The document discusses various aspects of analytical method validation including the key parameters to evaluate, strategies for validation, documentation requirements, and regulatory guidelines. It defines method validation as confirming the suitability of an analytical procedure for its intended use. Key parameters discussed include accuracy, precision, specificity, range, linearity, limits of detection and quantitation, ruggedness, and robustness. Validation involves experimental testing, documentation in a report, and approval. Methods must be validated before use and revalidated if changed. The document provides detailed guidance on conducting and documenting the method validation process.

1. ROSHAN H. KHETADE
Quality Assurance Department

2. Content
1. Introduction
2. Strategy for the validation of method.
3. Verification of standard method.
4. Validation of non-routine method.
5. Quality control plan.
6. Implementation of routine analysis
7. Transferring validated routine method
8. Revalidation.
9. Parameters for method validation.

3. Introduction
Method validation is the process used to confirm that the
analytical procedure employed for a specific test is suitable
for its intended use.
From method validation can be judge the quality, reliability
and consistency of analytical result.
It is an integral part of any good analytical practice.

4. Analytical method need to be validated or revalidation
 Before their introduction into routine use.
 Whenever the conditions change for which the method has
been validated
(e.g. instrument with different characteristic or sample with
different matrix.)
 Whenever the method changed and the change is outside the
original scope of the method.

5. The USP has published specific guidelines for
method validation for compound evaluation.
USP define eight step for validation
1. Accuracy
2. Precision
3. Specificity
4. Limit of detection
5. Limit of quantitation
6. Linearity and range
7. Ruggedness
8. Robustness

6. Strategy for the validation of method
The validity of a specific method should be demonstrated in
laboratory experiment using samples or standards that are similar
to unknown samples analyzed routinely.
The preparation and execution should follow a validation protocol,
preferably in a step-by-step instruction format.
Steps for complete method validation are as follows:

7. 1. Develop a validation protocol ,an operating procedure , or
a validation master plan for the validation.
2. For a specific validation project , define owners and
responsibilities.
3. Develop a validation project plan.
4. Define the application ,purpose and scope of the method.
5. Define the performance parameter and acceptance
criteria.
6. Define validation experiments.
7. Verify relevant performance characteristics of equipment.

8. 8. Quality material, e.g. standards and reagents for purity,
accurate amount, and sufficient stability.
9. Perform pre-validation experiment.
10. Adjust method parameter / Acceptance criteria if necessary.
11. Perform full internal(and external)validation experiment.
12. Develop SOPs for executing the method in the routine.
13. Define criteria for validation.
14. Define type and frequency of system suitability tests and
/analytical quality control checks for the routine.
15. Document validation experiments and result in the
validation report.

9. Validation Report
1. Objective and scope of the method.
2. Summary of methodology.
3. Type of compounds and matrix.
4. All chemical reagents, reference standards,QC sample with purity,
grade, their source, and detailed instruction on their preparation.
5. Procedure for quality check of standards and chemicals used.
6. Safety precautions.
7. A plan and procedure for method implementation from the method
development lab to routine analysis.
8. Method parameters.

10. 9. Critical parameter taken from robustness testing.
10. Listing of equipment and its function and performance
requirements. e.g.: cell dimensions, column temperature.
11. Detailed conditions on how the experiments were
conducted, including sample preparation.
12. Statistical procedures and representative calculations.
13. Procedures for Qc routine analysis. E.g. System
suitability.
14. Representative plots , e.g. chromatograms, spectra, and
calibration curve.
15. Method acceptance limit performance data.

11. 16. The expected uncertainty of measurement results.
17. Criteria for revalidation.
18. The person who developed and validated the method.
19. References
20. Summary and conclusion.
21. Approval with name, tittle, date, and signature of those
responsible for the review and approval of the
analytical test procedure.

12. Verification of standard method
There are two important requirement :
1. The standard’s method validation data are adequate and
sufficient to meet the laboratory’s method requirements.
2. The laboratory must be able to match the performance data
as described in the standard.

