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Review Article

Method Validation of Analytical Procedures

Prakash Chanda Gupta
QC Executive,
National Healthcare Pvt. Ltd., Nepal
p_c_gupta@yahoo.com

ABSTRACT
After the development of an analytical procedure, it is must important to assure that the procedure will
consistently produce the intended a precise result with high degree of accuracy. The method should give
a specific result that may not be affected by external matters. This creates a requirement to validate the
analytical procedures. The validation procedures consists of some characteristics parameters that makes
the method acceptable with addition of statistical tools.

Keywords: Validation, Analytical Procedure, Accuracy, Precision, Robustness

INTRODUCTION Common types of analytical procedure that can

Validation of an analytical procedure is the be validated [2]
process by which it is established, by laboratory Identification tests;
studies, that the performance characteristics of Quantitative tests for impurities content;
the procedure meet the requirements for the Limit tests for the control of impurities;
intended analytical applications.[1] Method Quantitative tests of the active moiety in
validation provides an assurance of reliability samples of drug substance or drug product or
during normal use, and is sometime referred to other selected component(s) in the drug
as the pro ess for providi g do u e ted product.
evidence that the method does what it is
i te ded to do. The ai o je tive of the Typical validation characteristics which should
validation is to demonstrate that the analytical be considered are listed below: [1]
method is suitable for its intended purpose, is Accuracy
accurate, specific and precise over the specified Precision
range that an analyte will be analyzed. Specificity
Analytical Method Validation is to be performed Detection Limit
for new analysis methods or for current Quantitation Limit
methods when any changes are made to the Linearity
procedure, composition of the drug product Range
and synthesis of the drugs substances. Robustness

The validation characteristics are to be evaluated on the basis of the type of analytical procedures.
Table 1: Evaluation of Validation Characteristics
Characteristics Type of Analytical Procedures
Identification Impurities Quantitative Tests
Quantitative Limit
How to cite this article: PC Gupta; Method Validation of Analytical Procedures; PharmaTutor; 2015; 3(1); 32-39

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Accuracy Not evaluated Evaluated Not evaluated Evaluated
Precision Not evaluated Evaluated Not evaluated Evaluated
Specificity Evaluated Evaluated Evaluated Evaluated
Detection Limit Not evaluated Not evaluated Evaluated Not evaluated
Quantitation Limit Not evaluated Evaluated Not evaluated Not evaluated
Linearity Not evaluated Evaluated Not evaluated Evaluated
Range Not evaluated Evaluated Not evaluated Evaluated

METHODS AND TERMINOLOGY

Accuracy Recovery = Analytical Result x 100%
The accuracy of an analytical method is the True Value
closeness of the test results obtained by that
method to the true value.[3] This is sometimes The recovery should be in the range of Control
termed trueness. It is recommended that limit.
accuracy should be determined using a
minimum of nine determinations over a The following method can be applied for
minimum of the three concentration levels, calculating the Upper Control Limit (UCL) and
covering the specified range (3 Lower Control Limit (LCL). The method involves
concentrations/3 replicates each of total the moving range, which is defined as the
analytical procedures).[4]
measurements ( xi  xi 1 ). These moving range
absolute difference between two consecutive

It is measured as the percent of analyte

recovered by assay. The recovery can be are averaged ( MR ) and used in the following
determined by the equation: formulae: [5]

UCL  x  3 LCL  x  3
MR MR
and
d2 d2

Where, xi is the individual analytical result, x is the sample mean, and d 2 is a constant commonly used
for this type of chart and is based on the number of observations associated with the moving range
calculation. Where n = 2 (two consecutive measurements), as here, d 2 = 1.128


 n 
( xi  x ) 2 
Precision

1/ 2

 
The precision of the analytical method
RSD (%) 
100 n1
x  n 1 
describes the closeness of repeated individual

 
measures of analyte. [6] The precision of an
analytical procedure is usually expressed as the  
standard deviation or relative standard
Where xi is an individual measurement in a set
deviation (coefficient of variation) of a series of
measurements. It is indicated by Relative of n measurement and x is the arithmetic mean
Standard Deviation, RSD, which is determined of the set. Generally, the RSD should not be
by the equation: more than 2%.

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Repeatability assay and impurity tests by chromatographic
Repeatability refers to the use of the analytical procedures, specificity can be demonstrated by
procedure within a laboratory over a short the resolution of the two components which
period of time using the same analyst with the elute closest to each other.[9]
[3]
same equipment. Repeatability should be
assessed using a minimum of nine It is not always possible to demonstrate that an
determinations covering the specified range for analytical procedure is specific for a particular
the procedure (i.e., three concentrations and analyte (complete discrimination). In this case a
three replicates of each concentration or using combination of two or more analytical
a minimum of six determinations at 100% of the procedures is recommended to achieve the
test concentration).[4] necessary level of discrimination.

