ISO/TS 5667-25:2022
(Main)Water quality — Sampling — Part 25: Guideline on the validation of the storage time of water samples
Water quality — Sampling — Part 25: Guideline on the validation of the storage time of water samples
The purpose of this document is to describe test plans and different operating methodologies of these test plans to define and verify the acceptable length of stability of a substance in a sample under specified conditions of preservation (temperature, matrix, light, addition of a stabilizer, where appropriate, type of preservation etc.) before starting analytical protocols (chemicals and physico-chemicals analysis). Biological and microbiological methods are excluded. It is necessary to have an analytical method with performances that have already been characterized (repeatability, intermediate precision, trueness, accuracy and uncertainty) in order to perform the stability study and implement its test plans.
Qualité de l'eau — Échantillonnage — Partie 25: Lignes directrices pour la validation de la durée de conservation des échantillons d'eau
Kakovost vode - Vzorčenje - 25. del: Smernice za validacijo roka uporabnosti vzorcev vode
Namen tega dokumenta je opisati testne načrte in različne metodologije delovanja teh testnih načrtov za opredelitev in preverjanje sprejemljivega trajanja stabilnosti snovi v vzorcu pod določenimi pogoji shranjevanja (temperatura, matrica, svetloba, dodatek stabilizatorja, kjer je to primerno, vrsta shranjevanja itd.) pred začetkom analitskih protokolov (kemijske in fizikalno-kemijske analize). Biološke in mikrobiološke metode so izključene.
Za izvedbo študije stabilnosti in izvajanje načrtov testiranja je treba imeti analitsko metodo z že opredeljenimi testi zmogljivosti (ponovljivost, vmesna natančnost, pravilnost, točnost in negotovost).
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-april-2023
Kakovost vode - Vzorčenje - 25. del: Smernice za validacijo roka uporabnosti
vzorcev vode
Water quality - Sampling - Part 25: Guideline on the validation of the storage time of
water samples
Qualité de l'eau - Échantillonnage - Partie 25: Lignes directrices pour la validation de la
durée de conservation des échantillons d'eau
Ta slovenski standard je istoveten z: ISO/TS 5667-25:2022
ICS:
13.060.45 Preiskava vode na splošno Examination of water in
general
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
TECHNICAL ISO/TS
SPECIFICATION 5667-25
First edition
2022-02
Water quality — Sampling —
Part 25:
Guideline on the validation of the
storage time of water samples
Qualité de l'eau — Échantillonnage —
Partie 25: Lignes directrices pour la validation de la durée de
conservation des échantillons d'eau
Reference number
© ISO 2022
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and symbols . 1
4 Principle . 5
5 Definition of the scope of the stability study . 6
5.1 Aim of the stability study . 6
5.2 Selection of the maximum acceptable delay before analysis and the acceptance
criteria . 6
5.3 Influence parameters of the maximum acceptable delay before analysis . 7
5.4 Duration of the study . 7
5.5 Concentration levels . 8
5.6 Definition of the matrices and selection of the representative samples . 8
5.7 Guidelines to the stability studies of transformation products . 9
6 Definition of the experimental plan .11
6.1 Determination of the value of the quantity of the material at T and at other times
during the study . 11
6.2 Test materials .12
6.2.1 Preparation .12
6.2.2 Characterization of the test materials . 13
6.2.3 MQD Evaluation . 14
6.3 Definition of the test plan . 14
6.3.1 Choice of the type of study . 14
6.3.2 Randomization . 14
7 Performance of the tests .15
7.1 General . 15
7.2 Chronological stability study . 15
7.3 Type 1 pseudo-isochronous stability study . 15
7.4 Type 2 pseudo-isochronous stability study . 16
7.5 Isochronous stability study . 17
8 Graphic representation of the data .17
9 Validation of the data .20
9.1 Initial validation of the study at T . 20
9.1.1 Calculation of the value assigned to T (VA ) . 20
0 T0
9.1.2 Verification of the accuracy . 21
9.2 Validation at times different from T . 22
10 Using the results . .22
10.1 Graphic view of all the data .22
10.2 Determination of the maximum acceptable delay before analysis by condition .23
10.3 Conclusion of the study . 33
11 Expression of stability .34
Annex A (informative) Measurement process .35
Annex B (informative) Fundamental notions.36
Annex C (informative) Study of stability and influence parameters .42
Annex D (informative) Example of study of atrazine stability in water . 44
Bibliography .50
iii
Foreword
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bodies (ISO member bodies). The work of preparing International Standards is normally carried out
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This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 6,
Sampling (general methods).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
This document addresses the need for harmonized and reliable data on stability, which is essential
for the expression of recommendations for both normative and regulatory purposes. It describes
a methodological framework that enables laboratories to produce quality data that can be shared
[7]
and even monetized .
It enables laboratories to study the stability of parameters when using the physico-chemical parameters
measurement system: organic micropollutants, inorganic and organometallic micropollutants, nutrients
and macropollutants in aqueous matrices (surface water, ground water, residual urban and industrial
water and drinking water). It covers the sampling, transport and laboratory storage operations.
NOTE This document does not cover solid matrices from aquatic environments (suspended solids,
sediments).
v
TECHNICAL SPECIFICATION ISO/TS 5667-25:2022(E)
Water quality — Sampling —
Part 25:
Guideline on the validation of the storage time of water
samples
1 Scope
The purpose of this document is to describe test plans and different operating methodologies
of these test plans to define and verify the acceptable length of stability of a substance in a sample
under specified conditions of preservation (temperature, matrix, light, addition of a stabilizer, where
appropriate, type of preservation etc.) before starting analytical protocols (chemicals and physico-
chemicals analysis). Biological and microbiological methods are excluded.
It is necessary to have an analytical method with performances that have already been characterized
(repeatability, intermediate precision, trueness, accuracy and uncertainty) in order to perform
the stability study and implement its test plans.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any amendments)
applies.
ISO/TS 21231, Water quality — Characterization of analytical methods — Guidelines for the selection of a
representative matrix
3 Terms, definitions and symbols
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
analytical process
detailed description of a measurement according to one or more measurement principles and to one
given measurement method, and including any calculations intended to obtain a measurement result
3.2
batch
definite amount of test material prepared by the laboratory at a given point in
time under supposedly identical conditions
3.3
chronological stability study
study in which individual samples prepared at the same time (i.e., as a batch), under identical conditions,
are measured as time elapses (e.g. one sample immediately, one after three months, the next one after
six months, etc.)
[SOURCE: ISO Guide 35:2017, 8.3.2.1]
3.4
homogeneity
uniformity of a specified property value throughout a defined portion of a reference material (RM)
[2]
Note 1 to entry: Tests for homogeneity are described in ISO Guide 35 .
Note 2 to entry: The “defined portion” may be, for example, an RM batch or a single unit within the batch.
