

Stability of Solutions and mobile phase. ( At least for 48 hours )

44

Table 1.6. Method Validation Requirements for Example (ICH) [18]

Method Validation requirements Acceptance Criteria

Precision

Assay repeatability

Intermediate precision (Ruggedness)

≤ 1% RSD

≤ 2% RSD

Accuracy

Mean recovery per concentration 100.0% ± 2.0%

Limit of detection

Signal to-to-noise ratio ≥ 3:1

Limit of quantification

Signal to-to-noise ratio ≥ 10:1

Linearity/Range

Correlation coefficient

y-Intercept

Visual

>0.99

± 10%

Linear

Robustness

System suitability met

Solution stability

yes

± 2% change from time zero

Specificity

Resolution from main peak >2 min. (retention time)

45

1.5. STATISTICAL PARAMETERS

Statistics consist of a set of methods and rules for organizing and interpreting

observations.

The precision or reproducibility of the analytical method was determined by repeating the

analysis and the following statistical parameters were calculated.

1.5.1 Mean

Best estimation of the population mean mcg/ml for random samples from a population.

x=∑i=1

Xi

n

Where

∑ = Sum of observations

x = Mean or arithmetic average (Ex/n)

x = Individual observed valve

n = Number of observation

1.5.2 Standard deviation

The positive square roof of the variance

S.D =

1.5.3 Relative standard deviation / Coefficient of variation

Measures of the spread of data compared with the mean

SD

RSD = ------ x 100

Mean

46

1.5.4 Standard error

SE = SD / n

E = Sum of observations

n = Number of observation

S.D = Standard deviation

1.5.5 Correlation: (Fit of regression line)

Purpose:

Measurement of the relation between two or more variables / measures

how close the points were to the regression line.

Correlation co-efficient can range from -1.00 + 1.00

Correlation value was denoted with the letter r

n(xy) – (x)( y)

r =

(nx2 – (x)2 (ny2 – (y)2

1.5.6 Regression

Purpose : 1. When the concentration range was so wide that the errors,

both random and systematic were not independent (which

was assumption).

2. When paring was inappropriate for other reason, notably a

long time span between two analysis (sample aging,

change in laboratory conditions etc.,)

47

Regression line

Y = mx + b

Where,

b = intercept of the line with the Y axis

m = Slope (tangent)

Slope m

n(xy) – (x)( y)

m = -------------------------

n( (x2)) – (x)2

Intercept b (y)( (x2) – (x)( xy)

b = -------------------------

n( (x2)) – (x)2

48

AIM AND OBJECTIVE OF THE STUDY

Aim

To develop an analytical method for dasatinib monohydrate tablets by RP – HPLC and to

partially validate the developed method as per ICH guidelines.

Objective

The scope of developing and validating an analytical method is to ensure a suitable method for a

particular analyte should be more specific, accurate and precise. The main objective for that is to

improve the conditions and parameters, which should be followed in the development and

validation.

The survey of literature reveals that good analytical methods are available for the drug dasatinib

monohydrate, but the existing methods are inadequate to meet the requirements. Hence it is

proposed to improve the existing methods and to develop new method for the estimation of

dasatinib monohydrate in pharmaceutical dosage forms.

49

PLAN OF WORK

To obtain thorough knowledge in practical HPLC method development.

A step-by-step procedure of method development to be implemented and initial

chromatographic conditions for assay of dasatinib monohydrate tablets was to be

established.

For the initial chromatographic conditions and trials, the methods to be optimized.

For the initial method, validation was to be performed by the developed RP – HPLC

method as per ICH guidelines.

50

2.1. SELECTION OF DRUG

Dasatinib an oral anti-cancer drug in the tablet dosage form was chosen for the

analytical method development and the method was validated.

2.1.1. DRUG PROFILE:

Dasatinib[21] is a 2-aminothiazole-derived inhibitor of Src family kinases.

Dasatinib is an oral multi- BCR/ABL and Src family tyrosine kinase inhibitor approved for

use in patient with chronic myelogenous leukaemia (CML) after imatinib treatment

and Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). It is being

evaluated for use in numerous other cancers, including advanced prostate cancer.

Structure

Systematic name -

N-(2-chloro-6-methylphenyl)-2-({6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-

methylpyrimidin-4-yl}amino)-1,3-thiazole-5-carboxamide

2.1.1.1. Physical and Chemical Properties:[22]

Colour- Off-white to pale yellow

Form -powder

Odour-none

Density ~ 1.408 g/cm3

Solubility- Soluble in Dimethyl Sulfoxide, Ethanol and Methanol

Partition coefficient- log Pow ~ 4.5 (n-octanol/water)1.8 pH 7.4

Dissociation constant-pK1 = 8.8 (acidic group(s))10.28

Melting temperature-280-2860c

51

Molecular Formula - C22H28ClN7O3S

Molecular Weight-506.02082 [g/mol]

CAS number- 863127-77-9

Storage -Store solid and solution at -20° C.

Category-Anti-Cancer.

2.1.1.2. PHARMACOLOGY OF DASATINIB[23]

Mechanism of action:

Dasatinib, at nanomolar concentrations, inhibits the following kinases: BCR-ABL,

SRC family (SRC, LCK, YES, FYN), c-KIT, EPHA2, and PDGFRß. Based on modeling studies,

dasatinib is predicted to bind to multiple conformations of the ABL kinase.

Dasatinib inhibited the growth of chronic myeloid leukemia (CML) and acute

lymphoblastic leukemia (ALL) cell lines overexpressing BCR-ABL. Under the conditions of the

assays, dasatinib was able to overcome imatinib resistance resulting from BCR-ABL kinase

domain mutations, activation of alternate signaling pathways involving the SRC family kinases

(LYN, HCK), and multi-drug resistance gene overexpression.

Pharmacokinetics:

The pharmacokinetics of dasatinib have been evaluated in healthy subjects and in

patients with leukemia.

Absorption

Maximum plasma concentrations (Cmax) of dasatinib are observed between 0.5 and 6

hours (Tmax) following oral administration.

