INTERNATIONAL ISO

STANDARD 25101

First edition
2009-03-01

Water quality — Determination of

perfluorooctanesulfonate (PFOS) and
perfluorooctanoate (PFOA) — Method for
unfiltered samples using solid phase
extraction and liquid
chromatography/mass spectrometry
Qualité de l'eau — Détermination du sulfonate de perfluorooctane
(PFOS) et de l'octanoate perfluoré (PFOA) — Méthode par extraction
en phase solide et chromatographie liquide/spectrométrie de masse
pour des échantillons non filtrés

Reference number
ISO 25101:2009(E)

© ISO 2009
ISO 25101:2009(E)

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ii © ISO 2009 – All rights reserved

 ISO 25101:2009(E)

Contents Page

Foreword............................................................................................................................................................ iv
1 Scope ..................................................................................................................................................... 1
2 Normative references ........................................................................................................................... 1
3 Principle ................................................................................................................................................. 1
4 Interferences ......................................................................................................................................... 2
5 Reagents ................................................................................................................................................ 3
6 Apparatus .............................................................................................................................................. 4
7 Sampling and sample pretreatment.................................................................................................... 4
8 Procedure .............................................................................................................................................. 4
9 Calibration ............................................................................................................................................. 6
10 Calculation............................................................................................................................................. 8
11 Expression of results ........................................................................................................................... 9
12 Test report ............................................................................................................................................. 9
Annex A (informative) Examples of suitable sorbents ................................................................................. 10
Annex B (informative) Suitable HPLC columns............................................................................................. 11
Annex C (informative) Examples of HPLC MS/MS chromatograms ............................................................ 12
Annex D (informative) Conditions for analysis of PFOS and PFOA using a single MS............................ 15
Annex E (informative) Precision data............................................................................................................. 16
Annex F (informative) Details of the samples used for the interlaboratory trial........................................ 17
Bibliography ..................................................................................................................................................... 19

© ISO 2009 – All rights reserved iii

ISO 25101:2009(E)

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.

International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.

The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.

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.

ISO 25101 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.

iv © ISO 2009 – All rights reserved

INTERNATIONAL STANDARD ISO 25101:2009(E)

Water quality — Determination of perfluorooctanesulfonate

(PFOS) and perfluorooctanoate (PFOA) — Method for unfiltered
samples using solid phase extraction and liquid
chromatography/mass spectrometry

WARNING — Persons using this International Standard should be familiar with normal laboratory
practice. This standard does not purport to address all of the safety problems, if any, associated with
its use. It is the responsibility of the user to establish appropriate safety and health practices and to
ensure compliance with any national regulatory conditions.

IMPORTANT — It is absolutely essential that tests conducted in accordance with this International
Standard be carried out by suitably qualified staff.

1 Scope
This International Standard specifies a method for the determination of the linear isomers of
perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) in unfiltered samples of drinking water,
ground water and surface water (fresh water and sea water) using high-performance liquid chromatography-
tandem mass spectrometry (HPLC-MS/MS). Other isomers may be reported separately as non-linear isomers
and qualified as such. The analytes specified in Table 1 can be determined by this method. The method is
applicable to a concentration range of 2,0 ng/l to 10 000 ng/l for PFOS and 10 ng/l to 10 000 ng/l for PFOA.
Depending on the matrix, the method may also be applicable to higher concentrations ranging from 100 ng/l to
200 000 ng/l after suitable dilution of the sample or reduction in sample size.

The user should be aware that particular problems could require the specification of additional conditions.

2 Normative references
The following referenced documents are indispensable for the application 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 3696:1987, Water for analytical laboratory use — Specification and test methods

ISO 5667-1, Water quality — Sampling — Part 1: Guidance on the design of sampling programmes and
sampling techniques

ISO 8466-1, Water quality — Calibration and evaluation of analytical methods and estimation of performance
characteristics — Part 1: Statistical evaluation of the linear calibration function

3 Principle
The analytes listed in Table 1 are extracted from the water sample by solid-phase extraction followed by
solvent elution and then determined by liquid chromatography with tandem mass-spectrometric detection.

NOTE This method is also applicable, with some limitations, to determination using high-performance liquid
chromatography with single mass-spectrometric (HPLC-MS) detection (see Annex D).

