PD ISO/TS 28581:2012

BSI Standards Publication

Water quality — Determination

of selected non-polar
substances — Method using
gas chromatography with mass
spectrometric detection (GC-
MS)
PD ISO/TS 28581:2012 PUBLISHED DOCUMENT

National foreword
This Published Document is the UK implementation of ISO/TS
28581:2012.
The UK participation in its preparation was entrusted to Technical
Committee EH/3/2, Physical chemical and biochemical methods.
A list of organizations represented on this committee can be
obtained on request to its secretary.
This publication does not purport to include all the necessary
provisions of a contract. Users are responsible for its correct
application.
© The British Standards Institution 2012. Published by BSI Standards
Limited 2012
ISBN 978 0 580 66221 8
ICS 13.060.50
Compliance with a British Standard cannot confer immunity from
legal obligations.
This Published Document was published under the authority of the
Standards Policy and Strategy Committee on 31 March 2012.
Amendments issued since publication
Date Text affected
 PD ISO/TS 28581:2012
TECHNICAL ISO/TS
SPECIFICATION 28581

First edition
2012-02-15

Water quality — Determination of

selected non-polar substances — Method
using gas chromatography with mass
spectrometric detection (GC-MS)
Qualité de l’eau — Détermination de substances non polaires
sélectionnées — Méthode par chromatographie en phase gazeuse avec
détection par spectrométrie de masse (CG-SM)

Reference number
ISO/TS 28581:2012(E)

© ISO 2012
PD ISO/TS 28581:2012
ISO/TS 28581:2012(E)

COPYRIGHT PROTECTED DOCUMENT

©  ISO 2012
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 [email protected]
Web www.iso.org
Published in Switzerland

ii  © ISO 2012 – All rights reserved

 PD ISO/TS 28581:2012
ISO/TS 28581:2012(E)

Contents Page

Foreword............................................................................................................................................................................. iv
Introduction......................................................................................................................................................................... v
1 Scope....................................................................................................................................................................... 1
2 Normative references.......................................................................................................................................... 1
3 Terms and definitions.......................................................................................................................................... 2
4 Principle.................................................................................................................................................................. 2
5 Interferences.......................................................................................................................................................... 4
5.1 Interferences with sampling, extraction and concentration..................................................................... 4
5.2 Interferences with gas chromatography........................................................................................................ 5
5.3 Interferences with GC-MS.................................................................................................................................. 6
6 Reagents................................................................................................................................................................. 6
7 Apparatus............................................................................................................................................................... 9
7.1 General requirements.......................................................................................................................................... 9
8 Sampling............................................................................................................................................................... 10
9 Procedure............................................................................................................................................................. 11
9.1 General considerations.................................................................................................................................... 11
9.2 Extraction............................................................................................................................................................. 11
9.3 Gas chromatography......................................................................................................................................... 12
9.4 Blank measurement........................................................................................................................................... 12
9.5 Mass spectrometric conditions...................................................................................................................... 12
10 Calibration............................................................................................................................................................ 13
10.1 General.................................................................................................................................................................. 13
10.2 Calibration by labelled internal standards.................................................................................................. 13
10.3 Calibration by internal standard..................................................................................................................... 13
11 Measurement of samples................................................................................................................................. 14
12 Identification........................................................................................................................................................ 14
13 Calculation........................................................................................................................................................... 17
13.1 Quantification by internal standards............................................................................................................ 17
13.2 Quantification by labelled internal standards............................................................................................ 18
13.3 Recovery of internal standards...................................................................................................................... 19
13.4 Concentration in the sample........................................................................................................................... 19
14 Expression of results........................................................................................................................................ 19
15 Test report............................................................................................................................................................ 20
Annex A (informative) Examples of GC-MS conditions........................................................................................... 21
Annex B (informative) Examples for the construction of special apparatus.................................................... 22
Annex C (informative) Silica clean-up.......................................................................................................................... 24
Bibliography...................................................................................................................................................................... 25

© ISO 2012 – All rights reserved  iii

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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.