[6]
Note 3 to entry: See also IUPAC Compendium on Analytical Nomenclature .
[SOURCE: ISO Guide 30:2015, 2.1.12]
3.5
influence parameter
intrinsic characteristic of the matrix, independent of the analyte concentration, a variation of which is
liable to modify the analytical result
[SOURCE: ISO/TS 21231:2019, 3.3.1]
3.6
influence parameter of the conditions of preservation
characteristic related to the conditions of storage and preservation of the sample, independent of the
analyte concentration
Note 1 to entry: E.g. container material, storage temperature, influence of light and/or relative humidity.
3.7
integrity
property that the parameter(s) of interest, information or content of the sample container has not been
altered or lost in an unauthorized manner or subject to loss of representativeness
[SOURCE: ISO 5667-3:2018, 3.1]
3.8
intermediate precision condition of measurement
condition of measurement, out of a set of conditions that includes the same measurement procedure,
same location, and replicate measurements on the same or similar objects over an extended period
of time, but may include other conditions involving changes
Note 1 to entry: The changes can include new calibrations, calibrators, operators, and measuring systems.
Note 2 to entry: A specification for the conditions should contain the conditions changed and unchanged,
to the extent practical.
Note 3 to entry: In chemistry, the term “inter-serial precision condition of measurement” is sometimes used
[8]
to designate this concept .
3.9
isochronous stability study
experimental study of “reference” material stability in which units exposed to different storage
conditions and times are measured in a short period of time
[SOURCE: ISO Guide 35:2017, 3.9]
3.10
matrix
all the constituents of the laboratory sample other than the analyte
Note 1 to entry: By extension, a matrix is defined by the analyst as waters characterized by a homogeneous
behaviour with regard to the analytical method used.
[SOURCE: ISO/TS 21231:2019, 3.3.3]
3.11
maximum acceptable delay before analysis
MaxADs
maximum acceptable delay between the end of the sampling process and the start of the analysis
operations, resulting from the stability study, that the laboratory uses to plan the analyses
3.12
maximum acceptable deviation for the stability study
MADs
maximum acceptable deviation relative to the assigned value of the sample at T , used to determine
the maximum acceptable delay before analysis
3.13
measurement precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as
standard deviation, variance, or coefficient of variation under the specified conditions of measurement.
Note 2 to entry: The "specified conditions" may be, for example, repeatability conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement
[1]
(see ISO 5725-1:1994 ).
Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
[8]
Note 4 to entry: Sometimes “measurement precision” is erroneously used to mean measurement accuracy .
3.14
measurement repeatability
measurement precision under a set of repeatability conditions of measurement
[SOURCE: JCGM 200:2012 (VIM), 2.21]
3.15
measurement reproducibility
measurement precision under reproducibility conditions of measurement
Note 1 to entry: I.e., condition of measurement, out of a set of conditions that includes different locations,
operators, measuring systems, and replicate measurements on the same or similar objects.
[SOURCE: JCGM 200:2012 (VIM), 2.25]
3.16
measurement trueness
closeness of agreement between the average of an infinite number of replicates measured quantity
values and a reference quantity value
Note 1 to entry: Measurement trueness is not a quantity and thus cannot be expressed numerically, but measures
[3]
for closeness of agreement are given in ISO 5725-1:1994 .
Note 2 to entry: Measurement trueness is inversely related to systematic measurement error, but is not related to
random measurement error.
Note 3 to entry: “Measurement accuracy” should not be used for ‘measurement trueness’.
[SOURCE: JCGM 200:2012 (VIM), 2.14]
3.17
minimum quantifiable deviation for the stability study (MQDs)
minimum deviation relative to the assigned value of the parameter in the sample at T , which can be
unequivocally imputed to the instability
Note 1 to entry: This deviation takes account of the inhomogeneity of the test material and the intrinsic variability
of all the measurement results over time used to determine the maximum acceptable deviations.
Note 2 to entry: The calculation of the minimum quantifiable deviation for the stability study depends on the
type of study (chronological, pseudo-isochronous or isochronous) (6.1.1).
3.18
pseudo-isochronous stability study
stability study in which some of the steps, in particular the preparation, are performed under
intermediate precision conditions, and in which the results of instrumental analyses are acquired
under repeatability conditions
3.19
repeatability condition
condition of measurement, out of a set of conditions that includes the same measurement procedure,
same operators, same measuring system, same operating conditions and same location, and replicate
measurements on the same or similar objects over a short period of time
Note 1 to entry: A condition of measurement is a repeatability condition only with respect to a specified set
of repeatability conditions.
Note 2 to entry: In chemistry, the term “intra-serial precision condition of measurement” is sometimes used
[8]
to designate this concept .
3.20
representative matrix
sample for which all the intrinsic characteristics are characteristics of a type of water or the source of
a group of samples
[SOURCE: ISO/TS 21231:2019, 3.3.2]
3.21
reproducibility condition
condition of measurement, out of a set of conditions that includes different locations, operators,
measuring systems, and replicate measurements on the same or similar objects
Note 1 to entry: The different measuring systems may use different measurement procedures.
[8]
Note 2 to entry: A specification should contain the conditions changed and unchanged, to the extent practical .
3.22
sample preservation
any procedure used to stabilize a sample in such a way that the properties under examination are
maintained stable from the collection step until preparation for analysis
[SOURCE: ISO 5667-3:2018, 3.2]
3.23
sample storage
process and the result of keeping a sample available under predefined conditions, usually for a specified
time interval between collection and further treatment of a sample
Note 1 to entry: Specified time is the maximum time interval [see ISO 5667-3].
3.24
stability
characteristic of an analyte in an aqueous matrix, when stored under specified conditions, to maintain
its property value within specified limits for a specified period from sampling to laboratory operations
3.25
stability interval
interval defined on the basis of the assigned value at T and the maximum acceptable deviation for the
stability study
3.26
storage time
period of time between filling of the sample container and further treatment of the sample in the
laboratory, if stored under predefined conditions
Note 1 to entry: Sampling finishes as soon as the sample container has been filled with the sample. Storage time
ends when the sample is taken by the analyst to start sample preparation prior to analysis.
Note 2 to entry: Further treatment is, for most analytes, a solvent extraction or acid destruction. The initial steps
of sample preparation can be steps complementary to the storage conditions for the maintenance of analyte
concentrations.
[SOURCE: ISO 5667-3:2018, 3.4]
4 Principle
The goal is to perform a series of tests, hereafter referred to as the “stability study”, to analyse
the variations in the value of a given parameter, between an initial time and a maximum time, on
samples representative of the scope of application of the measurement method of the parameter. The
conclusions of these tests are used to determine the maximum acceptable delay before analysis (3.11)
under the conditions of the study.
