Guide 35 Statistical principles for RM nota al pie.pdf

Esta presentaciEsta presentacióón es uso exclusivo de n es uso exclusivo de INTIINTI

General and statistical principles General and statistical principles for certification of RMfor certification of RM

ISO Guide 35 and Guide 34 ISO Guide 35 and Guide 34

INTI / REDELAC INTI / REDELAC International Seminar on RM / PTInternational Seminar on RM / PT

17 November 201017 November 2010Dan Dan TholenTholen, M.S., M.S.


TopicsTopics

Role of reference materials in traceabilityRole of reference materials in traceabilityDefinitionsDefinitionsDetermining the Assigned Property ValueDetermining the Assigned Property ValueUncertainty of the CRM Property ValueUncertainty of the CRM Property Value–– CharacterizationCharacterization–– HomogeneityHomogeneity–– StabilityStability–– Combined UncertaintyCombined Uncertainty


Role of reference materialsRole of reference materials

Provide Metrological TraceabilityProvide Metrological Traceability–– IdentityIdentity–– Presence or absencePresence or absence–– QuantificationQuantification

Quality ControlQuality ControlProficiency TestingProficiency TestingTransfer property values to other materialsTransfer property values to other materialsValidate measurement proceduresValidate measurement procedures


Definitions (Guide 34)Definitions (Guide 34)

reference material (RM)reference material (RM)

material, sufficiently homogeneous and stable with material, sufficiently homogeneous and stable with respect to one or more specified properties, which has respect to one or more specified properties, which has been established to be fit for its intended use in a been established to be fit for its intended use in a measurement processmeasurement process–– NOTE 1 RM is a generic term.NOTE 1 RM is a generic term.

–– NOTE 2 Properties can be quantitative or qualitative (e.g. NOTE 2 Properties can be quantitative or qualitative (e.g. identity of substances or species).identity of substances or species).

–– NOTE 3 Uses can include the calibration of a measurement NOTE 3 Uses can include the calibration of a measurement system, assessment of a measurement procedure, assigning system, assessment of a measurement procedure, assigning values to other materials, and quality control.values to other materials, and quality control.


Types of reference materialsTypes of reference materials

Calibration MaterialCalibration Material

Positive / negative controlsPositive / negative controls

Purity compoundsPurity compounds

Quality Control materialQuality Control material

Proficiency Test itemProficiency Test item

Material for method validationMaterial for method validation



certified reference material (CRM)certified reference material (CRM)

reference material reference material characterized by a characterized by a metrologicallymetrologically valid valid procedure for one or more specified properties, procedure for one or more specified properties, accompanied by a certificate that provides the value of accompanied by a certificate that provides the value of the specified property, its associated uncertainty, and a the specified property, its associated uncertainty, and a statement of metrological traceabilitystatement of metrological traceability–– NOTE 1 The concept of value includes qualitative attributes suchNOTE 1 The concept of value includes qualitative attributes such

as identity or sequence. Uncertainties for such attributes may bas identity or sequence. Uncertainties for such attributes may be e expressed as probabilities.expressed as probabilities.

–– NOTE 2 NOTE 2 MetrologicallyMetrologically valid procedures for the production and valid procedures for the production and certification of reference materials are given in, among others,certification of reference materials are given in, among others,ISO Guides 34 and 35.ISO Guides 34 and 35.


Types of Types of Certified Reference MaterialsCertified Reference MaterialsPrimary Standard Reference MaterialsPrimary Standard Reference Materials

Positive and Negative control materialsPositive and Negative control materials

Calibration materialsCalibration materials

Proficiency Testing Items Proficiency Testing Items –– using reference valuesusing reference values

Quality Control material Quality Control material –– if the value is knownif the value is known


Types of Types of CRMsCRMs

A CRM A CRM might bemight be a national Standard a national Standard

Reference Material (SRM)Reference Material (SRM)

A CRM does not need to be an SRMA CRM does not need to be an SRM

–– Certificate and property valueCertificate and property value

–– Traceability to a stated referenceTraceability to a stated reference

–– UncertaintyUncertainty


Definitions (VIM3)Definitions (VIM3)

