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METHOD DEVELOPMENT AND METHOD VALIDATION
FOR THE ESTIMATION OF DRGS BY USING RP-
HPLC
6/26/2016
1
VikasCollegeOfPharmacy
BY
@@@ Mr.S @@@
Pharmaceutical Analysis & Quality Assurance,
Vikas College Of Pharmacy,
Vissannapeta.
CONTENTS
 Introduction
 Principle
 Method Development
* Method Development
* Optimization
Techniques
 Method Validation
* Method Validation
* Validation Parameters
Conclusion
 References
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INTRODUCTION
 High Performance Liquid Chromatography
(HPLC) is the most widely used of all analytical
separation techniques.
 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 ,
causing different flow rates for the different
components and leading to the separation of the
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
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PRINCIPLE
“ 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
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.
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METHOD
DEVELOPMENT
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METHOD DEVELOPMENT
 HPLC method development and validation
play important role in the discovery,
development and manufacture of
pharmaceutical products, agro chemicals.
 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
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STEPS INVOLVED IN METHOD DEVELOPMENT:-
 Information on sample
 Define separation goals
 Special procedure requirement & pretreatment.
 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
Several reasons are available for the development of
a new method of analysis, they are :
 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
modified.
 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
exists that is similar to the sample of interest.
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OPTIMIZATION
The principle of optimization in
the method development is to reduce the
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
 The most commonly used
chromatographic methods are normal
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.
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SELECTION OF COLUMN
 Columns being the heart of HPLC for
optimum separation method.
 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
selection of mobile phase is very important for
the analysis of the drug .
 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 not
alters the pH of the sample.
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SELECTION OF DETECTORS
 Detectors are the eyes of the
chromatography system and measure the
compounds after separation of the 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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METHOD
VALIDATION
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METHOD VALIDATION
 Validation Establish a documented evidence which
provides a high degree of assurance that a specific
process will consistently produce a product meeting
its predetermined specifications and quality attributes.
 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
samples.
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NEED OF METHOD VALIDATION
 Before introduction of a new method in to
routine use.
 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
PROCEDURES INVOLVED IN VALIDATION:
1.Accuracy.
2.Precision.
3.Specificity/selectivity.
4.Limit of Detection.
5.Limit of Quantification.
6.Linearity.
7.Robustness.
8.System suitability.
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ACCURACY
 Accuracy The accuracy is the closeness of the test
results obtained by the method to he true value.
 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
the degree of agreement between a series of
measurements obtained from multiple sampling of the
same homogeneous sample. The RSD for all the
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.
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SPECIFICITY
 Specificity/Selectivity: The ability to assess
unequivocally the sample in the presence of
components that may be expected to be present. –
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
 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
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
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.
 This allows a comparison of the peak shape, peak
width, baseline resolution. Parameters to be
calculated to provide a system suitability test report.
6/26/2016
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Parameter Limit
Capacity factor K’>2
Injection Precision RSD < 1% for n ≥ 5
Resolution R>2
Tailing factor A≤2
Theoritical plates N>2000
PARAMETERS INVOLVED IN
VALIDATION
1.Mean. (Xi)
2.Standard deviation.(S.D)
3.Relative standard deviation.(RSD)
4.Correlation co-Efficient.(R)
5.Linear regression.
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VALIDATION PARAMETERS
MEAN :
The average result (ā) is calculated by
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
SD = [Σ (X- Xi) /n-1]1/2
Where,
Σ = sum of observations
Xi = mean
X = individual observation value
(X- Xi) = deviation of a value from mean
n =number of observations
6/26/2016
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RELATIVE STANDARD DEVIATION
Relative Standard Deviation (RSD) is
defined as the standard deviation expressed as the
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
estimate the relationship of two random variables.
 It provides a measure of the strength and
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
 A regression is a statistical analysis
assessing the association between any two
variables. It is used to find the relationship
between two variables.
y = a + b x
where ,
a = Intercept
b = Slope
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VikasCollegeOfPharmacy
CONCLUSION
 Whenever the method is changed and the
change is outside the scope of the original
method.
 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.
6/26/2016
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VikasCollegeOfPharmacy
REFERENCES
Instrumental methods of chemical
analysis By B.K. Sharma (Pg:286-385)
Instrumental methods of Chemical
Analysis By Gurudeep R Chathwal
(Pg: 2.624-2.639)
WIKIPEDIA.
6/26/2016
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6/26/2016
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HPLC Method Development & Method Validation (mr.s)

  • 1. METHOD DEVELOPMENT AND METHOD VALIDATION FOR THE ESTIMATION OF DRGS BY USING RP- HPLC 6/26/2016 1 VikasCollegeOfPharmacy BY @@@ Mr.S @@@ Pharmaceutical Analysis & Quality Assurance, Vikas College Of Pharmacy, Vissannapeta.
