ANALYTICAL

METHOD
VALIDATION

VALIDATION
means to
TO CHECK OR PROVE THE VALIDITY OF
According to FDA
The goal of validation is to establish a documented
evidence which provides a high degree of
assuarance that a specific process will consistently
produce a product meeting its predetermined
specifications and quality attributes .

ANALYTICAL METHOD
VALIDATION
Definition:
By ISO :
Confirmation by examination and provision of evidences that the
particular requirements for a specified intended use are fulfilled .
Another

definition as given in the Handbook for the Quality

Assurance of Metrological Measurements :
Method validation consists of documenting the quality of an
analytical procedure, by establishing adequate requirements for
performance criteria, such as accuracy, precision, detection
limit, etc. and by measuring the values of these criteria

Method validation has received

considerable attention in the literature
from industrial committees and
regulatory agencies :
A.

B.

C.

D.
E.
F.

The ISO/IEC Guide 25 includes a chapter on the validation of methods

with a list of nine validation parameters.
The International Conference on Harmonization (ICH) of Technical
Requirements for the Registration of Pharmaceuticals for Human Use
has given a text on the validation of analytical procedures, including
definitions for eight validation characteristics.
The U.S. Environmental Protection Agency (U.S. EPA) prepared a
guidance for methods development and validation under the Resource
Conservation and Recovery Act (RCRA).
The American Association of Official Analytical Chemists (AOAC)
The U.S Environmental Protection Agency (EPA)
The U.S. Food and Drug Administration (U.S. FDA)

THE ANALYTICAL METHOD

NEEDS TO BE VALIDATED AT
FOLLOWING STAGES
1. Before their introduction into routine use that is a new
method of analysis is going to be used in a laboratory or in
field analysis.
2. Whenever the conditions change for which the method has
been validated (e.g., instrument with different
characteristics)
3. Whenever the method is changed, and the change is
outside the original scope of the method.
4. When quality control indicates an established method is
changing with time.
5. In order to demonstrate the equivalence between two
methods (e.g., a new method and a standard)

PARAMETERS FOR METHOD

VALIDATION
as defined by the ICH

1.

Specificity

2.

Precision

3.

Accuracy

4.

Linearity

5.

Range

6.

Detection limit

7.

Quantitation limit

8.

Robustness

Specificity
The

terms selectivity and specificity are often used

interchangeably.

It

is
the ability to measure unequivocally the
desired analyte in the presence of interference, such
as synthetic precursors, excipients, enantiomers,
and known (or likely) degradation products that may
be expected to be present in the sample matrix.

Precision
Precision is the degree of agreement among
individual results
It is expressed as the relative standard deviation
(RSD).
The measured standard deviation can be
subdivided into three categories: repeatability,
intermediate precision, and reproducibility.

1. Repeatability: Repeatability is obtained when the analysis is

carried out in one laboratory by one operator using one piece of
equipment over a relatively short time span.
At least 5 or 6 determinations of ,three different matrices at
,two or three different concentrations should be done and the
relative standard deviation calculated.
2. Intermediate precision: Intermediate precision is long-term
variability of the measurement process and is determined by
comparing the results of a method run within-laboratory
variations :
usually on different days, by different analysts and with
different equipment.

The objective of intermediate precision validation is to

verify that in the same laboratory the method will provide
the same results once the development phase is over.

3. Reproducibility:
Reproducibility as defined by ICH represents the precision obtained
between laboratories .
The objective is to verify that the method will provide the same
results in different laboratories.
The reproducibility of an analytical method is determined by analyzing
aliquots from homogeneous lots in different laboratories with different
analysts and by using operational and environmental conditions that
may differ from but are still within the specified parameters of the
method (inter laboratory tests) .

Precision

True
value

Inaccurate but
precise

Accuracy
Definition :
Accuracy

is the degree of agreement of test

results generated by the analytical method with the

true value, or the closeness of the results obtained
by the procedure to the true value.

Accuracy should be established across the specified

range of the analytical procedure.

Accuracy

True value

Accurate but
imprecise

Precision and Accuracy

Accurate and
precise

Linearity
Definition :

The linearity of an analytical method is its

ability to elicit test results (response) that
are directly, or by means of well-defined
mathematical transformation, are proportional
to the concentration of analytes in samples
within a given range.

CALCULATION OF LINEARITY
(mathematically)

series of samples should be prepared in which the analyte

concentrations span 80120% of the expected concentration

range. A minimum of five concentrations should be used .

The

response should bedirectly or by means of a well-defined

mathematical calculation should be proportional to the

concentrations of the analytes. If there is a linear relationship, test
results should be evaluated by appropriate statistical methods.

CALCULATION OF LINEARITY
(graphically)
Graphical presentations of linearity plot of a caffeine sample using HPLC.

Response
versus amount

Response versus
log amount

Range
Range

is an expression of the lowest and highest

levels of analyte that have been demonstrated to be
determinable for the product .
The

specified range is normally derived from

linearity studies .
The

range is normally expressed in the same units as

the
test results (e.g., percentage, ppm) obtained
by the analytical method.