5.1 5.1 measurement standardmeasurement standard

realization of the definition of a given quantity, realization of the definition of a given quantity, with stated quantity value and associated with stated quantity value and associated measurement uncertainty, used as a referencemeasurement uncertainty, used as a reference



5.4 5.4 primary measurement standardprimary measurement standard

measurement standard established using a measurement standard established using a primary reference measurement procedure, or primary reference measurement procedure, or created as an artifact, chosen by conventioncreated as an artifact, chosen by convention

(Guide 30) 2.3 (Guide 30) 2.3 primary standardprimary standard: Standard that is : Standard that is designated or widely acknowledged as having the designated or widely acknowledged as having the highest metrological qualities highest metrological qualities and whose value is and whose value is accepted without reference to other Standards of the accepted without reference to other Standards of the same quantity, within a specified context.same quantity, within a specified context.



5.5 5.5 secondary measurement standardsecondary measurement standard

measurement standard established through measurement standard established through calibration with respect to a primary calibration with respect to a primary measurement standard for a quantity of the measurement standard for a quantity of the same kindsame kind

(Guide 30) 2.4 secondary standard: Standard whose (Guide 30) 2.4 secondary standard: Standard whose value is assigned by comparison with a primary value is assigned by comparison with a primary Standard of the same quantity.Standard of the same quantity.


Summary: Types of Summary: Types of Reference MaterialsReference Materials

NonNon--certified Reference Materialcertified Reference Material–– Sufficiently homogeneous and stableSufficiently homogeneous and stable

–– May have May have ““informationinformation”” valuesvalues

Certified Reference MaterialCertified Reference Material–– RM with certificate and property valueRM with certificate and property value

–– Statement of traceability and uncertaintyStatement of traceability and uncertainty

““AccreditableAccreditable”” CRMCRM–– Production meets Guide 34 (with 30 & 35)Production meets Guide 34 (with 30 & 35)

–– Certificate meets Guide 31Certificate meets Guide 31


ISO REMCO DocumentsISO REMCO Documents

ISO Guide 34 and 35 developed out of ISO Guide 34 and 35 developed out of step, but current versions are in harmony step, but current versions are in harmony –– Guide 34 1998, 2000, Guide 34 1998, 2000, 20092009

–– Guide 35 1985, 1989, Guide 35 1985, 1989, 20062006

))sort ofsort of


Guide 35 references in Guide 34Guide 35 references in Guide 34

Summary: Summary: Guide 35 is referenced for:Guide 35 is referenced for:–– 5.125.12 Metrological traceabilityMetrological traceability

–– 5.135.13 Assessment of homogeneityAssessment of homogeneity

–– 5.145.14 Assessment of stabilityAssessment of stability

–– 5.155.15 CharacterizationCharacterization

–– 5.165.16 Assignment of property values and their Assignment of property values and their uncertaintiesuncertainties


Guide 34 and 35 disharmonyGuide 34 and 35 disharmony

Guide 34 extends application of Guide 35 Guide 34 extends application of Guide 35 to include nonto include non--certified RMcertified RM–– Degree of homogeneityDegree of homogeneity

–– Long term stabilityLong term stability

Guide 34 raises an issue not covered in Guide 34 raises an issue not covered in Guide 35 Guide 35 –– determining equivalence of determining equivalence of replacement batchesreplacement batches

Guide 35 does not allow significant long Guide 35 does not allow significant long term instability, but Guide 34 says it can term instability, but Guide 34 says it can be addressed in the uncertaintybe addressed in the uncertainty


Determine Assigned ValuesDetermine Assigned Values

Called Called ““Characterization of Property Characterization of Property

ValuesValues””

Can be accomplished in several waysCan be accomplished in several ways

–– By definitionBy definition

–– By formulationBy formulation

–– By testingBy testing


Definitions of metrological Definitions of metrological traceability (VIM3)traceability (VIM3)

oo metrological traceability metrological traceability -- property of a property of a measurement result whereby the result measurement result whereby the result can be related to a reference through a can be related to a reference through a documented unbroken chain of documented unbroken chain of calibrations, each contributing to the calibrations, each contributing to the measurement uncertainty (+8 Notes)measurement uncertainty (+8 Notes)



NOTE 7 The ILAC considers the elements for NOTE 7 The ILAC considers the elements for confirming metrological traceability to be an confirming metrological traceability to be an unbroken metrological traceability chain to an unbroken metrological traceability chain to an international measurement standard or a international measurement standard or a national measurement standard, a national measurement standard, a documented measurement uncertainty, a documented measurement uncertainty, a documented measurement procedure, documented measurement procedure, accredited technical competence, accredited technical competence, metrological traceability to the SI, and metrological traceability to the SI, and calibration intervals (see ILACcalibration intervals (see ILAC--P10:2002[9]).P10:2002[9]).