Dasatinib exhibits dose proportional increases in AUC and linear elimination

characteristics over the dose range of 15 mg to 240 mg/day.

The overall mean terminal half-life of dasatinib is 3–5 hours.

52

Distribution

In patients, dasatinib has an apparent volume of distribution of 2505 L, suggesting that

the drug is extensively distributed in the extravascular space.

Binding of dasatinib and its active metabolite to human plasma proteins in vitro was

approximately 96% and 93%, respectively, with no concentration dependence over the

range of 100–500 ng/mL.

Metabolism

Dasatinib is extensively metabolized in humans, primarily by the cytochrome P450

enzyme 3A4. CYP3A4 was the primary enzyme responsible for the formation of the

active metabolite.

Flavin-containing monooxygenase3 (FMO-3) and uridine diphosphate-

glucuronosyltransferase (UGT) enzymes are also involved in the formation of dasatinib

metabolites.

In human liver microsomes, dasatinib was a weak time-dependent inhibitor of CYP3A4.

The exposure of the active metabolite, which is equipotent to dasatinib, represents

approximately 5% of the dasatinib AUC.

Elimination

Elimination is primarily via the feces. Following a single oral dose of [14C]-labeled

dasatinib, approximately 4% and 85% of the administered radioactivity was recovered in

the urine and feces, respectively, within 10 days.

Unchanged dasatinib accounted for 0.1% and 19% of the administered dose in urine and

feces, respectively, with the remainder of the dose being metabolites.

Drug-Drug Interactions:

53

Dasatinib is not an inducer of human CYP enzymes.

It is a time-dependent inhibitor of CYP3A4 and may decrease the metabolic clearance

of drugs that are primarily metabolized by CYP3A4. At clinically relevant

concentrations, dasatinib does not inhibit CYP 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, or

2E1.

Drugs That May Increase Dasatinib Plasma Concentrations

Drugs that inhibit dasatinib CYP3A4 are ketoconazole, itraconazole, erythromycin,

clarithromycin, atazanavir, indinavir, nefazodone, nelfinavir, ritonavir, saquinavir,

telithromycin may decrease metabolism and increase concentrations of dasatinib .

Drugs That May Decrease Dasatinib Plasma Concentrations

Drugs like antacids,,famotidine induce CYP3A4 enzyme and decrease the plasma

concentration of dasatinib.

Adverse Reactions:

The most frequently reported adverse events included fluid retention events such as

pleural effusion; gastrointestinal events including diarrhea, nausea, abdominal pain and

vomiting; and bleeding events.

Dosage and Adminstration:

The recommended dosage of dasatinib is 140 mg/day administered orally in two divided doses

(70 mg twice daily [BID]), one in the morning and one in the evening with or without a meal.

Tablets should not be crushed or cut; they should be swallowed whole.

Side Effects:

54

Headache, muscle pain, tiredness, weakness, dizziness ,joint pain, pain, burning or

tingling in the hands or the feet, rash, skin redness ,peeling skin, swelling, redness and pain

inside the mouth, mouth sores etc..,

2.1.2 ANALYTICAL PROFILE:

2.2 SELECTION OF METHOD

55

The selection of method depends upon the nature of the sample, its molecular

weight and solubility. Literature survey also helps for the selection of suitable method for the

analytical method development of dasatinib in its pharmaceutical dosage form.

2.2.1. LITERATURE REVIEW:

1.Sandra Roche et al., [24] studied Development of a high-performance liquid

chromatographic–mass spectrometric method for the determination of cellular levels of the

tyrosine kinase inhibitors lapatinib and dasatinib. His study includes Cellular samples were

extracted with a tert-butyl methyl ether:acetonitrile (3:1, v/v):1 M ammonium formate pH 3.5

(8:1, v/v) mixture. Separation was achieved on a Hyperclone BDS C18 (150 mm × 2.0 mm 3

μm) column with isocratic elution using a mobile phase of acetonitirile–10 mM ammonium

formate, pH 4 (54:46, v/v), at a flow rate of 0.2 mL/min. The limit of detection and limit of

quantification for lapatinib was determined to be 15 and 31 pg on column, respectively.

2. Haouala et al., [25] in the year 2009 studied Therapeutic Drug Monitoring of the new

targeted anticancer agents imatinib, nilotinib, dasatinib, sunitinib, sorafenib and lapatinib by

LC tandem mass spectrometry. His study includes Reverse-phase chromatographic

separation of TKIs is obtained using a gradient elution of 20 mM ammonium formate pH 2.2

and acetonitrile containing 1% formic acid, followed by rinsing and re-equilibration to the

initial solvent composition up to 20 min. The method was validated according to FDA

recommendations, The method is precise (inter-day CV%: 1.3–9.4%), accurate (−9.2 to

+9.9%) and sensitive (lower limits of quantification comprised between 1 and 10 ng/mL).

3 . Elisa pirro et al;[26]studied Development and validation of simple, rapid, and reliable

high-performance liquid chromatography (HPLC)-UV method for quantification of major

tyrosine kinase inhibitors, imatinib, dasatinib, and nilotinib, in human plasma is presented.

Chromatographic separation of the drugs is achieved on an RP-C18column at flow rate of 0.9

mL/min at 35°C; eluate is monitored at 267 nm. Mean intra-day and inter-day precision for

all compounds are 2.5 and 13.3%; mean accuracy is 13.9%; extraction recovery ranges

within 40.24 and 81.81 %. Calibration curves range from 10 to 0.005 µg/mL. Limits of

detection are 50 ng/mL for dasatinib; limits of quantification , 100 ng/mL for dasatinib.

56

4. Antonio D’Avolio et al,[27] studied A new method using high performance liquid

chromatography coupled with electrospray mass spectrometry is described for the

quantification of PBMC concentration of tyrosine kinase inhibitors imatinib, dasatiniband

nilotinib. A simple PBMC isolation and extraction procedure were applied on 10–14 mL of

blood aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline) was

achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse phase

analytical column with 25 min of analytical run, at flow rate of 0.25 mL/min. Mean intra-

and inter-day precision for all compounds were 8.76 and 12.20%; mean accuracy was

−3.86%; extraction recovery ranged within 79 and 91%. Calibration curves ranged from 50.0

to 0.25 ng. The limit of quantification was set at 0.25 ng for all the analyzed drugs.