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ISO 25101:2009(E)

Table 1 — Analytes determinable by this method

Analyte Formula a Abbreviation CAS b No.

Perfluoro-n-octanesulfonic acid
CF3(CF2)7SO3H PFOS 1763-23-1
(1,1,2,2,3,3,4,4,5,5,6,6,7,7,8,8,8-heptadecafluoro-n-octanesulfonic acid)
Perfluoro-n-octanoic acid
CF3(CF2)6COOH PFOA c 335-67-1
(pentadecafluoro-n-octanoic acid)
a The anion is the analyte.
b CAS = Chemical Abstract System.
c PFOA includes the acid and its salts.

4 Interferences

4.1 Interferences with sampling and extraction

Sampling containers shall consist of materials that do not change the composition of the sample during
sample storage. All types of fluoropolymer plastics, including polytetrafluoroethene (PTFE) and
fluoroelastomer materials, shall be avoided during sampling, sample storage and extraction. Glassware shall
be avoided for sampling due to potential analyte loss due to adsorption. Sample containers shall be rinsed
thoroughly with water (5.1) and methanol (5.5) prior to use. Sample containers shall be checked for possible
background contamination before use.

Commercially available adsorbent materials are often of varying quality. Considerable batch-to-batch
differences in quality and selectivity of these materials are possible. The recovery of a single substance may
vary with the concentration. Therefore, check analyte recovery periodically at different concentrations and
whenever new batches/lots of reagents or labware are used.

4.2 Interferences with HPLC-MS/MS

Substances with similar retention times and producing ions similar to those produced by the analytes of
interest may interfere with the determination.

These interferences may lead to incompletely resolved signals or additional signals in the chromatographic
pattern of target analytes, or both. Depending on their levels in the sample, such substances may affect the
accuracy and precision of the results.

Matrix interferences may be caused by contaminants that are co-extracted from the samples. The extent of
matrix interferences varies considerably, depending on the nature of the samples. In drinking water and
ground water, matrix interferences are usually negligible, whereas wastewater and sea water matrices can be
affected by matrix interferences that lead to ionization suppression or enhancement.

Interferences from instruments are significant for normal HPLC systems because many parts are made of
PTFE and other fluoropolymers. It is necessary to check for possible blank contamination from individual parts,
such as tubing, solvent inlet filters, valve seals and the degassing equipment, and replace these with materials
such as stainless steel and polyetheretherketone (PEEK), where possible. The HPLC-vial caps should
preferably be free of fluoropolymer material. The procedural blank including the instrumental blank should
preferably be at least 10-fold less than the expected concentrations in real samples.

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 ISO 25101:2009(E)

5 Reagents

Use certified or analytical-grade reagents and check contamination levels of target compounds by blank
determinations. If necessary, carry out additional cleaning steps to ensure background levels are minimized.

5.1 Water, complying with at least grade 3 as specified in ISO 3696:1987.

5.2 Acetic acid, w(CH3COOH) = 99,9 % mass fraction.

5.3 Ammonia solution, w(NH3) = 25 % mass fraction.

5.4 Ammonium acetate, w(CH3COONH4) = 97 % mass fraction.

5.5 Methanol (CH3OH), HPLC grade.

5.6 Internal-standard solutions:

1,2,3,4-13C4-PFOA, ρ = 1 ng/µl.

1,2,3,4-13C4-PFOS, ρ = 1 ng/µl.

Solutions of the internal standards are available commercially. They shall be diluted to the required
concentrations. If the standards are obtained as pure compounds, weigh 10 mg of each standard into a
separate 100 ml volumetric flask and make up to the mark with methanol (5.5). Dilute the solution thus
obtained initially by a factor of 100 with methanol (5.5).

Other internal standards, e.g. 13C5-PFNA [perfluoro-n-nonanoic acid, CF3(CF2)6COOH)], that meet the
internal-standard requirements are acceptable for use. However, the purity of some of these commercially
available standards is not adequate and, if such standards are used, the purity shall be determined in the
laboratory. Analysis of impurities in standards shall be carried out prior to using new batches of standards.

5.7 Solutions of reference compounds of the analytes listed in Table 1, 0,1 ng/µl, used as calibration
standards.

Weigh 10 mg of each reference compound into a separate 100 ml volumetric flask and make up to the mark
with methanol (5.5). Dilute this solution serially with methanol (5.5) to give an overall dilution of 1:1 000.
Standards may also be obtained as solutions if commercially available and diluted to the required
concentration.

