This document provides a summary of analytical method validation. It discusses the types of analytical procedures that should be validated, including identification tests, quantitative impurity tests, limit tests, and active moiety assays. It also summarizes key validation characteristics that should be considered, such as accuracy, precision, specificity, range, detection limit, quantitation limit, linearity, and robustness. The document provides definitions and methodology recommendations for validating analytical procedures. It emphasizes that the validation process verifies that an analytical method is suitable for its intended purpose.

1. • PRESENTD BY :-
• PATIL PRANJAY SADASHIV.
• FIRST YEAR M.PHARM.
• DEPARTMENT OF QUALITY ASSURANCE.
H. R. Patel Institute of Pharmaceutical Education
and Research, Shirpur
ANALYTICAL METHOD
VALIDATION

2. Introduction :
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.
This document does not necessarily seek to cover the
testing that may be required for registration in, or export
to, other areas of the world.
These terms and definitions are meant to bridge the
differences that often exist between various compendia
and regulators of the EC, Japan and USA.

3. Types of Analytical Procedures to be Validated
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..

4. A brief description of the types of tests considered
in this document is provided below.
- 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.

5. Typical validation characteristics which should be considered are
listed below:
Accuracy
Precision
• Repeatability
• Intermediate Precision
Specificity
Detection Limit
Quantitation Limit
Linearity
Range
Robustness
System suitability Testing

6. ANALYTICAL PROCEDURE :
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.
SPECIFICITY :
Specificity is the ability to assess unequivocally the
analytic in the presence of components which may be expected
to be present. Typically these might include impurities,
degrades, matrix, etc.

7. 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.

8. PRECISION :
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.

9. Methodology of Analytical Procedures
Identification
Suitable identification tests should be able to discriminate
between compounds of closely related structures which
are likely to be present.
Assay and Impurity Test(s)
For chromatographic procedures, representative
chromatograms should be used to demonstrate specificity
and individual components should be appropriately
labeled. Similar considerations should be given to other
separation techniques.

10. LINEARITY
A linear relationship should be evaluated across the range of the
analytical procedure. It may be demonstrated directly on the drug
substance (by dilution of a standard stock solution) and/or separate
weighing of synthetic mixtures of the drug product components,
using the proposed procedure.
Range:
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.
- 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.

11. ACCURACY
Accuracy should be established across the specified range
of the analytical procedure.
Assay :
Drug Substance :application of an analytical procedure to
an analyte of known purity (e.g. reference material).
Drug Product
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.

12. Impurities (Quantitation)
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.
Recommended Data
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).

13. PRECISION
Validation of tests for assay and for quantitative determination
of impurities includes an investigation of precision.
1. Repeatability
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.
2. Intermediate Precision
The extent to which intermediate precision should be
established depends on the circumstances under which the
procedure is intended to be used. Typical variations to be
studied include days, analysts, equipment, etc.

14. DETECTION LIMIT
Based on Visual Evaluation
Visual evaluation may be used for non-instrumental methods
but may also be used with instrumental methods .
Based on Signal-to-Noise
This approach can only be applied to analytical procedures
which exhibit baseline noise.

15. ROBUSTNESS :
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. Examples of typical
variations are:
- 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.

16. Analytical Method Validation Definition :
Validation of analytical method may be defined as
“The process by, which it is established , by laboratory
studies, that the performance characteristics of the method
meet the requirement for the intended analytical
application”.

17. Accuracy :
The closeness of test results obtained by that method to the
true value .
Precision :
The degree of agreement among individual test results when
the method is applied repeatedly to multiple sampling
of a homogeneous sample.
Specificity :
The ability to assess unequivocally the analyte in the
presence of components that may be expected to be present ,
such as impurities degradation product and matrix
components.

18. Limit of Detection :
The lowest amount of analyte in a sample that can be
detected ,but not necessarily quantitated ,under the stated
experimental conditions.
Limit Of Quantitation :
A characteristics of quantitative assays for low levels of
compounds in sample in sample matrices such as impurities
in bulk substances and degradation products in finished
pharmaceuticals. It is the lowest amount of analyte in a
sample that can be determined with acceptable precision
and accuracy under the stated experimental conditions.

19. Linearity :
Its ability to elicit tests that are directly or by a well defined
mathematical transformations proportional to the
concentration of analyte in a samples within a given range.
Range :
Interval between the upper and lower levels of analyte that
have been demonstrated to be determined with a suitable level
of precision ,accuracy and linearity using the method as
written.

20. Ruggedness :
The degree of reproducibility of test results obtained by the
analysis of the same sample under a variety of condition
such as different laboratories ,different analysts, different
instruments, different lot of reagents , different elapsed
assay times, different assay temperature, different days etc.
Robustness:
A measure of its capacity to remain unaffected by small
but deliberate variations in method parameters and
provides an indications of its reliability during normal
usage.

21. Analytical Method Validation :
Analytical Method Validation As Per USP

22. INTRODUCTION :
This guidance supersedes the draft of the same
name that published on February 19, 2014 and replaces
the 2000 draft guidance for industry on Analytical
Procedures and Methods Validation and the 1987
Guidelines for Submitting Samples and Analytical Data
for Methods Validation.
It provides recommendations on how you, the applicant,
can submit analytical procedures and methods validation
data to support the documentation of the identity, strength,
quality, purity, and potency of drug substances and drug
products. It will help you assemble information and
present data to support your analytical methodologies.