More on Traceability (ILAC P10)More on Traceability (ILAC P10)

P10: section 2a): P10: section 2a): )) Where such traceability is not technically Where such traceability is not technically possible or reasonable, the laboratory and the client and other possible or reasonable, the laboratory and the client and other interested parties may agree to using certified reference materiinterested parties may agree to using certified reference materials als provided by a competent supplier or using specified methods and/provided by a competent supplier or using specified methods and/or or consensus standards that are clearly described and agreed by allconsensus standards that are clearly described and agreed by allparties concerned;parties concerned;

Note 1: Note 1: It is It is recognisedrecognised by ILAC that, due to the nature of some by ILAC that, due to the nature of some

tests, it is not possible, realistic or relevant to expect tractests, it is not possible, realistic or relevant to expect traceability of eability of

measurement results to be demonstrated. ILAC Member Bodies measurement results to be demonstrated. ILAC Member Bodies

have agreed to investigate this issue and develop guidelines on have agreed to investigate this issue and develop guidelines on

such exceptions and areas where requirements for traceability arsuch exceptions and areas where requirements for traceability are e

difficult to apply.difficult to apply.


9.3 Approaches for characterization9.3 Approaches for characterization

Four approaches given in Guide 34:Four approaches given in Guide 34:

a) measurement by a single (primary) method in a single a) measurement by a single (primary) method in a single laboratory (laboratory (includes formulationincludes formulation););

b) measurement by two or more independent reference b) measurement by two or more independent reference methods in one laboratory;methods in one laboratory;

c) measurement by a network of laboratories using one c) measurement by a network of laboratories using one or more methods of demonstrable accuracy;or more methods of demonstrable accuracy;

d) a methodd) a method--specific approach giving only methodspecific approach giving only method--specific assessed property values, using a network specific assessed property values, using a network of laboratories.of laboratories.


Property Characteristics Property Characteristics -- IdentityIdentity

Can be accomplished by definition or Can be accomplished by definition or expert judgment expert judgment –– ““What is this material?What is this material?””

Should be confirmed by testingShould be confirmed by testing–– Presence (positive) or absence (negative, or Presence (positive) or absence (negative, or

blank) blank)

–– All measurements above Limit of DetectionAll measurements above Limit of Detection

Should be confirmed by independent partyShould be confirmed by independent party–– Property characteristicProperty characteristic


9.4.1 Single Laboratory9.4.1 Single Laboratory

Prefer primary methods (see definition in G35)Prefer primary methods (see definition in G35)

Prefer more than one method, or confirmPrefer more than one method, or confirm

Primary methods not always available, others Primary methods not always available, others are commonly usedare commonly used–– GravimetryGravimetry for gas mixtures and solutionsfor gas mixtures and solutions

–– Freezing point depression (for purity)Freezing point depression (for purity)

–– IDMS where applicableIDMS where applicable

Commercial Commercial CRMsCRMs usually use one laboratory, usually use one laboratory, may use one method (or use one method to may use one method (or use one method to confirm manufacture)confirm manufacture)


9.4.2 More than one Laboratory9.4.2 More than one Laboratory

Necessary basis of approachNecessary basis of approach::

a) there exists a set of laboratories that are a) there exists a set of laboratories that are equally capable in determining the equally capable in determining the characteristics of the RM to provide results characteristics of the RM to provide results with acceptable accuracy;with acceptable accuracy;

b) the differences between individual results, b) the differences between individual results, both within and between laboratories, are both within and between laboratories, are statistical in nature regardless of the causes.statistical in nature regardless of the causes.



