Razi

Vaccine & Serum

Research Institute

Analytical Method
Validation

A. Es-haghi
Ph.D.
Dept. of Physico chemistry
Razi vaccine and serum research institute
[email protected]

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 Introduction Razi
Vaccine & Serum
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ƒ Test procedures for assessment of the quality levels of

pharmaceutical products are subject to various requirements
ƒ The CGMP regulations [21 CFR 211.194(a)] require that test
methods, which are used for assessing compliance of
pharmaceutical products with established specifications, must meet
proper standards of accuracy and reliability
ƒ according to these regulations [21 CFR 211.194(a)(2)], users of
analytical methods described in the USP and the NF are not required
to validate accuracy and reliability of these methods, but merely
verify their suitability under actual conditions of use
ƒ Recognizing the legal status of USP and NF standards, it is
essential, therefore, that proposals for adoption of new or revised
compendial analytical methods be supported by sufficient laboratory
data to document their validity

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 What is Validation? Razi
Vaccine & Serum
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ƒ USP 30-NF 25, General Chapter <1225> Validation of

Compendial Methods
• “Validation of an analytical method is the process by which it is
established, by laboratory studies, that the performance
characteristics of the method meet the requirements for the
intended analytical applications.”
ƒ ICH Guideline Q2A – Text on Validation of Analytical
Procedures
• “The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intended purpose.”
ƒ FDA draft guidance – Analytical Procedures and Method
Validation
• “Methods validation is the process of demonstrating that
analytical procedures are suitable for their intended use.”

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 Analytical Method Validation Razi
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ƒ In actual practice, analytical methods validation involves

• Established analytical method

• Data from controlled samples
• Statistical evaluation
• Assessment of performance characteristics and applicability

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 USP Performance Characteristics Razi
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ƒ Performance Characteristics are discrete attributes, derived

by actual execution of an analytical method using controlled
samples, which demonstrate the suitability and proper use of
that method for a specific application

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 USP Performance Characteristics Razi
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ƒ SPECIFICITY
ƒ LINEARITY
ƒ RANGE
ƒ DETECTION LIMIT
ƒ QUANTITATION LIMIT
ƒ ACCURACY
ƒ PRECISION
ƒ RUGEDNESS
ƒ ROBUSTNESS
ƒ SYSYEM SUITABILITY

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 Specificity / Selectivity Razi
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ƒ ICH Q2A and USP

• the ability to assess unequivocally the analyte in the presence of
components that may be expected to be present, such as
impurities, degradation products, and matrix components
ƒ Lack of specificity of an individual analytical procedure may be
compensated by other supporting analytical procedure(s)
ƒ Other international authorities (IUPAC, AOAC) have preferred
the term “Selectivity” reserving “Specificity” for those
procedures that are completely selective

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 Specificity Razi
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ƒ Identification
• to ensure the identity of the analyte
ƒ Purity Test
• accurate statement of the content of impurities of an analyte
(related substances, heavy metals, residual solvents, etc.)
ƒ Assay
• an exact result which allows an accurate statement on the content
of potency of the analyte in a sample

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 Specificity Razi
Vaccine & Serum
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ƒ Some analytical procedures are not sufficiently specific for the

intended purpose
• Assay by titration
• Assay of enantiomer by achiral method
• Identification by UV absorbance
ƒ A combination of two or more analytical procedures is
recommended to achieve sufficient specificity

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 Specificity Razi
Vaccine & Serum
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ƒ Should be one of the first validation items

ƒ When the criteria are not met, this indicates that the method is
not sufficiently developed
ƒ Poor specificity can impact accuracy, precision and linearity

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 Linearity Razi
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ƒ The ability to generate responses which are directly

proportional to the concentration of an analyte in a
sample

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 Linearity Razi
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ƒ A linear relationship requires fewer standards to define the

response curve than a non-linear one
ƒ justifies the use of a single point standard to quantitate across
the range

