I.
PURPOSE :
To provide a procedure for the validation of analytical methods under use at
II.
SCOPE :
The procedure is to guide the analytical method validation studies of all the
analytical methods being developed and used for the analysis of different products
produced at Hetero Labs Limited. The procedure also provides the steps involved
in the qualification of the compendial methods.
III .
RESPONSIBILITY :
A.
Quality Control shall :
i. Prepare, Review and approve the method validation protocol with an assigned
protocol number.
ii. Carry out analysis & provide supporting data with a conclusive report on
method
validation carried out.
B.
Quality assurance shall :
i. Review and approve the method validation protocol.
ii. Should authorized any documents being used during the validation study
ii. Review and approve the method validation report
IV.
DEFINITIONS :
Method : A set of all the written procedures and instructions involved in the collection,
processing, storage and analysis of chemical matrix for an analyte.
System Suitability : Specific tests to ascertain the suitability and effectiveness of
the operating system when employing chromatographic methods such as pressurized
liquid chromatography and gas chromatography.
Validation: The obtaining and documenting of evidence to demonstrate that a
method can be relied upon to produce the intended result under stated conditions
within defined limits. It is the process of establishing that the performance
characteristics of a method (expressed in terms of analytical parameters) to meet
the requirements for the intended application of the method. The normal
analytical parameters used in method validation are defined as follows .
Protocol: A pre approved document ,which should define the scope, objective,
responsibilities, method details , experimental design and acceptance criteria with
consideration to the intended application of the study is being performed.
Report: An approved document ,which should explain the details found during the study
and also should be concluded the status of the study against to the predetermined acceptance
criteria
Specificity: The ability of a method to measure accurately and
specifically the
analyte (the constituent being tested or analysed) in the presence of components that
may be expected to be present in the sample. It is a measure of the degree of
interference in the analysis of complex sample mixtures such as blank, bulk drug
substances containing
degradation products, related substances etc.
Precision: The degree of agreement among individual test results when a method is applied
to multiple samplings of a homogeneous sample. It is a measure of either the degree of
reproducibility (agreement under different conditions) or of repeatability (agreement under
same conditions) of the method.
Limit of detection (LOD) : The lowest concentration of analyte in a sample that can
be detected, but not necessarily quantitated under the stated experimental conditions.
It is a parameter of limit tests.
Limit of Quantitation (LOQ) :: The lowest concentration of analyte in a sample that
can be determined with acceptable precision and accuracy under the stated
experimental conditions. It is a parameter of quantitative determinations of low levels
of compounds in sample mixtures, such as impurities in bulk drugs .
Linearity: The ability of a method to produce results that are directly or indirectly
proportional to the concentration of the analyte in samples within a given range.
Range : The interval between the upper and lower levels of analyte (including these
levels) that have been shown to be determined with precision, accuracy, and linearity
using the method as written..
Accuracy: The closeness of test results obtained by a method to the true value. It is
a measure of the exactness of the method.
Ruggedness ( Intermediate precision ) : The Intermediate precision of an analytical
method is the degree of reproducibility of test results obtained by the analysis of the
same samples under a variety of normal test conditions ( with the combination of
different analyst, different instrument, different lots of reagents, different day at normal
laboratory conditions etc.) Intermediate precision is normally expressed as the lack of
influence on test results of operational and environmental variables of the analytical
method.
Robustness : The robustness of an analytical procedure is a measure of its capacity to
retain unaffected by small, but deliberate variations in method parameters and provides
an indication of its reliability during normal usage.
V. PROCEDURE :
01. Before an analytical method is validated, the method must be completely developed
and a detailed procedure shall be documented.
02. A method must be validated for intended use, employing an approved protocol.
03. Based on the analyte, the technique, a specific procedure shall be identified to assure
the optimum operation of the system employed (as system suitability).
04. Validation studies shall be unique for each method, and shall be consistent with the
methods purpose, concentration range to be tested, the sample and the technique
used for analyte determination.
05. Validation experimental plans (protocols) shall be supported by appropriate
statistical methodologies to assist in the interpretation of the results obtained.
06. Any modification of an analytical method shall be subject to revalidation.
07. Documentation of the successful completion of laboratory studies is a basic
requirement for determining whether a method is suitable for its intended
applications.
08. The procedure used to evaluate each validation parameter as described in the approved
protocol will be focused into the following steps :
i. Identification of appropriate validation parameters.
ii.
Design of experimental plan for parameters evaluation (validation protocol).
iii.
Execution of the validation.
iv.
Documentation to support of the conclusion that the method has been validated.
v.
The documentation shall contain the following :
[
a. Approved protocol
b. Raw data – Analysis work books/test data sheet, Chromatograms,
Charts, Thermogram, Histograms (if any) etc.,
c. Validation report with conclusion of the study.
09. The experimental design and acceptance criteria of validation parameters can be altered
to other than the conditions specified in this SOP
VI. EXPERIMENTAL DESIGN:
A)
1.1
For Inhouse methods :
Determine the category (see Table I ) of the test/s which are to be validated. Prepare a
Method Validation Protocol in the specimen format given in the Annexure for each item.
