Analytical Method Validation
1.0 What
This document details the procedure for the
Analytical Method Validation.
Analytical Method Validation.
2.0
WHY
It is the policy
of your Company that the written procedure shall be followed
for the validation of analytical method used for the analysis for the
consistency of the method and must meet standards of accuracy and reliability
of test protocols with predefined norms.
of your Company that the written procedure shall be followed
for the validation of analytical method used for the analysis for the
consistency of the method and must meet standards of accuracy and reliability
of test protocols with predefined norms.
3.0
WHEN
This analytical method validation is applicable at the time
of
of
i)
Introduction
of New Product and validation data not provided by tie up company. (Other than
Pharmacopoeial products).
Introduction
of New Product and validation data not provided by tie up company. (Other than
Pharmacopoeial products).
ii)
Addition
of new Raw material (other than Pharmacopoeias product).
Addition
of new Raw material (other than Pharmacopoeias product).
iii)
Ruggedness
of pharmacopoeias drugs and drug product (if required).
Ruggedness
of pharmacopoeias drugs and drug product (if required).
4.0
Responsibility
Following persons are responsible for preparation, operation
and implementation of this procedure.
and implementation of this procedure.
|
S. No
|
Designation
|
Responsibility
|
|
01
|
Executive – Corporate Quality Assurance
|
Prepare the SOP for analytical method validation and
follow the SOP accordingly. |
|
02
|
Manager – Quality Control
|
Conducting the validation activity and maintain the
relevant record. |
|
03
|
Manager – Quality Assurance
|
To ensure the implementation of SOP
|
|
04
|
Manager – Corporate Quality Assurance
|
To ensure the implementation of SOP
|
5.0
PROCEDURE
5.1
Analytical Parameter
(Definition and Procedure)
5.1.1
Linearity and Range
Definition of Linearity: Linearity of an analytical method is its ability to elicit
test result that are directly or by a well defined mathematical transformation,
proportional to the concentration of analyte in samples within a given range.
test result that are directly or by a well defined mathematical transformation,
proportional to the concentration of analyte in samples within a given range.
Definition of Range : The range of an analytical method is the interval between
the upper and lower level of analyte that have been demonstrated to be
determined with a suitable level of precision, accuracy and linearity using the
method.
the upper and lower level of analyte that have been demonstrated to be
determined with a suitable level of precision, accuracy and linearity using the
method.
Procedure : The linearity should established the relationship between
the response of an analyte and its concentration. For assay method linearity and range shall be
done in the range of 80 to 120% (for content uniformity 80 to 120%) of standard
/ sample preparation.
the response of an analyte and its concentration. For assay method linearity and range shall be
done in the range of 80 to 120% (for content uniformity 80 to 120%) of standard
/ sample preparation.
For related substances linearity and range should be done in
the range of limit of Quantification and 120% of specification limits. A
minimum of 5 data points is required in the range as specified above.
the range of limit of Quantification and 120% of specification limits. A
minimum of 5 data points is required in the range as specified above.
5.1.2
Precision
System Precision: The system precision is the agreement of five or more
replicates that are obtained under identical conditions using some test
methods.
replicates that are obtained under identical conditions using some test
methods.
Method Precision: The precision of an analytical method is the degree of
agreement among individual test result when the method is applied repeatedly to
multiple sampling of a homogeneous sample.
agreement among individual test result when the method is applied repeatedly to
multiple sampling of a homogeneous sample.
Intermediate Precision: The
Intermediate Precision of the method is established by estimating the assay of
six different sample preparations of the same batch by different Chemist on a
different system, different column and on a different day.
Intermediate Precision of the method is established by estimating the assay of
six different sample preparations of the same batch by different Chemist on a
different system, different column and on a different day.
