IN MEASUREMENTS OF AMOUNT OF SUBSTANCE … · IN MEASUREMENTS OF AMOUNT OF SUBSTANCE ... the test...
Transcript of IN MEASUREMENTS OF AMOUNT OF SUBSTANCE … · IN MEASUREMENTS OF AMOUNT OF SUBSTANCE ... the test...
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Slide 41
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IN MEASUREMENTS
OF AMOUNT OF SUBSTANCE
©1992: P De Bievre
Slide 42
1. Results required to be related to stated references, such as Reference Materials (RMs)
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Slide 43
“Tested once, accepted everywhere”
Reliable and accurate measurements
Competent staff
Validated procedure
Comprehensive quality system
Traceability to stated reference
Comparability of results can be achieved through the link of all
individual measurement results to some common, stable reference or
measurement standard. This linking of results to a reference is termed
“traceability”.
02/03/55 Slide 44
Measurement in Measurement in Measurement in Measurement in
chemistrychemistrychemistrychemistry
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� Measurement is set of operations having the object of determining a value of a quantity
� Chemical analytical measurements are about measuring “something” in “something”
◦ Identifying and counting
◦ How much there is of a specified substance (chemical element, molecular species) in a simple, complex or very complex material (water, sediment, soil, blood)
� Number and its unit including an uncertainty
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quantity intended to be measured
� quantity we measure:
e.g, length, mass, temperature, volume, pressure, amount
concentration, mass concentration, mass fraction, electric current,
…
� the specification of the measurand results from the intention of the
analyst about what (s)he is going to measure
� it is a decisionmade before the measurement
� the measurand can be operationally defined, e.g.,
leachable Cd concentration in a ceramic plate, measured
according to a specified procedure
since we cannot specify perfectly any measurand, there is a ‘definitional
uncertainty’ associated with the measurand which must appear in our
uncertainty budget after the measurement
see ACQUAL 12(2007)561-562, 12(2007)613-61446
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ISO/IEC 17025 Clause 5.4.2
Method published in international, regional or national standards shall preferably be used
ISO/IEC 17025 Clause 5.4.4
The method developed shall have been validatedappropriately before use
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ISO defines Validation as:
‘is the confirmation by examination and provision of objective evidence that the particular requirements for a specific intended use are fulfilled’
ISO/IEC 17025 clause 5.4.5.1
From Eurachem:
“…the process of defining an analytical requirement, and
confirming that the method under consideration has
performance capabilities consistent with what the application
requires. Implicit in this is that it will be necessary to evaluate
the method’s performance capabilities. …The judgement of
method suitability is important; in the past method validation
has tended to concentrate on the process of evaluating the
performance parameters.”
-The fitness for Purpose of Analytical Methods, 3.1
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The latter definition identifies three discrete stages of methodvalidation:
1. Define the Analytical Requirement(s)
2. Determine (by collection and analysis of relevant data)the Appropriate Method Performance Metrices
3. Confirm the Adequacy (based on the determinedperformance metrics) of the Method to Meet the EstablishedAnalytical Requirement(s)
Method validation is a separate process from development andoptimization. It is an objective, systematic, evaluation that can onlybe undertaken AFTER development and optimization are complete.Unsatisfactory validation data may dictate the need for additionaldevelopment and optimization work, after which a new validationmust be executed.
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Validation may be either a single laboratoryvalidation (SLV) or a collaborative study (CS) involvingmultiple laboratories.
SLV- Provides estimates of all performance metrics
(except reproducibility) within a single laboratory
CS- Usually designed to estimate only repeatability
and combined intermediate precision/ reproducibility. Can
be designed to give explicit estimates of intermediate
precision and reproducibility.
Collaborative studies are virtually always preceded by a
complete SLV, which is required to evaluate readiness forundertaking a CS.