The stability study has six stages:
— Definition of the requirements (analytes, matrices, levels of concentration, storage conditions,
length of storage, maximum acceptable deviation for the stability study (3.12)), see Clause 5;
— Definition of the experiments plan (type of study, number of time intervals and total length
of the study), see Clause 6;
— Performance of the tests, see Clause 7;
— Validation of the data, see Clause 9;
— Using the results (based on a maximum acceptable deviation, 3.12), see Clause 10;
— Expression of the stability in the form of a maximum acceptable delay before analysis (3.11)
and the duration of stability corresponding to the conditions and the criteria (maximum acceptable
deviation for the stability study (3.12)) of the study, see Clause 10.
Since the stability study covers different stages of the data acquisition process according
to the organization of the measurement system, examples of the organization of the measurement
system are given in Annex A for reference.
The laboratory shall take the following factors into consideration:
— The method of determination used: for example, limit of quantification, repeatability, intermediate
precision, accuracy, influence parameters of the matrix (3.10) on the performance of the method;
— The “sample” material of the stability study: for example, homogeneity, physico-chemical properties;
— Suitability of the sample material for use as test material in accordance with storage time (3.26).
— Definition of the influence parameters of the preservation conditions (3.6) assessed as part
of the stability study: for example, time, temperature, addition of stabilizers;
— If studied, command of the storage and transportation conditions;
— Clearly defined acceptability criteria (maximum acceptable deviation, 3.12), with which the results
of the study will be compared.
5 Definition of the scope of the stability study
5.1 Aim of the stability study
Based on the scope of application of the method, the experimental plan shall clearly define the aim of the
stability study by specifying the measured analytes, the matrices (3.10) and the target concentration
levels.
5.2 Selection of the maximum acceptable delay before analysis and the acceptance
criteria
Respecting the maximum acceptable delay before analysis (3.11) may determine the quality of the
results of the analysis more than certain performance data of the measurement methods (bias). This is
the reason why the maximum acceptable delay before analysis shall be established and known before
the routine application of a laboratory analysis method.
The assessment of the results of a stability study with the intension of drawing a conclusion on the
stability, expressed as a maximum acceptable delay before analysis, of a given analyte in a representative
matrix of the scope of application shall be based on the interpretation of the results in perspective of
the requirements of the stability study. A maximum acceptable deviation (3.12) shall be set in order to
come to a conclusion. This maximum acceptable deviation shall be chosen before the start of the study,
because it determines the conditions of performance of the method and the admissibility of the data, in
particular with regard to the measurement method.
There are five ways to determine the maximum acceptable deviation. They are, in order of relevance:
a) The application of a regulatory requirement, if one exists;
b) Twice the repeatability standard deviation for isochronous or pseudo-isochronous type 1 studies
(Annex B), or twice the intermediate precision standard deviation for type 2 pseudo-isochronous
studies (Annex B) or chronological studies; the values of the standard deviation of repeatability
and intermediate precision being the values defined during the characterization of the method.
c) Use of the data from the stability study: dispersion at T of the stability study. See Formula (1):
Maximum acceptable deviation = Minimum quantifiable deviatiion (1)
d) The choice of an arbitrary value, determined according to the technical operational implementation
constraints (e.g., the best available method offering a precision of 15 %) or a value from a ring
trial (ISO 5667-3). In this case, the maximum acceptable deviation shall be greater than this value,
e.g., 25 %.
e) A value derived from the temporal operational implementation constraints (e.g., the impossibility
of performing an analysis before a given time due to the minimum transportation time). In this
case, the maximum acceptable delay before analysis is fixed and the maximum acceptable deviation
is based on the observations at the pre-determined time.
EXAMPLE If the temporal constraint is three days, (MaxADs =3 days) the maximum acceptable
deviation is estimated according to the observations (dispersion, for example) at T .
If the minimum quantifiable deviation is greater than the maximum acceptable deviation, it is
impossible to draw any conclusions about the stability. In this case, the laboratory should identify the
causes (e.g., lack of homogeneity of the materials, lack of performances of the method, need to stabilize
the materials) and repeat the study (e.g., with a different maximum acceptable deviation, another
method, etc.).
5.3 Influence parameters of the maximum acceptable delay before analysis
This protocol applies to conditions clearly defined by the laboratory, which defines the maximum
acceptable delay before analysis, the corresponding assessment criterion (see 5.2) and the factors likely
to impact it.
In particular, these factors may include:
— The container: ISO 5667-3 or the analysis standards applicable to the targeted parameters specify
the recommended containers for the transportation of samples based on current knowledge.
However, for certain classes of molecules, for which no normative guidelines exist, the compatibility
of these general recommendations on the container material (plastic, glass, etc.) that may cause
losses by adsorption, diffusion, the transfer of additives used in production, etc., should be verified;
— The storage temperature (ambient, refrigeration, freezing, etc.), which can impact the partition
of the substances between the dissolved and particulate phases, or the enantiomer form, or their
biological deterioration. ISO 5667-3 recommends that samples be preserved at 5 ± 3 °C during
transportation. Therefore, this temperature range is the reference temperature of the study,
in the absence of any other requirements. Other temperatures may be chosen for the study, for
example when demonstrating the stability of analytical extracts in the laboratory. The temperature
conditions of the study shall be clearly indicated, and they shall be monitored and documented
throughout the stability study;
— The influence parameters of the matrix: pH, suspended solids, etc;
— Light, which can cause the photodegradation of certain organic molecules, e.g.: Benzo[a]pyrene,
BDE209;
[15]
— The influence of sample pre-treatment on site, e.g. filtration ;
— The addition to the sample of stabilizing chemical agents (e.g., acid for metals, sodium hydroxide
for cyanides, solvents, etc.).
5.4 Duration of the study
The duration of the study shall cover the initially planned maximum acceptable delay before analysis.
The extrapolation of the MaxADs beyond T is not permitted. It is thus recommended to collect data
max
beyond the planned MaxADs,
Interpolation between the two ends of a time interval is not permitted in order to define the maximum
acceptable delay before analysis. Therefore, it is recommended to acquire additional information at
different intermediate intervals of time.
The laboratory shall adapt the number of the intervals of time (Figure 1) of the study according to its
initial knowledge of the stability of the analyte of concerned in order to minimize the risk of inconclusive
studies.
Key
1 time interval 1
2 time interval 2
3 time interval 3
4 time interval 4
Figure 1 — Example of a diagrammatic illustration of the notion of time laps
5.5 Concentration levels
The levels of concentration to be tested (p) are chosen according to the requirements of the
corresponding sector of activity, any regulatory requirements that may exist, the occurrence of data
in the environment, for example, and the performances of the analytical method and its scope of
application.
The experimental plan selects one of the two following approaches, depending on the complexity of the
matrices and their knowledge of the method:
— At least two levels of concentration (p≥ 2) by representative sample (n≥ 2) of the matrix of the scope
of application of the method shall be considered:
— One low concentration level, different from the LOQ, ≤ 25 % of the scope of the method for one-
decade methods or ≤ 10 % for more-decades methods,
— One high concentration level, in the second half of the scope of application of the method.