Store solutions 5.6 and 5.7 at a temperature of (4 ± 2) °C and bring them to room temperature prior to use
(i.e. before dilution or spiking or injection).

5.8 Acetate buffer, 0,025 mol/l, pH 4.

Mix 0,5 ml of acetic acid (5.2) with 349,5 ml of water (5.1). Dissolve 0,116 g of ammonium acetate (5.4) in
60 ml of water (5.1). Mix 200 ml of the diluted acetic acid with 50 ml of the ammonium acetate solution.

5.9 Ammonia/methanol solution, w = 0,1 % mass fraction.

Mix 0,4 ml of 25 % ammonia solution (5.3), with 99,6 ml of methanol (5.5).

5.10 Solid-phase extraction material, copolymer-based. Suitable materials are available commercially
(see Annex A).

5.11 Nitrogen (N2), purity > 99,996 %.

5.12 Sodium thiosulfate pentahydrate (Na2S2O3 ⋅ 5H2O).

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ISO 25101:2009(E)

6 Apparatus
Equipment of which any part may come into contact with the water sample or the extract shall be free from
interfering compounds.

Clean all labware and apparatus for solid-phase extraction by rinsing with water (5.1) and methanol (5.5).

6.1 Narrow-neck flat-bottomed polypropene bottles, capacity 1 000 ml, with conical shoulders and
screw caps.

The bottles and screw caps shall be washed, rinsed with methanol (5.5) and dried before use in order to
minimize contamination.

6.2 Solid-phase extraction cartridges, made of inert non-leaching plastic, e.g. polypropene.

The cartridges shall be packed with a minimum of 150 mg of solid-phase extraction material (5.10) as sorbent.
In general, 150 mg to 250 mg of sorbent (Annex A) in a single cartridge is sufficient for up to 1 000 ml of water.

6.3 Vacuum or pressure assembly, for the extraction step.

6.4 Volumetric flasks, with inert stoppers.

6.5 Graduated cylinder, capacity 500 ml.

6.6 Evaporation assembly, using a nitrogen (5.11) stream passing through a stainless-steel needle.

6.7 Vials, made of polypropene or polyethylene not containing fluoropolymer materials, capacity e.g. 1,5 ml,
depending on the auto-sampler.

6.8 High-performance liquid chromatograph, temperature-controlled and with all necessary accessories,
including gases, HPLC columns, injector and tandem mass spectrometer (6.9).

6.9 Tandem mass spectrometer, capable of determining the m/z values of selected precursor ions and
product ions of the target compounds listed in Table 2.

7 Sampling and sample pretreatment

Take, preserve and handle samples as specified in ISO 5667-1.

For sampling, use thoroughly cleaned bottles (6.1). Fill the bottle only to the shoulder with the water to be
sampled (approximately 1 000 ml). In the presence of free chlorine, immediately add approximately 80 mg of
sodium thiosulfate pentahydrate (5.12) or another suitable dechlorinating agent (e.g. sodium sulfite).

Store samples in a refrigerator at (4 ± 2) °C and analyse within two weeks. If the sample cannot be analysed
within two weeks of sampling, the sample may be frozen until analysis but its stability shall be checked during
storage, if necessary.

8 Procedure

8.1 Solid-phase extraction

8.1.1 General

In general, in this procedure samples are analysed without pretreatment, i.e. suspended solids are not
removed prior to analysis. Before starting the analysis, homogenize the sample by shaking.

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 ISO 25101:2009(E)

8.1.2 Conditioning of the solid-phase extraction material

The following procedure describes that used for commercially available 6 ml copolymer cartridges packed with
150 mg of sorbent sandwiched between two polyethylene frits.

Rinse the cartridge, in the following sequence, with 4 ml of ammonia/methanol solution (5.9), 4 ml of methanol
(5.5) and lastly 4 ml of water (5.1) prior to use. Make sure that the sorbent packing in the cartridge does not
run dry. Retain the water in the cartridge (with the water level just above the packing) to keep the sorbent
activated.

8.1.3 Sample extraction

Start the extraction immediately after conditioning the sorbent packing. Make sure that no air bubbles are
trapped in the sorbent bed when changing from conditioning to extraction. Maintain the sorbent material in the
cartridge immersed in water at all times.