5. Michael T. Furlong et al;[28] studied Dasatinib (Sprycel®) is a potent antitumor agent

prescribed for patients with chronic myeloid leukemia (CML). To enable reliable

quantification of dasatinib and its pharmacologically active metabolites in human plasma

during clinical testing, a sensitive and reliable liquid chromatography–tandem mass

spectrometry (LC–MS/MS) method was developed and validated. Samples were prepared

using solid phase extraction on Oasis HLB 96-well plates. Chromatographic separation was

achieved isocratically on a Luna phenyl–hexyl analytical column. Analytes and the stable

labeled internal standards were detected by positive ion electrospray tandem mass

spectrometry. The assay was validated over a concentration range of 1.00–1000 ng/mL

for dasatinib and its two active metabolites. Intra- and inter-assay precision values for

replicate QC control samples were within 5.3% for all analytes during the assay validation.

Mean QC control accuracy values were within ±9.0% of nominal values for all analytes.

Assay recoveries were high (>79%) .

6. Silvia De Francia et al,[29] studied A new method using high performance liquid

chromatography coupled with electrospray mass spectrometry is described for the

quantification of plasma concentration of tyrosine kinase inhibitors imatinib, dasatinib and

nilotinib. A simple protein precipitation extraction procedure was applied on 250 μl of

plasma aliquots. Chromatographic separation of drugs and Internal Standard (quinoxaline)

was achieved with a gradient (acetonitrile and water + formic acid 0.05%) on a C18 reverse

phase analytical column with 20 min of analytical run, at flow rate of 1 ml/min. Mean intra-

57

day and inter-day precision for all compounds were 4.3 and 11.4%; mean accuracy was

1.5%; extraction recovery ranged within 95 and 114%. Calibration curves ranged from

10,000 to 62.5 ng/ml. The limit of quantification was set at 62.5 ng/ml for dasatinib and

nilotinib.

7. Andrea Davies et al;[30] studied A high performance liquid chromatography (HPLC)

method that separates two of the currently licenced tyrosine kinase inhibitors (TKIs); nilotinib

(AMN107, Tasigna®) and imatinib (STI571, Glivec®), together with its main metabolite,

CGP-74588, from human plasma. After solid phase extraction the drug mix was separated

through a Gemini C6-phenyl column (150 mm × 4.6 mm, i.d.; 5 μm) (Phenomenex®, UK)

under isocratic mobile phase conditions of methanol:50 mM ammonium acetate (pH 8)

(65:35, v/v) with ultra-violet (UV) detection at 260 nm wavelength. For all compounds the

intra-day coefficient of variation and bias were <3% and <5% respectively; and inter-day

were <4% and <9%.

8. John Araujo et al.,[31] studied SRC is a tyrosine kinase that plays a role in oncogenic,

invasive and bone-metastatic processes. It has therefore been prioritized as a candidate

therapeutic target in patients with solid tumors. Several SRC inhibitors are now

in development, of which dasatinib has been most explored. Preclinical studies in a wide

variety of solid tumor cell lines, including prostate, breast and glioma, have shown that

that dasatinib acts as a cytostatic agent, inhibiting the processes of cell proliferation, invasion

and metastasis. Dasatinib also inhibits the activity of osteoclasts, which have a major role in

the development of metastatic bone lesions. Dasatinib has additive or synergistic activity in

combination with a number of other agents, including cytotoxic agents and targeted

therapies, providing a rationale for combination treatment in a clinical setting. Emerging

clinical data with dasatinib support experimental observations, with preliminary phase 1 and 2

data demonstrating activity, both as a single agent and as combination therapy, in a range of

solid tumors. Future clinical trials will further assess the clinical value of SRC inhibition

with dasatinib.

9. D.V.Mhaske et al;[32] studied Two sensitive and reproducible methods are described for

the quantitative determination of dasatinib in the presence of its degradation products. The

first method was based on high performance thin layer chromatography (HPTLC) followed

58

by densitometric measurements of their spots at 280 nm. The separation was on HPTLC

aluminium sheets of silica gel 60 F254 using toluene:chloroform (7.0:3.0, v/v). This system

was found to give compact spots for dasatinib after development (R F value of 0.23 ± 0.02).

The second method was based on high performance liquid chromatography (HPLC) of the

drug from its degradation products on reversed phase, PerfectSil column [C18 (5 μm,

25 cm × 4.6 mm, i.d.)] at ambient temperature using mobile phase consisting of

methanol:20 mM ammonium acetate with acetic acid (45:55, v/v) pH 3.0 and retention time

(t R  = 8.23 ± 0.02 min). Both separation methods were validated as per the ICH guidelines.

No chromatographic interference from the tablet excipients was found. Dasatinib was

subjected to acid–alkali hydrolysis, oxidation, dry heat, wet heat and photo-degradation. The

drug was susceptible to acid–alkali hydrolysis and oxidation. The drug was found to be stable

in neutral, wet heat, dry heat and photo-degradation conditions. As the proposed analytical

methods could effectively separate the drug from its degradation products, they can be

employed as stability indicating.

10. Eva Karlj et al;[33] studied Imatinib, dasatinib and nilotinib are three tyrosine kinase

inhibitors currently used to treat Bcr-Abl1 positive chronic myelogenous leukaemia (CML).

After the addition of isotopically labelled internal standard, the drug was extracted with 0.1%

formic acid in methanol. The collected extract (1 μL) was injected onto a Phenomenex

Kinetex 50 mm × 2.1 mm C18 column and eluted with acetonitrile gradient into a triple

quadrupole ESI–MS/MS Agilent 6460 operated in positive mode. The total run time was

only 2.6 min. The method was validated in terms of linearity, selectivity, specificity,

accuracy, precision, absolute and relative matrix effect and stability. The effect of

haematocrit (Hct) on the accurate concentration determination was also examined.

