Method Development &
Validation
By
Dr. Sajith Chandran
Jai Hind College, Churchgate, Mumbai-20
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ICH Guidelines
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ICH Guidelines
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ICH Guidelines
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ICH
International Council for Harmonisation of Technical Requirements for Pharmaceuticals
for Human Use.
Coordinated by the regulatory authorities of the European, Japanese and United States in
consultation with the pharmaceutical trade associations from these regions, to discuss and
agree the scientific aspects arising from product registration
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Need & Initiative
• It is an initiative that brings together regulatory authorities and pharmaceutical industry
to discuss scientific and technical aspects of pharmaceutical product development and
registration.
• The mission of the ICH is to promote public health by achieving greater harmonisation
through the development of technical Guidelines and requirements for pharmaceutical
product registration.
• Harmonisation leads to a more rational use of human, animal and other resources, the
elimination of unnecessary delay in the global development, and availability of new
medicines while maintaining safeguards on quality, safety, efficacy, and regulatory
obligations to protect public health.
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Purpose of an analysis
Purpose
What? & How much?
Qualitative
Quantitative
Nature of information required
Size of the sample available
Nature of the final measurement
Ø Proximate analysis – Elemental composition
Ø Partial analysis – only one of the components in the sample estimated
Ø Trace analysis – Trace amounts of substances analysed.
Ø Complete analysis / Total analysis – all components determined
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Sources of Method
Selection of a suitable analytical method can be made once the reason for carrying
out the analysis is well understood.
Ø In-house methods developed by one laboratory for their own special needs.
Ø Methods published in the open scientific literature (Eg: open access Journals).
Ø Methods supplied by trade organizations, e.g. the Institute of Petroleum .
Ø Methods in books published by professional organizations, e.g. The Royal Society of
Chemistry
Ø Methods from standards organizations, e.g. (UK) BSI, BP; (International) ISO; (Europe)
CEN, EP; (USA) ASTM, EPA, USP, etc.
Ø Methods from statutory publications, e.g. The Fertilisers (Sampling and Analysis)
Ø Regulations 1991 (SI No. 973) HMSO.
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Methods
§ Provides results which are fit for their intended purpose
§ Method satisfies some pre-defined criteria
§ Confidence required by the customer or other users of the results.
§ Screening methods - Screening methods must be extremely rapid, permitting a high
throughput of samples at low cost (contains false positives & negatives).
§ Surveillance methods – better selectivity
§ Methods usually represent a consensus view from a number of analytical laboratories
working in a particular application area.
§ Regulatory and reference methods are typically identified by an official body for use in
enforcement of a specific regulation
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Classification
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Factors that have to be considered
Factors that have to be considered when choosing between types of method include the
following:
Ø type of sample, matrix and measurands. Ø limit of quantitation
Ø time Ø working range
Ø cost Ø selectivity
Ø equipment availability Ø quantitative or semi-quantitative
Ø frequency of false negatives and false Ø performance requirements, including
positives extent of validation necessary
Ø limit of detection Ø acceptable measurement uncertainty.
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Cost
§ Major factors are the human resource and the cost of running and maintaining a
laboratory, the choice of method may have a small bearing on the total cost of the job.
§ Analysis requiring mass spectrometry or nuclear magnetic resonance spectroscopy will
be more expensive than classical techniques.
§ Capital cost of the equipment used and the seniority of the staff required to interpret
the data produced by such techniques.
§ Customer - customer may be prepared to accept the risk of making decisions based on
results with a large known uncertainty rather than incur the extra cost of obtaining
results that have a smaller uncertainty.
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Time
If a large number of samples have to be analysed, a method that is simple and rapid is to
be preferred so that data can be acquired quickly and with the minimum of effort and cost.
Two conditions to be considered:
Ø There is no problem and, therefore, no further work is required.
Ø There is some evidence that a particular analyte may be present but this needs to be
confirmed by further measurements.
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Equipment Required & Sample Size
• All items of equipment must be considered, including balances and volumetric
measuring devices, not just the expensive equipment and advanced instruments.