Procedure: The precision of an analytical method is by assaying a
sufficient number of aliquots of a homogeneous sample to be able to calculate
statistical estimates of standard deviation or relative standard deviation
(coefficient of variation) of a series of measurement. Precision may be
measured by the following three levels.
sufficient number of aliquots of a homogeneous sample to be able to calculate
statistical estimates of standard deviation or relative standard deviation
(coefficient of variation) of a series of measurement. Precision may be
measured by the following three levels.
i)
Repeatability or method precision :
It refers to use of the analytical procedure within a laboratory over a
short period time using the same analyst with of concern analytical method. The sample should be assessed using minimum
of nine determinations covering the specified range for the procedure (i.e.
three concentrations and three replicate of each concentration using minimum
six determinations at 100 % of the test concentration).
Repeatability or method precision :
It refers to use of the analytical procedure within a laboratory over a
short period time using the same analyst with of concern analytical method. The sample should be assessed using minimum
of nine determinations covering the specified range for the procedure (i.e.
three concentrations and three replicate of each concentration using minimum
six determinations at 100 % of the test concentration).
ii) Intermediate precision or ruggedness: The precision method on different days
by different analyst and using different instruments. The sample shall prepared and analysed as per
the method, six times by two different analysts on different days and on different
instruments.
by different analyst and using different instruments. The sample shall prepared and analysed as per
the method, six times by two different analysts on different days and on different
instruments.
iii) Reproducibility or system precision :
Reproducibility refers to the use of the analytical procedure in
different laboratories.
Reproducibility refers to the use of the analytical procedure in
different laboratories.
5.1.3
Accuracy
Definition: Accuracy of an
analytical method is the closeness of test results obtained by that method to
true values.
analytical method is the closeness of test results obtained by that method to
true values.
Procedure: For Active Pharmaceutical Ingredient (API), the accuracy is
inferred from specificity, linearity, and precision data.
inferred from specificity, linearity, and precision data.
For drug product the accuracy shall be derived by
performing, recovery experiments by spiking API in the placebo in triplicate at
minimum five different concentrations of assay sample preparation. For Related Substances (API and Drug Product)
the accuracy shall be shown by performing recovery experiment by spiking known
impurities in the same in triplicate at minimum five different concentrations.
performing, recovery experiments by spiking API in the placebo in triplicate at
minimum five different concentrations of assay sample preparation. For Related Substances (API and Drug Product)
the accuracy shall be shown by performing recovery experiment by spiking known
impurities in the same in triplicate at minimum five different concentrations.
5.1.4
Specificity
Definition:
The specificity is define as the ability to assess unequivocally the
analyte in the presence of components that may be expected to be present such
as impurities, degradation product and matrix components.
The specificity is define as the ability to assess unequivocally the
analyte in the presence of components that may be expected to be present such
as impurities, degradation product and matrix components.
Procedure :
Assay Method : In case of assay demonstration of specification requires
that it can be shown that the procedure is unaffected by presence of
impurities/related substances or excipient with the analyte peak. In practice this can be done by spiking the
drugs substances with appropriate level of impurity (1%) or excipient in
triplicate and demonstrating that the assay result is unaffected by the
presence of these extraneous materials.
Assay Method : In case of assay demonstration of specification requires
that it can be shown that the procedure is unaffected by presence of
impurities/related substances or excipient with the analyte peak. In practice this can be done by spiking the
drugs substances with appropriate level of impurity (1%) or excipient in
triplicate and demonstrating that the assay result is unaffected by the
presence of these extraneous materials.
Related Substances
method: This is done by spiking all known impurities
at 1% level and showing the separation of all impurities from the main peak and
from each other and demonstrating peak purity.
method: This is done by spiking all known impurities
at 1% level and showing the separation of all impurities from the main peak and
from each other and demonstrating peak purity.
5.1.5
Limit of detection
Definition: It is the lowest
amount of analyte in a sample that can be detected but not necessary to be
quantified. Several approaches for determining the detection limit are
possible, depending on whether the procedure is a non- instrumental. Approaches
other than those listed below may be acceptable.
amount of analyte in a sample that can be detected but not necessary to be
quantified. Several approaches for determining the detection limit are
possible, depending on whether the procedure is a non- instrumental. Approaches
other than those listed below may be acceptable.
