METHOD DEVELOPMENT AND METHOD VALIDATION

FOR THE ESTIMATION OF DRGS BY USING RP-

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HPLC

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CONTENTS

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 Introduction
 Principle

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 Method Development
* Method Development
* Optimization
Techniques
 Method Validation
* Method
Validation
* Validation
Parameters
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 Conclusion
 References
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INTRODUCTION

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 High Performance Liquid Chromatography
(HPLC) is the most widely used of all analytical
separation techniques.

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 It is a technique in analytical chemistry used
to separate the components in a mixture , to
identify each component, and to quantify each
component.
 It relies on pumps to pass a pressurized
liquid solvent containing the sample mixture
through a column filled with a solid adsorbent
material. 4
 Each component in the sample interacts
slightly differently with the adsorbent material ,

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causing different flow rates for the different
components and leading to the separation of the

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components as they flow out the column.

 The development of HPLC from classical

column chromatography can be attributed to the
development of smaller particle is important.

 They offer more surface area over the

conventional larger particle sizes.
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PRINCIPL
E
PRINCIPLE

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“ The principle of separation in normal phase mode and
reversed phase mode is adsorption. When a mixture of
components are introduced into a HPLC column, they travel

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according to their relative affinities towards the stationary
phase, more affinity towards stationary phase travels slower
and less affinity towards the stationary phase travels faster. No
two components have the same affinity towards the stationary
phase, the components are separated.”

In most of the cases in pharmaceutical industries –

RP-HPLC is used. Hence this gives detailing about RP-
HPLC. 7
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DEVELOPMENT
METHOD
METHOD DEVELOPMENT
 HPLC method development and validation
play important role in the discovery,

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development and manufacture of
pharmaceutical products, agro chemicals.

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 HPLC method should be developed within
the GMP and GLP environments using the
protocols set out in ICH guide lines.
 HPLC method development includes:
* Method Development
* Need for development
* Selection of suitable method
* Optimization 9
STEPS INVOLVED IN METHOD DEVELOPMENT:-
 Information on sample

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 Define separation goals

 Special procedure requirement & pretreatment.

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 Detector selecting and setting

 Optimization separation conditions

 Check for problems or needs of special procedure

 Recovery of purified material

 Quantitative calibration / Qualitative method

 Method validation for release to laboratories.

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NEED FOR DEVELOPING A NEW METHOD

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Several reasons are available for the development of
a new method of analysis, they are :

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 There may not be a suitable method for a
particular analyte in the
sample matrix.
 Existing may be too erroneous.
 Existing method may not provide adequate
sensitivity.
 Existing methods may be unreliable.

 Existing methods are too expensive and time

consuming. 11
SELECTION OF SUITABLE METHOD
 Using the available literature and previous
methodology, the methods are adopted

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and modified.
 Sample preparation and instrument conditions

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are adopted to make use of latest methods
and instrumentation.
 If no previous methods exist for the analyte in
the literature , work from analogy to
investigate compounds that are similar in
structure and properties.
 Usually a compound with analytical
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method exists that is similar to the sample of
interest.
OPTIMIZATION

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The principle of optimization in
the method development is to reduce the
cost, time and also to minimize the errors

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which are arising in the development.
The process of
optimization
includes:
* Selection of method
* Selection of mobile phase
* Selection of column
* Selection of detector 13
SELECTION OF METHOD

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 The most commonly used
chromatographic methods are normal
phase, reverse phase, reverse phase ion

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pair and ion exchange methods.
 In the selection of method for the
organic compounds primarily reverse phase
method should be tried.
 If not successful normal phase
should be tried, then reverse phase ion
pair method , ion exchange
chromatographic methods are at the end. 14
 SELECTION OF COLUMN

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 Columns being the heart of HPLC for
optimum separation method.

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 The selection of column involves the
following parameters:
* Column length
* Column diameter
* Column particle size
* Column particle shape

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SELECTION OF MOBILE PHASE

 In reversed phase chromatography the

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selection of mobile phase is very important for
the analysis of the drug .

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 The organic phase concentration
required for the mobile phase can be
estimated by “gradient elution method.”
 Separation can be optimized by
changing the initial mobile phase composition
according to the chromatogram obtained
from preliminary run.
 The pH of the mobile phase should
not16 alters the pH of the sample.
SELECTION OF DETECTORS
 Detectors are the eyes of the

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chromatography system and measure the
compounds after separation of the

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 column.
The must have certain characteristics
i.e, high Sensitivity, higher linear range,
application to most of the solutes,does
not contribute to band broadening, non-
destructive, faster response.

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VALIDATIO
METHOD

N
METHOD VALIDATION
 Validation Establish a documented evidence which
provides a high degree of assurance that a specific

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process will consistently produce a product meeting
its predetermined specifications and quality attributes.

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 Method validation is defined as the process of
proving (through scientific studies) analytical method
is acceptable for its intended use.
 The validation of analytical procedures is directed
to
the four most common types of analytical
procedures:
Identification tests,
Quantitative tests for impurity content,
samples. Limit tests for the control of 19

impurities,
Quantitative tests of the active moiety
in
 NEED OF METHOD VALIDATION

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 Before introduction of a new method in to
routine use.

