Validation of Analytical Methods: Rutendo Kuwana

Validation of analytical
methods
Rutendo Kuwana
Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
BACKGROUND-LAB METHOD FLOW
Method
Development
Method
Validation
Method
Transfer
Approved
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Assessment of Interchangeable Multisource Medicines, Kenya, August 2009
VALIDATION
THE PROCESS OF PROVIDING DOCUMENTED
EVIDENCE THAT SOMETHING DOES WHAT IT IS

INTENDED TO DO
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WHY VALIDATE?
To demonstrate that the method is suitable for its

intended use
Provides assurance of reliability
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WHO GMP - 4.11 Analytical methods,

computers and cleaning procedures
It is of critical importance that particular attention is paid to the
validation of analytical test methods, automated systems and
cleaning procedures.
Validation of analytical procedures used in the examination of
pharmaceutical materials (WHO Expert Committee on
Specifications for Pharmaceutical Preparations. 32nd Report.
Geneva, WHO, 1992 (WHO Technical Report Series, No. 823)
Text on Validation of Analytical Procedures Q2 (R1) Validation
of Analytical Procedures: Text and Methodology. ICH
Harmonized Tripartite Guidelines
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ANALYTICAL METHOD VALIDATION

PERFORMANCE CHARACTERISTICS
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TYPES OF ANALYTICAL METHODS TO BE

VALIDATED
Identification tests
Quantitative tests for impurities content
Limit tests for the control of impurities
Quantitative tests of the active in samples of the drug
substance (raw material), finished product or other
selected components in the drug
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ACCURACY
Expresses the CLOSENESS of
agreement BETWEEN the value,
which is accepted either as a
conventional TRUE VALUE or an
accepted REFERENCE VALUE and
the VALUE FOUND i.e. individual
observation or mean of
measurements
The closeness of test results to the
true value obtained by the method

(trueness).
Established across the range
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DETERMINATION OF ACCURACY
Drug Substance
Analysis of reference material
Compare results to a second, well-characterized method
Drug Product
Analysis of synthetic mixtures spiked with known quantities of
components
Compare results to a second, well-characterized method
Determined concurrently with precision, linearity and specificity
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DETERMINATION OF ACCURACY cont

Impurities (Quantitation)
Analysis of samples (Drug substances/Drug product) spiked with known
amounts of impurities
If impurities are not available, see specificity
Recommended Data
Minimum of 9 determinations over a minimum of 3 concentration levels
covering the specified range (e.g. 3 concentrations/3 replicates each)
Reported as % recovery of known added amount or difference between
the mean and true value, with confidence intervals
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PRECISION
Precision
The measure of the degree of

agreement (degree of scatter)
among test results when the
method is applied repeatedly

to multiple samplings of a
homogeneous sample
Expressed as %RSD for a
statistically significant number
of samples
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Precision (of any process)

Measured mean
Real mean
The precision (VARIABILITY) of an

analytical procedure is usually
expressed as the standard deviation
(S), variance (S2), or coefficient of
variation (= relative standard deviation,
R.S.D.) of a series of measurements.
The confidence interval should be

reported for each type of precision
investigated.
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PRECISION cont
May be considered at 3 levels:
Repeatability, a measure of variability under the same
operating conditions over a short interval (intra-assay
precision). Minimum of 9 determinations covering specified
range
Intermediate precision, a measure of within-laboratory
variations (different days, different analysts, different
equipment)
Reproducibility, expresses precision between laboratories (e.g.
in collaborative studies), also applies to method transfer
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Repeatability (of any process)

Measured
mean
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Repeatability expresses the

precision (spread of the data,
variability) under the same
operating conditions over a short
interval of time. Repeatability is also
termed intra-assay precision.
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Intermediate Precision and

Reproducibility
Measured
means
Intermediate precision expresses withinlaboratories variations. #1, #2 and #3:

different days, different analysts,
different (manufacturing) equipment,
etc.
Reproducibility expresses the precision
between laboratories #1, #2 and #3
(collaborative studies, usually applied to
standardization of methodology).
(Transfer of technology)
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ACCURACY & PRECISION
Inaccurate &
imprecise
Inaccurate but
precise
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Accurate but
imprecise
Accurate and precise
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SPECIFICITY/SELECTIVITY
The ability to measure accurately and
specifically the analyte in the presence of

components that may be expected to be
present in the matrix
The degree of interference

Active Ingredients
Excipients
Impurities (synthetic precursors, enantiomers)
Degradation Products
Placebo Ingredients
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SPECIFICITY/SELECTIVITY
Combination of 2 or more analytical procedures may be
required to achieve necessary level of discrimination
Stability indicating analytical methods should always be
specific
Analysts should ascertain whether the peaks within a sample
chromatogram are pure or consist of more than one compound.