Indicate in the protocol the experimental design comprising the tests or methods to be
validated, the relevant analytical performance parameters to be checked, on the basis of
the category given in the Table, the experimental details for carrying out these checks
and the acceptance criteria for each parameter. Guidelines for experimental designs for
some commonly done tests and assays are given below
Category of tests and selection of validation parameters for In-house methods
Category
of test
Specificity
Precision
Linearity Accuracy Intermediate
& range
precision
Robustness
LOQ
LOD
Assay by HPLC
Yes
Yes
Yes
No
Yes
Yes**
No
No
Chromatograph
ic purity by
HPLC
OVI &Residual
solvent test
Chromatograph
ic purity (By
TLC)
Assay
Potentiometry
Yes
Yes
Yes
Yes
Yes
Yes**
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
No
Yes
No
No
Yes
Yes
Yes
Yes
No
Yes
Yes
No
No
Particle size by
laser diffraction
Rinse analysis in
cleaning
validation
No
Yes
No
No
Yes
Yes
No
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Swab analysis in
cleaning
validation
** Carry out stability study for both mobile phase and test solution.
B) For Pharmacopoeial methods :
1.2 Determine the category (see Table I ) of the test/s which are to be validated.
Prepare a Method Validation Protocol in the specimen format given in the
Annexure for each item. Indicate in the protocol the experimental design
comprising the tests or methods to be validated, the relevant analytical
performance parameters to be checked, on the basis of the category given in the
Table, the experimental details for carrying out these checks and the acceptance
criteria for each parameter.
1.3
Guidelines for experimental designs for some commonly done tests and assays
are given below
Category of tests and selection of analytical parameters for Pharmacopoeial methods
Category
of test
Assay by HPLC
Specificity
Precision
Yes
Yes
Linearity Accuracy Intermediate
& range
precision
No
No
No
Robustness
LOQ
LOD
No**
No
No
Chromatographic
purity by HPLC
Yes
Yes
No
Yes
No
No**
Yes
Yes
Chromatographic
purity (By TLC)
Yes
Yes
No
No
No
No
No
Yes
Assay by
Potentiometry
Yes
Yes
Yes
No
Yes
No
No
No
** Carry out stability study for both mobile phase and test solution.
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
System
suitability
Experimental design
.
1. Establish the system suitability through out
the validation followed by 2 procedures.
Acceptance criteria
As applicable (As per method
details)
1 e.g. : For chromatographic
methods
suitable parameters such as
theoretical plates, tailing
factor must be determined.
2.Procedure1: Establish the system suitability
Whenever the chromatographic system
changed or disturbed.
2. As applicable (As per method
details)
3. Procedure2: When there is no disturbance in
chromatographic system , establish the online
system suitability between each parameter by
injecting the standard solution(single time) and
calculate the %RSD for the values obtained
from initial values and each online standard.
3. The cumulative RSD for the
values obtained from initial (system
suitability)values and the individual
online standard solution should be
less than 2.0%
Experimental design for an assay by HPLC method validation
Analytical
parameters
Experimental design
Acceptance criteria
Specificity 1. Diluent, impurity interference
2. Separate injection of individual impurities
for identification and assay in case of
chromatographic method.
2.The impurities of impurities should
not interfere at the retention time of
main compound
3. Compare the Relative retention times of
each component obtained from individual
solutions and blend solution.
3. The relative retention times (with
respect to drug substance) of each
impurity obtained from the
blend
solution should be comparable(within +
0.06) to the relative retention times
obtained from the individual solutions of
each impurity.
1 System suitability as applicable
1. Establish the system suitability based on the
condition
Precision
1. There should be no interference of
the diluent or impurities & the RT of
drug substance.
2. Repeatability of 5 replicate determination of
standard solution at assay level concentration.
3. Method precision of atleast 6 replicate assay
dermination of a sample. Determine the
%RSD of assays.
2 The % RSD for peak area response
of replicate standards should not be
more than 2.0 %
3 % RSD of assay should not be
more than 2.0 %
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Linearity & 1. Determination of minimum 7 levels of 1.
standard in the range of at least 50 to
range
150% (50%,80%,90%,100%,110%
120% and 150%) of the assay level
concentration .Carry out linearity for 2.
first & last level in five replicates & 3.
other levels in triplicate and take mean
response for calculation and also
calculate the % RSD for peak area
4.
response of three replicates of each level
.
1. Linearity co-efficient of mean response
of replicate determination plotted against
respective concentration should not be less
than 0.999.
The %y-intercept as obtained from the
linearity data should be within  2.0 %
determined using the formula :
100 x y intercept
= ------------------------------------Response of assay level
2. Determine linearity co-efficient and % y
intercept.
2. The % RSD for peak area response
of three replicates of each level
should not be more than 2.0 % .
3. Determine the precision of lower (1st
level) and higher level (last level) in
five replicates.
3. The % RSD for peak area responses
of five replicates of first & last level
should not be more than 2.0 %
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
1. Establish the system suitability
Ruggedness
(Intermediate
precision)
2.
Repeatability of 5 replicate determination of
Standard solution at assay level concentration.
Determine the % RSD of peak area response
& retention time.
1.System suitability as
applicable
2 . The % RSD for peak area
response of replicate standards
should not be more than 2.0 %
3. Intermediate precision of Six replicate
determination of assay of a sample (Which is
assayed under precision study) with the
combination of different analyst / day /
instrument / different column of same make.
Determine the %RSD of assays.
3 % RSD of assay should not be
more than 2.0 %
4. Determine the cumulative% RSD of assays of
precision study and Intermediate precision study
together.
4 Cumulative % RSD of assay of
precision study &intermediate
precision should not be more than
2.0 %
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Robustness
1. Deliberately alter any 2 critical
parameters(as per requirement) by
minor variation.
a
Mobile phase composition (higher
concentration by 2 % or lower
concentration by 5-10 %)
b
c
pH of mobile phase ( 0.2 )
Column oven temperature ( 2.0 °C)
d
Mobile phase flow rate
2. Establish the system suitability
3. Method precision of three replicate
determination of assay of a sample
(Which is analysed under precision
study) under each individual condition of
the robustness study . Determine the
%RSD of assays.
4.Determine the cumulative % RSD of
Assay of Precision study & Robustness
study together.
