SIST ISO 24293:2011

Water quality - Determination of individual isomers of nonylphenol - Method using solid phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS)

ISO 24293:2009 specifies a method for the determination of selected individual isomers of nonylphenol in non-filtered samples of drinking water, waste water, ground water and surface water. The method is applicable in concentrations between 0,001 µg/l and 0,1 µg/l for individual isomers and from 0,01 µg/l to 0,2 µg/l for the sum of 4-nonylphenol (mixture of isomers). Depending on the matrix, the method is also applicable to waste water in concentrations between 0,1 µg/l and 50 µg/l.

Qualité de l'eau - Détermination des isomères individuels de nonylphénol - Méthode par extraction en phase solide (SPE) et chromatographie en phase gazeuse/spectrométrie de masse (GC/MS)

Kakovost vode - Določevanje posameznih izomer nonilfenola - Metoda plinske kromatografije/masne spektrometrije (GC/MS) po ekstrakciji na trdni fazi (SPE)

Ta mednarodni standard opredeljuje metodo določevanja izbranih posameznih izomer nonilfenola v nefiltriranih vzorcih pitne vode, odpadne vode, podtalne vode in površinske vode. Ta metoda se uporablja pri koncentracijah med 0,001 µg/l in 0,1 µg za posamezen izomer in od 0,01 µg/l do 0,2 µg/l za vsoto 4-nonilfenola (mešanica izomerov). Glede na matrico se metoda uporablja tudi za odpadno vodo pri koncentracijah med 0,1 µg/l in 50 µg/1.

General Information

• Status: Published
• Public Enquiry End Date: 19-May-2011
• Publication Date: 15-May-2011
• 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: 03-May-2011
• Due Date: 08-Jul-2011
• Completion Date: 16-May-2011
• Ref Project: ISO 24293:2009 - Water quality - Determination of individual isomers of nonylphenol - Method using solid phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS)

Overview

ISO 24293:2009 specifies a validated analytical method for the determination of individual isomers of nonylphenol in non-filtered water samples using solid phase extraction (SPE) followed by gas chromatography/mass spectrometry (GC/MS). The standard covers drinking water, ground water, surface water and waste water and defines practical concentration ranges: 0.001–0.1 µg/L for individual isomers, 0.01–0.2 µg/L for the sum of 4‑nonylphenol isomers, and, depending on matrix, 0.1–50 µg/L for some waste water samples.

Key topics and requirements

• Target analytes: Thirteen 4‑nonylphenol isomers (eleven identified and two tentatively identified) that make up the majority of environmentally relevant isomers.
• Sampling & preservation: Non-filtered sampling per ISO 5667‑1; acidify to about pH 3.5 and quench residual chlorine (e.g., sodium thiosulfate) to avoid analyte degradation.
• SPE extraction: Conditioning and extraction using styrene‑divinylbenzene polymer SPE cartridges (typical packing ≥200 mg); extraction flow rates and procedures for high‑suspended‑solids samples are specified.
• Clean‑up & concentration: Solvent elution, drying (nitrogen), and concentration steps prior to GC/MS analysis.
• GC/MS analysis: Temperature‑programmed capillary GC coupled with MS, with quantification based on selected ions; operating conditions and suitable columns are described in informative annexes.
• Standards & calibration: Use of internal standard (example: 4‑n‑nonylphenol solution, 1 ng/µL) and calibration over the total procedure; procedures for blank determination, response factor calculation and recovery checks are provided.
• Documentation: Calculation methods, expression of results, performance data, and test report requirements (including limits of quantification and method performance) are included.

Applications and users

ISO 24293:2009 is intended for:

• Environmental and public‑health laboratories performing water quality monitoring for endocrine‑disrupting compounds.
• Regulatory bodies assessing compliance and contamination of drinking water, surface water, groundwater and waste water.
• Wastewater treatment operators, environmental consultants and research labs conducting source apportionment, trend monitoring or risk assessments for nonylphenol isomers. Practical benefits include isomer‑specific detection (important for toxicity profiling), low detection limits suitable for regulatory monitoring, and a reproducible SPE–GC/MS workflow.

Related standards

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

Keywords: ISO 24293:2009, nonylphenol, 4-nonylphenol, SPE, GC/MS, water quality, isomers, environmental monitoring, drinking water, waste water.