23. ANALYTICAL METHODS DEVELOPMENT :
An analytical procedure is developed to test a
defined characteristic of the drug substance or drug
product against established acceptance criteria for that
characteristic.
Early in the development of a new analytical procedure,
the choice of analytical instrumentation and methodology
should be selected based on the intended purpose and
scope of the analytical method.
Parameters that may be evaluated during method
development are specificity, linearity, limits of detection
(LOD) and limits of quantitation (LOQ), range, accuracy,
and precision.

24. CONTENT OF ANALYTICAL PROCEDURES :
You should describe analytical procedures in sufficient
detail to allow a competent analyst to reproduce the necessary
conditions and obtain results within the proposed acceptance
criteria.
You should also describe aspects of the analytical procedures that
require special attention. An analytical procedure may be
referenced from FDA-recognized sources (e.g., USP/NF,
Association of Analytical Communities (AOAC) International)12 if
the referenced analytical procedure is not modified beyond what is
allowed in the published method.
You should provide in detail procedures from other published
sources. The following is a list of essential information you should
include for an analytical procedure:

25. Principle/Scope :
A description of the basic principles of the
analytical test/technology (i.e., separation, detection);
target analytic and sample type (e.g., drug substance, drug
product, impurities or compounds in biological fluids).
Apparatus/Equipment :
All required qualified equipment and components
(e.g., instrument type, detector, column type, dimensions,
and alternative column, filter type).

26. Operating Parameters :
Qualified optimal settings and ranges (include
allowed adjustments supported by compendial sources or
development and/or validation studies) critical to the
analysis (e.g., flow rate, components temperatures, run
time, detector settings, gradient, head space sampler).
Reagents/Standards :
The following should be listed where applicable:
Description of reagent or standard
Grade of chemical (e.g., USP/NF, American Chemical
Society, High Performance or Pressure Liquid
Chromatography, or Gas Chromatography and
preservative-free)

27. Sample Preparation :
Procedures (e.g., extraction method, dilution or
concentration, desalting procedures and mixing by
sonication, shaking or sonication time) for the
preparations for individual sample tests.
A single preparation for qualitative and replicate
preparations for quantitative tests with appropriate units
of concentrations for working solutions (e.g., μg/ml or
mg/ml) and information on stability of solutions and
storage conditions.
Standards Control Solution Preparation.

28. System Suitability :
Confirmatory test(s) procedures and parameters to
ensure that the system (equipment, electronics, and
analytical operations and controls to be analyzed) will
function correctly as an integrated system at the time of
use.

29. Calculations :
The integration method and representative calculation formulas for
data analysis (standards, controls, samples) for tests based on label
claim and specification (e.g., assay, specified and unspecified
impurities and relative response factors).
Data Reporting:
A presentation of numeric data that is consistent with instrumental
capabilities and acceptance criteria. The method should indicate
what format to use to report results (e.g., percentage label claim,
weight/weight, and weight/volume) with the specific number of
significant figures needed.

30. REFERENCE STANDARDS AND MATERIALS :
Primary and secondary reference standards and materials
are defined and discussed in the following ICH guidance:
Q6B Specifications: Test Procedures and Acceptance
Criteria for Biotechnological/Biological Products, and Q7
Good Manufacturing Practice Guidance for Active
Pharmaceutical Ingredients.

31. ANALYTICAL METHOD VALIDATION :
A. Non compendial Analytical Procedures:
Analytical method validation is the process of
demonstrating that an analytical procedure is suitable for
its intended purpose. The methodology and objective of
the analytical procedures should be clearly defined and
understood before initiating validation studies. This
understanding is obtained from scientifically-based
method development and optimization studies.

32. • B. Validation Characteristics :
Although not all of the validation characteristics are applicable for
all types of tests, typical validation characteristics are:
• Specificity
• Linearity
• Accuracy
• Precision (repeatability, intermediate precision, and
reproducibility)
• Range
• Quantitation limit
• Detection limit

33. STATISTICALANALYSIS AND MODELS :
A. Statistics :
Statistical analysis of validation data can be used to
evaluate validation characteristics against predetermined
acceptance criteria. All statistical procedures and
parameters used in the analysis of the data should be
based on sound principles and appropriate for the intended
evaluation.

34. B. Models :
Some analytical methods might use chemo metric and/or
multivariate models. When developing these models, the
number of samples to provide adequate statistical power
and range for model development and validation should
be considered. Suitable software should be used for data
analysis. Model parameters should be deliberately varied
to test model robustness.

35. A. Revalidation :
Principles described in the validation section
(section VI) apply to revalidation. When a change is
made to an analytical procedure (e.g., a change in a piece
of equipment or reagent or because of a change in
manufacturing process or formulation), revalidation of all
or part of the analytical procedure should be considered.

36. B. Analytical Method Comparability Studies
Analytical method comparability study requests are
typically generated when you propose to substitute an
FDA-approved analytical procedure with an alternative
analytical procedure or when an analytical method is
transferred from one laboratory to the other.
1. Alternative Analytical Procedures
An alternative analytical procedure is an analytical
procedure that you use in place of the FDA- approved
analytical procedure. For an NDA or ANDA, you should
include any proposed alternate analytical procedures in
the application.

37. FDA METHODS VERIFICATION :
Part of the approval process for NDAs and ANDAs may
include FDA laboratory assessment to determine whether
the analytical procedures are acceptable for quality control
and suitable for regulatory purposes.
For certain biological products, samples representative of
the product for licensure along with summaries of results
of tests performed on the lots represented by these
samples should be submitted with the BLA.

38. REFERENCE:
Pharmaceutical Quality Assurance by Manohar A. Potdar,
Nirali Prakashan,Page no- 8.28-8.31

39. •THANK YOU