Use caution in the assumption of Use caution in the assumption of equivalence of results equivalence of results

Verify measurement uncertaintyVerify measurement uncertainty

Verify equivalence of methodsVerify equivalence of methods

Results must be Results must be metrologicallymetrologically traceable traceable to the same referenceto the same reference

Ideally, laboratories should be accreditedIdeally, laboratories should be accredited



Recommended number of labs variesRecommended number of labs varies–– Primary or wellPrimary or well--established methods: 2established methods: 2--3 labs3 labs

–– Less well established methods, but expect Less well established methods, but expect that all results will be valid: 6that all results will be valid: 6--8 labs8 labs

–– Many methods or chance of invalid results: Many methods or chance of invalid results: 1010--15 labs15 labs

–– Consider whether a balanced representation Consider whether a balanced representation of methods is neededof methods is needed



Recommended number of replicates:Recommended number of replicates:–– 2 units of RM2 units of RM

–– At least 6 replicates over 2 daysAt least 6 replicates over 2 days

–– Separate calibrations on every replicateSeparate calibrations on every replicate

–– If homogeneity will be determined by the experiment, If homogeneity will be determined by the experiment, need 3need 3--4 units per laboratory4 units per laboratory

Report each result, not meansReport each result, not means

Report uncertainty and method of uncertainty Report uncertainty and method of uncertainty determination (if appropriate)determination (if appropriate)


9.5 RM Property considerations9.5 RM Property considerations

9.5.2.1 Chemical properties certified for purity9.5.2.1 Chemical properties certified for purity–– Nothing is 100% pure Nothing is 100% pure -- all impurities and mass all impurities and mass

fractions should be listedfractions should be listed

–– Listed purity = 1Listed purity = 1--Sum of mass fractions of impuritiesSum of mass fractions of impurities

–– The combined standard uncertainty of the amount of The combined standard uncertainty of the amount of substance is the quadratic sum of uncertainties of the substance is the quadratic sum of uncertainties of the impuritiesimpurities



9.5.2.2 Chemical properties of synthetic 9.5.2.2 Chemical properties of synthetic RMsRMs --solutions and gas mixturessolutions and gas mixtures–– Often manufactured by Often manufactured by gravimetrygravimetry: :

Gravimetric value is basis of certificationGravimetric value is basis of certification

Verify value with suitable methodVerify value with suitable method

Homogeneity study for bottle variabilityHomogeneity study for bottle variability

Long term stability studyLong term stability study

–– Uncertainty due to verification and to bottle Uncertainty due to verification and to bottle homogeneity assumed to be small, but should homogeneity assumed to be small, but should be includedbe included



9.5.3 Conventional properties 9.5.3 Conventional properties –– materials materials defined by a method, test procedure, or defined by a method, test procedure, or particular piece of equipmentparticular piece of equipment

Often subject to large variabilityOften subject to large variability

Need very detailed method descriptionNeed very detailed method description

Need careful control of particular equipmentNeed careful control of particular equipment–– Can run a Can run a ““knownknown”” to verify equipment is OK at the to verify equipment is OK at the

time of the measurement, and provide traceabilitytime of the measurement, and provide traceability

Consider using more than one piece of Consider using more than one piece of equipment, laboratory, or operatorequipment, laboratory, or operator–– Assure independent calibrationsAssure independent calibrations


10.5.5 Treatment of Outliers10.5.5 Treatment of Outliers

Outliers different than Outliers different than ““stragglersstragglers”” (results on (results on limits of the distribution. Outliers should be limits of the distribution. Outliers should be eliminated, stragglers should be retainedeliminated, stragglers should be retained

–– Concern to underestimate uncertaintyConcern to underestimate uncertainty

Outliers can occur at any level Outliers can occur at any level –– single values, single values, means, variances, methods, laboratoriesmeans, variances, methods, laboratories

Choice of statistician whether to remove, based Choice of statistician whether to remove, based on tests and confirmation of assumptionson tests and confirmation of assumptions

Outliers are rarely replaced, and only if Outliers are rarely replaced, and only if conditions can be replicatedconditions can be replicated


10.8 Specific Issues10.8 Specific Issues

ANOVA based evaluation is recommended ANOVA based evaluation is recommended where possible, to assure assessment of key where possible, to assure assessment of key components in same mannercomponents in same manner

If collaborative study used to determine If collaborative study used to determine homogeneity, use 2homogeneity, use 2--way nested ANOVA with way nested ANOVA with balanced designbalanced design

Other procedures are possibleOther procedures are possible–– Robust meansRobust means

–– Weighted means Weighted means –– useful for combining uncertaintiesuseful for combining uncertainties

–– OtherOther


Uncertainty!Uncertainty!

