Fresenius J Anal Chem (1998) 360 : 359361

Springer-Verlag 1998

CONFERENCE CONTRIBUTION

A. Lamberty H. Schimmel J. Pauwels

The study of the stability of reference materials

by isochronous measurements

Received: 23 May 1997 / Accepted: 20 August 1997

Abstract An alternative method for stability testing of

candidate certified reference materials (CRMs) utilizing
isochronous measurements based on a storage design
(storing samples at different temperatures for different
time periods), which allows all measurements to be done
at the same time, i.e. at the end of the study, is compared
to classical designs of stability studies using reference
time or reference temperature. The execution of stability
studies using isochronous measurements only requires
repeatability conditions, whereas classical designs require
both repeatability and long term reproducibility conditions. Consequently, the new design allows the quality of
results to be improved.

1 Introduction
Stability testing is of the highest importance as Certified
Reference Materials (CRMs) may be sensitive to degradation by temperature, light, oxygen, humidity, microbiological activity etc.. Therefore, in most cases, a short-term
stability study of the possible behavior of the material
during transport as well as a long-term stability study of
the probable behavior of the material under controlled
storage conditions are an absolute necessity. In addition,
continuous stability monitoring during the life-time of the
reference material is required. All studies must be carried
out using highly repeatable and reproducible methods.
Short term degradation studies are carried out to simulate degradation during transport and to decide under
which conditions the material, once it is certified, has to
be dispatched. In addition they enable the decision whether
the material is stable enough to become a reference material. For this purpose storage under extreme conditions
(e.g. 40 C or higher) is compared to storage at lower tem-

A. Lamberty H. Schimmel J. Pauwels

European Commission, Joint Research Centre,
Institute for Reference Materials and Measurements (IRMM)
B-2440 Geel, Belgium

peratures (+18 C, +4 C, 20 C or lower) during relatively

short periods of time (3 to 6 months). If the parameters to
be certified change significantly compared to the expected
certified uncertainty, special arrangements for transport
may be required. However, if the decision to foresee special transport conditions is taken on the basis of insufficient evidence of stability as a consequence of poor repeatability, reproducibility and/or an insufficient number
of replicates, such a decision will influence the cost of the
CRM dramatically.
Long-term stability studies of the actual CRM upon
storage are generally an integrated part of the certification
project, and typically cover a storage period of two years.
Samples stored at various temperatures (e.g. room temperature, +4 C, 20 C, possibly also a lower temperature) are analyzed at regular time intervals (e.g. 0, 0.5, 1,
1.5 and 2 years). However, as CRMs are not yet required
to have an expiry date [1, 2] (due to the high cost of certification projects), these long term stability studies have to
be followed by long-term stability monitoring, which will
last the whole lifetime of the CRM. In order to demonstrate that the material remains stable within the certified
limits of each certified parameter, one needs analysis
methods which are highly reproducible over very long periods of time.

2 Classical stability studies

based on relative measurements
2.1 Multiple temperature studies
The first multiple temperature stability studies of BCR
CRMs were carried out on CRM 349-350, PCBs in cod
liver oil and mackerel oil [3]. In these studies samples
were stored at 18 C (the normal storage temperature of
samples in the fishery laboratory of Aberdeen), +20 C
(room temperature in a laboratory of Northern Europe in
1988) and +37 C (most critical temperature for microbial
growth) and conclusions were mainly obtained after long
and heavy phrasing. However, problems related to long-

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term reproducibility of the methods used could not be excluded in the way the results were presented and an analytical reason had to be given for the long term drift of the
method [4].
2.2 Measurements versus a reference time
A similar approach was used for CRM 187-188, organochlorine pesticides in two milk powders [5], but here the
ratio versus time zero was introduced. The results are expressed as the ratio between the concentration at a time t
and the concentration at time t = 0.
This method can be recommended for highly reproducible methods like IDMS or NAA, but is not satisfactory when method drifts constitute a problem.
2.3 Measurements versus a reference temperature
In the latter case, which is quite common in e.g. organic
determinations, temperature ratios may offer a better possibility. In 1989, Griepink and Maier concluded that, for
e.g. organic and organometallic compounds in matrix materials, problems related to long-term analytical fluctuations can be minimized by dividing results obtained on
samples stored at a given temperature by results obtained
on samples stored at a very low reference temperature [6].
The results are expressed as the ratio between the concentrations of samples stored at a temperature T and a reference temperature RT:
RT = XT/XRT
This procedure cancels out variations in analytical response as a function of time. However, to be able to prove
that samples are also stable at this reference temperature,
it is mandatory that samples stored at at least one higher
temperature are stable as well!
At the same time, also the combined uncertainty UT
was introduced, which takes into account the coefficients
of variation (CV) resulting from the measurements at both
temperatures [6]. This uncertainty is then expressed as:

number of measurements is carried out with a sufficiently

precise measurement method [7].
An attempt to increase the flexibility of this type of
studies to gain more information with less effort was published by U. Faure and P.J. Wagstaffe [8], but did not solve
the basic problem which is the need for highly reproducible measurements.