The method was linear in the range of 50–5000 μg/L for imatinib and nilotinib and in the

range of 2.5–250 μg/L for dasatinib, with correlation coefficient values higher than 0.997.

Lower limits of quantification (LLOQ) were 50 μg/L for imatinib and nilotinib and 2.5 μg/L

for dasatinib. The method proved to be accurate (% bias < 13.2) and precise (CV < 10.3%) on

intra- as well as on inter-day basis.

11. Lutz Götze et al;[34] studied A simultaneous test for six TKIs (erlotinib, imatinib,

lapatinib, nilotinib, sorafenib, sunitinib) was developed using liquid chromatography tandem

59

mass spectrometry in a multiple reaction monitoring mode. After protein precipitation the

specimens were applied to the HPLC system and separated using a gradient of acetonitrile

containing 1% formic acid with 10 mM ammoniumformiate on an analytic RP-C18

column.The calibration range was 10–1000 ng/mL for sunitinib and 50–5000 ng/mL for the

other TKIs with coefficients of determination ≥ 0.99 for all analytes. The intra- and inter day

coefficients of variation were ≤ 15% and the chromatographic run time was 12 min.

12. K. Micova et al;[35] studied Therapeutic drug monitoring is recommended for the optimal

several malignant diseases. The aim of this study was to develop and validate an isotope

dilution direct injection mass spectrometry method for the high throughput determination of

tyrosine kinase inhibitors in plasma from leukemic and cancer patients. The plasma for

analysis was deproteinated by methanol and the centrifuged supernatant was directly injected

to mass spectrometer without separation step. We developed a fast method with analysis time

of 55 s and 19 s in multiple injection setting. The method was successfully validated and

applied to the patient plasma samples. In order to overcome insufficient sensitivity of

dasatinib, multiple reaction monitoring cube mode in linear ion trap (MRM3) was

successfully applied. The limits of quantification were in the range 1.0–5.5 ng/ml.

Imprecisions were lower than 6.9% and the accuracy of the quality control samples ranged

between 99.0 and 107.9%.

13. Stephane Bouchate et al;[36] Tyrosine Kinase Inhibitors (TKIs) are a class of targeted

drugs for the treatment of malignant pathologies. Chromatography was performed on a

Waters Acquity-UPLC® system with BEH C18-50*2.1 mm column, under a gradient of

ammonium formate–acetonitrile. An Acquity-TQD® with electrospray ionization was used

for detection. Samples were prepared by solid phase extraction (Oasis® MCX μElution) and

eluate was injected in the system.Calibration curves ranged from 10 to 5000 ng/mL for

imatinib, its metabolite, nilotinib, lapatinib, erlotinib and sorafenib and from 0.1 to

200 ng/mL for dasatinib, axitinib, gefitinib and sunitinib. Peaks of each compound (retention

time from 0.76 to 2.51 min) were adequately separated. The mean relative extraction

recovery was in the range of 90.3–106.5%.

60

14. Zhongzhou Shen et al;[37] To characterize and enable efficient rat pharmacokinetic (PK)

screening in early drug discovery, automated sampling of blood time points are routinely

employed. With thedevelopment of dried blood spot (DBS) technology for drug level

quantitation, an opportunity exists for the automated collection of rat PK time points using

DBS. DBS, as an alternative sample collection technique has led to the increased collection

of PK study samples for the quantitative analyses of drug candidates in both pre-clinical and

clinical studies. However, the feasibility of using DBS samples for drug metabolite profiling

including both phase I and phase II metabolites has not been well established. This work

reports the study of metabolite profiling of dasatinibdosed to Wistar Han rats using automated

DBS collection. Automated DBS and plasma collection using a rat AccuSampler (VeruTech

AB, Sweden) was employed using dasatinib as a model compound. The DBS and plasma

samples were extracted by methanol and acetonitrile and both plasma and DBS extracts were

analyzed using a Sciex API4000 Qtrap mass spectrometer coupled to a Shimazdzu HPLC

system. Dasatinib and its metabolites were analyzed by multiple reaction monitoring (MRM)

and MRM trigger enhanced product ion scan (MRM-EPI). Both phase I oxidative

metabolites and phase II glucuronide conjugates and sulfate conjugates were detected from

both rat plasma and DBS samples. Overall, comparable metabolite profiles including phase I

oxidative and phase II glucuronide and sulfate conjugates were observed from both extracts

of plasma and DBS samples when using the untreated DBS cards for dasatinib. Chemically

treated DBS cards such as DMPK-A and DMPK-B cards may affect the dasatinibmetabolites.

Similar PK parameters were obtained for dasatinib from both plasma and DBS samples, after

correcting for blood to plasma ratio. The results obtained from this study suggest that

collection of study samples by DBS can be used for metabolite profiling, however, the

availability of limited samples may be a concern for multiple injections.

On literature survey it was found that, various analytical strategies have been used for

the measurement of Dasatinib either alone or in combination with various drugs in plasma and

pharmaceutical preparation using few spectrophotometric, High-performance liquid

chromatography (HPLC) and Reverse phase-high performance liquid chromatography (RP-

HPLC), LC-MS, HPLC-MS/MS, HPTLC, and LC-tandem mass spectrometry method.

61

In view of the need for a suitable method for routine analysis in formulation, attempts

are being made to develop and validate simple, precise and accurate analytical methods for the

estimation of Dasatinib and extend it for their determination in formulation. As chromatographic

method of analysis is a pre-requisite for the marketing of most of the formulation, one HPLC and

spectrophotometric methods were developed and validated for the determination of title drug.

The utility of the developed method to determine the content of drug in commercial tablet

is also demonstrated. Validation of the method was done in accordance with USP and ICH

guideline for the assay of active ingredients. The methods were validated for parameters like

accuracy, linearity, precision, specificity, and system suitability. These methods provide means

to simultaneously characterize and quantify the components of a mixture without prior separation

and derivatization. These proposed methods are suitable for the analysis in pharmaceutical

quality control laboratories.