Reproducibility Linearity
Reproducibility expresses the precision Linearity is the ability of the method to elicit
between laboratories (collaborative studies, test results that are directly, or by a well-
usually applied to standardisation of defined mathematical transformation,
methodology). Reproducibility is usually proportional to analyte concentration within a
demonstrated by means of an inter-laboratory given range.[10] It should be established initially
trial. [7] by visual examination of a plot of signals as a
function of analyte concentration of content. If
Intermediate Precision there appears to be a linear relationship, test
Intermediate precision is the results from within results should be established by appropriate
lab variations due to random events such as statistical methods. Data from the regression
different days, different analysts, different line provide mathematical estimates of the
equipment, etc.[8] degree of linearity. The correlation coefficient,
The standard deviation, relative standard y-intercept, and the slope of the regression line
deviation (coefficient of variation) and should be submitted.
confidence interval should be reported for each
type of precision investigated. It is recommended to have a minimum of five
concentration levels, along with certain
Specificity minimum specified ranges. For assay, the
Specificity is the ability to measure accurately minimum specified range is from 80% -120% of
and specifically the analyte of interest in the the target concentration.[11]

Regression line, y  ax  b
presence of other components that may be
expected to be present in the sample matrix
such as impurities, degradation products and Where, a is the slope of regression line and b
matrix components. It must be demonstrated is the y - intercept.
that the analytical method is unaffected by the
Here, x may represent analyte concentration
presence of spiked materials (impurities and/or
and y may represent the signal responses.
excipients).

 ( x  x)( y  x)
Correlation Coefficient,

 ( x  y)( y  y)
r
n
In case of identification tests, the method i i
should be able to discriminate between 1/ 2
compounds of closely related structures which i i
are likely to be present. Similarly, in case of

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x the determination of Detection Limit and
Where i is an individual measurement in a set
reliably quantified for the determination of
of n measurement and x is the arithmetic mean Quantitation Limit. A signal-to-noise ratio
y between 3 or 2:1 is generally considered
of the set, i is an individual measurement in a acceptable for estimating the detection limit
y
set of n measurement and is the arithmetic and A typical signal-to-noise ratio is 10:1 is
mean of the set. considered for establishing the quantitation
limit.
DETECTION LIMIT AND QUANTITATION LIMIT
The Detection Limit is defined as the lowest c. Standard Deviation of the response and the
concentration of an analyte in a sample that can Slope.

DL  3.3 / s
be detected, not quantified. The Quantitation The Detection Limit may be expressed as:
Limit is the lowest concentration of an analyte
in a sample that can be determined with

QL  10 / s
acceptable precision and accuracy under the The Quantitation Limit may be expressed as:
stated operational conditions of the analytical

Where,  is standard deviation of the response

procedures.[12] Some of the approaches to
determine the Detection Limit and Quantitation
Limit are: [13] and s is slope of the linearity curve.

a. Visual Evaluation The method used for determining the detection

Visual evaluation may be used for non- limit and the quantitation limit should be
instrumental methods. For non-instrumental presented. If DL and QL are determined based
procedures, the detection limit is generally on visual evaluation or based on signal to noise
determined by the analysis of samples with ratio, the presentation of the relevant
known concentrations of analyte and by chromatograms is considered acceptable for
establishing the minimum level at which the justification.
analyte can be reliably detected. And the
quantitation limit is generally determined by Range
the analysis of samples with known The range of an analytical procedure is the
concentrations of analyte and by establishing interval between the upper and lower levels of
the minimum level at which the analyte can be analyte (including these levels) that have been
determined with acceptable accuracy and demonstrated to be determined with a suitable
precision. Visual Evaluation approach may also level of precision, accuracy, and linearity using
be used with instrumental methods. the procedure as written. The range is normally
expressed in the same units as test results (e.g.,
b. Signal to Noise percent) obtained by the analytical
[10]
This approach can only be applied to analytical procedure.
procedures that exhibit baseline noise.
Determination of the signal-to-noise ratio is The following minimum specified ranges should
performed by comparing measured signals from be considered:[14]
samples with known low concentrations of For Assay of a Drug Substance (or a drug
analyte with those of blank samples and product) the range should be from 80% to 120%
establishing the minimum concentration at of the test concentration.
which the analyte can be reliably detected for

