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 Chapter 7

Validation of Analytical Methods

Tentu Nageswara Rao

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72087

Abstract
Method validation is a key element in the establishment of reference methods and
within the assessment of a laboratory’s competence in generating dependable analytical
records. Validation has been placed within the context of the procedure, generating
chemical data. Analytical method validation, thinking about the maximum relevant
processes for checking the best parameters of analytical methods, using numerous
relevant overall performance indicators inclusive of selectivity, specificity, accuracy,
precision, linearity, range, limit of detection (LOD), limit of quantification (LOQ), rug-
gedness, and robustness are severely discussed in an effort to prevent their misguided
utilization and ensure scientific correctness and consistency among publications.

Keywords: method validation, accuracy, precision, linearity, LOD, LOQ

1. Introduction

Analytical method validation is an essential requirement to perform the chemical evaluation

[1–3]. Method validation is a procedure of performing numerous assessments designed to
verify that an analytical test system is suitable for its intended reason and is capable of
providing beneficial and legitimate analytical data [4–8]. A validation examine includes testing
multiple attributes of a method to determine that it may provide useful and valid facts whilst
used robotically [9–11]. To accurately investigate method parameters, the validation test ought
to consist of normal test conditions, which includes product excipients [11–14]. Therefore, a
method validation examine is product-specific.

2. Procedure
2.1. Parameters to be checked for method validation

• Selectivity/Specificity
• Precision

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
132 Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

• Accuracy
• Linearity
• Range

• Stability
• Limit of Detection (LOD) and Limit of Quantitation (LOQ)

2.1.1. Selectivity/specificity
Selectivity of an analytical method is its ability to measure accurately an analyte in the
presence of interferences that may be expected to be present in the sample matrix.

Selectivity is checked by examining chromatographic blanks (from a sample that is known to

contain no analyte) in the expected time window of the analyte peak. And the raw data for
selectivity will be recorded in the raw data in approved formats.

2.1.2. Precision

Precision of a method is the degree of agreement among individual test results when the
procedure is applied repeatedly to multiple samplings.
Precision is measured by injecting a series of standards or analyzing series of samples from
multiple samplings from a homogeneous lot. From the measured standard deviation (SD) and
Mean values, precision as relative standard deviation (% rsd) is calculated.

SD
%rsd or CV ¼  100 (1)
Mean

The raw data for precision will be recorded in the approved format and the acceptance criteria
for precision will be given in the respective study plan or amendment to the study plan.
OR
Precision can be also calculated by using Horwitz equation:

The acceptable percent of relative standard deviation results for precision may be based on the
Horwitz equation, an exponential relationship between the among-laboratory relative stan-
dard deviation (RSDR) and Concentration (C): [15]

%RSDR ¼ 2ð1 0:5logCÞ

(2)

For estimation of repeatability (RSDr), is modified to:

%RSDr ¼ %RSDR  0:67 (3)

The Horwitz curve has been empirically derived and has been proven to be more or less
independent of analyte, matrix and method of evaluation over the concentration range C = 1
(100%) to C = 10 9 by the evaluation of vast numbers of method precision studies. The
 Validation of Analytical Methods 133
http://dx.doi.org/10.5772/intechopen.72087

modified Horwitz values for repeatability CV given under may be used for guidance. If
measured repeatability is outside those values, suggested explanation must be submitted for
consideration. The details were presented in Table 1.

2.1.3. Accuracy
The accuracy of an analytical method is the degree of agreement of test results generated by
the method to the true value.
Accuracy is measured by spiking the sample matrix of interest with a known concentration of
analyte standard and analyzing the sample using the “method being validated.” The proce-
dure and calculation for Accuracy (as% recovery) will be varied from matrix to matrix and it
will be given in respective study plan or amendment to the study plan.

2.1.4. Linearity
The linearity of an analytical method is its capability to elicit check consequences which might
be at once, or with the aid of well described mathematical adjustments, proportional to the
concentration of analytes in within a given range.
Linearity is determined by injecting a series of standards of stock solution/diluted stock
solution using the solvent/mobile phase, at a minimum of five different concentrations in the
range of 50–150% of the expected working range. The linearity graph will be plotted manually/
using Microsoft Excel or software of the computer (Concentration vs. Peak Area Response) and
which will be attached to respective study files.

2.1.5. Range

The range of an analytical method is the interval between the upper and lower levels that have
been demonstrated to be determined with precision, accuracy and linearity using the set
method. This range will be the concentration range in which the Linearity test is done.

Percent of analyte Proposed acceptable % RSDr

(Horwitz value  0.67)

100.00 1.340
50.00 1.490
20.00 1.710
10.00 1.900
5.00 2.100
2.00 2.410
1.00 2.680
0.25 3.300

Note: The unmodified Horwitz equation is used as a criterion of acceptability for methods collaboratively tested by
CIPAC.

Table 1. Details of Horwitz values.