5.1.5.1 Based on Standard Deviation of
the Response and the slope
the Response and the slope
For non-instrumental method the detection limit is generally
determined by the analysis of sample with known concentration of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
determined by the analysis of sample with known concentration of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
In case of instrumental analytical
procedures measure the magnitude of analytical back ground response by analysis
number of analyte samples of different concentrations at detection level (1:3
signal of noise ratio) and calculating limit of detection by following method.
procedures measure the magnitude of analytical back ground response by analysis
number of analyte samples of different concentrations at detection level (1:3
signal of noise ratio) and calculating limit of detection by following method.
LOD = 3.3.
x S.E.
x S.E.
Slope
Where S.E. (Standard Error) and slop are calculated by
number of concentration at LOD level.
number of concentration at LOD level.
5.1.5.2
Based on Visual Evaluation
Based on Visual Evaluation
Visual evaluation may be used for non –
instrumental methods but may also be used with instrumental methods.
instrumental methods but may also be used with instrumental methods.
The detection limit is determined by
the analysis of the samples with unknown concentrations of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
the analysis of the samples with unknown concentrations of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
5.1.5.3
Based on Signal to Noise
Based on Signal to Noise
This approach can only be applied to
analytical procedures which exhibit baseline noise.
analytical procedures which exhibit baseline noise.
Determination of the signal – to –noise
ratio is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. A signal –
to – noise ratio between 3:1 is generally considered acceptable for estimating
the detection limit.
ratio is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. A signal –
to – noise ratio between 3:1 is generally considered acceptable for estimating
the detection limit.
5.1.5.4
Based on the Standard Deviation of the
Blank
Based on the Standard Deviation of the
Blank
Measurement of the magnitude of
analytical background response is performed by analyzing an appropriate number
of blank samples and calculating the standard deviation of these responses.
analytical background response is performed by analyzing an appropriate number
of blank samples and calculating the standard deviation of these responses.
5.1.5.5
Based on the Calibration Curve
Based on the Calibration Curve
A specific calibration curve should be
studied using samples containing an nalyte in the range of detection limit. The
residual standard deviation of a regression line or the standard deviation of
y- intercepts of regression lines may be used as the standard deviation.
studied using samples containing an nalyte in the range of detection limit. The
residual standard deviation of a regression line or the standard deviation of
y- intercepts of regression lines may be used as the standard deviation.
5.1.6
Limit of Quantification
Definition: It is the lowest
amount of analyte in a sample at which that can be determined with acceptable precision and accuracy under
the stated experimental conditions. 5 to 10% precision is acceptable.
amount of analyte in a sample at which that can be determined with acceptable precision and accuracy under
the stated experimental conditions. 5 to 10% precision is acceptable.
5.1.6.1 Based on Standard Deviation of
the Response and the slope
the Response and the slope
For non-instrumental method the detection limit is generally
determined by the analysis of sample with known concentration of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
determined by the analysis of sample with known concentration of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
In case of instrumental analytical
procedures measure the magnitude of analytical back ground response by analysis
number of analyte samples of different concentrations at detection level (10:1 signal
of noise ratio) and calculating limit of detection by following method.
procedures measure the magnitude of analytical back ground response by analysis
number of analyte samples of different concentrations at detection level (10:1 signal
of noise ratio) and calculating limit of detection by following method.
LOD = 10.0 x
S.E.
S.E.
Slope
Where S.E. (Standard Error) and slop are calculated by
number of concentration at LOD level.
number of concentration at LOD level.
5.1.6.2
Based on Visual Evaluation
Visual evaluation may be used for non –
instrumental methods but may also be used with instrumental methods.
instrumental methods but may also be used with instrumental methods.
The detection limit is determined by
the analysis of the samples with unknown concentrations of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
the analysis of the samples with unknown concentrations of analyte and by
establishing the minimum level at which the analyte can be reliably detected.
5.1.6.3
Based on Signal to Noise
This approach can only be applied to
analytical procedures, which exhibit baseline noise.
analytical procedures, which exhibit baseline noise.
Determination of the signal – to –noise
ratio is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. A signal –
to – noise ratio between 10 :1 is generally considered acceptable for
estimating the detection limit.
ratio is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. A signal –
to – noise ratio between 10 :1 is generally considered acceptable for
estimating the detection limit.