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For Determination of an Impurity: from 50% to Different columns (different lots and/or
120% of the acceptance criterion. suppliers);
For Content Uniformity: a minimum of 70% to Temperature;
130% of the test concentration, unless a wider Flow rate.
or more appropriate range based on the nature
of the dosage form (e.g., metered-dose In the case of gas-chromatography, examples
inhalers) is justified. of typical variations are:
For Dissolution Testing: ±20% over the specified Different columns (different lots and/or
range suppliers);
(e.g., if the acceptance criteria for a controlled- Temperature;
release product cover a region from 20%, after Flow rate.
1 hour, and up to 90%, after 24 hours, the
validated range would be 0% to 110% of the System Suitability Testing
label claim). System suitability testing is an integral part of
many analytical procedures. The tests are based
Robustness on the concept that the equipment, electronics,
The robustness of an analytical procedure is a analytical operations and samples to be
measure of its capacity to remain unaffected by analyzed constitute an integral system that can
small but deliberate variations in procedural be evaluated as such. System suitability test
parameters listed in the procedure parameters to be established for a particular
documentation and provides and indication of procedure depend on the type of procedure
its suitability during normal usage. Robustness being validated. They are especially important
may be determined during development of the in the case of chromatographic procedures.[16]
[15]
analytical procedure.
If measurements are susceptible to variations in INTERPRETATION AND TREATMENT OF
analytical conditions, the analytical conditions VARIATION OF ANALYTICAL DATA
should be suitably controlled or a precautionary Analytical procedures are developed and
statement should be included in the procedure. validated to ensure the quality of drug
One consequence of the evaluation of products. The analytical data can be treated and
robustness should be that a series of system interpreted for the scientific acceptance. The
suitability parameters (e.g., resolution test) is statistical tools that may be helpful in the
established to ensure that the validity of the interpretation of analytical data are described.
analytical procedure is maintained whenever Many descriptive statistics, such as the mean
[16]
used. and standard deviation, are in common use.
Other statistical tools, such as calculating
Examples of typical variations are: confidence interval, outlier tests, etc. can be
Stability of analytical solutions; performed using several different, scientifically
Extraction time. valid approaches.

In the case of liquid chromatography, 1. Confidence Interval:

examples of typical variations are: A confidence interval for the mean may be
Influence of variations of pH in a mobile phase; considered in the interpretation of data. Such
Influence of variations in mobile phase intervals are calculated from several data points
composition; using the sample mean (x) and sample

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standard deviation (s ) according to the be performed on the same sample, if possible,
or on a new sample.[17]

 s 
formula: [17]

 x  t / 2,n1 , x  t / 2,n1 
s
 
When used appropriately, outlier tests are
valuable tools for pharmaceutical laboratories.
n n Several tests exist for detecting outliers such as
in which t / 2,n1 is a statistical number the Extreme Studentized Deviate (ESD) Test,
Dixon's Test, and Hampel's Rule.
number of degrees of freedom (n  1) , and the
dependent upon the sample size (n) , the

desired confidence level (1   ) .

Choosing the appropriate outlier test will
depend on the sample size and distributional
assumptions. Many of these tests (e.g., the ESD
Its values are obtained from published tables of Test) require the assumption that the data
the Student t-distribution. A confidence interval generated by the laboratory on the test results
provides limits around the experimentally can be thought of as a random sample from a
determined value of the mean within which the population that is normally distributed, possibly
true value lies with a given value of probability, after transformation.
usually 95%.
3. Generalized Extreme Studentized Deviate
2. Outlying Results: (ESD) Test
Occasionally, observed analytical results are This is a modified version of the ESD Test that
very different from those expected. Aberrant, allows for testing up to a previously specified
anomalous, contaminated, discordant, spurious, number, r, of outliers from a normally
suspicious or wild observations; and flyers, distributed population. Let r equal 1, and n
rogues, and mavericks are properly called equal 10.
outlying results. Like all laboratory results, these Normalize each result by subtracting the mean
outliers must be documented, interpreted, and from each value and dividing this difference by
managed. Such results may be accurate the standard deviation.
measurements of the entity being measured,

 
but are very different from what is expected. Take the absolute value of these results, select
Alternatively, due to an error in the analytical
the maximum value R1 , and compare it to a
previously specified tabled critical value 1
system, the results may not be typical, even
though the entity being measured is typical.
When an outlying result is obtained, systematic based on the selected significance level (for
laboratory and process investigations of the example, 5%). If the the maximum value is
result are conducted to determine if an larger than the tabled critical value, it is
assignable cause for the result can be identified as being inconsistent with the
established. Factors to be considered when remaining data. If the maximum value is less
investigating an outlying result include—but are than the tabled critical value, there is not an
not limited to—human error, instrumentation outlier. Sources for -values are included in many
error, calculation error, and product or statistical textbooks.
component deficiency. If an assignable cause
that is not related to a product or component CONCLUSION
deficiency can be identified, then retesting may Method Validation is an important analytical
tool to ensure the accuracy and specificity of

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the analytical procedures with a precise The validation of analytical methods not only
agreement. This process determines the requires the performance of characteristics
detection and quantitation limit for the parameter but also the statistical treatments of
estimation of drug components. The validation the analytical data. The acceptance of the
procedures are performed along with the variation of the analytical data is determined by
system suitability. Some statistical tools are also these treatments.
used to interpret the analytical results of the
validation characteristics.

↓ REFERENCES
1. Validation of Compendial Procedures <1225>, The United States Pharmacopeia, 32th Rev., and The
National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc., 2009; I:
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guidelines, International Conference on Harmonisation of Technical Requirements For Registration Of
Pharmaceuticals For Human Use, 2005; 1.
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National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc., 2009; I:
735.
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and The National Formulary, 27th Rev., Rockville, MD: The United States Pharmacopeial Convention Inc.,
2009; I: 402.
6. Guideline on bioanalytical method validation, European Medicines Agency, London, UK, 2011; I: 8.
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2013
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