Add the internal-standard solutions (5.6) to e.g. 500 ml of the water sample in the sample bottle (adding e.g.
100 µl of each, depending on the sample matrix) and mix thoroughly by shaking. Let this sample run through
the cartridge, conditioned as specified in 8.1.2, at a rate of one drop per second (3 ml/min to 6 ml/min). With
water samples containing more than 500 mg/l of suspended matter, carry out the extraction by passing 100 ml
of sample through the cartridge. Remove residual water in the sorbent packing by applying a vacuum to the
cartridge for 30 s. If the period of vacuum application is not enough to remove the water, repeat the vacuum
application several times, but not for more than 2 min because overuse of vacuum may lead to loss of target
compounds.

Reweigh the empty sample bottle with its original cap and calculate the net mass of sample, to the nearest 1 g,
from the difference in weight. Assuming a density of 1 g/ml, the value of the net mass (in grams) is equivalent
to the volume (in millilitres) of the water used in the extraction.

8.1.4 Elution

Add 4 ml of acetate buffer solution (5.8) to the dried cartridge and discard the eluate. Centrifuge the cartridge
at 1 500g for about 2 min or apply a vacuum to remove completely the residual solution from the cartridge.
Then elute the target analytes with 4 ml of methanol (5.5), followed by 4 ml of 0,1 % ammonia/methanol (5.9)
at a rate of one drop per second. Evaporate the eluate with a gentle stream of nitrogen gas (5.11) to a final
volume of 500 µl. The extract is now ready for HPLC-MS/MS analysis. The final extract volume may be
adjusted by dilution with methanol, depending on the concentrations of the target analytes in the sample. The
concentration of the sample should preferably be adjusted (by dilution or concentration) so that the
concentrations of the target analytes lies within the calibration range of the instrument.

8.2 HPLC-MS/MS operating conditions

Optimize the operating conditions of the HPLC-MS/MS system in the electrospray ionization (ESI) negative
mode in accordance with the manufacturer’s instructions. The appropriate HPLC gradient programme for the
mobile phase is determined experimentally during method development and validation. For optimum
sensitivity, selected ions for MS/MS transitions are listed in Table 2. An example of typical operating
conditions is given in Annex C.

8.3 Blank determination

Treat the blank in exactly the same manner as the samples, except that the sample is replaced by the
appropriate amount of water (5.1). Procedural blanks should preferably be analysed with each batch of
samples (a maximum of 10 samples).

8.4 Quality control samples

Analyse the quality control samples (procedural blank and spiked matrix sample) for each batch of samples to
ensure accuracy and reliability of the analytical process. Spike samples with solutions of reference
compounds (5.7), using volumes identical to those of the samples being analysed, and process them using
the same procedure used for real samples.

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ISO 25101:2009(E)

8.5 Identification

Peak identification is by comparison of the retention times and relative signal intensities observed for the ions
monitored (see Table 2) in the samples compared with those observed for the reference compounds.

The retention time for a target compound peak shall agree with the retention time observed for the reference
compound to within 0,5 % when measured under identical conditions. This applies to comparison by either
absolute or relative retention time. The relative abundance of diagnostic ions observed for samples shall
match the abundance observed for reference compounds to within 25 %. It is important that both of the above
criteria be satisfied in order to confirm the presence of a target compound.

Mixtures of PFOSs may contain branched isomers. Incomplete separation of the branched isomers from the
linear (n-octyl) isomer can lead to co-elution, which may interfere with the analysis. The linear (n-octyl) isomer
can be separated from the others by using specific chromatography columns (see Annex B) and by optimizing
the chromatographic conditions. The linear isomer of PFOS is the target compound of this method, and
separation of the linear isomer from the other, branched, isomers shall be demonstrated.

Table 2 — Selected diagnostic ions used in the determination

Selected diagnostic ions

m/z
No. Analyte Abbreviation
Precursor Product Qualifier
M1 a M2 a M3 a

1 Perfluoro-n-octanesulfonic acid PFOS 499 80 99

2 Perfluoro-n-octanoic acid PFOA 413 369 169
3 1,2,3,4-13C4-Perfluoro-n-octanesulfonic acid b 13C -PFOS
4 503 80 99
4 1,2,3,4-13C4-Perfluoro-n-octanoic acid b 13C -PFOA
4 417 372 169
a M2 is used as the product ion for the determination. M3 may be used for identification. M1 is the precursor ion used to obtain the
product ion.
b Internal standard.