The assigned property value is The assigned property value is meaningless without two things:meaningless without two things:–– Statement of metrological traceabilityStatement of metrological traceability

–– Estimate of uncertaintyEstimate of uncertainty

Applies to quantitative and qualitative Applies to quantitative and qualitative property valuesproperty values


6.16.1--6.2 Measurement uncertainty6.2 Measurement uncertainty

Basic principal:Basic principal:

The uncertainty of the Certified Reference The uncertainty of the Certified Reference Material must describe what is in a single test Material must describe what is in a single test portion taken by an CRM user, for a CRM that portion taken by an CRM user, for a CRM that has been stored and shipped according to has been stored and shipped according to specification, and is used within the claimed specification, and is used within the claimed shelf lifeshelf life

It is NOT the uncertainty of the mean of the It is NOT the uncertainty of the mean of the batch of material made by the RM producerbatch of material made by the RM producer

It is NOT the uncertainty of the mean of results It is NOT the uncertainty of the mean of results from a characterization study (from a characterization study (uucharchar))


Measurement UncertaintyMeasurement Uncertainty

Follow principals in GUM, prefer Type A Follow principals in GUM, prefer Type A

Must estimate uncertainty due toMust estimate uncertainty due to–– CharacterizationCharacterization

–– HomogeneityHomogeneityBetween bottle alwaysBetween bottle always

Within bottle where appropriateWithin bottle where appropriate

–– Transport (in excess of long term instability)Transport (in excess of long term instability)

–– Long term stability in storage by producerLong term stability in storage by producer

–– May need to add u for different methods / labsMay need to add u for different methods / labs



3.8 measurement uncertainty3.8 measurement uncertainty

nonnon--negative parameter characterizing the negative parameter characterizing the dispersion of the quantity values being dispersion of the quantity values being attributed to a attributed to a measurandmeasurand, based on the , based on the information usedinformation used

Applies only when measurements are usedApplies only when measurements are used

Does not apply to Does not apply to ““characteristicscharacteristics”” (nominal properties)(nominal properties)

Does apply to presence / absenceDoes apply to presence / absence



3.5 between3.5 between--bottle homogeneitybottle homogeneity

bottlebottle--toto--bottle variation of a property of a reference bottle variation of a property of a reference material [material [SSbbbb]]

–– NOTE It is understood that the term NOTE It is understood that the term ““betweenbetween--bottle bottle homogeneityhomogeneity”” applies to other types of packages (e.g. applies to other types of packages (e.g. vials) and other physical shapes and test pieces.vials) and other physical shapes and test pieces.

3.6 within3.6 within--bottle homogeneitybottle homogeneity

variation within one bottle of a property of a reference variation within one bottle of a property of a reference material [material [SSwbwb]]


Homogeneity for Qualitative RMHomogeneity for Qualitative RM

Can de determined by all tested samples Can de determined by all tested samples having the defined property (for example, having the defined property (for example, presence or absence)presence or absence)

Can be determined by calculating a Can be determined by calculating a confidence interval for the property value confidence interval for the property value –– For Positive For Positive -- lower limit is greater than the lower limit is greater than the

Limit of DetectionLimit of Detection

–– For Negative, Upper Limit is less than the For Negative, Upper Limit is less than the Limit of detectionLimit of detection


5.8 Project Design 5.8 Project Design -- HomogeneityHomogeneity

Always requiredAlways required–– When homogeneity can be reasonably When homogeneity can be reasonably

expected expected –– e.g. solutions and purified materiale.g. solutions and purified material

–– Inherent Inherent inhomogeneityinhomogeneity –– e.g. soilse.g. soils

Need representative sampleNeed representative sample–– Number of samples producedNumber of samples produced

–– Number to be tested Number to be tested -- generally 10generally 10--3030

–– Number of replicates Number of replicates –– generally 2generally 2--55

–– Selection process (random, systematic, etc.)Selection process (random, systematic, etc.)