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 Linearity Razi
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ƒ USP <1225>
• Evaluated across the RANGE
• Visual examination of a plot of signals as a function of
concentration
• Statistical evaluation of correlation coefficient, y-intercept, slope,
and residual sum of squares of regression line or after
mathematical transformation
ƒ ICH Guidance
• Performed directly on the drug substance (dilution of standard
stock solution)
• Separate weighing of synthetic mixtures of the drug components
• Minimum of five concentration is recommended

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 Linearity Razi
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ƒ Required for
• Assays (Category I)
• Impurity tests (Quantitative Category II)
• May be for Performance Tests (Category III-Dissolution, etc.)
ƒ Not required for
• Limit tests (Category II- Impurity Test)
• Identification

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 Range Razi
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ƒ UPS <1225>
• The interval between the upper and lower quantitation levels
of analyte (including these levels) demonstrated by suitable
precision, accuracy, and linearity
ƒ ICH Guidance
• The following minimum specified ranges should be
considered
• Assay of drug substance or finished drug product
– from 80% to 120% of the test concentration
• Content Uniformity
– minimum of 70% to 130% of the test concentration,
unless otherwise justified

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 Detection Limit/ Quantitation Limit Razi
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ƒ Characteristic for low level impurity assays

ƒ DL (LOD)
• the lowest amount of any analyte which can be detected but not
necessarily quantitated
ƒ QL (LOQ)
• the lowest amount of analyte that can be determined with
acceptable precision and accuracy

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 Determination of Detection Limit Razi
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ƒ Non-instrumental Methods
• analysis of samples with known concentrations of analyte to
establish the minimum concentration at which the analyte can be
reliably (the ability to reproducibly obtain a signal for the analyte)
detected
• Examples
– TLC, titrations, color compositions
ƒ Instrumental
• Same as for non-instrumental methods, plus estimation of the DL
can be made at a value at which the signal to noise ratio (S/N) is
2:1 or 3:1

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 Estimation of DL (ICH) Razi
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ƒ DL = 3.3 σ/S
• Where: σ = Standard deviation (“noise”) of response
• S = slope of the calibration curve for linearity
ƒ DL can be based on any of the following
• σ of the blank
• σ of the lowest level linearity point
• the calibration plot (regression)

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 Quantitation Limit
Razi
Definition Vaccine & Serum
Research Institute

ƒ USP <1225>
• the lowest amount of analyte in a sample that can be determined
with acceptable precision and accuracy under the stated
experimental conditions
ƒ Essentially identical to ICH Q2A
ƒ Quantitation Limit (QL) = Limit of Quantitation (LOQ)

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 Determination of QL Razi
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ƒ Non-Instrumental
• from analysis of samples with appropriate minimum level of
analyte quantified with acceptable accuracy and precision
ƒ Instrumental
• same as non-instrumental, plus estimation of the QL can be made
at a value at which the signals to noise is 10:1

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 Estimation of QL (ICH) Razi
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ƒ QL = 10 σ/S
• where σ = standard deviation of response
• S = slope of the calibration curve for linearity
• σ can be based standard deviation of the blank or of the lowest
level linearity point

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 Case Study Razi
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 Accuracy Razi
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ƒ Definition
• USP
• “The closeness of the result obtained by the method to the true
value.”
• ICH
• “The closeness of the result obtained by the method to a value
that is accepted as conventionally true value or as a reference
value.”