1. Select as per the applicability of the
method
2. System suitability as applicable.
( The % RSD for peak area response
of replicate standards should not be
more than 2.0 %
3 % RSD of assay should not be
more than 2.0 %
4. The cumulative % RSD of assay of
precision study along with the
results of Robustness study should
not be more than 2.0 %
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Solution
stability
1. Establish the system suitability
1 System suitability as applicable.
2. Prepare two samples of a batch and
calculate the assay of each preparation
and note down the result as initial assay
and store one sample is on bench top and
another one is in refrigerator simultaneously
for about 24 hours. After the specified time
interval evaluate the assay of each sample
using the same chromatographic
conditions(same mobile phase, column
&system) with freshly prepared standard
solution. Also monitor the pattern of
chromatogram of sample solution
2. a. The % variation between the initial
results and the results of solution
stability(individually) should be less
than 1.0%
3. When there is no solution stability for 24
hours in any of the above said conditions,
determine the solution stability at
predetermined time interval ( 2 hours, 4
hours, 8, hours,12 hours ) at laboratory
temperature. If sample is sensitive carry out
solution stability study by keeping the
sample solution in refrigerator at 2-8 °C at
same time interval of laboratory
temperature. Note: After finding the time
interval of solution at which is not stable no
need to evaluate solution stability for the
samples of remaining time intervals.
The time intervals can be reduced and
altered for sensitive drug substances .
3. Report the solution stability in hours
(solution stability is the last time
interval of the test solution is found
stable)
b .If any extraneous peak of impurities
or degradants are observed in
chromatogram of sample than that of
initial pattern consider the solution
is not stable.
Experimental design for the method validation of Assay by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Mobilephase
stability
1. Prepare mobile phase and store one
portion in refrigerator and the other
portion is at laboratory temperature for
at least two days
2. Establish the system suitability
3.
1. Establish the mobile phase stability
for 2 to 7 days at each condition
2. System suitability as applicable
Repeatability of 5 replicate determination 3 .The % RSD for peak area response
of replicate standards should not be
of Standard solution at assay level
more than 2.0 %
concentration.
4. Method precision of three replicate
determination of assay of a sample
(Which is assayed under precision study)
using each storage condition of mobile
phase. Determine the %RSD of assays.
5. Determine the cumulative% RSD of
assays of each condition of mobile phase
stability study and precision together.
4. % RSD of assay should not be
more than 2.0 %
5. Cumulative % RSD of assay of
precision study & each condition of
the mobile phase stability study
should not be more than 2.0 %
Experimental design for the method validation of Assay Titremetry
Analytical
parameters
Specificity
Experimental design
Check the blank interference of diluent.
.
Precision
Method precision of at least 6 replicate
assay determination of a sample.
Determine the % RSD of assays.
Acceptance criteria
The Burette reading value should be
negligible ( Around 0.1 ml ).
* Blank reading should be taken for the
calculation of assay .
% RSD of assay should not be
more than 2.0 %
Linearity &
range
Determination of minimum 6 levels of
standard in the range of at least 50 to 150%
of the assay level concentration including
assay level concentration. Carry out linearity
for first & last level in six replicates & other
levels in triplicate and take mean burette
reading for calculation. Determine linearity
co-efficient and % y intercept.
Determine the precision of assay content of
lower (1st level) and higher level (last level)
in six replicates.
Ruggedness Reproducibility of Six replicate
(Intermediate determination of assay of a sample
(Which is assayed under precision study)
precision)
by a different analyst / day at normal
laboratory condition / instrument (if
available)Determine the cumulative %
RSD of assays of precision study and
Intermediate precision study together.
Linearity co-efficient of mean burette
reading of replicate determination plotted
against respective concentration should not
be less than 0.999.The %y-intercept as
obtained from the linearity data (without
extrapolation through origin 0,0) should be
within  2.0 % determined using the formula
:
100 x y intercept
= ------------------------------------B.R. of assay level
The % RSD for % assay of
replicate (1st level) and higher level
(last level) should not be more than 2.0 %.
1 System suitability as per method
2 % RSD of assay should not be
more than 2.0 %
3 Cumulative % RSD of assay of
precision study & intermediate
precision should not be more than
2.0 %
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
System
suitability
Experimental design
.
1. Establish the system suitability through
out the validation followed by 2
procedures.
2.Procedure1:Establish the system
suitability when ever the chromatographic
system changed or disturbed.
Acceptance criteria
1. As applicable (As per method details)
e.g. : For chromatographic methods
suitable parameters such as
resolution, RRT , Peak to valley ratio
must be determined. If not specified keep
following criteria .
2. As applicable (As per method
details)
3. Procedure2: When there is no 3. Should complies the system suitability
disturbance in chromatographic system ,
establish the online system suitability
between each parameter and observe the
compliance of system suitability.
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
Specificity
Experimental design
Acceptance criteria
1.Blank interference
Inject the blank solution and conclude the
interference due to blank at the retention
times corresponding to each known
impurity and the main compound
1.There should not be interference due to
blank at the at the retention times
corresponding to each known impurity and
the main compound
Inject the individual standard solutions of
each impurity and the main compound at
limit level concentration. And Establish the
RRT for each component with respective
to the drug substance retention time
obtained from the blend solution.
2. The elution order and the RRT of each
component obtained from individual
solutions and the blend solution should be
comparable (with in + 0.06)
Prepare the blend solution containing all
components (each impurity and the main
compound) and inject.