5.1.6.4
Based on the Standard Deviation of the Blank
Based on the Standard Deviation of the Blank
Measurement of the magnitude of
analytical background response is performed by analyzing an appropriate number
of blank samples and calculating the standard devition of these responses.
analytical background response is performed by analyzing an appropriate number
of blank samples and calculating the standard devition of these responses.
5.1.6.5
Based on the Calibration Curve
Based on the Calibration Curve
A specific calibration curve should be
studied using samples containing an nalyte in the range of detection limit. The
residual standard deviation of a regression line or the standard deviation of
y- intercepts of regression lines may be used as the standard deviation.
studied using samples containing an nalyte in the range of detection limit. The
residual standard deviation of a regression line or the standard deviation of
y- intercepts of regression lines may be used as the standard deviation.
5.1.7
Robustness
Definition: It to show the
ability of method to remain unaffected by small but deliberate variations in
the method parameters.
ability of method to remain unaffected by small but deliberate variations in
the method parameters.
Procedure: The robustness of method shall be done by following
deliberately change in the method.
deliberately change in the method.
The stability of analyte in solution should be demonstrated
to show the robustness of the method.
to show the robustness of the method.
i) Change in
flow rate ± 0.2 ml/min.
flow rate ± 0.2 ml/min.
ii) Change in
pH buffer/mobile phase by ± 0.2 unit.
pH buffer/mobile phase by ± 0.2 unit.
iii) Change in
organic content of mobile phase by ± 5%.
organic content of mobile phase by ± 5%.
iv) Change in
wavelength by ± 2 nm.
wavelength by ± 2 nm.
5.2
VALIDATION PLAN
5.2.1 Following analytical parameter shall be validated before
implementation of analytical method.
i) Assay
ii) Determination of related
substances.
substances.
iii) Dissolution Rate tests.
5.2.2 If the assay procedure for content uniformity is different from the
composite assay the test method shall be validated.
5.2.3 For pharmacopoeial drugs and drugs products only robustness
parameter shall be validated.
5.2.4 For non-pharmacopoeial drugs and drug product, the method shall be
validated for the following parameters.
5.2.5
Raw Material
Assay
i)
System
Suitability
System
Suitability
ii)
Specificity
Specificity
iii)
Linearity
& Range
Linearity
& Range
iv) Precision – System
precision
precision
Method
precision
precision
Intermediate precision
v) Accuracy
vi) Robustness
Related substances
i) System
Suitability
Suitability
ii)
Specificity
Specificity
iii)
Linearity
& Range
Linearity
& Range
iv)
Limit
of Detection
Limit
of Detection
v)
Limit
of Quantification
Limit
of Quantification
vi) Precision – System
precision
precision
Method
precision
precision
Intermediate precision
vii) Accuracy
5.2.6
Finished Product
Assay of active material(s) and
preservative (s) (if any)
preservative (s) (if any)
iv)
System
Suitability
System
Suitability
v)
Specificity
Specificity
vi)
Linearity
& Range
Linearity
& Range
iv) Precision – System
precision
precision
Method
precision
precision
Intermediate
precision
precision
v) Accuracy
vi) Robustness
5.2.7
Dissolution Rate
i) System suitability ii) Specificity iii) linearity & range
iv) Precision – System precision
Method
precision
precision
Intermediate precision
v) Accuracy
vi) Robustness
5.3
Revalidation
A partial or complete revalidation of the analytical method
shall be done according to following defined change and revalidation criteria.
shall be done according to following defined change and revalidation criteria.