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 Whenever condition change for which
method has been validation e.g. instrument
with different characteristics.
 Whenever the method is changed and the
change is outside the scope of the original
method.

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DISCRIPTION OF METHOD
VALIDATION

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PROCEDURES INVOLVED IN VALIDATION:
1.Accuracy.

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2.Precision.
3.Specificity/selectivity
.
4.Limit of Detection.
5.Limit of Quantification.
6.Linearity.
7.Robustness.
8.System suitability. 21
ACCURACY

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 Accuracy The accuracy is the closeness of the test
results obtained by the method to he true value.

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 Accuracy should be established across its range.

 Accuracy assessed using a minimum of 9 determinations

over a minimum of 3 concentration levels
 The acceptance criterion for accuracy is the Relative
Standard Deviation (RSD) for all the recovery
values should not be more than 2%.

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PRECISION & REPEATABILITY
 Precision : The precision of an analytical method is

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the degree of agreement between a series of
measurements obtained from multiple sampling of the
same homogeneous sample. The RSD for all the

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assays of sample preparations should not be more
than 2%.
 Repeatability : Repeatability expresses the precision
under the same operating conditions over a short
interval of time. Repeatability is also termed intra-
assay precision . a minimum of 9 determinations
covering the specified range for the procedure ( e.g.,
3 concentrations/3 replicates each); or a minimum
of
6 determinations at 100% of the test concentration. 23
SPECIFICITY

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 Specificity/Selectivity: The ability to assess
unequivocally the sample in the presence of
components that may be expected to be present. –

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Impurities – degrading agents – excipients ,
Specificity must be demonstrated for: –
Identification – Impurities Test – Assay Test.
 The RSD for all the assays of sample preparations
should not be more than 2%.

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LINEARITY

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 Linearity: The linearity of an analytical procedure is
its ability to obtain test results that are directly
proportional to the concentration of the analyte in

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the sample. Linearity is usually demonstrated by
the analysis of various concentrations of the analyte
(s) across the indented range and represented
graphically.
 The relationship between the concentration (in %)
of drug in sample area of should be linear in the
specified range and the correlation should not be
less than 0.9.
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 Robustness: The robustness of an analytical
procedure is a measure of its capacity to remain
unaffected by small but deliberate variations in

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method parameters and provides an indication of its
reliability during use. The RSD for the samples
should not be more than 2%.

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 System suitability: The simplest form of an HPLC
system suitability test involves a comparison of
chromatogram trace with a standard trace.

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 This allows a comparison of the peak shape, peak
width, baseline resolution. Parameters to be
calculated to provide a system suitability test report.
Parameter Limit
Capacity factor K’>2
Injection Precision RSD < 1% for n ≥ 5
Resolution R>2
Tailing factor A≤2
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Theoritical plates N>2000
PARAMETERS INVOLVED IN
VALIDATION

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1.Mean. (Xi)
2.Standard deviation.(S.D)

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3.Relative standard deviation.(RSD)
4.Correlation co-Efficient.(R)
5.Linear regression.

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 VALIDATION PARAMETERS

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MEAN :
The average result (ā) is calculated by

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summing the individual results and dividing the
sum by the number (n) of individual values.

Xi= x1 + x2 + x3 ........ /n
Where,
x1 ,x2 ,x3...... = values of
individual results n = Number of
individual results.
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STANDARD DEVIATION
 It is the root mean square deviation of values
from their average

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SD = [Σ (X- Xi) /n-1]1/2
Where,

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Σ = sum of observations
Xi = mean
X = individual
observation value
(X- Xi) = deviation of a
value from mean
n =number of
observations 30
RELATIVE STANDARD DEVIATION

Relative Standard Deviation is

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(RSD)
defined as the standard deviation expressed as the

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percentage of mean
RSD = (SD/XI) × 100
Where,
SD = Standard
Deviation XI = Mean

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CORRELATION CO-EFFECIENT

 The correlation co-efficient (R) is used to

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estimate the relationship of two random
 variables.

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It provides a measure of
 the strength and direction of
the correlation varying from +1 to -1.
Positive values indicate the two variables are
positivelyvalues
 Negative correlated, meaning
indicate that thethe
twotwo variables
variables are
vary in the correlated,
negatively same direction .
meaning the two variables
vary in the contrary direction.

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 Values close to +1 or -1 reveal the two
variables are highly related

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LINEAR REGRESSION
A regression is a statistical analysis

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
assessing the association between
variables. It is used to any two the

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find between two
variables. relationship
y=a+bx
where , a = Intercept
b = Slope

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CONCLUSION
 Whenever the method is changed and the

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change is outside the scope of the original
method.

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 If no previous methods exist for the analyte in
the literature , work from analogy to
investigate compounds that are similar in
structure and properties.
 HPLC method should be developed within the
GMP and GLP environments using the
protocols set out in ICH guide lines.
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REFERENCES

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 Instrumental methods of chemical

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analysis By B.K. Sharma (Pg:286-385)
 Instrumental methods of Chemical
Analysis By Gurudeep R
Chathwal (Pg: 2.624-2.639)
 WIKIPEDIA.

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