Iso Guide 80 2014

GUIDE 80
First edition
2014-08-15
Guidance for the in-house preparation

of quality control materials (QCMs)
Lignes directrices pour la préparation interne des matériaux de
référence utilisés pour le contrôle qualité
Reference number
ISO GUIDE 80:2014(E)
© ISO 2014
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© ISO 2014
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ii © ISO 2014 – All rights reserved

Contents Page
Foreword .......................................................................................................................................................................................................................................... v
Introduction ................................................................................................................................................................................................................................ vi
1 Scope ................................................................................................................................................................................................................................. 1
2 Normative references ...................................................................................................................................................................................... 1
3 Terms and definitions ..................................................................................................................................................................................... 1
4 Quality control materials (QCMs) ....................................................................................................................................................... 2
5 Applications of quality control materials (QCMs) ............................................................................................................. 2
6 Steps in the in-house preparation of quality control materials (QCMs) ..................................................... 3
7 Material specification ...................................................................................................................................................................................... 4
7.1 Matrix type, matching and commutability ...................................................................................................................... 4
7.2 Properties and property values ................................................................................................................................................ 4
7.3 Unit size ......................................................................................................................................................................................................... 4
7.4 Total bulk amount o f material ................................................................................................................................................... 4
8 Preparation of quality control materials (QCMs) ............................................................................................................... 5
8.1 Sourcing o f bulk material ............................................................................................................................................................... 5
8.2 Material processing............................................................................................................................................................................. 5
8.3 Sub-division and packaging ......................................................................................................................................................... 7
9 Homogeneity ............................................................................................................................................................................................................ 9
9.1 Overview ...................................................................................................................................................................................................... 9
9.2 Analytical approach ............................................................................................................................................................................ 9
9.3 Statistical treatment o f homogeneity data................................................................................................................... 10
10 Characterization and value assignment.................................................................................................................................... 12
11 Stability ....................................................................................................................................................................................................................... 12
11.1 Overview ................................................................................................................................................................................................... 12
11.2 Assessing stability ............................................................................................................................................................................. 12
11.3 Assigning an expiry date to a QCM ...................................................................................................................................... 13
12 Transportation .................................................................................................................................................................................................... 13
13 Documentation for quality control materials (QCMs) ................................................................................................ 13
13.1 General ........................................................................................................................................................................................................ 13
13.2 Information to be available with quality control materials (QCMs) ...................................................... 13
13.3 Labelling o f QCM units .................................................................................................................................................................. 14
13.4 Use ful information to be retained ....................................................................................................................................... 14
14 Storage ......................................................................................................................................................................................................................... 15
14.1 General ........................................................................................................................................................................................................ 15
14.2 Monitoring o f storage conditions ......................................................................................................................................... 15
15 Using quality control materials (QCMs) .................................................................................................................................... 15
15.1 General ........................................................................................................................................................................................................ 15
15.2 Minimum sample size .................................................................................................................................................................... 15
15.3 Mixing procedure ............................................................................................................................................................................... 15
15.4 Dry mass correction ........................................................................................................................................................................ 16
15.5 Storing opened containers o f QCMs .................................................................................................................................. 16
Annex A (informative) Case study 1 — Preparation of a QCM from coal ...................................................................... 17
Annex B (informative) Case study 2 — Preparation of geological or metallurgical quality control
materials (QCMs) .............................................................................................................................................................................................. 19
Annex C (informative) Case study 3 — Preparation o f a wheat flour fortified with folic acid quality
control material (QCM) ............................................................................................................................................................................... 26
© ISO 2014 – All rights reserved iii

Annex D (informative) Case study 4 — Bauxite quality control material (QCM) ................................................. 32
Annex E (informative) Case study 5 — Pharmaceutical reference standards ........................................................ 37
Annex F (informative) Case study 6 — Preparation of testing materials for “bromate in water” ...... 42
BIBLIOGRAPHY ...................................................................................................................................................................................................................... 49
iv © ISO 2014 – All rights reserved

Foreword
ISO (the International Organization for Standardization) is a worldwide federation o f national standards
bodies (ISO member bodies). The work o f preparing International Standards is normally carried out
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electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the di fferent approval criteria needed for the
di fferent types o f ISO documents should be noted. This document was dra fted in accordance with the
editorial rules o f the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some o f the elements o f this document may be the subject o f
patent rights. ISO shall not be held responsible for identi fying any or all such patent rights. Details o f any
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the ISO list o f patent declarations received (see www.iso.org/patents).
Any trade name used in this document is in formation given for the convenience o f users and does not
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For an explanation on the meaning o f ISO specific terms and expressions related to con formity
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to Trade (TBT) see the following URL: Foreword - Supplementary in formation
The committee responsible for this document is ISO/REMCO, Committee on referen ce m aterials (which
has the task to prepare guidance documents for the preparation, characterization, certification and use
o f re ference materials (RMs) and the competence assessment o f re ference material producers.
© ISO 2014 – All rights reserved v

Introduction
Re ference materials (RMs) are widely used in measurement laboratories for a variety o f purposes and it
is important to recognize that the material most appropriate for a particular application should be used.
Certified re ference materials (CRMs), i.e. those which have property values and associated uncertainties
assigned by metrologically valid procedures are primarily used for method validation and calibrations
providing metrological traceability.
The preparation o f re ference materials for metrological quality control (i.e. control o f the quality o f
measurements not products) is an important activity which provides materials suitable for the day-to-
day demonstration that a particular (part o f a) measurement system is under statistical control. Such
materials do not require characterization by metrologically valid procedures, and can be prepared “in-
house”, i.e. by laboratory sta ff familiar with their behaviour, to fulfil specific quality control requirements.
Re ference materials which are su fficiently homogeneous and stable are necessary for metrological
quality control purposes, such as demonstrating a measurement system is under statistical control,
per forms as expected and provides reliable results; where the trueness o f the measurement result is
not critical. Di fferent industries use various terminologies to describe such materials (e.g. in-house
re ference materials, quality control materials, check samples, etc.). For the purposes o f this Guide, the
term “Quality Control Materials” (QCMs) will be used to simpli fy repeated citation.
While CRMs are produced by established re ference material producers and are commercially available,
QCMs are o ften prepared by a laboratory for its own internal use. Frequently, QCMs are characterized
only for a limited scope (a limited number o f property values) and for specific laboratory applications.
The rationale for preparing quality control materials can be one or a combination o f the following factors:
— to have an RM representing as closely as possible routine samples, suitable for quality control;
— to have a suitable day-to-day RM to complement a commercially available CRM;
— no suitable CRM exists;
— the application does not require a material having the full characteristics o f a CRM (e.g. traceability
and uncertainty for specified property values).
QCMs are RMs and as such the requirements o f ISO Guide 34[1] for the production o f RMs apply. However,
i f the material is only used in-house by the preparing laboratory, some requirements (e.g. for transport
stability) can be relaxed. The preparation o f a QCM is related to that o f a CRM and those preparing QCMs
may wish to consult ISO Guides 34[1] and 35 [2] for further guidance. Where appropriate, this Guide will
re fer to relevant parts o f these Guides.
It is recognized that the aim o f many laboratories requiring QCMs is to minimize the time and e ffort
needed to prepare the materials. To this end, many laboratories use samples o f real products for which
there is a body o f analytical data available. A number o f case studies are included as annexes o f this
guidance document to provide examples o f how such data may be processed to confirm fitness for
purpose o f the materials.
vi © ISO 2014 – All rights reserved

GUIDE ISO GUIDE 80:2014(E)
Guidance for the in-house preparation of quality control

materials (QCMs)
1 Scope
T h i s Gu ide outl i ne s the e s s enti a l charac teri s tics o f re ference materia l s for qua l ity control (QC ) pur p o s e s ,
and de s crib e s the pro ces s es b y wh ich they c an b e prep are d b y comp e tent s ta ff with i n the faci l ity in
wh ich they wi l l b e u s e d (i . e . where i n s tabi l ity due to tran s p or tation cond ition s i s avoide d) . T he content
o f th i s Gu ide a l s o appl ie s to i n herently s table materi a l s , wh ich c an b e tran s p or te d to o ther lo c ation s
without ri s k o f a ny s ign i fic ant cha nge i n the prop er ty va lue s o f i ntere s t.
T he pri mar y aud ience for th i s Gu ide i s lab orator y s ta ff who a re re qui re d to prep are and u s e materi a l s for
s p e c i fic i n-hou s e qua l ity control appl ic ation s . P rep aration o f QC M s , where tran s p or tation i s a ne ce s s a r y
comp onent o f the s upply chai n, s uch as lab orator y s ite s at d i fferent lo c ation s or for pro ficienc y te s ti ng
s cheme s , s hou ld con form to the relevant re qu i rements o f I S O Gu ide s 3 4 [1] and 3 5 . [2]
T he des c rip tion o f the pro duc tion o f re ference materia l s (RM s) , as de tai le d i n I S O Gu ide 3 4 [1] and
ISO Guide 35[2] i s a l s o appl icable to the prep aration o f qua l ity control materi a l s (QC M s) . H owever, the
re qu i rements for “i n-hou s e” QC M s are les s demand i ng tha n tho s e for a cer ti fie d re ference materia l
(C RM ) . T he prep a ration o f QC M s shou ld i nvolve homo geneity a nd s tabi l ity a s s e s s ments , and a l i m ite d
cha rac teri z ation o f the materi a l to provide a n i nd ic ation o f its relevant prop er ty va lue s a nd thei r
variation, prior to u s e . T h i s do c u ment provides the qua l ity c riteri a that a materia l shou ld fu l fi l to
b e con s idere d fit- for-pu rp o s e for demon s trati ng a me as u rement s ys tem i s u nder s tati s tic a l control .
Gu idance on u s es o f s uch materia l s , for e xample s e tti ng up a QC char t, i s ade quately covere d el s ewhere
[3] [4] [5] [ ]
, , , 6
and i s no t i nclude d i n th i s Gu ide .
T he layout a nd s truc tu re o f th i s Gu ide provide s genera l i n formation on the prep aration o f QC M s i n the
mai n chap ters , with s p e ci fic c as e s tud ie s coveri ng a range o f s e c tors i n the an nexe s . T he c as e s tud ie s
are no t comple te “pro ce s s manua l s ” but are i nclude d to h igh l ight s ome o f the key con s ideration s when
prep ari ng QC M s . T he c a s e s tud ie s var y i n complexity and de ta i l , i nclud i ng s e c tor s p e c i fic term i nolo g y,
but provide a range o f i n formation for lab orator y s ta ff to d raw from .
I t i s exp e c te d that tho s e i nvolve d i n QC M prep a ration wi l l have s ome knowle dge o f the typ e o f materi a l
to b e prep are d and b e aware o f a ny p o tenti a l problem s due to matri x e ffe c ts , contam i nation, e tc .
2 Normative references
T he fol lowi ng do c uments , i n whole or i n p ar t, are normatively re ference d i n th i s do c u ment and are
i nd i s p en s able for its appl ic ation . For date d re ference s , on ly the e d ition c ite d appl ie s . For u ndate d
re ference s , the late s t e d ition o f the re ference d do c u ment (i nclud i ng any a mend ments) appl ie s .
I S O Gu ide 3 0 , Reference materials — Selected terms and definitions

I S O/I E C Guide 9 9, Intern ation al vocabulary of m etrology — Basic an d gen eral con cepts an d associated
term s (VIM)
I S O 3 5 3 4 -1 , Statistics — Vocabulary an d symbols — Part 1 : Gen eral statistical term s an d term s used in
probability
3 Terms and definitions

For the pu rp o s e s o f th i s do c ument, the term s and defi n ition s i n I S O Gu ide 3 0 [7] ISO/IEC Guide 99 [8] a nd
ISO 3534-1[9] and the fol lowi ng apply.
© ISO 2014 – All rights reserved 1

3.1
indicative value
va lue o f a quantity or prop er ty, i nclude d i n the cer ti fic ate o f a C RM or o ther wi s e s uppl ie d, wh ich i s
provide d for i n formation on ly (i . e . i s no t cer ti fie d b y the pro ducer or the cer ti fyi ng b o dy)
N o te 1 to entr y: Va lue s a s s igne d to qu a l ity control m ater i a l s (QC M s) c a n o n l y b e i nd ic ati ve i n th at the y h ave no
me tro lo gic a l trace ab i l ity. I S O Gu ide 3 0 : 19 9 2 [7] u s e s the term “u ncer ti fie d va lue” to de s c rib e a va lue o f a qu a ntity
pro vide d fo r i n fo r m ation on l y.
4 Quality control materials (QCMs)

T he term “qua l ity control materi a l” or “QC M ” ha s b e en devi s e d for the pu rp o s e s o f th i s Gu ide s olely to
s i mpl i fy rep e ate d re ference to materi a l s u s e d routi nely to a s s e s s the pre ci s ion o f te s t pro ce du re s . I t i s
no t i ntende d to defi ne a new cl as s o f re ference materia l s . Such materia l s are variou s ly re ferre d to i n the
op en l iterature a s “i n-hou s e re ference materia l s ”, “qua l ity control s ample s ”, “che ck s ample s ”, “s e t up
s a mples ”, e tc .
Where no s uitable C RM e xi s ts , lab oratorie s may u s e QC M s to provide a n a s s e s s ment o f the rep e atabi l ity
/ i nterme d iate pre ci s ion / repro ducibi l ity o f a me a s urement re s u lt. QC M s ca n no t b e u s e d to e s tabl i sh
me trolo gic a l trace abi l ity or truene s s o f a me as u rement re s u lt.
QC M s shou ld a lways comply with the b as ic re qu i rements o f a ny re ference materia l, i . e . they shou ld b e
s u ffic iently homo gene ou s and s table with re s p e c t to the prop er ties o f i ntere s t. T he level o f he tero geneity
shou ld b e le s s th an the e xp e c te d s tanda rd deviation o f the me a s u rement pro ces s or an e s tabl i s he d
criterion va lue agai n s t wh ich the a s s e s s ment o f lab orator y p er formance or the “norma l i z ation” of
re s u lts i s accep table . T he QC M s hou ld b e s table for a p erio d o f ti me that i s at le as t a s long as that du ri ng
which it is intended to be used.

5 Applications of quality control materials (QCMs)
T he pri ncip a l fu nc tion o f QC M s i s to provide lab oratorie s with an e conom ic a l me an s o f che cki ng thei r
routi ne te s t pro ce dure s for pre c i s ion on a re gu la r b as i s (e . g. dai ly, we ekly or month ly) .
Wh i le C RM s ca n i n a l l c as e s replace QC M s , QC M s a re no t repl acements for C RM s; they a re complementar y
to them h avi ng a s p e ci fic, l i m ite d pu rp o s e i n the me as u rement pro ce s s . C RM s pro duce d accord i ng to
the principles of ISO Guide 34[1] are e s s entia l to e s tabl i s h the concep t o f me trolo gic a l trace abi l ity i n a
me a n i ng fu l ma nner, and provide the h ighe s t s tand ard with re s p e c t to re ference materia l s . T here i s no
re qu i rement for QC M s to h ave me trolo gic a l ly trace able a s s igne d va lues; con s e quently, QC M s c an no t
b e u s e d to e s tabl i s h me trolo gica l trace abi l ity or to e s ti mate u ncer tai nty. For me tho d va l idation and
uncer tai nty e s ti mation, QC M s may b e u s e d to a l i m ite d ex tent (e . g. for e s tabl i s h ment o f a pre c i s ion
es ti mate a s p ar t o f the to ta l me as u rement u ncer ta i nty) .
Us e s o f QC M s i nclude ( but a re not l i m ite d to) :
— prep aration o f QC char ts – to demon s trate control o f a me a s u rement pro ce s s with i n a lab orator y or
to con fi rm the e ffe c tivene s s o f a lab orator y’s qua l ity control pro ce s s or to demon s trate control o f a
me a s u rement pro ce s s over a p erio d o f ti me;
— comp ari s on o f re s u lts (e . g. from two or more s erie s o f relate d s ample s either i n a shor t p erio d o f
ti me or over an ex tende d p erio d o f ti me when a me as u rement pro ce s s i s known to var y) ;
— me tho d development – to e s tabl i s h con s i s tenc y (for va l idation a cer ti fie d re ference materia l s hou ld
b e u s e d) ;
— i n s tru ment p er forma nce checks;
— rep e atabi l ity and repro ducibi l ity s tud ies – rep e ate d use over an e xtende d p erio d of ti me,
i n s tru ments , op erators , e tc . , to e s ti mate long-term repro duc ibi l ity or robu s tne s s o f a me a s u rement
pro ce s s or lab orator y;
2 © ISO 2014 – All rights reserved

— as check samples – for example, to confirm the degree o f equivalence o f measurement results from
two or more laboratories (e.g. provider and user), where the materials are inherently stable;
— operator variability;
— impact o f any changes to the environmental conditions (e.g. temperature, humidity).
When confirming that a measurement process is under statistical control, [3] , [4] , [5] , [6 ] the acceptability
o f laboratory per formance is generally assessed by comparing either the standard deviation or the
range o f the individual results for the QCM against a pre-established criterion. I f a lack o f control o f the
measurement process is identified, the laboratory needs to take action. In the simplest case, this may
require repeating the “suspect” measurements, perhaps following a re-calibration o f instruments.
A more in-depth discussion o f the uses o f quality control materials can be found in ISO Guide 33. [10]
Regardless o f the intended use, it is necessary to assess homogeneity and stability o f a QCM. [11]
6 Steps in the in-house preparation of quality control materials (QCMs)

The fundamental purpose o f QCMs is to detect change. In general, more pragmatic and less rigorous
protocols can be used for stability and homogeneity steps to strike a balance between material
development costs on the one hand and the intended use o f the material on the other.
The production o f any re ference material requires a level o f technical and organizational competence.
It is acknowledged that in many cases “in-house” QCMs will be prepared by technically competent sta ff
that is knowledgeable about the materials/processes being used.
The key steps involved in the in-house preparation o f a typical QCM are summarized in the flow chart
in Figure 1 and are described in more detail in References [12 ] and [13 ]. Materials can be sourced
from, processed, sub-divided and packaged by third parties, where they have specialized equipment
and/or expertise. Materials may even be products which are commercially available and meet the user’s
specification (e.g. food products available in appropriately sized units from a single production batch).
Figure 1 — Key steps in the preparation of a typical QCM
NOTE Any o f these steps may be subcontracted to a technically competent subcontractor.