• Sample size is linked to the limit of detection
• Food and agriculture - amount of sample is not normally a limiting factor.
• Clinical chemistry - amount of sample is not normally a limiting factor.
• Organic matter - has to be destroyed using oxidizing acids, the smaller the mass of
sample taken the better, as the digestion takes less time and uses smaller volumes of
acids, thus giving lower blank values.
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Accuracy
§ Accuracy is often used to describe the overall doubt about a measurement result. It is
made up of contributions from both bias and precision.
§ The definition of accuracy in ISO 5725-1:1994, is ‘The closeness of agreement between a
test result and the accepted reference value’ (single result).
§ The term ‘accuracy’, when applied to a set of observed values, describes the
consequence of a combination of random variations and a common systematic error or
bias component. Preferable to express the ‘quality’ of a result as its uncertainty, for range
of values.
% Error = obtained result – expected result expected result × 100
expected result
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Bias/Recovery
§ Bias is the difference between the mean value of a large number of test results and an
accepted reference value for the test material.
§ The bias is a measure of ‘trueness’ of the method. It can be expressed in a number of
ways, i.e. simply as a difference or as a ratio of the observed value to the accepted value.
§ When expressed as a percentage, is often termed recovery.
§ This represents how much of the analyte of interest has been extracted from the matrix
and measured.
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Precision
§ Precision is the closeness of agreement between independent test results obtained
under stipulated conditions.
§ Precision depends only on the distribution of random errors and does not relate to the
true value. It is calculated by determining the standard deviation of the test results from
repeat measurements.
§ In numerical terms, a large number for the precision indicates that the results are
scattered, i.e. the precision is poor.
§ Quantitative measures of precision depend critically on the stipulated conditions.
§ Repeatability and reproducibility are the two extreme conditions.
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Repeatability & Reproducibility
Repeatability (r) is the value below which the absolute difference between two single test
results obtained with the same method on identical test material, under the same
conditions (same operator, same apparatus, same laboratory and a short interval of time)
may be expected to lie, with a specified probability; in the absence of other indications, a
probability of 95% is used.
Reproducibility (R) is the value below which the absolute difference between two single
tests results obtained by the same method on identical test material, under different
conditions (different operators, different apparatus, different laboratories and/or different
time) may be expected to lie, with a specified probability; in the absence of other
indications, the probability is again taken as 95%.
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Selectivity
Selectivity refers to the extent to which a method can be used to determine particular
analytes in mixtures or matrices without interference from other components of similar
behaviour.
The degree of discrimination between the measurand and other substances present in,
or
extracted from, the matrix must be carefully considered.
Attention will have to be paid to the clean-up procedures used and the discriminating
power of the detection system.
Required to carry out tests where potential interferents, that may be present in some
samples, are added to the matrix and their influence measured.
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Sensitivity
§ The ability to demonstrate that two samples have different amounts of analyte is an
essential part of many analyses.
§ A method’s sensitivity is a measure of its ability to establish that such differences are
significant. Sensitivity is often confused with a method’s detection limit.
§ Sensitivity is the change in signal per unit change in the amount of analyte.
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Limit of Detection - LoD
The limit of detection of an individual analytical procedure is the smallest amount of
an analyte in a sample which can be detected but not necessarily quantified with an
acceptable uncertainty.
The limit of detection is derived from the smallest concentration, xL that can be
detected with reasonable certainty for a given procedure.
The method will indicate that there is some measurand present, at the stated level of
significance, but the amount cannot be specified. The value xL is given by equation:
xL = xbl + kSbl
where xbl is the mean of the results obtained by measuring blank solutions, Sbl is the
standard deviation of the blank measures and k is a numerical factor chosen
according to the confidence level required.
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Robustness and Ruggedness
Methods are subject to a variety of chemical and physical interferences that
contribute uncertainty to the analysis.
When a method is relatively free from chemical interferences, it can be applied to the
determination of analytes in a wide variety of sample matrices. Such methods are
considered Robust.
Random variations in experimental conditions also introduce uncertainty.
