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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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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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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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
1
100%
1.3
10
10-1
10%
2.8
1
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
A
Bq
C
D
Accuracy
X
X
X
Precision
X
X
X
X
X
X
X
X
X
Robustness
X
Linearity and range
Specificity
X
X
X
X?
X
Limit of detection
Limit of quantitation
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Bd
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X
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 equipment—its 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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