Friday, July 20, 2012
INTERNATIONAL CONFERENCE ON HARMONISATION
OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE
ICH Harmonised Tripartite Guideline
Validation
of Analytical Procedures:
Tex
Q2(R1)
Current Step
4 version
Parent Guideline dated 27 October 1994
(Complementary Guideline on Methodology
dated 6 November 1996
incorporated in November 2005)
incorporated in November 2005)
This
Guideline has been developed by the appropriate ICH Expert Working Group and
has been subject to consultation by the regulatory parties, in accordance with
the ICH Process. At Step 4 of the
Process the final draft is recommended for adoption to the regulatory bodies of
the European Union, Japan
and USA
Q2(R1)
Document History
Document History
|
First Codification
|
History
|
Date
|
New Codification
November
2005
|
Parent Guideline: Text on Validation of Analytical
Procedures
|
Q2
|
Approval by
the Steering Committee under Step 2 and release for public
consultation.
|
26 October 1993
|
Q2
|
|
Q2A
|
Approval by
the Steering Committee under Step 4
and recommendation for adoption to the three ICH regulatory bodies.
|
27 October
1994
|
Q2
|
Guideline on Validation of Analytical Procedures: Methodology
developed to complement the Parent Guideline
developed to complement the Parent Guideline
|
Q2B
|
Approval by
the Steering Committee under Step 2
and release for public consultation.
|
29 November 1995
|
in Q2(R1)
|
|
Q2B
|
Approval by
the Steering Committee under Step 4 and recommendation for adoption to
the three ICH regulatory bodies.
|
6 November 1996
|
in Q2(R1)
|
Current Step 4 version
|
Q2A and Q2B
|
The parent
guideline is now renamed Q2(R1) as the guideline Q2B on methology has been
incorporated to the parent guideline.
The new title is “Validation of Analytical Procedures: Text and
Methodology”.
|
November 2005
|
Q2(R1)
|
VALIDATION OF ANALYTICAL
PROCEDURES:
Text and Methodology
Text and Methodology
ICH Harmonised Tripartite Guideline
TABLE OF CONTENTS
PART I:
PART II:
Text on Validation of
Analytical Procedures
ICH Harmonised Tripartite
Guideline
Having reached Step 4 of the ICH Process at
the ICH Steering Committee meeting on 27 October 1994, this guideline is
recommended for adoption
to
the three regulatory parties to ICH
This document
presents a discussion of the characteristics for consideration during the
validation of the analytical procedures included as part of registration
applications submitted within the EC, Japan
and USA Japan
and USA
The objective
of validation of an analytical procedure is to demonstrate that it is suitable
for its intended purpose. A tabular
summation of the characteristics applicable to identification, control of
impurities and assay procedures is included.
Other analytical procedures may be considered in future additions to
this document.
The discussion
of the validation of analytical procedures is directed to the four 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 or
other selected component(s) in the drug product.
Although there
are many other analytical procedures, such as dissolution testing for drug
products or particle size determination for drug substance, these have not been
addressed in the initial text on validation of analytical procedures. Validation of these additional analytical
procedures is equally important to those listed herein and may be addressed in
subsequent documents.
A brief
description of the types of tests considered in this document is provided
below.
- Identification
tests are intended to ensure the identity of an analyte in a sample. This is normally achieved by comparison of a
property of the sample (e.g., spectrum, chromatographic behavior, chemical
reactivity, etc) to that of a reference standard;
- Testing
for impurities can be either a quantitative test or a limit test for the
impurity in a sample. Either test is
intended to accurately reflect the purity characteristics of the sample.
Different validation characteristics are required for a quantitative test than
for a limit test;
- Assay
procedures are intended to measure the analyte present in a given sample. In the context of this document, the assay
represents a quantitative measurement of the major component(s) in the drug
substance. For the drug product, similar
validation characteristics also apply when assaying for the active or other
selected component(s). The same
validation characteristics may also apply to assays associated with other
analytical procedures (e.g., dissolution).