1. Determine the signal-to-noise ratio at
Limit of
quantitation 0.01% level for each component and
calculate the S/N ratio. If the S/N ratio
(LOQ)
is less than 10.0 for 0.01% solution,
then establish the LOQ concentration
based on the S/N ratio which should
give the S/N ratio between 10 and 15
Method-1: Using soft ware
Procedure: as appropriate by respective
software
1.System suitability as applicable
2. The 0.01%(with respective to sample
concentration) concentration is acceptable
as LOQ level if the signal-to-noise ratio is
more than 10 . or the solution which should
give the S/N ratio is between 10 and 15
Method-2: Manual
Experimental design for the method validation of chromatographic purity/
Signal to noise ratio can be calculated by
substances
using software or theRelated
following
formula. by HPLC
Analytical
2 HExperimental design
parameters S/N = -----------H
Acceptance criteria
where H = height of peak corresponding to
the component concerned in the
chromatogram obtained with the prescribed
reference solution, measured from the
maximum of ht peak to the extrapolated
baseline of the signal observed over a
distance equal to 20 times the width at half
height.
h = Range of the background noise in the
chromatogram obtained after injection or
applicable of a blank, observed over a 2. The % RSD for peak area response
of replicate LOQ solution should not
distance equal to 20 times the width at half
be more than 5.0 % & 1.0 % for
height of the peak in the chromatogram
retention time.
obtained with the prescribed reference
Limit of
quantitation
(LOQ)
Method-2: Manual
Signal to noise ratio can be calculated
by using software or the following
formula.
2H
S/N = -----------h
where H = height of peak corresponding
to the component concerned in the
chromatogram obtained with the
prescribed reference solution, measured
from the maximum of ht peak to the
extrapolated baseline of the signal
observed over a distance equal to 20
times the width at half height.
h = Range of the background noise in the
chromatogram obtained after injection or
applicable of a blank, observed over a
distance equal to 20 times the width at
half height of the peak in the
chromatogram obtained with the
prescribed reference solution & if
possible, situated equally around the
place where this peak would be found.
1. The 0.01%(with respective to sample
concentration) concentration is acceptable
as LOQ level if the signal-to-noise ratio is
more than 10 . or the solution which should
give the S/N ratio is between 10 and 15
Experimental design for the method validation of chromatographic purity/
2. Determine the Related
% RSD for
peak area by HPLC
substances
Analytical
parameters
Limit of
quantitation
(LOQ)
responses & retention time.
Experimental design
Note: Can be used either one of the
methods mentioned above for the
evaluation
of S/N
ratio.
2.
Determine
the %
RSD for peak area
responses .
Note: Can be used either one of the
methods mentioned above for the
evaluation of S/N ratio.
Acceptance criteria
2. The % RSD for peak area response
of replicate LOQ solution should not
be more than 5.0 % .
Limit of
detection
(LOD)
1. Determine the signal-to-noise ratio of
solutions prepared at appropriate
concentrations based on the signalto-noise ratio obtained during the
determination of LOQ.
1.The concentration is acceptable as LOD
level if the signal-to-noise ratio is between 3
to 5
Calculate the Signal to noise ratio as
choosen for LOQ study
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
Experimental design
parameters
Linearity &
1.Determination of minimum 6 levels of
range
all impurities standards for known
impurities and drug solution for unknown
impurities in the range of atleast 25 to
150% of the limit level concentration
including limit of quantitation as first
level and limit level concentration.
2.Carry out linearity for first & last level
in six replicates & other levels in triplicate
and take mean response for calculation.
Note: Determine the precision at each
level and calculate the % RSD ( which
should not be more than 5.0%) before
considering the mean value of each
3.Determine linearity co-efficient and %
y intercept using Excel macros. (Continue
linearity with LOQ chapter if possible).
4.Determine the precision of lower (1st
level)and higher level (last level) for 6
replicates 6 replicates.
Acceptance criteria
1.Linearity co-efficient of mean response
of replicate determination plotted against
respective concentration should not be less
than 0.998.
2.The %y-intercept as obtained from the
linearity data should not be more than 
5.0 % determined using the formula :
100 x y intercept value
= ------------------------------------Response of limit level
Note: When the % Y intercept is not
meeting the acceptance criteria, establish
the linearity study separately at LOQ
concentration considering the LOQ as
level –1 and evaluate the linearity study
separately and confirm the range.
3. The % RSD for peak area response
of replicate first & last level should
not be more than 5.0 %
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
Precision
Accuracy
Experimental design
Acceptance criteria
A. System precision:
1 System suitability as applicabe
1. Repeatability of 6 replicate determination
of Impurity solution for known impurities
and drug for unknown impurities at the
limit level concentration.
2 The % RSD for peak area response
2.Determine % RSD of the peak area
of replicate standards should not be
responses of individual known impurity and
more than 5.0 %
drug substance .
B.Method precision:
1.Reproducibility of at least 6 replicate
determination of a sample of a batch..
2.Determine the % RSD of content of
known impurities, single maximum
unknown impurity and total impurities.
3 The % RSD of known, single
maximum unknown impurity & Total
impurities should not be more than
15.0 %.
To a sample in which content of
impurities is already known (Analysed
under precision study), individual linearity
levels of known impurities are added and
the recovery is calculated. Carry out this
exercise in minimum three replicates for
atleast four levels each covering the
LOQ,50,100 & 150 % of limit level
concentration.
Recovery should be between 80% and 120
% at LOQ level and for the other levels
should be between 85% and 115%.
Report % recovery in range at each level .
Experimental design for the method validation of chromatographic purity
Related substances by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Ruggedness 1 Carry out system suitability
(Intermediate
2. Repeatability of 6 replicate determination of
precision)
Impurity solution for known impurities and
drug for unknown impurities at the limit level
concentration. Determine % RSD of the peak
area responses & RT of individual
known impurity and the drug
1 System suitability as applicable
2 The % RSD for peak area response
of replicate standards should not be
more than 5.0 %
3 .Intermediate precision of six samples &
inject once (which is analysed under
precision study) by a different analyst / day /
instrument
/ different lot column of same
make.
4.Determine the % RSD of known, single
maximum unknown impurity & Total
impurities.
3. The % RSD of known, single
maximum unknown impurity & Total
impurities should not be more than
15.0 %.
