Accred Qual Assur (2001) 6 : 20–25

Q Springer-Verlag 2001 GENERAL PAPER

Thomas P. J. Linsinger Homogeneity and stability of reference

Jean Pauwels
Adriaan M. H. van der Veen materials
Heinz Schimmel
Andrée Lamberty

Received: 25 April 2000

Accepted: 12 September 2000
Presented at Analytica Conference 2000,
11–14 April 2000, Munich, Germany
T.P.J. Linsinger 7 J. Pauwels (Y)
H. Schimmel 7 A. Lamberty
European Commission, Joint Research
Centre, Institute for Reference Materials
and Measurements (IRMM), 2440 Geel,
Belgium
e-mail: pauwels6irmm.jrc.be
Tel.: c32–14–571 722
Fax: c32–14–590 406
A.M.H. van der Veen
Nederlands Meetinstituut,
Schoemakerstraat 97, 2600 AR Delft,
The Netherlands
Abstract Homogeneity and stabili-
ty are two crucial characteristics of
any certified reference material
(CRM). Utmost care must be tak-
en during preparation to create
materials as homogeneous and sta-
ble as possible. Degradation can
generally be minimised by reducing
the water activity of the material to
a level between 0.15 and 0.35.
However, careful preparation by it-
self is not enough. Positive demon-
stration of homogeneity and stabil-
ity is required from the perspective
of implementing uncertainty cal-
culus according to the Guide to the
Expression of Uncertainty in Meas-
urement (GUM). In many cases,
homogeneity and stability studies
fail to give sufficient quantitative
information on homogeneity and
stability, mainly because of a lack
of measurement repeatability and
insufficient number of replicates.
In this work, some solutions to
these problems and their implica-
tions are presented.
Keywords Uncertainty 7
Homogeneity 7 Stability 7
Certified reference material

This not only includes the uncertainty of the batch

Introduction
In the past, certification of reference materials was
largely understood as a process of assigning a value to a
material. Little emphasis was given to characteristics
like homogeneity or stability and very few attempts
have been made to include possible effects of inhomo-
geneity and degradation in certified reference material
(CRM) uncertainty, even if this was suggested [1]. With
increasing emphasis on uncertainty estimations for ana-
lytical measurements, there is also a clear need for un-
certainty estimations for CRMs. As the number of
CRMs with limited homogeneity and/or stability also
increases, there is also from the viewpoint of the CRM
producer an additional reason to establish a complete
uncertainty statement, including the contributions from
homogeneity and stability. From a perspective of the
Guide to the Expression of Uncertainty in Measurement
(GUM) [2], the uncertainty of a CRM should take into
account all uncertainty sources relevant to the user.
characterisation (uchar), but also all uncertainties re-
lated to possible between-bottle variation (ubb), insta-
bility upon long-term storage (ults) and instability dur-
ing transport to the customer (usts). The combined
standard uncertainty for a CRM can thus be expressed
as shown in Eq. (1) [3].
uCRM p [u 2charcu 2bbcu 2ltscu 2sts] 1/2 (1)
Existing ISO Guides like ISO Guide 31 [4] and ISO
Guide 34 [5] explicitly require inclusion of uncertainties
connected to homogeneity and stability in the com-
bined standard uncertainty. They urge certifying bodies
to state shelf-lives for their CRMs, in order to inform
the user about the expected lifetime of the CRM. Prac-
tical models exist for estimation of the uncertainty of
the batch characterisation, based on the analytical re-
sults submitted [1, 6] or an approach based on the full
uncertainty budgets submitted by the participants [7].
Some approaches have also been suggested for the

