Principles of Analytical Method

Validation
Danielle T. Matuginas, RPh
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 Identification Quantitation Limit Potency

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 Deviation 0.90
 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
 Analytical Method Validation
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.