— A minimum concentration level of p =1, ≤ 25 % of scope of the method for one-decade methods,
resp. ≤ 10 % for more-decades methods, with several representative samples of the matrix (p x n
≥ 4). In this case two time laps should be taken.
The laboratory shall substantiate its choice of n=2 by demonstrating that its scope of application
is restricted and, therefore, sufficiently described by two representative samples, in accordance with
ISO/TS 21231.
NOTE 1 Performing stability studies at the limit of quantification (LOQ) is not generally relevant.
The uncertainty of the methods at the LQ does not allow for the unequivocal interpretation of the data.
NOTE 2 When representative samples free of any background contamination of analyte cannot be found,
the environmental background noise should be considered to determine the lowest level of contamination tested
in the stability study.
5.6 Definition of the matrices and selection of the representative samples
The experimental plan defines the matrices or group of matrices for the study by referring
to ISO/TS 21231. Consequently, the analyst selects representative samples of these matrices or group
of matrices. The laboratory shall substantiate its choice.
The representativeness of the samples for the stability study is critically important. Two strategies
can be considered:
— the use of natural samples, or
— the use of synthetic samples, which are natural samples whose physico-chemical characteristics
are varied using the recipes in ISO/TS 21231; or the addition of influence parameters.
Each type of matrix in the scope of application of the method shall be studied. The influence parameters
of each sample shall be measured and recorded at least at T .
To guarantee the representativeness of the study and cover the entire range of the influence parameters
of the matrix for the parameter(s) of interest, these samples shall have different intrinsic characteristics
by type of matrix (surface water, ground water, for example), and include the extreme values of the
influencing parameters. For example, the content of suspended solids and the pH for the methods
used to analyse organic analytes, or the content of suspended solids and the conductivity for nutrients
(see ISO/TS 21231).
5.7 Guidelines to the stability studies of transformation products
Whenever a stability study is made of a parameter that is known to be a transformation product
(metabolite, by-products of oxidation or hydrolysis, for example) of a parent compound (analyte)
that may be present in the sample, it is imperative to include the measurement of the parent compound
(analyte) in the stability assessment of the transformation product. Transformation products usually
have longer half-lives than the parent compound. Consequently, the stability study may erroneously
conclude to the stability (Figure 2, b) of the transformation product. In real sample, where the parent
compound is present, stability study will lead to instability conclusion of the transformation product
together with the parent compound (Figure 2, c).
2a) Stability study – parent compound
2b) Stability study – transformation product (alone)
2c) Stability study – parent compound/transformation product mixtures
Key
X time
1 instability
2 stability
Figure 2 — Stability study of parent compound/transformation product mixtures
The maximum acceptable delay before analysis set for the transformation product cannot be longer
than the maximum acceptable delay before analysis of the parent compound, irrespective of the results
achieved in the stability study of the transformation product. If this condition is not met, there is a risk
of bias in the estimate of the concentration of the transformation product in the sample. The maximum
acceptable delay before analysis will be set at the shortest period of time. If this period of time
is incompatible with the imperatives of the laboratory, the laboratory shall use a means of stabilizing
the parent compound.
6 Definition of the experimental plan
Stability studies shall be associated with a homogeneity study of the material.
There are three possible approaches to stability studies: chronological, isochronous and an intermediate
approach, known as pseudo-isochronous (Annex B). Table 1 compares these approaches.
The study shall be documented (Annex C). In particular, every laboratory should substantiate its
choices in the corresponding records of this study.
Table 1 — Comparative description of the different approaches to stability studies
* Chronological stability Isochronous stability Pseudo-isochronous
study study stability study
Ease of implementation Performance of measure- A flexible approach that is
ments under repeatability compatible with the require-
conditions ments of the study and the
operational constraints of
Acquistion of the results in Ver y d i s c r im in a t or y
the laboratory and the scope
Advantages the course of the study in order to highlight inho-
of application
mogeneity and instability
Production of the test material Production of the test ma- Production of the test mate-
in one batch terial in one batch rial in one batch or in several
batches
Need for a measurement Need to temporarily store Availability of the results at
method with a very good in- the samples under condi- the end of the study
termediate precision tions of stability
L e s s d i s c r i m i na t o r y Availability of the results at Need for a highly repro-
Drawbacks
in order to highlight inho- the end of the study ducible method to prepare
mogeneity and instability the test materials in order
to achieve good intermediate
precision
The admissibility of the stability study requires reliable results. Consequently, internal quality checks
are necessary throughout the study: blanks, yield, limit of quantification check, for example.
Moreover, all the equipment shall imperatively be calibrated and checked before use.
6.1 Determination of the value of the quantity of the material at T and at other times
during the study
Table 2 exemplifies the number of measurements of a study.
Table 2 — Examples of number of tests to be performed
Number of tests
Number of time inter- 1 1 2 3 3 4 4 2 1 1 7 7 4 2 2
vals
Key
m total number of measurements to be made under repeatability or precision conditions for the stability study
n number of representative samples
p number of concentration levels
q number of measurements to be made under repeatability conditions for each representative sample at each time
interval T
i
r number of measurements to be made under repeatability conditions for each representative sample at the initial point
in time T
Table 2 (continued)
Number of tests
Number of representa- 2 4 4 2 4 2 4 5 6 3 4 2 5 6 3
tive samples (materi-
als): n
Number of concentra- 2 1 1 2 1 2 1 1 1 2 1 2 1 1 2
tions: p
Characterization at T : 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
Number of measure-
ments to be performed
under repeatability
conditions at T : r
Number of measure- 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2
ments to be performed
under repeatability
conditions
at each day T: q
Total numbers 24 24 36 36 36 44 44 35 30 30 68 68 55 42 42
of measurements to be
performed: m
Key
m total number of measurements to be made under repeatability or precision conditions for the stability study
n number of representative samples
p number of concentration levels
q number of measurements to be made under repeatability conditions for each representative sample at each time
interval T
i
r number of measurements to be made under repeatability conditions for each representative sample at the initial point
in time T
The data at T influences the acceptance and the interpretation of the stability study, so this data
shall be reliable. The measurements at T are used to determine the so-called reference value of the
concentration levels chosen in the study materials and their homogeneity. To this end, at least three
replicates r shall be made per material at the initial point in time T , i.e., r=12 at T .
0 0
The laboratory shall check the homogeneity of the material prior to the characterization at T .
For the other time intervals, the minimum number of measurements q to be made under repeatability
conditions will depend on the test plan chosen by the laboratory (Table 2):
If there are at least three-time intervals and n × p = 4, at least two measurements under repeatability
conditions (q = 2) are taken per condition (material, time interval), therefore:
— at T , the number of measurements performed is n × p × r with r=3, i.e. n × p × r = 12;
— for each time interval after T , n × p × q = 8 measurements;
— for the 3-time intervals, n × p × q × 3 = 24 measurements.
