STANDARD OPERATING PROCEDURE

Department : QUALITY CONTROL

Title : ANALYTICAL METHOD VALIDATION

1.0 PURPOSE
The purpose of this SOP is to lay down a detailed procedure for analytical Method
validation Quality control Department.

2.0 SCOPE
This SOP is applicable for defining and addressing Analytical Method Validation in
Quality Control Department.

3.0 RESPONSIBILITY
All personnel working in Quality Control department and Quality Assurance

4.0 ACCOUNTABILITY
 Quality Control Officer/Executive/QC Designee / QA designee /QC-QA Head

5.0 DEFINITION(S)
 Analytical method validation:
Analytical method validation (AMV) is the systematic evolution of a method by means
of experiment testes in order to confirm and provide objective evidence that the specific
requirement for its intended use are met. It demonstrates that the analytical method
produce reliable results and is appropriate to the intended purpose, in a documented
manner and by objective criteria.
 Analytical method verification:
The verification process for compendia test procedure is the assessment of whether the
procedure can be used for its intended purpose, under the actual condition of use for a
specified drug substance and / or drug product.

6.0 PROCEDURE
6.1 Analytical method validation:
6.1.1 Types of analytical procedures to be validated:
AMV shall be performed for the following analytical procedure:
 Identification test:
 Quantitative test for impurities content (for e.g Test for Related
substance, residual solvents)
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

 Limit tests for the control of impurities.

 Quantitative test of the active substance in sample in sample of drug
substance or drug product or other selected component (s) in the drug
product (for assay test).
Parameter to be considered in AMV:
6.1.2 The following parameters must be covered while performing Analytical
Method Validation.
Sr. Type of Identific Impurity test Quantitative
No analytical ation test for
Limit Quantitative
procedure test active
test test for
substance
impurities
1 Accuracy - + - +

2 precision - + - +

a repeatability - + - +

b Intermediate - + (1) - +(1)

Precision
(raggedness)
3 Specificity + + + +
(2)
4 Limit of - - (3) + -
Detection
5 Limit of - + - -
quantitation
6 Linearity - + - +

7 Robustness - + - +

- signifies that this characteristic is not normally evaluated.

+ signifies that this characteristic is normally evaluated
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

1) In cases where reproducibility (see glossary) has been performs,

intermediate precision is not needed.
2) Lack of specification of one analytical procedure could be compensated
by other supporting analytical procedure (s)
3) May be needed in some cases.
6.1.3 Data element required for validation:
 Considering a wide variety to test method (Assay, Related substance,
residual solvent, etc.) the test methods are classified in different categories
based on validation data requirement as follow:
 Category I:
Analytical method for quantitation of major components of bulk drug
substance or active ingredient (including preservation) in finished
pharmaceutical product (assay).
 Category II:
Analytical method for determination of impurities in bulk substance or
degradation compound in finished pharmaceutical product. These method
include related substance and residual solvents test.
 Category III:
Identification tests.
For each category, different analytical information is needed which is as follows:
Analytical Category-I Category-II Category-III
performance Quantitative Limit Tests
parameter
Accuracy Yes Yes A No
Precision Yes Yes No No
Specificity Yes Yes Yes Yes

Detection No No Yes No
Limit
Quantitation No Yes No No
Limit
Linearity Yes Yes No No
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

Range Yes Yes a No

 A: may be required considering the nature of the test.

 Physical method may also be classified into the four validation categories.
For example, validation of a quantitative spectroscopy method may
involve evolution of Category I or Category II Analytical performance
Characteristics, depending on the method requirement. Category III
analytical performance characteristic usually applies to validation of
qualitative identification spectroscopic method. However the various
techniques may be used for different purpose, and the specific use of the
method and characteristics of the material being analysed should be
considered when definitively applying a category to a particular type of
method.
6.2 Analytical Method verification:
6.2.1 Types of analytical procedure to be verified:
All the method which have been previously at another site of company
procedure which are official in any pharmacopeia need not be validated
but should be verified.
6.2.2 Parameter to considered in analytical method validation:
 Precision
 Intermediate precision
 Specificity
 Limit of Detection
 Limit of quantitation
Other method verification can be supported by other parameter as mentioned
in the table in section 6.1.2
6.2.3 Data elements required for verification:
 Considering a variety of test method (Assay, RS by HPLC) it is only;
logical that different test method require different test method require
different verification schemes. The most common categories of method
for which verification data should be required. These categories are as
follows:
 Category I: Analytical method for quantitation of major components of
active ingredient (including preservatives) in Finished pharmaceutical
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

product (Assay)
 Category II: Analytical method for determine of impurities in bulk substance
or degradation compound in finished product. These methods include related
compound test.
Analytical Category-I Category-II
performance (Assay) Quantitative Limit Tests
characteristic
Accuracy No No No

