VALIDATION OF ANALYTICAL TEST METHOD

What is method validation?

Validation is the documented act proving that any procedure, process equipment , material,
activity or system actually leads to the expected results.
Method validation is the process of defining an analytical requirement and conforming that the
method under consideration has performance capabilities consistent with what the application
requires

When should methods be validated ?

A method should be validated when it is necessary to verify that its performance parameters are
adequate for a particular analytical problem, For example
 Non standard method
 Laboratory designed/developed method
 Amplification and modification of standard methods to confirm that the methods are
fit for intended use
 Established method use in different laboratory or with different analyst or with
different instrumentation
 To demonstrate the equivalence between two methods e.g. new method and a
standard
Types of Analytical Procedures to be validated
The discussion of the validation of analytical procedure in this document is directed to the
following most common types of analytical procedures:
 Identification tests.
 Quantitative tests for impurities' content
 Limit tests for the control of impurities.
 Quantitative tests of the active moiety in samples of drug substance or drug product.
 Analytical procedures used for determination of performance characteristics of finished
dosage form ( e.g. dissolution)
There are many other analytical procedures such as particles size determination for drug substance
etc. , validation of these additional analytical procedures is equally important.

Requirements of analytical procedure validation

Validation of analytical procedures require-
 Qualified and calibrated instruments.
 Documented methods
 Reliable reference standards
 Qualified analysts
 Sample integrity
Typical validation characteristics used in Method validation
Accuracy
Precision
- Repeatability
 - Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness

Recommended validation characteristics of the various types of test

Type of test characteristics Identification Testing for Impurities Assay Specific test
Dissolution
Quantitative Limit (Measurement
only)
Content/Potency

Accuracy - + - + +4
Precision- Repeatability - + - + +4
Precision -Intermediate - +1 - +1 +4
Precision
Specificity +2 + + +5 +4
Detection Limit - -3 + - -
Quantitation Limit - + - - -
Linearity - + - + -
Range - + - + -
Robustness - + -3 + +4

Note
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated
1 In cases where reproducibility has been performed, intermediate precision is not needed
2 Lack of specificity of one analytical procedure may be compensated for by the addition
of a second analytical procedure(s)
3 May be needed in some cases
4 May not be needed is some cases
5 Lack of specificity for an assay for release may be compensated for by impurities testing

Extent of Validation
 New method require complete validation
 Pharmacopoeial methods require partial validation ( or verification )
 Significant changes means partial revalidation

When Revalidation is required

 Changes in the synthesis of the drug substance
 Changes in the composition of the finished product
 Changes in the analytical procedure
The degree of revalidation required depends on the nature of the changes

DEFINATION AND METHODOLOGY

1. Precision:
The precision of an analytical procedure express the closeness of agreement between a series of
measurements obtained from multiple sampling of the same homogeneous sample under the
prescribed conditions .
Precision should be investigated using homogeneous, authentic samples. If it is not possible to
obtain a homogeneous sample it may be investigated using artificially prepared samples or a
sample solution. The precision of an analytical procedure is usually expressed as the variance,
standard deviation or co-efficient of variation of a series of measurements

There are three types of precision:

1.1 Repeatability : Expresses the precision under the same operating conditions over a short
interval of time (Same method ,same laboratory, same operator and same equipment)

Repeatability should be assessed using -

a] a minimum of 9 determinations covering the specified range for the procedure
e.g 3 concentrations/3 replicates each or
b] a minimum of 6 determinations at 100% of the test concentration.

1.2 Intermediate precision : express within laboratory variations

Typical variations to be studied include different days, different analyst, different
equipments, different source of reagents etc.

1.3 Reproducibility : Reproducibility express the precision between laboratories.

Reproducibility should be considered in case of standardization of an analytical
procedure, for instances for inclusion of procedure in pharmacopoeia

Assessing parameters-
Effectivity of procedure in multiple laboratory with acceptable CV ( Co-efficient of
variation ) requirement
Types of Analytical Procedure CV requirement
Assay Procedure Not more than 2%
Impurities analytical procedure:
Specification limit : 1- 10% Not more than 2%
0.01% Not more than 10%
1 ppm Not more than 20%