Standards Content (Sample)

SLOVENSKI STANDARD
01-junij-2011
.DNRYRVWYRGH'RORþHYDQMHSRVDPH]QLKL]RPHUQRQLOIHQROD0HWRGDSOLQVNH
NURPDWRJUDILMHPDVQHVSHNWURPHWULMH*&06SRHNVWUDNFLMLQDWUGQLID]L63(
Water quality - Determination of individual isomers of nonylphenol - Method using solid
phase extraction (SPE) and gas chromatography/mass spectrometry (GC/MS)
Qualité de l'eau - Détermination des isomères individuels de nonylphénol - Méthode par
extraction en phase solide (SPE) et chromatographie en phase gazeuse/spectrométrie
de masse (GC/MS)
Ta slovenski standard je istoveten z: ISO 24293:2009
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 24293
First edition
2009-07-15
Water quality — Determination of
individual isomers of nonylphenol —
Method using solid phase extraction
(SPE) and gas chromatography/mass
spectrometry (GC/MS)
Qualité de l'eau — Détermination des isomères individuels de
nonylphénol — Méthode par extraction en phase solide (SPE) et
chromatographie en phase gazeuse/spectrométrie de masse (GC/MS)

Reference number
©
ISO 2009
PDF disclaimer
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accepts no liability in this area.
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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
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 2
4 Reagents. 2
5 Apparatus . 3
6 Sampling and sample pretreatment. 4
7 Procedures . 4
7.1 Solid phase extraction. 4
7.2 Clean up. 5
7.3 GC/MS operating conditions . 5
7.4 Blank determination . 5
7.5 Identification. 5
8 Calibration . 7
8.1 General requirements. 7
8.2 Calibration over the total procedure with internal standard . 7
9 Calculation. 8
9.1 Calculation of contribution of individual isomers of nonylphenol in technical mixture. 8
9.2 Calculation of relative response factor of individual isomers of nonylphenol . 8
9.3 Quantification of individual isomers of nonylphenol using relative response factor . 9
9.4 Calculation of internal standard recovery. 9
10 Expression of results . 10
11 Test report . 10
Annex A (informative) Sorbent example . 11
Annex B (informative) Suitable capillary column. 12
Annex C (informative) Examples of chromatograms. 13
Annex D (informative) Example of FID chromatogram and composition ratio (%) of isomers
in 4-nonylphenol standard. 16
Annex E (informative) Method performance data . 18
Annex F (informative) Description of the matrices of the samples used for the interlaboratory trial. 21
Bibliography . 24

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 24293 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
iv © ISO 2009 – All rights reserved

Introduction
The user should be aware that particular problems could require the specifications of additional marginal
conditions.
INTERNATIONAL STANDARD ISO 24293:2009(E)

Water quality — Determination of individual isomers of
nonylphenol — Method using solid phase extraction (SPE)
and gas chromatography/mass spectrometry (GC/MS)
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 selected individual isomers of
nonylphenol in non-filtered samples of drinking water, waste water, ground water and surface water. The
method is applicable in concentrations between 0,001 µg/l and 0,1 µg/l for individual isomers and from
0,01 µg/l to 0,2 µg/l for the sum of 4-nonylphenol (mixture of isomers). Depending on the matrix, the method is
also applicable to waste water in concentrations between 0,1 µg/l and 50 µg/l.
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, 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
Extraction of the analytes listed in Table 1 from the acidified water sample by solid phase extraction, solvent
elution and determination by gas chromatography with mass spectrometric detection.
The thirteen isomers listed (eleven identified isomers and two isomers with tentative identification) constitute
more than 90 % of the 4-nonylphenol isomers that are detectable in technical products and in environmental
samples in general. Water samples containing more than 500 mg/l of suspended matter and waste water
samples are extracted by passing 100 ml of the sample through the solid phase extraction cartridge (5.2).
Table 1 — Analytes determinable by this method
Analyte Formula Abbreviation
4-(2,4-Dimethylheptan-4-yl)phenol C HO NP1
15 24
4-(2,4-Dimethylheptan-2-yl)phenol C H O NP2

15 24
4-(3,6-Dimethylheptan-3-yl)phenol C H O NP3

15 24
a
4-(3,5-Dimethylheptan-3-yl)phenol C HO NP4
15 24
4-(2,5-Dimethylheptan-2-yl)phenol C H O NP5

15 24
a
4-(3,5-Dimethylheptan-3-yl)phenol C HO NP6
15 24
4-(3-Ethyl-2-methylhexan-2-yl)phenol C HO NP7
15 24
b c
4-(3,4-Dimethylheptan-4-yl)phenol C HO NP8
15 24
e
4-(3,4-Dimethylheptan-3-yl)phenol C HO NP9
15 24
c
4-(3,4-Dimethylheptan-4-yl)phenol C HO NP10
15 24
4-(2,3-Dimethylheptan-2-yl)phenol C HO NP11
15 24
4-(3-Methyloctan-3-yl)phenol C HO NP12
15 24
d e
4-(3,4-Dimethylheptan-3-yl)phenol C H O NP13

