VALIDATION OF ANALYTICAL

METHODS

1
 Analytical Method Development
• Prerequisites for analytical method
validation
– Six “M”s
Man Machine Methods
qualified calibrated characterised

robust documented
skilled qualified suitable
Quality of the
Reference analytical method
Vibrations Time
standards Irradi- Analysts´
ations support
Tempe-
Quality rature Humidity Supplies

Material Milieu Management

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 Analytical Method Development
• Validation should verify the suitability of an
analytical method for its intended purpose

• Validation should be founded on method

development performed beforehand that
suggest the suitability and robustness of the
method

• Validation may be performed in different

ways (individual purpose) according to
common standards
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 Validation Protocol
– Method principle / objective
– Listing of responsibilities
• Laboratories involved and their role in the validation
– Method categorization
– List of reagents (including test lots) and standards
– Test procedures to evaluate each validation parameter
and proposed acceptance criteria
– Plan or procedure when acceptance criteria are not met
– Requirements for the final report

• The validation process cannot proceed until the protocol

and all parties involved approve the acceptance criteria

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 Analytical Method Development
• Validation Characteristics
Identification Impurities Assay
quantitative limit
Accuracy - + - +
Precision - + - +
Specificity + + + +
Detection Limit - - + -
Quantitation Limit - + - -
Linearity - + - +
Range - + - +
Robustness + + + +

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 Analytical Method Development
• Accuracy and precision

Accurate &
precise
Accurate &
imprecise
Inaccurate &
precise Inaccurate & imprecise
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 Analytical Method Development
• Precision
– Expresses the closeness of agreement between a
series of measurements obtained from multiple
sampling of the same homogenous sample
– Is usually expressed as the standard deviation (S) or
coefficient of variation (RSD) of a series of
measurements
– Precision may be considered at three levels
• Repeatability (intra-assay precision)
• Intermediate Precision (variability within a laboratory)
• Reproducibility (precision between laboratories)

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• Normal distribution, probability function [P(x)]
and confidence interval [CI]
– Probability (P), that measures from a normal distribution fall within [µ-x n, µ+xn]

for xn = nis described by the “erf-function” µ mean)

xn P
 0.6826895

Number of times each value occurs

 0.9544997
 0.9973002
 0.9999366
 0.9999994

• An interval of ± 3 
covers 99.73% of values
Values
 
 
 
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• Relationship of variability, probability and
reliability of data
– High variability of data (large s) generate large
confidence intervals and thus lower the reliability of
the mean
– Low variability of data (small s) generate small
confidence intervals and thus increase the reliability
of the mean

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• Repeatability
– Six replicate sample preparation steps from a homogenously prepared
tablet mixture (nominal value of API 150 mg)

Mean ± 3 SD =
Confidence interval of 99.73%

98.96 ± 3x1.32% = 95% -

102.92%

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• Intermediate precision
– Expresses within-laboratories variations (different days, different analysts,
different equipment etc.)
• Intermediate precision
– Expresses within-laboratories variations (different days, different analysts,
different equipment etc.)
Peak Peak Peak
Injection area area area
analyst 1 analyst 2 analyst 3 Mean ± 3 SD: (177252  100%)
1 173865 175656 177965 Analyst 1: 98.96% ± 3 x 1.32%
2 174926 175878 178556 Analyst 2: 99.12% ± 3 x 0.28
3 172933 176004 177342
Analyst 3: 100.70% ± 3 x 0.51
4 175011 176344 178011
5 179557 175332 179466 Average of 3 analysts ± 3SD:
6 176425 174959 179688
Mean 175453 175695 178504 95% - 102.23%
SD () 2329 495 918
RSD 1.32% 0.28% 0.51%
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• Reproducibility
– Expresses the precision between laboratories
• Collaborative studies, usually applied to
standardisation of methodology
– Transfer of technology
– Compendial methods

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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
• Sometimes referred to as TRUENESS

mean true

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To find out whether a method is accurate:
• Drug substance (assay)
– Application of the method to an analyte of known purity (e.g. reference
substance)
– Comparison of the results of one method with those of a second well-
characterised method (accuracy known)

• Drug product (assay)

– Application of the method to synthetic mixtures of the drug product
component to which known quantities of the analyte have been added
• Drug product may exceptionally be used as matrix

• Drug substance/Drug product (Impurities)

– Application of the method to samples spiked with known amounts of
impurities

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• When one can expect Accuracy problems
– Insufficient selectivity of the method
• Impurity peaks are not resolved and account for assay
value
– Recovery is < 100%
• Irreversible adsorption of analyte to surfaces of the
system
– Incorrect assay value of a reference standard
• Due to decomposition of reference standard
– Incorrect assay value due to change in matrix
• Analytical laboratory still uses the preceding matrix as
standard

