Analytical method validation

PRESENTED BY
P. SAI PRAVEEN
ANALYTICAL DEPT.
1

 Introduction
 System suitability
 Specificity
 Linearity and Range
 Precision
 Accuracy
 LOD & LOQ
 Robustness
 Solution stability
 Validation Report
 Time management in validation
 Revalidation
 Conclusion
 References
2

Method
Validation
Method
Transfer
Method
Development
Approved
3

 Method validation means establishing through
documented evidence, a high degree of assurance that an
analytical method will consistently yield results that
accurately reflect the quality characteristics of the
products tested.
4

The currently available guidelines for analytical method
validation were
 ICH (Q2R1)
 USP (Chapter <1225>)
 ANVISA (RE 899)
 AOAC
 IUPAC
 FDA (CDER, CBER - Draft guideline Feb 2014 )
5

 Four most common types of analytical procedures to be
validated:
• Identification tests
• Quantitative tests for impurities content
• Limit tests for the control of impurities
• Quantitative tests of the active moiety
 Compendial Vs Non-compendial methods
6

 For identification of source of
potential errors.
 Determination if Method is
Acceptable for Intended Use.
 Establish that test data obtained
was consistent, reliable &
accurate.
 Validation of analytical methods
is also required by most
regulations.l
 Establish proof that method can
be used for decision making.
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Characteristic Identific
ation
Impurities Testing Assay
Quantitative Limit
Accuracy _ + _ +
Precision
a. Repeatability _ + _ +
b. Intermediate precision _ + _ +
Specificity + + + +
LOD _ _ + _
LOQ _ + _ _
Linearity _ + _ +
Range _ + _ +
- signifies that this characteristic is not normally evaluated
+ signifies that this characteristic is normally evaluated
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 What analyte should be detected?
 Should information be qualitative or quantitative?
 What are expected concentration levels?
 What are sample matrices?
 What precision and accuracy is required?
 Are there any specific requirements?
 What are required detection and quantitation limits?
10

 Suitability of Instrument - Status of Qualification and
Calibration.
 Suitability of Materials - Status of Reference Standards,
Reagents, Sample, Placebo Lots.
 Suitability of Analyst - Status of Training and Qualification
Records.
 Suitability of Documentation - Written analytical procedure
and proper approved study plan with pre-established
acceptance criteria.
 Stability of samples & standard.
11

System suitability testing is an integral part of many
analytical procedures.
To verify proper functioning of the operating system
Parameters Recommendation
Resolution R ≥ 1.5, between the peak of interest and
the closest potential interferent
(degradant, Impurity, excipient, etc.)
Tailing T ≤ 2
Theoretical plates (N) In general N > 2000
Repeatability % CV ≤ 2.0% (n ≥ 5)
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“No sample analysis is acceptable unless the suitability of the
system have been demonstrated.” (USP Chapter 621)

 The ability to assess unequivocally the analyte in the
presence of components which may be expected to be
present.
 Interfering substances
- Active ingredients
- Excipients
- Impurities
- Degradation products
13

Forced degradation study (for stability indicating methods)Forced degradation study (for stability indicating methods)
 Should include samples stored under relevant stress conditions: light, heat,
humidity, acid/base hydrolysis and oxidation.(Target : 5-20% Degradation)
Purpose:
 Stability indicating power of analytical method
 Address the stability of compound
 Establish the degradation pathway
 Identify the degradation products (if required)
Specificity Evaluation:
 Identification test
 Assay
 PDA – peak purity
14

 Ability to obtain test results that are directly (or by a well-
defined mathematical transformation) proportional to the
concentration of analyte in samples within a given range.
 The range of an analytical procedure is the interval
between the upper and lower levels of analyte (including
these levels) that have been demonstrated to be determined
with a suitable level of precision, accuracy, and linearity.
15

 For establishment of linearity, minimum 5 concentrations
are recommended.
 Linearity results should be established by appropriate
statistical methods.
 Assay:- 80% to 120% of test concentration is recommended
16

 The following parameters should be determined:
 correlation coefficient (> 0.99)
 y-intercept
 slope of the regression line
 Residual sum of squares
 The range of the procedure is validated by verifying that the
analytical procedure provides acceptable precision, accuracy,
and linearity when applied to samples containing analyte at
the extremes of the range as well as within the range.
17

 The precision of an analytical procedure expresses the
closeness of agreement (degree of scatter) between a
series of measurements obtained from multiple sampling
of the same homogeneous sample under the prescribed
conditions.
18

Concentration Absorbance
10 µg/ml
Mean
0.21
0.32
0.39
0.53
0.36
Less Variation More Variation
High Precision Low Precision
Concentration Absorbance
10 µg/mL
Mean
0.28
0.31
0.29
0.30
0.29
19