In other circumstances, particularly when there is an urgent market requirement for such documents, a technical
committee may decide to publish other types of document:

— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in
an ISO working group and is accepted for publication if it is approved by more than 50 % of the members
of the parent committee casting a vote;

— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
committee and is accepted for publication if it is approved by 2/3 of the members of the committee
casting a vote.

An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further
three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed,
it is reviewed again after a further three years, at which time it must either be transformed into an International
Standard or be withdrawn.

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/TS 28581 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 2, Physical,
chemical and biochemical methods.

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Introduction

Non-polar substances occur in nearly all types of water. These substances are adsorbed on solids (sediments,
suspended matter) as well as dissolved in the liquid phase.

A large group of non-polar substances are polycyclic aromatic hydrocarbons (PAH). Some PAH are known or
suspected to cause cancer. Maximum acceptable levels have been set in a number of countries. For instance,
the European Council Directive 98/83/EC[10] on the quality of water intended for human consumption set the
maximum acceptable level for benzo[a]pyrene at 0,010 µg/l, and for the sum of four specified PAH (benzo[b]
fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, indeno[1,2,3-cd]pyrene) at 0,100 µg/l.

There are further International Standards for the analytical determination of PAH in water and waste water.

ISO  6468 specifies methods for the determination of certain organochlorine insecticides, polychlorinated
biphenyls and chlorobenzenes in drinking water, ground water, surface water and waste water.

ISO 17993[6] specifies methods for the determination of 15 PAH by high performance liquid chromatography in
drinking water, ground water and surface water.

ISO 7981[2] specifies methods for the determination of 6 PAH by high performance thin layer chromatography
or by high performance liquid chromatography in drinking water and ground water.

ISO 17858[5] specifies methods for the determination of dioxin-like polychlorinated biphenyls in waters and
waste waters.

ISO 28540 [9] specifies the determination of PAH using gas chromatography with mass spectrometric
detection (GC-MS).

© ISO 2012 – All rights reserved  v

PD ISO/TS 28581:2012
 PD ISO/TS 28581:2012
TECHNICAL SPECIFICATION ISO/TS 28581:2012(E)

Water quality — Determination of selected non-polar

substances — Method using gas chromatography with mass
spectrometric detection (GC-MS)
WARNING  — The use of this Technical Specification may involve hazardous materials, operations
and equipment.

Persons using this Technical Specification should be familiar with normal laboratory practice. This
document 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 according to this Technical Specification
be carried out by suitably trained staff.

1 Scope
This Technical Specification specifies a method for the determination by gas chromatography with mass
spectrometric detection (GC-MS) of polycyclic hydrocarbons and pesticide residues in drinking water and ground
water at mass concentrations above 0,005 µg/l and surface water and waste water at mass concentrations
above 0,01 µg/l (for each single compound).

This method can apply to non-polar substances other than polycylic aromatic hydrocarbons (PAH) and pesticide
residues. However, it is necessary to verify the applicability of this method for these compounds.

NOTE 1 A potentially suitable method for this verification is specified in ISO/TS 13530.[3]

This Technical Specification can be used for samples containing up to 150 mg/l of suspended matter.

NOTE 2 Determination of PAH using GC-MS lies within the scope of ISO 28540.[9]

2 Normative references
The following document, in whole or in part, are normatively referenced in this document and are indispensable
for its application. For dated references, only the edition cited applies. For undated references, the latest edition
of the referenced document (including any amendments) applies.

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

ISO 5667-3, Water quality — Sampling — Part 3: Preservation and handling of water samples

ISO 6468, Water quality — Determination of certain organochlorine insecticides, polychlorinated biphenyls and
chlorobenzenes — Gas-chromatographic method after liquid-liquid extraction

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

© ISO 2012 – All rights reserved  1

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ISO/TS 28581:2012(E)

3 Terms and definitions

For the purposes of this document, the following terms and definitions apply.

3.1
analyte
substance to be determined

[SOURCE: ISO 15089:2000,[4] definition 3.2]

Note 1 to entry Substances covered by this specification are listed in Table 1.