7 M a te r i a l s p e c i fi c a ti o n
The key criteria in the specification and selection o f a QCM are for the material to be as close as possible
to real samples and available in appropriate quantities.
7.1 Matrix type, matching and commutability

In general terms, the uncertainties associated with a measurement result arise from the two main
stages o f the measurement procedure:
— the preparation o f a sample comprising digestion, extraction, clean-up, etc.;
— the measurement o f the property in the prepared sample by a suitable technique.
The scope and applicability o f a matrix re ference material is an important consideration for both the
production and use o f all re ference materials.
The matrix o f the QCM should be the same or as similar as possible to the matrix o f the routine test
samples, so that a satis factory result for the QCM is genuinely indicative o f satis factory results for the
test samples. This matrix matching requires some knowledge o f the analytical procedure used on the
routine samples, so that a judgment can be made as to the degree o f variation o f the physical/chemical
properties o f the sample and test matrices that may cause them to respond di fferently to a particular
measurement procedure. For example, a freeze-dried food matrix may behave di fferently during analysis
to a similar foodstu ff with higher moisture content.
Generally, QCMs are prepared for specific purposes and the materials’ properties can be closely matched
to the samples under analysis.
Commutability has particular significance in clinical chemistry and has been described elsewhere. [14]
In practice, the impetus for the preparation o f a QCM may o ften be the fact that adequate matrix CRMs are
not available and there fore the QCM producer is likely to use the specific matrix/property combination
in question and matching is not an issue.
7.2 Properties and property values

As for any re ference material, the QCM should be characterized for those properties that are o f particular
importance in the measurement o f the routine test samples. The properties o f the QCM should be as
similar as possible to those expected in the test samples. This may require some preliminary screening
measurements to be carried out on a number o f candidate materials, to enable the most appropriate to
be selected.
7.3 Unit size
Unit size is the amount o f material that comprises a single bottled unit o f the QCM. When preparing a
QCM, the size o f individual units should be based on the likely use, i.e. the amount o f material required
for the measurements concerned and whether the units are to contain su fficient material for a single
analysis or for multiple measurements.
7.4 Total bulk amount of material

An estimate is required o f the total bulk amount o f candidate material that should be sourced. In
principle, this may be estimated by considering
— the number o f units per year required by the laboratory,
— the unit size,
— the preparation yield,

— the quantity o f material that can readily be homogenized,

— the length o f time the supply is to be maintained and the assumed stability o f the material,
— the type and size o f the required storage facility.
8 Preparation of quality control materials (QCMs)

8.1 Sourcing of bulk material
Sourcing and processing o f bulk materials for QCM preparation may at first seem di fficult especially in
those cases where large quantities o f material are required. However, there are a number o f options that
may be available including:
— excess sample material;
— accurate gravimetric formulation.
Processing the bulk material can have significant cost implications for the preparation o f QCMs and
simple, straightforward processing methods should be used to ensure cost-e ffective QCM preparation.
The exact preparation procedures required for a particular QCM will depend on the nature o f the matrix
and the properties o f interest.
In general, liquid matrix QCMs are much easier to produce than their solid counterparts. The main reason
for this is that homogeneous liquids can easily be achieved even with fairly rudimentary equipment (e.g.
large mixing containers equipped with paddle or magnetic stirrers). A liquid is easily spiked, filtered or
mixed with additives and stabilizers. The corresponding processes for solid materials, milling, grinding,
mixing and sieving are much more di fficult to accomplish homogeneously, especially for quantities
greater than 20 kg. These techniques require a significant investment in major capital equipment when
large-scale preparation is envisaged.
During preparation o f both liquid and solid materials it is important to prevent contamination by
substances which can potentially inter fere with the intended measurement process (e.g. a similar
material or contamination o f a blank material). Hence, all bottles, vials or flasks to be used for final
containment must be care fully cleaned and dried be fore filling to remove possible contaminants.
When sourcing biological materials for example, for control o f measurement procedures for medical
laboratories, the following specific issues need to be considered:
— ethics o f the retention and use o f residual patients’ samples for the preparation o f QCMs;
— legal liabilities o f retention and use o f residual patients’ samples purchased for the preparation o f
QCMs;
— medical laboratories creating QCMs need to have a high degree o f confidence in the trueness o f the
material selected, to avoid use o f misidentified organisms;
— materials sourced for QCM preparation should be screened for potential risks for health hazards,
especially i f the preparation includes the use o f contaminated sharps or has the potential for aerosol
formation.
8.2 Material processing

8.2.1 General
Once the bulk material has been sourced there are a number o f processing stages which may need to
be carried out to ensure the material has the appropriate homogeneity and stability for its intended
purpose. Some o f the more common processes are described in the following sub clauses.

8.2.2 Drying
Remova l o f water ma ke s matri x materi a l s far e a s ier to ha nd le and a l s o i mprove s b o th thei r s hor t and
long term s tabi l ity. D r yi ng o f s oi l s a nd s i m i lar matrice s may b e c arrie d out at ambient or elevate d
temp eratu re, dep end i ng on the prop er ties o f i ntere s t, s i nce the more volati le comp onents may b e p ar tly
lo s t at h igher temp erature s . Water remova l a l s o re duce s the l i kel i ho o d o f m icrobi a l grow th formation,
wh ich i s a p a r tic u lar problem with biolo gic a l materia l s . Fre e ze - d r yi ng i s a te ch n ique wh ich i s u s e fu l
with temp eratu re s en s itive prop er tie s or matrice s .
8.2.3 Milling and grinding

For s ol id s , s ome form o f cru s h i ng , m i l l i ng , gri nd i ng and p a r ticle s i ze re duc tion i s o ften ne ce s s ar y to
en s u re u ni form p ar ticle s i z e and to i mprove homo geneity. For la rge bu l k quantitie s , the s e pro ce s s e s a re
slow and may ta ke s evera l days to comple te . C are s hou ld b e ta ken no t to i ntro duce contam i nation from
the app a ratu s duri ng the gri nd i ng pro ce s s . T he he a lth a nd s a fe ty a s p e c ts o f gri nd i ng large quantitie s
o f p ar tic u late matter, wh ich may have toxic comp onents , shou ld a l s o b e con s idere d . C r yo gen ic gri nd i ng
at −78 ° C (s ol id C O 2 ) or −19 6 ° C ( l iqu id N 2 ) may b e ne ce s s ar y for p olymers , biolo gic a l, oi ly/ fatty a nd
therma l ly labi le materia l s .
8.2.4 Sieving
Sievi ng i s o ften c arrie d out a fter m i l l i ng and gri nd i ng to i mprove materia l homo geneity. Par tic u late
materia l s s uch as s oi l s , ore s , as he s and ground biolo gic a l materia l s are p as s e d th rough a s tanda rd s ieve
to remove large p a r ticle s that a re ab ove a pre s crib e d s i ze .
Sievi ng however change s the matri x comp o s ition . I f a large frac tion i s remove d b y s ievi ng , the ana lyte
concentration may change a nd the matri x may no longer refle c t the comp o s ition o f re gu la r tes t s ample s .
8.2.5 Mixing and blending

B u l k s ol id materi a l s hou ld b e homo gen i ze d b y thorough m i xi ng , us i ng for e xa mple a rol l-m i xer, s ha ker
or end- over- end m i xer. Such m i xi ng i s c arried out a fter m i l l i ng , gri nd i ng and s ievi ng.
B lend i ng o f two or more materia l s with s u ffic iently s i m i l ar matri x comp o s ition s and d i fferi ng prop er ty
va lue s may enable the prep aration o f QC M s with a de s i re d prop er ty va lue s , a s e t o f s i m i lar QC M s coveri ng
a range o f prop er ty va lue s , or the prep aration o f QC M s from a n exi s ti ng re ference materi a l .
I n order to ob tai n homo gene ou s m i xtu res , the materia l s to b e m i xe d s hou ld have s i m i la r den s itie s and
p ar ticle s i ze d i s tribution s .
8.2.6 Filtration
Fi ltration o f s olution s b e fore b o ttl i ng remove s any p ar tic u late a nd fibrou s s ol id s th at wou ld comprom i s e
the homo geneity o f the bu l k materia l . H owever, s ome l iquid s c an no t b e fi ltere d due to i) vi s co s ity, i i)
p o tentia l lo s s o f ac tive i ngre d ients b y ad s orp tion to the fi lter or i i i) the i ntro duc tion o f contam i nation .
Qua l i fic ation o f the fi lter i s critic a l to avoid ing lo s s o f ac tive i ngre d ients .
Typic a l ly, l iqu id s , waters and le ach ate s are fi ltere d th rough a 0 ,45 µm fi lter prior to b o ttl i ng or amp ou l i ng.
8.2.7 Stabilization
C er tai n a na lyte s a re un s table i n s olution and as a con s e quence ne e d to b e s tabi l i z e d at the bu l k s tage
o f the prep a ration pro ce du re . Me ta l s , for example, c an pre cipitate out o f neutra l or a l ka l i ne s olution s
b e c au s e o f hyd rolys i s or oxidation and adj u s tment o f the pH o f the s olution to b elow 2 cou nterac ts
th i s problem . C opp er at a concentration of 1 mg· l −1 has b e en used to cou nterac t a lga l grow th in
aque ou s s olution s . D i fferent materi a l s m ay re qu i re o ther appro ache s s uch a s add ition o f a ntioxidants ,
pre s er vative s , tex ture s tabi l i z ers , e tc .

8.2.8 Sterilization
Prepared soils, sewage sludges and biological materials may contain persistent pathogens that are
potentially harm ful to humans. They may also contain spores that cause fungal moulds to develop on
storage, which could initiate changes in either the composition o f the bulk material or the individual
units. Such organisms need to be destroyed be fore the final units are prepared and packaged.
Be fore sterilizing any candidate QCM, it is important to consider the impact o f the proposed sterilization
process on the material, particularly those which degrade at elevated temperatures.
Autoclaving is an inexpensive and convenient means o f sterilization that can be used for materials that
are temperature resistant, for example metals in sediments. Autoclaving can be done on the bulk material
prior to final homogenization and unit preparation or on the final samples. However, it is important to
ensure that the core o f the material reaches 121 °C.
Irradiation can be used on the final packaged units (e.g. ampoules, bottles or pouches). Gamma irradiation
is a convenient means o f sterilization at ambient temperature so changes in matrix composition are
less likely than with autoclaving. Dose values need to be determined such that they are e ffective in
removing pathogens but do not adversely a ffect the material by, for example, raising the temperature
to unacceptable levels (e.g. chocolate). However, gamma irradiation is beyond the means o f most
laboratories, requiring specialist sub-contractors.
8.3 Sub-division and packaging

8.3.1 General
Once the bulk material has been processed it will need to be sub-divided and packaged. The following
sub clauses describe some o f the key considerations for the sub-division process and choice o f containers
to ensure the QCM is su fficiently homogeneous and stable for its intended purpose.
8.3.2 Choice of containers

For QCMs to be produced cost-e ffectively one aspect that needs care ful consideration is the choice o f
appropriate containers for the individual units. I f unsuitable containers are used, a material may quickly
degrade to the extent that time-consuming and expensive sourcing and preparation work on the bulk
material may have to be repeated. The type o f container used depends on the inherent stability o f the
material and the length o f time it is required to remain stable. For particularly susceptible materials, two
forms o f containment (e.g. a vial within a polyethylene bag) can provide additional protection against
degradation and contamination.
The following examples serve to illustrate the need for care ful consideration o f the container and its
closure.
— Organic materials can either lose or pick up moisture i f the container is not securely closed. Glass
containers with screw-caps fitted with “polycone” inserts 1) are pre ferable to simple screw caps.
Sealed cans, foil pouches or septum-lined crimp-top vials o ffer more security.
— Oxygen sensitive materials should be prepared and sub sampled under an inert gas atmosphere
(nitrogen or argon).
— For water samples containing low concentrations o f metals (e.g. mg/kg or below), glass containers
are not recommended because o f possible adsorption o f the metals onto the walls over time. High-
density polyethylene (HDPE) bottles with screw-caps are more suitable for this application, but
they themselves have the potential problem o f loss o f water by evaporation through the bottle walls.
This can be minimized by storage in a re frigerator (rather than at ambient temperature) or by the
use o f fluorine-treated polyethylene bottles.
1) Polycone liners are cone-shaped polyethylene cap liners that provide a better seal than simple wadded cap
closure.
— The possibility o f contamination o f the QCM by the leaching of impurities from the container
should also be considered. For example, the iron content o f canned foodstu ff QCMs may be subject
to unpredictable increases on a can-by-can basis, as iron leaches from the can wall into the food
matrix. Bottles (whether glass or HDPE) containing aqueous acid solutions may also give rise to
leaching problems. As a general rule, containers that might interact with the QCM should be care fully
evaluated be fore use by suitable leaching trials.
— For relatively inert matrices, such as soils and other dried environmental or biological materials,
screw-cap glass jars are usually satis factory. Amber glass gives additional protection against
degradation induced by light.
— QCMs comprising relatively volatile components susceptible to evaporation, such as some organic
solvents, will normally require a septum-lined crimp-top, glass vial or flame-sealed glass ampoules.
Vials and ampoules should pre ferably be amber to reduce the impact o f light.
Some preliminary experimental work, including blank studies, may be required to identi fy the most
suitable container type to use for a particular QCM.
The e ffect o f repeated opening and closing o f the sample containers may also be assessed i f repeated use
o f the material is anticipated.
Tamper evident closures should be considered i f the unit should only be used once.
8.3.3 Sub-division procedures
Once a homogeneous bulk material has been produced, the essential requirement o f any sub-division
process is that the homogeneity o f the material is maintained. That is, the sub-division process itsel f, or
the time taken to complete the sub-division o f a bulk material, should not re-introduce heterogeneity
into the material. This may conceivably occur in a number o f ways.
Matrices comprised o f mixtures o f liquids o f di ffering volatilities (e.g. ethanol in water) may undergo
selective evaporation o f one component during a prolonged sub-division run, causing a rising or falling
trend in property value from the first to the last units produced. E ffects o f this sort may be minimized
by protecting the bulk material from evaporation and by completing the sub-division in as short a time
as is consistent with accurate dispensing.
All liquids and solutions should be stirred continuously while individual aliquots are being dispensed.
Solutions should be filtered be fore dispensing commences i f particulates are likely to be present to an
extent that could a ffect the properties o f interest.
Care should be taken with solid particulate matrices such as soils, sediments, industrial products, etc.
to ensure that segregation o f finer particles does not occur during sub-division. Special care should be
taken when sampling bulk material from a large drum, to ensure that there is no vertical segregation.
Ri ffling is a process for representatively subdividing free-flowing powdered materials so that each
aliquot receives similar particulate fractions. When operated e ffectively, ri ffling minimizes flow
segregation and produces units with low between-unit variation. Commercial ri ffling devices can be
used to sub-divide such materials without introducing heterogeneity. Sampling and sub-division o f
particulate materials are described in more detail in ISO 14488:2007. [15]
In food matrices with a high fat content (e.g. mackerel paste), there may be a tendency for the fat to
separate as a discrete phase. I f such e ffects occur, the matrix should be stirred continuously during
dispensing and/or additives included in the matrix to slow down the separation process.
As a general principle, sub-division o f a bulk material should be completed as quickly as possible to
minimize the opportunities for the matrix to revert to heterogeneity. Where appropriate, steps should
be taken to maintain a homogeneous bulk material during the sub-division process. It may be necessary
to discard the first and/or last portions dispensed from the bulk material, especially o f complex matrices
that are especially prone to segregation e ffects.