Therefore should know how many compounds are in the
sample or use procedures to detect peak purity
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LOD, LOQ and SNR
Limit of Quantitation (LOQ)

Limit of Detection (LOD)
Signal to Noise Ratio (SNR)
Peak B
LOQ
Peak A
LOD
Baseline
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noise
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LIMIT OF DETECTION
The lowest concentration of an analyte in a sample that
can be detected, not quantified
Expressed as a concentration at a specified signal:noise
ratio
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LIMIT OF QUANTIFICATION
The lowest concentration of analyte in a sample that can
be determined with acceptable precision and accuracy
under stated operational conditions
Expressed as concentration of analyte
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21
LINEARITY
The Ability of the method to obtain test results that are directly
proportional to concentration within a given range
Method: dilution of stock solution/separate weighings
Expressed as the variance of the slope of the regression line

Correlation coefficient, y-intercept, slope of regression line and
residual sum of squares should be presented together with plot of
the data
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22
Linearity
Linearity expresses differences in precision at different points of a
given range.
The linearity of an analytical procedure is its ability (within a given
range) to obtain test results, which are directly proportional to the
concentration (amount) of analyte in the sample
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RANGE
Interval between upper and lower levels of analyte demonstrated
by the method
Confirms that the analytical procedure provides acceptable degree
of linearity, accuracy and precision when applied to samples

containing amounts of analyte within or at the extremes of the
specified range
Minimum 5 concentrations
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Range (minimum requirements)

Assay of an API or a FPP: 20% of the test concentration.
Content uniformity: 30% of the test concentration (unless a wider more
appropriate range, based on the nature of the dosage form (e.g., metered
dose inhalers), is justified).
Dissolution testing: 20 % over the specified range.

Impurity: from the reporting level of an impurity to 120% of the
specification. (Unusually potent or toxic impurities, LOD and LOQ should be
commensurate with ICH requirement.)
If assay and purity are performed together as one test and only a
100% standard is used, linearity should cover the range from the
reporting level of the impurities to 120% of the assay specification
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Typical expected results for Analyte concentration vs. Precision

(The AOAC manual for the Peer-Verified Methods program)
Analyte Conc (%)
Analyte Ratio
Unit
RSD%
100
100%
1.3
10
10-1
10%
2.8
10-2
1%
2.7
0.1
10-3
0.1%
3.7
0.01
10-4
100 ppm
5.3
0.001
10-5
10 ppm
7.3
0.0001
10-6
1 ppm
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0.00001
10-7
100 ppb
15
0.000001
10-8
10 ppb
21
0.0000001
10-9
1 ppb
30
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RUGGEDNESS
Ruggedness
Degree of reproducibility of test
results under a variety of
conditions
Different Analysts
Different Laboratories
Different Instruments
Different Reagents
Different Days
Etc.
Expressed as %RSD
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ROBUSTNESS
Measure of the capacity to

remain unaffected by small
(deliberate) variations in
method parameters
Indication of reliability
during normal use
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Sensitivity and robustness
Input-output
relationship
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Stability of analytical solution

Solutes may readily decompose prior to chromatographic investigations e.g. during
sample preparation , extraction, cleanup, phase transfer or storage of prepared vials
(refrigerators or automatic sampler). Method development should investigate the
stability of the analytes AND standards.
System stability
stability of the samples being analyzed in a sample solution.
Measure of the bias in assay results generated during a preselected time
interval e.g. 1 48 hours using a single solution
should be determined by replicate analysis of the sample solution.
considered appropriate when the RSD, calculated on the assay results obtained
at different time intervals, Less than 20 percent of the corresponding value of
the system precision
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SYSTEM SUITABILITY
The checking of a system, before or during analysis of unknowns, to
ensure system performance.
No sample analysis is acceptable unless the requirements for system
suitability have been met. (USP Chapter 621)
Plate Count, Tailing, Resolution
Determination of reproducibility (%RSD)
For %RSD < 2.0%, Five replicates
For %RSD > 2.0%, Six replicates
System Suitability "Sample - A mixture of main components and
expected by-products utilized to determine system suitability
Whenever There is a Significant change in Equipment or Reagents
System Suitability Testing Should be Performed (USP Chapter 621)
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Classes of analytical tests