The objective
of the analytical procedure should be clearly understood since this will govern
the validation characteristics which need to be evaluated. Typical validation characteristics which
should be considered are listed below:
Accuracy
Precision
Repeatability
Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Each of these
validation characteristics is defined in the attached Glossary. The table lists
those validation characteristics regarded as the most important for the
validation of different types of analytical procedures. This list should be considered typical for
the analytical procedures cited but occasional exceptions should be dealt with
on a case-by-case basis. It should be
noted that robustness is not listed in the table but should be considered at an
appropriate stage in the development of the analytical procedure.
Furthermore revalidation may be necessary
in the following circumstances:
- 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. Certain other
changes may require validation as well.
|
Type of
analytical procedure
|
Identification
|
Testing for impurities
|
Assay
- dissolution
(measurement only)
- content/potency
|
|
characteristics
|
|
quantitat. limit
|
|
|
Accuracy
|
-
|
+ -
|
+
|
|
Precision
Repeatability
Interm.Precision
|
-
-
|
+ -
+ (1) -
|
+
+ (1)
|
|
Specificity (2)
|
+
|
+ +
|
+
|
|
Detection Limit
|
-
|
- (3) +
|
-
|
|
Quantitation Limit
|
-
|
+ -
|
-
|
|
Linearity
|
-
|
+ -
|
+
|
|
Range
|
-
|
+ -
|
+
|
- signifies that this characteristic is not
normally evaluated
+ signifies
that this characteristic is normally evaluated
(1) in
cases where reproducibility (see glossary) has been performed, intermediate
precision is not needed
(2) lack of
specificity of one analytical procedure could be compensated by other
supporting analytical procedure(s)
(3) may be
needed in some cases
The analytical procedure refers to the way
of performing the analysis. It should describe in detail the steps necessary to
perform each analytical test. This may include but is not limited to: the
sample, the reference standard and the reagents preparations, use of the apparatus,
generation of the calibration curve, use of the formulae for the calculation,
etc.
2. SPECIFICITY
Specificity is the ability to assess
unequivocally the analyte in the presence of components which may be expected
to be present. Typically these might include impurities, degradants, matrix,
etc.
Lack of specificity of an individual
analytical procedure may be compensated by other supporting analytical
procedure(s).
This definition has the following
implications:
Identification: to
ensure the identity of an analyte.
Purity Tests: to
ensure that all the analytical procedures performed allow an accurate statement of the
content of impurities of an analyte, i.e. related substances test, heavy
metals, residual solvents content, etc.
Assay
(content or potency):
to
provide an exact result which allows an accurate statement on the content or potency of the analyte in a
sample.
3. ACCURACY
The accuracy of an analytical procedure
expresses the closeness of agreement between the value which is accepted either
as a conventional true value or an accepted reference value and the value
found.
This is sometimes termed trueness.
4. PRECISION
The precision of an analytical procedure
expresses the closeness of agreement (degree of scatter) between a series of
measurements obtained from multiple sampling of the same homogeneous sample
under the prescribed conditions. Precision may be considered at three
levels: repeatability, intermediate
precision and reproducibility.
Precision
should be investigated using homogeneous, authentic samples. However, 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 coefficient of
variation of a series of measurements.
4.1. Repeatability
Repeatability expresses the precision under
the same operating conditions over a short interval of time. Repeatability is
also termed intra-assay precision .
4.2. Intermediate precision
Intermediate precision expresses
within-laboratories variations: different days, different analysts, different
equipment, etc.
4.3. Reproducibility
Reproducibility expresses the precision
between laboratories (collaborative studies, usually applied to standardization
of methodology).
5. DETECTION LIMIT
The
detection limit of an individual analytical procedure is the lowest
amount of analyte in a sample which can be detected but not necessarily
quantitated as an exact value.
6. 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 sample matrices, and is used particularly for the determination of
impurities and/or degradation products.
7. LINEARITY
The linearity of an analytical procedure is
its ability (within a given range) to
obtain test results which are directly
proportional to the concentration (amount) of analyte in the sample.
8. RANGE
The range of an analytical procedure is the
interval between the upper and lower concentration (amounts) of analyte in the
sample (including these concentrations) for which it has been demonstrated that
the analytical procedure has a suitable level of precision, accuracy and
linearity.
9. 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.