15 24
a
Possible enantiomer.
b [6]
Information from MAKINO et al.
c
Possible enantiomer.
d [5]
Information from KATASE et al.
e
Possible enantiomer.
4 Reagents
Use reagents with negligible concentrations of the compounds of interest compared with the concentrations to
be determined. Verify by blank determinations and, if necessary, apply additional cleaning steps.
4.1 Water, grade 1, as specified in ISO 3696.
4.2 Acid, e.g. hydrochloric acid, w(HCl) = 37 %, or sulfuric acid, c(H SO ) = 1 mol/l.
2 4
4.3 Acetone, C H O.
3 6
4.4 Methanol, CH OH.
4.5 Hexane, C H .
6 14
4.6 Sodium sulfate, anhydrous, Na SO , powdered.
2 4
2 © ISO 2009 – All rights reserved

13 13
4.7 Internal standard solution, 4-n-Nonylphenol (ring- C ), C H - C H -OH solution, ρ = 1 ng/µl.
6 9 19 6 4
Weigh 10 mg of 4-n-nonylphenol in a 100 ml measuring flask and bring to volume with methanol (4.4). Dilute
this solution with methanol in the ratio of 1:100. Acetone is not suitable for preparation of standard solution in
this method. Alternative internal standards [e.g. 4-n-nonylphenol (deuterium label)] may be used if internal
standard requirements can be met.
4.8 4-nonylphenol solution, ρ = 1 ng/µl (calibration standard).
Weigh 10 mg of 4-nonylphenol, C H O (technical mixture of isomers), CAS No 25154-52-3, in a 100 ml
15 24
measuring flask and bring to volume with hexane (4.5). Dilute this solution in the ratio of 1:100 with hexane if a
calibration over the total procedure is applied.
4.9 Solid phase material, on styrene-divinylbenzene polymer basis, e.g. commercially available packing
material (see Annex A).
4.10 Nitrogen, N , purity W 99,996 % volume fraction, for drying of the sorbent packing after sample
extraction and for concentration of extracts by evaporation.
4.11 Sodium thiosulfate pentahydrate, Na S O · 5 H O.
2 2 3 2
4.12 Ethyl acetate, C H O .
4 8 2
4.13 Diethyl ether, C H O.
4 10
4.14 Corresponding internal standard solution for syringe spike, phenanthrene (d ), C D solution,
10 14 10
CAS No 85-01-8, ρ = 0,1 ng/µl. Weigh 10 mg of phenanthrene (d ) in a 100 ml measuring flask and bring to
volume with hexane (4.5). Dilute this solution with hexane in the ratio of 1:1 000.
5 Apparatus
Equipment or parts which may come into contact with the water sample or the extract should be free from
interfering compounds.
Clean all glasswares by rinsing with acetone (4.3). Avoid detergents when using a labware washing machine.
Alternatively, heat all glassware, except volumetric wares, to at least 400 °C for at least 2 h prior to use.
5.1 Narrow-neck flat-bottomed glass bottles, conical shoulders, preferably brown glass, of capacity
1 000 ml, with glass stoppers or with PTFE-lined screw caps (PTFE = polytetrafluoroethene).
Keep samples away from light if brown glass bottles are not available. The bottle and cap liner or glass
stopper should be rinsed with acetone (4.3) and dried before use in order to minimize contamination.
5.2 Solid phase extraction cartridges, inert non-leaching plastic, e.g. polypropene or glass.
The cartridges should be packed with a minimum of 200 mg of sorbent (4.9). The commercially available disk
type may be used provided there is enough information available concerning the sample volume and the
required quantity of elution solvent. These cartridges are used for extraction.
5.3 Vacuum or pressure assembly, for the extraction step.
5.4 Volumetric flasks, with inert stopper.
5.5 Quartz wool, rinsed with acetone (4.3).
5.6 Muffle furnace, capable of being maintained at a temperature of 400 °C.
5.7 Evaporation assembly, e.g. rotary evaporator with vacuum stabilizer and water bath.
5.8 Vials, brown glass with PTFE-lined septa, capacity e.g. 1,5 ml, according to the autosampler.
5.9 Gas chromatograph/mass spectrometer. The gas chromatograph shall be temperature-programmable,
with all required accessories including gases, capillary columns, capillary injector and mass spectrometric
detector.
The mass spectrometer should be capable of operating over the mass range of interest and it should be
equipped with a data system capable of quantifying ions using selected m/z values.
5.10 Clean up cartridge column, inert non-leaching plastic, e.g. polypropene or glass.
The cartridges should be packed with a minimum of 200 mg of sorbent (reverse phase, silica). These
cartridges are used for clean up.
5.11 Flame ionization detector.
6 Sampling and sample pretreatment
Take samples as specified in ISO 5667-1.
Use carefully cleaned bottles for sampling (5.1). Fill each bottle only to its shoulder with water to be sampled
(approximately 1 000 ml). In the presence of free chlorine, immediately add approximately 80 mg of sodium
thiosulfate pentahydrate (4.11). Other non-interfering substances may be used for dechlorination as well (e.g.
sodium sulfite). Acidify the samples with acid (4.2) to pH 3,5.
If necessary, store the samples in a refrigerator (2 °C to 5 °C) and analyse them as soon as possible, but not
later than 2 weeks after sampling.
7 Procedures
7.1 Solid phase extraction
7.1.1 General
In general, samples are examined without pretreatment; in other words, suspended solids are not removed
prior to analysis. Before starting the analysis, homogenize the samples. If blocking of the cartridge packing is
likely to occur, use a filter aid, e.g. quartz wool (5.5).
7.1.2 Conditioning of the solid phase material
The following procedures are described for commercially available 6 ml polypropylene cartridges (5.2) packed
with 200 mg of sorbent (4.9) sandwiched between two polyethylene frits. The manufacturer’s guidance for
other materials of the SPE cartridge shall be preferred.
Rinse the cartridge with two 10 ml aliquots of acetone (4.3) and let the cartridge drain dry after the first rinsing.
Before the acetone level of the second aliquot falls below the top edge of the packing, add 10 ml of water (4.1),
acidified with acid (4.2) to pH 3,5, to the cartridge, and make sure that the sorbent packing in the cartridge
does not run dry. Retain the water in the cartridge (water level just above the packing) to keep the sorbent
activated.
7.1.3 Sample extraction
Start the extraction immediately after conditioning. 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.
4 © ISO 2009 – All rights reserved