13. Evaluation and validation of standard
method:
Define scope of user method
Define validation parameter and limits
Standard method
fits scope,
parameter and limit
Perform part or full validation
Define and perform system suitability
testing
No
yes

14. Step for validating complete analytical procedure
Sampling
Sample preparation
Analysis
Calibration
Data evaluation
Reporting

15. Quality control plan
For any method that will be used for routine analysis, a QC plan
should be developed. The plan should ensure that the method,
together with the equipment, delivers consistently accurate result.
Plan may include recommendation for the following:
1. Selection, handling and testing of QC standards.
2. Type and frequency of equipment checks and calibrations
3. Type and frequency of system suitability testing
4. Type of frequency of QC sample
5. Acceptance criteria for equipment checks, system suitability test
and QC sample analysis.

16. Transferring validated routine method
Validated routine method are transferred between laboratories at
the same or different sites when contract laboratories offer services
for routine analysis in different area or when product are
manufactured in different area.
When validated routine methods are transferred between
laboratories and sites. Their validated state should be maintained
to ensure the same reliable result in the receiving laboratory.

17. The transfer should be controlled by a procedure. The
recommended step are
 Designate a project owner
 Develop a transfer plan
 Define transfer tests and acceptance criteria
 Describe rationale for tests
 Train receiving lab operators in transferring lab on equipment,
method , critical parameters, and troubleshooting
 Repeat two critical method validation test in routine lab
 Analyze at lest three sample in transferring and receiving lab
 Document transfer result

18. Revalidation
Revalidation is necessary whenever a method changed and the
new parameter lies outside the operating range.
For example the operating range of the column temperature has
been specified to be between 30°C and 40°C, the method should be
revalidated if, for whatever reason, the new operating parameter is
41°C.

19. Define and document change
Change fits the
method’s scope,
parameter and limit
Perform part or full revalidation
Define and perform system
suitability testing
yes
No
Flow diagram for revalidation

20. Precision
Accuracy
Limit of detection
Limit of quantitation
Specificity
Linearity
Range
Robustness
Ruggedness
Stability
Method
validation

21. PRECISION
 The precision of on analytical procedure express the closeness of
agreement (degree of scatter) between a sampling of the same
homogenous sample under the prescribed condition.
 Precision may be considered at three level : repeatability,
intermediate precision, reproducibility.
 Precision should be investigated using homogeneous authentic
sample.
 The precision of on analytical procedure is usually expressed as the
various standard deviation of coefficient of variation of a serious of
measurement.

22. REPEATABILITY
Repeatability expressed the precision under interval of time
repeatability is also term termed intra-assay precision.
INTERMEDIATE PRECISION
Intermediate precision express within laboratories variation
different days, different analysts, different equipment etc.
REPRODUCABILITY
Reproducibility express the precision between laboratories
collaborative studies usually applied to standardization of
methodology.

23. ACCURACY
The agreement between the test result obtained by the proposed
method and the true value.
It expresses the correctness of the method.
It is expressed as percentage by the assay of known amount of
substance.
Accuracy also evaluated by recovery studies in which amount of
drug is added to previously analyzed pharmaceutical preparation
of the drug and tested for the recovery of the added drug.

24. Should be established across specified range of analytical procedure.
Should be assessed using a minimum of 3 concentration levels. Each
in triplicate (total of 9 determination)
Should be reported as
 Percent recovery of known amount or
 The difference between the mean assay result and the accepted
value
 The absolute error is a measure of the accuracy of the measurement,
it is then calculated as
Absolute error = mean error
(true value – measured value )/true value x 100

25. LIMIT OF DETECTION
It can be defined as the lowest amount of the analyte in a sample that can be
detected but not necessarily quantified under the standard experiment
condition.
How is it determined ?
Based on visual inspection
Detection limit is determined by the analysis of sample with known
concentration of analyte and by establishing the minimum level at
which the analyte can be reliably detected.
Based on standard deviation of the black
Measurement of the magnitude of analytical background response is
performed by analyzing an appropriate number of black sample and
calculating the standard deviation of these response

26. Based on the slop of the calibration curve
A specific calibration curve is study using sample containing and
analyte in the range of limit of detection the residual standard
deviation of a regression line or the standard deviation of y-intercept
of regression lines may be used as the standard deviation.

