Validation of Spectrophotometric
and Spectrometric Methods
Ian Ken D. Dimzon, Ph.D.
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Validation of Spectrophotometric and
Spectrometric Methods:
Process Overview
Ian Ken D. Dimzon, Ph.D.
Quality Assurance
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Method Validation
- makes use of a set of tests that both (1) test any
assumptions on which the analytical method is based and
(2) establish and document the performance
characteristics of a method thereby demonstrating
whether the method is fit for a particular analytical
Analytical Problem
purpose.
IUPAC Define requirements for a method to solve
this problem
COMPARE
Fit-for-Purpose
• Satisfies an agreed requirement (what the customer wants to Analytical Method (SOP)
know)
Evaluate performance characteristics of the
• Provided on time, in an appropriate form and at a minimum
method
cost and effort
• Allows critical decisions to be made
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Method Validation Process
1. Purpose
Set a clear objective. What does the analytical method
trying to achieve?
2. Performance Criteria
What performance parameters are critical? What values
should they have to achieve the objectives? What is the
uncertainty of each method?
3. Test Plan
What available methods fit the set criteria? Which of them
will be least time-consuming and least expensive?
4. Interpretation
How should the data be interpreted? Does it answer
the objectives?
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When is full method validation necessary?
• non-standard methods;
• laboratory-designed/developed methods;
• standard methods used outside their intended scope;
• amplifications and modifications of standard methods.
For standard methods of analysis (e.g. from AOAC, ISO or DIN), full method validation
is not necessary. However, the laboratory must verify that it is able to fulfill the
reported performance characteristics of the method as indicated in the standard.
Method Verification
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How is Method Validation done?
Two Approaches:
- Interlaboratory Comparison
- Single-Laboratory
Extent of Validation:
- Full Validation
- Partial (Method Verification)
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Questions to be asked about the method
• Does the method unambiguously identify the analyte/s of interest?
• How responsive is the instrument/method to the presence of the
analyte?
• Is the signal of the analyte differentiable from noise? How low can
the analyte concentration be for detection? for quantification?
• Is the relationship between signal and analyte concentration
established using a reasonable mathematical function?
• Is the method accurate?
• What critical steps in the method must be controlled to maintain
the method accuracy?
Method Performance Characteristics
Parameter Qualitative Tests Qualitative Trace Quantitative Quantitative Physical
(Pure (Detected not Major Trace Properties
Substance) Detected) Component
Selectivity x x x x (x)
Sensitivity x x x x
LOD/LOQ x x
Working Calibration x x x
Range
Accuracy (Trueness and Reliability Reliability x x x
Precision
Ruggedness x x x x (x)
Applicability to Other NA X x x NA
Matrices
Extent: Full Validation or Verification?
Illustration: Determination of Phosphate-P in Surface Water
Parameter Method 1: Standard Method for Water Method 2: In-House developed method for a
(Vanadate Method) different matrix
Selectivity Standard Method Documentation states Experiment to establish that the method is
possible interferences free from interferences
No need for the laboratory to repeat 1. Identify interferences
experiments done to identify interferences 2. Verify that the identified interferences
Laboratory verifies that the identified are not present in the reagents and
interferences listed addressed by the samples
laboratory (reagents and samples)
Sensitivity Standard Method guarantees that there is a Laboratory verifies if their instrument
signal produces signal
Laboratory verifies if their instrument produces
signal
Extent: Full Validation or Verification?
Illustration: Determination of Phosphate-P in Surface Water
Parameter Method 1: Standard Method for Water Method 2: In-House developed method for a
(Vanadate Method) different matrix
LOD/LOQ Standard Method Documentation states the Laboratory studies instrumental noise and
achievable LOD and LOQ the signal of the analyte
Laboratory verifies that the LOD and LOQ can Laboratory explores different methods to
be achieved using the available instrument; estimate LOD/LOQ
if stated LOD/LOQ is not achievable, Laboratory verifies LOD/LOQ
laboratory defines their own LOD/LOQ
Working Standard Method states the linear range or Laboratory determines the linear range of the
Calibration Range working range, proposes a calibration method
method Laboratory defines working range
Laboratory verifies that the linear range or Laboratory explores the different calibration
working range is doable using the available techniques and decide which is most
instrument appropriate for a particular problem
Laboratory verifies the working range
Extent: Full Validation or Verification?
Illustration: Determination of Phosphate-P in Surface Water
Parameter Method 1: Standard Method for Water (Vanadate Method 2: In-House developed method for a
Method) different matrix
Accuracy Standard Method Documentation states the Laboratory performs full study to determine
achievable precision (includes both repeatability repeatability precision and trueness.
and reproducibility) and trueness Reproducibility cannot be determined
Laboratory verifies that it can achieve comparable unless the lab participates in
precision and trueness. Trueness can only be interlaboratory comparison. Trueness can
determined by analyzing CRM and participating only be determined by analyzing CRM
in proficiency testing. and participating in proficiency testing.