Add the internal standard solution (4.7), in a known amount (e.g. 50 µl) dependent on the sample matrix, to
the water sample (e.g. 1 000 ml) in the sample bottle and mix thoroughly. Let this sample run through the
cartridge, conditioned as specified in 7.1.2, at a flow rate of 5 ml/min to 10 ml/min. Extract samples containing
more than 500 mg/l of suspended matter and waste water samples by passing a 100 ml sample through the
cartridge. Rinse the cartridge with 10 ml of water (4.1), acidified with acid (4.2) to pH 3,5.
Remove the residual water in the sorbent packing by passing nitrogen through the cartridge at a flow rate of
about 500 ml/min for about 1 h.
NOTE Depending on the colour of the moist adsorbent, the end of the removal of water from the cartridge can be
recognized by the change of colour of sorbent material. The colour of the moist adsorbent is brown; the dry material is light
orange. The end of the removal of water from the cartridge can usually be recognized by brightening of the sorbent
packing.
Reweigh the empty sample bottle with its original cap or stopper and calculate the net weight of sample by
difference to the nearest 1 g. For an assumed density of 1 g/ml, this net weight (in grams) is equivalent to the
volume (in millilitres) of water extracted. The amount of the added volume of acid (4.2) to acidify the sample is
negligible.
7.1.4 Elution
Add 1 ml of acetone (4.3) to the completely dried cartridge, allow to equilibrate for e.g. 10 min and elute
through the cartridge, followed by adding five 1 ml aliquots of acetone (4.3) to the cartridge, but do not allow
the acetone to elute below the top of the sorbent packing during the elution steps.
7.2 Clean up
Concentrate the eluate using a gentle stream of nitrogen to almost dryness. Add 1 ml of hexane (4.5) and
transfer all into a clean up cartridge column (5.10). In general, 500 mg of silica in the cartridge requires the
following extraction procedure. Wash with 10 ml of ethyl acetate (4.12) and subsequently with 15 ml of hexane.
Add the sample and immediately elute with 15 ml of hexane followed by 10 ml of 30 % of diethyl ether (4.13)
in hexane.
To confirm elution profiles of 4-nonylphenol, carry out a separation test using 4-nonylphenol for each batch of
cartridges before analysis. Evaporate the cleaned extract using the evaporation device, concentrate the
extract to a volume of approximately 2 ml and spike 50 µl to 100 µl of corresponding internal standard
substance for syringe spike (4.14) into the extract, then subsequently concentrate the extract further to a
volume of 50 µl to 100 µl using a gentle flow of nitrogen. Transfer the extract to a suitable vial.
7.3 GC/MS operating conditions
Optimize the operating conditions of the GC/MS system in electron ionization mode in accordance to the
manufacturer’s instructions. Determine the appropriate GC oven temperature programme experimentally
during method development and validation. For the sake of sensitivity, selected ions (Table 2) are detected.
An example of operating conditions is given in Annex C.
7.4 Blank determination
Treat the blank in exactly the same way as the sample, but replace the sample by the appropriate amount of
pure water (4.1).
7.5 Identification
Identify the sample component by matching both the retention times and relative intensities of the diagnostic
ions (Table 2) of sample components and calibration standard (4.8). It is a critical issue to identify individual
isomers in order to obtain a similar chromatogram to Annex C that enables accurate identification. Old column
material and inadequate temperature control may result in shifting of retention time between isomers. Reliable
measurements of the thirteen peaks can be enabled by using isomer-specific single ion monitoring. Because
of the absence of all of the fragment ions, 4-n-NP is not suitable. Major ions obtained from 4-n-NP are 107
and 220; detectable ions are obtained for 121, 135 and 149. It is difficult to use 4-n-NP to obtain ions 163 and
191. It is necessary to use a specific pair of ions (target M and qualifier M in Table 2) for the quantification of
1 2
each resolved peak.
The target compound is present (identified) in the sample if:
⎯ the relative or the absolute sample component retention time measured in the selected ion current
chromatogram matches the relative or absolute retention time of the authentic compound within ± 0,2 %
(or a maximum of ± 6 s) in the chromatogram of the latest calibration standard, measured under identical
conditions;
⎯ the selected diagnostic ions (see Table 2) are present at the substance specific retention time;
⎯ the relative intensities of all selected diagnostic ions measured in the sample do not deviate by more than
± (0,1 Q + 10) % from the relative intensities determined in the external standard solution. (Q is the
relative intensity of the diagnostic ion in the external standard solution.)
Table 2 — Selected diagnostic ions for identification and quantification
Selected diagnostic ions
No Analyte Abbreviation
Target Qualifier
a a
M M
1 2
1 4-(2,4-Dimethylheptan-4-yl)phenol NP1 121 163
2 4-(2,4-Dimethylheptan-2-yl)phenol NP2 135 220
3 4-(3,6-Dimethylheptan-3-yl)phenol NP3 135 107 or 121
b
4 4-(3,5-Dimethylheptan-3-yl)phenol NP4 149 191
5 4-(2,5-Dimethylheptan-2-yl)phenol NP5 135 163
b
6 4-(3,5-Dimethylheptan-3-yl)phenol NP6 149 191
7 4-(3-Ethyl-2-methylhexan-2-yl)phenol NP7 135 220
c d
8 4-(3,4-Dimethylheptan-4-yl)phenol NP8 163 121