How many Bottles and How many How many Bottles and How many Replicates?Replicates?

No easy formulaNo easy formula

–– Between Bottle variance Between Bottle variance σσbbbb

–– Within Bottle varianceWithin Bottle variance σσwbwb

–– Repeatability Repeatability σσrr

–– Number of samples producedNumber of samples produced

–– Need for uncertainty of estimates Need for uncertainty of estimates ssbbbb sswbwb


7.17.1--7.6 Homogeneity Study7.6 Homogeneity Study

Assure sufficient subsample for testingAssure sufficient subsample for testing

Measure every element of RM?Measure every element of RM?–– If not measured, element must be If not measured, element must be

demonstrated to be correlated with tested demonstrated to be correlated with tested elementelement

–– Can come from literature or other sourceCan come from literature or other source

Randomized measurementsRandomized measurements–– Check for trend in measurement orderCheck for trend in measurement order

–– Check for trend in manufacture orderCheck for trend in manufacture order


7.8 Homogeneity Study7.8 Homogeneity Study

Estimating betweenEstimating between--bottle variancebottle variance

Two models discussed:Two models discussed:–– SubsamplingSubsampling is possibleis possible

–– SubsamplingSubsampling is not possibleis not possible

Statistical analysis the same for both (fully Statistical analysis the same for both (fully nested one factor design)nested one factor design)

(reference Figures 1 and 2, clause 7.8)(reference Figures 1 and 2, clause 7.8)


7.87.8--7.9 Homogeneity Study7.9 Homogeneity Study

ss22bbbb = s= s22

AA = (= (MSMSamongamong –– MSMSwithinwithin)/n)/n00

ssbbbb = = uubbbb = = √√ ss22AA

NOTE: If NOTE: If MSMSwithinwithin > > MSMSamongamong then sthen s22bbbb = 0= 0


7.9 7.9 uubbbb when when ssrr is largeis large

When measurement has poor repeatability When measurement has poor repeatability or for other reasons, if or for other reasons, if MSMSwithinwithin > > MSMSamongamong

then we cannot say then we cannot say uubbbb = 0= 0

This is actually fairly commonThis is actually fairly common

It is necessary to use an upper limit for It is necessary to use an upper limit for uubbbb

uubbbb = = √√((MS((MSwithinwithin/n)/n)√√(2/ (2/ ѵѵMSwithinMSwithin))))

where where ѵѵMSwithinMSwithin == Degrees of Freedom for Degrees of Freedom for MSMSwithinwithin

(usually the number of bottles * (n(usually the number of bottles * (n00--1))1))

nn00 = number of replicates= number of replicates


uubbbb when when ssrr is largeis large

This is fairly common situation, and alternative This is fairly common situation, and alternative limits may be usedlimits may be used

Upper limit of confidence interval could be usedUpper limit of confidence interval could be used

((MSMSamongamong –– MSMSwithinwithin)/n)/n ≤≤ uu22bbbb ≤≤ ss22

bbbb + s+ s22rr/n/n00

Could use Could use uubbbb = = √√(s(s22bbbb + + MSMSwithinwithin /n/n00))

With With ssbbbb ≥≥ 0 , as calculated0 , as calculated


Homogeneity questionHomogeneity question

Should it be possible that Should it be possible that uubbbb = 0?= 0?

Should there always be a component due Should there always be a component due to between bottle differences?to between bottle differences?

If a RM Producer knows If a RM Producer knows ssrr and the and the experiment shows experiment shows ssrr is in control at the is in control at the time, can we allow stime, can we allow s22

bbbb = 0 ?= 0 ?