134 Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

2.1.6. Stability
Many analytes readily decompose prior to chromatography investigations, for example during
the preparation of the sample solutions, during extraction, clean-up, phase transfer, and
during storage of prepared vials. Under these circumstances, method development should
investigate the stability of the analyte. Accuracy test takes care of stability. It is required to
mention in the method how long a sample after extraction can be stored before final analysis,
based on the duration taken for accuracy test.

2.1.7. Limit of detection and limit of quantitation

The term LOD is defined as the lowest concentration at which the instrument is able to detect
but not quantify and the noise to signal ratio for LOD should be 1:3. The term LOQ is defined
as the lowest concentration at which the instrument is able to detect and quantify. The noise to
signal ratio for LOQ should be 1:10.
Determination of Limit of Detection (LOD) and Limit of Quantitation (LOQ) from Detector
Linearity experiments (applicable to only instrument sensitivity).
LOD and LOQ values are calculated manually by taking Noise to signal ratio of a lowest/
known concentration of linearity samples and it will be expressed in μg/ml or ppm. To
calculate in %, values of LOD and LOQ will be multiplied by 100/lowest or known concentra-
tion of test item (mg/L) taken for analysis of that particular a.i. or impurity analysis.
Calculations of LOD and LOQ values for instrument sensitivity:

Noise
LOD ðmg=LÞ ¼ 3   Lowest concentration of the linearity samples
Signal
Noise
LOQ ðmg=LÞ ¼ 10   Lowest concentration of the linearity samples
Signal

Calculations of LOD and LOQ values for method:

LOD ðmg=LÞ
LOD ð%Þ ¼  100
Test item conc:used for quantification
LOD ðmg=LÞ
LOQ ð%Þ ¼  100
Test item conc:used for quantification

OR

2.1.8. Mathematical derivations

2.1.8.1. Determination of limit of detection (LOD) and limit of quantitation (LOQ)

Prepare a series of standard solutions (minimum five concentrations covering working con-
centrations used for routine analysis) and analyze each solution minimum twice and record
the instruments response.
 Validation of Analytical Methods 135
http://dx.doi.org/10.5772/intechopen.72087

• Using the concentrations and corresponding instrument response, LOD and LOQ can be
calculated as follows:

Let the linear regression equation be Y ¼ a þ bX.

Where, X and Y are the variables (data of two parameters). Generally, X is called the indepen-
dent variable and Y, the dependent variable.
Take concentration on X-axis and instrument response on Y-axis.

“a” and “b” are the regression constants. Further, “a” is known as the intercept and “b,” the
slope of the line.

Let (X1, Y1), (X2, Y2), (X3, Y3)…(Xn, Yn) be the set of values required to be fit in the linear
equation.

a. Method of arriving at “a” and “b” y

i. Tabulate as given below:

X1 Y1
X2 Y2

. .
. .
. .

Xn Yn
____________________________________

Mean, ¼ X ¼ Σ X=n Y ¼ Σ X=n

____________________________________
ii. Calculate the following parameters:

2
Σ xx ¼ Σ X X ¼ ΣX2 ðΣXÞ2 =n

2
Σ yy ¼ Σ Y Y ¼ ΣY2 ðΣYÞ2 =n
Σ xy ¼ ΣXY ð ΣXÞ ðΣYÞ=n

iii. Calculate the slope “b,” and intercept “a” as given below:
P
xy
b¼P
xx

a¼Y bX

b. Method of calculation r (correlation coefficient)

136 Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

P
xy
r ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P P ffi
xx: yy

c. Method of calculation standard deviation for “a” and “b”

The standard deviation of the individual deviations of measured values in Y, above and below
the linear line (fitted line) is:

vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
uP nP o
2 P
u
t yy ð xy Þ = xx
Sy:x ¼
n 2

From this, the standard deviation for “a” and “b” are calculated.

Standard deviation
rffiffiffiffiffiffiffiffiffiffiffi
P 2ffi
X
for “a,” represented = Sy:x n P xx

as Sa

Standard deviation.
qffiffiffiffiffiffiffiffiffiffiffiffi
1
For “b,” represented = Sy:x n P xx

as Sb

2.1.8.2. Application of a, b, and Sa to obtain limit of detection and limit of quantitation

When Sa is obtained for a linear calibration line, then it provides a clear information on the
standard deviation of the “Blank” (or Control) response from the instruments.

The LOD and LOQ can be worked out, as given below:

jaj þ 3 Sa
LOD ¼
b
jaj þ 10 Sa
LOQ ¼
b

Note:

• The above calculations can be programmed in a computer but before every use, the
computer program must be validated using the example given in section

• The above procedure can also be used for obtaining LOD and LOQ of the method from
recovery test results by taking fortified concentration on X-axis and obtained concentra-
tions on Y-axis.
 Validation of Analytical Methods 137
http://dx.doi.org/10.5772/intechopen.72087

3. Example

In this example, the linear regression equation is employed to find out the extent of linear
response of an Detector to a reference analytical standard in the concentration range of about
0.2–3.0 ppm.
Each of these working standards is injected thrice (1 μl per injection), and the peak area counts
corresponding to the active ingredient peak are given below.
From the peak areas corresponding to each concentration level, the mean, standard deviation
(SD) and coefficient of variation (%CV) are also calculated. The details were presented in
Table 2.
Fitting the data of concentration of standard solution and mean detector response (peak
area counts) in a linear equation
Let the equation be Y ¼ a þ bX.