The total number of measurements is m = 12 + 24 = 36, and m= r + q = 36 at least.
6.2 Test materials
6.2.1 Preparation
This step shall be properly controlled to avoid:
i. insufficient homogeneity of the test material that prevents the results from being interpreted and
requires the test to be repeated;
----------
...
TECHNICAL ISO/TS
SPECIFICATION 5667-25
First edition
2022-02
Water quality — Sampling —
Part 25:
Guideline on the validation of the
storage time of water samples
Qualité de l'eau — Échantillonnage —
Partie 25: Lignes directrices pour la validation de la durée de
conservation des échantillons d'eau
Reference number
© ISO 2022
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
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Published in Switzerland
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms, definitions and symbols . 1
4 Principle . 5
5 Definition of the scope of the stability study . 6
5.1 Aim of the stability study . 6
5.2 Selection of the maximum acceptable delay before analysis and the acceptance
criteria . 6
5.3 Influence parameters of the maximum acceptable delay before analysis . 7
5.4 Duration of the study . 7
5.5 Concentration levels . 8
5.6 Definition of the matrices and selection of the representative samples . 8
5.7 Guidelines to the stability studies of transformation products . 9
6 Definition of the experimental plan .11
6.1 Determination of the value of the quantity of the material at T and at other times
during the study . 11
6.2 Test materials .12
6.2.1 Preparation .12
6.2.2 Characterization of the test materials . 13
6.2.3 MQD Evaluation . 14
6.3 Definition of the test plan . 14
6.3.1 Choice of the type of study . 14
6.3.2 Randomization . 14
7 Performance of the tests .15
7.1 General . 15
7.2 Chronological stability study . 15
7.3 Type 1 pseudo-isochronous stability study . 15
7.4 Type 2 pseudo-isochronous stability study . 16
7.5 Isochronous stability study . 17
8 Graphic representation of the data .17
9 Validation of the data .20
9.1 Initial validation of the study at T . 20
9.1.1 Calculation of the value assigned to T (VA ) . 20
0 T0
9.1.2 Verification of the accuracy . 21
9.2 Validation at times different from T . 22
10 Using the results . .22
10.1 Graphic view of all the data .22
10.2 Determination of the maximum acceptable delay before analysis by condition .23
10.3 Conclusion of the study . 33
11 Expression of stability .34
Annex A (informative) Measurement process .35
Annex B (informative) Fundamental notions.36
Annex C (informative) Study of stability and influence parameters .42
Annex D (informative) Example of study of atrazine stability in water . 44
Bibliography .50
iii
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
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 different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 6,
Sampling (general methods).
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
iv
Introduction
This document addresses the need for harmonized and reliable data on stability, which is essential
for the expression of recommendations for both normative and regulatory purposes. It describes
a methodological framework that enables laboratories to produce quality data that can be shared
[7]
and even monetized .
It enables laboratories to study the stability of parameters when using the physico-chemical parameters
measurement system: organic micropollutants, inorganic and organometallic micropollutants, nutrients
and macropollutants in aqueous matrices (surface water, ground water, residual urban and industrial
water and drinking water). It covers the sampling, transport and laboratory storage operations.
NOTE This document does not cover solid matrices from aquatic environments (suspended solids,
sediments).
v
TECHNICAL SPECIFICATION ISO/TS 5667-25:2022(E)
Water quality — Sampling —
Part 25:
Guideline on the validation of the storage time of water
samples
1 Scope
The purpose of this document is to describe test plans and different operating methodologies
of these test plans to define and verify the acceptable length of stability of a substance in a sample
under specified conditions of preservation (temperature, matrix, light, addition of a stabilizer, where
appropriate, type of preservation etc.) before starting analytical protocols (chemicals and physico-
chemicals analysis). Biological and microbiological methods are excluded.
It is necessary to have an analytical method with performances that have already been characterized
(repeatability, intermediate precision, trueness, accuracy and uncertainty) in order to perform
the stability study and implement its test plans.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies.
For undated references, the latest edition of the referenced document (including any amendments)
applies.
ISO/TS 21231, Water quality — Characterization of analytical methods — Guidelines for the selection of a
representative matrix
3 Terms, definitions and symbols
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
analytical process
detailed description of a measurement according to one or more measurement principles and to one
given measurement method, and including any calculations intended to obtain a measurement result
3.2
batch
definite amount of test material prepared by the laboratory at a given point in
time under supposedly identical conditions
3.3
chronological stability study
study in which individual samples prepared at the same time (i.e., as a batch), under identical conditions,
are measured as time elapses (e.g. one sample immediately, one after three months, the next one after
six months, etc.)
[SOURCE: ISO Guide 35:2017, 8.3.2.1]
3.4
homogeneity
uniformity of a specified property value throughout a defined portion of a reference material (RM)
[2]
Note 1 to entry: Tests for homogeneity are described in ISO Guide 35 .
Note 2 to entry: The “defined portion” may be, for example, an RM batch or a single unit within the batch.
[6]
Note 3 to entry: See also IUPAC Compendium on Analytical Nomenclature .
[SOURCE: ISO Guide 30:2015, 2.1.12]
3.5
influence parameter
intrinsic characteristic of the matrix, independent of the analyte concentration, a variation of which is
liable to modify the analytical result
[SOURCE: ISO/TS 21231:2019, 3.3.1]
3.6
influence parameter of the conditions of preservation
characteristic related to the conditions of storage and preservation of the sample, independent of the
analyte concentration
Note 1 to entry: E.g. container material, storage temperature, influence of light and/or relative humidity.
3.7
integrity
property that the parameter(s) of interest, information or content of the sample container has not been
altered or lost in an unauthorized manner or subject to loss of representativeness
[SOURCE: ISO 5667-3:2018, 3.1]
3.8
intermediate precision condition of measurement
condition of measurement, out of a set of conditions that includes the same measurement procedure,
same location, and replicate measurements on the same or similar objects over an extended period
of time, but may include other conditions involving changes
Note 1 to entry: The changes can include new calibrations, calibrators, operators, and measuring systems.
Note 2 to entry: A specification for the conditions should contain the conditions changed and unchanged,
to the extent practical.
Note 3 to entry: In chemistry, the term “inter-serial precision condition of measurement” is sometimes used
[8]
to designate this concept .
3.9
isochronous stability study
experimental study of “reference” material stability in which units exposed to different storage
conditions and times are measured in a short period of time
[SOURCE: ISO Guide 35:2017, 3.9]
3.10
matrix
all the constituents of the laboratory sample other than the analyte
Note 1 to entry: By extension, a matrix is defined by the analyst as waters characterized by a homogeneous
behaviour with regard to the analytical method used.