f
9 4-(3,4-Dimethylheptan-3-yl)phenol NP9 149 107

d
10 4-(3,4-Dimethylheptan-4-yl)phenol NP10 163 121

11 4-(2,3-Dimethylheptan-2-yl)phenol NP11 135 220
12 4-(3-Methyloctan-3-yl)phenol NP12 191 163
e f
13 4-(3,4-Dimethylheptan-3-yl)phenol NP13 135 107
4-n-Nonylphenol (ring- C) — 113 —
a
M is used for quantification; M may be used for identification.
1 2
b
Possible enantiomer.
c [6]
Information from MAKINO et al.
d
Possible enantiomer.
e [5]
Information from KATASE et al.
f
Possible enantiomer.
6 © ISO 2009 – All rights reserved

----------------------
...

INTERNATIONAL ISO
STANDARD 24293
First edition
2009-07-15
Water quality — Determination of
individual isomers of nonylphenol —
Method using solid phase extraction
(SPE) and gas chromatography/mass
spectrometry (GC/MS)
Qualité de l'eau — Détermination des isomères individuels de
nonylphénol — Méthode par extraction en phase solide (SPE) et
chromatographie en phase gazeuse/spectrométrie de masse (GC/MS)

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
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Principle. 2
4 Reagents. 2
5 Apparatus . 3
6 Sampling and sample pretreatment. 4
7 Procedures . 4
7.1 Solid phase extraction. 4
7.2 Clean up. 5
7.3 GC/MS operating conditions . 5
7.4 Blank determination . 5
7.5 Identification. 5
8 Calibration . 7
8.1 General requirements. 7
8.2 Calibration over the total procedure with internal standard . 7
9 Calculation. 8
9.1 Calculation of contribution of individual isomers of nonylphenol in technical mixture. 8
9.2 Calculation of relative response factor of individual isomers of nonylphenol . 8
9.3 Quantification of individual isomers of nonylphenol using relative response factor . 9
9.4 Calculation of internal standard recovery. 9
10 Expression of results . 10
11 Test report . 10
Annex A (informative) Sorbent example . 11
Annex B (informative) Suitable capillary column. 12
Annex C (informative) Examples of chromatograms. 13
Annex D (informative) Example of FID chromatogram and composition ratio (%) of isomers
in 4-nonylphenol standard. 16
Annex E (informative) Method performance data . 18
Annex F (informative) Description of the matrices of the samples used for the interlaboratory trial. 21
Bibliography . 24

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
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International Standard requires approval by at least 75 % of the member bodies casting a vote.
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rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 24293 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.
iv © ISO 2009 – All rights reserved

Introduction
The user should be aware that particular problems could require the specifications of additional marginal
conditions.
INTERNATIONAL STANDARD ISO 24293:2009(E)