27. LIMIT OF QUANTITATION
The quantitation limit of an indivial analytical procedure is the lowest
amount of analyze in a sample which can be quantitatively determine
with suitable precision and accuracy.
The quantitative limit is parameter of quantities assay for low week
of compound in sample matrices and is used particularly for the
determination of impurities and /or degradation product.
It express as the concentration of analyte (e.g. part per million) in the
sample

28. SPECIFICITY
Is the ability to measure the analyte in the present of other
component which may be expected to be present typically this
might include impurities, degradants, matrix etc.
Lack of specificity of an individual procedure may be
compensated by other supporting analytical procedure.
An investigation of specificity should be conducted the validation
of identification tests, the determination of impury and assay.

29. LINEARITY
It indicates the ability to produce the result that are directly
proportional to the concentration of analyte in sample within a
given range (Y =mx+c).
The following parameter should be determine
 Correlation coefficient
 y-intercept (c)
 Slop of the regression line (m)

30. RANGE
The range of analytical procedure is the interval between the upper
and lower level of analyte (including these level) that have been
demonstrated with a suitable level of precision, accuracy and
linearity.
The range is normally expressed in the same units as the test
results(E.g. percentage, parts per million) obtained by the
analytical method.

31. The following minimum specified range should be consisted:
1) For the assay of a drug substance or a finished (drug) product:
normally from 80-120 % of the test concentration.
2) For content uniformity, covering a minimum of 70-130 % of
the test concentration ,unless a wider more appropriated range,
based on the nature of the dosage form (e.g. metered dose
inhalers),is justified.
3) For dissolution testing +/- % over the specified range.

32. ROBUSTNESS
The robustness of analytical method is measure of its capacity to remain
unaffected by small but deliberated variation in method parameter and provide an
indicated of it reliably during normal usage.
Robustness examines the effect of variation in operational parameter on the
analysis result for the determination of a method robustness, parameter like
variation in detector wavelength are varied within a realistic range and the
quantitative influence of the variable id determine.

33. If the influence of the parameter is within a previously specified
tolerance , the parameter is said to be within the method
robustness range.
Acceptance criteria % RSD should not be more than 2%.

34. RUGGEDNESS
It is a measure of method reproducibility under variable conditions
within specified test parameter of test method.
The following are the typical method parameter need to tested
during method validation.
 Analyst to analyst variability.
 Column to column variability.
 System to system variability.
 Different days different laboratory.
 Stability of solution of mobile phase.(at least for 48 hours)

35. STABILITY
Stability testing is an integral part of many analytical procedure.
the tests are based on the concept that the equipment, electronics,
analytical operation and sample to be analyzed constituent an
integral system that can be evaluated as such.
Determination: Reputability, Tailing factor (T), Capacity factor
(k’),Resolution(R), and Theoretical plates(N).

36. ICH/ USP VALIDATION REQUIRMENT
AND PARAMETER
 USP
 Specificity
 Linearity and range
 Accuracy
 Precision
 Limit of detection
 Limit of quantitation
 Ruggedness
 robustness
 ICH
 Specificity
 Linearity
 Range
 Accuracy
 Precision
Repeatability
Intermediate precision
Reproducibility
 Limit of detection
 Limit of quantitation

37. Reference
Pharmaceutical process validation An
international third edition revised and
expanded volume 129 edited by Robert A.
Nash ,Alfred H . Watcher page no. 542-559.