[SOURCE: ISO/TS 21231:2019, 3.3.3]
3.11
maximum acceptable delay before analysis
MaxADs
maximum acceptable delay between the end of the sampling process and the start of the analysis
operations, resulting from the stability study, that the laboratory uses to plan the analyses
3.12
maximum acceptable deviation for the stability study
MADs
maximum acceptable deviation relative to the assigned value of the sample at T , used to determine
the maximum acceptable delay before analysis
3.13
measurement precision
closeness of agreement between indications or measured quantity values obtained by replicate
measurements on the same or similar objects under specified conditions
Note 1 to entry: Measurement precision is usually expressed numerically by measures of imprecision, such as
standard deviation, variance, or coefficient of variation under the specified conditions of measurement.
Note 2 to entry: The "specified conditions" may be, for example, repeatability conditions of measurement,
intermediate precision conditions of measurement, or reproducibility conditions of measurement
[1]
(see ISO 5725-1:1994 ).
Note 3 to entry: Measurement precision is used to define measurement repeatability, intermediate measurement
precision, and measurement reproducibility.
[8]
Note 4 to entry: Sometimes “measurement precision” is erroneously used to mean measurement accuracy .
3.14
measurement repeatability
measurement precision under a set of repeatability conditions of measurement
[SOURCE: JCGM 200:2012 (VIM), 2.21]
3.15
measurement reproducibility
measurement precision under reproducibility conditions of measurement
Note 1 to entry: I.e., condition of measurement, out of a set of conditions that includes different locations,
operators, measuring systems, and replicate measurements on the same or similar objects.
[SOURCE: JCGM 200:2012 (VIM), 2.25]
3.16
measurement trueness
closeness of agreement between the average of an infinite number of replicates measured quantity
values and a reference quantity value
Note 1 to entry: Measurement trueness is not a quantity and thus cannot be expressed numerically, but measures
[3]
for closeness of agreement are given in ISO 5725-1:1994 .
Note 2 to entry: Measurement trueness is inversely related to systematic measurement error, but is not related to
random measurement error.
Note 3 to entry: “Measurement accuracy” should not be used for ‘measurement trueness’.
[SOURCE: JCGM 200:2012 (VIM), 2.14]
3.17
minimum quantifiable deviation for the stability study (MQDs)
minimum deviation relative to the assigned value of the parameter in the sample at T , which can be
unequivocally imputed to the instability
Note 1 to entry: This deviation takes account of the inhomogeneity of the test material and the intrinsic variability
of all the measurement results over time used to determine the maximum acceptable deviations.
Note 2 to entry: The calculation of the minimum quantifiable deviation for the stability study depends on the
type of study (chronological, pseudo-isochronous or isochronous) (6.1.1).
3.18
pseudo-isochronous stability study
stability study in which some of the steps, in particular the preparation, are performed under
intermediate precision conditions, and in which the results of instrumental analyses are acquired
under repeatability conditions
3.19
repeatability condition
condition of measurement, out of a set of conditions that includes the same measurement procedure,
same operators, same measuring system, same operating conditions and same location, and replicate
measurements on the same or similar objects over a short period of time
Note 1 to entry: A condition of measurement is a repeatability condition only with respect to a specified set
of repeatability conditions.
Note 2 to entry: In chemistry, the term “intra-serial precision condition of measurement” is sometimes used
[8]
to designate this concept .
3.20
representative matrix
sample for which all the intrinsic characteristics are characteristics of a type of water or the source of
a group of samples
[SOURCE: ISO/TS 21231:2019, 3.3.2]
3.21
reproducibility condition
condition of measurement, out of a set of conditions that includes different locations, operators,
measuring systems, and replicate measurements on the same or similar objects
Note 1 to entry: The different measuring systems may use different measurement procedures.
[8]
Note 2 to entry: A specification should contain the conditions changed and unchanged, to the extent practical .
3.22
sample preservation
any procedure used to stabilize a sample in such a way that the properties under examination are
maintained stable from the collection step until preparation for analysis
[SOURCE: ISO 5667-3:2018, 3.2]
3.23
sample storage
process and the result of keeping a sample available under predefined conditions, usually for a specified
time interval between collection and further treatment of a sample
Note 1 to entry: Specified time is the maximum time interval [see ISO 5667-3].
3.24
stability
characteristic of an analyte in an aqueous matrix, when stored under specified conditions, to maintain
its property value within specified limits for a specified period from sampling to laboratory operations
3.25
stability interval
interval defined on the basis of the assigned value at T and the maximum acceptable deviation for the
stability study
3.26
storage time
period of time between filling of the sample container and further treatment of the sample in the
laboratory, if stored under predefined conditions
Note 1 to entry: Sampling finishes as soon as the sample container has been filled with the sample. Storage time
ends when the sample is taken by the analyst to start sample preparation prior to analysis.
Note 2 to entry: Further treatment is, for most analytes, a solvent extraction or acid destruction. The initial steps
of sample preparation can be steps complementary to the storage conditions for the maintenance of analyte
concentrations.
[SOURCE: ISO 5667-3:2018, 3.4]
4 Principle
The goal is to perform a series of tests, hereafter referred to as the “stability study”, to analyse
the variations in the value of a given parameter, between an initial time and a maximum time, on
samples representative of the scope of application of the measurement method of the parameter. The
conclusions of these tests are used to determine the maximum acceptable delay before analysis (3.11)
under the conditions of the study.
The stability study has six stages:
— Definition of the requirements (analytes, matrices, levels of concentration, storage conditions,
length of storage, maximum acceptable deviation for the stability study (3.12)), see Clause 5;
— Definition of the experiments plan (type of study, number of time intervals and total length
of the study), see Clause 6;
— Performance of the tests, see Clause 7;
— Validation of the data, see Clause 9;
— Using the results (based on a maximum acceptable deviation, 3.12), see Clause 10;
— Expression of the stability in the form of a maximum acceptable delay before analysis (3.11)
and the duration of stability corresponding to the conditions and the criteria (maximum acceptable
deviation for the stability study (3.12)) of the study, see Clause 10.
Since the stability study covers different stages of the data acquisition process according
to the organization of the measurement system, examples of the organization of the measurement
system are given in Annex A for reference.
The laboratory shall take the following factors into consideration:
— The method of determination used: for example, limit of quantification, repeatability, intermediate
precision, accuracy, influence parameters of the matrix (3.10) on the performance of the method;
— The “sample” material of the stability study: for example, homogeneity, physico-chemical properties;
— Suitability of the sample material for use as test material in accordance with storage time (3.26).
— Definition of the influence parameters of the preservation conditions (3.6) assessed as part
of the stability study: for example, time, temperature, addition of stabilizers;
— If studied, command of the storage and transportation conditions;
— Clearly defined acceptability criteria (maximum acceptable deviation, 3.12), with which the results
of the study will be compared.