VALIDATION OF ANALYTICAL
PROCEDURES: METHODOLOGY
ICH Harmonised Tripartite Guideline
Having
reached Step 4 of the ICH Process at the ICH Steering Committee meeting on 6
November 1996, and incorporated into the core guideline in November 2005, this
guideline is recommended for adoption to the three regulatory parties to ICH
INTRODUCTION
This document is complementary to the
parent document which presents a discussion of the characteristics that should
be considered during the validation of analytical procedures. Its purpose is to
provide some guidance and recommendations on how to consider the various
validation characteristics for each analytical procedure. In some cases (for example, demonstration of specificity), the
overall capabilities of a number of analytical procedures in combination may be
investigated in order to ensure the quality of the drug substance or drug
product. In addition, the document provides an indication of the data which
should be presented in a registration application
.
All relevant data collected during
validation and formulae used for
calculating validation characteristics should be submitted and discussed as
appropriate.
Approaches other than those set forth in
this guideline may be applicable and acceptable. It is the responsibility of
the applicant to choose the
validation procedure and protocol most suitable for their product. However it
is important to remember that the main objective of validation of an analytical
procedure is to demonstrate that the procedure is suitable for its intended
purpose. Due to their complex nature, analytical procedures for biological and
biotechnological products in some cases may be approached differently than in
this document.
Well-characterized reference materials,
with documented purity, should be used throughout the validation study. The
degree of purity necessary depends on the intended use.
In accordance with the parent document, and
for the sake of clarity, this document considers the various validation
characteristics in distinct sections. The arrangement of these sections
reflects the process by which an analytical procedure may be developed and
evaluated.
In practice, it is usually possible to
design the experimental work such that the appropriate validation
characteristics can be considered simultaneously to provide a sound, overall
knowledge of the capabilities of the analytical procedure, for instance:
specificity, linearity, range, accuracy and precision.
An investigation of specificity should be
conducted during the validation of identification tests, the determination of
impurities and the assay. The procedures used to demonstrate specificity will depend
on the intended objective of the analytical procedure.
It is not always possible to demonstrate
that an analytical procedure is specific for a particular analyte (complete
discrimination). In this case a combination of two or more analytical procedures
is recommended to achieve the necessary level of discrimination.
Suitable identification tests should be
able to discriminate between compounds of closely related structures which are
likely to be present. The discrimination of a procedure may be confirmed by
obtaining positive results (perhaps by comparison with a known reference
material) from samples containing the analyte, coupled with negative results
from 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 confirm that a positive response is not obtained. The choice of
such potentially interfering materials should be based on sound scientific
judgement with a consideration of the interferences that could occur.
For chromatographic procedures,
representative chromatograms should be used to demonstrate specificity and
individual components should be appropriately labelled. Similar considerations
should be given to other separation techniques.
Critical separations in chromatography
should be investigated at an appropriate level. For critical separations,
specificity can be demonstrated by the resolution of the two components which
elute closest to each other.
In cases where a non-specific assay is
used, other supporting analytical procedures should be used to demonstrate
overall specificity. For example, where a titration is adopted to assay the
drug substance for release, the combination of the assay and a suitable test
for impurities can be used.
The approach is similar for both assay and
impurity tests:
1.2.1 Impurities are available
For the assay , this should involve
demonstration of the discrimination of the analyte in the presence of
impurities and/or excipients; practically, this can be done by spiking pure
substances (drug substance or drug product) with appropriate levels of
impurities and/or excipients and demonstrating that the assay result is
unaffected by the presence of these materials (by comparison with the assay
result obtained on unspiked samples).
For the impurity test, the discrimination
may be established by spiking drug substance or drug product with appropriate
levels of impurities and demonstrating the separation of these impurities
individually and/or from other components in the sample matrix.
1.2.2 Impurities are not available
If impurity or degradation product
standards are unavailable, specificity may be demonstrated by comparing the
test results of samples containing impurities or degradation products to a
second well-characterized procedure e.g.: pharmacopoeial method or other
validated analytical procedure (independent procedure). As appropriate, this should include samples stored under relevant
stress conditions: light, heat, humidity, acid/base hydrolysis and oxidation.
- for
the assay, the two results should be compared;
- for
the impurity tests, the impurity profiles should be compared.
Peak purity tests may be useful to show
that the analyte chromatographic peak is not attributable to more than one
component (e.g., diode array, mass spectrometry).