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‘is the confirmation by examination and provision of evidence that specified requirements have been met’
ISO/IEC 17025
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� There are minimum procedures which are fundamental to
any laboratory, and include the following:
◦ Documentation procedures
◦ Procedures for labeling, preparation and storage of
chemicals, solutions and reagents
◦ Environmental monitoring (measuring temperature-
critical items such as lab refrigerators, freezers,
incubators, etc.)
◦ General lab housekeeping (cleaning, waste disposal)
◦ Calibration of instrument and equipment with
measurable components (including balances,
thermometers, pipettes, etc.)
◦ Laboratory instrument and equipment maintenance
procedures
◦ Analyst training
Basic laboratory procedureBasic laboratory procedureBasic laboratory procedureBasic laboratory procedure
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� Work to an agreed customer requirement
� Use validated methods and calibrated equipment
� Staff are qualified and competent
� Participate in independent assessments of technical performance
� Use sound quality assurance and quality control practices
� metrological traceability (Ensure comparability with measurements made in other laboratories)
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VAM, LGC, UK
� Documented evidence
� High degree of assurance
� Specific process
� Consistently
� Predetermined specifications
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� IUPAC Technical Report (2002) (International Union of
Pure and Applied Chemistry)
� NATA Technical Note 17 (National Association of
Testing Authorities)
� ICH Guidelines (2005) (International Conference on
Harmonization)
� Joint FAO / WHO Food Standards Programme (Food
and Agriculture Organization, World Health
Organization)
� USFDA Bioanalytical Method Validation Guidance for
Industry (2001) (Food and Drug Administration)
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� Non-standard methods
� Laboratory-designed/develop methods
� Standard methods used outside of their intended
scope
� Amplifications and modifications of standard
methods
ISO/IEC 17025 clause 5.4.5.2
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◦ New method developed for particular problem
◦ Established method revised to incorporate improvements or extended to a new problem
◦ When quality control indicates and established method is changing with time
◦ Established method used in a different laboratory, or with different analyst or different instrumentation
◦ To demonstrate the equivalence between two methods
◦ Major instruments replaced
◦ New batches of standards
◦ Used of validated method after out-of use for a long time
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Fit for Purpose?
�Demonstration that a method is “fit for purpose” is the
ultimate goal of the validation process.
� One widely utilized measure of acceptable method
performance (precision) in a SLV is the Horwitz Ratio.
Although most often, and appropriately used for evaluating
data from collaborative studies, it is sometimes applied to
SLVs. For repeatability:
Horratr = RSDr
PRSDr
Where RSDr = repeatability relative standard deviation from validation data and
PRSDr = repeatability relative standard deviation predicted by the Horwitz
equation:
PRSDr = C-0.15
Only precision
is addressed/
RSDs tend to
be overestimated
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Acceptance criteria
� HorRat = Horwitz Ratio =
Reference HORRAT
AOAC
Codex, EU
ectedexp
obs
RSD
RSD
2≤
2<
59
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� This equation was defined by Horwitz et al1 from a practical consideration of a number of collaborative studies done by AOAC over many years.
� Reproducibility, CVR
� Repeatability, CVr
C = concentration of analyte in term of decimal fraction
1505.0R
)Clog5.01(R
C2CV
2CV
−
−
=
=
)Clog5.01(r 266.0CV −
×=
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Horwitz noted that values for RSDr (the repeatability CV) were usually between half and two-thirds that of RSDR.
For this reason, repeatability acceptabilities are proposed as the Horwitz values for RSDR
x 0.67.
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� Confirmation of identity
� Specificity/Selectivity
� Limit of detection (LOD)
� Limit of quantitation (LOQ)
� Linearity and working range
� Sensitivity
� Accuracy
� Precision
� Ruggedness and Robustness
� Measurement Uncertainty
� Metrological Traceability
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Key performance characteristic
Major compounds-quantitative
Major compounds and traces-
quantitative
Traces-qualitative
Traces-quantitative
LOD
LOQ
Linearity
Range
Precision
Accuracy
Specificity
Ruggedness
N
N
Y
Y
Y
Y
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
N
N
N
N
N
Y
N
N
Y
Y
N
Y
Y
Y
Y
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From: Validation and Qualification in Analytical Laboratories by Ludwig Huber
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Confirmation is an analytical process where a measurement is performed by more than onetechnique, to conform the presence of an analyte
That the entity you are observing and measuring some quantity is what you think it is!