3.2
calibration standard
solution prepared from a secondary standard and/or stock solutions and used to calibrate the response of the
instrument with respect to analyte concentration

[SOURCE: ISO 18073:2004,[7] definition 3.1.2]

3.3
diagnostic ion
selected fragment ion, molecular ion or other characteristic ion from the mass spectrum of the target compound
with the highest possible specificity

[SOURCE: ISO 22892:2006,[8] definition 3.6]

3.4
injection standard
standard mixture added to a sample before injection into the GC-MS apparatus, to monitor variability of
instrument response and to calculate internal standard recovery

3.5
internal standard
isotopically labelled standard or a non-polar substance added to samples prior to extraction, unlikely to be
present in the sample, against which the concentrations of native substances are calculated

Note 1 to entry The substance is added to the sample before extraction and is used for quantification of the components
to be measured. Recoveries of these standards are also calculated and used to check the performance of the procedure.

3.6
native compound
non-labelled compound

3.7
selected ion mode
SIM
selected ion recording
SIR
measuring the intensity of selected diagnostic ions only

[SOURCE: ISO 22892:2006,[8] definition 3.8, modified — the last two synonyms have been added.]

4 Principle
The non-polar substances determinable by the method specified in this Technical Specification are listed in Table 1.

The non-polar substances present in the aqueous sample are extracted from the water sample by liquid-
liquid extraction with hexane. An internal standard mixture is added to the sample prior to extraction. The
extract is concentrated by evaporation and the residue taken up in a solvent appropriate for clean-up or gas
chromatography (GC).

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Other volatile solvents can also be used if it is proven that there is equal or better recovery (recovery between
70 % and 110 %).

NOTE Other possible suitable solvents are: isohexane C6H15 (CAS: 107-83-5); cyclohexane: C6H12 (CAS: 110-82-7);
pentane: C5H12 (CAS: 109-66-0); petroleum ether: boiling range 40 °C to 60 °C.

The liquid-liquid extraction method shall not be used with samples containing more than 150  mg/l of
suspended matter.

If necessary, extracts of surface water or waste water samples can be cleaned by column chromatography
prior to analysis. Prior to injection, injection standards are added to each extract, and an aliquot of the extract
is injected into the gas chromatograph.

The non-polar substances are separated on a suitable fused silica capillary column, coated with a film of cross-
linked non-polar polysiloxane or slightly polar modified polysiloxane with an efficient separation. The column
shall be suitable for separating critical and isomeric pairs of substances. Identification and quantification is
performed by means of mass spectrometry (MS) using electron impact ionization (EI).

Table 1 — Non-polar substances determinable that can be determined

by using this Technical Specification

Molar mass
Name Molecular formula CAS number
g/mol
PAH
Naphthalene C10H8 128,17 91-20-3
Acenaphthylene C12H8 152,20 208-96-8
Acenaphthene C12H10 154,21 83-32-9
Fluorene C13H10 166,22 86-73-7
Phenanthrene C14H10 178,23 85-01-8
Anthracene C14H10 178,23 120-12-7
Pyrene C16H10 202,26 129-00-0
Fluoranthene C16H10 202,26 206-44-0
Chrysene C18H12 228,29 218-01-9
Benzo[a]anthracene C18H12 228,29 56-55-3
Benzo[b]fluoranthene C20H12 252,32 205-99-2
Benzo[k]fluoranthene C20H12 252,32 207-08-9
Benzo[a]pyrene C20H12 252,32 50-32-8
Dibenzo[a,h]anthracene C22H14 278,35 053-70-3
Benzo[ghi]perylene C22H12 276,34 191-24-2
Indeno[1,2,3-cd]pyrene C22H12 276,34 193-39-5
PCB    
PCB-28: 2,4,4′-trichlorobiphenyl C12H7Cl3 257,54 7012-37-5
PCB-52: 2,2′,5,5′-tetrachlorobiphenyl C12H6Cl4 291,99 35693-99-3
PCB-101: 2,2′,4,5,5′-pentachlorobiphenyl C12H5Cl5 326,43 37680-73-2
PCB-118: 2,3′,4,4′,5-pentachlorobiphenyl C12H5Cl5 326,43 31508-00-6
PCB-138: 2,2′,3,4,4′,5′-hexachlorobiphenyl C12H4Cl6 360,88 35065-28-2
PCB-153: 2,2′,4,4′,5,5′-hexachlorobiphenyl C12H4Cl6 360,88 35065-27-1
PCB-180: 2,2′,3,4,4′,5,5′-heptachlorobiphenyl C12H3Cl7 395,33 35065-29-3
OCP    
Hexachlorobenzene (HCB) C6Cl6 284,78 118-74-1
α-Hexachlorocyclohexane (α-HCH) C6H6Cl6 290,83 319-84-6
β-Hexachlorocyclohexane (β-HCH) C6H6Cl6 290,83 319-85-7