A linear relationship should be evaluated
across the range (see section 3) of the analytical procedure. It may be
demonstrated directly on the drug substance (by dilution of a standard stock
solution) and/or separate weighings of synthetic mixtures of the drug product
components, using the proposed procedure. The latter aspect can be studied
during investigation of the range.
Linearity should be evaluated by visual
inspection of a plot of signals as a function of analyte concentration or
content. If there is a linear relationship, test results should be evaluated by
appropriate statistical methods, for example, by calculation of a regression
line by the method of least squares. In some cases, to obtain linearity between
assays and sample concentrations, the test data may need to be subjected to a
mathematical transformation prior to the regression analysis. Data from the
regression line itself may be helpful to provide mathematical estimates of the
degree of linearity.
The correlation coefficient, y-intercept,
slope of the regression line and residual sum of squares should be submitted. A
plot of the data should be included. In addition, an analysis of the deviation
of the actual data points from the regression line may also be helpful for
evaluating linearity.
Some analytical procedures, such as
immunoassays, do not demonstrate linearity after any transformation. In this
case, the analytical response should be described by an appropriate function of
the concentration (amount) of an analyte in a sample.
For the establishment of linearity, a
minimum of 5 concentrations is recommended. Other approaches should be
justified.
The specified range is normally derived
from linearity studies and depends on the intended application of the
procedure. It is established by confirming that the analytical procedure
provides an acceptable degree of linearity, accuracy and precision when applied
to samples containing amounts of analyte within or at the extremes of the
specified range of the analytical procedure.
The following minimum specified ranges
should be considered:
- for
the assay of a drug substance or a finished (drug) product: normally from 80 to
120 percent of the test concentration;
- for
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;
- for
dissolution testing: +/-20 % 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.
- for
the determination of an impurity: from the reporting level of an impurity1 to 120% of the specification;
-
for impurities known to be
unusually potent or to produce toxic or unexpected pharmacological effects, the
detection/quantitation limit should be commensurate with the level at which the
impurities must be controlled;
Note: for validation of
impurity test procedures carried out during development, it may be necessary to
consider the range around a suggested (probable) limit.
- 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 the
impurities[1] to
120% of the assay specification.
Accuracy should be established across the
specified range of the analytical procedure.
4.1.1 Drug Substance
Several methods of determining accuracy are
available:
a) application
of an analytical procedure to an analyte of known purity (e.g. reference
material);
b) comparison
of the results of the proposed analytical procedure with those of a second
well-characterized procedure, the accuracy of which is stated and/or defined
(independent procedure, see 1.2.);
c) accuracy
may be inferred once precision, linearity and specificity have been
established.
4.1.2 Drug Product
Several methods for determining accuracy
are available:
a) application
of the analytical procedure to synthetic mixtures of the drug product
components to which known quantities of the drug substance to be analysed have
been added;
b) in
cases where it is impossible to obtain samples of all drug product components ,
it may be acceptable either to add known quantities of the analyte to the drug
product or to compare the results obtained from a second, well characterized
procedure, the accuracy of which is stated and/or defined (independent
procedure, see 1.2.);
c) accuracy
may be inferred once precision, linearity and specificity have been
established.
Accuracy should be assessed on samples
(drug substance/drug product) spiked with known amounts of impurities.
In cases where it is impossible to obtain
samples of certain impurities and/or degradation products, it is considered
acceptable to compare results obtained by an independent procedure (see 1.2.).
The response factor of the drug substance can be used.
It should be clear how the individual or
total impurities are to be determined e.g., weight/weight or area percent, in
all cases with respect to the major analyte.
Accuracy should be assessed using a minimum
of 9 determinations over a minimum of 3 concentration levels covering the
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 the
difference between the mean and the accepted true value together with the
confidence intervals.
Validation of tests for assay and for
quantitative determination of impurities includes an investigation of precision.
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.
The extent to which intermediate precision
should be established depends on the circumstances under which the procedure is
intended to be used. The applicant should
establish the effects of random events on the precision of the analytical
procedure. Typical variations to be studied include days, analysts, equipment,
etc. It is not considered necessary to study these effects individually. The
use of an experimental design (matrix) is encouraged.