2. Accuracy:
The accuracy of an analytical procedure expresses the closeness of agreement between the value
which is accepted either as conventional true value or an accepted reference value and the value
found . This is sometimes termed trueness.
There are several methods for determination of accuracy in assay of drug substance and drug
product are available.
( a) Application of an analytical procedure to an analyte of known purity ( e.g. reference
material ) for drug substance.
(b) Spiked placebo studies in specified ranges of analytical procedures. Active material
or impurity added at required level to placebo matrix ( for drug product only)
(c) If no placebo obtainable , accuracy may be verified by using standard addition
techniques, i.e. to add known quantity of the analyte to the drug product ( for drug
product only)
(d) Spiked drug substance / drug product with known amounts of impurities ( for
impurities only)
(e) Comparison of two methods for equivalence i.e. to compare the result obtained from
a second well characterized procedure, the accuracy of which is stated and or
defined ( for drug substance, drug product and quantitation of impurities)
Accuracy should be assessed using a minimum of 9 determinations over a minimum of 3
concentration levels covering specified range ( e.g. 3 concentrations /3 replicates each of the total
analytical procedure)
Accuracy should be reported as percent recovery by the assay of known added amount of analyte
in the sample or as difference between the mean and the accepted true value together with the
confidence intervals.

3. Specificity:
Specificity is the ability to measure accurately and specifically the analyte of interest in the
presence of other components that may be expected to be present in the sample matrix
Investigation of specificity should be conducted during the validation of identification test, the
determination of impurities, degradation matrix and the assay. In the case of lack of specificity of
an individual analytical procedure may be compensated by other supporting analytical
procedure(s)

Above definition has the following implications

3.1 Identification test : to ensure the identify of an analyte

3.2. Impurity test : to ensure that all of the analytical procedures performed allow an accurate
statement of the content of impurities of an analyte i.e related substances test , heavy metal test ,
residual solvents etc.
3.3 Assay : to provide an exact result which allows an accurate statement on the content of
potency of the analyte in a sample.

Approaches for determination of specificity:

Identification: Suitable identification tests should be able to discriminate compounds of closely
related structures which are likely to be present. The discrimination of a procedure may be
confirmed by obtaining positive results form samples containing the analyte coupled with
negative results from the samples which do not contain the analyte. In addition the identification
test may be applied to materials structurally similar to or closely related to the analyte to conform
that a positive response is not obtained.

Impurities; The discrimination may be established by spiking drug substance with appropriate
label of impurities and demonstrating the separation of these impurities individually from other
components in the sample matrix.

Assay : The discrimination of the analyte in the presence of impurity and or excipients can be
done by spiking pure substance with appropriate labels of impurities and demonstrating that the
assay result is unaffected by presence of those material.

If impurity or degradation product standards are unavailable , specificity may be demonstrated

by comparing the test results of a sample containing impurities or degradation products to a
second well characterized procedure
For chromatographic procedures , representative chromatogram should be used to demonstrate
specificity and individual components should be appropriately labeled. Similar consideration
should be given to other separation technique
For critical separation , specificity can be demonstrated by the resolution of the two components
which elute closet to each other.
In case where a non- specific assay is used, other supporting analytical procedure should be used
to demonstrate overall specificity.

4. Detection limit:

The detection limit of an individual analytical procedure is the lowest amount of an analyte in a
sample which can be detected but not necessarily quantitated as an exact value.

Several approaches for determining the detection limit are possible, depending on whether the
procedure is a non instrumental or instrumental.
4.1 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 samples with known
concentration of analyte and by establishing the minimum level at which the analyte can be
reliably detected.

4.2 Based on signal- to- noise

This approach can only be applied to analytical procedures which exhibit baseline noise.
Determination of signal to noise ratio is performed by comparing measured signal from samples
and establishing the minimum concentration at which the analyte can be reliably detected. A
signal- to- noise ratio between 3 or 2:1 is generally considered acceptable for estimating the
detection limit.

4.3 Based on standard deviation of response and slope

The limit of detection may be expressed as

3.3σ
LOD =----------------------
S
Where, σ = the standard deviation of the response
S = the slope of calibration curve of the analyte.

5. Quantitation limit :
The quantitation limit of an individual analytical procedure is the lowest amount of analyte in a
sample which can be quantitatively determined with suitable precision and accuracy.
The quantitation limit is a parameter of quantitative assays, for low levels of compounds in a
sample matrix and is used particularly for the determination of impurities and / or degradation.
Several approaches for determining the quantitation limit are possible depending on whether the
procedure is a non- instrumental or instrumental.

5.1 Based on visual evaluation

Visual evaluation may be used for non-instrumental methods but may also be used with
instrumental methods. The quantification limit is determined by the analysis of samples with
known concentration of analyte and by establishing the minimum level at which the analyte can
be quantified with acceptable accuracy and precision..

5.2 Based on signal- to- noise

This approach can only be applied to analytical procedures which exhibit baseline noise.
Determination of signal to noise ratio is performed by comparing measured signal from sample
with known low concentration of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably quantified. A typical signal-to- noise
ratio 10:1.