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 Accuracy Razi
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ƒ The accuracy of an analytical method should be

established across its range

ƒ ICH recommends a minimum of 9 determinations over a

minimum of 3 concentrations covering the stated
quantitation range (3 test at 3 conc.)
ƒ Accuracy is calculated as
• the percentage of recovery by the assay of the known added
amount of analyte in the sample
• the difference between the mean and the accepted true
value, together with confidence intervals

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 Accuracy Razi
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ƒ 5 ways to determine the Accuracy

• Testing of a Reference Standard
• Mixture with Excipients (Spiked placebo)
• Standard addition (Spiked sample)
• Inferred from linearity and specificity data
• Comparison with a method known to be accurate (reference
method)

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 Accuracy Razi
assay of a drug substance Vaccine & Serum
Research Institute

ƒ Testing of a Reference Standard

ƒ Comparison with a reference standard method

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 Recovery of Drug F Ref Std at different levels Razi
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 Comparison with Reference Method Razi
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There is no difference with p < 0.05

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 Precision Razi
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ƒ The degree of agreement among results from individual

samplings of a homogeneous sample

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 Precision Razi
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ƒ Precision is expressed as RSD

ƒ Precision should be evaluated across the specified
Quantitation range of the method
ƒ Determination
• Assay individual samples of a homogeneous preparation
• Calculate Standard Deviation or Relative Standard Deviation
ƒ Method precision should include all sources of variation from
sample preparation to rounding the final test result

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 Precision Razi
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ƒ ICH recommends a minimum of 9 determinations over a

minimum of 3 concentrations covering the stated quantitation
range (3 test at 3 conc.)

OR

ƒ Using a minimum of 6 determinations at 100% of the test

concentration

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 ICH-USP Level of Precision Razi
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ƒ Repeatability
• agreement within a short period or the same analyst and
instrumentation
ƒ Intermediate precision
• agreement in results intra-laboratory but from different days,
analysts and equipment (as appropriate)
ƒ Reproducibility
• agreement in results between laboratories (as in a collaboration
study)

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 Precision vs. Ruggedness Razi
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ƒ In USP, Reproducibility and intermediate precision are

also considered together with Ruggedness

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 Precision
Razi
choice of acceptance criteria Vaccine & Serum
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ƒ Practical Considerations
• Ability to meet criteria
• may be instrument dependent
• Maximum acceptance criteria for precision should be linked to
acceptance criteria for claim or specification to be able to obtain a
reliable result (reduce Out-Of Specifications)

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 Precision Razi
Vaccine & Serum
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ƒ Repeatability (5 replicate injections)

Assay Limit (%) Max, % RSD
99.0 – 101.0 0.37
98.5 – 101.5 0.55
98.0 – 102.0 0.73
95.0 – 105.0 1.9
90.0 – 110.0 3.9
Impurities (depending on level) 5 to 25

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 Precision
Razi
choice of acceptance criteria Vaccine & Serum
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ƒ Intermediate Precision
• FDA recommends a minimum of 2 different days with different
analysts
• Acceptance criteria
• Perform F-test
• results between labs should not be statistically different
• Typically about 2x repeatability (2 x RSD)
– However, a statistical difference may still not be
significant (practical consideration). Need to evaluate
suitability for methods intended use

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 Reproducibility Precision Razi
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ƒ FDA
• at least 2 laboratories within the company
ƒ FDA
• for multi-laboratory follow ASTM 691
• Standard practice for conducting an laboratory study to
determine the precision of a test method
ƒ AOAC Protocol
• 8 samples, 8 laboratories

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 Ruggedness Razi
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ƒ Reproducibility with the same samples using different

laboratories, analysts, days, reagent lots (same brand), and
environmental conditions
ƒ Precision should be evaluated across the specified
Quantitation range of the method

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 Ruggedness vs. Robustness Razi
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ƒ Reproducibility of the test results under conditions normally

expected from laboratory to laboratory and from analyst to
analyst
ƒ Method parameters are kept constant (difference with
robustness)

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 Ruggedness Razi
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ƒ Determination
• Analysis of an homogeneous sample in different laboratories, by
different analysts, under prevalent environmental conditions using
the specified parameters
• The degree of reproducibility of test results is determined as
function of the variables

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 Ruggedness Razi
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ƒ study normal variables

• Analyst A, B, C
• Day 1, 2, 3
• Lab L, M N
• Column X, Y, Z

ƒ Selected comparisons are evaluated since evaluating all

possible variations would require 81 experiments (3 x 3 x 3 x
3)
ƒ The influence of each variable may be evaluated using a multi-
factor experimental design (Plackett- Burman, Hendrix, etc.)