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• Specificity
– Is the ability to assess unequivocally the analyte in the presence of
components which may be expected to be present (impurities,
degradants, matrix…)
Identity testing
– To ensure the identity of an analyte
Purity testing
– To ensure accurate statement on the content of impurities of an
analyte
Assay
– To allow an accurate statement on the content of an analyte in a
sample

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 • Specificity: Overlay chromatogram of an impurity solution
with a sample solution

From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman
Lam, Y.C. Lee and Xue-Ming Zhang, ISBN 0-471-25953-5, Wiley & Sons

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Specificity and stability

• Stress stability testing to ensure the stability indicating potential

of an analytical method

Assure that the API can be assessed specifically in the presence of

known and unknown (generated by stress) impurities

Assure that known impurities/degradants can be specifically

assessed in the presence of further degradants

By peak purity assessment and (overlay of) chromatograms

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• Stress stability studies versus forced
degradation studies

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• 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
– If there is a linear relationship test results should be
evaluated by appropriate statistical methods
• Correlation coefficient (r)
• Y-intercept
• Slope of regression line
• Residual sum of squares
• PLOT OF THE DATA

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• Usual acceptance criteria for a linear calibration
curve
– r > 0.999; y-intercept a < 0 to 5% of target concentration
RSD (wrt calibration curve) < 1.5-2%

r < 0.997
r > 0.997

From: Analytical Method Validation and Instrument Performance Verification, Edited by Chung Chow Chan,Herman Lam, Y.C. Lee and
Xue-Ming Zhang, ISBN 0-471-25953-5, Wiley & Sons

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• Range
– The range of an analytical procedure is the interval
between the upper and lower concentration
(amounts) of analyte in the sample for which it has
been demonstrated that the analytical procedure has
a suitable level of precision, accuracy and linearity

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• Range
– Assay
• 80 to 120% of test concentration
– Content uniformity
• 70 to 130% of test concentration
– Dissolution
• Q-20% to 120%
– Impurities
• Reporting level – 120% of specification limit (with
respect to test concentration of API)
– Assay & Impurities
• Reporting level to 120% of assay specification

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• Limit of Detection (LOD, DL)
– The LOD of an analytical procedure is the lowest amount of analyte in
sample which can be detected but not necessarily quantitated as an exact
value

• Determination is usually based on

– Signal to noise ratio (~3:1) (baseline noise)

Or

– Standard deviation of response () and Slope (S)

• 3.3 /S

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• Limit of Quantitation (LOQ, QL)
– The LOQ is the lowest amount of analyte in a sample
which can be quantitatively determined with
suitable precision and accuracy
• The quantitation limit is used particularly for the
determination of impurities and/or degradation
products
• Determination is usually based on
– Signal to noise ratio (~10:1) (baseline noise)
or
– Standard deviation of response () and Slope (S)
• 10 S

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LOD, LOQ and Signal to Noise Ratio
(SNR)
Signal to Noise = 10:1
LOQ

Signal to Noise = 3:1 LOD

Noise

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 LOD and LOQ
Concentration Absorbance (Mean
(µg/ml) ± SD)

*(n= 6)
0 0.000
2 0.101 ± 0.0017
4 0.218 ± 0.0036
6 0.348 ± 0.0040
8 0.459 ± 0.0053
10 0.568 ± 0.0066
12 0.681 ± 0.0046
14 0.799 ± 0.0054
16 0.901 ± 0.0069
18 0.998 ± 0.0073
20 1.098 ± 0.0065

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• LOQ and impurities
– In determination of impurities in APIs the LOQ
should be determined in the presence of API
• LOQ should be NMT reporting level
• LOQ should be given relative to the test concentration
of API

– Specificity of impurity determination should

always be demonstrated in the presence of API at
API specification levels
• Spiking of test concentration (API) with impurities at
levels of their specification range

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• Spiking
– API test concentration (normalised)
• 0.1 mg/ml (100%)
– Impurity spiking concentrations
• 0.001 mg/ml (1%) – specification limit
• 0.0001 mg/ml (0.1%) – limit of quantitation (minimum
requirement)

API at test concentrations

API below test concentrations

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• Robustness
– Robustness of an analytical procedure should show
the reliability of an analysis with respect to
deliberate variations in method parameters
– The evaluation of robustness should be considered
during the development phase
– If measurements are susceptible to variations in
analytical conditions the analytical conditions
should be suitably controlled or a precautionary
statement should be included in the procedure

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• System suitability testing
– Based on the concept that equipment, electronics,
analytical operations and samples to be analysed
constitute an integral system that can be evaluated
as such
– Suitability parameters are established for each
analytical procedure individually
• Depend on the type of analytical procedure

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Summary
• Analytical procedures play a critical role in pharmaceutical
equivalence and risk assessment/management
– Establishment of product-specific acceptance criteria
– Assessment of stability of APIs

– Validation of analytical procedures should demonstrate

that they are suitable for their intended use
• Validation of analytical procedures deserves special
attention during assessment of dossiers for prequalification

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 THANK YOU

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