5.3 Based on standard deviation of response and slope

 Quantitation limit(LOQ) may be expressed as
10 σ
LO Q =----------------------
S
Where, σ = the standard deviation of the response
S = the slope of calibration curve of the analyte.
6. Linearity :
The linearity of an analytical procedure is its ability ( with a given range) to obtain test results
which are directly proportional to the concentration (amount) of analyte in the sample.

Linearity may be demonstrated directly on the drug substance by diluting of standard stock
solution and or separate weighing of synthetic mixtures of the drug product component using
proposed procedure.
Linearity should be evaluated by visual inspection of plot of signals as a function of analyte
concentration . If there is a linear relationship, test result should be evaluated by appropriate
statistical methods, for example, by the calculation of a regression line by the method of least
squares
The correlation coefficient, y intercept, slope of the regression line should be determined,
correction coefficient should be >0.98
For the establishment of linearity a minimum of 5 concentration is recommended
7. Range :
The range of an analytical procedure is the interval between the upper and lower concentration
(amounts) of analyte in the sample for which it has been demonstrated that the analytical
procedure has a suitable level of precision, accuracy and linearity.
The following minimum specified ranges should be considered:
(i) Assay of drug substance or drug product: normally from 80 to 120 percent of the test
concentration.
(ii) Content uniformity, covering a minimum of 70 to 130 percent of the test concentration,
unless a wider more appropriate range, based on the nature of the dosage form ( e.g.
metered dose inhalers) is justified.
(iii) Dissolution testing: ± 20 percent over the specified range (e.g. if the specifications for
a controlled released product cover a region from 20% after 1 hour up to 90% , after 24
hours, the validated range would be 0-110% of the label claim.)
(iv) Determination of impurities : from reporting level of impurity to 120% of the
specification.
(v) If assay and purity are performed together as one test and only a 100% standard is used,
linearity should cover the range from the reporting level of impurities to 120% of the
assay specification.
8. 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 normal usage.

Examples of typical variations are:

- stability of analytical solutions,
- extraction time
In the case of liquid chromatography, examples of typical variations are
- influence of variations of pH in a mobile phase,
- influence of variations in mobile phase composition,
- different columns (different lots and/or suppliers),
- temperature,
- flow rate.
In the case of gas-chromatography, examples of typical variations are
- different columns (different lots and/or suppliers),
- temperature,
- flow rate.

9. System suitability testing:

Systems suitability testing is an integral part of many analytical procedures. The test are based on
the concept that the equipment, electronics, analytical operations and samples to be analysed
constitute an integral system that can be evaluated as such. System suitability test parameters to
be established for a particular procedure depend on the type of procedure being validated.

Verifications of compendial procedure:

Complete re-validation of a compendial method is not required, some of the analytical

performance characteristics may be used for the verification process. Only those characteristics
that are considered to be appropriate for the verification of the particular method need to be
evaluated. The degree and extents of the verification process may depend on the type of
procedure and its associated equipment or instrumentation, on the specific procedural steps, and
on which article are being tested.
Some useful equations
N
Σ ( xi – x) 2
100 i= l
RSD(%) = --------- [ ------------------------]½
_ N-1
x
Where xi is an individual measurement in a set of N measurement and x is the arithmetic
mean of the set.
N
Σ (xi -x) ( yi - y )
i= l
Correlation co-efficient, r = ------------------------------------------------
[ Σ (xi – x) Σ (yi - y ) ]½
i= l i= l

Where xi is the an individual measurement in a set of N measurement and x is the arithmetic

mean of the set yi is an individual true value in a set of N true value and y is the
arithmetic mean of the set
 Typical Validation Protocol for Assay Method

1. Objective
To demonstrate in a laboratory study that the performance characteristics of an assay make it fit
for the intended analytical application.
To record the information and data needed to establish the performance specifications for the
assay.

2. Scope
To be performed for new assay , or for current assays when any changes are made to the
procedure , composition of the finished product and synthesis of the drug substances .
All equipment must be calibrated before being used for the validation of an analytical assay.

3. Responsibility
Person trained and responsible for performing the analytical test will perform the validation
study and record the information.
The supervisor will plan the study, write the protocol, supervise the performance, and verify the
completion of the records.
QA will review and approve the protocol before the validation study, and review and approve the
data in validation report.

4. Materials, Equipment, Documents

-SOP and Data record forms for the assay under test.
- Materials and equipment as described in the SOP.
- Reference to documents which provide evidence that the equipment to be used is calibrated.

5. Procedure
5.1 Performance characteristics
Specify the conditions for the performance of the test, the analysis to be made on the data
collected and the acceptance criteria to be met. Different types of validation studies are needed
for different types of analytical test.