62

2.3. SELECTION OF INSTRUMENT

Dasatinib is polar in nature the RP-HPLC method was preferred because of its

simplicity and suitability.

The reasons for developing RP-HPLC method for determination of the drug in tablet

dosage forms are as follows:

1. To develop newer RP-HPLC Method by Isocratic Mode.

2. To reduce the run time as compared with previously reported literature.

3. To develop a method for drug in its dosage form.

Advantages of less run time in HPLC:

It’s beneficial to the company economically.

To estimate the different compounds with less time in different formulations like tablets,

capsules, syrups, expectorants and injections.

Utilisation of minimum solvent.

Reduce the cost.

Less utilisation of men, machine and materials.

As RP-HPLC was chosen as the instrument wide variety of equipment, coloumns, eluent and

operational parameters are involved in it.

2.3.1. Coloumns:

The column was one of the most important components of the HPLC because the

separation of the sample components was achieved when those components pass through the

column. Trials were done on different coloumns and the optimized one is Cosmicil BDS

coloumn.

Cosmicil BDS coloumn:[37]

BDS is a base deactivated silanol in which the residual silanol groups are deactivated

which is suitable for the basic,acidic and neutral analytes.

63

This coloumn is suitable for elution with different eluents like

methanol,acetonitrile,water,disodium hydrogen phosphate,potassium dihydrogen

phosphate, acetic acid etc.,

These coloumns shows wide pH range i.e. 2-9.

The flow-rate is 0.8-2.0ml/min.

The UV-detection range is 220-330nm.

Column Dimensions

2.3.2. Mobile Phase:

Mobile phases used for HPLC are typically mixtures of organic solvents and waters or

aqueous buffers. These are chosen based on the following points:

1. The drug must be stable in moile phase for atleast duration of analysis.

2 .Excesive salt concentration must be avoided which otherwise lead to damage of the

equipment.

3. Minimize the absorbance of buffer.

Considering the above points methanol and acetonitrile are used as mobile phase .

2.3.2.1. Methanol:[38]

It is also known as methyl alcohol.

It is a polar mobile phase which is used for RP-HPLC.

The UV-cutoff range 205nm.

64

TypeLength

(mm)

Width

(mm)

Particle Size

(µm)

C-18 BDS 150 4.6 5

Toxicity:

Methanol has a high toxicity in humans. If ingested, for example, as little as 10 mL of pure

methanol can cause permanent blindness by destruction of the optic nerve, and 30 mL is

potentially fatal, although the median lethal dose is typically 100 mL (4 fl oz) (i.e. 1–2 mL/kg of

pure methanol. Toxic effects take hours to start, and effective antidotes can often prevent

permanent damage. Because of its similarities in both appearance and odor to ethanol (the

alcohol in beverages), it is difficult to differentiate between the two (such is also the case

with denatured alcohol).

Methanol is toxic by two mechanisms. First, methanol (whether it enters the body

by ingestion, inhalation, or absorption through theskin) can be fatal due to its CNS

depressant properties in the same manner as ethanol poisoning. Second, in a process

of toxication, it is metabolized to formic acid (which is present as the formate ion)

via formaldehyde in a process initiated by the enzyme alcohol dehydrogenase in

the liver. Methanol is converted to formaldehyde via alcohol dehydrogenase (ADH) and

formaldehyde is converted to formic acid (formate) via aldehyde dehydrogenase (ALDH). The

conversion to formate via ALDH proceeds completely, with no detectable formaldehyde

remaining. Formate is toxic because it inhibits mitochondrial cytochrome c oxidase, causing the

symptoms of hypoxia at the cellular level, and also causing metabolic acidosis, among a variety

of other metabolic disturbances.

2.3.2.2. Acetonitrile:[39]

It is also known as methyl cyanide

Its low viscosity and low chemical reactivity make it a popular choice for liquid

chromatography.

65

Toxicity:

Acetonitrile has only a modest toxicity in small doses. It can be metabolised to

produce hydrogen cyanide, which is the source of the observed toxic effects. Generally the onset

of toxic effects is delayed, due to the time required for the body to metabolize acetonitrile to

cyanide (generally about 2–12 hours).

Acetonitrile poisoning in humans (or, to be more specific, of cyanide poisoning after

exposure to acetonitrile) are rare but not unknown, by inhalation, ingestion and (possibly) by

skin absorption.[13] The symptoms, which do not usually appear for several hours after the

exposure, include breathing difficulties, slow pulse rate, nausea, and vomiting: Convulsions and

coma can occur in serious cases, followed by death from respiratory failure. The treatment is as

for cyanide poisoning, with oxygen, sodium nitrite, and sodium thiosulfate among the most

commonly-used remedies.

66

3.1. CHEMICALS AND INSTRUMENTS

3.1.1 MATERIALS:

A. Chemicals used:

B. Drug:

S. No. Drug name Manufactured by

1. Dasatini b(std) NATCO Pharma Pvt. Ltd.

2. Dasatini b(sample) NATCO Pharma Pvt. Ltd.

67

S. No. Chemical name Grade

1. Acetonitrile HPLC

2. Methanol HPLC

3. Purfied Water MilliQ HPLC

4. Triethylamine HPLC

5. Orthophosporic acid HPLC

3.1.2. Instruments used

S. No. Name of the instrument Make

1. HPLC

Column

Waters

Cosmicsil BDS C-18,5microns,

(150X4.6mm)

2. Orbital shaker

3. Analytical balance Afcoset

4. Membrane filter Smart Labtech Pvt. Ltd., BV - 40

5. pH meter

6. Centrifuge apparatus

68

3.2. MATERIALS AND METHODS

3.2.1. Buffer preparation:

Add 4.0ml of triethylamine to 100ml water and adjut the pH to 6.5±0.05 diluted with

orthophosphoric acid add 10ml of methanol and mix well.