A method is Rugged if a method’s sensitivity is highly independent on experimental
conditions, such as temperature, acidity, or reaction time, then slight changes in those
conditions may lead to significantly different results
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Safety
§ Need for special facilities for work involving neutron activation analysis and
§ radiochemical measurements
§ Methods which require toxic solvents, such as benzene and certain chlorinated
hydrocarbons, or toxic reagents, such as potassium cyanide.
§ Statutory Methods have to be used, there may be no alternative
§ Appropriate safety assessment must be carried out before the work is started.
§ Required safety protocols are followed
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Scale of Operation
Three limitations are
Ø Amount of sample available for the analysis,
Ø Concentration of analyte in the sample,
Ø Absolute amount of analyte needed to obtain a measurable signal.
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Making Your Choice
Choice of method will depend on several factors.
Above all, ‘fitness for purpose’ must be uppermost in your mind.
Will the method you have selected be adequate for the decision you and/or your
customer has to take when the result is available?
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Validation
§ Before a procedure can provide useful analytical information, it is necessary to
demonstrate that it is capable of providing acceptable results.
§ Validation is an evaluation of whether the precision and accuracy obtained by
following the procedure are appropriate for the problem.
§ validation ensures that the written procedure has sufficient detail so that different
analysts or laboratories following the same procedure obtain comparable results.
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Reasons for Incorrect Analytical Results
Ø Method used
Ø Incompetence
Ø Calculation/transcription errors
Ø Unsuitable method used
Ø Contamination
Ø Interferences
Ø Calibration errors
Ø Sampling errors
Ø Losses/degradation
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Method Validation
Method validation is defined in the international standard, ISO/IEC 17025 as, the
confirmation by examination and provision of objective evidence that the particular
requirements for a specific intended use are fulfilled.
This means that a validated method, if used correctly, will produce results that will be
suitable for the person making decisions based on them.
Method validation is a planned set of experiments to determine these values. The
method performance parameters that are typically studied during method validation
are selectivity, precision, bias, linearity working range, limit of detection, limit of
quantitation, calibration and ruggedness.
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Method Validation
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Method Validation
If no method exists for the analysis required, then either an existing method has to be
adapted or a new method developed.
The adapted or developed method will need to be optimized and the controls required
identified, hence ensuring that the method can be used routinely in the laboratory.
Evidence is then collected so as to demonstrate that the method is ‘fit for purpose’.
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Method Validation & Verification
This limited validation where all that is done is confirming that the established
published performance can be achieved is called verification.
The continued satisfactory performance of the method will need to be checked by
using adequate control procedures.
Where the validation of a standard method is thought to be inadequate, further
validation will be required along the lines detailed in the following sections.
The laboratory needs to demonstrate that it can meet the requirements specified
in the measurement specification.
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References
1. Quality in the analytical chemistry laboratory, E Prichard, John Wiley and sons N.Y 1997.
2. Quality Assurance In Analytical Chemistry, Elizabeth Prichard and Vicki Barwick LGC,
Teddington, UK, John Wiley and sons N.Y 2007, Chapter – 1, 3 & 4.
3. Modern Analytical Chemistry, David Harvey, McGraw-Hill Higher Education, Chapter-3 &
7.
4. Quality assurance in analytical Chemistry, W Funk, V Dammann, G. Donnevert VCH
Weinheim1995.
5. Pharmaceutical Manufacturing Handbook - Regulations And Quality - Shayne Cox
6. Amit S. Patil et. al., Quality by Design (QbD) : A new concept for development of Quality
pharmaceuticals, International Journal of Pharmaceutical Quality Assurance; 4(2); 13-19.
7. Lalit Singh and Vijay Sharma, Quality by Design (QbD) Approach in Pharmaceuticals:
Status, Challenges and Next Steps, Drug Delivery Letters, 2015, 5, 2-8.
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THANK YOU
[email protected] 9819844994
Acknowledgements: The author acknowledges the adoption of graphs, diagrams and data from various web sites
and books under fair use practice. This lecture presentation is compiled for the larger interests of undergraduate
students of Chemistry and therefore can be downloaded or printed free for educational purpose only.
Disclaimer: This study material is not intended for commercial purpose and no part of this document or the
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