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Table 1 (continued)

Molar mass
Name Molecular formula CAS number
g/mol
γ-Hexachlorocyclohexane (γ-HCH) C6H6Cl6 290,83 58-89-9
δ-Hexachlorocyclohexane (δ-HCH) C6H6Cl6 290,83 319-86-8
ε-Hexachlorocyclohexane (ε-HCH) C6H6Cl6 290,83 6108-10-7
Aldrin C12H8Cl6 364,93 309-00-2
Dieldrin C12H8Cl6O 380,91 60-57-1
Endrin C12H8Cl6O 380,91 72-20-8
Heptachlor C10H5Cl7 373,32 76-44-8
Heptachlor epoxide (exo-, cis- or b-isomer) C10H5Cl7O 389,30 28044-83-9
Heptachlor epoxide (endo-, trans- or a-isomer) C10H5Cl7O 389,30 1024-57-3
α-Endosulfan C9H6Cl6O3S 406,92 959-98-8
β-Endosulfan C9H6Cl6O3S 406,92 33213-65-9
p,p′-DDE C14H8Cl4 318,02 72-55-9
o,p′-DDD C14H10Cl4 320,04 53-19-0
o,p′-DDT C14H9Cl5 354,49 784-02-6
p,p′-DDD C14H10Cl4 320,04 72-54-8
o,p′-DDE C14H8Cl4 318,02 3424-82-6
p,p′-DDT C14H9Cl5 354,49 50-29-3
Methoxychlor C16H15Cl3O2 345,65 72-43-5
Chlorobenzenes  
1,2,4-Trichlorobenzene C6H3Cl3 181,45 120-82-1
1,2,3-Trichlorobenzene C6H3Cl3 181,45 87-61-6
1,3,5-Trichlorobenzene C6H3Cl3 181.45 108-70-3
1,2,3,4-Tetrachlorobenzene C6H2Cl4 215,89 634-66-2
1,2,3,5-Tetrachlorobenzene C6H2Cl4 215,89 634-90-2
1,2,4,5-Tetrachlorobenzene C6H2Cl4 215,89 95-94-3
Pentachlorobenzene C6HCl5 250,34 608-93-5
Pentachloronitrobenzene C6Cl5NO2 295,34 82-68-8
Organophosphorus  
Azinphos-ethyl C12H16N3O3PS2 345,40 2642-71-9
Bromofenvinphos-ethyl C12H14BrCl2O4P 404,02 33399-00-7
Chlorofenvinphos C12H14Cl3O4P 359,57 470-90-6
Chloropyriphos-ethyl C9H11Cl3NO3PS 350,59 2921-88-2
Chloropyriphos-methyl C7H7Cl3NO3PS 322,53 5598-13-0
Heptenophos C9H12ClO4P 250,02 23560-59-0

5 Interferences

5.1 Interferences with sampling, extraction and concentration

Use sampling containers of materials that do not affect the analyte content during the contact time (preferably
of stainless steel or glass). Avoid plastics and organic materials other than polytetrafluoroethene (PTFE) during
sampling, sample storage or extraction. Care should be taken with the use of surfactants for cleaning sample
containers because they may lead to the formation of emulsions during liquid-liquid extraction.