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 Ruggedness Razi
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Degree of reproducibility of test results as a function of

the variables

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 Robustness Razi
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ƒ The ability to remain unaffected by small but deliberate

variations in method parameters- evaluates reliability
ƒ Provides an indication of the reliability of the method
during normal usage

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 Robustness Razi
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ƒ Evaluated by varying method parameters and determining the

effect (if any) on the results of the method
ƒ It should be considered early in the development of a method
ƒ If the results are susceptible to parameter variations, these
parameters should be adequately controlled and a
precautionary statement included in the method

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 Robustness Razi
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ƒ Two approaches
• Uni-variable
• Systematically varying each parameter sequentially
• Typically, each parameter may be varied by 5 – 10% above
and below the value set in the method
• Multi-variable
• Use of the statistical design
– Central Composites
– Fractional factorials,
– Two-factor Plackett-Burman statistical approach
– Hendrix

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 System Suitability Razi
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ƒ Ensures that both methodology and instrumentation are

performing within expectation prior to the analysis of the
test samples

ƒ USP General Chapter <1225>

• “System suitability tests are based on the concept that the
equipment, electronics, analytical operations, and samples to be
analyzed constitute an integral system that can be evaluated as
such. System suitability test parameters to be established for a
particular procedure depend on the type of procedure being
evaluated. They are especially important in the case of
chromatographic procedures.”

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 System Suitability Razi
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ƒ Provides assurances that the system is working at the

time of analysis
ƒ Ensures that both methodology and instrumentation are
performing within expectations prior to the analysis of the
test samples
ƒ Should be monitored during run time to verify that criteria
remain realistic and achievable

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 System Suitability Razi
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ƒ Parameters for chromatographic method

• Resolution – specificity
• Column efficiency – specificity
• Relative standard deviation – precision
• Tailing factor – accuracy and precision
• QL – sensitivity
• Capacity factor – specificity
• Reference Standard Check - analyte

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 USP Method Categories Razi
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ƒ Category I
• Quantitative test for major active ingredients (chromatographic
and titration)
ƒ Category II
• Impurities or degradation compounds; quantitative or limit tests
ƒ Category III
• performance tests (dissolution, drug release, tapped density,
particle size)
ƒ Category IV
• Identification tests (TLC, IR, UV, Chemical ID <191>

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 USP <1225>
Razi
Data Elements for Validation Vaccine & Serum
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 ICH Q2B
Razi
Data Elements for Validation Vaccine & Serum
Research Institute

May be required, depending on the nature of the specific test

 Is not needed if reproducibility has been performed

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 Choice of Performance Characteristics Razi
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ƒ Not all Performance Characteristics are needed to validate

an analytical method or validated in the same way
• Determine the expected use of the test procedure (e.g., assay,
dissolution, limit test, ID test)
• Take into account the type of analytical technique used
(chromatographic, spectrophotometric, titration, etc.)
ƒ Establish the requirements of the analytical application
• Degree of precision and accuracy needed
• The concentration range of the analyte
• The level of tolerable interference, if any

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 Protocol for Final Validation Studies Razi
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ƒ Initial analytical methods are often developed in R&D

areas with some preliminary validation data
ƒ These procedures can be slightly modified when
transferred to QC areas for complete validation including
multi-site collaborative studies

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 Validation Protocols Razi
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ƒ The validation protocol document contains

• Description of the analytical method and its intended use
(purpose)
• List of applicable performance characteristics
• Appropriate acceptance criteria
• Review and approval signatures

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 Pre-validation Assessment Razi
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ƒ Equipment
• Suitable for expected accuracy?
ƒ Reference Materials
• Suitability characterized?
ƒ Analytical Method
• Is procedure finalized?
ƒ Validation Protocol
• Management / QA approved?