evaluation of homogeneity studies [8, 9] that give useful
results for studies in which the observed inhomogeneity
is at least of the same order of magnitude as the repea-
tability of measurement. As for the estimation of shelf-
life, Pauwels et al. [10] concluded that approaches used
in industry are not suitable for the estimation of shelf-
life of CRMs because measurement variation is usually
large compared to the effect (degradation) to be deter-
mined.
Preparation of materials
Preparation should be done in a way to ensure minim-
isation of heterogeneity and instability, in conjunction
with improvement of the mechanical properties of the
material. Care should be taken to avoid contamination,
which could introduce inhomogeneities to an otherwise
homogeneous material. The material should then be
transformed into an optimal physical and chemical
form. To keep the material in this form, it should be
sampled in a tight and inert container. Experience at
the Institute for Reference Materials and Measure-
ments (IRMM) shows that very few containers stay air-
tight over longer periods of time, especially at very low
temperatures. To minimise degradation the water con-
tent of the material is frequently reduced to a very low
level. However, water activity, not total water content
is the relevant parameter, especially for microbiological
degradation. Water activity is defined as the relative
humidity with which a material is in equilibrium. A de-
piction of various degradation rates versus water activi-
ty is shown in Fig. 1.
Fig. 1 Schematic representation of different possibilities of dete-
rioration with respect to the equilibrium relative humidity at
Tpconst., whereby the axis “water content” is only with respect
to the sorption isotherm; Figure taken from [13]
21
Obviously, microbiological degradation will not oc-
cur at water activities below 60%. Degradation due to
enzymatic activity and non-enzymatic browning are
very low at water activities below 30%. The autoxida-
tion rate is lowest for water activities between 15 and
40%. This means that degradation actually increases if
the material is dried too much. Apart from autoxida-
tion, other effects like condensation reactions are possi-
ble at too low water activities. Taking these considera-
tions into account , water activities between 0.15 and
0.30 appear to ensure the minimum possible degrada-
tion rate. Adjusting the material to a higher water ac-
tivity than technically possible has another advantage:
the material is not so hygroscopic, thus facilitating han-
dling of the material. Care should be taken to store the
material at the correct temperature from a very early
point in the production process. Doing so minimises the
probability of starting degradation processes.
Homogeneity testing
In homogeneity testing, there are two types of homo-
geneity of importance. First, there is the within-bottle
homogeneity, which dictates the minimum sample in-
take, for which the established uncertainty is still valid.
Secondly, there is the between-bottle homogeneity,
which deals with the bottle-to-bottle (or, more general,
unit-to-unit) variation. The common element in both
types of experiments is that a separation between the
effect (heterogeneity) and the variability of measure-
ment should be established. In order to do so, it is most
advantageous to carry out a within-bottle homogeneity
study with a very small amount of sample, so that the
between-portions effect can be quantified. After com-
pleting the test, the minimum sample intake can then
be calculated.
For a between-bottle homogeneity test, it is most ad-
vantageous to take the optimal sample intake for the
chosen method to minimise analytical variation. It
stands to reason that this intake should be larger than
the minimum sample intake described above. When be-
tween-unit variation should be quantified, the two
effects can be separated by analysis of variance
(ANOVA) [9] or an ANOVA-like approach [8] as
shown in Eq. (2) [9].
u 2c,bb p s 2bbcs 2meas (2)
In Eq. (2), uc,bb is the combined uncertainty of the
between-unit experiment, expressed as the uncertainty
on a single unit; sbb is the variation between units and
smeas is the variation of the measurement. smeas is the
intrinsic variability of the method (smethod) divided by
the square root of n, the number of replicates per unit.
sbb is the estimate for the variation in the material.
22
Homogeneity contribution to CRM uncertainty
The concepts for the separation of between-unit varia-
bility from an analytical variability require that both
are at least of the same order of magnitude. This is in
principle always feasible, as measurement variability
can be decreased to any level desired by performing
several replicates on an individual unit (increasing n).
However, sometimes this is not possible due to limited
resources (amount of material, time, labour, etc.). If sbb
as obtained from an ANOVA-approach is found to be
(close to) zero, two conclusions are possible: ubb
(Eq. .1) can be assumed to be negligible (mathematical-
ly: 0) if the material is known to be homogeneous, thus
accepting the value of sbb. This might be the case for
solutions, and for matrix CRMs from which experience
has shown that they indeed can be prepared in such a
way that heterogeneity can effectively be reduced to
(approximately) zero.
Otherwise, a more conservative estimate for ubb may
be considered. This has been proposed by Pauwels et
al. [8] to be the uncertainty of sbb (named ubetw in [8])
and an approximation for u(sbb) was given. Although
this approximation yields reasonable results, it is not
consistent with the definition of sbb and is likely to be
too optimistic.
There are however other alternatives. The simplest
option is to adopt uc,bb from Eq. (2) as estimator for ubb
in Eq. (1). This option is very straightforward, and it is
easily seen that it fully complies with the definition of a
(combined) standard uncertainty. The greatest draw-
back is that this estimator does not take into account
the fact, that a substantial part of uc,bb will come from
the measurement, rather than from heterogeneity.
The problem of obtaining variances from ANOVA
close to zero, or even negative, has been discussed in
literature. This problem is closely associated to the
problem outlined already: the variance is obtained as a
difference of two mean squares, and so there is a de-
pendence of sbb on smeas. One possible way to appre-
ciate this is to use an expression from Federer [11]:
" MS PMSwithin MSwithin P MS among
among
ubb p c e MSwithin (3)
n n
where MSamong represents the mean squares ‘among
groups’ and MSwithin represents mean squares within
groups. For cases where MSamong 1 MSwithin, within in-
creasing MSamong, Federer’s estimator for ubb tends to
the value obtained from ANOVA. For small values of
MSamong, a “correction” takes place, primarily to avoid
negative estimators for the among-group variance, in
the present case, sbb 2.
Here, a new estimate of the impact of analytical var-
iability on sbb, which can be used as estimator for ubb
will be given. In the cases considered here, this estima-
tor for ubb avoids the problems associated with using
sbb from ANOVA directly as estimator.
Assuming a homogeneity study with a fully nested
design, in which k units of the batch are analysed in n
replicates each. The experiment is evaluated through
ANOVA as described by Van der Veen et al. [10] and
sbb can be estimated as:
sbb p " MS among PMSwithin
n
(4)
Now, the impact of MSwithin on sbb must be deter-
mined. The uncertainty of MSwithin can be expressed
as:
u (MSwithin) p " 2 MS 2within
nMSwithin
, (5)
with nMSwithin being the degrees of freedom of MSwithin,
as the variance of a variance is equal to 2s 4/n [14]. Us-
ing standard uncertainties, the maximum value MSamong
is therefore MSwithincu(MSwithin) in case where
MSamongpMSwithin. This leads to a “corrected”
MSamong
MS*amongpMSwithinc " 2 MS 2within
nMSwithin
(6)
Inserting the result from Eq. (6) in Eq. (4) gives:
4
u*bbp " MSwithin
n "n 2
MS within
(7)
Equation (7) can therefore be used to estimate max-
imum between-bottle variability that could be masked
by method variation. Unless additional knowledge
about CRM homogeneity is available or the certifica-
tion was organised in a way that the influence of meas-
urement variability was included in other components
of CRM uncertainty, Eq. (7) can be used as an estimate
for the impact of analytical variability on sbb as ob-
tained from ANOVA, which can be used as the lower
limit for ubb.
Frequently, a different set-up for homogeneity stud-
ies is used (BCR-type): N units are analysed once (esti-
mation of uc,bb) and one unit is analysed in n-replicates
(estimation of smethod). Using the method shown above,
the following result is obtained:
4
u*
bbpsmeas "n 2 smeas
(8)
smeasis in this case equal to the smethod, as only one repli-
cate per bottle was performed. nsmeas is n-1, the degrees
of freedom for the determination of this standard de-
viation. As ;(MSwithin/n) is equal to smeas, the two re-
sults are obviously the same.
 23