In the case o f QCMs intended for trace analysis, special care must be taken not to introduce additional
impurities (e.g. from the air, apparatus, laboratory vessels, etc.) during subdivision o f the material as
this could change the property value being measured.
9 Homogeneity
9.1 Overview
Homogeneity is a relative concept. The required level o f homogeneity o f a QCM is dependent on an
understanding o f the expected variation o f the amount o f sample used in the measurement process
under investigation (see 7.3). In all cases, the level o f inhomogeneity should result in a smaller e ffect on
the measurement result than the expected variation o f the measurement process or should be below an
established criterion value.
Once a candidate QCM has been sub-divided into individual aliquots it is important to establish whether
there are any variations in its property values between aliquots. For certain QCM matrices, such as
true solutions which have been prepared by procedures such as filtration (to remove particulates) and
thorough mixing, formal homogeneity testing is, in principle, not necessary. Such materials may be
formally regarded as being inherently homogeneous. Nevertheless, because o f the risk o f contamination
(e.g. introduced due to packaging) or imper fect subdivision, it is recommended to carry out a simple
homogeneity study.
For more complex matrices such as foodstu ffs, soils and solid matrices that are inherently heterogeneous,
a formal experimental investigation o f homogeneity is required. A su fficient number o f units,
representative o f the entire batch o f the QCM should be chosen and analysed for selected properties.
[1] In certain instances, one property can be chosen to represent and quanti fy the homogeneity o f
several properties o f a similar general type. This should be based on scientific evidence or on previous
experience that certain properties exhibit similar behaviour [13] or are known to have a strong tendency
to homogeneous distribution in the sample (e.g. some metals in alloys).
A statistical evaluation o f the data and a test for su fficient homogeneity are carried out, which can be
readily achieved using spreadsheet so ftware (see 9.3). ISO Guide 35 [2] details the requirements for the
assessment o f homogeneity and gives full details and examples o f recommended approaches for the
design o f homogeneity studies and the statistical treatment o f homogeneity data.
Homogeneity has two aspects, between-unit homogeneity and within-unit homogeneity. Between-unit
homogeneity reflects the variation in the measurement results in each unit o f the material. Within-unit
homogeneity is reflected in the minimum size o f subsample that is representative for the whole unit.
It should be confirmed that sample sizes typically used in the day-to-day analysis are larger or at least
equal to this size.
9.2 Analytical approach

A validated analytical method having a su fficient degree o f repeatability should be selected for evaluation
o f the homogeneity. The selected units should be representative o f the entire batch and the number o f
units is dictated by the total number o f units produced.
Sampling guidelines [16] for the homogeneity testing o f multi-unit batches recommend that for a stock
comprising “n ” individual units o f material the number o f units to be analysed for homogeneity should
be three times the cubed root o f “n ”. For a stock o f 600 to 1 000 units this equates to between 27 and 30
units to be analysed in duplicate. This represents a considerable analytical e ffort which will be time-
consuming and expensive. A study o f the impact o f reducing the number o f units selected for homogeneity
assessment o f a QCM [2] concluded that in certain circumstances 10 units analysed in duplicate were
su fficient. For the preparation o f a QCM there fore, there may be scope for cost savings by reducing the
number o f units selected for homogeneity testing, although this should be assessed on a case by case
basis.

ISO Guide 35 [2] acknowledges the fact that, in some circumstances, it may not be feasible technically or
economically to determine the homogeneity o f all properties o f interest. Where only selected properties
are assessed for homogeneity, these must be representative o f the other properties o f interest (e.g. on
the basis o f established physical or chemical relationships).
The following represents a suitable approach for homogeneity assessment.
— Select a number g o f the samples in their final packaged form at random, where g ≥ 10.
— Prepare two test portions from each sample using techniques appropriate to the test material to
minimize between-test-portion di fferences.
— Taking the 2 g test portions in a random order, obtain a measurement result on each, completing the
whole series o f measurements under repeatability conditions.
— Calculate the arithmetic mean x , within-sample standard deviation Sw, and between-sample
standard deviation Ss , as shown in 9.3.
The measurements should be carried out such that measurement dri ft can be distinguished from any
trend in the batch o f samples. This can be achieved by measuring the replicates in randomized order or
by reversing the order o f the replicates.
9.3 Statistical treatment of homogeneity data

The principles of ISO Guide 35 [2] should be applied with respect to the statistical evaluation of the results
or homogeneity.
f
Adapting example B.3 from ISO Guide 35:2006 [2] (chromium in soil) to a homogeneity study o f 10 units
in duplicate, the data become:
Table 1 — Homogeneity study of 10 units

Unit number Result 1 Result 2 Mean Variance
mg·kg−1 mg·kg−1 mg·kg−1 mg·kg−1
1 121,3 128,74 125,02 27,68
2 120,87 121,32 121,10 0,10
3 122,4 122,96 122,68 0,16
4 117,60 119,66 118,63 2,12
5 110,65 112,34 111,50 1,43
6 117,29 120,79 119,04 6,12
7 115,27 121,45 118,36 19,10
8 118,96 123,78 121,37 11,62
9 118,67 116,67 117,67 2,00
10 126,24 123,51 124,88 3,73
with an overall mean o f 120,02 mg·kg−1 .

This data need to be assessed to determine whether the material is su fficiently homogeneous for use
as a QCM. To be o f use as a QCM, the between–bottle standard deviation should be no greater than
one third o f the within–laboratory reproducibility standard deviation (which can be obtained from
existing control chart data, where available, or from existing reproducibility and repeatability data for
the method). This is analogous to the approach recommended for the homogeneity acceptance criteria
for proficiency testing materials. [17]
When reviewing this data, consideration should be given to the nature o f the material (in this example
- a soil) and whether such variation is within acceptable limits.

A use ful first step is to review the data graphically. This enables any discordant features (such as outlying
samples, trends or other systematic e ffects) to be readily identified.
The data from Table 1 are plotted in Figure 2 and while there are no obvious trends, there is variability
o f the within unit duplicate results and the data from unit 5 requires investigation.
Figure 2 — Data plot
This graphical analysis o f the data are complemented by statistical analysis to determine the within unit
and between-unit standard deviations. Single factor analysis o f variance using spreadsheet so ftware
gives the following values in Table 2 .
Table 2 — Results of single factor analysis of variance

Sum of squares Degrees of Mean squares
Source of variation (SS) freedom (MS)
Between-units 284,94 9 31,67
Within-unit 74,05 10 7,41
Total 358,99 19
The between-unit variance is estimated using:
MS between − MS within 31 , 67 − 7 , 41
2
sA = = = 12 , 13 mg 2 ⋅ kg −2
n0 2
The between-unit standard deviation is the square root o f this variance:

s bb = 12 , 13 = 3 , 48 mg ⋅ kg −1
The repeatability standard deviation can be computed as:

sr = MS within = 7 , 41 = 2 , 72 mg ⋅ kg −1
The result shows a rather high between-bottle heterogeneity which is mainly caused by unit 5. I f the
heterogeneity is above pre-set limits and i f it makes the QCM unsuitable for use, further manipulation o f

the material may be necessary in order to improve homogeneity. In this case, it would also be advisable
to per form an in-depth analysis o f the data for point 5 to determine i f there are any technical reasons to
question the validity o f this data which could lead to its exclusion from the data set.
Other examples for assessing the homogeneity o f QCMs are described in the case studies in Annexes A
through F.
10 Characterization and value assignment
The purpose o f QCMs is to monitor measurement processes for change. In order to achieve this e ffectively
an indication o f the property values o f the QCM used to monitor the process is needed. It is also necessary
to have an indication o f the likely variation in values due to heterogeneity between di fferent aliquots.
An e ffective way o f determining an indicative property value is to use the overall mean derived from the
homogeneity study. The range within which the property values may reasonably be expected to lie can
be estimated by the deviation from this overall mean value. This deviation from the mean can be used
to establish control chart warning limits. Conventionally, warning and action limits (also described as
lower and upper action limits) are established at two and three times the standard deviation respectively.
11 Stability
11.1 Overview
Di fferent materials will exhibit di fferent types o f (in)stability, some o f which may be excluded from
consideration given long-term historic data and knowledge; some may be undetectable but cannot be
excluded from consideration; some can be detected and should be assessed and some follow certain, and
well-established, chemical or physical principles.
In principle, the approaches to stability testing described in ISO Guides 34[1] and 35 [2] apply. However, i f
the material is not to be transported beyond the confines in which it was prepared, no short-term tests
for degradation need to be per formed. For stability during storage, stability assessment o f all properties
o f the material can be both costly and time-consuming and may not be necessary i f su fficient checks are
in place to distinguish between an out o f specification result due to a faulty test sample, measurement
instrument dri ft or QCM degradation. However, those responsible for QCM preparation have to be aware
o f the trade-o ff between costs for stability testing and the costs incurred by investigating a method
dri ft, which is in reality caused by degradation o f the material.
It should be pointed out that as some QCMs are made for repeated use, investigation o f the stability o f
opened units may be particularly use ful.
Adequate stability o f QCMs is required; so that users can be confident they will not undergo any
significant change a ffecting the property value(s) during the period o f its intended use, which in some
cases could be a number o f years.
Care ful choice o f the container used for the material can address some intrinsic stability issues (e.g.
sensitivity to oxygen, light, moisture; susceptibility to evaporation o f components).
Some materials (e.g. metallurgical materials) are inherently stable, while for others storage at a
precautionary low temperature may lengthen the period o f stability.
Failures in preparation or unexpected contamination/impurities may impair the stability significantly.
11.2 Assessing stability

Full stability assessment for any re ference material is a costly, time consuming and involved process,
and is inappropriate for QCMs as described in this Guide, which are prepared in the laboratory where
they are intended to be used. It is likely that the laboratory will have previous experience o f the stability

o f the types o f matrix and property values it is preparing as QCMs, or there may be well established
background in formation from similar materials.
However, the financial implications o f using QCMs whose property values have changed significantly
can be large (e.g. release o f out-o f-specification products, or non-release o f in-specification products)
and laboratories using QCMs should have procedures in place which describe the actions to be taken
in the event o f a QCM giving an unexpected result. Possible actions include use o f freshly prepared
calibrants, comparison with certified re ference materials and frequent sensitivity checks to confirm
any deviations or trends in the QCM results. More in formation can be found in ISO standards on control
charts (e.g. ISO 7870-1 [3] and ISO/IEC 17025 [18] ).
Should a formal assessment o f stability be deemed necessary, the principles o f ISO Guide 35 [2] should be
followed. An example o f this approach is given in Annex F (Case study 6).
11.3 Assigning an expiry date to a QCM

The quality systems o f many laboratories require standard materials and reagents to be labelled with
expiry dates. [19] The assignment o f a formal expiry date is one o f the most problematic tasks in re ference
material production. It involves making a prediction o f the future behaviour o f a material based on an
extrapolation o f its past behaviour. Any stated expiry date for a QCM should be based upon previous
experience o f the stability o f the types o f matrix and property values and any background in formation.
The stated expiry date should not be regarded as absolute and the analyst using the QCM should follow
laboratory procedures in the event o f a QCM giving an unexpected result (see 11.2).
Materials which are considered to be inherently stable do not need an expiry date, but a statement
justi fying the basis for the assumption o f inherent stability is required. A typical statement for a
metallurgical material might read – “this is a m etallurgical m aterial used as a QC m aterial in th e an alysis of
elem ents in ore an d is con sidered in h erently stable provided it is stored an d h an dled un der th e recomm en ded
con dition s ”.
12 Transportation
In general, this guidance relates to materials which are prepared and used in the same locality (i.e. not
for wider distribution). However, it is recognized that some materials are inherently stable and their
property values will not be adversely impacted by transportation (controlled or otherwise).
Should there be a requirement to transport the material and there is any concern about its stability, a
stability assessment following the principles o f ISO Guide 35 [2] should be carried out.
13 Documentation for quality control materials (QCMs)

13.1 General
As with laboratory reagents, QCMs should be appropriately labelled and have instructions for their sa fe
and e ffective use. ISO Guide 31 [20] provides details o f the requirements o f the contents o f certificates
and labels for re ference materials.
13.2 Information to be available with quality control materials (QCMs)

The following in formation should be readily available to the QCM user: [10] , [20]
— name and description o f the material;
— re ference number and/or batch number;
— date o f preparation;

— i ntende d u s e o f the materi a l and any s p e ci a l i n s tr uc tion s for its u s e (e . g. s p e c i fic d r yi ng pro ce du re s
to fol low or i n s truc tion on d r y mas s corre c tion shou ld b e given, i f appl ic able) ;
— i nd ic ative va lue(s) , i f appl ic able;
— the m i ni mum amou nt o f materia l re qu i re d to ach ieve con s i s tent re s u lts (th i s wi l l dep end up on the
quantitie s u s e d for the homo geneity s tudy) , i . e . m i n i mu m s a mple i nta ke;
— s torage i n s truc tion s;
— i n formation on exp e c te d shel f-l i fe;
— any s p e c ia l s a fe ty pre c aution s to b e appl ie d by u s ers .
13.3 Labelling of QCM units

E ach u n it o f the QC M s hou ld b e cle arly lab el le d, i n a man ner that enable s it to b e u nambiguou s ly l i n ke d to
the i n formation for the materi a l . T he lab el s hou ld there fore contai n the fol lowi ng e s s enti a l i n formation:
— name a nd de s crip tion o f the materi a l;
— re ference nu mb er and/or b atch nu mb er;
— ha z ard and s a fe ty l ab el s , where appropri ate;
— s torage a re a envi ron menta l cond ition s i nclud i ng temp eratu re a nd hum id ity;
— prep aration date;
— e xp e c te d expi r y date .
I t may b e u s e fu l to provide the fol lowi ng add itiona l i n formation on the lab el:
— u n it s i ze (e . g. 2 0 g) ;
— i nd ividua l u n it nu mb er (th i s may help to identi fy a ny b o ttl i ng trend s i n the event o f a n u ne xp e c te d
re s u lt b ei ng ob ta i ne d) ;
13.4 Useful information to be retained

I n formation relati ng to the prep aration o f the QC M wi l l be re qui re d if a quer y ari s e s regard i ng
the materia l du ri ng u s e, or i f a new b atch o f the materia l h as to b e pro duce d . I t i s go o d prac tice to
re tai n/arch ive data relati ng to the fol lowi ng a s p e c ts o f the prep a ration o f the QC M i n a n acce s s ible form:
— releva nt s p e ci fic ation s;
— s ou rce and prep aration o f the bu l k materia l;
— typ e s o f contai ners u s e d for the i nd ividua l u n its;
— s ub - d ivi s ion pro ce du re s us e d;
— any s p e c ia l tre atments u s e d to s tabi l i z e or s teri l i z e the materi a l;
— s upplementa r y data o n the materia l (e . g. p ar ticle s i z e, moi s ture content, e tc .) ;
— fu l l de tai l s o f a l l me tho d s u s e d to pro duce the materi a l;
— a l l data from me tho d s u s e d to pro duce the materi a l;
— s p e ci fic e xp erience u s e fu l to avoid pit fa l l s and co s tly errors du ri ng prep a ration o f new b atche s or
relate d QC M s .