Precision Yes Yes No

Specificity Yes Yes Yes
Detection Limit No No Yes
Quantitation No Yes No
Limit
Linearity No No No

Robustness No No No

6.3 Analytical Parameters used in Validation/ verification are elaborated

below:
6.3.1 Accuracy:
 Definition:
 A procedure is deemed accurate I, on the average, the method provide the
true answer. It is also defined as closeness of these result obtained by that
method to the true value.
 It may be expressed as percent recovery by the assay of known added
amount of impurities.
 Accuracy is a measure of the exactness of the analytical method.
 Design appropriate experiment and generate data. Study and assess
whether it is in support of accuracy of the method.
 Procedure
 This study is performed on LOQ, 80.0%, 100.0%, and 120.0% impurity
level concentration in related substance/ residual solvent test and 80.0 %,
100.0 %, and 120.0% test level concentration for assay test.
 The amount of added or spiked analyte/impurity depend on the limit of
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

particular impurities in the product, e.g. if the limit of particular impurity

is NMT 0.10% then we have to spike the impurity to 0.1% level with
respect to test concentration for 100.0 % level
 Acceptance Criteria:
 The recovery should be between 80.0% to 120.0% for levels of 80.0%,
100.0% and 120.0% and 70.0% to 130.0% for LOQ. The % RSD of
recovery for three replicate injections should be between 2.0% to 10.0%
for related substance test, between 10.0 to 15.0% for residual tests and
2.0% for assay test.
6.3.2 Precision:
 Definition:
 The precision of an analytical method is the degree of agreement among
individual test result when the procedure is applied repeatedly to multiple
sampling of an analytical method is usually expresses as standard
deviation n or relative standard deviation.
 By assaying a sufficient number of aliquots of a homogenous sample to be
able to calculate standard deviation or relative standard deviation,
precision can be determined.
 The precision types :
 System precision
 Method precision
 Intermediate precision
6.3.3 System precision :
 Definition:
 Replicate injections of standard preparation are compared to ascertain
precision test, over a short interval of time on the same equipment and
analyst.
 Procedure:
 In this study standard solution or system suitability solution test is injected
in six replicates and the % RSD is established.
 Acceptance Criteria:
 Resolution between tow peaks should be maximum or as specified in
method.
 % RSD for RT should be NMT 1,2 or as specified in method
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

6.3.4  % RSD for area should be NMT 2,5,15 or as specified in method.

Method precision:
 Definition:
 The precision of analytical method is the degree of agreement among
individual test results when the method is applied repeatedly to multiple
sampling of a homogenous sample. It is a measure of either
reproducibility or repeatability of the method.
 If known impurities are not detected in the test sample consideration for
validation, method precision shall be justified by spiking of known
concentration of impurities.
 Procedure:
 In this study six different test samples prepared separately shall be
injected and the % RSD of these six result is determined.
 Acceptance Criteria:
 The limit is between 2.0% to 10.0% for related substance test between
10.0 to 15.0% for residual solvent test and 2.0% for assay tests. This
6.3.5 depends on the being carried out.
Intermediate Precision:
 Definition:
 Intermediate precision (Ruggedness) of an analytical method is the degree
of repeatability of test results obtained by the analysis of same sample by /
different analyst/different days/ different instrument / columns etc.
 Procedure:
 In this study six different test sample prepared separately are injected and
the % RSD of tests six result is determined by changing the different
physical parameter like different analyst/ different days/ different
instrument / columns etc. without changing any analytical parameter. Also
the %RSD of results obtained from different sets are determined
 Acceptance Criteria:
 The limit is between 2.0 % to 10.0% for related substance test, between
10.0% to 15.0% for residual solvent and 2.0% for assay tests. This
depends on the test being carried out.
6.3.6
Specificity:
 The term specific refers to a method, which produces a response foe only
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

