Principles of Analytical

Method Validation
Ian Ace A. Pacquiao, RPh,
MSPharm
 Introduction
⦿ Quality should be built into the product
and testing alone cannot be relied on to
ensure product quality
⦿ The validation process of an analytical
procedure demonstrates that a
procedure is suitable for its intended
purpose.
⦿ The validation process is an essential but
time-consuming activity for most
analytical development laboratories.
 Objectives
⦿ At the end of the activity, the students are
expected to:
• Discuss the basic principles of analytical method
validation
• Discuss the process of analytical method
validation
• Identify the information needed in an analytical
procedure
• Discuss the different analytical method
validation characteristics
 Basic Principles of Analytical
Method Validation
⦿ There are many reasons for the validation
of analytical procedures, which includes:
• Regulatory requirements
• Good Science
• Quality Control Requirements
Cycle of Analytical Methods
 Process of Analytical Method
Validation
⦿ Typical process that is followed in an
analytical method validation is
chronologically listed
• Planning and deciding on the method validation
experiments
• Writing and approval of method validation
protocol
• Analysis of the method validation data
• Reporting the analytical method validation
• Finalizing the analytical method procedure
 Process of Analytical Method
Validation
⦿ A normal validation protocol should
contain the following contents at a
minimum
• Objective of the protocol
• Validation parameters that will be evaluated
• Acceptance criteria for all validation parameters
evaluated
• Details of the experiments to be performed
• Draft analytical procedure
 Process of Analytical Method
Validation – Validation Parameters
Type of Analytical Testing for Impurities Assay –
Procedure Content/
Characteristics Identificati Quantitatio Limit Potency
on n
Accuracy - + - +
Precision -
Repeatability - + - +
Intermediate Precision + - +
Specificity + + + +
Detection Limit - - + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
 Information Required in an
Analytical Procedure
⦿ Minimal information that should be
included in the final analytical procedure
are as follows:
• Rationale of the analytical procedure and
description of the capability of the method
• Proposed analytical procedure
• List of permitted impurities and its levels in an
impurity assay
• Validation data
 Analytical Method Validation
Characteristics
⦿ Typical Validation Characteristics include:
• Accuracy
• Specificity
• Precision
• Linearity
• Range
• Robustness
• Detection limit
• Quantitation limit
• Sample and standard stability
 Analytical Method Validation
Characteristics
⦿ Accuracy
• International Convention on Harmonization
(ICH) defines accuracy as:
● Closeness of agreement between the values that are
accepted either as conventional true values or an
accepted reference value and the value found (in the
analytical procedure*)
• Also defined as trueness
● Closeness of agreement between value found using
the method and either an accepted reference value or
established true value
 Analytical Method Validation
Characteristics
⦿ Accuracy
• Evaluation method example: Spike and Recovery
Experiments
 Analytical Method Validation
Characteristics
⦿ Accuracy
• ICH recommends assessing a minimum of nine
determinations over a minimum of three
concentration levels covering the specified
range
• This is usually reported as percent recovery by
the assay of known added amount of analyte in
the sample
• Typical accuracy of the recovery of the drug
substance is expected to be at about 99-101%
 Analytical Method Validation
Characteristics
⦿ Method Precision
• The precision of an analytical procedure
expresses the closeness of agreement between a
series of measurements obtained from multiple
samples of the same homogenous sample under
prescribed conditions
• This is usually investigated at three levels mainly
● Repeatability
● Intermediate precision
● Reproducibility
 Analytical Method Validation
Characteristics
⦿ Repeatability (intra-assay precision)
• Measure of precision under:
● Same operating conditions
● Short interval of time
● Normal operating conditions
● Same equipment
 Analytical Method Validation
Characteristics
⦿ Assessment of Repeatability according to
ICH
• Use of a minimum of nine determinations over a
minimum of three concentration levels
• Use a minimum of six determinations at 100% of
the test concentration
 Analytical Method Validation
Characteristics – Repeatability Data
Replicate Percentage Label Claim
1 100.6
2 102.1
3 100.5
4 99.4
5 101.4
6 101.1
Mean 100.9
Percentage Relative Standard 0.90
Deviation
 Analytical Method Validation
Characteristics
⦿ Intermediate Precision
• This is defined as the variation within the same
laboratory
• Parameters investigated include:
● Day to day variation
● Analyst variation
● Equipment variation
• Experimental design is encouraged
 Analytical Method Validation
Characteristics
⦿ Intermediate Precision
• ICH recommends the reporting of standard