3.2.2. Solvent mixture:

Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively.

3.2.3. Mobile phase:

Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v

respectively.

3.2.4. Preparation of standard solution:

Accurately weighed quantity of the drug was transfered about 68.0 mg of Dasatinib

monohydrate (working standard) into 50ml volumetric flask .

Add about 30ml of solvent mixture and sonicate to dissolve.

Cool the solution to room temperature and dilute to volume with solvent mixture.

Transfer 1.0ml of the above solution into a 10ml volumetric flask and dilute to volume

with mobile phase.

3.2.5. Preparation of working standard solution:

From the standard stock solution, 2.0 ml, 3.0 ml ,4.0 ml, 5.0 ml and 6.0 ml was

transferred into a 10 ml volumetric flask and made up to the mark to produce

20,30,40,50,60 µg/ml respectively with mobile phase.

69

3.2.6. Preparation of sample solution:

Weigh and finely powder not fewer than 10 tablets.

Transfer an accurately weighed portion of powder, equivalent to 68.0mg of Dasatinib

into a 100ml volumetric flask.

Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate

for 30min with occasional shakings.

Cool the solution to room temperature and dilute to volume with solvent mixture.

Centrifuge the solution at 3000RPM for 15min.

Transfer 1ml of the above solution into a 10ml volumetric flask, dilute to volume with

mobile phase.

3.2.7. Preparation of Placebo

The amount of powdered inactive ingredient supposed to be present in 10 tablets was

accurately weighed and transferred in to 100 ml volumetric flask.

Add about 60ml of solvent mixture , shake on orbital shaker for 15min and sonicate

for 30min with occasional shakings.

Cool the solution to room temperature and dilute to volume with solvent mixture.

Centrifuge the solution at 3000RPM for 15min.

Transfer 1ml of the above solution into a 100ml volumetric flask, dilute to volume

with mobile phase.

70

3.3. METHOD DEVELOPMENT

The objective of this study was to optimize the assay method for estimation

of Dasatinib based on the literature survey made.

Trial-1

Chromatographic conditions

Flow rate : 1.2 ml / min

Column : Devosil ODS C18, 150 mm X 4.6 mm, 5 m

Detector wavelength : 315 nm

Column temperature : 35 0C

Injection volume : 10 l

Run time : 10 min

Mobile phase

Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 50:50 v/v is used.

Mobile phase was pumped at a flow rate of 1.2ml/min.

Observation

The tailing factor of the peaks obtained was high & fails the system suitability. The

chromatogram for trial-1 was shown in .

71

Fig:2 Trial -1 chromatograph

.

Trail-2

Chromatographic conditions

Flow rate : 1.2 ml / min

Column : Cosmicsil ODS C18, 150 mm X 4.6 mm, 5 m

Detector wavelength : 311 nm

Column temperature : 35 0C

Injection volume : 10 l

Run time : 10 min

Mobile phase

Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 60:40v/v at a

flow rate of 1.0ml/min. Cosmicsil ODS C-18, 150 mm X 4.6 mm, 5 m column was used. The

column temperature was maintained at 35 gc.

72

Observation

The tailing factor of the peaks obtained was high & fails the system suitability. The

chromatogram for trial-2 was shown.

Fig:3 Trial -2 chromatograph

Trial -3

Chromatographic conditions

Flow rate : 1.0 ml / min

Column : Cosmicsil BDS, C18, 150 mm X 4.6 mm, 5 m

Detector wavelength : 315 nm

Column temperature : 25 0C

Injection volume : 10 l

Run time : 10 min

73

Mobile phase

Solution of phosphate buffer (pH-6.5), acetonitrile and methanol in the ratio of 50:50v/v at a

flow rate of 1.0ml/min. Cosmicsil BDS C-18, 150 mm X 4.6 mm, 5 m column was used. The

column temperature was maintained at 25 gc.

Observation

Peak of Dasatinib was well resolved with the retention time of 6.46 min. The chromatogram for

trial -3 (optimized method) was shown.

Fig:4 Trial-3 chromatograph

3.3.1. OPTIMIZED METHOD FOR ASSAY

Buffer preparation:

Add 4.0ml of triethylamine to 100ml water and adjut the pH to 6.5±0.05 diluted with

orthophosphoric acid add 10ml of methanol and mix well.

Solvent mixture:

Prepare a mixture of methanol and acetonitrile in the ratio of 50:50 v/v respectively.

Mobile phase:

74

Prepare a filtered and degassed mixture of buffer and solvent in the ratio of 50:50 v/v

respectively.

Preparation of standard solution:

Accurately weighed quantity of the drug was transfered about 68.0 mg of Dasatinib

monohydrate (working standard) into 50ml volumetric flask .Add about 30ml of solvent

mixture and sonicate to dissolve.Cool the solution to room temperature and dilute to volume

with solvent mixture.Transfer 1.0ml of the above solution into a 10ml volumetric flask and

dilute to volume with mobile phase.

Preparation of working standard solution:

From the standard stock solution, 2.0 ml, 3.0 ml ,4.0 ml, 5.0 ml and 6.0 ml was

transferred into a 10 ml volumetric flask and made up to the mark to produce 20,30,40,50,60

µg/ml respectively with mobile phase.

Preparation of sample solution:

Weigh and finely powder not fewer than 10 tablets. Transfer an accurately weighed

portion of powder, equivalent to 68.0mg of Dasatinib into a 100ml volumetric flask. Add about

60ml of solvent mixture , shake on orbital shaker for 15min and sonicate for 30min with

occasional shakings. Cool the solution to room temperature and dilute to volume with solvent

mixture. Centrifuge the solution at 3000RPM for 15min.Transfer 1ml of the above solution into a

10ml volumetric flask, dilute to volume with mobile phase.

Preparation of Placebo

75

The amount of powdered inactive ingredient supposed to be present in 10 tablets was

accurately weighed and transferred in to 100 ml volumetric flask. Add about 60ml of solvent

mixture , shake on orbital shaker for 15min and sonicate for 30min with occasional shakings.