5. Determine the cumulative % RSD of
known, single maximum unknown impurity &
Total impurities obtained from the results of
precision study & ruggedness study.
4. The cumulative % RSD of the results
obtained from precision study and
Ruggedness study should not be more
than 15.0 %
Experimental design for the method validation of chromatographic purity/Related substances by HPLC
Analytical
Experimental design
Acceptance criteria
parameters
1. Deliberately alter any 2 critical parameters(as
Robustness
per requirement) by minor variation.
Select as per the applicability of the
method
a Mobile phase composition (higher
concentration by 2 % or lower concentration
by 5-10 %)
b pH of mobile phase ( 0.2 )
c Column oven temperature ( 5.0 °C)
d Mobile phase flow rate ( 0.2 ml/min)
1. Establish the system suitability
2.Repeatability of 6 replicate determination of 2
Impurity solution for known impurities and
drug for unknown impurities at the limit
level concentration. Compare the RRT’S of
individual known impurities &Determine %
RSD of the peak area responses of individual
known impurity and the drug.
1. System suitability as applicable
2.The RRT’Sof known impurities should
be comparable with the RRT’S obtained
from the specificity study
3.The % RSD for peak area response
of replicate standards should not be
more than 5.0 %
3. Method precision of three replicate determination of a4. The % RSD of known, single
sample (Which is analysed under precision study) under maximum unknown impurity &
Total impurities should not be more
each individual condition of the robustness study .
than 15.0 %.
Determine the %RSD of known impurity, single max
unknown impurity and total impurities.
5. The cumulative % RSD of the results
4. Determine the cumulative % RSD of known,
obtained from precision study and
single maximum unknown impurity & Total
robustness study should not be more
impurities obtained from the results of precision
than 15.0 %
study & robustness study.
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Solution
stability
1.Prepare 2 sets of samples of a batch in
1. System suitability as applicable.
triplicate and store one set of samples is on
bench top and another set is in refrigerator
simultaneously for about 24 hours.
2.After completion of the specified time
interval establish the system suitability and
inject both the conditions of the solution
2.a. The cumulative % RSD of the
results obtained from precision study
and robustness study should not be
more than 15.0 %
stability samples individually and calculate
the % RSD between the results obtained
b.If any extraneous peak of
from precision study and each condition of
impurities or degradants are
solution stability study individually .
observed in chromatogram of
Also monitor the pattern of chromatogram
sample than that of initial pattern
of sample solution.
consider the solution is not stable.
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Solution
Stability
3.When there is no solution stability for 24 hours
3.Report the solution stability in
in any of the above said conditions, determine the
hours(solution stability is the last
solution stability at predetermined time interval
time interval of the test solution is
( 2 hours, 4 hours, 8, hours,12 hours ) at laboratory
found stable)
temperature. If sample is sensitive carry out
solution stability study by keeping the sample
solution in refrigerator at 2-8 °C at same time
interval of laboratory temperature. Note: After
finding the time interval of solution at which is not
stable no need to evaluate solution stability for the
samples of remaining time intervals.
The time intervals can be reduced and altered
based on the intended application..
Experimental design for the method validation of chromatographic purity/
Related substances by HPLC
Analytical
parameters
Experimental design
Acceptance criteria
Mobilephase
stability
1. Prepare mobile phase and store one
portion in refrigerator and the other
portion is at laboratory temperature for
at least 2 to7 days
1. Establish the mobile phase stability
for 2 to 7 days at each condition
2. Establish the system suitability
2. System suitability as applicable
3. Repeatability of 6 replicate determination
of Impurity solution for known impurities and 3 . The % RSD for peak area response
drug for unknown impurities at the limit level of replicate standards should not be
concentration. Determine % RSD of the peak more than 5.0 %
area responses of individual known impurity
and the drug
4 Method precision of three replicate
determination of a sample (Which is analysed
under precision study) under each individual
condition of the robustness study . Determine
the %RSD of known impurity, single max
unknown impurity and total impurities.
4. The % RSD of known, single
maximum unknown impurity & Total
impurities should not be more than
15.0 %.
5. Determine the cumulative % RSD of known,
single maximum unknown impurity & Total
impurities obtained from the results
of
precision study & each mobile phase stability
study
5.The cumulative % RSD of the results
obtained from precision study
precision study and each mobile
phase stability study should be less
than 15.0%
Experimental design for the method validation of chromatographic purity by TLC
Analytical
parameters
Specificity
Experimental design
Acceptance criteria
1. System suitability determination.
1 The impurity should be well separated
from drug substance.
2 .Check interference of the diluent / 2 There should be no interference of the
blank
diluent / blank at the Rf value of
drug substance and impurities
Limit of
Detection
(LOD)
Prepare a different concentrations of
solutions
of
impurity
&
drug
The intensity of the spot corresponding
to lowest concentration , which is
clearly visible is detection level .
substances and check the lowest
detection level .
Precision
Method precision of at least 6 sample The intensities of all impurities should
preparations Check the impurities and be comparable .
compare the intensity of impurity spots
visually .
Ruggedness
(Intermediate
precision)
Prepare the LOD solution and observe.
1.The intensity of the spot corresponding
to LOD solution should be visible clearly
(for the concentration of LOD derived )
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
System
suitability
Experimental design
Acceptance criteria
1. Establish the system suitability through out 1. As applicable (As per method details)
the validation followed by 2 procedures.
2.Procedure1: Establish the system suitability 2. As applicable (As per method
When ever the chromatographic system
details)
changed or disturbed.
3. Procedure2: When there is no disturbance 3. Should comply with the system
suitability requirements
in chromatographic system , establish the
Specificity
online system suitability between each
parameter and observe the compliance of
system suitability.
4. a) Resolution must be determined.