Example :
Method of analysis for assay.
Specification limit : 90.0% -110.0% of label claim
Label claim: 500mg per tablet
Related substance : not more than 0.3%
Assay Procedure: HPLC
Sample: Material with known purity added to drug product.
Concentration of sample: 5 levels, range 80%-120%.
Replication : 3 each replicate should be analyzed duplicate, at the minimum.
Analysis done by different analyst on different days in the same laboratory.
 Validation Requirement
Validation parameter Indicator Requirement
Specificity Resolution between adjacent two peak Not less than 1.5
Accuracy Percent recovery 98.0%-102.0%
Precision Relative Standard Deviation (RSD) Not more than 2%
Linearity Linear relationship between analytical Regression line correlation
response and concentration of analyte in coefficient > 0.98
the range of analysis
Range Comply accuracy and precision RSD < 2.0%
requirement in the range of analytical Recovery 98.0%-102.0%
procedure
Robustness Complies with accuracy and precision RSD < 2.0%
requirement in range of internal variability Recovery 98.0%-102.0%
of analytical procedure parameter such as
different in instrument type and
manufacturer, reagent manufacturer, pH
and composition of mobile phase, analyst
and day in the same laboratory.
System suitability Resolution R : >1.5
RSD of six injection of standard solution RSD < 2.0%
Tailing factor T < 1%
Theoretical plate count N < 1000
Column capacity factor Rt : >2
Relative retention time State in procedure

5.2 Documentation
(a) Table of analytical response and analyte concentration ;
(b) Chromatograms of sample solution, standard solution and system suitability solution, if
appropriate ; and
(c) Any calculation, mathematical transformation and statistical analysis use in the validation
program
5.3 Procedure
This include sample preparation and details of assay procedure
Example
5.3.1 Sample preparation
Samples are prepared by adding known quantities of the analyte to the product.
-Use analyte (active ingredient) of know purity(e.g. reference material)
-Determine the active ingredient content of a drug product e.g tablets by carrying out a well
characterized assay procedure on at least 3 different samples of 20 tablets each, taken from the
same batch. Determine the average tablet weight in each sample (w1, w2 and w3)and the content
of active ingredient (a1, a2 and a3 mg per tablet)
-Add a known quantity (e.g. b mg where b=20 d and d is the label claim per tabet) of the analyte
to at least 3 different sample of 20 pulverized tablets each (samples c1, c2 and c3)
-Weigh 3 replications (using c1, c2 and c3) of 5 samples each containing 80%,90%,100%,110%,
and 120% of d.
No. Average weight of c1sample Average weight of c2 sample Average weight of c3 sample
1 80% of d 80% of d 80% of d
2 90% of d 90% of d 90% of d
3 100% of d 100% of d 100% of d
4 110% of d 110% of d 110% of d
5 120% of d 120% of d 120% of d
Distribute the sample to at least three analysts. The sample should be analyzed in duplicate ,
Each analyst analyses a series of sample c1, c2 and c3

Describe the step by step procedure of test method ( Assay solution , standard solution
chromatographic system etc.)
Analytical pattern on validation of analytical procedure:
Sample Analyst Day-1 Day-2 Day-3 Remarks
Analyst-1 Analyst-2 Analyst-3
C.1. 1 C.1.1 C.2.1 C.3.1
C.2. 2 C.1.2 C.2.2 C.3.2
C.3. 3 C.1.3 C.2.3 C.3.3
C.1.4 C.2.4 C.3.4
C.1.5 C.2.5 C.3.5
5.3.2 Determination of performance characteristics
Specificity (Resolution of two peaks)
Accuracy (Recovery)
Precision (RSD)
Linearity (Regression line, y= a+bx ; correlation coefficient,
6. Evaluation
- Attach all data record and charts
- Performs all the predetermined calculations and statistical analyses
- Compare to acceptance criteria
7. Prepare the deviation report
Including the justification and impact on the validity of the assay
8. Prepare the Assay Procedure Validation Report
This should include : date study initiated; date completed; observations made; problems
encountered; completeness of information collected ; summary of the deviation report ;results of
test and statistical analyses; whether results of each run of the assay meet acceptance criteria ;
whether the variation between the assay repeats meet the specified criteria; location of original
data; and other information relevant to the study.
Conclusions will be made on the validity of the assay for individual results and for the replicates
9. Approval
Submit the Assay Procedure Validation documents to QA for evaluation and approval.
10. Calculations and Statistical Analyses
11. Deviation Report
Deviation; Justification for acceptance; Impact on Process
12. Assay Procedure Validation Report
Result; conclusion