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If automatic samplers are used, avoid the use of silicone or rubber material for the tubes. If these materials are
present, ensure that the contact time is minimized. Rinse the sampling line with the water to be sampled before
taking the test sample. Use ISO 5667-1 and ISO 5667-3 for guidance.

Keep the test samples away from direct sunlight and prolonged exposure to light. Store the samples in coloured
containers. Clear glass bottles are also suitable, but then the samples shall be kept in a dark box.

During storage of the test samples, loss of components may occur due to adsorption on the walls of the
containers. The extent of the losses may depend on the storage time.

Concentration of organic solvents can lead to loss of volatile components like naphthalene, chlorobenzenes
and phosphorous containing pesticides.

5.2 Interferences with gas chromatography

Non-polar substances are separated on a suitable fused silica capillary column, coated with a film of cross-
linked non-polar polysiloxane or slightly polar modified polysiloxane with an efficient separation. The column
shall be suitable for the separation of benzo[a]pyrene and benzo[e]pyrene. Identification and quantification
is performed by means of MS using electron-impact ionization (EI). Sufficient resolution (e.g. not less than
R = 0,8) between the peaks of benzo[b]fluoranthene and benzo[k]fluoranthene as well as of benzo[a]pyrene
and benzo[e]pyrene is to be set as a quality criterion for the capillary column. Benzo[ j]fluoranthene cannot
be separated from benzo[k]fluoranthene and benzo[b]fluoranthene. It is possible that triphenylene is not
completely separated from benzo[a]anthracene and chrysene. If this occurs, state this fact in the test report.

NOTE Benzo[ j]fluoranthene, benzo[e]pyrene and triphenylene are not part of the 16 target PAH analytes.

Chromatographic separation between the following pairs can be critical. Due to their molecular mass differences,
quantification can be made by mass selective detection. When incomplete resolution is encountered, peak
integration shall be checked and, when necessary, corrected.

— PCB 52 – PCB 73;

— PCB 101 – PCB 89/PCB 90;

— PCB 118 – PCB 106;

— PCB 138 – PCB 164/PCB 163.

Interferences between the following isomeric pairs of chlorobiphenyls can also be critical as they have the
same mass and fragmentation pattern. Therefore, the resolution between the compounds should be R > 0,8.

PCB Ballschmitter No.

— Trichloro PCB 28 – PCB 31

— Tetrachloro PCB 52 – PCB 43

— Pentachloro PCB 101 – PCB 113

  PCB 118 – PCB 149

— Hexachloro PCB 153/PCB 168 – PCB132

  PCB 138/PCB 164/PCB163 – PCB PCB160

— Heptachloro PCB 180 – PCB 193

Adsorptions and disruption of selected parameters, for example 4,4′-DDT (p,p′-DDT); 2,4′-DDT (o,p′-DDT)
and/or endrin, can occur in the injector.

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5.3 Interferences with GC-MS

Substances that co-elute with the target components may interfere with the determination. These interferences
may lead to incompletely resolved signals and may, depending on their magnitude, affect accuracy and
precision of the analytical results. Non-symmetrical peaks and peaks that are broader than the corresponding
peaks of the reference substance suggest interferences.

Chromatographic separation between dibenzo[a,h]anthracene and indeno[1,2,3-cd]pyrene is mostly critical. Due

to their molecular mass differences, quantification can be made by mass selective detection. When incomplete
resolution is encountered, peak integration shall be checked and, if necessary, the baseline corrected.

6 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade, “for residue
analysis” or “for GC analysis”, where appropriate, and distilled or demineralized water or water of equivalent
purity. Pay extra attention that each batch of solvents does not contain blank concentrations affecting the results.