-Speciation-Interferences and impurities
CITAC/Eurachem Guide to Quality in Analytical
Chemistry: An Aid to Accreditation (2002)
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� Specific: a method that produces a response for a target analyte only
� Selective: a method that provides responses for a number of chemical entities that may or may not be distinguished from each other
- The ability to measure accurately an analyte in the presence of interferences (USP)
- Analyze at least five independent sources of control matrix to assess interference effects
How to improve selectivity
- Good sample preparation
- Selective instrument e.g. mass spectrometer vs. UV spectrometer
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Procedure
◦ Analyze samples and reference materials by
candidate and other independent methods
◦ Analyze samples containing various suspected
interferences in the presence of the analyte of
interest
◦ If detection or quantification is inhibited by the
interferences, further method development will be
required
Eurachem Guide
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What is the smallest concentration that can be detected?
� The smallest amount or concentration of an analyte that can be reliably distinguished from zero with a specified level of confidence
� The lowest value measured by a method that is greater than the uncertainty associated with it (NATA technical Note 17, 2006)
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Sample blank produce signal
◦ Single measurement of each 10 independent sample blank, then calculate mean and standard deviation, s
No signal from sample blank
◦ Single measurement of each 10 independent sample blank fortified at lowest acceptable concentration,
then calculate standard deviation (s)
LOD = mean of sample blank +3s LOD = 0+3s
LOD = 0+4.65s
(Eurachem Guide page 17-18)
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� LOD determined from calibration curve
◦ is the standard error of the regression
◦ is the slope of the linear calibration
xys /
b
b
sc
xy
dl
/3=
∑ −++=
−
i
i
xyn
dlcc
c
nmb
stc
)(
1122
2
/2,'05.0 From IUPAC
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� Sample blanks spiked with the analyte at a
range of concentration levels
� Measure 10 independent replicates at
each concentration level
� Calculate % positive or % negative at each
concentration level
� LOD is the lowest concentration at which
the test becomes unreliable
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Spiked conc, mg/kg No. of replicates Positive/negative results
200 10 10/0
100 10 10/0
80 10 8/2
60 10 5/5
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If the criteria is 100% positive: cut-off (threshold) is 100 mg/kg =LOD
Limit of Detection (LOD) for Limit of Detection (LOD) for Limit of Detection (LOD) for Limit of Detection (LOD) for
Qualitative MeasurementQualitative MeasurementQualitative MeasurementQualitative Measurement
What is the smallest concentration that can be
measured with a given measurement uncertainty?
� The lowest concentration of analyte that can be
determined with an acceptable level of
repeatability precision and trueness
(Eurachem Guide)
� The concentration of analytes that can be
determined with an acceptable uncertainty (NATA technical Note 17)
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� Sample blank produce signal◦ Single measurement of each 10 independent sample blank
� No signal from sample blank◦ Single measurement of each 10 independent sample blank
fortified at different concentration close to LOD
Calculate standard deviation (s) at each concentration level that can be quantify with the acceptable uncertainty
LOQ = mean sample blank value +5s (or 6s or 10s)LOQ = the lowest analyte conc. which can be
determined with acceptable uncertainty(Eurachem Guide page 19)
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What is the range over which the calibration model holds?