9 Calibration

9.1 General requirements

For practical reasons, calibration uses a solution containing the analytes of interest and internal standards
(see Table 2).

Ensure there is a linear dependence between signal and concentration. Determine the linear working range
using at least five measurements at different concentrations (see ISO 8466-1).

The calibration function for a compound is valid only for the measured concentration range. Additionally, the
calibration function depends on the condition of the instrument, which shall be checked regularly. For routine
analysis, a regular check of the calibration function by means of two-point calibration is sufficient.

For routine analysis, a single calibration shall be carried out with internal standards over the complete
analytical procedure. As the calibration is performed over the complete procedure with internal standards,
determination of the recoveries is not necessary, but should preferably be recorded for quality control
purposes.

Table 3 gives an explanation of the subscripts used in the following equations and text.

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 ISO 25101:2009(E)

Table 3 — Explanation of subscripts

Subscript Meaning

i Indicates the identity of the target compound

e Indicates calibration
I Indicates the identity of the internal standard
g Indicates the overall procedure

9.2 Calibration over the complete procedure with internal standards

When using labelled internal standards, the determination of the concentration is independent of any errors
made during injection. Also, errors caused by sample losses during particular steps of sample pre-treatment
or the adjustment of the final sample extract volume, as well as by matrix effects in the sample, are minimized.
The recovery of the internal standard calculated using Equation (1) shall be between 70 % and 125 % for the
internal-standard batch to be considered acceptable.

Add the internal standards prior to extraction of the target compounds from the samples. The mass of internal
standard to be added depends on the sample volume and on the expected concentration of the target
compounds in the sample. The mass concentration ρI of the internal standard shall be the same for calibration
and for sample measurement.

ρ I g ⋅ Vsam
wrec = ⋅ 100 (1)
ρ I ⋅VI

where

wrec is the percent recovery of internal standard I from the spiked sample;

ρIg is the mass concentration of internal standard I in the spiked sample, expressed in nanograms per
litre;

ρI is the mass concentration of internal standard I in the internal-standard solution used to spike the
sample, expressed in nanograms per litre;

Vsam is the volume of the sample, expressed in litres;

VI is the volume of the internal-standard solution added to the sample, expressed in litres.

Use the same solvent composition and internal-standard concentrations for the working standard solutions
and for the extracts.

Prepare a plot of the results, plotting the values of the ratio yie/yIe (see below) (using peak areas, peak heights
or integration units) for each target compound as the ordinate and the associated ratio of the mass
concentrations ρie/ρIe as the abscissa.

Determine the linear regression function using the corresponding pairs of values of yie/yIe and ρie/ρIe in the
measurement series in accordance with Equation (2):

y ie ρ
= a iI e ie + biI e (2)
y Ie ρ Ie

where

yie is a dependent variable corresponding to the measured response, expressed in units which will
depend on the method of measurement used, e.g. area, for a given value of ρie of target compound i
in the calibration;

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ISO 25101:2009(E)

yIe is a dependent variable corresponding to the measured response, expressed in units which will
depend on the method of measurement used, e.g. area, for a given value of ρIe of internal standard I
in the calibration;

ρie is an independent variable corresponding to the mass concentration, expressed in nanograms per
litre, of target compound i in the calibration solution;

ρIe is an independent variable corresponding to the mass concentration, expressed in nanograms per
litre, of internal standard I in the calibration solution;

aiIe is the slope of the calibration curve of yie/yIe as a function of the mass concentration ratio ρie/ρIe,
often called the response factor;

biIe is the ordinate intercept of the calibration curve.

10 Calculation

10.1 Use of the calibration curve to determine the result

Determine the result for each sample using the calibration curve prepared as described in 9.2. The same
procedure shall always be used for calibration, but the concentrations of the internal-standard solutions used
will depend on the samples being analysed. The concentrations used to prepare the calibration curve shall
straddle the concentrations in the samples. Discard, if necessary, high or low points on the curve to provide a
better linear fit over the concentration range most appropriate to the set of samples being analysed. Inject
internal standards regularly to check the stability and linearity of the HPLC-MS/MS system. Prepare a new
calibration curve if the HPLC-MS/MS conditions change or if the results of a calibration check differ by more
than 20 % from the original calibration curve.