Water quality — Determination of individual isomers of
nonylphenol — Method using solid phase extraction (SPE)
and gas chromatography/mass spectrometry (GC/MS)
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 selected individual isomers of
nonylphenol in non-filtered samples of drinking water, waste water, ground water and surface water. The
method is applicable in concentrations between 0,001 µg/l and 0,1 µg/l for individual isomers and from
0,01 µg/l to 0,2 µg/l for the sum of 4-nonylphenol (mixture of isomers). Depending on the matrix, the method is
also applicable to waste water in concentrations between 0,1 µg/l and 50 µg/l.
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, 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
Extraction of the analytes listed in Table 1 from the acidified water sample by solid phase extraction, solvent
elution and determination by gas chromatography with mass spectrometric detection.
The thirteen isomers listed (eleven identified isomers and two isomers with tentative identification) constitute
more than 90 % of the 4-nonylphenol isomers that are detectable in technical products and in environmental
samples in general. Water samples containing more than 500 mg/l of suspended matter and waste water
samples are extracted by passing 100 ml of the sample through the solid phase extraction cartridge (5.2).
Table 1 — Analytes determinable by this method
Analyte Formula Abbreviation
4-(2,4-Dimethylheptan-4-yl)phenol C HO NP1
15 24
4-(2,4-Dimethylheptan-2-yl)phenol C H O NP2

15 24
4-(3,6-Dimethylheptan-3-yl)phenol C H O NP3

15 24
a
4-(3,5-Dimethylheptan-3-yl)phenol C HO NP4
15 24
4-(2,5-Dimethylheptan-2-yl)phenol C H O NP5

15 24
a
4-(3,5-Dimethylheptan-3-yl)phenol C HO NP6
15 24
4-(3-Ethyl-2-methylhexan-2-yl)phenol C HO NP7
15 24
b c
4-(3,4-Dimethylheptan-4-yl)phenol C HO NP8
15 24
e
4-(3,4-Dimethylheptan-3-yl)phenol C HO NP9
15 24
c
4-(3,4-Dimethylheptan-4-yl)phenol C HO NP10
15 24
4-(2,3-Dimethylheptan-2-yl)phenol C HO NP11
15 24
4-(3-Methyloctan-3-yl)phenol C HO NP12
15 24
d e
4-(3,4-Dimethylheptan-3-yl)phenol C H O NP13

15 24
a
Possible enantiomer.
b [6]
Information from MAKINO et al.
c
Possible enantiomer.
d [5]
Information from KATASE et al.
e
Possible enantiomer.
4 Reagents
Use reagents with negligible concentrations of the compounds of interest compared with the concentrations to
be determined. Verify by blank determinations and, if necessary, apply additional cleaning steps.
4.1 Water, grade 1, as specified in ISO 3696.
4.2 Acid, e.g. hydrochloric acid, w(HCl) = 37 %, or sulfuric acid, c(H SO ) = 1 mol/l.
2 4
4.3 Acetone, C H O.
3 6
4.4 Methanol, CH OH.
4.5 Hexane, C H .
6 14
4.6 Sodium sulfate, anhydrous, Na SO , powdered.
2 4
2 © ISO 2009 – All rights reserved

