The analytical results you communicate can have far-reaching consequences and can

form the basis for taking decision on safety of use of commercial products, foods and
natural resources in addition to decisions involving legal matters.

As a responsible analyst before you undertake analysis in the laboratory you should make
sure that your results are authentic and are universally acceptable. This key objective can
be realized only if the method selected for the purpose is duly validated.

Analytical method validation establishes documented evidence that the procedure adopted for a test
is fit for the intended purpose in terms of quality, reliability and consistency of results.
What Is Analytical Method Validation?
Analytical method validation establishes documented evidence that the procedure
adopted for a test is t for the intended purpose in terms of quality, reliability and
consistency of results.

In other words, if the same method is adopted in any other laboratory across the world,
under the same set of conditions and control parameters, the results should be in
agreement.

Each and every method requires validation:

• Before putting to routine use

• When analytical conditions are changed such as change of technique, change in

desired concentration range or change of sample matrix

• Whenever changes are made to an existing procedure

It is important to collect relevant information on analysis requirements before you

plan validation of the procedure

• Components to be detected

• Expected concentration levels

• Required level of detection and quanti cation

• Nature of sample matrix

• Type of analytical technique to be used

• Required degree of precision and accuracy

Parameters of Method Validation

Before undertaking the method validation procedure, it should be ascertained using
reference standards that the response of the analytical measurement is on account of the
analyte of interest and not due to any other chemical species.

After this has been established, you may proceed with the validation which evaluates the
following method-speci c features. Here are the various method validation parameters.

• Selectivity and speci city

• Linearity

• Range

• Accuracy

• Precision

• Limit of quanti cation

• Ruggedness

• Robustness

We shall discuss each of the analytical method validation parameters in detail so that you
can better understand analytical method validation.

Selectivity and Specificity

Selectivity and speci city are often used synonymously but these are di erent terms.
Speci city is the unequivocal response of the technique to a speci c analyte present in
the sample whereas selectivity can be applicable to a collective response to a group of
analytes having similar chemical and physical characteristics.

The method selected should meet the criteria for selectivity and sensitivity in presence of
deliberately added interferents that can be expected in the sample under investigation.

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 Selectivity is more frequently used as analytical techniques and is seldom speci c to only
one analyte. Further, selectivity should not be a ected by interfering species such as
degradation products, impurities and other matrix components.

Linearity
Linearity refers to the ability of analytical procedures to produce results in direct
proportion to the concentration range of analyte in samples within the required
concentration levels.

• Linearity should be determined using a minimum of 6 standards whose

concentration spans from 80% to 120% of expected concentration level.

• Linearity report should include slope of line, linear range and correlation coe cient
data. Correlation coe cient r should be greater than or equal to 0.99 in the
working range

Range
Operating range is deduced from the calibration plot. It is the interval between the upper
and lower concentration of analyte falling in the linear range. The results corresponding to
this range demonstrate acceptable levels of precision, accuracy and linearity.

Accuracy
• Degree to which the determine value of analyte corresponds to the true value.

• Accuracy can vary over the expected concentration range.

• It should be determined using a working or reference standards in the 80% – 120%

level of expected range

• Accuracy is determined by :

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 1. Analyzing a sample of known concentration and comparing with the true value

2. Spiking a blank (Sample having all components except the analyte) and comparing
with the expected result.

3. Standard addition method in which the sample concentration is determined. A

known amount of analyte is added and the concentration is once again
determined. The di erence of the two concentration values is compared with the
actual value of added analyte.

• Accuracy is also de ned by the comparison of test results with those obtained
using another validated test procedure

Precision
• Precision expresses closeness of a series of measurements of the same sample
under identical conditions

• High degree of precision does not necessarily means a high degree of accuracy

• Precision is expressed as variance, standard deviation or as coe cient of variation

of a series of measurements

• Minimum of ve replicate sample determinations should be carried out

Limit of Detection
• Lowest amount of an analyte that can be detected but not necessarily quanti ed

• Lowest concentration of calibration standard which produces a peak response

corresponding to the analyte should be measured at least 6 to 10 times. Average
response (X) and standard deviation (SD) are required to calculate limit of detection

Limit of detection = X + (3SD)

• Signal to noise ratio at limit of detection should be at least 3:1

Signal to noise ratio should be greater than 3 at limit of detection and greater than 10 at limit of
quanti cation
Limit of Quantitation
• Lowest amount of the analyte that can be quantitatively determined with de ned
precision under the stated experimental conditions

• 6 – 10 observations should be made for average response and standard deviation

Limit of quantitation = X + (10SD)

• Signal to noise ratio should be at least 10:1 at the limit of quantitation

Ruggedness
Ruggedness measures reproducibility of test results under following conditions :

• Results generated for same sample under identical conditions by di erent

laboratories

• Results generated for sample under identical conditions by di erent analysts

• Same analysis using di erent instruments

• Same analysis under di erent environmental conditions

• Same analysis using test materials from di erent sources

Robustness
Robustness examines the e ect of operational parameters changes on the analytical
results

• pH

• Temperature

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 • Operational conditions such as ow rate, injection volume, detection wavelength or
mobile phase composition in chromatographic analysis

Variation should be deliberate but within realistic range to study the robustness of the
method. The results of the analysis after making the deliberate changes should be within
the method’s speci ed tolerance limits.