� Working or measuring range is the set if values of measurands for which the error of a measuring instrument is intended to lie within specified limits
(Eurachem Glossary A18)
� Linear range is by inference the range of analytical concentrations over which the method gives test results proportional to the concentration of the analyte
(Eurachem Glossary A12)
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0.2
0.4
0.6
0.8
1.0
0.2 0.4
0.6
0.8 1.0 1.20
LOD
LOQ
Working range
Linear range
Concentration
Response
Linearity and Working RangeLinearity and Working RangeLinearity and Working RangeLinearity and Working Range
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1. Blank plus RMs or fortified sample blanks at
various concentrations (at least 6 conc. Plus
blank), single measurement at each
concentration
◦ Plot the responds against concentration of standard
◦ Different concentrations should be prepared
independently, and not from aliquots of the
same master solution
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2. at least 6 different concentrations within the linear range, 3 replicate measurements at each conc.
� Plot the responds against concentration of standard
� Calculate regression coefficient
� Calculate and plot the residues at each concentration
Random distribution of residual about the straight line confirms linearity. Systematic trends indicate non-linearity
If non-linear curve is necessary, functions higher than quadratic are not advised
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The change in the response of a measuring instrument divided by the corresponding change in the stimulus
No specification for sensitivity, consider the uncertainty of a
result
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Do we get the right answer?
Accuracy is the quality of the result in terms of “Trueness” and “ Precision” in relation to the requirements of its use
Trueness is the closeness of agreement between the average value obtained from a large set of test results and accepted reference value
Bias is the different between the expectation of the test results and an accepted reference value
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1. Analyze reagent blank and CRM using
candidate method
� 10 replicates
� Calculate the average concentration of CRM
(with blank subtraction) determined by candidate method
� Difference between values obtained by
candidate method and certify value can be used to estimate method bias
“Method’s Bias”
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2. Analyze reagent blank and RM using candidate and independent method preferably primary method
- 10 replicates
- Calculate the average concentration of RM (with blank subtraction)
determined by candidate method
- Calculate the average concentration of RM (with blank subtraction) determined by independent/primary method
- Difference between results obtained from these 2 methods can be used to estimate method bias
“Method’s Bias” relative to independent/primary method
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The fraction of analyte added to a test sample (fortified or
spiked sample) prior to analysis, the unfortified and fortified samples, percentage recover (%R) is calculated as follows:
% Recovery = (C1-C2)x100/C3
C1= concentration determined in fortified sample
C2 = concentration determined in unfortified sample
C3 = concentration of fortification
Analyse matrix blank, CRM or sample fortified with interested
analyte at different concentration level (low mid high) 6
replicates at each concentration and calculate % recovery
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Analyte % Analyte ratio Unit Mean recovery
(%)
100 1 100% 98-102
10 0.1 10% 98-102
1 0.01 1% 97-103
0.1 0.001 0.1% 95-105
0.01 0.0001 100 ppm 90-107
0.001 0.00001 10 ppm 80-110
0.0001 0.000001 1 ppm 80-110
0.00001 0.0000001 100 ppb 80-110
0.000001 0.00000001 10 ppb 60-115
0.0000001 0.000000001 1ppb 40-120
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AOAC manual for the Peer-Verified Methods Program (November 1993)
Can we reproduce the result?