a single analyst. The terms specificity and selectivity are often used
interchangeably. Selectivity refers to a method which provides response
for a numbers of chemical entities which may or may not be distinguished
from ll other response the method is said to be selective.
 For the tests discussed below, the above definition has the following
implications identification. : ensure the identity of the analyst.
 Purity test: ensure that all of the analytical procedure performed allow an
accurate statement of the content of impurities of an analyte ( e.g. related
substance test, heavy metals limit, organic volatile impurities).
 Assay: provide an exact result, which allows an accurate statement on the
content or potency of the analyte in a sample.
 Procedure:
 In the case of qualitative (identification tests) the ability to select between
compound of closely related structure that are likely to be present should
be demonstrated. This should be confirmed by obtaining positive results
(perhaps by comparison to a known reference material) from sample
containing the analyst coupled with negative results from sample that do
not contain the analyst and by confirming that a positive response is not
obtained from material structural to or closely related to the analyte.
 In case of analytical procedure for impurities, specified may be
established by spiking the drug substance or product with appropriate
levels of demonstrating that these impurities are determined with
appropriate precision.
 In the case of the assay, demonstration pf specificity requires that it can
be shown that the procedure is unaffected by the presence of impurities or
excipients. In practice, this can be done by spiking the drug substance or
product with appropriate level of impurities or excipients and
demonstrating that the assay results is unaffected by the presence of these
extraneous materials . if impurity or degradation product standards are
unavailable, specificity may be demonstrated by comparing the test results
of sample containing impurities or degradation by comparing the test
results of sample containing impurities or degradation products to a
second well- characterized procedure (e,g pharmacopeia or other validated
procedure) these comparisons should include sample store under relevant
stress conditions (e.g light heat, humidity, acid/ base hydrolysis and
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

oxidation)
 In case of the assay, the result should be compered; in the case of
chromatographic impurity tests the impurity profiles should be compared
 In this study for related substance and residual solvent test individual
impurities are injected and there retention times are determined and
proved that the retention times are specified and there is on interference
with each other
 For assay studies limit impurities are spiked in the product and the
specificity of the test is proved by determining the assay which should be
in Limit
 Acceptance criteria:
 RT should be distinct for different impurities
6.3.7  Resolution between two closest peaks should be as high as possible
 Foe Titrimetric assay the assay value should be within limit.

Limits of detection:
 The ability to detect, lowest or smallest concentration of analyte in a
sample is expressed as the limit of detection (LOD).
 Limit tests substantiate that the analyte concentration is above or below a
certain limit.
 For instrumental methods the lowest concentration of impurities which
gives a recognizable peak is limit of detection.
 Based on visual evaluation:
 In case of non- instrument analytical method and also with instrument
method the limit of detection (LOD) is determined by analysis of sample
with known concentrations of analyte and by establishing the minimum
level at which the analyte can be reliably detected. In case , if repeatability
(%RSD) is well within the acceptance limit, however peak shape is not
proper this level should be considered as limit of detection. For
instrumental method, different techniques are used such as keeping signal
to noise ratio of 2:1 or 3:1. Alternatively, the magnitude of analytical
background response is measured by analysing a number of blank sample
and calculating standard devotion of this response.
 Standard deviation, multiplied by defector, usually 2 or 3 provides an
estimate of limit of detection.
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

 Any one of the concept can be used to ascertain OD

 Procedure:
 In this study the impurities are serially injected by lowering their
concentrations and their LOD value are determined based on the % RSD
criteria.
 Acceptance criteria:
 The lowest concentration of impurities which gives a recongranizable
6.3.8
peak is limit of detection
The Quantitation limit of an individual analytical procedure is the lowest
amount of analyte in a sample, which can be quantitative determined with
suitable precision and accuracy. The quantitative limit is a parameter of
quantitation assays for low levels of compounds in sample matrices, and is
used particularly for the determination of impurities and / or degradation
product. It may be establishing the minimum level at which the analyte
and be quantified with acceptable accuracy and precision.
 In this study the impurities are serially injected by lowering their
concentration and their LOQ values are determined.
 Based on visual evolution:
 For non-instrumental analytical method and also with instrument method,
the limit of quantitation is determined by the analysis of sample with
known concentration of analyte and by establishing the minimum level at
which the analyte and be detected with acceptable accuracy and precision.
 Procedure:
 In this study the impurities are serially injected by lowering their
concentration and their LOQ values are determined based on the % RSD
criteria.
 Acceptance criteria:
 The lowest concentration of the % RSD less than the limit gives in the
individual protocol is the limit of Quantitation for instrument procedures
the same method may be used as for non-instrumental. The quantitation
limit is shown concentration of analyte above and below the quantitation
limit. Determination of signal to noise ratio is performed by comparing
measured signals from sample with known concentration of analyte with
those of blank samples and by establishing the minimum consternation at
which the analyte can be reliable quantified. A typical signal to noise ratio
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