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 Pre-validation Assessment Razi
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ƒ Equipment

• Demonstrate that equipment used in validation studies is suitable

for use and is comparable to equipment used for routine analysis
• Calibrated (as applicable)
• Qualification should have been performed
– Installation Qualification
– Operation Qualification
– Performance Qualification
• Routine maintenance performed- proper working order

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 Pre-validation Assessment Razi
Vaccine & Serum
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ƒ Reference Materials
• evaluate suitability of use

• FDA requires no additional evaluation of USP Reference

Standards
• Standardization of reagents vs. Primary standards when available
(i.e., use of reagent grade chemical need qualification)
• Principal analyte (purity and homogeneity)
• Impurity Standards
– chemical purity is very critical if used to determine
relative response
– Solution stability of standard preparations

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 Pre-validation Assessment Razi
Vaccine & Serum
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ƒ Analytical Method

• must be final before validation

• Individual procedure steps may impact overall method accuracy
or precision
• Solution preparations
– concentration of analyte in solution for analysis (suitable
range)
• System suitability
– necessary parameters and acceptance criteria must be
specified
– Changes may negatively impact validation studies
previously performed
– Later changes in tailing factor or resolution criteria from
robustness evaluation may have a significant impact on
integration in accuracy evaluations

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 Standard and Sample Solution Preparation Razi
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ƒ Overall method precision and accuracy are also impacted by

sampling, weighing, and quantitative dilutions
ƒ No instrumental technology can overcome these errors
• The precision and accuracy of a final analytical method can be
better than the precision and accuracy of the preparation of the
test solutions

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 Revalidation Razi
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ƒ Revalidation may be necessary in the following cases

• a submission to the USP of a revised analytical method
• use of an established general method with a new product or
raw material

ƒ The ICH documents give guidance on the necessity for

revalidation in the following circumstances
• changes in the synthesis of the drug substance
• changes in the composition of the drug product
• changes in the analytical procedure

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 RVSRI deffects in Analytical Method Validation Razi
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ƒ Equipment
ƒ Personnel
ƒ Reference Materials
ƒ Analytical Methods
ƒ Validation Protocol
ƒ Change control
ƒ Weak links between different chain to final product be
completely assured

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 Quick Quiz
Razi
Cumulative errors in serial dilution Vaccine & Serum
Research Institute

ƒ To prepare 100 fold dilution of a stock solution, what is the

most precise procedure when considering the necessary
individual Class A pipette and volumetric flask tolerances?

A. 1.0 mL/10 mL; 1.0 mL/10 mL

B. 5.0 mL/50 mL; 5.0 mL/50 mL
C. 10.0 mL/100 mL; 10.0 mL/100 mL
D. 1.0 mL/100 mL
E. 2.0 mL/200 mL
F. 5.0 mL/500 mL

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 Tolerance of Class A Glassware
Razi
(see USP <31> Volumetric apparatus) Vaccine & Serum
Research Institute

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 Quick Quiz
Razi
Cumulative errors in serial dilution Vaccine & Serum
Research Institute

Sum of step errors

ƒ A. 1.0/10; 1.0/10L 0.6 + 0.2 + 0.6 + 0.2 = ± 1.6%

ƒ B. 5.0/50; 5.0/50 0.2 + 0.1 + 0.2 + 0.1 = ± 0.6%
ƒ C. 10.0/100; 10.0/100 0.2 + 0.08 + 0.2 + 0.08 = ± 0.43%
ƒ D. 1.0/100 0.6 + 0.08 = ± 0.68%
ƒ E. 2.0/200 0.3 + 0.05 = ± 0.35%
ƒ F. 5.0/500 0.2 + 0.03 = ± 0.23%

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