14 Storage
14.1 General
Completed batches o f QCMs should be stored under conditions that will ensure they remain unchanged.
Generally, this entails ensuring that the individual containers are securely closed and are stored away
from extremes o f heat, light and humidity.
Storage in dark conditions at the appropriate temperature (e.g. room temperature, 4 °C or −18 °C) is
usually the key aspect o f ensuring long-term stability. For QCMs comprised o f matrices where stability
is/might be considered questionable, such as food matrices and aqueous solutions, it is recommended
that long-term storage is carried out at sub-ambient temperatures. For matrix/property types where
stability is more ensured, such as soils containing metals or PCBs, long-term storage may usually be
carried out at ambient temperature.
14.2 Monitoring of storage conditions

The relevant storage conditions should be monitored at regular intervals, to ensure that, for example,
the appropriate storage temperature is being maintained. A recommended procedure is to check the
temperatures o f re frigerators/freezers, etc. on a daily basis, so that i f a failure does occur, the user
can take appropriate action (e.g. trans fer the material to another storage location, i f available, or carry
out an assessment o f the continued fitness for purpose o f the material). A record should be kept o f the
temperature readings o f re frigerators/freezers, etc.
15 Using quality control materials (QCMs)

15.1 General
All re ference materials, whether from commercial suppliers or in-house producers, should have
instructions for their use. These instructions should include details on how the material should be
stored and handled, including specific instructions for sub-sampling and dry-mass correction.
15.2 Minimum sample size

All re ference materials (with the exception o f homogeneous solutions and gases) are inherently
heterogeneous but for practical purposes taking su fficiently large samples reduces the heterogeneity
to an acceptable value.
The minimum sample size to be taken is the smallest sub-sample that is still representative o f the whole
unit for the target property value. Frequently, this is estimated as the minimum sample size used in the
assessment o f the homogeneity o f the material. The minimum sample size stated to be used is there fore
a conservative estimate and not the absolute minimum.
15.3 Mixing procedure

Prolonged storage o f multi-component mixtures and matrix materials may cause settling and separation
o f the material. It is there fore important that aliquots are adequately mixed be fore a new sub-sample
is withdrawn. This can o ften be achieved by simple shaking o f the units. Where stirring is required to
re-mix the material su fficiently for use, care should be taken not to introduce any impurity that will
inter fere with the measurement process.
Where possible, simple visual inspection o f the material be fore sub-sampling for use, can o ften identi fy
heterogeneity, such as agglomeration.

15.4 Dry mass correction

M any prop er ty va lue s a re pre s ente d as content per dry mass of sample. T h i s ha s the advantage that
p o tentia l moi s tu re up ta ke do e s no t cha nge the va lue, but ha s the d i s advantage th at the prop er ty va lue i s
then dep endent on the me tho d for the de term i nation o f the d r y mas s . Stud ie s [21] have shown that re s u lts
from d i fferent me tho d s (e . g. d r yi ng oven, Karl Fi s cher titration, vac uum d r yi ng oven) d i ffer s ign i fic antly
– no t le a s t b e c au s e they me a s u re d i fferent th i ngs .
For moi s tu re s en s itive materia l s , appropri ate p ackagi ng wou ld b e the pre ferre d me tho d o f control l i ng
moi s tu re content. Where th i s i s no t p o s s ible and the moi s tu re content o f the materia l may ch ange over
ti me, a con s i s tent me tho d for d r y-mas s corre c tion s hou ld b e appl ie d e ach ti me the materia l i s u s e d .
15.5 Storing opened containers of QCMs

Rep e ate d op en i ng a nd clo s i ng o f re ference materi a l contai ners i ncre a s e s the ri sk o f contam i nation .
I n the pro ce s s o f prep ari ng the QC M the e ffe c t o f op en i ng and clo s i ng o f the contai ner a nd rep e ate d
fre e z i ng/thawi ng o f u nits i n term s o f s tabi l ity and homo geneity shou ld b e con s idere d
2) .
Where p o s s ible the u n it s i ze shou ld b e cho s en s uch that it refle c ts the amou nt o f s ample ne e de d for the
typ e o f ana lys i s for wh ich it i s de s igne d . I n th i s way, once op ene d e ach un it i s enti rely u s e d a s s o on as
p o s s ible a fter op en i ng to avoid p o tenti a l degradation . P rep ari ng QC M s a s a numb er o f s ma l l, s i ngle u s e
un its may b e adva ntage ou s i n th i s c as e .
I f op ene d contai ners o f QC M s a re to b e s tore d , s torage cond ition s shou ld b e s ele c te d u nder wh ich
the materi a l ha s b e en demon s trate d to b e s table . T he s e cond ition s s hou ld pro te c t the materi a l from
acc identa l contam i nation and b e i nclude d i n the u s er i n s truc tion s .
2) This is no t always easy as illus trated by the re ference material B C R- 5 2 2 (haemo glo b incyanide in b ovine b lo o d
lys ate) , which do es no t re- dis s o lve p ro p erly a fter it has b een fro zen.

Annex A
(informative)
Case study 1 — Preparation of a QCM from coal3)
A.1 Objective
A coal testing laboratory uses a QCM for daily quality control for proximate and ultimate analysis in
accordance with the applicable ISO standards. One can o f 1 l, holding approximately one kg coal, is
su fficient for checking the analysis results for a week. The laboratory would like to use the material for
one year, and calculates that it needs 100 kg o f starting material. The starting material, as delivered, is
50 mm top size.
The laboratory is interested in a QCM that represents blended coal o f the type used in power plants.
A.2 Sampling
The samples are mechanically removed from a conveyor belt, crushed and sieved to a top size o f 10 mm
and split into 6 portions o f 10 kg per sample. In total, 12 samples are taken from the blend. The laboratory
receives 12 plastic bags, each containing 10 kg blended coal.
A.3 Checking of the suitability of the material

The laboratory takes a sample from one o f the bags and prepares it for analysis. It determines the volatile
matter, and ash contents, gross calorific value (proximate analysis), as well as the contents o f carbon,
hydrogen, nitrogen and sul fur (elemental analysis). These results confirm the suitability o f the material
with respect to the content levels and calorific value.
A.4 Sample preparation

The coal is dried in air at ambient temperature to remove the excess o f water, sieved, split into 10
portions and subdivided.
For the subdivision, a laboratory ri ffler is used with 10 tubes. In order to eliminate possible di fferences
between the bags, the subdivision scheme shown in Table A.1 is used.
3) This case study was provided by Adriaan M.H. van der Veen, NMi Van Swinden Laboratorium B.V., Thijsseweg
11, 2629 JA Del ft, The Netherlands.
Table A.1 — Subdivision scheme

01 02 03 04 05 06 07 08 09 10
↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓ ↓
01.01 02.02 03.03 04.04 05.05 06.06 07.07 08.08 09.09 10.10 → A
01.02 02.03 03.04 04.05 05.06 06.07 07.08 08.09 09.10 10.01 → B
01.03 02.04 03.05 04.06 05.07 06.08 07.09 08.10 09.01 10.02 → C
01.04 02.05 03.06 04.07 05.08 06.09 07.10 08.01 09.02 10.03 → D
01.05 02.06 03.07 04.08 05.09 06.10 07.01 08.02 09.03 10.04 → E
01.06 02.07 03.08 04.09 05.10 06.01 07.02 08.03 09.04 10.05 → F
01.07 02.08 03.09 04.10 05.01 06.02 07.03 08.04 09.05 10.06 → G
01.08 02.09 03.10 04.01 05.02 06.03 07.04 08.05 09.06 10.07 → H
01.09 02.10 03.01 04.02 05.03 06.04 07.05 08.06 09.07 10.08 → I
01.10 02.01 03.02 04.03 05.04 06.05 07.06 08.07 09.08 10.09 → J
Starting with the 10 bags (top row), a 100 subsamples are made by dynamic ri ffling. The numbering o f
the subsamples reveals the sample from which it is subdivided (first pair o f digits) and from which tube
o f the ri ffler it stems (second pair o f digits). The subsamples are combined in such a fashion, that each
composite sample A through J contains one subsample from each bag and one subsample from each tube
o f the dynamic ri ffler.
In a second step, the 10 composite samples A to J are ri ffled again to give a 100 samples. The samples
are put into small plastic bags in cans. From 10 cans, chosen at random, 2 subsamples are taken for a
homogeneity test. The samples for the between-bottle homogeneity study are analysed for moisture and
ash content, and gross calorific value.
The cans containing the 100 samples are closed and labelled with the date o f blending, and the sequence
number obtained from the second sub-sampling. Composite sample A delivered cans 1 to 10, and so on.
The laboratory considers preservation o f the history from the sample preparation essential to support
a root cause analysis, i f required later.
A.5 Between-bottle homogeneity study

The between-bottle homogeneity study is carried out with two replicates on 10 cans from the batch
o f 100. One-way analysis o f variance is used to determine the between-bottle standard deviation. [22]
Previous experience has shown that for the selected parameters (ash content and gross calorific value)
the between-bottle standard deviation should not be greater than the repeatability standard deviation
o f the tests. For both parameters, this objective is achieved in the homogeneity study.
A.6 Characterization
The laboratory monitors its quality using a Shewhart chart. The standard deviation is taken from a
previous chart from a similar blend. The mean value is obtained from 10 measurements from one can,
taken over 10 consecutive days. On days 1 and 10, a CRM was analysed as well to confirm the laboratory
results. The QCM was put to use with the mean from these 10 measurements, a fter care fully scrutinizing
the data. The data analysis indicated no irregularities.

Annex B
(informative)
Case study 2 — Preparation of geological or metallurgical quality
control materials (QCMs) 4)
B.1 General
The materials produced include various geological or metallurgical particulate materials sourced from
customers o f the analytical facility (matrix matched), typically in the order o f 600 kg each. This includes,
but is not restricted to, ores, concentrates, feeds, tails, slags and un-mineralized rock, soils or sediments.
B.2 Project initiation

The need for in-house re ference material preparation generally stems from the di fficulty in sourcing a
suitable commercial re ference material for the analysis o f a material o f a unique sample matrix.
B.3 Sourcing of material

The following factors are taken into account.
— The matrix o f the material must be as close as practically possible to the samples for which it
will be used as a quality control. By mixing mineralized ore with barren material/ lower grade
ore o f similar overall composition, materials o f di fferent grades and a predetermined matrix can
be manu factured. Once the material is o f the requisite composition it can be prepared into one
homogenous bulk re ference material.
— Materials are stored and prepared in separate facilities according to their grade to prevent cross
contamination. Precious metal concentrates may require additional sa fekeeping procedures.
— The quantity o f material should be su fficient to last for the duration o f an analytical campaign or
at least long enough that it can be replaced in good time, allowing for some overlap period during
which subsequent materials are used in conjunction.
— The value or toxicity o f the material may be such that it would not be sa fe to handle large quantities,
nor feasible to obtain large quantities.
B.4 Preparation of reference material

The preparation encompasses the crushing, pulverization, screening, splitting and packaging o f solid
geological / metallurgical materials into “homogenous” pulps. The term “homogenous” is used with
caution. These materials normally consist o f 100 % sub 75 µm or 43 µm particles below which the
material consists o f discrete mineralogical grains. The term “homogeneity” naturally has a mass
constraint and is o ften assessed purely upon a “fit for purpose” basis.
Equipment used:
a) jaw crusher capable o f crushing to 100 % passing 5 mm;
b) closed circuit mill capable o f producing 50 % 43 µm or greater;
4) This case study is based upon information provided by Vicky Anderson o f Anglo Research, 8 Schonland Street
Theta Johannesburg, P O Box 106, Crown Mines, 2025 Republic o f South Africa.
c) u ltras on ic s ieve s ha ker with 75 µm or 43 µm s c re en;
d) la rge 10 c up ro tar y s pl itter;
e) 10 0 l pla s tic d ru m s;
f) V-blender or e qu iva lent;
g) 5 0 l plas tic d r um s;
h) la rge b a lance (s c a le) ;
i) p acki ng mach i ne;
j) a s s or te d ma rki ng p en s , p olythene b ags , lab el s , p acki ng tap e .
B.5 Crushing, blending and milling source material

P rior to s a mpl i ng , the comp onent materi a l s for grade de term i nation pur p o s e s , the materia l mu s t b e
cru s he d to 5 m m or fi ner to m i n i m i ze the s a mpl i ng error. C ru s h i ng i s u s ua l ly accompl i she d u s i ng a
lab orator y j aw cru s her but o ther cr u shers may b e u s e d to e xp e d ite the pro ce s s .
S ampl i ng s to ckpi le s i s i n herently d i ffic u lt and prone to s a mpl i ng error due to s e gregation . T he fol lowi ng
pro ce dure has b e en adop te d to ob tai n an approxi mate grade .
— S a mple 3 0 s ample s o f ab out 5 0 0 g e ach at random i nter va l s from the s to ckpi le at random heights .
Tr y to s ample the i n s ide o f the s to ckpi le a s b e s t as p o s s ib le .
— C omp o s ite the s ample and ri ffle s pl it it down to ab out 5 kg.
— M i l l the ri ffle d comp o s ite and ana lys e i n the u s ua l man ner.
T he pla n ne d fi na l grade o f a re ference materi a l i s c a lc u late d from the grade o f its comp onents . T he
acc u rac y o f the grade ca lc u l ation o f the comp onents , and thu s the m i x ture, i s pre dom i nately de term i ne d
by the s ampl i ng error. T he comp o s ition o f the m i xtu re i s s i mply de term i ne d as fol lows . T he concentration
c of element i,
ci = Σ cij wj
where
cij i s the concentration o f element i in component j;
wj i s the mas s frac tion o f comp onent j .

For e xample, i f a low grade materia l contai n s 0 , 5 0 g/t Cu and a fe e d 4, 0 0 g/t Cu, a grade c a lc u lation for a
m i x ture o f 10 0 kg bla n k and 4 0 0 kg fe e d wi l l b e a s fol lows:
ci = 0 , 5 0 × 10 0/(10 0 + 4 0 0) + 4, 0 0 * 4 0 0/(10 0 + 4 0 0) = 3 , 3 0 g/t Cu
P rior to m i l l i ng the materi a l, the c ru s her pro duc ts a re blende d in a m i xer or V blender add i ng
approxi mately the re qu i re d fi na l prop or tion s o f the d i fferent c ru s her pro duc ts . I f more materi a l i s
re qu i re d to b e m i xe d than the m i xer c an accom mo date, a nu mb er o f b atche s are made up compri s i ng
approxi mately the s a me prop or tion s o f the comp onents .
C lo s ed c i rc u it m i l l s a re pre ferre d for thei r blend i ng prop er tie s . D r y m i l l i ng i s pre ferre d to avoid le ach i ng
or a lteri ng the oxidation s tate o f the s ou rce materi a l . M i l l p o ts a re thorough ly cle ane d prior to m i l l i ng a
new re ference materi a l but no t b e twe en m i l l i ng c ycle s o f the s ame materia l . O nce approxi mately 10 0 kg
o f m i l le d materia l ha s acc u mu late d , s cre en i ng com mence s wh i le the m i l l i ng i s comple te d . M ateri a l s are
s cre ene d at the re qu i re d top s i z e s i nce more ma l le able me ta l p a r ticle s may tend to res i s t s i ze re duc tion

unlike the associated silicate or sulfide gangue. Oversized particles are recycled to the mill. The material
passing the screen is used for manu facturing re ference materials. Be fore using the large ultrasonic
screen, it is inspected for holes exceeding the specification and for damage at the edge o f the screen.
The screen is thoroughly cleaned be fore use. The process is continued until less than 1 kg o f oversize
material remains. This is then discarded in an environmentally responsible manner.
B.6 Homogenizing milled material

The final screened fraction is divided into a number o f equal mass portions such that each portion can
be accommodated in the V-blender. These portions are designated Drum 1, Drum 2, Drum 3, etc. Note: i f
these do not fit into the blender then they must be split further. The blender must not be full; otherwise,
it will not function adequately.
Prior to commencing the blending step, the material must not contain excess moisture that would cause
it to form clumps that do not disaggregate in the V blender. A sub-sample is tested for moisture by
drying a portion at 60 °C overnight. I f the sample contains > 2 % moisture, the entire batch is dried at
60 °C be fore further preparation. The moisture content is recorded. Samples are not dried at a higher
temperature lest minerals such as pyrhotite or gypsum alter into other species.
Each o f the original drums Drum 1, Drum 2, Drum 3, etc. (which are o f equal mass) must be homogenized
for at least four hours in the V blender that is not filled beyond the mark. For a 600 kg RM, there should
be four drums o f about 150 kg each for blending in a V-blender o f 100 l capacity (50 l to the mark). The
material in each o f the homogenized drums is split using a cleaned, large 10 cup rotary splitter. The feed
rate and rotation rate o f the rotary splitter are adjusted so that each cup receives a minimum o f 30, but
pre ferably 35 increments. Each split is weighed. I f the per cent relative standard deviations (%RSD) o f
these weights exceed 2 %, the splits must be recombined, blended and split again. Each acceptable split
from each drum is designated A, B, C….to J. The A’s, B’s, C’s … J’s are combined into 10 new composites.
V-blend each o f the new composites (A to J) for 4 h. Rotary split each homogenized drum using the large
10 cup rotary splitter again checking the % RSD o f splitting. Each split from each drum (A, B, C, etc.)
is designated 1 through to 10. Splits 1 to 10 from drums A to J are combined into 10 new composites
(repeat o f earlier procedure). These 10 new composites relabelled, for example, D1 to D10, are each
blended in the V-blender for 4 hours (see Figure B.1).
Homogeneity tests are per formed on the 10 composites. I f homogeneity testing fails and blending is
implicated, repeat the rotary splitting and recombination o f each drum and blend for 4 h (another repeat
o f the earlier procedure). Repeat the homogeneity test again.