Consequently, Eqs. (7) and (8) can be used as mini-

mum uncertainty contributions from the homogeneity
study to the combined standard uncertainty of the ref-
erence material. This does not apply for cases in which
additional knowledge proves homogeneity or the in-
fluence of measurement variability was taken into con-
sideration elsewhere, or additional evidence can be
provided that inhomogeneity in the system could have
been removed effectively. It may be argued that the in-
*
fluence of measurement variability (u bb ) should always
be added to sbb rather than following the approach of
Eqs. (7) and (8). For nested designs, ubb would then be
estimated as Eq. (9):

"
4

ubb p s 2bb c
MSwithin
n
7 "n 2
MS within
(9)

As can be seen in Figs. 2 and 3, differences are negli-
gible apart from cases where sbb;smeas. Taking into
consideration that Eqs. (7) and (8) are conservative es-
timations anyway, it does not seem necessary to do so,
although certification bodies can of course opt for this
slightly more conservative option, which has the advan-
tage to be continuous in contrast to the change from sbb
to u*b b depending on what is larger. The most important
argument for using Eq. (9) is found in the fact that it is
easier to implement in software environments as
Eq. (9) is still valid for sbb 1 0, whereas the approxima-
tions given by Eqs. (7) and (8) will loose their validity
with increasing sbb.
As can be seen in Fig. 3, u*b b can be decreased in
BCR-type homogeneity studies by performing more
Fig. 3 Estimated sbb vs. true sbb for BCR-type homogeneity stud-
ies. Standard deviation of the method (smethod)p1; The dotted line
shows estimated sbb for nmethodp5 if the contribution of method
repeatability is always added to sbb. Dashed lines show lower
boundaries for the estimated sbb for nmethodp9 and 19
measurements for the determination of smethod. Fully
nested ANOVA gives generally smaller uncertainty es-
timates: assume a case where Np10 bottles are ana-
lysed once and one bottle is analysed in 10 replicates
(nsmeasp9). With smethodp1, u*b b is 0.69. However, if 20
measurements are performed in a way that 10 bottles
are analysed in 2 replicates each, minimum estimate for
sbbp 0.47. For the same number of measurements, per-
forming the study in a way that can be evaluated by
ANOVA gives a lower minimum uncertainty, because
of the decreased smeas (smethod/;n). Still, the approxi-
mation presented here for estimating ubb by u*b b should
not be used as a replacement for proper homogeneity
studies. Figure 3 clearly shows that investments in ho-
mogeneity studies pay off in terms of better reliability
of the results, and smaller uncertainties, even in cases
where despite all efforts a proper quantification of the
between-bottle homogeneity variance is not possible.
In those cases, the more conservative approach as pro-
posed is a better alternative than to forget altogether
about certification due to problems in the homogeneity
study.

