SIST ISO 25101:2010

Water quality - Determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) - Method for unfiltered samples using solid phase extraction and liquid chromatography/mass spectrometry

ISO 25101:2009 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 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.

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

Kakovost vode - Določevanje perfluorooktansulfonata (PFOS) in perfluorooktanoata (PFOA) - Metoda za nefiltrirane vzorce z ekstrakcijo na trdni fazi in s tekočinsko kromatografijo z masno selektivnim detektorjem

Ta mednarodni standard določa metodo določevanja linearnih izomerov  perfluorooktansulfonata (PFOS)in perfluorooktana (PFOA) v nefiltriranih vzorcih pitne, podzemne in površinke vode (sladke in morske vode) z visoko zmogljivo tekočinsko kromatografijo z masno selektivnim detektorjem (HPLC-MS/MS). Druge izomere se lahko priglasi ločeno kot nelinerane izomere in so določeni kot taki. Analiti, opredeljeni v Preglednici 1, se lahko določijo po tej metodi. Metoda se uporablja za razpon koncentracije od 2.0 ng/l do 10 000 ng/l za PFOS in od 10 ng/l do 10 000 ng/l za PFOA. Odvisno od matrice se ta metoda lahko uporabi tudi za višje koncentracije v razponu od 100 ng/l do 200 000 ng/l po primernem razredčevanju vzorca ali zmanjševanju velikosti vzorca.

General Information

• Status: Published
• Public Enquiry End Date: 19-Jul-2009
• Publication Date: 05-Jul-2010
• ICS: 13.060.50 - Examination of water for chemical substances
• Technical Committee: KAV - Water quality
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 12-May-2010
• Due Date: 17-Jul-2010
• Completion Date: 06-Jul-2010
• Ref Project: ISO 25101:2009 - Water quality - Determination of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) - Method for unfiltered samples using solid phase extraction and liquid chromatography/mass spectrometry

Overview

ISO 25101:2009 specifies a validated analytical method for the determination of the linear isomers of perfluorooctanesulfonate (PFOS) and perfluorooctanoate (PFOA) in unfiltered water samples. The standard targets drinking water, ground water and surface water (fresh and sea water) and combines solid‑phase extraction (SPE) sample preparation with high‑performance liquid chromatography–tandem mass spectrometry (HPLC‑MS/MS) for selective, sensitive quantification. The method reports other isomers separately as non‑linear isomers where applicable.

Key topics and technical requirements

• Analytes and scope
  • Linear PFOS and PFOA are the primary analytes. Non‑linear isomers may be reported separately.
  • Typical working ranges: PFOS 2.0 ng/L to 10 000 ng/L; PFOA 10 ng/L to 10 000 ng/L. Higher concentrations (100–200 000 ng/L) can be accommodated after dilution or reduced sample size.
• Sample handling
  • Use inert, non‑fluoropolymer sample bottles; avoid glassware for sampling to prevent adsorption losses.
  • If free chlorine is present, add a dechlorinating agent (e.g., sodium thiosulfate).
  • Store at (4 ± 2) °C and analyse within two weeks or freeze with verified stability.
• Solid‑phase extraction (SPE)
  • Copolymer‑based SPE sorbents are recommended (typical cartridges contain 150–250 mg sorbent).
  • Conditioning sequence and careful wetting of sorbent are required; extraction is performed on unfiltered (whole) samples.
• Instrumental analysis
  • Separation by HPLC followed by tandem mass spectrometry (MS/MS) for selected precursor → product ion monitoring.
  • Annex D describes limited use with single MS; Annexes A–C give sorbent, column and chromatogram examples.
• Quality control
  • Use 13C‑labelled internal standards (e.g., 13C‑PFOA, 13C‑PFOS).
  • Check for matrix effects (ion suppression/enhancement), especially in seawater and wastewater.
  • Avoid fluoropolymer contamination in instrument plumbing and vial caps; procedural blanks should be substantially lower than expected sample concentrations.