Figure B.1 — Flow diagram of splitting and blending
B.7 Homogeneity testing

From each o f the ± 60 kg splits (D1 to D10), a minimum o f five random samples are submitted to the
laboratory, pre ferably for analysis by the method that will ultimately be the routine method o f choice for
the material. The samples are taken at di fferent points across the sur face o f the sample and at di fferent
depths using a clean auger sampler. A random systematic or stratified random technique is acceptable.
When these samples are submitted to the laboratory, the order is stratified on the worksheets. In other
words, the first sample from each split / drum is analysed in sequence, followed by the second sample
from each split / drum, etc. I f the samples from the splits (1 to 10) were analysed sequentially, bias and

instrumental dri ft could produce statistical di fferences that are not a function o f the composition o f the
samples, rather a result o f the analytical process. The samples are submitted in the required sequence.
The data received from the laboratory are trans ferred to an Excel 5) spreadsheet for manipulation and
statistical analysis. Outliers for the full array o f data are first identified using Chauvenet’s principle (see
Table B.1).
Table B.1 — Use of Excel to determine Chauvenet outliers
Objective Excel Formulae
Mean =AVERAGE(array)
Standard deviation =STDEV(array)
Count =COUNT(array)
Rejection probability (RP) =1/(2*count)
Range =-NORMSINV(0.5*RP)
Rejection limit (RL) =range*stdev
Chauvenet outlier =i f(ABS(value-mean)>RL, fail, pass)
Outliers are hence selected based upon the standard deviation o f the data. By removing outliers from a
data set, the standard deviation is altered and a second set o f outliers is identified and subsequently a
third. The successive sets o f outliers are only removed with caution, as at this stage the objective is to
determine the spread o f the data and not to assign an accurate mean. As a rough guide, i f it is necessary
to reject > 10 % o f the data in order to obtain an acceptable % RSD, repeat analyses are requested be fore
a final decision is made (rejected data are those suspected o f poor analyses).
I f the material shows unacceptable overall precision (the overall variance is higher than expected for the
material, the grade and the laboratory / method requirements), the preparation process is stopped and
the cause o f the poor precision determined. Material in-homogeneity may be the result o f insu fficient
particle size reduction or poor mixing. In the former case further mixing o f the material will not result
in an improvement o f the overall error. Poor overall precision may, for example, be attributed to: poor
screening, insu fficient grinding/crushing, excessive grinding/crushing or poor quality assays. Further
progress in preparing the material depends on the cause o f the poor precision. A simple skewness
test is used to indicate i f the distribution formed by the pooled data are positively skewed or not.
Positively skewed data arising from underlying Poisson or log-normal distributions can complicate the
certification process and may indicate insu fficient grinding/crushing. Limits for significantly skewed
table are determined at a 90 % confidence limit.
A single factor ANOVA is used to for testing the hypothesis that means from two or more samples (in the
statistical sense) are equal, i.e. the samples are drawn from populations with the same mean. At a 95 %
confidence limit, di fference between the batches will be indicated, by chance alone, 5 % o f the time. I f
the ANOVA indicates that there is a di fference in the population means between the drums, the sample
is re-homogenized (see Figure B.2 ).
5) Excel is an example o f a suitable product available commercially. This in formation is given for the convenience
o f users o f this document and does not constitute an endorsement by ISO o f this product.
Drum 1 Drum 2 Drum 3 Drum 4 Drum 5 Drum 6 Drum 7 Drum 8 Drum 9 Drum 10
0.245 0.245 0.237 0.251 0.265 0.228 0.250 0.244 0.242
0.254 0.234 0.252 0.240 0.240 0.248 0.248 0.255 0.249 0.255
0.276 0.245 0.249 0.255 0.250 0.253 0.265 0.243 0.245 0.258
0.261 0.245 0.268 0.254 0.259 0.244 0.250 0.262 0.275
0.254 0.269 0.249 0.236 0.257 0.257 0.237 0.256 0.269 0.253
Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Drum 1 5 1.29 0.258 0.000134
Drum 2 5 1.238 0.2476 0.000166
Drum 3 5 1.255 0.251 0.000124
Drum 4 5 1.236 0.2472 7.47E-05
Drum 5 5 1.271 0.2542 9.17E-05
Drum 6 5 1.23 0.246 0.000126
Drum 7 4 1 0.25 0.000133
Drum 8 5 1.266 0.2532 5.07E-05
Drum 9 4 1.007 0.25175 0.000137
Drum 10 5 1.283 0.2566 0.000142
ANOVA
Source of Varia on SS df MS F P-value F crit
Between Groups 0.000722 9 8.02E-05 0.686767 0.716084 2.137528
Within Groups 0.00444 38 0.000117
Total 0.005162 47
Comment: F<Fcrit (Between Drum Homogeneity Acceptable)
Figure B.2 — Microsoft Excel ANOVA to determine homogeneity
Should the material pass all o f the required homogeneity criteria detailed above, it is important to note
that the material is only considered su fficiently homogenous, at the sample mass used for analysis, to be
fit for use for the method used to determine its homogeneity (as the %RSD determined will be unique
to the testing method). For further guidance on how to compute the between-bottle heterogeneity,
two scenarios are possible using ANOVA. I f MS between groups is larger than the MS within groups,
the between bottle variance is computed by subtracting MS between groups from MS within groups
and dividing by the number o f replicates made per unit (in this case 5). The between-bottle standard
deviation is then the square root o f this variance as also given under 9.3 . I f MS between groups is smaller
than MS within groups the between-bottle heterogeneity is computed as given for case study 4. Further
explanation can be found in ISO Guide 35. [2]
B.8 Packaging
Once initial homogeneity tests have been completed, the material is ready for packaging. Generally for
internal laboratory use, each drum is split into 10 kg buckets using the rotary splitter. Should the material
require long term storage, the material may be packaged according to method requirements which
could include sealed and nitrogen-purged foil packets o f 150 g each or sealed glass bottles depending

up on s ample matri x. S hou ld fi na l p ackagi ng b e re qu i re d, a m i n i mu m o f 10 random p acke ts / b o ttle s are
s ele c te d for ana lys i s to veri fy that the p ackagi ng pro ce du re ha s no t re s u lte d i n s egregation or a lo s s o f
fi ne s . T he % RS D b e twe en ana lys e s ( p acke ts) i s comp are d to re qui re d me tho d % RS D a nd th at ob tai ne d
du ri ng i n iti a l homo geneity tes ti ng.
For a proven prep aration pro ce du re for a re gu l arly ha nd le d (con s i s tent) matri x, the % RS D o f a na lys e s
o f random ly s ele c te d fi na l p acke ts m ay b e u s e d as s tand- a lone evidence that the materi a l i s s u ffic iently
homogeneous for its intended purpose.

B.9 Assigning accepted performance limits
For a materi a l i ntende d to mon itor con s i s tenc y / rep e atabi l ity o f a me tho d on ly, it may b e con s idere d
s u fficient to a s s ign va lue s b a s e d up on trace abi l ity to o ther C RM s , or even o ther re ference materi a l s and
long term me tho d rep e atabi l ity. I n s uch a c a s e, the materia l wou ld b e u s e d i n conj u nc tion with a s i mple,
one p age de s crip tion o f de term i ne d me a n and p er formance gate s b a s e d up on two s tand ard deviation s
i n either d i re c tion, s igne d o ff b y the L ab orator y Qua l ity M anager.

Annex C
(informative)
Case study 3 — Preparation o f a wheat flour fortified with folic
acid quality control material (QCM) 6)
C.1 Introduction
Folic acid fortification o f wheat flour for bread making became mandatory in Australia in September
2009. The level o f fortification required is between 2 mg and 3 mg o f folic acid per kilogram o f wheat
flour. The analytical methods used by laboratories measuring folic acid need to detect and accurately
quanti fy added folic acid near the regulated levels. The results from laboratories need to be comparable
to enable a consistent assessment o f industry compliance with the Food Standards Code. The material
described here was prepared as a proficiency test (PT) study sample and has subsequently been used as
an in-house QC re ference material. It has been used for analytical method development purposes and to
ensure comparability o f results between analytical batches. The purity o f the commercial folic acid used
to prepare the sample and the adsorption o f folic acid onto the walls o f the mixing containers were two
important issues that were identified in the preparation o f this material.
In this example, development o f an analytical method for the determination o f folic acid in flour
commenced at the same time as the preparation o f this material. Consequently there was no reliable
method to determine homogeneity o f folic acid in the material and an alternative approach was used.
Barium carbonate was used to demonstrate (validate) that the mixing protocol ensures a homogeneous
mix at the expected fortification level. Barium carbonate was mixed into a separate flour sample in
similar concentrations to the anticipated folic acid concentration, the idea being to demonstrate that
a solid i.e. barium carbonate mimicking folic acid can be evenly distributed within the flour matrix.
Subsamples o f the flour were analysed for barium by inductively coupled plasma mass spectrometry
(ICPMS) and found to be homogeneously distributed throughout the flour with the concentration o f
the barium in the subsamples at the expected concentration. It was there fore assumed that the mixing
protocol produced homogeneous materials at the expected concentration – i.e. it was validated.
The folic acid analytical method when developed was used to analyse other similar folic acid/flour PT
study samples produced using the described procedure. However it was found that although the folic acid
was homogeneously distributed in the flour it was not actually present at the expected concentration.
Eventually a fter a full mass balance experiment it was determined that the folic acid was sticking to
the sides o f the stainless steel mixing vessels and this was why the concentration o f the folic acid in the
study samples was less than the expected fortification level. The fact that folic acid is pale yellow made
this easier to determine. Further experiments determined that the stainless steel vessels were probably
the least suitable for mixing and polypropylene the best option.
C.2 Material description and specification
C.2.1 White wheat flour fortified with folic acid at 1,86 mg/kg.
C.3 Preparation
The steps in the production o f this QC material are shown in Figure C.1
6) This case study was provided by Meg Cro ft and Stephen Davies, National Measurement Institute Australia. PO
Box 138, North Ryde NSW 1670, Australia.
Figure C.1 — Production steps

C.3.1 Issues
The following issues were considered:
— Purity determination o f the commercial folic acid used to forti fy the flour;
— adsorption o f folic acid onto the sur face o f mixing containers;
— degradation o f folic acid due to light sensitivity (literature);
— possible risk o f deterioration o f the matrix (flour) in storage by insects (e.g. weevils);
— fortification o f a solid (powder) material with another solid (powder);
— uni formity o f particle size; and
— sub-sampling.
C.3.2 Approaches
The approaches taken for each o f these issues are listed below.
— Purity of the commercial folic acid used to fortify the flour: A commercially sourced bottle o f folic
acid had a label stating the folic acid content as being “approx. 98 %” from which it was not clear i f
this re ferred to mass fraction or mole fraction. The manu facturer’s “certificate o f analysis” stated
the presence o f 8 % moisture, which suggested that the statement “approx. 98 %” re fers to the
purity o f the organic component (i.e. folic acid and impurities o f similar structure) and not the total
purity o f folic acid in the so-called pure material. In this case, an in-house assessment o f purity was
conducted (quantitative NMR, thermogravimetric analysis and Karl Fischer titration) to confirm
the total purity o f the folic acid. A standard comparison was also per formed using folic acid sourced
from alternate commercial suppliers.
— Light sensitivity:Sample preparation was per formed in foil-covered containers or inside other
containers that blocked exposure to light.
— Minimize risk of deterioration of the matrix (flour) by insects: The final product was stored overnight
at −80 °C to destroy any insect larvae that may have been present.
— A concentrated pre-mix was
Fortification of a solid (powder) material with another solid (powder):
gravimetrically prepared by combining and thoroughly mixing sieved folic acid and sieved white
flour. The final material was prepared by dilution o f an appropriate sub-sample o f this pre-mix
with un fortified sieved flour. To ensure homogeneity, the mixing process was done in stages.
The concentrated flour pre-mix was added to approximately 200 g un fortified flour and mixed
thoroughly by tumbling in a mini drum hoop mixer. Every 2 h approximately 200 g o f un fortified
flour was added to the mix and the process continued until the desired concentration was achieved.
— Adsorption onto surface of mixing containers: The concentration o f the folic acid in the fortified flour
as determined by replicate analysis was less than the expected gravimetric fortification level. A
rigorous mass balance approach involving analysis o f solutions used to wash the empty container
walls at each stage during the preparation o f folic acid fortified flour samples confirmed the
adsorption o f folic acid onto the sur faces o f the various mixing vessels employed in the preparation
process. While various di fferent containers (perspex, metal, polypropylene) were tested to minimize
sample adsorption, this problem could not be completely eliminated. Accordingly, the gravimetric
fortification level was not used to assign the mass fraction o f folic acid in the flour.
— Uniformity of particle size: Both the unspiked flour and folic acid were passed through a 180 μm
sieve be fore mixing.
— Sub-sampling: The folic acid fortified material was divided into 50 g portions using a Retsch PT 100
Rotary Sample Divider 7) .
7) A Retsch PT 100 Rotary Sample Divider is an example o f a suitable product available commercially. This
C.4 Sub-division and packaging (e.g. any contamination issues, preferential

evaporation, special sealing requirements)
C.4.1 Issues
The following issues were considered:
— adsorption on containers;
— light sensitivity.
C.4.2 Approaches
The issues were addressed by:
— use o f polypropylene screw cap containers to minimize adsorption and provide ease o f access to the
material;
— recommending storage in the dark at room temperature.
C.5 Homogeneity
C . 5 . 1 Ac h i e vi n g a n d c o n fi r m i n g h o m o g e n e i ty
Homogeneity was achieved through serial dilution and thorough mixing o f a concentrated pre-mix (see
C.3.2).
Statistical analysis determined the preparation procedure produced homogeneous samples containing
the expected (gravimetric) concentration o f barium.
Testing the methodology using barium carbonate made the assumption that folic acid and barium
carbonate would behave similarly during the preparation process. This was not found to be the case as
folic acid was found to adsorb onto the walls o f the mixing containers. (See discussion above.)
The homogeneity o f samples produced using the procedure has since been tested multiple times for
subsequent folic acid in flour PT studies. In these studies, the homogeneity o f samples was confirmed by
analysis o f 7 × 1 g subsamples in duplicate for folic acid. The folic acid mass fraction was determined using
exact-matching isotope dilution with liquid chromatography tandem mass spectrometry (LC-MS/MS) in
Selected Reaction Mode for detection.
ANOVA was used to determine within (analytical) and between bottle variance. [17]
C.5.2 Examples of data and data treatment
The in-house QC material was produced be fore an analytical method for folic acid was available (see
previous discussion re validation o f the homogeneity using barium carbonate). The data provided
below were obtained for a similar PT study material fortified at a similar concentration, confirming the
homogeneity o f the folic acid fortified wheat flour QC materials produced using the described procedure.
See Figure C.2.
in formation is given for the convenience o f users o f this document and does not constitute an endorsement by ISO
of this product.
Study S3 Folic acid

Bottle No. A B
301 2,39 2,36
306 2,39 2,30
307 2,35 2,37
31 4 2,29 2,45
31 7 2,37 2,36
323 2,32 2,27
325 2,36 2,42
Anova: Single Factor
SUMMARY
Groups Count Sum Average Variance
Row 1 2 4,757 2,3785 0,00042
Row 2 2 4,685 2,3425 0,003784
Row 3 2 4,723 2,361 5 0,000265
Row 4 2 4,741 2,3705 0,01 3285
Row 5 2 4,728 2,364 3,2E-05
Row 6 2 4,584 2,292 0,001 1 52
Row 7 2 4,78 2,39 0,001 352
ANOVA
Source of Variation SS df MS F P-value F crit
Between Groups 0,01 2476 6 0,0020793 0,71 7365 0,6492 3,865968853
Within Groups 0,02029 7 0,0028986
Total 0,032766 13
Prepared by
Checked by
Figure C.2 — Production steps
For further guidance on how to compute the between-bottle heterogeneity, two scenarios are possible
using ANOVA. I f MS between groups is larger than the MS within groups, the between bottle variance
is computed by subtracting MS between groups from MS within groups and dividing by the number o f
replicates made per unit (in this case 2). The between-bottle standard deviation is then the square root
o f this variance as also given under 9.3 . I f MS between groups is smaller than MS within groups the
between-bottle heterogeneity is computed as given for case study 4. Further explanation can be found
in ISO Guide 35.[2]
C.5.3 Achieving and confirming stability
Initially, the stability o f folic acid was assumed based on literature precedents.
The stability o f the flour was also a concern and this was addressed by treatment at −80 °C o f the final
product.
The stability o f the material was demonstrated by analysis o f the material with batches o f samples over
a period o f time – no trends were observed.