7.10 Within Bottle Homogeneity7.10 Within Bottle Homogeneity

Not relevant in some casesNot relevant in some cases

Not always necessary even when possibleNot always necessary even when possible

Within bottle always mixed with Within bottle always mixed with repeatability at some levelrepeatability at some level

ssrr ≤≤ sswbwb

Estimated as Estimated as √√MSMSwithinwithin in most casesin most cases



3.10 short3.10 short--term stabilityterm stability

stability of a property of a reference material stability of a property of a reference material during transport under specified during transport under specified transporttransport

conditions [conditions [SSstssts]]

3.11 long3.11 long--term stabilityterm stability

stability of a property of a reference material stability of a property of a reference material under specified under specified storagestorage conditions at the CRMconditions at the CRM--producer [producer [SSltslts]]


5.9 Project Design 5.9 Project Design -- StabilityStability

Need to describe conditions that affect Need to describe conditions that affect stability of materialstability of material

Choose storage temperature that is best Choose storage temperature that is best for long term stabilityfor long term stability

Study short term stability (transport):Study short term stability (transport):–– Under stressed conditionsUnder stressed conditions

–– Under specified conditionsUnder specified conditions

Plan to retain sufficient samples for long Plan to retain sufficient samples for long term monitoring under storage conditionsterm monitoring under storage conditions


Project Design Project Design -- StabilityStability

Stability testing may require many Stability testing may require many samples samples –– at least 2 bottles at every at least 2 bottles at every temperature and every time temperature and every time –– Short term, 3Short term, 3--5 points in 2 weeks5 points in 2 weeks

–– Long term, 3Long term, 3--4 points for regression4 points for regression


8.1 Stability Studies8.1 Stability Studies

Two types of stability are of interestTwo types of stability are of interest–– Long term (shelf life) in storage at RM Long term (shelf life) in storage at RM

ProducerProducer

–– Short term when shipped to user Short term when shipped to user –– in addition in addition to stability in storageto stability in storage

If it is not possible to maintain stability If it is not possible to maintain stability during shipment, add a component to during shipment, add a component to uncertaintyuncertainty


Stability StudiesStability Studies

Guide 35 states (8.1) Guide 35 states (8.1) ““It is often equally It is often equally important to know what might happen to the important to know what might happen to the sample if proper transport conditions are not sample if proper transport conditions are not maintainedmaintainedV”V”

That is, the RM Producer should test the effects That is, the RM Producer should test the effects of shipment conditions that are possible but not of shipment conditions that are possible but not likely if reasonable precautions are takenlikely if reasonable precautions are taken

Recommendation is to assist in developing Recommendation is to assist in developing guidelines for shipmentguidelines for shipment

Should unexpected conditions be included in the Should unexpected conditions be included in the uncertainty claim?uncertainty claim?


8.2 Long term Stability Studies8.2 Long term Stability Studies

Classical:Classical:–– Store under defined conditionsStore under defined conditions

–– Test periodically for change, withinTest periodically for change, within--lab conditionslab conditions

–– Retain records, to develop experience and detect Retain records, to develop experience and detect deteriorationdeterioration

IsochronusIsochronus::–– Controlled experiment with some samples stressed Controlled experiment with some samples stressed

by heat (or other suspected conditions)by heat (or other suspected conditions)

–– Mathematical model to predict long term change due Mathematical model to predict long term change due to conditionsto conditions

Guide 35 prefers Guide 35 prefers isochronusisochronus estimatesestimates


8.3 Evaluation of Stability Data8.3 Evaluation of Stability Data

Prefer to have a kinetic model for instability, Prefer to have a kinetic model for instability, based on understandingbased on understanding

Can evaluate change with a linear model Can evaluate change with a linear model ––simple linear regressionsimple linear regression

YYijij = = ββ00 ++ ββ11XXi i + + εεijij

YYijij = stability result at time i for sample j (if > 1)= stability result at time i for sample j (if > 1)

XXii = = time itime i

ββ00 is the regression coefficients for interceptis the regression coefficients for intercept

ββ11 is the regression coefficient for slopeis the regression coefficient for slope

εεijij is random error for time i and sample jis random error for time i and sample j



ββ1 1 is expected to be zero for a stable RM (no is expected to be zero for a stable RM (no change over time)change over time)

ββ00 is the intercept; this has little real is the intercept; this has little real interpretation, but should be close to the interpretation, but should be close to the assigned value assigned value –– the mean at time 0the mean at time 0

εεijij is expected to be random, normal distributedis expected to be random, normal distributed

Need to test statistical significance of regression, Need to test statistical significance of regression, (F test) same as significance of (F test) same as significance of ββ1 1 ((t test)t test)

Reference Guide 35 8.3, page 23Reference Guide 35 8.3, page 23



If the regression is significant there is a If the regression is significant there is a trend and trend and “V“Vthis usually means the RM this usually means the RM cannot be certified.cannot be certified.””