� Repeatability: ◦ Same lab, same analyst, same instrument, relative short time span
◦ At least 6 determinations of 3 different matrices at 2 or 3 different concentrations the calculate the RSD (ICH)
◦ At least 9 replicates covering the complete specified range e.g. 3 concentrations with 3 injections at each concentration (ICH)
◦ At least 10 injections with RSD <1% (US EPA)
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� 95% confidence interval on the difference between
duplicate results measured under repeatability
conditions
rr ssr 8.222 ==
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Analyte % Analyte Ratio Unit RSD (%)
100 1 100% 1.3
10 10-1 10% 1.8
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 11
0.00001 10-7 100 ppb 15
0.000001 10-8 10 ppb 21
0.0000001 10-9 1 ppb 30
86AOAC manual for the Peer-Verified Methods Program (November 1993)
Expected %RSDr calculated from Horwitz’s equation = 0.66x2 (1-0.5logC)
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� Intermediate precision (ICH)◦ The long term variability of the measurement
process
◦ Comparing the results of a method run within a
single lab over a number of weeks
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� Reproducibility (ICH)
- Precision obtained from different labs, different analysts, different operational and environmental conditions
- Analyzing aliquots from homogeneous lots
“Inter-laboratory tests”
Reproducibility Limit
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RR ssR 8.222 ==
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Precision
Intermediate Precision
Reproducibility
Instrument same different different
Batches of accessories e.g. columns
same different different
Analyst same different different
Sample matrices same different different
Concentration same different different
Batches of material e.g. reagents
Same different different
Environmental conditions Same different different
Laboratory same same different
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When there are small changes in conditions do we get the same answers?Robustness of an analytical procedure is a measure
of its capability to remain unaffected by small, but
deliberate variations in method parameters and
provided an indication of its reliability during normal
usage (Eurachem Glossary A25.2)
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� Identify parameters that will affect performance of test method
� Study the effect by RMs or CRMs
� Evaluate the effect of each parameters
� Improve the test method or control the effect
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� Human factors◦ Control via education and on the job training
� Technical factors◦ Stability of analytical solutions◦ Sample preparation◦ Influence of variation of pH, temperature, flow rate, etc
� Environmental factors◦ Temperature, pressure, humidity, pollution, contamination
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Techniques used for ruggedness testing◦ Significance testing, F-test� Are the sets of data significantly different?
◦ Factorial design� Allow investigation of the interaction of variables� More economical when variables are less than 5
◦ Plackett-Burman method design� Assumes interaction effects are negligible � Calculate the effect of each factor on a measurable
quantity
◦ Youden-Steiner testing� Assumes interaction effects are negligible � Calculate the effect of each factor on a measurable
quantity
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� JCGM 100:2008 – Guide to the Expression of Uncertainty in Measurement – The GUM (2008)◦ Available for free from the BIPM (www.bipm.org)
� Eurachem Guide – Quantifying Uncertainty in Analytical Measurement (2000) (http://www.eurachem.ul.pt/)
� NATA Technical Note 33 - Guidelines for Estimating and Reporting Measurement Uncertainty in Chemical Test Results
� Nordtest Report – Handbook for Calculation of Measurement Uncertainty in Environmental Laboratories (2004)
� Eurolab Technical Report No. 1/2007 – Measurement Uncertainty Revisited: Alternative approaches to uncertainty evaluation (2007)
� http://www.measurementuncertainty.org/mu/examples/
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Degree of external validation
1. The method is externally validated in a method-performance study
2. The method is externally validated but is used on a new matrix or using new instrument(s)
3. Well established but not tested method
Recommended internal validation
1. Verification of trueness and precision
2. Verification of trueness and precision, possibly also detection limit
3. Verification, possibly a more extensive validation
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Recommended internal validation
4. Verification, possibly a more extensive validation
5. The method needs to be fully validated
6. The method needs to be fully validated
Degree of external validation
4. The method is published in the scientific literature and states importance characteristics
5. The method is published in scientific literature without presentation of performance characteristics
6. The method was internally developed
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1. Design a validation or protocol: - The needs of the client- What is analytical achievable- Conditions of lab (environment, equipment)
2. Determine specificity and standard curve3. Determine precision, expressed as repeatability and
reproducibility4. Determine trueness5. Determine working range/measuring range6. Determine detection limit7. Determine limit of quantification/limit of determination8. Determine robustness9. Determine sensitivity10. Evaluate the result from step2-8 against the protocol11. Documented the work in a report
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� Blanks: reagent blanks and sample blanks
� Fortified/Spiked samples
� Incurred materials
� Reference materials (RMs)
� Certified reference materials (CRMs)
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Reagent blanks (solvent blanks)◦ Reagents used during the analytical process (including
solvents used for extraction or dissolution) are analyzed in isolation in order to see whether they contribute to the measurement signal. The measurement signal arising from the analyte can then be corrected accordingly
Sample blanks (matrix blanks)◦ The matrices with no analyte. They are difficult to obtain but
such materials are necessary to give a realistic estimate of interferences that would be encountered in the analysis of test samples
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Validation ToolsValidation ToolsValidation ToolsValidation Tools
Fortified samples
◦ These are materials or solutions which have been fortified with the analyte(s) of interest.