Cool the solution to room temperature and dilute to volume with solvent mixture. Centrifuge the

solution at 3000RPM for 15min.Transfer 1ml of the above solution into a 100ml volumetric

flask, dilute to volume with mobile phase.

Test Procedure

10 µl of the standard, sample, blank and placebo preparations in duplicate were

injected separately into HPLC system and the peak responses for Dasatinib were measured. The

quantities from the peak area in mg of Dasatinib were calculated per tablet taken.

Optimized Chromatographic conditions

Flow rate : 1.0 ml / min

Column : Cosmicsil BDS, C18, 150 mm X 4.6 mm, 5 m

Detector wavelength : 315 nm

Column temperature : 25 0C

Injection volume : 10 l

Run time : 10 min

Retention time : 6.467

Observation:

The peak shape of Dasatini b was good and also optimum plate count and tailing.

Conclusion:

76

Hence this method was finalized for the estimation of Dasatinib

Calculation: The amount of drug was calculated by using the following formula:

AT WS DT P Avg. Wt

Assay % = -------------- x ----------x --------- x ----------x------------------ X 100

AS DS WT 100 LC

Where

At = Average area of sample

As = Average area of standard

Ws = Weight of standard

Ds = Dilution factor of standard

Dt = Dilution factor of sample

Wt = Weight of sample

P = Purity of working standard used

Aw = Average weight of tablets taken for analysis

Table 3.1. Assay data by HPLC

77

3.4. VALIDATION PARAMETERS

78

DASATINIB

Standard Area 1 311353

2 311363

3 311343

4 311323

5 311345

6 311333

Average 311343.33

Sample area 1 311297

2 311287

3 311277

4 311307

5 311267

6 311259

Average 311282.33

Tablet average weight 0.04889

Standard weight 50

std.purity 99.8

Sample weight 489.6

Label amount 50

%Assay 99.8

Validation of analytical method was a process of establishing documental

evidence which provides a high of assurance that a specific process will consistently produce a

product of predetermined specifications and quantity attributes.

The following parameters have been validated.

1. System suitability

2. Linearity

3. Accuracy

4. Precision

5. Robustness

6. LOD & LOQ

3.4.1. System Suitability:

Chromatograph the standard preparations (six replicate injections) and measure the peak

area responses for the analyte peak and evaluate the system suitability parameters as directed.

Table 3.2. System suitability data by HPLC

System suitability Parameters Mirtazapine

%RSD 0.78 %

Tailing factor 1.24

No. of theoretical plates 7620

Acceptance Criteria:

79

The number of theoretical plates for Dasatinib peak should be NLT 2000.

% RSD for six replicate injectionsof peak area response for Dasatinib peak from the

standard preparation should not be morethan 2.0.

The tailing factor for Dasatinib should not be morethan 2.0.

From the system suitability studies it was observed that all the parameters were within limit.

3.4.2. Linearity:

Linearity of the proposed HPLC method for determination of Dasatinib were evaluated

by analysing a series of different concentrations of standard drug. In this study Six

concentrations were chosen ranging between 20-60µg mL-1 for Dasatinib. Each concentration

was injected six times and obtained information on variation in the peak area response of pure

analyte was plotted against corresponding concentrations and result was shown in Table . The

linearity of the calibration graph was validated by the high value of correlation coefficient, slope

and the intercept value was shown

Table:3.3. Linearity range and average area values

Solution Concentration Peak area*

80

No. (µg / ml)

1 20 153482

2 30 228347

3 40 311353

4 50 388054

5 60 460767

*- average of 6 replicate injections for each concentration

15 20 25 30 35 40 45 50 55 60 650

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

Y=7985X-11196R2=0.999

Calibration curve of Dasatinib

Acceptance criteria

Correlation coefficient should be not less than 0.999.

Table 3.4. Calibration parameters for Dasatinib

81

Observation

The linearity Correlation coefficient for dasatinib is 0.999

3.4.3. Precision

Precision of the analytical method was studied by analysis of multiple sampling of

homogeneous sample. It was demonstrated by repeatability and intermediate precision

measurements of peak area and peak symmetry parameters of HPLC method for the title

ingredient. The repeatability (within-day in triplicates) and intermediate precision (for 3 days)

were carried out at six concentration levels for compound. Triplicate injections were made and

the obtained results within and between the days of trials were in acceptable range. The precision

expressed as % RSD is given .

3.4.3.1. Repeatability

82

Parameter Results

Slope 7985

Intercept -11196

Correlation co-efficient 0.999

Percentage curve fitting 99.9%

Six sample solutions were prepared and injected into the HPLC system as per test

procedure.

Table 3.5. Results of repeatability

*ˉaverage of 6 replicate injections for each concentration

Acceptance criteria

Relative standard deviation of percentage assay results should not be more than 2.0 %.

Observation

The Relative standard deviation was found to be 0.78% .

83

Conc. of dasatinib

(g/mL)

Peak Area % RSD*

40

309400

0.78

312247

305016

307219

307467

308121

3.4.3.2. Intermediate precession (analyst to analyst variability)

Two analysts as per test method conducted the study. For Analyst-1 refer

precision (Repeatability) results and the results for Analyst-2 were discussed below.

Table 3.6. Results of intermediate precession

Conc. of dasatinib

(g/mL)

Peak Area

% RSD*

50

308344

0.80

307467

307219

305017

312247

309402

*-average RSD of 6 replicate injections

Acceptance criteria

Relative standard deviation of % assay results should not more than 2.0 % by both the analysts.

Observation

The Relative standard deviation was found to be 0.80% .

3.4.4. Accuracy

Accuracy of an analytical method is the closeness of test results obtained by that

method to the true value. The accuracy of an analytical method should be established across its

linearity range. Accuracy was performed in three different levels, each level in triplicate for

84

Capecitabine using standards at 50%, 100% and 150%.Each sample was analysed in triplicate for

each level.