CHANGE PARAMETER
TO BE VALIDATED
TO BE VALIDATED
i) Change in composition of drugs – Specifications
– Linearity
–
Precision
Precision
ii) Revision in specification – Linearity
– Accuracy
iii) Change in Master Formula (Excipients) – Specifications
– Linearity
– Precision
– Precision
iv) Change in analytical Procedure – Complete
validation
validation
5.4
Acceptance Criteria
5.4.1
Acceptance criteria for HPLC analysis
The acceptance
criteria for the analytical method validation will be followed:
criteria for the analytical method validation will be followed:
|
Characteristics
|
No.
of replicates to be Injected |
Acceptance
Criteria |
|
System Suitability
|
||
|
Peak Symmetry
|
Standard X 6 replicates
|
NMT 2.0
|
|
Theoretical Plates
|
NLT 1500
|
|
|
% RSD ( for assay and dissolution rate tests)
|
NMT 2.0
|
|
|
% RSD (for Impurity Profile)
|
NMT 10.0
|
|
|
Linearity
|
5 Concentrations X 3
replicates |
R2 > 0.99, similar response ratio
|
|
Precision – System
|
Standard X 6
replicates |
RSD NMT 2%
|
|
Precision – Method
|
Standard X 6
replicates
Sample X 6
replicates |
RSD NMT 2% (For
Assay)
RSD NMT 10% (For RS)
|
|
Precision –
Intermediate |
Standard X 6
replicates
Sample X 6
replicates |
RSD < 2% (For
Assay)
RSD < 15% (For
Related Substances) |
|
Accuracy
|
Placebo X 1
injection
5 Standard X 3
replicates
5 spiked samples at
different concentration X 3 replicates |
97 to 103 % (for
Assay)
80 to 120 % (for RS)
|
|
Characteristics
|
No.
of replicates to be Injected |
Acceptance
Criteria |
|
Specificity
|
Blank X 3 replicates
Placebo X 3
replicates
Standard X 3
replicates
Placebo spiked with
analyte X 3 replicates |
No interference
|
|
Quantification
|
Standard X 6
replicates |
Signal to noise ratio 10:1
|
|
Detection
|
Standard X 6
replicates |
Signal to noise
ratio 3:1 |
|
Range
|
5 Concentrations X 3
replicates |
Concentration
where data can be reliably determine (97 to 103 % recovery) |
|
Robustness
|
For each parameter:
Standard X 3
replicates
Sample X 3
replicates |
Overall RSD < 2%
(for Assay)
Overall RSD < 15%
(for RS) |
5.4.2
Acceptance criteria
for ICP-MS analysis
The acceptance
criteria for the analytical method validation will be followed:
criteria for the analytical method validation will be followed:
|
Characteristics
|
No.
of replicates to be Injected |
Acceptance
Criteria |
|
System Suitability
|
||
|
% RSD
|
Standard X 6
replicates |
NMT 2.0
|
|
Limit of Detection
(Intensity / Blank Response)
|
Standard X 6
replicates |
Signal
noise ratio 3:1 |
|
Limit of Quantification
(Intensity / Blank Response)
|
Standard X 6
replicates |
Signal noise ratio 10:1
|
|
Specificity
|
Blank X 3 replicates
Standard X 3
replicates
Sample X 3
replicates
Sample + Satndard X
3 replicates |
No Interference
|
|
Precision
|
||
|
System Precision
|
Standard X 6
replicates |
RSD NMT
10.0% |
|
Method Precision
|
Standard calibration
curve
Sample X 6
replicates |
RSD NMT
10.0% |
|
Intermediate
Precision |
Standard calibration
curve
Sample X 6
replicates |
RSD NMT
15.0% |
|
Linearity
|
5 Concentrations
X 3 replicates |
R2 <0.99,
similar response ratio |
|
Range
|
5 Concentrations
X 3 replicates |
Concentration where data can be reliably
determine (80 to 120 % recovery) |
|
Accuracy
|
Standard calibration
curve
5 spiked samples
at different concentration X 3 replicates |
80 to 120%
|
|
Characteristics
|
No.
of replicates to be Injected |
Acceptance
Criteria |
|
Dilution Integrity
(%RSD &
Accuracy) |
Dilution 2 Times x 3 Replicates
Dilution 4 Times x 3 Replicates
|
NMT
15.0 %
%
Accuracy – 85 – 115% |
|
Stability
(%RSD &
Accuracy) |
Stability (for
0, 4, 6… hours)
Standard x 3 replicates
Sample x 3
replicates |
NMT 10.0 %
%
Accuracy – 90 – 110% |