10.2 Calculation of results after calibration with internal standards

Calculate the mass concentration ρig of target compound i in accordance with Equation (3) after solving
Equation (2).

⎛ y ig ⎞ ρ Ig
ρ ig = ⎜ − biI e ⎟ ⋅ (3)
⎜ y Ig ⎟ a iI e
⎝ ⎠

where

yig is the measured value, expressed in units which will depend on the method of measurement used,
e.g. area, for target compound i in the sample;

yIg is the measured value, expressed in units which will depend on the method of measurement used,
e.g. area, for internal standard I in the sample;

ρig is the mass concentration of target compound i in the sample, expressed in nanograms per litre;

ρIg see Equation (1);

biIe see Equation (2);

aiIe see Equation (2).

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10.3 Treatment of results lying outside the calibration range

If the concentrations of the target compounds in the sample lie outside the range of the calibration curve, use
a smaller volume of sample for the extraction or dilute the final extract by a suitable factor with the internal-
standard solutions (5.6) in methanol.

11 Expression of results
Report the results for the compounds listed in Table 1, in nanograms per litre, ng/l, to two significant figures.
Results for branched isomers may be reported, but shall be identified as such.

12 Test report
The test report shall include at least the following information:

a) a reference to this International Standard (ISO 25101);

b) identification of the sample;

c) the sample storage and pre-treatment protocol;

d) the results obtained for the individual compounds, expressed in accordance with Clause 11;

e) the recoveries obtained of the internal standards;

f) details of any deviation from the procedure specified and of all circumstances that may have influenced
the results;

g) the date of the analysis.

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ISO 25101:2009(E)

Annex A
(informative)

Examples of suitable sorbents

Table A.1 — Examples of sorbents suitable for solid-phase extraction of analytes

Sorbent Product name (supplier) 1) Amount of sorbent in cartridge

Copolymer Oasis®WAX (Waters) 100 mg to 200 mg

Copolymer Oasis®HLB (Waters) 60 mg to 200 mg
Copolymer [Si(C18H37)] Sep-Pak®tC18 (Waters) 1 g to 10 g
Chromabond®HR-P
Copolymer 100 mg to 200 mg
(Macherey-Nagel)

Sorbents from other suppliers may be suitable, but they have not been evaluated for use in this method.

1) Oasis®WAX, Oasis®HLB, Sep-Pak®tC18 and Chromabond®HR-P are examples of suitable products available
commercially. This information is given for the convenience of users of this International Standard and does not constitute
an endorsement by ISO of these products.

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 ISO 25101:2009(E)

Annex B
(informative)

Suitable HPLC columns

Table B.1 — Examples of suitable HPLC columns

Column Supplier

Betasil® C18 2) Thermo Hypersil-Keystone

ACE® 3 C18 2) Advanced Chromatography Technologies

HPLC columns from other suppliers may be suitable, but they have not been evaluated for use in this method.

2) ACE® 3 C18 and Betasil® C18 are examples of suitable products available commercially. This information is given for
the convenience of users of this International Standard and does not constitute an endorsement by ISO of these products.

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ISO 25101:2009(E)

Annex C
(informative)

Examples of HPLC MS/MS chromatograms

Table C.1 — Conditions used to produce the chromatograms in Figures C.1 and C.2 (for which an
Agilent HP1100 3) liquid chromatograph interfaced with a Micromass Quattro Ultima Pt 3) was used)

HPLC conditions

Injection volume: 10 µl
Flow rate: 0,3 ml/min
Mobile phase: 9:1 2 mM aqueous ammonium acetate solution/methanol (A)
methanol (B)
Guard column: stationary phase ACE 3 C18
inner diameter 2,1 mm
particle size 3 µm
Main column: stationary phase ACE 3 C18
length 150 mm
inner diameter 2,1 mm
particle size 3 µm
Column temperature: 30 °C
Gradient programme (% of B) 30 % at 0,0 min, increase to 85 % at 18 min and keep at this level until 20 min, then
revert to original conditions
MS conditions