Stability testing

Two types of stability tests should be performed: one

Fig. 2 Estimated sbb vs. true sbb for analysis of variance
(ANOVA)-type homogeneity studies. Standard deviation of the
methodpMSWp1. The dotted line shows estimated sbb for np2
if the contribution of method repeatability is always added to sbb
(Eq. 9) Dashed lines show lower boundaries for the estimated sbb
for np3 and np4
study at elevated temperature to elucidate whether any
degradation can be expected during transport. This
study will usually be of short duration, typically not
longer than 4 weeks. Based on the results of this study,
transport conditions must be chosen that ensure that
the contribution of short-term stability is negligible. In
24
these cases, usts can be eliminated from Eq. (1), al-
though the same case should be taken as in the case of
the homogeneity study. The approaches as presented
for the homogeneity study are equally valid to stability
studies, but also the “clause” that the context in which
the measurements take place as well as the true trans-
port conditions should govern the choice made.
A second study must be performed at storage tem-
perature to obtain information about the stability dur-
ing storage. Simulation of long-term storage by harsh
conditions is usually not appropriate, as the degrada-
tion mechanism might change. The attempt to estimate
stability data by extrapolating data from higher temper-
atures via the Arrhenius-equation is therefore not rec-
ommended, as usually the processes underlying stabili-
ty problems are too complex to be modelled. Usually
stability should therefore be evaluated at storage tem-
perature.
Usually, a stability study consists of a series of meas-
urements performed at different times. If the temporal
trend is significant, uncertainty of stability is evaluated
by regression analysis. For insignificant trends, the
measurements can be evaluated through ANOVA [15]
or through regression analysis. However, in many cases
these evaluations will not yield quantitative results, es-
pecially if the material was prepared in a way to ensure
optimal stability as described above. The frequency of
this situation can be avoided by choosing highly repeat-
able methods, higher number of replicates or isochron-
ous studies [12]. Still, for several studies the problems
of how to estimate the uncertainty contribution of sta-
bility if no degradation can be shown will remain. One
possible solution is described in detail by Linsinger et
al. [16]: Following the same line of reasoning as for the
homogeneity study, the influence of the measurement
variability on the estimate of the degradation rate is
calculated. This is in this case the standard deviation of
the slope of the regression line. Using this standard de-
Fig. 4 Results of a simulated stability study. The data shown are,
the assumed regression line with a slope of 0 and the ults-“trian-
gle”. The latter was obtained by multiplication of B the standard
deviation of the slope with the time. Depending on the chosen
shelf-life, ults decreases or increases
viation as conservative estimate of the degradation
rate, an uncertainty “triangle” such as shown in Fig. 4
is calculated.
A shelf-life is chosen and ults is directly obtained
from this uncertainty triangle. In the absence of real de-
gradation, the size of ults depends on between-bottle
and analytical variation, because both might mimic de-
gradation. Between-bottle variation can be minimised
by careful preparation. Analytical variation itself can
be reduced by using either a method with less variation
or by the isochronous measurement scheme [12], in
which repeatability rather than reproducibility condi-
tions are applied. The influence of analytical variation
can be reduced further by performing higher numbers
of replicates per time and by increasing the length of
the stability study.
Conclusion
Certification of a material consists of four compo-
nents:
1) Careful preparation and bottling of the material
2) Homogeneity testing using an design optimised to
give positive proof of homogeneity
3) Stability testing using a design optimised to give po-
sitive proof of stability
4) Value assignment to the batch.
These four components are equally important and
putting less emphasis on any one of them will result in a
significant decrease of the quality of the material.
When evaluating CRM uncertainty, uncertainties de-
rived from stability and homogeneity must be included,
and sometimes even the analytical variation in these ex-
Fig. 5 Contribution of individual uncertainty components to cer-
tified reference materials (CRM) uncertainty for total glucosino-
late in CRM BCR 190R (TG/CRM 190R), arsenic in CRM
BCR278R (As/CRM 278R) and manganese in CRM BCR 185R
(Mn/CRM278R). Shown are individual uncertainty components
and combined uncertainty
 25

periments should be appreciated when estimating these
uncertainty components. These contributions are fre-
quently more important than uncertainty from the
characterisation of the batch, as can be seen typical ex-
amples in Fig. 5.
For homogeneity testing and stability testing, highly
repeatable methods should be chosen. If these are not
available, repeatability can be improved by increasing
the number of measurements. The work clearly demon-
strates the direct relationship between the combined
standard uncertainty of the candidate CRM and the in-
vestments done in the homogeneity and stability stud-
ies. The work also shows that if – despite all efforts –
the analytical variance plays a dominant role in this
stage of the certification project, there are still options
available, but at a higher ‘price’ (puncertainty).

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