5 Definition of the scope of the stability study
5.1 Aim of the stability study
Based on the scope of application of the method, the experimental plan shall clearly define the aim of the
stability study by specifying the measured analytes, the matrices (3.10) and the target concentration
levels.
5.2 Selection of the maximum acceptable delay before analysis and the acceptance
criteria
Respecting the maximum acceptable delay before analysis (3.11) may determine the quality of the
results of the analysis more than certain performance data of the measurement methods (bias). This is
the reason why the maximum acceptable delay before analysis shall be established and known before
the routine application of a laboratory analysis method.
The assessment of the results of a stability study with the intension of drawing a conclusion on the
stability, expressed as a maximum acceptable delay before analysis, of a given analyte in a representative
matrix of the scope of application shall be based on the interpretation of the results in perspective of
the requirements of the stability study. A maximum acceptable deviation (3.12) shall be set in order to
come to a conclusion. This maximum acceptable deviation shall be chosen before the start of the study,
because it determines the conditions of performance of the method and the admissibility of the data, in
particular with regard to the measurement method.
There are five ways to determine the maximum acceptable deviation. They are, in order of relevance:
a) The application of a regulatory requirement, if one exists;
b) Twice the repeatability standard deviation for isochronous or pseudo-isochronous type 1 studies
(Annex B), or twice the intermediate precision standard deviation for type 2 pseudo-isochronous
studies (Annex B) or chronological studies; the values of the standard deviation of repeatability
and intermediate precision being the values defined during the characterization of the method.
c) Use of the data from the stability study: dispersion at T of the stability study. See Formula (1):
Maximum acceptable deviation = Minimum quantifiable deviatiion (1)
d) The choice of an arbitrary value, determined according to the technical operational implementation
constraints (e.g., the best available method offering a precision of 15 %) or a value from a ring
trial (ISO 5667-3). In this case, the maximum acceptable deviation shall be greater than this value,
e.g., 25 %.
e) A value derived from the temporal operational implementation constraints (e.g., the impossibility
of performing an analysis before a given time due to the minimum transportation time). In this
case, the maximum acceptable delay before analysis is fixed and the maximum acceptable deviation
is based on the observations at the pre-determined time.
EXAMPLE If the temporal constraint is three days, (MaxADs =3 days) the maximum acceptable
deviation is estimated according to the observations (dispersion, for example) at T .
If the minimum quantifiable deviation is greater than the maximum acceptable deviation, it is
impossible to draw any conclusions about the stability. In this case, the laboratory should identify the
causes (e.g., lack of homogeneity of the materials, lack of performances of the method, need to stabilize
the materials) and repeat the study (e.g., with a different maximum acceptable deviation, another
method, etc.).
5.3 Influence parameters of the maximum acceptable delay before analysis
This protocol applies to conditions clearly defined by the laboratory, which defines the maximum
acceptable delay before analysis, the corresponding assessment criterion (see 5.2) and the factors likely
to impact it.
In particular, these factors may include:
— The container: ISO 5667-3 or the analysis standards applicable to the targeted parameters specify
the recommended containers for the transportation of samples based on current knowledge.
However, for certain classes of molecules, for which no normative guidelines exist, the compatibility
of these general recommendations on the container material (plastic, glass, etc.) that may cause
losses by adsorption, diffusion, the transfer of additives used in production, etc., should be verified;
— The storage temperature (ambient, refrigeration, freezing, etc.), which can impact the partition
of the substances between the dissolved and particulate phases, or the enantiomer form, or their
biological deterioration. ISO 5667-3 recommends that samples be preserved at 5 ± 3 °C during
transportation. Therefore, this temperature range is the reference temperature of the study,
in the absence of any other requirements. Other temperatures may be chosen for the study, for
example when demonstrating the stability of analytical extracts in the laboratory. The temperature
conditions of the study shall be clearly indicated, and they shall be monitored and documented
throughout the stability study;
— The influence parameters of the matrix: pH, suspended solids, etc;
— Light, which can cause the photodegradation of certain organic molecules, e.g.: Benzo[a]pyrene,
BDE209;
[15]
— The influence of sample pre-treatment on site, e.g. filtration ;
— The addition to the sample of stabilizing chemical agents (e.g., acid for metals, sodium hydroxide
for cyanides, solvents, etc.).
5.4 Duration of the study
The duration of the study shall cover the initially planned maximum acceptable delay before analysis.
The extrapolation of the MaxADs beyond T is not permitted. It is thus recommended to collect data
max
beyond the planned MaxADs,
Interpolation between the two ends of a time interval is not permitted in order to define the maximum
acceptable delay before analysis. Therefore, it is recommended to acquire additional information at
different intermediate intervals of time.
The laboratory shall adapt the number of the intervals of time (Figure 1) of the study according to its
initial knowledge of the stability of the analyte of concerned in order to minimize the risk of inconclusive
studies.
Key
1 time interval 1
2 time interval 2
3 time interval 3
4 time interval 4
Figure 1 — Example of a diagrammatic illustration of the notion of time laps
5.5 Concentration levels
The levels of concentration to be tested (p) are chosen according to the requirements of the
corresponding sector of activity, any regulatory requirements that may exist, the occurrence of data
in the environment, for example, and the performances of the analytical method and its scope of
application.
The experimental plan selects one of the two following approaches, depending on the complexity of the
matrices and their knowledge of the method:
— At least two levels of concentration (p≥ 2) by representative sample (n≥ 2) of the matrix of the scope
of application of the method shall be considered:
— One low concentration level, different from the LOQ, ≤ 25 % of the scope of the method for one-
decade methods or ≤ 10 % for more-decades methods,
— One high concentration level, in the second half of the scope of application of the method.
— A minimum concentration level of p =1, ≤ 25 % of scope of the method for one-decade methods,
resp. ≤ 10 % for more-decades methods, with several representative samples of the matrix (p x n
≥ 4). In this case two time laps should be taken.
The laboratory shall substantiate its choice of n=2 by demonstrating that its scope of application
is restricted and, therefore, sufficiently described by two representative samples, in accordance with
ISO/TS 21231.
NOTE 1 Performing stability studies at the limit of quantification (LOQ) is not generally relevant.
The uncertainty of the methods at the LQ does not allow for the unequivocal interpretation of the data.
NOTE 2 When representative samples free of any background contamination of analyte cannot be found,
the environmental background noise should be considered to determine the lowest level of contamination tested
in the stability study.
5.6 Definition of the matrices and selection of the representative samples
The experimental plan defines the matrices or group of matrices for the study by referring
to ISO/TS 21231. Consequently, the analyst selects representative samples of these matrices or group
of matrices. The laboratory shall substantiate its choice.
The representativeness of the samples for the stability study is critically important. Two strategies
can be considered:
— the use of natural samples, or
— the use of synthetic samples, which are natural samples whose physico-chemical characteristics
are varied using the recipes in ISO/TS 21231; or the addition of influence parameters.