Type of equipment: tandem quadrupole

Ionization: ESl negative
Mode: selected ion monitoring (SRM)
Capillary voltage: 1 kV
Temperatures: MS source 120 °C
desolvation temperature 450 °C
Flow rates: cone gas flow rate 60 l/h
desolvation gas flow rate 740 l/h
Cone voltage: for PFOS 90 V
for PFOA 35 V
Collision energy: for PFOS 35 eV
for PFOA 10 eV

3) The Agilent HP1100 liquid chromatograph interfaced with a Micromass Quattro Ultima Pt is an example of a suitable
combination available commercially. This information is given for the convenience of users of this International Standard
and does not constitute an endorsement by ISO of these instruments.

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 ISO 25101:2009(E)

a) PFOS (5 pg/µl)

b) Isotope-labelled PFOS (5 pg/µl)

c) PFOA (5 pg/µl)

d) Isotope-labelled PFOA (5 pg/µl)

Key
X time, min
Y relative response, in percent

Figure C.1 — Chromatograms of internal-standard solutions in methanol

© ISO 2009 – All rights reserved 13

ISO 25101:2009(E)

a) PFOS (4,4 pg/µl)

b) Isotope-labelled PFOS (1 pg/µl)

c) PFOA (1,2 pg/µl)

d) Isotope-labelled PFOA (1 pg/µl)

Key
X time, min
Y relative response, in percent

Figure C.2 — Chromatograms of a sea water extract in methanol

14 © ISO 2009 – All rights reserved

 ISO 25101:2009(E)

Annex D
(informative)

Conditions for analysis of PFOS and PFOA using a single MS

Use of a single MS may cause overestimation of target compounds in some matrices because of interferences
which cannot be removed without MS/MS determination. However, a single MS is widely used as a screening
method and is a possible alternative method to MS/MS provided the method is carefully validated and the
results qualified. The single-MS method is applicable to concentrations ranging from 200 ng/l to 200 000 ng/l
for PFOS and 500 ng/l to 200 000 ng/l for PFOA. Confirmation of the identification of target compounds using
MS/MS (see Annex C) or direct measurement with MS/MS prior to use is necessary.

Table D.1 — Typical conditions

Instruments: Waters/Micromass Quattro Ultima

Type: quadrupole
Ionization: ESI negative
Mode: single-ion monitoring (SIM)
Capillary voltage: 3 kV
Temperatures: MS source 150 °C
Desolvation temperature 350 °C
Flow rates: Cone gas flow rate 140 l/h
Desolvation gas flow rate 460 l/h

Table D.2 — Ions monitored

Component Mass to charge ratio Dwell time Cone voltage

m/z
Da s V

PFOA 369 a 0,20 30

13C a
4 PFOA 372 0,20 30
PFOS 499 0,20 30
13C PFOS 503 0,20 30
4
a Under these conditions, PFOA is predominantly observed as the decarboxylated fragment ion.

© ISO 2009 – All rights reserved 15

ISO 25101:2009(E)

Annex E
(informative)

Precision data

An international interlaboratory trial was performed in February 2007. 24 laboratories from 9 countries took
part (Australia 1, Belgium 1, Canada 3, Germany 7, Italy 1, Japan 2, Switzerland 1, United Kingdom 2, United
States 6). PFOS and PFOA were analysed in sea water (sample 1), river water (sample 2), river water with a
high concentration (sample 3), two different concentrations in distilled water (sample 4, sample 5) and a
standard solution in methanol. Laboratories which did not provide internal-standard recovery results were
rejected as outliers. Results for which the internal-standard recovery did not lie between 70 % and 125 %
were also rejected as outliers as required in 9.2. The precision data are summarized in Table E.1.

