This document discusses analytical method validation. It defines analytical method validation as providing assurance that an analytical method can consistently and accurately determine the presence or quantity of attributes. The objectives of validation are to obtain consistent, reliable and accurate data. Key parameters that are assessed in validation include specificity, accuracy, precision, linearity, range, limits of detection and quantification, ruggedness and robustness. The validation process involves planning, testing method performance characteristics, selecting validation acceptance criteria, and documenting results in a validation report. Validation is important for analytical methods used in pharmaceutical analysis.

1. PREPARED BY:
DEVIPRIYA PV
M PHARM
Department of Pharmaceutical Analysis
ANALYTICAL METHOD
VALIDATION

2. DEFINITION
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“Documented programme which provides a high degree
of assurance that an analytical assay method will
consistently determine the presence, absence or quality
of one or more attributes with accuracy and precision”

3. OBJECTIVE
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 To obtain consistent , reliable and accurate data.
 The results from method validation can be used to
judge the quality, reliability and consistency of
analytical results, which is an integral part of any good
analytical practice.

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 Analytical methods need to be validated, verified, or
revalidated in the following instances:
1. Before initial use in routine testing.
2. When transferred to another laboratory.
3. Whenever the conditions or method parameters for
which the method has been validated change and the
change is outside the original scope of the method.

5. ICH
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 Requirements:
 Analytical methods should be validated unless the method
employed is included in the relevant pharmacopoeia or other
recognized standard reference.
 The degree of analytical validation performed should reflect
the purpose of the analysis and the stage of the API
production process.
 Appropriate qualification of analytical equipment should be
considered before starting validation of analytical methods.

6. USP
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Chapters for method validation
Chapter <1225>
“Validation of
Compendial
Methods”
Chapter <1226>
“Verification of
Compendial
Methods”
Chapter <621>
“Chromatography”.

7. PARAMETERS FOR METHOD
VALIDATION
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SPECIFICITY
LINEARITY
RANGE
ACCURACY
PRECISION
LIMIT OF DETECTION
LIMIT OF QUANTITATION
• AS PER ICH
SPECIFICITY
LINEARITY AND RANGE
ACCURACY
PRECISION
LIMIT OF DETECTION
LIMIT OF QUANTITATION
RUGGEDNESS
ROBUSTNESS
• AS PER USP

8. SPECIFICITY
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 Ability to assess unequivocally the analyte in the
presence of components which may be expected to be
present.
 Include impurities, degradants, matrix etc.
 Procedures used to demonstrate specificity:
Identification test
Assay(content or potency).
Purity test.

9. ACCURACY
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 ICH defines the accuracy of an analytical procedure as
the closeness of agreement between the conventional
true value or an accepted reference value and the value
found.
 The extent to which test results generated by the
method and the true value agree.
 Should be assessed using a minimum of 3
concentration levels, each in triplicate.

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 Should be reported as:
Percent recovery of known amount added
The difference between the mean assay result and the
accepted value.

11. PRECISION
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 The closeness of agreement 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
 Reproducibility

12. LINEARITY
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 ICH defines linearity of an analytical procedure as its
ability to obtain test results that are directly
proportional to the concentration of analyte in the
sample.
 Evaluated by:
Visual inspection of plot.
Statistical methods.
Acceptance criteria

13. RANGE
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 ICH defines the range of an analytical procedure as the
interval from the upper to the lower concentration of
analyte in the sample for which it has been
demonstrated that the analytical procedure has a
suitable level of precision, accuracy and linearity.

14. LIMIT OF DETECTION(LOD)
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 ICH defines the detection limit of an individual
analytical procedure as the lowest amount of analyte in
a sample which can be detected but not necessarily
quantitated as an exact value.
 Evaluated by:
Signal to noise ratio
Visual evaluation
Standard deviation of the response and the slope.

15. LIMIT OF QUANTITATION(LOQ)
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 ICH defines the LOQ of an individual analytical
procedure as the lowest amount of analyte in a sample
which can be quantitatively determined with suitable
precision and accuracy.

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17. RUGGEDNESS
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 The degree of reproducibility of results obtained under
a variety of conditions, such as different laboratories,
analysts, instruments, environmental conditions,
operators and materials.
 Expressed as %RSD.
 Determined by the analysis of aliquots from
homogeneous lots in different laboratories.

18. ROBUSTNESS
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 ICH defines the robustness of an analytical procedure
as a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters.
 It provides an indication of the procedure’s reliability
during normal usage.

19. METHOD VALIDATION PROCESS
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 The validity of a specific method should be
demonstrated in laboratory experiments using samples
or standards that are similar to unknown samples
analyzed routinely.
 The process is broken down in phases because of the
length of time and complexity.

20. VALIDATION PHASES
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22. VALIDATION PLANNING
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 The master plan should include:
 Purpose and scope.
 Glossary.
 Responsibilities, like user departments, management, QA
 Method performance characteristics and approaches for
testing.
 Steps for method validation.
 Selection of tests and acceptance criteria.
 Approach and parameters for system suitability testing.

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 Modification and revalidation methods.
 Verification of Compendial and standard methods.
 Transfer of analytical methods.
 List of required SOPs.
 Approval process, documentation and archiving.
 Templates for project plans, test protocols and validation
reports.

24. TESTING FOR PERFORMANCE
CHARACTERISTICS
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 PREPARATION:
 Any chemicals used to determine critical validation
parameters, should be available in sufficient quantities,
accurately identified, sufficiently stable and checked for
exact composition and purity according to specifications.
 Any other materials and consumables, should be new and
qualified.

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 Analytical equipment should be clearly defined, well
characterized, qualified or calibrated
 Operators should be sufficiently familiar with the
technique and equipment.
 TEST EXECUTION:
 Specificity/selectivity
 Repeatability of retention times and peak areas
 Linearity, LOQ, LOD , range
 Accuracy at different concentrations
 Intermediate precision
 Reproducibility

26. SELECTING VALIDATION
PARAMETERS AND LIMITS
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Validation report and documentation.
Method Adjustments.
Changes.
Revalidation.
Verification of Standard and Compendial Methods

28. VALIDATION OF BIOLOGICALS
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 A substance that is produced by or extracted from a
biological source and that needs for its characterization
and the determination of its quality a combination of
physical-chemical-biological testing, together with the
production process and its control.

29. CONTENT OF ANALYTICAL
PROCEDURES
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 Principle/scope.
 Apparatus/equipment.
 Operating parameters.
 Reagents/standards.
 Sample preparation.
 Standards control solution preparation.
 Procedure.
 System Suitability.
 Calculations.
 Data reporting.

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 For biologicals follow a two-tired approach for reference
standard and materials.
 Comparison of each new reference standard with a primary
reference standard so that it is linked to clinical trial material
and the current manufacturing process.
 Statistical analysis of validation data can be used to evaluate
validation characteristics against predetermined acceptance
criteria.

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