◦ The fortification is usually made by spiking. These materials or solutions may already contain the analyte of interest
◦ Fortification with a known amount of analyte enables the increase in response to the analyte to be measured and calculated in terms of the amount added
Spiked samples
- Similar to the fortified sample but spiking does notnecessary have to be restricted to the analyte of interest
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Validation ToolsValidation ToolsValidation ToolsValidation Tools
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Incurred samples◦ The materials in which the analyte of interest may be
essentially alien, but has been introduced to the bulk at
some point prior to the material being sampled
◦ The analyte is thus more closely bound in the matrix than it
would be by spiking
1. Herbicides in flour from cereal sprayed with herbicides
during its growth.
2. Growth promoters in meat derived from beast fed with
feeds containing the promoters
3. Active ingredients in pharmaceutical formulations added at
the formulation stage
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Reference materials (RMs)◦ Any materials used as a basis for reference, and could
include laboratory reagents of known purity, industrial
chemicals, or other artifacts
◦ The property or analyte of interest needs to be stable and
homogenous
◦ The material does not need to have the high degree of
characterization, traceability and certification more
properly associated with certified reference materials
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103
3.1 reference material RM Material, sufficiently homogeneous and stable with respect to one or more specified properties, which has been established to be fit for its intended use in a measurementprocess.
NOTE 1 RM is a generic term
NOTE 2 Properties can be quantitative or qualitative, e.g., identity of substances or species.
NOTE 3 Uses may include the calibration of a measurement system, assessment of a measurement procedure, assigning values to other materials, and quality control.
NOTE 4 An RM can only be used for a single purpose in a given measurement ISO REMCO
VIM, 3rd Edition103
Certified reference materials (CRMs)
◦ The characterization of the parameter of interest in a certified reference material is generally more strictly controlled than for a reference material
◦ The characterized value is certified with a stated uncertainty by a recognized institution
◦ Characterization is normally done using several different methods, so that as far as possible,
◦ Any bias in the characterization is reduced or even eliminated
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105
3.2 certified reference material CRM reference material, characterized by a metrologically valid procedure for one or more specified properties, accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a statement of metrological traceability
NOTE 1 The concept of value includes qualitative attributes such as identity or sequence. Uncertainties for such
attributes may be expressed as probabilities.
NOTE 2 Metrologically valid procedures for the production and certification of reference materials are given in,
among others, ISO Guides 34 and 35.
NOTE 3 ISO Guide 31 gives guidance on the contents of certificates.
VIM, 3rd Edition
ISO REMCO
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� RM � Homogeneous� Stable� Specified properties value
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• CRM � Homogeneous
� Stable
� Specified property value
� Certificate (acc. ISO Guide 31)
+ Uncertainty + Traceability
���� Metrologically valid procedure
RM
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� Method development and validation– evaluation of trueness
– uncertainty estimation
� Calibration of instrument
� Proof of method performance– statistical quality control
– establishing traceable results
– equipment qualification
� Proficiency testing– training and verification of competence
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Use of reference material in chemical analysis
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� Eurachem Guide “The Fitness for Purpose of Analytical Methods” LGC, UK, December 1998
� NATA Technical Note 17 “Guidelines for the validation and verification of chemical test methods”, Australia, April 2009
� Train-the –trainer course “Analytical Method Validation”, National Measurement Institute, Australia, September, 2010
� The Association of Official Analytical Chemists (AOAC international), “Peer-Verified Methods Program”, November, 1993
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Thank YouThank YouThank YouThank You