4. System suitability determination.
(Resolution should be more than 1.0
(Repeatability of 6 replicate injections of
between each solvent).
solvent standard at the working level.
Determine the % RSD of peak area response)
b) The % RSD for peak area response
should not be more than 15 %
1.
1. Diluent interference.
2. 1.There should be no interference of the
diluent. If interference is observed, the
response of interference should not be
2. Separate injection of individual solvents for more than 5.0 % to the response of
Individual identification.
standard concentration. Consider the
interference observed for calculation.
22. Compared the retention time of
individual solvents with respective
retention time of solvents in standard
solution.
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Experimental design
Acceptance criteria
1. Determine the signal-to-noise ratio at
lower levels (equal to the first level of
linearity & higher than LOD)
Method-1: Using soft ware
Procedure: as appropriate by
respective
software
Method-2: Manual
Signal to noise ratio can be calculated by using
software or the following formula
Signal to noise ratio can be calculated by using
the following formula.
2H
Limit of
quantitation
(LOQ)
1.System suitability as applicable
2. The concentration is acceptable
as LOD level if the signal-to-noise
ratio is between 10 to 15
S/N = -----------h
where H = height of peak corresponding to the
component concerned in the chromatogram
obtained with the prescribed reference solution,
measured from the maximum of ht peak to the
extrapolated baseline of the signal observed over
a distance equal to 20 times the width at half
height.
h = Range of the background noise in the
chromatogram obtained after injection or
applicable of a blank, observed over a distance
equal to 20 times the width at half height of the
peak in the chromatogram obtained with the
prescribed reference solution & if possible,
situated equally around the place where this peak
would be found.
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Limit of
quantitation
(LOQ)
Experimental design
Acceptance criteria
2. The % RSD for peak area
Determine the % RSD for peak area responses response
2. The % RSD for peak area
& retention time from 6 replicate injections.
response
of replicate
LOQ solution
of replicate
LOQ solution
should
should
not
be
more
than
15.0
%
not
Note: Can be used either one of the methods
be more than 5.0 %
mentioned above for the evaluation of S/N ratio.
Limit of
detection
(LOD)
1. Determine the signal-to-noise ratio of solutions 1.The concentration is acceptable
prepared at appropriate concentrations based on
as LOD level if the signal-to-noise
the signal-to-noise ratio obtained during the
ratio is between 3 to 5
determination of LOQ.
2. Calculate the Signal to noise ratio as
detailed in LOQ study
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Linearity &
range
Experimental design
1 Determination of minimum 6 levels of all
solvents in the range of atleast 50 to 150% of
the limit level concentration including limit of
quantitation as first level and limit level
concentration.
2.Carry out linearity for first & last level in six
replicates & other levels in triplicate and take
mean response for calculation.
Note: Determine the precision at each level and
calculate the % RSD ( which should not be more
than 15.0%) before considering the mean value
of each
3.Determine linearity co-efficient and % y
intercept using Excel macros. (Continue
linearity with LOQ chapter if possible).
4.Determine the precision of lower (1st level)and
higher level (last level) for 6 replicates 6
replicates.
Acceptance criteria
1. Linearity co-efficient of mean
response of replicate determination
plotted against respective
concentration should not be less
than 0.995.
The %y-intercept as obtained
from the linearity data (without
extrapolation through origin 0)
should not be more than  5.0 %
determined using the formula :
100 x y intercept value
= ------------------------------------Response of limit level
Note: When the % Y intercept is
not meeting the acceptance criteria,
establish the linearity study
separately at LOQ concentration
considering the LOQ as level –1
and evaluate the linearity study
separately and confirm the range.
2. The % RSD for peak area
response of replicate first & last
level should not be more than
15.0 % .
Experimental design for the method of Residual solvents by GC
Analytical
parameters
Precision
Experimental design
Acceptance criteria
1. Repeatability of 6 replicate injections of
solvent standard at the standard level.
Determine the % RSD of peak area
response & retention time of individual
solvent.
1 System suitability as applicable
2. Method precision of atleast 6 replicate
determination of a sample. Determine
% RSD of the content of each solvents.
3. The % RSD for content of
solvents should not be more than
15.0%.
2.The RSD should not be more
than 15.0% for peak area response
3. If solvents are not detectable in sample then
4. The % RSD of % recovery of
spike working level concentration in sample. (Six solvents should not be more than
sample preparation & inject once). To be
15.0%.
established the method precision. (Consider this
level as a level 3 of Accuracy).
Accuracy
To a sample in which content of solvents are
already known (analysed in precision study),
individual linearity levels are added and the
recovery is calculated. Carry out this exercise in
minimum three replicates for at least four levels
each covering LOQ, 50,100 & 150 % of
working level concentration.
Recovery should be between 85
and 115 % at each level
Report % recovery in range at each
level .
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Experimental design
Acceptance criteria
Ruggedness
(Intermediate
precision)
1. Carry out system suitability
1 System suitability as applicable
2. Repeatability of 6 replicate determination
of each solvent at the limit level
concentration. , determine % RSD of the
peak area responses of individual solvent
2.The % RSD for peak area response
of replicate standards should not be
more than 15.0 %
3.Diluent interference
4.Intermediate precision of six replicate
determination of trace solvents of a sample
(which is analysed under precision study) by
a different analyst / day / instrument /
different column of same make, calculate
the % RSD of solvents present in sample
5.Calculate the cumulative % RSD of
content of solvents in precision study and
Intermediate precision study together.
3.There should be no interference of
the diluent. If interference is
observed, the response of interference
should not be more than 5.0 % to the
response of standard concentration.
Consider the interference observed for
calculation.