Method validation may appear to be a tedious and time-consuming activity but once the
method is validated before adoption, it will not let you down under any circumstances.

What Is the Need for Analytical Method Validation?

Before you plan an analysis of a sample in your laboratory, you should be aware of the
essential details of the sample like its homogeneity, physical state, available quantity and
stability.

It is also essential to know the requirements of analysis such as:

• Components to be detected and quanti ed

• Expected concentration levels

• Nature of the sample matrix. In other words, the nature of interferences that can be
present that can complicate the determination of required components

• Required degree of accuracy and precision

• Appropriate analytical technique for the analysis

• Material and apparatus required for the analysis

• Availability of required resources

• Cost of analysis

The above considerations require a judicious selection of the method. Let us see what
could be those useful reference sources that you can access. You can consider yourself
fortunate if your laboratory has already a validated method available which can be
adopted without applying a second thought. However, this may not be the case every
time, and you would feel the need to search for a suitable method due to the following
constraints:

• The sample matrix is di erent from routine samples and requires a di erent set of
extraction procedures or can introduce di erent interferences

• Non-availability of required reagents or glassware

• Non-functional or un-calibrated instruments

In such situations, you will have to look for other alternative methods so you will have to
search for the method through the available options such as:

• Standard textbooks

• Reference analytical journals

• Pub Med

• Google search

The more you search, chances are that you may get confused further. Let us assume that
after going through the references you have zeroed down on a particular method.

Now comes the important stage of validating the method before it is put into routine use.
You have to keep in mind the fact that the analytical results reported by you can have far-
reaching consequences concerning the safety of use of the products, e cacy of drugs,
toxic limits for environmental samples, or verdict on legal matters.

As a responsible analyst, you have to make sure that the data reported by you is reliable
and would be accepted universally. Such objectives can be achieved only by following
the rigorous validation process.

Validation becomes necessary:

•  Before adopting the method in routine use

• Whenever there is a requirement of analysis of components in the di erent sample

matrix

• Sample analysis requires di erent analytical procedures

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As a regular practice, any method needs to be validated before adoption. However, it
would need to be revalidated under the following situations:

• Any changes made in the established procedure

• Any new accessory is added to the existing system

• The system has undergone major operational problems which have been recti ed.

Why Is It Essential to Adopt Validated Analysis Methods?

Analytical chemistry is a science which guides you on how to get the desired information
from your sample. The method validation guidelines provided comprise of

• Selection of appropriate technique

• Procedure to be followed

• Analysis and Interpretation of data

• Environmental controls and other precautions to be adopted

You can consider yourself lucky if a validated method meeting your analysis requirements
is already available in your laboratory. Alternately, you will have to identify the appropriate
method through a literature search.

Your literature search will be successful if you have clarity on your analysis objectives. The
search can be initiated using Internet search engines or referring to documented sources
such as research journals. The next step is to validate the method before adopting it.

Benefits of Method Validation

Method validation is a practice which can prove to be bene cial in a number of situations
such as:

• Publication of your ndings in international journals of repute or presentation in

professional conferences

• Instils a degree of self-esteem and con dence in yourself as well as in those who
use your results

• Acceptance of procedures and results by accreditation bodies and external

auditors

• Arriving at critical decisions concerning the use of data with regards to the safe use
of products by consumers

• Freedom from disputes concerning non-agreement of test results with other

laboratories

Critical Role of Method Validation in Chemical Analysis

Each and every analysis requires the choice of a method that will help you achieve the
required objectives. In case an established method is available, you should consider
yourself fortunate as this will save you the e ort and time required to validate the method
before adopting it.

Several options are available to you for the selection of the method to suit your analysis
requirements. Such options include a survey of standard analytical textbooks, research
journal publications, Google search, etc.

You will come across several methods in your Google search but this can add to further
confusion as the method selected may not be suitable for your analysis requirements.

In making your method choice, you also have to keep into consideration other
factors such as:

• Cost of analysis

• Availability of required reagents and standards

• Availability of required instrumental facilities

• Availability of trained manpower

• Time requirement for analysis

• Simplicity of method

It is essential to keep in mind that the simplest method may not be the ideal one so you
have to carefully evaluate the choice keeping in view the considerations listed above.
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