  • 2. CONTENTS  Introduction  Principle  Method Development * Method Development * Optimization Techniques  Method Validation * Method Validation * Validation Parameters Conclusion  References 6/26/2016 2 VikasCollegeOfPharmacy
  • 4. INTRODUCTION  High Performance Liquid Chromatography (HPLC) is the most widely used of all analytical separation techniques.  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. 6/26/2016 4 VikasCollegeOfPharmacy
  • 5.  Each component in the sample interacts slightly differently with the adsorbent material , causing different flow rates for the different components and leading to the separation of the 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. 6/26/2016 5 VikasCollegeOfPharmacy
  • 7. PRINCIPLE “ 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 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. 6/26/2016 7 VikasCollegeOfPharmacy
  • 9. METHOD DEVELOPMENT  HPLC method development and validation play important role in the discovery, development and manufacture of pharmaceutical products, agro chemicals.  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 6/26/2016 9 VikasCollegeOfPharmacy
  • 10. STEPS INVOLVED IN METHOD DEVELOPMENT:-  Information on sample  Define separation goals  Special procedure requirement & pretreatment.  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. 6/26/2016 10 VikasCollegeOfPharmacy
  • 11. NEED FOR DEVELOPING A NEW METHOD Several reasons are available for the development of a new method of analysis, they are :  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. 6/26/2016 11 VikasCollegeOfPharmacy
  • 12. SELECTION OF SUITABLE METHOD  Using the available literature and previous methodology, the methods are adopted and modified.  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 exists that is similar to the sample of interest. 6/26/2016 12 VikasCollegeOfPharmacy
  • 13. OPTIMIZATION The principle of optimization in the method development is to reduce the 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 6/26/2016 13 VikasCollegeOfPharmacy
  • 14. SELECTION OF METHOD  The most commonly used chromatographic methods are normal 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. 6/26/2016 14 VikasCollegeOfPharmacy
  • 15. SELECTION OF COLUMN  Columns being the heart of HPLC for optimum separation method.  The selection of column involves the following parameters: * Column length * Column diameter * Column particle size * Column particle shape 6/26/2016 15 VikasCollegeOfPharmacy
  • 16. SELECTION OF MOBILE PHASE  In reversed phase chromatography the selection of mobile phase is very important for the analysis of the drug .  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 not alters the pH of the sample. 6/26/2016 16 VikasCollegeOfPharmacy
  • 17. SELECTION OF DETECTORS  Detectors are the eyes of the chromatography system and measure the compounds after separation of the 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. 6/26/2016 17 VikasCollegeOfPharmacy
  • 19. METHOD VALIDATION  Validation Establish a documented evidence which provides a high degree of assurance that a specific process will consistently produce a product meeting its predetermined specifications and quality attributes.  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 samples. 6/26/2016 19 VikasCollegeOfPharmacy
  • 20. NEED OF METHOD VALIDATION  Before introduction of a new method in to routine use.  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. 6/26/2016 20 VikasCollegeOfPharmacy
  • 21. DISCRIPTION OF METHOD VALIDATION PROCEDURES INVOLVED IN VALIDATION: 1.Accuracy. 2.Precision. 3.Specificity/selectivity. 4.Limit of Detection. 5.Limit of Quantification. 6.Linearity. 7.Robustness. 8.System suitability. 6/26/2016 21 VikasCollegeOfPharmacy
  • 22. ACCURACY  Accuracy The accuracy is the closeness of the test results obtained by the method to he true value.  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%. 6/26/2016 22 VikasCollegeOfPharmacy
  • 23. PRECISION & REPEATABILITY  Precision : The precision of an analytical method is the degree of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample. The RSD for all the 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. 6/26/2016 23 VikasCollegeOfPharmacy
  • 24. SPECIFICITY  Specificity/Selectivity: The ability to assess unequivocally the sample in the presence of components that may be expected to be present. – 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%. 6/26/2016 24 VikasCollegeOfPharmacy
  • 25. LINEARITY  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 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. 6/26/2016 25 VikasCollegeOfPharmacy
  • 26.  Robustness: The robustness of an analytical procedure is a measure of its capacity to remain 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%. 6/26/2016 26 VikasCollegeOfPharmacy
  • 27.  System suitability: The simplest form of an HPLC system suitability test involves a comparison of 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. 6/26/2016 27 VikasCollegeOfPharmacy Parameter Limit Capacity factor K’>2 Injection Precision RSD < 1% for n ≥ 5 Resolution R>2 Tailing factor A≤2 Theoritical plates N>2000
  • 28. PARAMETERS INVOLVED IN VALIDATION 1.Mean. (Xi) 2.Standard deviation.(S.D) 3.Relative standard deviation.(RSD) 4.Correlation co-Efficient.(R) 5.Linear regression. 6/26/2016 28 VikasCollegeOfPharmacy
  • 29. VALIDATION PARAMETERS MEAN : The average result (ā) is calculated by 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. 6/26/2016 29 VikasCollegeOfPharmacy
  • 30. STANDARD DEVIATION  It is the root mean square deviation of values from their average SD = [Σ (X- Xi) /n-1]1/2 Where, Σ = sum of observations Xi = mean X = individual observation value (X- Xi) = deviation of a value from mean n =number of observations 6/26/2016 30 VikasCollegeOfPharmacy
  • 31. RELATIVE STANDARD DEVIATION Relative Standard Deviation (RSD) is defined as the standard deviation expressed as the percentage of mean RSD = (SD/XI) × 100 Where, SD = Standard Deviation XI = Mean 6/26/2016 31 VikasCollegeOfPharmacy
  • 32. CORRELATION CO-EFFECIENT  The correlation co-efficient (R) is used to estimate the relationship of two random variables.  It provides a measure of the strength and 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. 6/26/2016 32 VikasCollegeOfPharmacy
  • 33.  Values close to +1 or -1 reveal the two variables are highly related 6/26/2016 33 VikasCollegeOfPharmacy
  • 34. LINEAR REGRESSION  A regression is a statistical analysis assessing the association between any two variables. It is used to find the relationship between two variables. y = a + b x where , a = Intercept b = Slope 6/26/2016 34 VikasCollegeOfPharmacy
  • 35. CONCLUSION  Whenever the method is changed and the change is outside the scope of the original method.  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. 6/26/2016 35 VikasCollegeOfPharmacy
  • 36. REFERENCES Instrumental methods of chemical analysis By B.K. Sharma (Pg:286-385) Instrumental methods of Chemical Analysis By Gurudeep R Chathwal (Pg: 2.624-2.639) WIKIPEDIA. 6/26/2016 36 VikasCollegeOfPharmacy