C.6 Storage and handling

Store i n a d r y envi ron ment, away from l ight at ro om temp eratu re . T he de ci s ion to s tore at ro om
temp erature wa s made b a s e d up on l iteratu re pre ce dents and con fi rme d over ti me .
T he m i n i mum s ample s i ze re qu i rement was 1 g b a s e d on homo geneity te s ti ng.
S a fe ty i n formation: Non-ha z ardou s . For lab orator y u s e on ly. D o no t con s u me .

Annex D
(informative)
Case study 4 — Bauxite quality control material (QCM) 8)
D.1 Introduction
Aluminium industry laboratories use bauxite quality control materials to monitor day-to-day and batch-
to-batch analytical per formance. The stability o f the analytical process is checked and the magnitude
o f the time-and-operator-di fferent intermediate precision standard deviation is evaluated by applying
the control chart method[23] to the analysed values o f available alumina9) , reactive silica10) and major
oxides content in a bauxite quality material.
D . 2 M a te r i a l d e s c r i p ti o n a n d s p e c i fi c a ti o n
D.2.1 Washed bauxite sample with high content of gibbsite .
D.3 Preparation
D.3.1 Issues
The following issues are addressed:
— material in su fficient quantity, as to be available for analysis over a given period o f time, but also
adequate to be handled in a chemical laboratory facility;
— particle size less than 150 μm;
— avoid material contamination;
— minimize heterogeneity between units o f re ference material.
D.3.2 Approaches
The approaches taken are listed below.
— A batch o f 5 kg o f washed bauxite is oven-dried, crushed, ground and sieved to pass a 150 μm screen.
The product is mixed and then bottled in 200 g units using a rotary sample divider.
— Use inert equipment and handle the material in order to sa feguard against contamination.
D.3.3 Packaging requirements
Packaging requirements are:
— plastic or glass bottles with screw cap;
— units o f the re ference material clearly labelled.
8) This example was provided by Dr Maria Alice de Goes, CETEM, Rio de Janeiro - RJ – Brazil, 21941-908.
9) Amount o f alumina that is digested in a caustic solution (150 ºC) at similar conditions o f Bayer Process.
10) Amount o f silica that reacts with sodium hydroxide (150 ºC) at similar conditions o f Bayer Process.
D.4 Homogeneity
D . 4 . 1 Ac h i e ve a n d c o n fi r m i n g h o m o g e n e i ty
Materials such as ores are heterogeneous in composition by nature. Much depends on the options
available during preparation to reduce the batch inhomogeneity.
To assess homogeneity, a subset o f the batch o f units o f the re ference material is selected by a stratified
random sampling scheme. For each selected unit, measurements for available alumina, reactive silica
and major oxides are carried out in triplicate, under repeatability conditions.
A one-way analysis o f variance approach[22] is per formed on the data to compute the within and
the between-unit standard deviations. The uncertainty component due to sampling, expressed as a
percentage o f the grand mean, is evaluated.
D.4.2 Examples of data and data treatment
Measurements for available alumina content (% m/m) were carried out under repeatability conditions.
In order to separate a trend in the measurements from a trend in the batch o f bottles, the replicates were
measured in a randomized order. See Table D.1 .
Table D.1 — Measurements for available alumina content

Bottle no. Available alumina
% m /m
3 50,2 50,0 49,9
5 50,0 50,2 50,0
6 50,0 50,2 49,8
7 50,0 50,2 49,9
11 50,0 50,2 50,0
13 50,0 50,1 49,9
15 50,0 50,2 49,9
21 50,0 49,9 50,0
23 50,0 50,2 49,9
24 50,1 50,0 49,9
Analytical method:
Alkali digestion [NaOH (80 g/l); digestor 150 °C + addition (C 6H 11O 7 Na) + KF] / Titrimetry (HCl)
Sample size – 0,65 g
The data was evaluated for consistency using h and k statistics. [22] The h and k plots in Figure D.1
indicate that no specific bottle exhibit patterns o f results that are markedly di fferent from others in the
homogeneity study.

Con si ste n cy w i th i n -bottl e

k- va lu es
1 .60 1 . 48
1 .40
1 .20 1 .1 3 1 .1 3 1 .1 3 1 .1 3
1 .00 0. 85 0. 85
0.80 0. 74 0. 74
0.60
0. 43
0.40
0.20
0.00
3 5 6 7 11 13 15 21 23 24
Bo t t l e s
Con si ste n cy be tw e e n -bottl e

h - val u e s
2.0
1 . 37 1 . 37
1 .5
1 .0
0.5 0. 32 0. 32 0. 32 0. 32
0.0
-0.5
-1 .0 -0. 74 -0. 74 -0. 74
-1 .5
-2.0 -1 . 79
-2.5
3 5 6 7 11 13 15 21 23 24
Bo t t l e s
Figure D.1 — Plots of consistency within- and between -bottle
A one-way analysis o f variance approach[22] was per formed on the data to compute the within and
the between-unit standard deviations which are, respectively, estimations o f the analytical standard
deviation (san) and the sampling standard deviation (ssam). See Tables D.2 and D.3.

Table D.2 — ANOVA: Single factor

SUMMARY
Group Count Sum Average Variance
bottle 3 3 150,1 50,033 33 0,023 333
bottle 5 3 150,2 50,066 67 0,013 3 33
bottle 6 3 150 50 0,04
bottle 7 3 150,1 50,033 33 0,023 333
bottle 11 3 150,2 50,066 67 0,013 333
bottle 13 3 150 50 0,01
bottle 15 3 150,1 50,033 33 0,023 333
bottle 21 3 149,9 49,966 67 0,003 333
bottle 23 3 150,1 50,033 33 0,023 333
bottle 24 3 150 50 0,01
Table D.3 — ANOVA: Source of variation

ANOVA
Source of
variation SS df MS F P-value F crit
Between
groups 0,027 9 0,003 0,163 636 0,995 792 2,392 814
Within
groups 0,366 666 7 20 0,018 333
Total 0,393 666 7 29

san = 0,018
ssam =0
For further guidance on how to compute the between-bottle heterogeneity, two scenarios are possible
using ANOVA. I f MS between groups is larger than the MS within groups, the between bottle variance
is computed by subtracting MS between groups from MS within groups and dividing by the number
o f replicates made per unit (in this case 3). The between-bottle standard deviation is then the square
root o f this variance as also given under 9.3 . I f MS between groups is smaller than MS within groups,
the between-bottle heterogeneity is computed as given below. Further explanation can be found in
ISO Guide 35. [2]
The uncertainty component due to sampling estimate that accounts for insu fficient repeatability o f the
analytical method was calculated using the following formula: [24]
2 MS within 2
u sam = s sam +
n ν MS within

u sam = 0 , 044
where
MSwithin is the within mean square;
vMS,within is the respective degrees of freedom.
The uncertainty component due to sampling, expressed as a percentage o f the grand mean, is less than
0,1 %. There fore, the re ference material could be considered su fficient homogeneous.
D . 5 Ac h i e vi n g a n d c o n fi r m i n g s ta b i l i ty
Based on the nature o f the material, deterioration is not anticipated provided the material is properly
handled and stored.
D.6 Storage and handling

D.6.1 Temperature and other environmental conditions for storage
Units o f the re ference material should be stored at ambient temperature in a dry place.
D.6.2 Minimum sample size requirements
The minimum sample size was that used for the homogeneity assessment, i.e. 0,65 g.
D.6.3 Safety information
Avoid dispersion o f, exposure to dust by inhalation, eye contact or skin contact.
Dispose residual material in accordance with regulations pertaining for inorganic chemical and
mineralogical waste.

Annex E
(informative)
Case study 5 — Pharmaceutical reference standards 11)
E.1 Introduction
Establishment o f re ference standards during the drug development and commercialization process is
very complex, and needs a thorough understanding. From very early stage in the process, pharmaceutical
industries should consider establishing re ference standards to evaluate the raw materials, process
impurities, intermediates, metabolites, degradation products and Active Pharmaceutical Ingredients
(APIs). [25]
Re ference standards are needed almost at every stage in the development o f a drug candidate in order
to make sure the final drug product is o f the highest quality. In the absence o f available o fficial re ference
standards, manu facturers o ften establish in-house re ference standards.
Sourcing and preparation o f bulk materials for re ference material production can at first seem a di fficult
and daunting task, and large quantities may sometimes be required. High quality re ference materials
(RMs) are demanding and costly to produce and i f materials are available from other sources it is not
normally cost e ffective for laboratories to make their own. However, should this be necessary, this
case study provide guidance for non-specialist laboratories to prepare their own re ference materials
for quality control. Some o f the key issues that need to be considered are: selection o f materials
(appropriateness, native material versus spikes, material preparation, etc.), traceability, testing,
preparation and packaging (homogeneity, contamination, etc.), stability testing, value assignment
exercises, uncertainty estimation, documentation, mechanism for the approval o f the assigned value,
storage, and distribution.
Note that biologics (large molecules) are beyond the scope o f this guidance.
E.2 General selection criteria used in establishing a reference standard by a

pharmaceutical company
E.2.1 General
The criteria used in selecting a material to use as a re ference standard is complex and depends on many
factors. There is no magic bullet and every situation is di fferent. The following is a general guideline in
selecting a re ference standard followed by a specific example o f a standard selected by a pharmaceutical
industry.
Once a bulk material has been sourced there are a number o f processing stages which may need to be
carried out to ensure the material has the appropriate level o f homogeneity and stability for its intended
purpose.
Some o f the more common processes are drying to remove water or solvents so that the material is easier
to handle and its stability improves. Water removal also reduces the likelihood o f microbial growth
formation, which is a particular problem with biological materials, and for such materials, freeze drying
is employed to remove water. Please note that our scope in this paper is restricted to smaller organic
re ference materials only.
11) This case study is based on information provided by I ffaaz Salahudeen, PhD, and Frank Hu, PhD, Bristol-Myers
Squibb Pharmaceuticals, New Brunswick, NJ, USA.
Sieving is an additional process carried out a fter milling and grinding to improve material homogeneity.
Bulk solid materials must be homogenized by thorough mixing.
Certain analytes can be unstable and appropriate salt would be selected in order to stabilize the material.
E . 2 . 2 S p e c i fi c a ti o n o f
m a te r i a l
A re ference standard should be made using a best known synthetic process yielding the highest purity
with minimum amount o f other external components such as residual solvents and heavy metals. The
physical properties o f the material are also important. It should be less hygroscopic, with free flowing
crystalline structure with minimum aggregation or lumping.
Impurities classified as organic (process and drug related), inorganic, or residual solvents can be
introduced during the manu facturing process for the drug substance, drug product, or excipient
and/or through storage o f the material. Impurities should be controlled throughout the manu facturing
process. Impurities that are process-related should be kept to a minimum to avoid degradation and
unwanted pharmacological e ffects. Compounds that are susceptible to hydrolysis, for example, should
be thoroughly dried to remove moisture and then stored in a desiccator. Re ference standards that
contain a high percentage o f organic volatile impurities may experience purity changes over time as the
solvents evaporate.
I f the re ference standard is in a salt form, the amount o f salt present must be determined so that the
purity can be corrected for content. Applying the molecular weight to the correction will not account for
residual salt that may be produced during synthesis.
E.2.3 Sourcing of material
Re ference-standard materials that are synthesized by the user or supplied by a contract manu facturer
or secondary company must be characterized. Both the re ference standards and drug substance may
be synthesized initially using the same process. The re ference standard should be o f the highest purity
possible; the drug substance may require further purification to become a re ference standard (additional
purification steps used for a drug substance should be fully described and included in any regulatory
filing).
E.2.4 Processing
I f needed, the material selected can be dried, solvent removed, re-crystallized or purified. I f the material
has a tendency to form lumps, a delumping procedure can be employed. Large particles can be reduced
by grinding. In order to increase its stability, a di fferent salt can be selected or certain additives can be
added to the material. For materials with < 95 % purity, or mixtures, thorough mixing or homogenization
may be necessary in order to make sure the mixture is homogeneous. An old or API lot also can be used
a fter fresh testing as long as its properties are within the acceptable range.
E . 2 . 5 Q u a l i fi c a ti o n
For the initial lot, an example requalification period may be 3, 6 or 12 months for the first year and
annually therea fter. In this scenario, it is recommended that during development, the re ference standard
be assessed a fter 3 months at the intended storage condition and at an accelerated storage condition.
Validation o f the analytical method for organic impurities should occur a fter the full accelerated storage
condition has been evaluated. The total length o f the requalification program will depend on the
intended li fe o f the re ference standard and the length o f the stability and clinical programs. I f the initial
lot is proven to be stable for at least one year, then subsequent lots will require annual requalification
only. In all study scenarios, a protocol is required to outline the re ference-standard material, lot, storage
conditions, frequency o f test, analytical procedures, acceptance criteria, and reporting criteria. [26]
Organic impurities. Determination of organic impurities is the most challenging aspect of developing
a suitable analytical method because these impurities are unique to the parent compound and because
various degradation pathways can lead to various impurities. Actual and potential organic impurities
that arise during synthesis, purification, and storage must be identified and quantified. The synthesis

o f the re ference standard should be evaluated to predict and identi fy potential impurities from raw
materials. Potential degradation product also can occur as a result o f storage. Short-term (forced
degradation) and long-term (evaluation under accelerated conditions) stress testing, there fore, should
be evaluated during development. The design o f the long-term stress test depends on the intended
storage condition.
The quantity o f organic impurities present can be determined with high-per formance liquid
chromatography (HPLC) and ultraviolet (UV) detection. Degradation products and compounds related
to the product can be evaluated by the area per cent or from the relative response o f the standard
being used. The technique used to obtain this data will depend on the amount o f impurities and related
compounds present and the decomposition pathway o f the re ference-standard material.
To consider the impact on the purity evaluation using area per cent versus relative response factor,
the following scenario may be considered. I f analysis shows an impurity at 0,05 % and the relative
response factor o f the impurity is hal f o f the standard (i.e. the amount o f impurity present shows a 50 %
detector response compared with the equivalent amount o f standard), then there could be 0,1 % o f
actual impurity. This level may be insu fficient to a ffect overall purity results. I f there was 1 % impurity
based on area per cent present, however, then there would be 2 % o f actual impurity that could a ffect
overall purity.
The approach to determining the relative-response factor for each impurity is a more accurate process,
but potential pitfalls should be considered. The relative-response factor approach requires additional
development because the component needs to be isolated and the relative response factor must be
determined. In addition, as the re ference standard ages, new unknown impurities may be detected.
The relative-response factor o f these new impurities must be determined, and the method updated i f
the new unknown is significant enough to alter the purity. Much o f this in formation may be ascertained
during the development o f the drug substance.
Impurities that arise from raw materials, synthesis, purification, and storage require care ful
consideration because they may not produce detector responses that are related to the re ference-
standard material. Quantification by area per cent would not be appropriate in such cases. Rather, the
impurities must be isolated and identified so that an appropriate re ference standard can be used, or
a relative response factor determined. For example, i f the re ference-standard material is a salt, then
the cation response would not be equivalent to the re ference standard. In such instances, a specific
re ference standard is required for the cation, and a separate analytical method for quantification may
be needed.
Inorganic impurities. Inorganic impurities such as metals and non-combustible materials are typically
evaluated using compendial procedures. I f inorganic impurities are proven to be less than the reporting
threshold at initial characterization, then further analysis is not required.
Residual solvents. The potential for residual solvents should be evaluated during development of the
drug substance and can be estimated by reviewing the synthesis pathway. USP General Chapter Residual
Solvents [1] details a generic procedure for this evaluation. Residual solvents, however, may be specific
to the manu facturing process and require a specific test procedure. An additional specific test procedure
may be required i f the USP procedure is not suitable for the re ference standard being evaluated, or i f the
solvents used during synthesis are not included in USP. I f residual solvents (previously re ferred to as
organic volatile impurities, or OVIs, by USP) are proven to be less than the reporting threshold at initial
characterization, further analysis is generally not required at subsequent intervals. I f the amount o f
residual solvents present a ffects the purity, however, they should be evaluated at each requalification
interval.
E.2.6 Sub-division and packaging
Re ference materials are subdivided from the bulk material by either manually or by automated means.
Many materials can either lose or pick up moisture i f the container is not securely closed. There fore,
septum lined crimp-top vials are more suitable. Vials should pre ferably be amber to reduce the light
impact. The amount per vial depends on its application. Repeated opening and closing o f re ference
material containers increases the risk o f contamination. Stability may also be a ffected by repeated