6.1 Solids

6.1.1 Sodium sulfate, Na2SO4, anhydrous, precleaned by heating to 500 °C for 4 h or free of interfering compounds.

6.2 Solvents

6.2.1 Hexane, C6H14.

6.2.2 Acetonitrile, CH3CN.

6.2.3 Acetone, C3H6O.

6.2.4 Decane, C10H22.

6.2.5 Isooctane, C8H18.

6.2.6 Dichloromethane, CH2Cl2.

6.3 Gases

6.3.1 Nitrogen, volume fraction 99,999 %, for evaporating the extracts.

6.4 Standards

6.4.1 Reference substances (see Table 2) and internal standards.

Choose internal standards with physical and chemical properties (such as extraction behaviour, retention time)
that are similar to those of the compounds to be analysed.

Use an internal standard for every class of compounds for the GC-MS method to evaluate results. Use at least
two internal standards per class of substance. Verify the stability of the internal standards regularly. Table 2
contains compounds that can be used. The internal standards are added to the sample to be extracted and are
therefore dissolved in a water-soluble solvent.

NOTE 13C isotopically labelled standards can also be used as internal standard.

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Certified solutions of non-polar substances of certified purity are available from a limited number of suppliers,
e.g. the Institute for Reference Materials and Measurements (IRMM)1), the National Institute of Science and
Technology (NIST)2) or other commercial providers. Because of the dangerous nature of the substances used,
commercially available, preferably certified, standard solutions should be used. Skin contact should be avoided.

Table 2 — Native and deuterated non-polar substances

Native reference substances Labelled internal standard substances

PAH PAH
Naphthalene Naphthalene-d8 (CAS No. 1146-65-2)
Acenaphthene Acenaphthene-d10 (CAS No. 15067-26-2)
Acenaphthylene Acenaphthylene-d8 (CAS No. 93951-97-4)
Fluorene Fluorene-d10 (CAS No. 81103-79-9)
Anthracene Anthracene-d10 (CAS No. 1719-06-8)
Phenanthrene Phenanthrene-d10 (CAS No. 1517-22-2)
Fluoranthene Fluoranthene-d10 (CAS No. 93951-69-0)
Pyrene Pyrene-d10 (CAS No. 1718-52-1)
Benzo[a]anthracene Benzo[a]anthracene-d12 (CAS No. 1718-53-2)
Chrysene Chrysene-d12 (CAS No. 1719-03-5)
Benzo[b]fluoranthene Benzo[b]fluoranthene-d12 (CAS No. 93951-98-5)
Benzo[ j]fluoranthenea (CAS No. 205-82-3)  
Triphenylenea (CAS No 217-59-4)  
Benzo[k]fluoranthene Benzo[k]fluoranthene-d12 (CAS No. 93952-01-3)
Benzo[a]pyrene Benzo[a]pyrene-d12 (CAS No. 63466-71-7)
Benzo[e]pyrenea (CAS No. 192-97-2) d12 Available (CIL)b
Indeno[1,2,3-cd]pyrene Indeno[1,2,3-cd]pyrene-d12 (CAS No. 203578-33-0)
Dibenzo[a,h]anthracene Dibenzo[a,h]anthracene-d14 (CAS No. 13250-98-1)
Benzo[ghi]perylene Benzo[ghi]perylene-d12 (CAS No. 93951-66-7)
PCB-28: 2,4,4′-trichlorobiphenyl PCB-28: 13C-2,4,4′-trichlorobiphenyl
PCB-52: 2,2′,5,5′-tetrachlorobiphenyl PCB-52: 13C-2,2′,5,5′-tetrachlorobiphenyl
PCB-101: 2,2′,4,5,5′-pentachlorobiphenyl PCB-101: 13C-2,2′,4,5,5′-pentachlorobiphenyl
PCB-118: 2,3′,4,4′,5-pentachlorobiphenyl PCB-118: 13C12- 2,3′,4,4′,5-pentachlorobiphenyl
(CAS No. 104130-40-7)
PCB-138: 2,2′,3,4,4′,5′-hexachlorobiphenyl PCB-138: 13C-2,2′,3,4,4′,5′-hexachlorobiphenyl
(CAS No. 35065-28-2)
PCB-153: 2,2′,4,4′,5,5′-hexachlorobiphenyl PCB-153: 13C-2,2′,4,4′,5,5′-hexachlorobiphenyl
PCB-180: 2,2′,3,4,4′,5,5′-heptachlorobiphenyl PCB-180: 13C-2,2′,3,4,4′,5,5′-heptachlorobiphenyl
OCP OCP
α-Hexachlorocyclohexane (α-HCH) (α-HCH) 13C
6H 6Cl 6 (CAS No. 222966-66-7)
β-Hexachlorocyclohexane (β-HCH) 13C available (CIL)b
γ-Hexachlorocyclohexane (γ-HCH) (γ-HCH) 13C6H6Cl6 (CAS No.104215-85-2)
δ-Hexachlorocyclohexane (δ-HCH) 13C Available (CIL)b
ε-Hexachlorocyclohexane (ε-HCH)  

