Method Validation

CHEM323.1: Analytical Chemistry 4

LABORATORY ACTIVITY 4

Ma. Karla Barela – Carmona | makarla.barela@ustp.edu.ph | 09358408004 | Chem323.1 | 2nd semester | SY 2022-2023
Method Validation
Please visit:
https://www.complianceonline.com/
resources/analytical-method-
validation-questions-and-
answers.html

Ma. Karla Barela – Carmona | makarla.barela@ustp.edu.ph | 09358408004 | Chem323.1 | 2nd semester | SY 2021-2022
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), ruggedness, and
robustness are severely discussed in an effort to prevent their
misguided utilization and ensure scientific correctness and
consistency among publications.
I. Introduction
• Analytical method validation is an essential requirement to
perform the chemical evaluation.
• 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.
• A validation examine includes testing multiple attributes of a method
to determine that it may provide useful and valid facts whilst used
robotically. To accurately investigate method parameters, the
validation test ought to consist of normal test conditions, which
includes product excipients.
• Therefore, a method validation examine is product-specific.
II. Procedure
• Parameters to be checked for method validation
– Selectivity/specificity
– Precision
– Accuracy
– Linearity
– Range
– Stability
– Limit of detection and limit of quantitation
– Mathematical derivations
II.1 Parameters to be checked for
method validation

▪ Selectivity/Specificity
▪ Precision
▪ Accuracy
▪ Linearity
▪ Range
▪ Stability
▪ Limit of Detection (LOD) and Limit of
Quantitation (LOQ)
II.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.
II.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.
II.1.2 Precision
• From the measured standard deviation (SD)
and Mean values, precision as relative
standard deviation (% rsd) is calculated.

• 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 -
II.1.2 Precision
• 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 standard
deviation (RSDR) and Concentration (C):

• For estimation of repeatability (RSDr), is

modified to:
II.1.2 Precision
• 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 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.
II.1.2 Precision
Table 1. Details of Horwitz values.

Note: The unmodified Horwitz equation is used as a criterion of

acceptability for methods collaboratively tested by CIPAC.
II.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 procedure 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.
II.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.
II.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.
II.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.
II.1.7 Limit of detection (LOD) and
limit of quantitation (LOQ)

• Limit of Detection (LOD) – 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.

• Limit of Quantitation (LOQ) – lowest

concentration at which the instrument is able to
detect and quantify. The noise to signal ratio for
LOQ should be 1:10.
II.1.7 Limit of detection (LOD) and
limit of quantitation (LOQ)
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
concentration of test item (mg/L) taken for
analysis of that particular a.i. or impurity
analysis.
II.1.7 Limit of detection (LOD) and
limit of quantitation (LOQ)
Determination of Limit of Detection (LOD) and Limit of
Quantitation (LOQ) from Detector Linearity experiments
(applicable to only instrument sensitivity).

• Calculations of LOD and LOQ values for

instrument sensitivity:
II.1.7 Limit of detection (LOD) and
limit of quantitation (LOQ)
Determination of Limit of Detection (LOD) and Limit of
Quantitation (LOQ) from Detector Linearity experiments
(applicable to only instrument sensitivity).

• Calculations of LOD and LOQ values for

method:
II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ):

Prepare a series of standard solutions (minimum

five concentrations covering working
concentrations used for routine analysis) and
analyze each solution minimum twice and record
the instruments response.
▪ Using the concentrations and
corresponding instrument response, LOD
and LOQ can be calculated as follows:
II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ): (cont.)

➢ Let the linear regression equation be Y = a + bX.

➢ Where, X and Y are the variables (data of two
parameters). Generally, X is called the
independent 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.
II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ): (cont.)
a) Method of arriving at “a” and “b”
i. Tabulate as given below:
X1 Y1
X2 Y2
. .
. .
. .
Xn Yn
𝜮𝑿 𝜮𝒀
ഥ=
𝑴𝒆𝒂𝒏 = 𝑿 ഥ=
𝑴𝒆𝒂𝒏 = 𝒀
𝒏 𝒏
ii. Calculate the following parameters:
II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ): (cont.)
iii. Calculate the slope “b,” and intercept “a” as given
below:

b) Method of calculation r (correlation coefficient)

II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ): (cont.)

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:

From this, the standard deviation for “a” and “b” are calculated.
II.1.8 Mathematical derivations
• Determination of limit of detection (LOD) and limit of
quantitation (LOQ): (cont.)

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

• Standard deviation for “a” represented

as Sa:

• Standard deviation for “b” represented

as Sb:
II.1.8 Mathematical derivations
• 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:

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 concentrations on Y-axis.
Laboratory Activity #1:
By Group, look for one (1) Method Validation scientific journal that
includes the parameters, and make a short report with the following
format, and present in the class next meeting.
Note:
• Laboratory Report should not be more than 5 pages
(except if there are more table and figures that needs to
be included in your report).
• Only a representative will submit in USTeP.
• Title = 10 %
• Objectives = 20%
• Procedure = 20%
• Parameter = 20%
• Results = 20%
• Conclusion = 10%
• Total = 100%
Ma. Karla Barela – Carmona | makarla.barela@ustp.edu.ph | 09358408004 | Chem323 | 2nd semester | SY 2020-2021
References

• Rao, Tentu Nageswara (April 25th 2018). Validation of

Analytical Methods. DOI: 10.5772/intechopen.72087

Ma. Karla Barela – Carmona | makarla.barela@ustp.edu.ph | 09358408004 | Chem323 | 2nd semester | SY 2020-2021