METHOD DEVELOPMENT AND METHOD VALIDATION

FOR THE ESTIMATION OF DRGS BY USING RP-

6/26/2016
HPLC

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BY
@@@ Mr.S @@@
Pharmaceutical Analysis & Quality Assurance,
Vikas College Of Pharmacy,
Vissannapeta.

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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
 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.
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 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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PRINCIPLE
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.

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

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modified.

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 Sample preparation and instrument conditions
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 method
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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

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cost, time and also to minimize the errors
which are arising in the development.
The process of optimization
includes:
* Selection of method
* Selection of mobile phase
* Selection of column
* Selection of detector
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SELECTION OF METHOD

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 The most commonly used
chromatographic methods are normal

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phase, reverse phase, reverse phase ion
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 column.

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 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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VALIDATION
METHOD
METHOD VALIDATION
 Validation Establish a documented evidence which

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provides a high degree of assurance that a specific
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,
Limit tests for the control of impurities,
Quantitative tests of the active moiety in
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samples.
 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

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proportional to the concentration of the analyte in
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

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unaffected by small but deliberate variations in
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

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chromatogram trace with a standard trace.
 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

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their average
SD = [Σ (X- Xi) /n-1]1/2

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Where,
Σ = sum of observations
Xi = mean
X = individual observation value
(X- Xi) = deviation of a value from mean
n =number of observations

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RELATIVE STANDARD DEVIATION

Relative Standard Deviation (RSD) is

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defined as the standard deviation expressed as the
percentage of mean

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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.
 It provides a measure of the strength and

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direction of the correlation varying from +1 to -1.
 Positive values indicate the two variables are
positively correlated, meaning the two variables
vary in the same direction .
 Negative values indicate that the two variables are
negatively correlated, 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

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 A regression is a statistical analysis
assessing the association between any two
variables. It is used to find the relationship

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between two variables.
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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