deviation, relative standard deviation and
confidence interval of the data
 Analytical Method Validation
Characteristics
⦿ Reproducibility
• Measures the precision between laboratories
• This parameter is considered in the
standardization of an analytical procedure
• Validation of this analytical procedure the
following should be done
● Similar studies performed at different laboratories
using the same sample and same experimental
designs
 Analytical Method Validation
Characteristics
⦿ Reproducibility
• Is measured during method transfers between
laboratories
● Originating laboratory
● Receiving laboratory
• Direct method transfer
● Protocol be initiated with:
● Details of the experiment to be performed
● Acceptance criteria
 Characteristics – Method
Transfer Data
Runs Average Percent
Originating Laboratory 12 100.7
Receiving Laboratory 4 100.2
 Analytical Method Validation
Characteristics
⦿ Specificity
• ICH defines this as the ability to assess
unequivocally an analyte in the presence of
components that may be expected to be present
• Identity Tests should recognize compounds of
closely related structures which are likely to be
present should be discriminated from each other
 Analytical Method Validation
Characteristics
⦿ Specificity
• Identity tests
● Can be confirmed by obtaining positive results from
samples containing the analyte coupled with negative
results from samples that do not contain the analyte
● May also be applied material structurally similar or
closely related to the analyte to confirm a positive
response is not obtained
 Analytical Method Validation
Characteristics
⦿ Detection Limits
• This is a characteristic for the limit test only
• This is the lowest amount of analyte in a sample
that can be detected but not necessarily
quantitated under specified experimental
conditions
• Usually expressed in concentration of the
analyte in the sample
● Percentage, parts per million (ppm), parts per billion
(ppb)
 Analytical Method Validation
Characteristics
⦿ Detection Limits
• Approaches to determine detection limits
● Visual examination
● Instrumental methods
● Analysis of a series of samples with known concentrations
and establishing minimum level at which analyte can be
reliably detected
 Analytical Method Validation
Characteristics
⦿ Quantitation Limits
• Characteristic of quantitatve assays for low
levels of compounds in sample matrices such as
● Impurities in bulk drug substance
● Degradation products in finished pharmaceuticals
• The QL of a method is affected by both the
detector sensitivity and the accuracy of sample
preparation at the low concentration of
impurities
 Analytical Method Validation
Characteristics
⦿ Quantitation Limit
• ICH recommends three approaches to the estimation
of QL
● Visual evaluation
● Determination of signal to noise ratio (instrumental
method)
● Estimation of QL using an equation
• Whatever approach is applied, the QL should be
subsequently validated by the analysis of a suitable
number of samples prepared at the QL and
determining the precision and accuracy at this level
 Analytical Method Validation
Characteristics
⦿ Linearity
• This is the ability of an analytical procedure to
obtain test results of variable data which are
directly proportional to the concentration in the
sample
• Approaches for determination of linearity
● Weigh different amounts of standard to prepare
linearity solutions at different concentrations
● Prepare a stock solution of high concentration and
dilution of the stock solution
 Analytical Method Validation
Characteristics
⦿ Range
• This is the interval between the upper and the
lower concentration of analyte in the sample for
which it has been demonstrated that the analyte
has a suitable level of precision, accuracy and
linearity
 Analytical Method Validation
Characteristics
⦿ Range
• For assay of drug substances or finished
products, it is normally recommended to have a
range of 80-120% of nominal concentration
• For content uniformity, a normal range would
cover 70-130% of the nominal concentration
unless a wider and more appropriate range is
justified
• For dissolution testing, a normal range is +20%
over the specified range
 Analytical Method Validation
Characteristics
⦿ Robustness
• This is a measure of the analytical method to
remain unaffected by small but deliberate
variations in method parameters and provides
an indication of its reliability during normal
usage
• This is normally considered during the
development phase of the drug and depends
upon the procedure under study
 Analytical Method Validation
Characteristics
⦿ Robustness
• Common method parameters that can affect the
analytical procedure should be considered
based on the analytical technique and the
properties of the samples. Specifically:
● Sample preparation
● Extraction time
● Sample solvent
● Membrane filters
● Sample and standard stability
 Analytical Method Validation
Characteristics
⦿ Robustness
• When results are affected by critical
experimental parameters, precautionary
statements should be included in the analytical
procedure should be explicitly written.