freeze/thaw cycle. There fore, su fficient quantity o f material needed for analysis should be vialed
separately, instead o f using bulk containers for repeated use.
Light sensitive materials are sub-divided into amber vials, and heat sensitive materials are stored at
lower temperatures. Any material with a significant amount o f solvent needs to be stored care fully.
Prolonged storage o f inherently heterogeneous matrix materials may cause settling and separation
o f the material. It is there fore important the units are adequately mixed be fore a new sub-sample is
withdrawn. This can o ften be achieved by simple shaking o f the units (bottles).
For moisture-sensitive materials, appropriate packaging would be the pre ferred method o f controlling
moisture content.
E.2.7 Homogeneity assessment
For materials with < 95 % purity, or mixtures, thorough mixing or homogenization may be necessary in
order to making sure the mixture is homogeneous. These mixtures need to be assessed for homogeneity.
E.2.8 Documentation requirements
Ideally, a document complying with ISO Guide 31 [20] and a report covering the characterization and
value assignment will be produced.
For quantitative standards, purity values should be assigned, and based on past history and stability, a
re-test date should also be provided. However, for qualitative standards (e.g. retention time markers or
impurity mixtures), assigned purity values are not needed, as long as a chromatographic verification is
per formed to prove that the impurities are detected (above the detection limits).
Upon testing a Certificate o f Analysis (COA) will be issued. The COA will have all the necessary
in formation including the chemical name, catalogue #, Lot #, test date, re-test date (most re ference
standards can be used upon re-certification and therefore, they do not need any expiry date, but a re-
test date). Any special handling instruction such as hazardous category, light or heat sensitivity) also
should be provided in the COA.
E.2.9 Storage requirements
Re ference-standard materials are o ften expensive to manu facture and are generally o f limited supply. It
is important, there fore, to consider how the material will be stored, distributed, and controlled. Once the
storage conditions are ascertained, the re ference-standard material should be monitored continually
using a suitable environmental monitoring system. It is advisable to store the material in at least two
di fferent locations in case there is a prolonged excursion from the storage condition. The material should
be stored in a secure environment with controlled access and distribution. Even i f the material is proven
to be stable at room temperature, most o f the solid or powdered materials can be re frigerated or frozen
so that their shel f lives can be extended a fter re-testing.
E.2.10 Quantity
In the early phase o f product development (prior to Phase 3), batches o f 10 g to 100 g quantity would be
qualified as re ference standard. A fter the program moves into the Phase 3, a larger quantity (500 g to
1 000 g) from one pilot-scale batch would be qualified as re ference standard.
E.3 Specific example o f the selection criteria used in establishing a re ference

standard
E.3.1 General
The criteria described in the example below are appropriate for the specific standard under discussion
and should be considered as a guide instead o f general requirement for selecting a re ference standard.

E.3.2 Specification o f material

Compound X is a monohydrate and mono propylene glycol solvate. A representative batch o f Compound
X prepared with the best-known synthetic process available at the time was selected. The batch has a
high purity (>99,0 %) and contains < 0,1 % o f residual solvent. Compound X is a free flowing crystalline
material with minimum aggregates or lumps.
E.3.3 Sourcing of material
The material was supplied in house early on from laboratory-scale productions and later on from pilot-
scale productions.
E.3.4 Processing
Compound X is prepared as a crystalline material with an average particle size o f 10 to 20 microns
through a final crystallization step o f the synthesis. The material has a tendency to form so ft lumps so
delumping procedure was occasionally employed. No additional processing is required.
E.3.5 Sub-division and packaging
Re ference materials are subdivided from the bulk material by either manually or by automated means.
The amount per vial is 120 mg. Re ference material is subdivided into amber vials, stoppered, then
crimped, labelled, and stored. Vialed re ference material is stored in a secure re frigerator. A minimum o f
two per cent o f each batch (maximum o f 500 mg) o f re ference material will be retained in a secure cold
temperature storage unit for at least three years a fter its last use date.
E.3.6 Homogeneity assessment
The manu facturing process o f Compound X is optimized to produce highly pure and homogeneous
material. The homogeneity o f the re ference material is continuously monitored through usage by
various analysts and laboratories at various sites. A separate homogeneity assessment is not considered
necessary.
E.3.7 Documentation requirements
A Certificate o f Analysis (COA) was issued upon initial testing. The re ference standard batch is tested
annually for re-certification. A new COA is issued a fter annual re-certification. In-house MSDS (material
sa fety data sheet) is provided along with the re ference standard material when shipped to customers.
The compound is neither light sensitive nor hygroscopic. No special handling instruction is required.
E.3.8 Storage requirements
Bulk material is stored in secure freezer. Vialed re ference material is stored in a secure re frigerator.
E.3.9 Quantity
In the early phase o f product development (prior to Phase 3), batches o f 10 g to 100 g quantity were
qualified as re ference standard. A fter the program moved into the phase 3, a 500 g supply from one
pilot-scale batch was qualified as re ference standard.

Annex F
(in fo rmative)
Case study 6 — Preparation of testing materials for “bromate in

water” 12)
F.1 Introduction
T h i s i s a c a s e s tudy for the prep aration o f l iqu id te s ti ng materi a l s . I t may s e em trivia l but c are fu l
exam i nation o f the ta sk reve a l s that s evera l i mp or tant i s s ue s mu s t b e tackle d . T he a i m was to prep are
d i fferent typ e s o f water s pi ke d with low level s o f bromate .
B romate i s forme d a s a b y-pro duc t from the re ac tion o f the d i s i n fe c tant o z one with natura l con s tituents
pre s ent i n water and it i s regu late d i n the D ri n ki ng Water D i re c tive (DWD) o f the Europ e an Un ion
(ma xi mu m a l lowe d l i m it o f 10 µg· l −1 ). [27] T he i nge s tion o f large amou nts o f bromate may c au s e s ymp tom s
s uch as nau s e a, vom iti ng , d iarrho e a and ab dom i na l p a i n s . B romate i s a l s o an ac tive oxidant i n biolo gica l
s ys tem s and h as b e en s hown to cau s e an i ncre as e o f tu mou rs i n an i m a l exp eri ments .
T he ne e d for rel i able ana lytica l me tho d s at the concentration level s s tipu late d by the Eu rop e a n
legi sl ation i s there fore ob viou s . T h i s c a s e s tudy concern s the pro ces s i ng a nd eva luation o f homo geneity
and s tabi l ity o f five d i fferent typ e s o f water with re s p e c t to bromate, na mely: s o ft d ri n ki ng water, ha rd
d ri n ki ng water, m i nera l water, s wi m m i ng p o ol water and raw water. I n add ition a blan k a nd s tandard
i n u ltra-pu re water was prep are d . T he cas e s tudy materia l s were u s e d i n a n i nter- comp a ri s on e xerc i s e
for the I nternationa l O rgan i z ation for Stand ard i z ation for a me tho d up date . [28] T h i s c as e s tudy ha s a l s o
b e en de s crib e d i n gre ater de tai l i n Re ference s [ 29 ] and [ 30].

F. 2 M a te r i a l d e s c r i p ti o n a n d s p e c i fi c a ti o n
F.2.1 Soft drinking water.
F.2.2 Hard drinking water.
F.2.3 Mineral water.
F.2.4 Swimming pool water.
F.2.5 Raw water.
F.2.6 Synthetic bromate standard solution in ultra pure water.
F.2.7 Ultra pure water used as blank (Typ e 1 water as s p ecified in F.4).
12) This cas e s tudy is b as ed o n in fo rmatio n p rovided by Fernando C o rdeiro , I nge Verb is t, H åkan E mteb o rg, J ean
C haro ud- Go t, M aria C . C o ntreras , Philip Taylo r, and B eatriz de la C alle ( J RC - I ns titute fo r Re ference M aterials and
M eas urements , Reties eweg 1 1 1 , B - 2 440 Geel/B elgium) and Franz S chmitz (Landes b etrieb , H es s is ches Landes lab o r,
Wies b aden, Germany) .

F.3 Important considerations for preparation of liquid materials by spiking

F.3.1 Gravimetric preparation of spiking solutions
The following should be taken into consideration:
— use o f checked and calibrated balances (traceability to SI);
— use o f balances with su fficient resolution;
— uncertainty equations o f the balances;
— purity and its uncertainty o f the commercially available bromate;
— molar fraction o f bromate in potassium bromate.
F.3.2 Homogeneity tests of inherently homogeneous liquids
The following should be taken into consideration:
— the need to veri fy the absence o f contamination from final containers or anywhere in the handling
sequence;
— the need to veri fy the absence o f trends in filling order;
— the need to veri fy the absence o f carry-over or memory e ffects between samples;
— it is mandatory to care fully mix the bulk during whole filling sequence;
— the need to prevent evaporation during long sequences o f work, especially important for volatile
components and solvents.
F.4 Preparation of the test samples

A concentrated stock solution was gravimetrically prepared from potassium bromate, KBrO 3 of ACS
ISO Reagent grade > 99,8 % purity (Merck, KGaA, Darmstadt, Germany13)) in Type 1 water, (18 MΩ cm,
0,056 μS·cm−1 ; < 10 µg·l−1 TOC; Millipore N.V., Belgium). By dissolving 0,638 8 g o f KBrO 3 (mole fraction
o f 76,58 % BrO3 in KBrO 3 ) in 486,50 g o f Type 1 water a concentrated bromate stock solution o f
1 005,5 mg·l−1 was prepared. An intermediate stock o f 100,27 mg·l−1 was therea fter prepared by taking
53,414 g o f the concentrated stock solution and diluting it with Type 1 water up to 535,62 g.
The purity o f the bromate salt (99,8 %) was considered to be adequate in relation to the ± 25 %
precision and trueness criterion o f the measurements stipulated by the Drinking Water Directive. [27]
A moderate bias between the stated and actual purity o f a few % would still be well within these 25 %.
The uncertainty o f purity was based on expert judgment (Type B uncertainty) given the in formation
provided by the supplier concerning the impurities present. This uncertainty component was judged
to be U = 0,2 % (k = 2) since the purity of the bromate salt cannot be above 100 %. In addition, when
purchasing the bromate standard, the best quality available was chosen to prepare a good quality spike.
All water types were filtered through a 0,45 µm membrane filter prior to spiking (Pall, Versaflow filter
PN 12131 14)). At the time of spiking, aliquots of the intermediate stock solution were accurately weighed
and added to the exactly known sample volumes o f about 25 l to obtain the final bromate concentrations
in the di fferent water types. The obtained concentrations were subsequently set as the assigned
13) This in formation is given for the convenience o f users o f this document and does not constitute an endorsement
by ISO o f this product.
14) The trademarks mentioned in this case study are examples o f suitable products available commercially. This
in formation is given for the convenience o f users o f this document and does not constitute an endorsement by ISO
of these products.
(re ference) values for the di fferent spiked water types. In the case o f the swimming pool water, no spike
was added.
All solutions, including the blank solution, contained ethylenediamine at a concentration o f 50 mg·l−1
which was added as stabilizer to avoid the formation o f additional bromate from bromine in the presence
o f ozone a fter preparation o f the solution. The blank solution was measured using liquid chromatography
followed by inductively coupled plasma mass spectrometry (LC-ICP-MS) to check for the presence o f
bromate. The concentration o f bromate was below the limit o f quantification (LOQ = 0,5 µg·l−1), thus the
material could be used as a blank for the purpose o f this exercise.
Filling was taking place in an ampouling machine, Rota R910/PA (Wehr Baden, Germany) that has a very
accurate piston pump. Consequently, slightly more than 50 ml o f the di fferent water types was filled into
60 ml amber narrow mouth polyethylene bottles (PN 2004-0002, Nalgene, USA) and stored at 4 °C until
dispatch. The amber coloured plastic prevents light exposure o f the content o f the bottle. Approximately
400 units o f each water material were produced. Between each water type the trans fer line from pump
to bottle was rinsed with 1 000 ml Type 1 water and > 300 ml o f each solution was discarded be fore
actual filling o f each water type took place. The drums containing the di fferent spiked waters were
under continuous mixing using a stainless steel propeller mounted on an electrical motor. The amber
polypropylene screw caps were removed and placed manually be fore and a fter the filling, respectively.
F.5 Homogeneity
Homogeneity studies were carried out for all water samples except the blank using a method based on
high per formance liquid chromatography, coupled with inductively coupled plasma mass spectrometry
(HPLC-ICP-MS). The HPLC-ICP-MS system consisted o f a Perkin Elmer quaternary pump with vacuum
degasser. The analytical column was a Dionex® anion exchange column IonPac AS16 250x4 mm I.D. A
sample volume o f 250 µl was injected. Isocratic elution was applied using 35 mM NaOH as eluent with a
flow rate o f 0,3 ml.min−1 .
The experimental design used for the assessment o f the homogeneity o f the test samples complied with
the requirements set by the ISO 13528 [32] and by the IUPAC Harmonized Protocol. [17]
ISO 13528 describes the tests to determine i f the samples are to be considered adequately homogeneous
to be used in a proficiency testing exercise. These tests compare the between-bottle standard deviation
with the target standard deviation o f an exercise. In this particular case, it should be checked i f the
material is su fficiently homogeneous within the per formance limits dictated by legislation. There fore,
the target standard deviation was set to 25 % o f each assigned value (for each water test sample), which
is the precision stipulated in the DWD. Both tests indicated that all water test samples were su fficiently
homogeneous for the bromate analysis (Table F.1).
The between-bottle relative standard uncertainty (u bb ,) ranged from 1,0 % to 6,0 %. ubb was estimated
using methods described in ISO Guide 35. [2]

Table F.1 — Homogeneity tests

Soft Hard Mineral Swimming Raw Standard Sol.
Measurement results (µg l -1)
Bottle No. R1 R2 R1 R2 R1 R2 R1 R2 R1 R2 R1 R2
22 3 ,00 2 ,82 10 , 3 9 9 ,9 7 3 ,7 7 3 ,68 8 , 62 8,08 7,71 7, 49 2 ,09 2 , 19
63 2 ,66 2 ,64 9 ,71 1 0 , 42 3 , 52 3,27 8, 37 8, 52 7, 4 0 6 ,9 0 2 ,24 2 , 31
119 3 ,1 2 2 ,76 10 , 01 9 , 74 3 ,7 8 3 , 59 8,0 4 8 ,1 3 7, 3 3 7, 4 8 2 ,02 2 ,21
135 2 ,55 2 ,37 10.18 9, 8 5 3 , 41 3 , 59 9 , 42 8, 52 7,74 7,7 7 2 ,1 2 2 ,1 1
167 2 , 41 2 ,86 9, 6 8 9 , 62 3,25 3,36 8 , 61 7, 8 4 7, 1 8 7, 74 2 ,1 2 2 ,01
240 2 , 83 2 ,9 9 1 0 , 42 10 , 3 2 2 ,9 6 3 , 41 7,9 9 7,9 7 7, 2 6 7, 61 2 ,05 1 ,9 9
251 2 , 51 2 ,4 4 10 , 2 2 1 0 , 43 3,25 3 ,82 8,24 8,56 8,37 7,70 2 ,02 2 ,1 3
299 2 , 62 2 , 52 10 , 2 2 10, 2 5 3 ,08 3 , 57 9 ,1 0 8, 59 7, 3 9 7, 5 8 2 ,1 5 2 ,07
325 2 ,83 2 , 52 1 0 ,1 5 10,0 4 3 , 52 3 ,66 8,24 9 ,1 0 7,7 9 7,70 2 ,03 2 ,02
370 3 ,32 2 ,7 9 10, 2 1 10,0 6 3,28 3,34 8 ,46 8,33 7, 5 0 7, 2 5 2 ,14 2 ,22
Mean 2,73 10,09 3,46 8,44 7,54 2,11

σ ( 2 5 % ) 0,682 2,524 0,864 2,109 1,886 0,528
Homogeneity test according to the ISO 13528 (values in µg l -1)
0.3 σ 0,205 0 ,7 5 7 0,259 0,633 0, 5 66 0 ,1 5 8
Sx 0 , 2 10 0 , 2 14 0 ,1 7 9 0,315 0, 248 0 , 0 76
Sw 0, 2 03 0,218 0 , 2 17 0, 377 0,256 0,068
Ss 0 ,1 52 0 ,147 0,090 0 ,1 6 8 0 ,1 70 0,059
Ss ≤ σ ? Yes Yes Yes Yes Yes Yes

Test Result Passed Passed Passed Passed Passed Passed
Homogeneity test according to IUPAC International Harmonised Protocol (values in µg l -1)

S2 an 0 , 0 41 0,048 0 , 0 47 0 ,142 0,066 0,005
S2 S am 0,023 0,02 2 0,009 0,02 8 0,02 9 0,003
σ All 2 0 , 0 51 0, 573 0 , 0 67 0 ,40 0 0, 320 0,02 5
C r i ti c a l va lue 0 ,1 3 7 1 ,1 2 6 0 ,1 74 0,896 0,668 0,052
S2 S am ≤ C r i tic a l ? Yes Yes Yes Yes Yes Yes

Test Result Passed Passed Passed Passed Pas sed Passed
R1 deno te s repl ic ate 1 ; R2 deno tes repl ic ate 2 . T he s tanda rd deviation for the I LC a s s e s s ment (σ) that
i s u s e d i n th i s table wa s ca lc u late d a s a frac tion (2 5 %) o f the me an c a lc u late d from the homo geneity
s tud ie s , no t a s a frac tion o f the re ference va lue .
Following ISO 13528:2005: [32]

S x i s the s tandard devi ation o f the s a mple s average;
Sw i s the with i n- s ample s/ b o ttle s tandard devi ation;
Ss i s the b e twe en- s ample/ b o ttle s tanda rd deviation .