4.% RSD for content of solvents
should not be more than 15.0 %
1. 5.Cumulative % RSD for content of
2. solvents of precision study &
intermediate precision together
should not be more than 15.0 %
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Experimental design
Acceptance criteria
Robustness
1. Deliberately alter any 2 critical
parameters(as per requirement) by minor
variation.
a. Carrier gas flow rate
b.Initial column oven temperature ( 2.0 C)
1. Select as per the applicability of the
method
2.Establish the system suitability
2. System suitability as applicable
3. Repeatability of 6 replicate determination of 3. The % RSD for peak area response
of replicate standards should not be
each solvent at the limit level concentration
more than 15.0
in each condition , determine % RSD of the
peak area responses of individual solvent.
4. There should be no interference of
4. Diluent interference
the diluent. If interference is
observed, the response of interference
should not be more than 5.0 % to the
response of standard concentration.
Consider the interference observed for
calculation.
5. precision of three replicatedetermination
5. % RSD for content of solvents
of trace solvents of a sample (which is
should not be more than 15.0.
analysed under precision study) in each
condition Calculate the % RSD of solvents
present in sample
3. 6.Cumulative % RSD for content of
6. Calculate the cumulative % RSD of
solvents of precision study &
content of solvents in precision study and
robustness study together
each condition of robustness study together.
should not be more than 15.0 .
Experimental design for the method validation of Residual solvents by GC
Analytical
parameters
Experimental design
Acceptance criteria
4. Cumulative % RSD for the
content of solvents obtained from
precision study & each condition
of robustness
study together should not be
more
than 15.0 %.
Robustness
1. Deliberately alter any 2 critical
parameters(as per requirement) by minor
variation.
a Carrier gas flow rate
b Initial column oven temperature ( 2.0 C)
1. Select as per the applicability of the
method
2.Establish the system suitability
2. System suitability as applicable
3 Repeatability of 6 replicate determination of 3. The % RSD for peak area response
of replicate standards should not be
each solvent at the limit level concentration
more than 15.0 %
in each condition , determine % RSD of the
peak area responses of individual solvent.
4. Diluent interference
5. precision of three replicate determination
of trace solvents of a sample (which is
analysed under precision study) in each
condition Calculate the % RSD of solvents
present in sample
4.There should be no interference of
the diluent. If interference is
observed, the response of interference
should not be more than 5.0 % to the
response of standard concentration.
Consider the interference observed for
calculation.
5. % RSD for content of solvents
should not be more than 15.0 %.
5. 6.Cumulative % RSD for content of
solvents of precision study &
6. Calculate the cumulative % RSD of
robustness study together
content of solvents in precision study and
should not be more than 15.0 %.
each condition of robustness study together.
Experimental design for the method validation of rinse sample analysis
Analytical
Experimental design
Acceptance criteria
parameters
System suitability determination.
System suitability as applicable
Specificity
6. Cumulative
% RSD
for the of the
1. There
should be no
interference
content of solvents obtained from
diluent.
precision
study
& each condition
2. For UV
methods
the interference
due to
of robustness
blank/blank
swab should not be more
studyattogether
be
Diluent interference
than 0.005
specifiedshould
wave not
length.
more
than 15.0 %.
1. Determine the signal-to-noise ratio
Limit of
quantitation for 1 ppm or higher levels .Carry out
6 replicate determinations at this
(LOQ)
concentration.
2. Determine the % RSD for peak area
response/ absorbance
Evaluation of S/N ratio
A) For HPLC methods:
Method-1: Using soft ware
Procedure:
as
appropriate
by
respective software
Method-2: Manual
Signal to noise ratio can be calculated
by using software or the following
formula
Signal to noise ratio can be calculated
by using the following formula.
1. The1ppm solution is acceptable as LOQ
level if the signal-to-noise ratio is more than
10 . or the solution which should give the
S/N ratio is between 10 and 15
2. The % RSD for peak area response
/ Absorbance of replicate LOQ
solution should not be more than
5.0 %
2H
S/N = -----------h
Experimental design for the method validation of rinse sample analysis
Analytical
Experimental design
Acceptance criteria
parameters
Limitof
quantitation
(LOQ)
where H = absorbance at specified wave
length due to standard sample.
-----
h = absorbance at specified wave length
due to blank.
consider always the h=0.005 (worst case)
as default.
C.For analytical techniques where
signal-to-noise ratio cannot be measured,
then calculate the quantation limit by
thefollowing formula :
10 
QL = ---S
where  = standard deviation of the
responses as estimated by measuring
magnitude by analysing 3 replicates at
lower levels (lower or equal to the first
linearity level and higher than LOD) and
calculating standard deviation of these
responses.
S = slope of the calibration curve of the
analyte as determined in the linearity
and range study
Analytical
parameters
Limit of
detection
(LOD)
Experimental design for the method validation of rinse sample analysis
Experimental design
Acceptance criteria
1. Determine the signal-to-noise ratio of
solutions prepared at appropriate
concentrations based on the signal-tonoise ratio obtained during the
determination of LOQ.
3. Follow the procedure as choosen for
LOQ study
1. The concentration is acceptable as LOD
if the signal-to-noise ratio is should be
between 3-5).
Linearity &
range
1. Determination of minimum 6 levels
of drug solution in the range of
atleast 50 to 150% of the limit level
concentrationi including limit of
quantitation as first level and limit
level concentration.
Linearity co-efficient of mean response of
replicate determination plotted against
respective concentration should not be less
than 0.995.
2. Carry out linearity for first & last
level in six replicates & other levels
in triplicate and take mean response
/ absorbance for calculation. and
determine linearity co-efficient.
3. Determine the precision of lower
(1stlevel) and higher level (last level)
for 6 replicates.
Analytical
parameters
Precision
Accuracy
The % RSD of first & last level for peak
area response / Absorbance of replicate
standards should not be more than 5.0
Experimental design for the method validation of rinse sample analysis
Experimental design
Acceptance criteria
Reproducibility of % content of trace analyte
obtained in the working level concentration
of Accuracy level in six replicates. (100 %
concentration)
Prepare standard stock solutions.