Reproducibility is assessed by means of an
inter-laboratory trial. Reproducibility should be considered in case of the
standardization of an analytical procedure, for instance, for inclusion of
procedures in pharmacopoeias. These data are not part of the marketing authorization
dossier.
The standard deviation, relative standard
deviation (coefficient of variation) and confidence interval should be reported
for each type of precision investigated.
Several approaches for determining the
detection limit are possible, depending on whether the procedure is a
non-instrumental or instrumental. Approaches other than those listed below may
be acceptable.
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 concentrations of analyte and by establishing
the minimum level at which the analyte can be reliably detected.
This approach can only be applied to
analytical procedures which exhibit baseline noise.
Determination of the signal-to-noise ratio
is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. A
signal-to-noise ratio between 3 or 2:1 is generally considered acceptable for
estimating the detection limit.
The detection limit (DL) may be expressed
as:
|
DL =
|
3.3 σ
|
|
|
S
|
where σ = the standard deviation of the response
S = the slope of the calibration
curve
The slope S may be estimated from the
calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways, for example:
6.3.1 Based on the Standard Deviation of the
Blank
Measurement of the magnitude of analytical
background response is performed by analyzing an appropriate number of blank
samples and calculating the standard deviation of these responses.
6.3.2 Based on the Calibration Curve
A specific calibration curve should be
studied using samples containing an analyte in the range of DL. The residual
standard deviation of a regression line or the standard deviation of
y-intercepts of regression lines may be used as the standard deviation.
The detection limit and the method used for
determining the detection limit should be presented. If DL is determined based
on visual evaluation or based on signal to noise ratio, the presentation of the
relevant chromatograms is considered acceptable for justification.
In cases where an estimated value for the
detection limit is obtained by calculation or extrapolation, this estimate may
subsequently be validated by the independent analysis of a suitable number of
samples known to be near or prepared at the detection limit.
Several approaches for determining the
quantitation limit are possible, depending on whether the procedure is a
non-instrumental or instrumental. Approaches other than those listed below may
be acceptable.
Visual evaluation may be used for
non-instrumental methods but may also be used with instrumental methods.
The quantitation limit is generally
determined by the analysis of samples with known concentrations of analyte and
by establishing the minimum level at which the analyte can be quantified with
acceptable accuracy and precision.
This approach can only be applied to
analytical procedures that exhibit baseline noise.
Determination of the signal-to-noise ratio
is performed by comparing measured signals from samples with known low
concentrations of analyte with those of blank samples and by establishing the
minimum concentration at which the analyte can be reliably quantified. A
typical signal-to-noise ratio is 10:1.
The quantitation limit (QL) may be
expressed as:
|
QL =
|
10 σ
|
|
|
S
|
where σ = the standard deviation of the response
S = the slope of the calibration
curve
The slope S may be estimated from the
calibration curve of the analyte. The estimate of σ may be carried out in a variety of ways for example:
7.3.1 Based on Standard Deviation of the Blank
Measurement of the magnitude of analytical
background response is performed by analyzing an appropriate number of blank
samples and calculating the standard deviation of these responses.
7.3.2 Based on the Calibration Curve
A specific calibration curve should be
studied using samples, containing an analyte in the range of QL. The residual
standard deviation of a regression line or the standard deviation of
y-intercepts of regression lines may be used as the standard deviation.
The quantitation limit and the method used
for determining the quantitation limit should be presented.
The limit should be subsequently validated
by the analysis of a suitable number of samples known to be near or prepared at
the quantitation limit.
The evaluation of robustness should be
considered during the development phase and depends on the type of procedure
under study. It should show the reliability of an analysis with respect to
deliberate variations in method parameters.
If measurements are susceptible to
variations in analytical conditions, the analytical conditions should be
suitably controlled or a precautionary statement should be included in the
procedure. One consequence of the evaluation of robustness should be that a
series of system suitability parameters (e.g., resolution test) is established
to ensure that the validity of the analytical procedure is maintained whenever
used.
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.
System suitability testing is an integral
part of many analytical procedures. The tests are based on the concept that the
equipment, electronics, analytical operations and samples to be analyzed
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. See Pharmacopoeias for additional information.
[1] see chapters “Reporting Impurity Content of Batches” of the
corresponding ICH-Guidelines: “Impurities in New Drug Substances” and
“Impurities in New Drug Products”