Table 3.7. Percent recovery results for Dasatinib

Sample Concentration % of spiked level

Amount of drug added in mg

Amount of drug found in mg

Percent recovery

Statistical analysis of %recovery

1. 50

50

50.20 100.4 Mean- 100.16

2. 50 50.15 100.3 S.D- 0.109

3. 50 49.90 99.8 %RSD- 0.108

1. 100

100

100.50 100.5 Mean- 100.26

2. 100 100.10 100.1 S.D- 0.152

3. 100 100.20 100.2 %RSD- 0.151

1. 150

150

150.0 100.0 Mean- 99.96

2. 150 149.85 99.7 S.D- 0.247

3. 150 150.09 100.19 %RSD- 0.247

Acceptance criteria

The mean % recovery of the Dasatinib monohydrate at each spike level should be not less than

98.0 % and not more than 102.0 %.

Observation:

The mean % recovery levels were found to be 100.1.

85

3.4.5. Specificity

Specificity is the ability to asses unequivocally the analyte in the presence of

components which may be expected to be present.Lack of specificity of an individual analytical

procedure may be compensated by other supporting analytical procedures. Solutions of standard

and Sample are prepared as per test method and injected into the chromatographic system.

Blank interference:

A study to establish the interference of blank was conducted. Mobile phase was injected

as per the test method. Chromatogram of blank should not show any peak at the retention time of

analyte peak.

Fig 6 Standard chromatogram for Dasatinib identification

86

Fig 7 Chromatogram for blank interference

Fig 8Chromatogram for placebo interference

87

3.4.6. Robustness

The robustness of the proposed method was determined by analysis of aliquots

from homogenous lots by differing physical parameters like flow rate and mobile phase

composition which may differ but the responses were still within the specified limits of the

assay.

3.4.6.1. Effect of variation of flow rate

A study was conducted to determine the effect of variation in flow rate.

Standard solution was prepared and injected into the HPLC system by keeping flow rates 1.0

ml/min and 1.2 ml/min. The effect of variation of flow rate was evaluated.

3.4.6.2. Effect of variation of temperature

A study was conducted to determine the effect of variation in temperature.

Standard solution prepared as per the test method was injected into the HPLC system at 25, 27

and 35ºC temperature.

The system suitability parameters were evaluated and found to be within the limits for a

temperature changes.

88

Table 3.8.Results of robustness

ParametersOptimum

range

Conditions in

procedureRemarks

Flow rate

ml/min1.0,1.2 1.2

At lower flow rates the asymmetry factor was

increased and at higher flow rates the relative

retentions was decreased.

Temperature 25,35oC AmbientBeyond the optimum range there is a change

in symmetry.

3.4.7. Limit of detection (LOD)

Calibration curve was repeated for 5 times and the standard deviation (SD) of the

intercepts was calculated.

The LOD was determined by the formula:

LOD = 3.3 σ / S

= 3.3 (98.1936 / 7985)

= 0.0405

Detection limit was 0.0405 µg / ml.

89

3.4.8. Limit of quantification (LOQ)

Calibration curve was repeated for 5 times and the standard deviation (SD) of the

intercepts was calculated The LOQ was determined by the formula:

LOQ = 10 σ / S

= 10 (98.1936 / 7985)

= 0.1229

Quantification limit was 0.1229 µg / ml.

90

RESULTS & DISCUSSION

The objective of the proposed work was to develop a method for the determination of

Dasatinib monohydrate to validate the methods according to USP and ICH guidelines and the

methods developed was found to be rapid, simple, precise, accurate and economic and then

applied on pharmaceutical dosage form.

In the method development, HPLC conditions were optimized to obtain, an adequate

separation of eluted compound. Various ratios of mobile phase systems were prepared and used

to provide an appropriate of of buffer (pH-6.5±0.05):solvent mixture [acetonitrile:

methanol(50:50 v/v)] in the ratio of 50:50v /v is used gave a better resolution and sensitivity.

Mobile phase and flow rate selection was based on peak parameters (height, tailing, theoretical

plates, capacity or symmetry factor), run time.

The optimum wavelength for detection was 315 nm at which better detector response for

the title drug was obtained. The retention time for Dasatinib monohydrate was found to be 6.467

min . The calibration was linear in concentration range of 20-60 µg mL -1 with regression 0.9999,

intercept -11196 and slope 7985 for Dasatinib monohydrate . The low values of % R.S.D

indicate the method is precise and accurate.

Sample to sample precision and accuracy were evaluated using t samples of different

concentrations, which were prepared and analyzed on same day. These results show the accuracy

and repeatibility of the assay. The % R.S.D. reported was found to be less than 2 %.The

proposed method was validated in accordance with ICH parameters and the applied for analysis

of the same in laboratory prepared mixtures.

91

The Limit of Quantification and Limit of Detection were calculated from the linearity

curve method using slope and standard deviation of intercepts of calibration curve. Limit of

Quantification and Limit of Detection were found to be 0.1229 µg / ml and 0.0405 µg / ml

respectively.

The proposed methods are accurate, simple, rapid and selective for the estimation of

Dasatinib monohydrate in laboratory prepared mixtures. Hence, these methods can be

conveniently adopted for the routine analysis of Dasatinib monohydrate in quality control

laboratories.

92

SUMMARY & CONCLUSION

A HPLC method was developed for the estimation of Dasatinib in tablet dosage form using

reverse phase high performance liquid chromatography.HPLC Waters (Model.No:2690) with

UV\VIS detector and Cosmicsil BDS C- 18 with ambient temperature, injection volume of 10µl

is injected and eluted with mobile phase of phosphate buffer (pH-6.5), Acetonitrile and methanol

in the ratio 50:50 v/v, which was pumped with a flow rate 1.0ml/min and detected by UV at

315nm. The peak of Dasatinib was found at 6.4675min.The developed method was validated for

various parameters as per ICH guidelines like accuracy, precision, linearity, LOD, LOQ,

ruggedness and robustness.The proposed method was applied for the determination of Dasatinib

in tablets. Hence the proposed method was found to be satisfactory and could be used for the

routine analysis of Dasatinib in the tablets.

93

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97