13 13
4.7 Internal standard solution, 4-n-Nonylphenol (ring- C ), C H - C H -OH solution, ρ = 1 ng/µl.
6 9 19 6 4
Weigh 10 mg of 4-n-nonylphenol in a 100 ml measuring flask and bring to volume with methanol (4.4). Dilute
this solution with methanol in the ratio of 1:100. Acetone is not suitable for preparation of standard solution in
this method. Alternative internal standards [e.g. 4-n-nonylphenol (deuterium label)] may be used if internal
standard requirements can be met.
4.8 4-nonylphenol solution, ρ = 1 ng/µl (calibration standard).
Weigh 10 mg of 4-nonylphenol, C H O (technical mixture of isomers), CAS No 25154-52-3, in a 100 ml
15 24
measuring flask and bring to volume with hexane (4.5). Dilute this solution in the ratio of 1:100 with hexane if a
calibration over the total procedure is applied.
4.9 Solid phase material, on styrene-divinylbenzene polymer basis, e.g. commercially available packing
material (see Annex A).
4.10 Nitrogen, N , purity W 99,996 % volume fraction, for drying of the sorbent packing after sample
extraction and for concentration of extracts by evaporation.
4.11 Sodium thiosulfate pentahydrate, Na S O · 5 H O.
2 2 3 2
4.12 Ethyl acetate, C H O .
4 8 2
4.13 Diethyl ether, C H O.
4 10
4.14 Corresponding internal standard solution for syringe spike, phenanthrene (d ), C D solution,
10 14 10
CAS No 85-01-8, ρ = 0,1 ng/µl. Weigh 10 mg of phenanthrene (d ) in a 100 ml measuring flask and bring to
volume with hexane (4.5). Dilute this solution with hexane in the ratio of 1:1 000.
5 Apparatus
Equipment or parts which may come into contact with the water sample or the extract should be free from
interfering compounds.
Clean all glasswares by rinsing with acetone (4.3). Avoid detergents when using a labware washing machine.
Alternatively, heat all glassware, except volumetric wares, to at least 400 °C for at least 2 h prior to use.
5.1 Narrow-neck flat-bottomed glass bottles, conical shoulders, preferably brown glass, of capacity
1 000 ml, with glass stoppers or with PTFE-lined screw caps (PTFE = polytetrafluoroethene).
Keep samples away from light if brown glass bottles are not available. The bottle and cap liner or glass
stopper should be rinsed with acetone (4.3) and dried before use in order to minimize contamination.
5.2 Solid phase extraction cartridges, inert non-leaching plastic, e.g. polypropene or glass.
The cartridges should be packed with a minimum of 200 mg of sorbent (4.9). The commercially available disk
type may be used provided there is enough information available concerning the sample volume and the
required quantity of elution solvent. These cartridges are used for extraction.
5.3 Vacuum or pressure assembly, for the extraction step.
5.4 Volumetric flasks, with inert stopper.
5.5 Quartz wool, rinsed with acetone (4.3).
5.6 Muffle furnace, capable of being maintained at a temperature of 400 °C.
5.7 Evaporation assembly, e.g. rotary evaporator with vacuum stabilizer and water bath.
5.8 Vials, brown glass with PTFE-lined septa, capacity e.g. 1,5 ml, according to the autosampler.
5.9 Gas chromatograph/mass spectrometer. The gas chromatograph shall be temperature-programmable,
with all required accessories including gases, capillary columns, capillary injector and mass spectrometric
detector.
The mass spectrometer should be capable of operating over the mass range of interest and it should be
equipped with a data system capable of quantifying ions using selected m/z values.
5.10 Clean up cartridge column, inert non-leaching plastic, e.g. polypropene or glass.
The cartridges should be packed with a minimum of 200 mg of sorbent (reverse phase, silica). These
cartridges are used for clean up.
5.11 Flame ionization detector.
6 Sampling and sample pretreatment
Take samples as specified in ISO 5667-1.
Use carefully cleaned bottles for sampling (5.1). Fill each bottle only to its shoulder with water to be sampled
(approximately 1 000 ml). In the presence of free chlorine, immediately add approximately 80 mg of sodium
thiosulfate pentahydrate (4.11). Other non-interfering substances may be used for dechlorination as well (e.g.
sodium sulfite). Acidify the samples with acid (4.2) to pH 3,5.
If necessary, store the samples in a refrigerator (2 °C to 5 °C) and analyse them as soon as possible, but not
later than 2 weeks after sampling.
7 Procedures
7.1 Solid phase extraction
7.1.1 General
In general, samples are examined without pretreatment; in other words, suspended solids are not removed
prior to analysis. Before starting the analysis, homogenize the samples. If blocking of the cartridge packing is
likely to occur, use a filter aid, e.g. quartz wool (5.5).
7.1.2 Conditioning of the solid phase material
The following procedures are described for commercially available 6 ml polypropylene cartridges (5.2) packed
with 200 mg of sorbent (4.9) sandwiched between two polyethylene frits. The manufacturer’s guidance for
other materials of the SPE cartridge shall be preferred.
Rinse the cartridge with two 10 ml aliquots of acetone (4.3) and let the cartridge drain dry after the first rinsing.
Before the acetone level of the second aliquot falls below the top edge of the packing, add 10 ml of water (4.1),
acidified with acid (4.2) to pH 3,5, to the cartridge, and make sure that the sorbent packing in the cartridge
does not run dry. Retain the water in the cartridge (water level just above the packing) to keep the sorbent
activated.
7.1.3 Sample extraction
Start the extraction immediately after conditioning. 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.
4 © ISO 2009 – All rights reserved