Detection limit (limit of

detection)
Definition :

Limit of Detection is the smallest quantity of

an analyte that can be detected, but not
necessarily quantified.
Approaches to calculation :
1.
visual evaluation
2.
signal to noise ratio
3.
standard deviation of the response and the
slope of the calibration curve

Detection limit (limit of

detection)
Calculation

1. visual evaluation- DL is determined by the analysis of a series of samples with known

concentrations and establishing the minimum level at which the analyte can be reliably detected.

2. signal to noise ratio- For instrumental procedures that exhibit background noise, it is common
to compare measured signals from samples with known low concentrations of analyte with those of
the blank samples. The minimum concentration at which the analyte can reliably be detected is
established using an acceptable signal - to - noise ratio of 2 : 1 or 3 : 1.

3. standard deviation of the response and the slope of the calibration curve
DL=3/S
where is the standard deviation of the response and S is the slope of the calibration curve

Quantitation limit (limit of

quantitation)
Definition :
Quantitation limit is the lowest concentration of an analyte in a sample that may be
determined with acceptable accuracy and precision

Approaches to calculation :
1.
2.
3.

visual evaluation
signal to noise ratio
standard deviation of the response and the slope of the calibration curve
QL=10/S

where is the standard deviation of the response and S is the slope of the calibration curve

Robustness
Definition :

Robustness is the ability of the procedure to provide analytical results

of acceptable accuracy and precision under a variety of conditions .
e.g., different flow rate, column temperature, injection volume,
detection wavelength, or mobile phase composition in case of HPLC

Robustness

tests examine the effect , operational parameters have on

the analysis results .

If

the influence of the parameter is within a previously specified

tolerance the parameter is said to be within the methods robustness

range

Steps in Method
Validation
1. Develop a validation protocol or operating procedure for the
validation.
2. Define the application, purpose, and scope of the method.
3. Define the performance parameters and acceptance criteria.
4. Define validation experiments.
5. Verify relevant performance characteristics of equipment.
6. Qualify materials (e.g., standards and reagents).
7. Perform prevalidation experiments.
8. Adjust method parameters or/and acceptance criteria if
necessary.

Steps in Method
Validation
continued

9. Perform full internal (and external) validation

experiments.
10. Develop SOPs for executing the method in the routine.
11. Define criteria for revalidation.
12. Define type and frequency of system suitability tests
and/or analytical quality control (AQC) checks for the
routine.
13. Document validation experiments and results in the
validation report.

The scope of the method and its validation parameters and

acceptance criteria should be defined early in the process . These can
be defined by finding answers to the following questions :

What analytes should be detected?

What is the expected concentration range?

What are the sample matrices?

Are there interfering substances expected and if so, should they

be detected and quantified?

Are there any specific legislative or regulatory requirements?

Should information be qualitative or quantitative?

Continued..

What are the required detection and quantitation limits?

What precision and accuracy is expected?

How robust should the method be? For example, should the
method work at a specific room temperature or should it run
independently from room temperatures?

Which type of instrument should be used?

Will the method be used in one specific laboratory or should it

be applicable all laboratories in your organization?

What skills should the anticipated users of the method have?

Contents of a Validation
Report
1.

Description of the method.

2.

Objective and scope of the method (applicability, type).

3.

Summary of methodology, including sampling procedures.

4.

Type of compounds and matrix.

5.

All chemicals, reagents, mobile phases, reference standards,

quality control samples with purity, grade, their source, or
detailed instructions on their preparation.

6.

Procedures for quality checks of standards and chemicals used

Safety precautions.

Contents of a validation report

continued..1
7. A plan and procedure for method implementation from method
development lab to routine.
8. Method parameters.
9. Critical parameters taken from robustness testing.
10.Listing of equipment and its functional and performance
requirements (e.g., cell dimensions, baseline noise, column
temperature range). For a complex equipment a picture or schematic
diagrams may be useful.
11.Detailed conditions on how the experiments were conducted,
including sample preparation. The report must be detailed enough to
ensure that it can be reproduced by a competent technician with
comparable equipment.
12.Statistical procedures and representative calculations.
13.Procedures for quality control in the routine (e.g., system suitability
tests) with acceptance criteria.

Contents of a validation report continued..2

14. Representative plots (e.g., chromatograms, spectra, and
calibration curves).
15.Method-acceptance limit performance data.
16.The expected uncertainty of measurement results.
17.Criteria for revalidation.
18.The person who developed and initially validated the method.
19.Approval with names, titles, date, and signature of those
responsible for the review and approval of the analytical test
procedure.
20.The expected uncertainty of measurement results.
21.Criteria for revalidation.
22.The person who developed and initially validated the method.
23.References (if any).
24.Approval with names, titles, date, and signature of those
responsible for

THANK YOU..
PRESENTED BY :
Sambhunath Saha
Sudhanshu Gorai