SOP on Analytical Method Validation

Analytical Method Validation 

1.0         What

This document details the procedure for the
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.

3.0         
WHEN
           

This analytical method validation is applicable at the time
of

i)      
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).

iii)    
Ruggedness
of pharmacopoeias drugs and drug product (if required).

4.0         
Responsibility

Following persons are responsible for preparation, operation
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.

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.

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.

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.

         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.

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.

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.

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.

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).

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.

iii) Reproducibility or system precision : 
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.

Procedure: For Active Pharmaceutical Ingredient (API), the accuracy is
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.

          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.

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.

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.

         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.

          5.1.5.1 Based on Standard Deviation of
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.

           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.

                       LOD    =          3.3.
x S.E. 

                                                            Slope

Where S.E. (Standard Error) and slop are calculated by
number of concentration at LOD level.

             5.1.5.2   
Based on Visual Evaluation

Visual evaluation may be used for non –
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.

              5.1.5.3   
Based on Signal to Noise

This approach can only be applied to
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.

              5.1.5.4   
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.

              5.1.5.5   
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.

          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.

           5.1.6.1 Based on Standard Deviation of
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.

           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.

               LOD            =          10.0 x
S.E. 

                                                             Slope

Where S.E. (Standard Error) and slop are calculated by
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.

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.

              5.1.6.3    
Based on Signal to Noise

This approach can only be applied to
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.

             5.1.6.4    
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.

             5.1.6.5    
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.

          5.1.7   
Robustness

Definition:  It to show the
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.

The stability of analyte in solution should be demonstrated
to show the robustness of the method.

i)          Change in
flow rate ± 0.2 ml/min.

ii)         Change in
pH buffer/mobile phase by  ± 0.2 unit.

iii)         Change in
organic content of mobile phase by ±  5%.

iv)        Change in
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.

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

ii)            
Specificity

iii)           
Linearity
& Range

iv)        Precision                    System
precision

                                                Method
precision

                                              
Intermediate precision

v)         Accuracy

vi)        Robustness

Related substances

i)          System
Suitability

ii)            
Specificity

iii)           
Linearity
& Range

iv)          
Limit
of Detection

v)           
Limit
of Quantification

vi)        Precision                    System
precision

                                                Method
precision

                                               Intermediate precision

vii)         Accuracy

5.2.6     
Finished Product

            Assay of active material(s) and
preservative (s)  (if any)

iv)          
System
Suitability

v)           
Specificity

vi)          
Linearity
& Range

iv)        Precision                    System
precision

                                                Method
precision

                                               Intermediate
precision

v)         Accuracy

vi)        Robustness

5.2.7     
Dissolution Rate

            i)          System suitability            ii)          Specificity            iii)         linearity & range
iv)         Precision                   System precision
                                                Method
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.

                         CHANGE                                            PARAMETER
TO BE VALIDATED

i) Change in composition of drugs                             Specifications

                                                                                    Linearity

                                                                        
          Precision

ii) Revision in specification                                         Linearity         

                                                                                    Accuracy

iii) Change in Master Formula (Excipients)                Specifications

          Linearity
                                                                            Precision

iv) Change in analytical Procedure                            Complete
validation

5.4         
Acceptance Criteria

5.4.1     
Acceptance criteria for  HPLC analysis

The acceptance
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:

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

Rangearea)%een two different analysisalyst / d
main peak of the product.l volumetric flask. Dissolve and dilute upto the
mark with

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%