Table E.1 — Precision data

l lo n no X η sR CVR sr CVr
Sample Analyte
% ng/l % ng/l % ng/l %

Sea water PFOS 19 9 56 50 40,4 90 8,2 20,2 1,6 3,9

(sample 1) PFOA 17 7 50 54 9,4 97 1,5 16,4 0,4 3,8

River water PFOS 19 6 55 42 9,1 90 2,4 26,8 0,3 3,8

(sample 2) PFOA 18 7 54 39 18,7 91 3,7 19,5 0,6 3,0
River water with high PFOS 16 10 48 65 469 96 92 19,6 20,6 4,4
concentration
(sample 3) PFOA 16 8 48 54 4 440 91 976 22,0 186 4,2

Distilled water with low PFOS 17 7 47 40 2,6 91 0,7 27,0 0,1 3,3
concentration
(sample 4) PFOA 17 2 51 24 16,8 98 3,2 18,8 1,2 7,3

Distilled water with PFOS 18 5 55 31 47,8 96 7,7 16,1 2,1 4,4

high concentration
(sample 5) PFOA 18 5 54 33 359 96 55 15,5 18 5,0

where
l is the number of laboratories;
lo is the number of outlier laboratories;
n is the number of analytical values;
no is the percentage of outlier values;
X is the mean of the values remaining after outlier rejection;
η is the mean internal-standard recovery;
sR is the reproducibility standard deviation;
CVR is the reproducibility coefficient of variation;
sr is the repeatability standard deviation;
CVr is the repeatability coefficient of variation.

16 © ISO 2009 – All rights reserved

 ISO 25101:2009(E)

Annex F
(informative)

Details of the samples used for the interlaboratory trial

F.1 Sea water

The sea water sample for the interlaboratory trial (see Table E.1) was taken off Daikoku Futo which overlooks
Tokyo Bay (Japan). The sample was homogenized by gentle hand shaking. PFOS and PFOA were
determined (see Table F.1). This was the sample with the highest amount of suspended-particle matter
(8,1 mg/l).

Table F.1 — Sea water sample

Analyte Amount of native Result

standard added
ng/l

PFOS 0 40,4
PFOA 0 9,4

F.2 River water

The first river water sample for the interlaboratory trial (sample 2 in Table E.1) was taken from the Arakawa
river in Tokyo (Japan). The sample was homogenized by gentle hand shaking. PFOS and PFOA were
determined (see Table F.2).

Table F.2 — River water sample

Analyte Amount of native Result

standard added
ng/l

PFOS 0 9,1
PFOA 0 18,7

F.3 River water with high concentration

The second river water sample for the interlaboratory trial (sample 3 in Table E.1) was produced from the first
(see Clause F.2) by spiking it, after homogenization by gentle hand shaking, with PFOS and PFOA standards.
PFOS and PFOA were determined (see Table F.3).

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ISO 25101:2009(E)

Table F.3 — River water sample spiked with native standards

Analyte Amount of native Result for river water Result

standard added (sample 2) + amount
added by spiking
ng/l ng/l ng/l

PFOS 439 448 469

PFOA 3 900 3 920 4 440

F.4 Distilled water with low concentration

The first distilled-water sample used for the interlaboratory trial (sample 4 in Table E.1) was spiked with low
levels of PFOS and PFOA standards. PFOS and PFOA were determined (see Table F.4).

Table F.4 — Distilled water spiked with low levels of native standards

Analyte Amount of native Result

standard added
ng/l ng/l

PFOS 2,1 2,6

PFOA 21,0 16,8

F.5 Distilled water with high concentration

The second distilled-water sample used for the interlaboratory trial (sample 5 in Table E.1) was spiked with
high levels of PFOS and PFOA standards. PFOS and PFOA were determined (see Table F.5).

Table F.5 — Distilled water spiked with high levels of native standards

Analyte Amount of native Result

standard added
ng/l ng/l

PFOS 43,2 47,8

PFOA 374 359

18 © ISO 2009 – All rights reserved

 ISO 25101:2009(E)

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[2] TANIYASU, S., et al: Analysis of fluorotelomer alcohols, fluorotelomer acids, and short- and long-chain
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[3] W EREMIUK, A., et al: Quantitative determination of perfluorinated surfactants in water by

LC-ESI-MS/MS, J. Sep. Sci., 29 (2006), pp. 2251-2255

[4] TSENG, C., et al: Analysis of perfluorooctanesulfonate and related fluorochemicals in water and
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1105 (2006), pp. 119-126

[5] LANGLOIS, I., et al: Structural identification of isomers present in technical perfluorooctane sulfonate by
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[6] VILLAGRASA, M., LÓPEZ DE ALDA, M., and BARCELÓ, D.: Environmental analysis of fluorinated alkyl
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[7] VAN LEEUWEN, S., et al: Struggle for quality in determination of perfluorinated contaminants in
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ISO 25101:2009(E)

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