Applications and users

ISO 25101:2009 is used for:

• Regulatory compliance testing and environmental monitoring of PFOS/PFOA in water.
• Drinking‑water utilities, environmental laboratories and contract testing labs.
• Research institutions, consultants and regulatory agencies performing occurrence, fate and risk assessments.
• Situations requiring trace‑level quantification of perfluoroalkyl substances (PFAS) in unfiltered waters.

Keywords: ISO 25101:2009, PFOS, PFOA, perfluorooctanesulfonate, perfluorooctanoate, HPLC‑MS/MS, solid phase extraction, water quality, unfiltered samples.

Related standards

• ISO 3696 (Water for analytical laboratory use)
• ISO 5667‑1 (Sampling - guidance on sampling programmes)
• ISO 8466‑1 (Calibration and evaluation of analytical methods)

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2010
.DNRYRVWYRGH'RORþHYDQMHSHUIOXRURRNWDQVXOIRQDWD3)26LQ
SHUIOXRURRNWDQRDWD3)2$0HWRGD]DQHILOWULUDQHY]RUFH]HNVWUDNFLMRQDWUGQL
ID]LLQVWHNRþLQVNRNURPDWRJUDILMR]PDVQRVHOHNWLYQLPGHWHNWRUMHP
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
Ta slovenski standard je istoveten z: ISO 25101:2009
ICS:
13.060.50 3UHLVNDYDYRGHQDNHPLþQH Examination of water for
VQRYL chemical substances
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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 2009
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2009
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2009 – All rights reserved

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

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).
Table 1 — Analytes determinable by this method
a b
Analyte Formula Abbreviation CAS No.
Perfluoro-n-octanesulfonic acid
CF (CF ) SOH PFOS 1763-23-1
3 2 7 3
(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
c
CF (CF )COOH PFOA 335-67-1
3 2 6
(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.
2 © ISO 2009 – All rights reserved

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(CH COOH) = 99,9 % mass fraction.
5.3 Ammonia solution, w(NH ) = 25 % mass fraction.
5.4 Ammonium acetate, w(CH COONH ) = 97 % mass fraction.
3 4
5.5 Methanol (CH OH), HPLC grade.
5.6 Internal-standard solutions:
1,2,3,4- C -PFOA, ρ = 1 ng/µl.
1,2,3,4- C -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. C -PFNA [perfluoro-n-nonanoic acid, CF (CF ) COOH)], that meet the
5 3 2 6
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 (N ), purity > 99,996 %.
5.12 Sodium thiosulfate pentahydrate (Na S O ⋅ 5H O).
2 2 3 2
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.
4 © ISO 2009 – All rights reserved

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 ionizatio
...

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 2009
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2009
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail [e-mail]
Web www.iso.org
Published in Switzerland
ii © ISO 2009 – All rights reserved

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

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).
Table 1 — Analytes determinable by this method
a b
Analyte Formula Abbreviation CAS No.
Perfluoro-n-octanesulfonic acid
CF (CF ) SOH PFOS 1763-23-1
3 2 7 3
(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
c
CF (CF )COOH PFOA 335-67-1
3 2 6
(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.
2 © ISO 2009 – All rights reserved

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(CH COOH) = 99,9 % mass fraction.
5.3 Ammonia solution, w(NH ) = 25 % mass fraction.
5.4 Ammonium acetate, w(CH COONH ) = 97 % mass fraction.
3 4
5.5 Methanol (CH OH), HPLC grade.
5.6 Internal-standard solutions:
1,2,3,4- C -PFOA, ρ = 1 ng/µl.
1,2,3,4- C -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. C -PFNA [perfluoro-n-nonanoic acid, CF (CF ) COOH)], that meet the
5 3 2 6
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 (N ), purity > 99,996 %.
5.12 Sodium thiosulfate pentahydrate (Na S O ⋅ 5H O).
2 2 3 2
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.
4 © ISO 2009 – All rights reserved

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 batc
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