The objective of validation of an analytical procedure is to
demonstrate that it is suitable for its intented purpose.
Class A: To establish identity
Class B: To detect (Bd) and quantitate (Bq) impurities

Class C: To determine quantitatively the concentration, or assay
Class D: To assess characteristics
Other classes not covered in the guides
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Criteria for analytical classes

Criteria
Bq
Accuracy
Precision
Robustness
Linearity and range

Specificity
X
X?
Limit of detection
Limit of quantitation
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Bd
X
X
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General requirements
Qualified and calibrated instruments
Documented methods
Reliable reference standards
Qualified analysts
Sample integrity
Change control (e.g., synthesis, FPP composition)
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General requirements (2)
Analytical methods should be used within GMP and GLP

environments, and must be developed using the protocols
and acceptance criteria set out in the ICH guidelines Q2
(R1)
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General requirements (3)

Validation Protocol important
Revalidation should accompany
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formulation changes (new samples with new compounds or new matrices)
manufacturing batch changes
new analysts with different skills,
new instruments with different characteristics,
new location with different environmental conditions,
new chemicals and/or reference standards and
modification of analytical parameters.
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Validation Report
Objective and scope of the method (applicability, type).
Summary of methodology.
Type of compounds and matrix.
All chemicals, reagents, reference standards, QC samples with purity, grade, their source
or detailed instructions on their preparation.
Procedures for quality checks of standards and chemicals used.
Method parameters.
Critical parameters taken from robustness testing.
Listing of equipment and its functional and performance requirements, e.g., cell
dimensions, baseline noise and column temperature range.
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Validation Report (2)
Detailed conditions on conduct of experiments, including sample preparation
Statistical procedures and representative calculations.
Procedures for QC in routine analyses, e.g., system suitability tests.
Representative plots, e.g., chromatograms, spectra and calibration curves.
Method acceptance limit performance data and expected uncertainty of

measurement results.
Criteria for revalidation.
The person(s) who developed and validated the method.

References (if any).
Summary and conclusions.
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Use and Validation of Pharmacopoeial methods

The degree of validation of a pharmacopoeial method should be adequate for
required purpose, and the laboratory be able to match any stated performance data.
Following product specific attributes should be considered
the type of compounds to be analyzed,
matrices,
the type of information required (qualitative or quantitative),
detection and quantitation limits,
Concentration range for analysis based on own product
precision and accuracy (sample preparation critical) as specified by the client of the
analytical data and
the type of equipmentits location and environmental conditions.
Therefore PQP three parameters required for PQP Specificity, Accuracy and
Precision
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Main Points Again

Validation of analytical procedures is a critical
requirement in risk assessment and management:
establishment of product-specific acceptance criteria, and
stability of APIs and FPPs.
Validation should demonstrate that the analytical

procedure is suitable for its intented purpose.
HPLC systems and method validation deserves special
attention during the inspection of QC laboratories.
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Validation of Analytical Methods: Rutendo Kuwana

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Validation of analytical

Training workshop: Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

BACKGROUND-LAB METHOD FLOW

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

THE PROCESS OF PROVIDING DOCUMENTED

EVIDENCE THAT SOMETHING DOES WHAT IT IS

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

To demonstrate that the method is suitable for its

Provides assurance of reliability

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

WHO GMP - 4.11 Analytical methods,

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

ANALYTICAL METHOD VALIDATION

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

TYPES OF ANALYTICAL METHODS TO BE

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

true value obtained by the method

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

DETERMINATION OF ACCURACY cont

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

The measure of the degree of

method is applied repeatedly

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Precision (of any process)

The precision (VARIABILITY) of an

The confidence interval should be

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Repeatability (of any process)

Repeatability expresses the

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Intermediate Precision and

Intermediate precision expresses withinlaboratories variations. #1, #2 and #3:

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

ACCURACY & PRECISION

Accurate and precise

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

specifically the analyte in the presence of

The degree of interference

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

chromatogram are pure or consist of more than one compound.

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

LOD, LOQ and SNR

Limit of Quantitation (LOQ)

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Expressed as the variance of the slope of the regression line

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

of linearity, accuracy and precision when applied to samples

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Range (minimum requirements)

Dissolution testing: 20 % over the specified range.

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Typical expected results for Analyte concentration vs. Precision

Analyte Conc (%)

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Measure of the capacity to

Assessment of Interchangeable Multisource Medicines, Kenya, August 2009

Sensitivity and robustness