Add the internal standard solution (4.7), in a known amount (e.g. 50 µl) dependent on the sample matrix, to
the water sample (e.g. 1 000 ml) in the sample bottle and mix thoroughly. Let this sample run through the
cartridge, conditioned as specified in 7.1.2, at a flow rate of 5 ml/min to 10 ml/min. Extract samples containing
more than 500 mg/l of suspended matter and waste water samples by passing a 100 ml sample through the
cartridge. Rinse the cartridge with 10 ml of water (4.1), acidified with acid (4.2) to pH 3,5.
Remove the residual water in the sorbent packing by passing nitrogen through the cartridge at a flow rate of
about 500 ml/min for about 1 h.
NOTE Depending on the colour of the moist adsorbent, the end of the removal of water from the cartridge can be
recognized by the change of colour of sorbent material. The colour of the moist adsorbent is brown; the dry material is light
orange. The end of the removal of water from the cartridge can usually be recognized by brightening of the sorbent
packing.
Reweigh the empty sample bottle with its original cap or stopper and calculate the net weight of sample by
difference to the nearest 1 g. For an assumed density of 1 g/ml, this net weight (in grams) is equivalent to the
volume (in millilitres) of water extracted. The amount of the added volume of acid (4.2) to acidify the sample is
negligible.
7.1.4 Elution
Add 1 ml of acetone (4.3) to the completely dried cartridge, allow to equilibrate for e.g. 10 min and elute
through the cartridge, followed by adding five 1 ml aliquots of acetone (4.3) to the cartridge, but do not allow
the acetone to elute below the top of the sorbent packing during the elution steps.
7.2 Clean up
Concentrate the eluate using a gentle stream of nitrogen to almost dryness. Add 1 ml of hexane (4.5) and
transfer all into a clean up cartridge column (5.10). In general, 500 mg of silica in the cartridge requires the
following extraction procedure. Wash with 10 ml of ethyl acetate (4.12) and subsequently with 15 ml of hexane.
Add the sample and immediately elute with 15 ml of hexane followed by 10 ml of 30 % of diethyl ether (4.13)
in hexane.
To confirm elution profiles of 4-nonylphenol, carry out a separation test using 4-nonylphenol for each batch of
cartridges before analysis. Evaporate the cleaned extract using the evaporation device, concentrate the
extract to a volume of approximately 2 ml and spike 50 µl to 100 µl of corresponding internal standard
substance for syringe spike (4.14) into the extract, then subsequently concentrate the extract further to a
volume of 50 µl to 100 µl using a gentle flow of nitrogen. Transfer the extract to a suitable vial.
7.3 GC/MS operating conditions
Optimize the operating conditions of the GC/MS system in electron ionization mode in accordance to the
manufacturer’s instructions. Determine the appropriate GC oven temperature programme experimentally
during method development and validation. For the sake of sensitivity, selected ions (Table 2) are detected.
An example of operating conditions is given in Annex C.
7.4 Blank determination
Treat the blank in exactly the same way as the sample, but replace the sample by the appropriate amount of
pure water (4.1).
7.5 Identification
Identify the sample component by matching both the retention times and relative intensities of the diagnostic
ions (Table 2) of sample components and calibration standard (4.8). It is a critical issue to identify individual
isomers in order to obtain a similar chromatogram to Annex C that enables accurate identification. Old column
material and inadequate temperature control may result in shifting of retention time between isomers. Reliable
measurements of the thirteen peaks can be enabled by using isomer-specific single ion monitoring. Because
of the absence of all of the fragment ions, 4-n-NP is not suitable. Major ions obtained from 4-n-NP are 107
and 220; detectable ions are obtained for 121, 135 and 149. It is difficult to use 4-n-NP to obtain ions 163 and
191. It is necessary to use a specific pair of ions (target M and qualifier M in Table 2) for the quantification of
1 2
each resolved peak.
The target compound is present (identified) in the sample if:
⎯ the relative or the absolute sample component retention time measured in the selected ion current
chromatogram matches the relative or absolute retention time of the authentic compound within ± 0,2 %
(or a maximum of ± 6 s) in the chromatogram of the latest calibration standard, measured under identical
conditions;
⎯ the selected diagnostic ions (see Table 2) are present at the substance specific retention time;
⎯ the relative intensities of all selected diagnostic ions measured in the sample do not deviate by more than
± (0,1 Q + 10) % from the relative intensities determined in the external standard solution. (Q is the
relative intensity of the diagnostic ion in the external standard solution.)
Table 2 — Selected diagnostic ions for identification and quantification
Selected diagnostic ions
No Analyte Abbreviation
Target Qualifier
a a
M M
1 2
1 4-(2,4-Dimethylheptan-4-yl)phenol NP1 121 163
2 4-(2,4-Dimethylheptan-2-yl)phenol NP2 135 220
3 4-(3,6-Dimethylheptan-3-yl)phenol NP3 135 107 or 121
b
4 4-(3,5-Dimethylheptan-3-yl)phenol NP4 149 191
5 4-(2,5-Dimethylheptan-2-yl)phenol NP5 135 163
b
6 4-(3,5-Dimethylheptan-3-yl)phenol NP6 149 191
7 4-(3-Ethyl-2-methylhexan-2-yl)phenol NP7 135 220
c d
8 4-(3,4-Dimethylheptan-4-yl)phenol NP8 163 121

f
9 4-(3,4-Dimethylheptan-3-yl)phenol NP9 149 107

d
10 4-(3,4-Dimethylheptan-4-yl)phenol NP10 163 121

11 4-(2,3-Dimethylheptan-2-yl)phenol NP11 135 220
12 4-(3-Methyloctan-3-yl)phenol NP12 191 163
e f
13 4-(3,4-Dimethylheptan-3-yl)phenol NP13 135 107
4-n-Nonylphenol (ring- C) — 113 —
a
M is used for quantification; M may be used for identification.
1 2
b
Possible enantiomer.
c [6]
Information from MAKINO et al.
d
Possible enantiomer.
e [5]
Information from KATASE et al.
f
Possible enantiomer.
6 © ISO 2009 – All rights reserved

8 Calibration
8.1 General requirements
For practical reasons, the calibration is based on a solution containing the calibration standard (4.8).
Ensure that 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 substance is valid only for the measured concentration range. Additionally, the
calibration function dep
...