Ruggedness Standard Method states in the procedure part Laboratory performs full ruggedness study.
which steps must be done carefully. Laboratory determines the applicability
Standard Method defines applicability domain. domain.
Laboratory strictly follows the method. Laboratory drafts an SOP that reflects the
If laboratory deviates from the method, ruggedness results of the ruggedness study.
study must be done on the modified step. Laboratory strictly follows the SOP.
Types of Methods
• Primary methods
• Standard methods
• In-house developed methods
• Methods from scientific publications
• partial methods – sample preparation only or instrumental parameters
only
• whole methods – from sampling to measurement
• Methods from scratch
Use of Standard Methods:
Deutsches Institut
für Normung
European Committee for
Standardization
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US-EPA Approved Methods
for Drinking Water
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Standard Methods:
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ISO
Is this the same matrix as the ...
- groundwater near the landfill?
- landfill leachate?
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References
Method Validation:
Definition of the problem and setting
the analytical requirements
Ian Ken D. Dimzon, Ph.D.
• “The laboratory shall use test and/or calibration methods, including methods for
sampling, which meet the needs of the customer and which are appropriate for
the tests and/or calibrations it undertakes...”
• “When the customer does not specify the method to be used, the laboratory shall
select appropriate methods...”.
ISO/IEC 17025
The laboratory defines the problem
• Talk to the customer (user of information)
• The “customer” has the problem and is the beneficiary of the analytical
information
• Restate the customer problem into an analytical problem
• Set criteria or requirements
• Present initial list candidate methods to be evaluated
[Link] Activity
Customer: Drinking Water Treatment Facility
Problem: Does the water contain acceptable heavy metal level?
slido
Formulate the analytical problem
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Enumerate analytical requirements
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What are the candidate methods?
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Analytical Requirement
• Analyte (e.g., copper, creatinine, hexavalent chromium)
• Measurand
• State the measurand (the quantity intended to be measured). e.g. is
it the ‘total’ concentration of the analyte(s) present that is of interest,
the ‘amount extracted’ under specified conditions, or the result
obtained from a specified (standard) measurement procedure?
• State the units in which the measurement results will be reported.
• State required range (e.g., concentration range in samples).
• Matrix and form
• Purpose of measurement (e.g., to check compliance with a particular
regulation or a manufacturing specification)
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Analytical Requirement
• Extent of validation to be done (e.g., verification only, development+validation)
• Target performance characteristics – List all performance characteristics to be
evaluated; justify any omission
Type of Analysis
Parameter Major Trace Physical
Qualitative
Component Analysis Properties
Precision / / /
Selectivity / / / /
Bias / / /
Ruggedness / / / /
Linear Range / / /
LOD / /
LOQ /
Analytical Requirement
• Performance requirements
• How does the method need to perform to deliver results that are fit for
purpose?
• Summarize the performance target values for the performance
characteristics to be evaluated during the study.
• State and justify how the performance requirements were defined.
• Performance target values may be:
• Defined in standards/regulations
• Stated in a published standard method (can the stated performance be achieved?)
• Related to a product specification in manufacturing quality control
• Based on performance of similar procedures that are known to be fit for purpose
• Defined as the current state-of-the-art (what is the method capable of?)
Analytical Requirements
• Other considerations
• Is there any historical data on method performance available?
• Is sampling/subsampling required (and will this be done within the laboratory)?
• Are there any restrictions on sample size or availability?
• Is the analyte dispersed or localised within the samples?
• Are there any known interferences?
• List any CRMs that are commercially available with a matrix and property
values that are similar to the test samples.
• Identify any other (C)RMs that may be used during the validation study (e.g.
pure substance reference materials used for preparing spiked samples).
Planning the method validation study
Points to consider:
• The method to be studied
• Critical steps in the method and instrument requirements
• Supporting information
• Extent of validation
• Order of evaluation of performance characteristics
• Materials to be analyzed
• Experimental design
• simple replication
• linear calibration
• netted design
• fractional factorial design
Validation Plan and Report
1. Title
a. Nature of the method: International standard or developed
in-house
b. Scope of the method: sample, analyte, measurand,
measurement unit, intended use
c. Description of the method: sampling and subsampling,
sample preparation, instrumental technique
2. Planning
a. Type of validation – Full validation or only Verification? Is
there an intention to extend the method? (other matrices?
wider calibration range? new reagents?)
b. Extent of Validation – Which performance characteristics
are to be tested?
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Validation Plan and Report
3. Performance Characteristics (one subsection per figure of merit)
a. Brief explanation of the performance characteristics
b. Description of the experiment done to obtain the result
c. Description of how will the results be evaluated (statistical tests)
4. Summary
a. Summary of the important results
b. Implication regarding routine use, internal and external quality control
c. Answer to the question: Is the method fit-for-purpose?
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Sample Format
Sample Format
Sample Format