Where, Y = Mean peak area counts and X = Concentration of standard solution, μg/ml.
The calculations were presented in Table 3.

Conc. of standard solution (μg/ml) Peak area Mean SD %CV

(n 1)

1 2 3

0.1956 32,827 33,299 32,731 32,952 304 0.923

0.4890 87,783 88,480 87,446 87,903 527 0.600
0.9780 176,037 174,675 177,203 175,972 1265 0.719
1.467 246,212 250,786 246,849 247,949 2477 0.999
1.956 319,143 319,615 315,316 318,025 2358 0.741
2.934 415,059 410,773 418,407 414,746 3827 0.923

%CV = SD  100/Mean: The coefficient of variation (CV) shows that the Injection variation is less than 1%.

Table 2. Calculation details of mean, SD, and %CV.

Sl. no. Y X

1. 32952 0.1956
2. 87903 0.4890
3. 175972 0.9780
4. 247949 1.4670
5. 318025 1.9560
6. 414746 2.9340

Table 3. Calculation details of additional parameters.

138 Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

P P P
Y ¼ 1277547 X ¼ 8:0196 XY ¼ 2424193:441
Y ¼ 212924:5 X ¼ 1:3366 n¼6
P 2
Y ¼ 3:7441177  1011
P 2
X ¼ 15:820245

Using the above parameters, calculate the following

X2 XÞ2 =n
P P P
xx ¼ ð
¼ 15:820245 ð8:0196Þ2 =6
¼ 5:101248

Y2 YÞ2 =n
P P P
yy ¼ ð
¼ 3:7441176  1011 ð1277547Þ2 =6
¼ 1:0239070  1011
P P P P
xy ¼ XY ð XÞð YÞ=n
¼ 2424193:441 ð1277547Þð8:0196Þ=6
¼ 716624:12

Calculation of a, b, and r
P
xy
b¼P
xx
716624:12
¼
5:101248
¼ 140480:16
P
xy
b¼P
xx
716624:12
¼
5:101248
¼ 140480:16

a ¼ Y bX
¼ 212924:5 140480:16  1:3366
¼ 25158:718
P
xy
r ¼ pP ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiP
ffiffiffiffiffiffiffiffiffiffi
xx
yy
716624:12
r ¼ pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi11
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi ¼ 0:99157
1:0239070X10 X5:101248

Note: Sometimes r2 is also used to express the goodness of fit.

Calculation of standard deviation for a and b:
 Validation of Analytical Methods 139
http://dx.doi.org/10.5772/intechopen.72087

sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
nP P o
ð xyÞ2 = xx
P
yy
Sy:x ¼
n 2
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 n offi

11 2
1:0239070X10 ð716624:12Þ =ð5:101248Þ
¼
6 2
¼ 20731:806

The standard deviation for a is calculated as:

sffiffiffiffiffiffiffiffiffiffiffiffiffiffi
P 2ffi
X
Sa ¼ Sy:x P
n xx
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
15:820245
¼ 20731:806
6  5:101248
¼ 14905

The standard deviation for b is calculated as

rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
Sb ¼ Sy:x P
n
xx
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
1
¼ 20731:806
6  5:101248

Note: Assay procedures vary from highly exacting analytical determinations to subjective
evaluations of attributes. Therefore different test methods require different validation schemes.

Category I
Analytical methods for quantitation of major excipients and/or active ingredients, and pre-
servatives in finished goods.

Category II
Analytical methods for determination of impurities or degradation compounds in finished
goods. These methods include quantitative assays and limit tests, titrimetric and bacterial
endotoxin tests.

Category III

Analytical methods for determination of performance characteristics, e.g., sterility testing,

dissolution and drug release for pharmaceutical products.

Data Elements Required for Assay Validation.

Details of required validation parameters of assay presented in Table 4.
140 Calibration and Validation of Analytical Methods - A Sampling of Current Approaches

Analytical parameters Assay Assay category 2 Limit Test Assay

category 1 quantitative category III

Assay accuracy Yes Yes * *

Precision Yes Yes No Yes
Specificity Yes Yes Yes *
Limit of detection Yes Yes Yes *
Limit of quantitation Yes Yes No *
Linearity Yes Yes No *
Range Yes Yes * *
Robustness * * * *

*May be required depending on the specific test.

Table 4. Validation parameters of assay [16].

4. Conclusions

Analytical validation data playing a fundamental role in pharmaceutical industry, pesticide

industry for releasing the economic batch and long term stability information consequently,
the records must be produced to suited regulatory authority requirements.

Author details

Tentu Nageswara Rao

Address all correspondence to: [email protected]

Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India

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