3 Isochronous measurements
The proposed method can be used when the total duration
of the stability study is known. Consequently it is applicable to the short-term study of possible degradation during transport as well as to the long-term study of storage
conditions. It is based on a storage design of the samples
at different temperatures for different time intervals allowing all measurements to be done at the same time i.e.
at the end of the study. The storage designs for a typical
short and long term study are shown in Figures 1 and 2.
Samples stored at a given temperature (T) are stored
for various times, and either before or afterwards they are
stored at a very low (reference) temperature at which their
stability is supposed to be good. At the beginning (t = 0),
all samples reserved for the stability study are transferred
to a very low storage temperature (20 C or even lower if
deep freeze storage is to be expected for long-term conservation). For each of the storage temperatures studied
(+40 C, +18 C, +4 C, -20 C, ) samples are moved
from this very low reference temperature to the corresponding studied storage temperature at different times
(e.g. t = 0, 6, 12, 18 months for the long-term study and t
= 0, 3, 4, 5 months for the short-term study). At the defined end time the samples are immediately analyzed or
put back (for a short time) at reference temperature before
analysis. The samples that remained at the reference tem-

UT = (CVT2 + CVRT2)1/2 RT/100

Stability was then considered to be guaranteed if RT +/
UT is not significantly different from 1. It should, however, be noted that the uncertainty of the results obtained
after a given time interval t for samples stored at a given
temperature T should be expressed in the form of a 95%
confidence interval rather than as a coefficient of variation (as is correctly done in homogeneity studies), because for stability studies the relevant property is the
mean value describing the state of the material at a given
time t. In fact, to evaluate the real outcome of a stability
study, the measured (relative) concentration should be fitted versus time, and both the slope and the 95% C.I. of the
regression line should be compared to the certified uncertainty range of the CRM. Significant extrapolation to
longer storage periods is then only possible if a sufficient

Fig. 1 Typical storage design for isochronous measurements short

term study

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Fig. 2 Typical storage design for isochronous measurements

long term study

perature for the entire study give the starting value for
t = 0.
All samples are then analyzed under repeatability conditions (i.e. comparable as for a homogeneity study) in as
short as possible a period of time. With such a procedure
the risk of having deviations in the response of the analytical equipment, the analysis method, and/or the calibration are minimized.
This method has several advantages: 1) stability studies can be performed under repeatability conditions similar to those needed to carry out homogeneity studies; this
largely facilitates their execution as long-term reproducibility is no longer required; 2) the evaluation can be
made temperature by temperature, starting with the samples stored at the highest temperature. If instability is detected after a given time t, one may decide not to analyze
anymore the samples stored for much longer times and to
start analyzing samples stored at the next lower temperature. If, on the other hand, stability is demonstrated for the
full period at a given temperature, no further analyses of
samples stored at lower temperatures are required; and 3)
contrary to classical stability testing on the basis of RT
as a function of t, it is not required to analyze samples
stored at two (or more) different temperatures including
the reference temperature.
A possible disadvantage could be that a larger number
of samples is to be measured within a relatively short period of time. This can however be avoided by freezingin the status of those samples which cannot be measured

immediately by storing them (for a short time) at reference temperature until the analysis can be carried out. Another disadvantage may be that the results of the longterm stability study are only available after 2 years, which
may possibly cause delay in the certification of the CRM.
Therefore it is advisable to extend the short-term scheme
from 3 to 6 months so that sufficient pre-information is
available. Moreover, the bulk of the CRM should be
stored at a sufficiently safe temperature until the longterm storage temperature is decided.
At IRMM this method is currently being applied to different recertification projects of sold out BCR reference
materials.
Acknowledgements The authors would like to thank Dr. A.
Boenke and Dr. E. Maier, European Commission, S, M & T Programme, for their critical comments.

References
1. Quality system guidelines for the production of reference materials (1996) ISO Guide 34
2. Guidelines for the production and certification of BCR reference
materials (1997) BCR/01/97
3. Griepink B, Wells DE, Frias Ferreira M (1988) EUR 11 520 EN
4. Maier E (1997) private communication
5. Griepink B, Mulders EJ, Van der Paauw CG, Quirijns JK (1989)
EUR 12 319 EN
6. Griepink B, Maier E, Muntau H, Wells DE (1990) EUR 12 823
EN
7. Pauwels J, Lamberty A, Schimmel H, Fresenius J Anal Chem (in
preparation)
8. Faure U, Wagstaffe PJ (1993) Fresenius J Anal Chem 345 :
124126