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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.
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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.
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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.
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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.
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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)
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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).
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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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 %.
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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%.
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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).
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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 )
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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)
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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
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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.,)
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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
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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.
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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.
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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
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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.
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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:
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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:
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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
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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.
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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-
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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
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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
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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%.
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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.
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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.
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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.
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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
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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.
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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.
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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
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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
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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.
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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.
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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 .
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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.
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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
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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:
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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
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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:
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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
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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
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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:
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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*
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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
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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%
Page 83
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
Page 84
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
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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.
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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
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Fig 7 Chromatogram for blank interference
Fig 8Chromatogram for placebo interference
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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.
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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.
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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.
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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.
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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.
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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.
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