Each type of matrix in the scope of application of the method shall be studied. The influence parameters
of each sample shall be measured and recorded at least at T .
To guarantee the representativeness of the study and cover the entire range of the influence parameters
of the matrix for the parameter(s) of interest, these samples shall have different intrinsic characteristics
by type of matrix (surface water, ground water, for example), and include the extreme values of the
influencing parameters. For example, the content of suspended solids and the pH for the methods
used to analyse organic analytes, or the content of suspended solids and the conductivity for nutrients
(see ISO/TS 21231).
5.7 Guidelines to the stability studies of transformation products
Whenever a stability study is made of a parameter that is known to be a transformation product
(metabolite, by-products of oxidation or hydrolysis, for example) of a parent compound (analyte)
that may be present in the sample, it is imperative to include the measurement of the parent compound
(analyte) in the stability assessment of the transformation product. Transformation products usually
have longer half-lives than the parent compound. Consequently, the stability study may erroneously
conclude to the stability (Figure 2, b) of the transformation product. In real sample, where the parent
compound is present, stability study will lead to instability conclusion of the transformation product
together with the parent compound (Figure 2, c).
2a) Stability study – parent compound
2b) Stability study – transformation product (alone)
2c) Stability study – parent compound/transformation product mixtures
Key
X time
1 instability
2 stability
Figure 2 — Stability study of parent compound/transformation product mixtures
The maximum acceptable delay before analysis set for the transformation product cannot be longer
than the maximum acceptable delay before analysis of the parent compound, irrespective of the results
achieved in the stability study of the transformation product. If this condition is not met, there is a risk
of bias in the estimate of the concentration of the transformation product in the sample. The maximum
acceptable delay before analysis will be set at the shortest period of time. If this period of time
is incompatible with the imperatives of the laboratory, the laboratory shall use a means of stabilizing
the parent compound.
6 Definition of the experimental plan
Stability studies shall be associated with a homogeneity study of the material.
There are three possible approaches to stability studies: chronological, isochronous and an intermediate
approach, known as pseudo-isochronous (Annex B). Table 1 compares these approaches.
The study shall be documented (Annex C). In particular, every laboratory should substantiate its
choices in the corresponding records of this study.
Table 1 — Comparative description of the different approaches to stability studies
* Chronological stability Isochronous stability Pseudo-isochronous
study study stability study
Ease of implementation Performance of measure- A flexible approach that is
ments under repeatability compatible with the require-
conditions ments of the study and the
operational constraints of
Acquistion of the results in Ver y d i s c r im in a t or y
the laboratory and the scope
Advantages the course of the study in order to highlight inho-
of application
mogeneity and instability
Production of the test material Production of the test ma- Production of the test mate-
in one batch terial in one batch rial in one batch or in several
batches
Need for a measurement Need to temporarily store Availability of the results at
method with a very good in- the samples under condi- the end of the study
termediate precision tions of stability
L e s s d i s c r i m i na t o r y Availability of the results at Need for a highly repro-
Drawbacks
in order to highlight inho- the end of the study ducible method to prepare
mogeneity and instability the test materials in order
to achieve good intermediate
precision
The admissibility of the stability study requires reliable results. Consequently, internal quality checks
are necessary throughout the study: blanks, yield, limit of quantification check, for example.
Moreover, all the equipment shall imperatively be calibrated and checked before use.
6.1 Determination of the value of the quantity of the material at T and at other times
during the study
Table 2 exemplifies the number of measurements of a study.
Table 2 — Examples of number of tests to be performed
Number of tests
Number of time inter- 1 1 2 3 3 4 4 2 1 1 7 7 4 2 2
vals
Key
m total number of measurements to be made under repeatability or precision conditions for the stability study
n number of representative samples
p number of concentration levels
q number of measurements to be made under repeatability conditions for each representative sample at each time
interval T
i
r number of measurements to be made under repeatability conditions for each representative sample at the initial point
in time T
Table 2 (continued)
Number of tests
Number of representa- 2 4 4 2 4 2 4 5 6 3 4 2 5 6 3
tive samples (materi-
als): n
Number of concentra- 2 1 1 2 1 2 1 1 1 2 1 2 1 1 2
tions: p
Characterization at T : 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
Number of measure-
ments to be performed
under repeatability
conditions at T : r
Number of measure- 3 3 3 2 2 2 2 2 2 2 2 2 2 2 2
ments to be performed
under repeatability
conditions
at each day T: q
Total numbers 24 24 36 36 36 44 44 35 30 30 68 68 55 42 42
of measurements to be
performed: m
Key
m total number of measurements to be made under repeatability or precision conditions for the stability study
n number of representative samples
p number of concentration levels
q number of measurements to be made under repeatability conditions for each representative sample at each time
interval T
i
r number of measurements to be made under repeatability conditions for each representative sample at the initial point
in time T
The data at T influences the acceptance and the interpretation of the stability study, so this data
shall be reliable. The measurements at T are used to determine the so-called reference value of the
concentration levels chosen in the study materials and their homogeneity. To this end, at least three
replicates r shall be made per material at the initial point in time T , i.e., r=12 at T .
0 0
The laboratory shall check the homogeneity of the material prior to the characterization at T .
For the other time intervals, the minimum number of measurements q to be made under repeatability
conditions will depend on the test plan chosen by the laboratory (Table 2):
If there are at least three-time intervals and n × p = 4, at least two measurements under repeatability
conditions (q = 2) are taken per condition (material, time interval), therefore:
— at T , the number of measurements performed is n × p × r with r=3, i.e. n × p × r = 12;
— for each time interval after T , n × p × q = 8 measurements;
— for the 3-time intervals, n × p × q × 3 = 24 measurements.
The total number of measurements is m = 12 + 24 = 36, and m= r + q = 36 at least.
6.2 Test materials
6.2.1 Preparation
This step shall be properly controlled to avoid:
i. insufficient homogeneity of the test material that prevents the results from being interpreted and
requires the test to be repeated;
ii. an under-estimation or an over-estimation of the stability.
and, therefore, a wrong decision resulting in poor data quality.
Usually, the test materials for these studies are natural samples, synthetic materials or natural samples
that are spiked.
The notion of the representativeness of a sample includes the delay before the preparation of the
stability test materials at T .
The materials used in the stability study shall be prepared as soon as possible after the samples are
[15]
taken in order to optimally preserve their representativeness . The means of preservation between
taking the sample and its preparation shall not alter the representativeness of the sample, e.g., if the
study is dedicated to whole water samples, on site filtration is not allowed.
The experimental plans required to conduct the stability studies may require large volumes of samples
that, in a large majority of cases, shall be adjusted to meet the need for the representativeness of the
matrix. In particular, this may involve making additions (spiking) of the studied parameter. This step
involves significant technical particularities, such as the need for a large container that is compatible
with the requirements of the analysis of micropollutants in the form of traces (nature of the container,
cleanin
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