 Precision may be considered at three levels:
20

Repeatability
• Repeatability expresses the precision under the same
operating conditions (with-in a laboratory over a short
period of time).
• Repeatability should be assessed
using a minimum of 6 determinations at 100% level
(or)
9 preparations covering the specified range (3 preparations
each at 3 levels).
 Evaluation:
Standard deviation
Relative standard deviation (coefficient of variation)
21

• Intermediate precision expresses variations within
laboratories, such as
• different days,
• different analysts,
• different equipment,
• different columns etc.
 Evaluation:
Standard deviation
Relative standard deviation (coefficient of variation)
22

• Reproducibility expresses the precision between
laboratories. It is assessed by means of an inter-laboratory
trial. (Defined as ruggedness in USP)
23

 Closeness of agreement between the conventional true
value or an accepted reference value and the value found.
 Accuracy = trueness + precision
24
Low Accuracy
High Precision

1. Assay
a)Known amount of drug substance spiked with synthetic
mixtures of drug product components (excipients)
b)Accuracy should be assessed using a minimum of 9
determinations over a minimum of 3 concentration levels
covering the specified range (e.g., 3 concentrations/3
replicates each of the total analytical procedure).
Evaluation
% recovery from the amount added
to amount found
% CV at each level (for 3 replicates)
DRUG PRODUCT
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 Limit of Detection:
It is the lowest amount of analyte in a sample which can
be detected but not necessarily quantitated.
 Limit of Quantitation:
It is the lowest amount of analyte in a sample which can
be quantitatively determined with suitable precision and
accuracy.
26

Method
Based on visual evaluation
Based on standard deviation of response and slope
Signal to noise ratio 2:1 or 3:1
 Limit of Detection  Limit of Quantitation
LOD = 3.3 σ / Slope LOQ = 10 σ / Slope
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 The robustness of an analytical procedure is a
measure of its capacity to remain unaffected
by small, but deliberate variations in method
parameters and provides an indication of its
reliability during normal usage.
 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.
 Typical variations:
Stability of analytical solution
Extraction time
28

 In the case of liquid chromatography, examples of typical
variations are:
 pH of a mobile phase (± 0.2 units)
 mobile phase composition (Organic ± 2-5%)
 buffer concentration (± 10%)
 Temperature (± 5°C)
 flow rate (± 10%)
 Wavelength (± 2 nm)
Evaluation:
 System suitability criteria should meet at each variable
condition
 Comparison of results under varying conditions with precision
under normal conditions.
 Expressed as % CV (or) as ratio of results.
29

 Standard and sample solutions were analyzed at initial and
at regular intervals by storing at Room temperature/
refrigerator conditions (2-8°C).
 Determine the period of time, a solution can be held before
analysis without compromising accuracy.
 Evaluation
The difference b/w initial response to specified interval
(expressed as % difference or as ratio).
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VALIDATION REPORT
Generally method validation report shall have (but not limited to)
Objective and scope of the method (applicability, type).
Standard, Sample and Placebo details.
Detailed list of chemicals, reagents and equipment used
Summary of methodology.
Validation data (parameter wise – procedure, results, conclusion etc.,)
Summary of data (results in brief – parameter wise)
Summary of report (overall view of validation exercise, any critical
issues, recommendations etc., for the application of method)
Representative plots, e.g., chromatograms, spectra, peak purity data and
calibration curves.
31

 There are no official guidelines on the sequence of validation
experiments and the optimal sequence can be depending on the
method itself. Based on experience, for HPLC method the following
sequence has been proven to be useful for time management.
If the method is proved as
stable and robust under
pre-validation program
1.Specifity
2.Linearity
3.LOD,LOQ*(if required)
4.Precision
5.Accuracy
6.Range
7.Solution stability
8.Robustness
In case if stability and
robustness data is not
available
1. Solution stability
2. Robustness
3.Specifity
4.Linearity
5.LOD,LOQ*(if required)
6.Precision
7.Accuracy
8.Range
32

 Change in the analytical procedure, drug substance, drug
product may necessitate revalidation of the analytical
procedures.
 “The degree of revalidation depends on the nature of the
change.”
 Revalidation should accompany
 formulation changes (eg. new matrices)
 manufacturing changes (eg. Synthetic route)
 modification of analytical parameters.
REVALIDATION
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 When the method is properly validated – consistent,
reliable and accurate results are obtained.
 METHOD VALIDATION =ERROR ASSESSMENT
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1) ICH harmonised tripartite guideline - Validation of Analytical
Procedures : Text and methodology - ICH Q2(R1)
2) United States Pharmacopoeia, Chapter <1225> Validation of
compendial methods
3) http://www.anvisa.gov.br/hotsite/genericos/legis/resolucoes/20
03/899_03re_e.pdf
4) http://www.aoac.org/imis15_prod/AOAC_Docs/StandardsDev
elopment/SLV_Guidelines_Dietary_Supplements.pdf
5) http://www.iupac.org/publications/pac/
2002/pdf/7405x0835.pdf
1) http://www.labcompliance.com/tutorial/
methods/default.aspx? sm=d_d
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