Options:Options:–– Do not certify the materialDo not certify the material

–– Shorten the shelf lifeShorten the shelf life

–– Make the uncertainty larger (in new Guide 34) Make the uncertainty larger (in new Guide 34)


8.4.1 Monitoring Stability 8.4.1 Monitoring Stability --evaluationevaluation

Possible for instability to occur suddenly, Possible for instability to occur suddenly, not as gradual trendnot as gradual trend

Monitoring assures uncertainty estimates Monitoring assures uncertainty estimates are still valid are still valid

Can be done with isochronous design (see Can be done with isochronous design (see References)References)


8.5 Determine Shelf Life in relation 8.5 Determine Shelf Life in relation to Long Term Stabilityto Long Term Stability

Even if there is no significant trend of instability, Even if there is no significant trend of instability, there is a need to make allowance for long term there is a need to make allowance for long term degradationdegradation

To do so, describe a model where it is assumed To do so, describe a model where it is assumed that the property value Y decreases linearly from that the property value Y decreases linearly from the initial value Ythe initial value Y00 with a constant relative with a constant relative degradation rate b' as a function of time Xdegradation rate b' as a function of time X

Y(bY(b00,b,b′′,X)=Y,X)=Y00(1+b(1+b′′X)X)

See reference 25 See reference 25


Combined UncertaintyCombined Uncertainty

After determining uncertainty components, After determining uncertainty components, combine as Root Sum of Squarescombine as Root Sum of Squares

uuCRMCRM==√√(u(u22charchar+u+u22

bbbb+u+u22ltslts+u+u22

stssts))

May need to add other components for a specific May need to add other components for a specific Certified Reference MaterialCertified Reference Material


6.2 Measurement uncertainty6.2 Measurement uncertainty

xxCRMCRM==xxcharchar++δδxxbbbb++δδxxltslts++δδxxstssts

With care, With care, δδxxbbbb δδxxltslts δδxxstssts are zero, are zero,

so so xxCRMCRM = = xxcharchar

Even if Even if δδxx.. = 0, uncertainties are >0= 0, uncertainties are >0


Notes on uncertaintyNotes on uncertainty

SSbbbb and and SSltslts must always be included in the must always be included in the uncertainty estimate as components > 0uncertainty estimate as components > 0

SSwbwb and and SSstssts need to be considered, but these need to be considered, but these components can be = 0components can be = 0


Expanded uncertaintyExpanded uncertainty

Convention is for 95% coverageConvention is for 95% coverage–– kk=2 is acceptable=2 is acceptable

–– Can use t statisticCan use t statistic

–– Use confidence interval if distribution is not Use confidence interval if distribution is not symmetricsymmetric

Check Check –– all homogeneity and stability test all homogeneity and stability test results should be within the expanded results should be within the expanded uncertainty intervaluncertainty interval

(can expand for laboratory(can expand for laboratory’’s measurement s measurement uncertainty)uncertainty)


Data exercises Data exercises

Conduct the analyses in Annex B.5, B.6, Conduct the analyses in Annex B.5, B.6, B.7 and match the Annex B resultsB.7 and match the Annex B results

B.5 Simple Linear Regression, B.5 Simple Linear Regression, uultslts per 8.5 per 8.5 and and uultslts per 8.3.2 (nextper 8.3.2 (next--toto--last paragraph)last paragraph)

B.6 Characterization using ANOVA; grand B.6 Characterization using ANOVA; grand mean, uncertainty associated with grand mean, uncertainty associated with grand mean, and mean, and ssrr

B.7 Weighting; weights, final weights, B.7 Weighting; weights, final weights, mean, and uncertainty of meanmean, and uncertainty of mean


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Guide 35 Statistical principles for RM nota al pie.pdf

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