Transfer known quantity of stock solution on
SS plate having curved edges to hold the
solution. Clean the plate with diluent used for
cleaning in production. Collect the combined
washings in a dish. and reconstitute to the
known volume and carry out determination.
Prepare standard solutions of each level as
above. Carry out recovery for at least three
levels covering 50 to 150 % of limit level for
at least 3 replicate each. and calculate %
recovery . Report the least recovery (out of
all the levels )result as the recovery factor of
the method .
% RSD of the content of trace
analyte should not be more than 10.0
%.
Recovery should be between 75%
and 125%.
Note: If the recovery factor is
obtained between 85 to 115 %,there
is no need to consider the recovery
factor for the trace level
determination.
Ruggedness Repeat the precision study on a different
(Intermediate instrument (if available) /different day / with
different analyst and determine the
precision)
reproducibility of replicate determinations.
Calculate together the %RSD of recoveries
obtained from precision and intermediate
precision study.
% RSD of the content of trace
analyte should not be more than
10.0 %.
Cumulaqtive % RSD of content of
trace analyte obtained from
intermediate precision study and the
precision study should not be more
than 15%.
Experimental design for the method validation of swab sample analysis
Analytical
Experimental design
Acceptance criteria
parameters
System suitability determination.
As applicable
Specificity
There should be no interference of the
Diluent / Swab interference
diluent.
For UV methods the interference due
to blank/blank swab should not be
more than 0.005 at specified wave
length
Reproducibility of % content of trace analyte %
RSD of the content of trace analyte
Precision
obtained in the working level concentration should not be more than 10.0 %.
of Accuracy level in six replicates on each
surface (100 % concentration)
Accuracy
(Recovery
from
surface)
Prepare standard stock solutions. Transfer
known quantity on SS plate, evaporate to
dryness on water bath. Swab the surface of
plate with swab. Transfer the swab in
volumetric flask. Add known amount of
diluent, sonicate for about 2 minutes and
carry out determination. Prepare standard
solutions of each level as above without
evaporation. [In case of heat sensitive
material, do not evaporate, add known
volume of stock solution (about 1 ml) on SS
plate, swab the plate and proceed as above].
Carry out recovery study for atleast 50 to 150
% of each level l in atleast three replicate and
calculate % recovery. Report the least
recovery (out of all the levels )result as the
recovery factor of the method .
Recovery should be between 75 and
125%.
Note: If the recovery factor is obtained
between 85 to 115 %,there is no need
to consider the recovery factor for the
trace level determination.
Experimental design for the method validation of swab sample analysis
Analytical
Experimental design
Acceptance criteria
parameters
Swab
Releasing
capacity
(Recovery
from
swab)
Transfer a swab stick into a test tube. Add
Recovery should be between 85 % to
1 ml of the standard stock solution of
115 %
highest Concentration study on swab
surface. Allow the swab to soak the
solution. Add known amount of diluent,
swirl the flask and carry out determination.
Prepare the standard by diluting quantity of
the same standard stock solution to known
volume as above. Calculate % recovery
using the values
Obtained from swab solution and standard
solution(directly prepared)
Carry out the above exercise using the
standard stock solution of the 10ppm
concentration and determine the %
recovery.
Ruggedness Repeat the precision study on a different
(Intermediate instrument(if available) /different day/
precision)
with different analyst and determine the
reproducibility of replicate(6times)
determinations.
Calculate together the RSD of the results
obtained for precision study and
intermediate precision study.
% RSD of the content of trace analyte
should not be more than 10.0 %.
Cumulative % RSD of content of trace
analyte obtained from intermediate
precision study and the precision study
should not be more than 15%.
Experimental design for the method validation of particle size analysis by lazer technique
Analytical
Experimental design
Acceptance criteria
parameters
Precision
Determine the stability of sample dispersion
1. RSD should not be more than 15
for 6 timesat different time intervals( with 5
or 25 % (decide based on the
For Dry methods minutes interval) and calculate the % RSD of
requirement)
(Stability of
d(10) , d(50) & d(90) values.
dispersion)
2. RSD should not be more than 15
Method precision of atleast 6 sample
Forliquid
or 25 % (decide based on the
preparation ,determine the % RSD of d(10)
methods
requirement)
, d(50) & d(90) values.
Ruggedness
Intermediate precision of atleast 6 sample 1.RSD should not be more than or
(Intermediate
preparation, determine the % RSD of d(10) , 25 % (as decided in precision study)
precision)
d(50) & d(90) values. Also determine the
cumulative %RSD of precision study and 2. Cumulative RSD of precision
study and reproducibility study
reproducibility study together .
together should not be more than
25 %
Robustness
Deliberately alter any 2 critical parameters(as
per requirement) by minor variation.
Change in RPM speed of pump / magnetic
stirrer/pressure / different obscuration/
change in sonication time for sample
Method precision from 3 sample preparation
in each condition, Determine the % RSD of
of d(10) , d(50) & d(90) values. Also
determine the cumulative %RSD of precision
study and each condition of robustness study
together .
1..RSD should not be more than or
25 % (as decided in precision study)
2. Cumulative RSD of precision
study and robustness study together
should not be more than 25 %
Calculations :
Standard deviation :
Definition: . The standard deviation is a measure of how widely values are dispersed from the
average value (the mean).
Formula:
Standard deviation (s) =
∑(Xi –X )2
n-1
Where n = no of observations in the set , X i = i th observation , X = average
% Relative standard deviation
Definition :The variability of data may often be better described as a relative variation rather
than an absolute variation. This can be accomplished by calculating the coefficient of variation
(CV) that is the ratio of the Standard deviation to the mean
Formula
RSD = standard deviation X 100
mean