Following the IUPAC Harmonized protocol: [17]

2
S an is estimated analytical variance (within-sample/bottle);
2
S Sam is the estimated sample variance (between-sample/bottle);
2
σ All is the maximum allowed variance (0,3 σ) 2 .
Critical value C = F1 + F2 , with F1 , F2 being constants derived from F-test tables corresponding to the
number o f bottles used to the homogeneity study.
F.6 Stability
It is necessary to obtain in formation about the stability o f the analyte-matrix combination that has been
prepared. Stability studies can be made in di fferent ways but an isochronous stability study[2] , [33] offers
distinct advantages with respect to significance and precision. Three temperatures (4 ° C, 18 ° C and
60 ° C) were tested with the aim to:
— measure all samples under repeatability conditions (thus avoiding the need to combine the
repeatability with long term reproducibility conditions);
— find suitable temperature conditions for sample dispatch (linear regression o f the stability data
indicated su fficient stability at all temperatures for the investigated time);
— quanti fy the potential degradation during the entire inter-laboratory comparison study
(approximately two months, i.e. the estimated li fe time o f the material).
The evaluation o f the stability o f the test materials was made using the So ftCRM 2.0 so ftware, but it can
also be made in Excel.
The materials proved to be stable at 18 °C for a length covering the whole time frame o f the exercise.
The relative standard uncertainty due to the stability ranged from 2,5 % to 7,3 %.
Table F.2 shows the standard uncertainty (ust) obtained from a stability study carried out at 18 °C
considering a shel f li fe o f nine weeks for so ft drinking water. Data for the other waters was calculated in
the same way and is presented in Table F.3 under the column stability. The users o f this material were
instructed to store the material at 4 °C a fter receipt.

Table F.2 — Stability tests

Soft drinking water
Results in µg·l − 1
Weeks
Bottle 0 3 5 7
1 3 ,1 1 2 , 69 3 ,1 8 2 ,88
2 3 ,03 2 ,9 4 3 ,19 2 ,83
Slope = − 0 , 0 14
SE Slope = 0 ,02 6
Intercept = 3 ,032
SE Intercept = 0 ,1 17 u St µg· l −1 0 ,1 2
C o r rel atio n C o e ffic ient = 0 , 0 45 u St (%) 4,1
S l o p e o f t h e l i n e a r r e g r e s s i o n s i g n i fi c a n t l y < > 0 ( 9 5 %) : No
S l o p e o f t h e l i n e a r r e g r e s s i o n s i g n i fi c a n t l y < > 0 ( 9 9 % ) : No
Test results Stable
F.7 Reference values and estimation of expanded uncertainty

T he ma xi mum a l lowe d concentration o f bromate i n d ri n ki ng water accord i ng to the DWD i s 10 µg· l −1 . [27]
T he bromate concentration s i n the d i fferent water s ample s were prep a re d accord i ngly, i . e . te s t water
s ample s were s pi ke d up to a level no t exce e d i ng th i s va lue . Swi m m i ng p o ol water, wh ich had i ncipient
bromate, wa s no t s pi ke d . T he re ference va lue for the bromate concentration level i n s wi m m i ng p o ol
water was e s tabl i she d from the homo geneity s tudy.
No bromate cou ld b e de te c te d i n the blan k s ample ( b elow the de te c tion l i m it o f 0 , 5 µg· l−1 ) . T h i s s a mple
was u s e d to de te c t fa l s e p o s itive s .
T he bromate concentration s i n the s ample s u s e d , X ref, a re pre s ente d i n Table F. 3 .

Xref was u s e d for the as s e s s ment o f the ana lytic a l re cover y on the bromate de term i nation i n the d i fferent
te s t s ample s .
T he s tandard u ncer tai ntie s (u ref ) a s s o ciate d with tho s e concentration s were c a lc u late d b y prop agati ng
contribution s for ch arac teri z ation, i.e. from the s pi ki ng pro ce du re s (u ch a r) , homo geneity (u bb ) and
s tabi l ity s tud ie s (u st ) as fol lows:
u ref
= u 2
char
+u +u 2
bb
2
st
(F.1)
where
u ch a r i s the s tandard u ncer tai nty o f the cha rac teri z ation (gravi me tric prep aration) ;
u bb i s the s tandard u ncer tai nty ari s i ng from the homo geneity s tud ie s ( b e twe en-b o ttle va ri a -
tion) ;
u st i s the s tandard u ncer tai nty ari s i ng from the s tabi l ity s tud ie s .
T he s tanda rd u ncer tai ntie s for ch arac teri z ation (u ch a r) were e s ti mate d for the s pi ke d materi a l s
following the Guide to th e expression of un certainty in m easurem ent (GUM) [31] combi n i ng the u ncer ta i nty
derive d from the prep a ration o f the s to ck s olution s , i . e . u s i ng the u ncer tai nty e quation s o f the b a la nce s
used, from s pi ki ng with pip e tte s and from the u ncer tai nty o f the pu rity o f the KB rO 3 s tandard materi a l .

T he e xp ande d u ncer tai nty Uref wa s c a lc u late d applyi ng a coverage fac tor o f 2 , repre s enti ng a con fidence
level o f approxi mately 9 5 % .
For the s wi m m i ng p o ol water, u ch a r wa s b as e d on ac tua l me as u rements . C on s e quently, the u ncer tai nty
contribution from cha rac teri z ation i s much larger tha n the gravi me tric a l ly prep are d s pi ke s as c an b e
seen in Table F. 3 .
Table F.3 — Concentrations and their uncertainties of bromate in the different test materials
Gravimetric Homogeneity Stability Combined
(spike) u ref Uref
Sample Xref u u bb u sf u ref
cha r
µg· l −1 µg· l −1 µg· l −1 µg· l −1 µg· l −1 % %
S o ft d ri n ki ng water 2 ,68 0 , 01 0 ,1 5 0 ,1 2 0 ,19 7, 2 14,4
H ard d ri n ki ng water 10 , 0 0 0 , 02 0 ,1 5 0 , 51 0,53 5,3 10 , 5
M i nera l water 3 ,00 0 , 01 0,09 0 ,16 0 ,19 6, 3 1 2 ,7
Swimming pool 8 ,4 4 0,60 0 ,17 0,21 0,66 7, 8 15 ,6
water
Raw water 7,9 5 0,02 0 ,17 0,2 9 0,33 4, 2 8 ,4
Stand ard s olution 1 , 67 0 , 01 0,06 0 ,1 5 0 ,17 9,9 19, 8

BIBLIOGRAPHY
[1] ISO Guide 34:2009, General requirements for the competence of reference material producers
[2] ISO Guide 35, Reference materials — General and statistical principles for certification
[3] ISO 7870-1, Control charts — Part 1: General guidelines
[4] ISO 7873, Control charts for arithmetic average with warning limits
[5] ISO 7870-3:2012, Control charts — Part 3: Acceptance control charts
[6] ISO 7870-2:2013, Control charts – Part 2: Shewhart control charts
[7] ISO Guide 30, Terms and definitions used in connection with reference materials
[8] ISO/IEC Guide 99:2007, International vocabulary of metrology — Basic and general concepts and
associated terms (VIM)
[9] ISO 3534-1:2006, Statistics — Vocabulary and symbols — Part 1: General statistical terms and
terms used in probability
[10] ISO Guide 33:2000, Uses of certified reference materials

[11] EMONS, H, LINSINGER, T.P.J., GAWLIK, B.M. Re ference Materials: terminology and use. “Can’t one
see the forest for the trees?”, Trends in Analytical Chemistry, 2004, Vol. 23, p. 442 to 449
[12] IAEA-TECHDOC-1350. Development and Use o f Re ference Materials and Quality Control Materials.
International Atomic Energy Agency (IAEA), 2003, ISBN 92–0–103303–6
[13] LAWN, R., ROPER, R., HOLCOMBE, G. STUART, B. Low-cost QC Laboratory Re ference Materials –
Investigation o f Cost-e ffective Production Procedures. Laboratory o f the Government Chemist
(LGC), Teddington, 2001, LGC/VAM/2001/009
[14] Guideline A. C53-A. “Characterization and Qualification of Commutable Reference Materials for
Laboratory Medicine”, Clinical and Laboratory Standards Institute. CSLI, 2010
[15] ISO 14488:2007, Particulate materials — Sampling and sample splitting for the determination of
particulate properties
[16] BS 5309-1:1993, Sampling Chemical Products — Part 1: Introduction and General Principles
[17] THOMPSON, M., ELLISON, S.L.R. and WOOD, R. The International Harmonized Protocol for the
Proficiency testing o f Analytical Chemistry Laboratories. Pure and Appl. Chem , 2006, Vol. 78, p.
145 to 196
[18] ISO/IEC 17025:2005, General requirements for the competence oftesting and calibration laboratories
[19] ILAC G19. Guidelines for forensic science laboratories. International Laboratory Accreditation
Corporation. ILAC. Silverwater, 2002, pp. 15.
[20] ISO Guide 35, Reference materials — Contents of certificates and labels
[21] RUCKOLD, S., GROBECKER, K. H. and ISENGARD, H.-D. Determination o f the contents o f water and
moisture in milk powder. Fresenius’ J. Anal. Chem., 2000, Vol. 368, p. 522 to 527
[22] ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2:
Basic method for the determination of repeatability and reproducibility of a standard measurement
method

[23] ISO 5725-6:1994, Accuracy (truen ess an d precision) of m easurem ent m eth ods an d results — Part 6:
Use in practice of accuracy values
[24] LINSINGER, T.P.J., PAUWELS, J., VAN DER VEEN, A.M.H., SCHIMMEL, H. and LAMBERTY, A.
Homogeneity and stability o f re ference materials. Accreditation an d Quality A ssuran ce, 2001, Vol.
6, p. 20 to 25
[25] USP Stimuli article by the USP Council o f Experts. Primary and Secondary Re ference Materials
for Procedures to Test the Quality o f Medicines and Foods. USP Re ference Standards Committee,
2011
[26] BROWNE, D. Re ference-Standard Material Qualification. Pharmaceutical Technology, 2009, 33(4),
p. 66 to 73
[27] Council Directive 98/83/EC. O fficial Journal o f the European Communities o f the 3rd November
1998 (L330) on the quality o f the water intended for human consumption
[28] ISO 11206:2011, Water quality — Determin ation of dissolved brom ate — Meth od usin g ion
chrom atography (IC) an d post colum n reaction (PCR)
[29] IMEP-25b:2009, Determination o f bromate in drinking water. ISBN 978-92-79-14684-8

[30] CORDEIRO RAPOSO, F., ROBOUCH, P., DE LA CALLE, M. B., EMTEBORG, H., CHAROUD-GOT J. AND
SCHMITZ, F. Determination o f Dissolved Bromate in Drinking Water by Ion Chromatography and
Post Column Reaction: Interlaboratory Study. Journ al of AOAC Intern ation al, 2011, Vol. 94, pp.
1592 to 1600
[31] ISO/IEC Guide 98-3:2008, Un certainty of m easurem ent — Part 3: Guide to th e expression of
un certainty in m easurem ent (GUM:1 995)
[32] ISO 13528, Statistical methods for use in pro ficiency testing by interlaboratory comparisons
[33] L ambert y A., S chimmel H., Pauwels J. The study o f the stability o f re ference materials by
isochronous measurements. Fresenius J. An al. Ch em . 1998, 360 pp. 359–361

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GUIDE 80

Guidance for the in-house preparation

COPYRIGHT PROTECTED DOCUMENT

ii © ISO 2014 – All rights reserved

© ISO 2014 – All rights reserved iii

iv © ISO 2014 – All rights reserved

© ISO 2014 – All rights reserved v

vi © ISO 2014 – All rights reserved

Guidance for the in-house preparation of quality control

o f th i s Gu ide a l s o appl ie s to i n herently s table materi a l s , wh ich c an b e tran s p or te d to o ther lo c ation s

but provide a range o f i n formation for lab orator y s ta ff to d raw from .

I S O Gu ide 3 0 , Reference materials — Selected terms and definitions

3 Terms and definitions

ISO 3534-1[9] and the fol lowi ng apply.

© ISO 2014 – All rights reserved 1

pro vide d fo r i n fo r m ation on l y.

4 Quality control materials (QCMs)

me trolo gic a l trace abi l ity or truene s s o f a me as u rement re s u lt.

shou ld b e le s s th an the e xp e c te d s tanda rd deviation o f the me a s u rement pro ces s or an e s tabl i s he d

which it is intended to be used.

Wh i le C RM s ca n i n a l l c as e s replace QC M s , QC M s a re no t repl acements for C RM s; they a re complementar y

es ti mate a s p ar t o f the to ta l me as u rement u ncer ta i nty) .

Us e s o f QC M s i nclude ( but a re not l i m ite d to) :

me a s u rement pro ce s s over a p erio d o f ti me;

ti me or over an ex tende d p erio d o f ti me when a me as u rement pro ce s s i s known to var y) ;

— i n s tru ment p er forma nce checks;

pro ce s s or lab orator y;

2 © ISO 2014 – All rights reserved

6 Steps in the in-house preparation of quality control materials (QCMs)

Figure 1 — Key steps in the preparation of a typical QCM

NOTE Any o f these steps may be subcontracted to a technically competent subcontractor.

© ISO 2014 – All rights reserved 3

7.1 Matrix type, matching and commutability

7.2 Properties and property values

7.4 Total bulk amount of material

4 © ISO 2014 – All rights reserved

— the quantity o f material that can readily be homogenized,

8 Preparation of quality control materials (QCMs)

8.2 Material processing

© ISO 2014 – All rights reserved 5

with temp eratu re s en s itive prop er tie s or matrice s .

8.2.3 Milling and grinding

therma l ly labi le materia l s .

to remove large p a r ticle s that a re ab ove a pre s crib e d s i ze .

8.2.5 Mixing and blending

pre s er vative s , tex ture s tabi l i z ers , e tc .

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8.3 Sub-division and packaging

8.3.2 Choice of containers

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9.2 Analytical approach

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9.3 Statistical treatment of homogeneity data

Table 1 — Homogeneity study of 10 units

with an overall mean o f 120,02 mg·kg−1 .

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Figure 2 — Data plot

Table 2 — Results of single factor analysis of variance

The between-unit standard deviation is the square root o f this variance:

The repeatability standard deviation can be computed as:

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11.2 Assessing stability

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11.3 Assigning an expiry date to a QCM