is 10:1 based on the standard deviation of response and the slope the
quantitation limit can be expressed as :
10 x a
QL= ------------
S
 Where ‘S’ is the slope of the calibration curve and ‘a’ is the standard
6.3.9
deviation the response.
Linearity:
 Linearity of an analytical method is its ability to put across test results that
are directly or by well-defined mathematical transformation, proportional
to the concentration of analyte in sample with in a given range.
 Linearity is expressed in term of the variance around slop of the
regression line, calculated according to an established mathematical
relationship from test results obtained by the analysis of sample with
varying concentration of analyte.
 The linearity of an analytical method is determined by mathematical
treatment of test results obtained by analysis of sample with analyte
concentration across the claimed range of the method. The slop of the
regression line and its variance provide a mathematical measure of
linearity. Plotting the test result graphically as a function of analyte
concentration is also acceptable.
 Procedure:
 In this study solution ranging from 80.0% to 120.0%. i.e 80.0%, 90.0%,
100.0% , 110.0% & 120.0% for assay and LOQ, 50.0% to 120.0% i.e
LOQ 50.0%,60.0%, 80.0%,100.0% & 120.0% for impurities and residual
solvent are analyte in triplicate and then % RSD of individual levels with
correlation coefficient is determined.
 Acceptance criteria:
6.3.10  The %RSD at each level should not be more than 50 or as specified in the
method for each level and the correlation coefficient should be NLT 0.99.
Robustness:
 It is a measure of the capacity of analytical procedure to remain
unaffected by small but deliberate variations in the method parameter and
provide an indication of its reliability during normal usage.
 Procedure:
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

 In this study we make small but deliberate variations in analytical test

parameters are made. For e.g validation in pH, Mobile phase composition,
flow rate, oven temperate etc re made. Same batch material is analysed by
making variations in analytical parameters.
 Acceptance criteria:
 The % variance of results from the standard condition result should be
NMT 10.0. The parameters vary from method to method.
 Procedure:
 In this study standard solution or system suitability solution test is injected
in six replicates and the %RSD is established. The system suitability
parameters very from method to method.
 Acceptance criteria :
 Resolution between two peaks should be maximum or as specified in
method
 % RSD for RT should be NT 1 or 2 or as specified in method.
 % RSD for area should be NMT 25,15 or as specified in method
 Note: the acceptance criteria may vary from method to method based on
6.3.11
the nature of method or as mention in individual monogram.

System suitability:
 System suitability is an integral part of many analytical procedure. System
suitability tests are based on the concept that the requirement, electronic,
analytical operations are sample to be analysed constitute an integral
system that can be evaluated such.
 System suitability tests are used to verify that their solution and
reproducibility of the chromatographic system are adequate for the
analysis to be done.
 The resolution is the function of column efficiency and it is specified to
ensure that closely eluting compound are resolved from each other. To
establish the general resolving power of the system and to ensure that
internal standards are resolved from the drug. Column efficiency, tailing
factor, theoretical plate, capacity factor and relative standard deviation are
6.4 the test parameter to be established to the particular test procedures
depending on the type of procedure being validated.
6.4.1
Method:
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

Analytical method validation/ verification activity shall be carried out

6.4.2 based on approved protocol (By QA). All the parameters that are to be
performed shall be included in the protocol
Analytical method validation protocol and report shall be numbered
Analytical method validation shall be performed for finished product
testing methods the analytical method validation of AI (but not limited to)
shall be done by analytical development lab. In case of finished product
analytical method validation is carried out by ADL then the same shall be
6.4.3 discussed with cross-functional team and unanimous decision shall be
taken followed by appropriate QMS documentation. After validation from
ADL method shall be transferred. The analytical method validation
involving gas chromatographic instrument shall be performed on case to
6.4.4 case basis at ADL or QC department of company.
During the performance pf validation / verification activity, all the data
6.4.5 should be record in Analytical method validation / verification data sheet.
Recording: record the findings of validation exercise, inclusive of
experiments designed analytical results deviations and other related
supporting documents, with remarks and signatures of participates, data
6.4.6
and proper authorization and compile it as validation master file.
After execution of protocol based method validation/ verification, report
6.4.7
shall be paraded.
The analytical method validation/ verification data sheet and report shall be
reviewed for each parameter and the conclusion shall be derived on the
6.4.8
results.
The analytical method is deemed to be validation/ verified if all the results
fall in the acceptance criteria of each parameter as mentioned in the
6.5
approved protocol.
6.5.1 Re-validation:

Re-validation shall be carried out to each & every validated method after 5
6.5.2
years.
STANDARD OPERATING PROCEDURE
Department : QUALITY CONTROL
Title : ANALYTICAL METHOD VALIDATION

During Re-validation of method validation, precision & accuracy test to be

6.5.3 carried but not limited too with respect to the method.
Re-validation should be report based study & protocol for the same is to be
taken from the original validation method only.

7.0 ATTACHMENTS

Annexure No. Title of the Annexure

QG025/01-01 Index of method validation

8.0 REFERENCE(S)

Document Number Title of Document

SOP on SOP
Employee Training
Handling of Change Control
Document Control and Numbering Procedure

9.0 ABBREVIATION(S)

QA Quality Assurance
SOP Standard Operating Procedure
QC Quality Control
Revision History

New Effective Change Control

Revision History of change
revision date Number
01 New Standard Operating 01 NA
Procedure.