1) Institute for Reference Materials and Measurements (IRMM), Geel, Belgium is an example of a suitable supplier. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.
2) National Institute of Science and Technology (NIST), Washington, DC, USA, is an example of a suitable supplier. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this
supplier.

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Table 2 (continued)

Native reference substances Labelled internal standard substances

Aldrin 13C available (CIL)b
Dieldrin 13C available (CIL)b
Endrin 13C available (CIL)b
Heptachlor 13C available (CIL)b
Heptachlor epoxide (exo-, cis- or a-isomer) 13C available (CIL)b
Heptachlor epoxide (endo-, trans- or b-isomer)  
α-Endosulfan d4 and 13C available (CIL)b
β-Endosulfan d4 and 13C available (CIL)b
p,p′-DDE 13C available (CIL)b
o,p′-DDD 13C available (CIL)b
o,p′-DDT 13C available (CIL)b
p,p′-DDD 13C available (CIL)b
o,p′-DDE 13C available (CIL)b
p,p′-DDT 13C available (CIL)b
Methoxychlor 13C available (CIL)b
1,2,4-Trichlorobenzene d3 available (CIL)b
1,2,3-Trichlorobenzene d3 available (CIL)b
1,3,5-Trichlorobenzene d3 available (CIL)b
1,2,3,4-Tetrachlorobenzene 13C available (CIL)b
1,2,3,5-Tetrachlorobenzene  
1,2,4,5-Tetrachlorobenzene d2 and 13C6 available (CIL)b
Pentachlorobenzene 13C available (CIL)b
Pentachloronitrobenzene 13C available (CIL)b
Hexachlorobenzene (HCB) HCB 13C6Cl6
Organophosphorus Organophosphorus
Azinphos-ethyl d10 available (Ehrenstorfer)
Bromofenvinphos-ethyl  
Chlorofenvinphos d10 available (Ehrenstorfer)
Chloropyriphos-ethyl d10 available (CIL, Ehrenstorfer)b
Chloropyriphos-methyl d6 available (Ehrenstorfer)
Heptenophos  
a Not part of the 16 target analytes, but only for checking whether resolution is sufficient.
b Cambridge Isotope Laboratory (CIL) and Dr. Ehrenstorfer are examples of suitable suppliers. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of these suppliers.

The most commonly used internal standards are isotopically labelled substances. They are highly recommended.
They are used for the evaluation of the results and quantification of the individual substances (Clauses 11 and 12).

6.4.2 Injection standard

Add an isotopically labelled non-polar substance to the final extract and to the calibration solutions (6.5.3)
before GC-MS injection to check the recovery of the internal standards.

Prepare a stock solution of the injection standard in an appropriate solvent, e.g. acetonitrile (6.2.2) or hexane
(6.2.1), with a mass concentration, ρ ≈ 10 µg/ml.

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6.5 Solutions

6.5.1 Single substance stock solutions

Prepare solutions of the single native substances and internal standards (see Table 2) in an appropriate solvent,
e.g. acetonitrile (6.2.2) or hexane (6.2.1), with mass concentration, ρ ≈ 200 µg/ml.

These solutions can be used for confirmation and identification of single components in the chromatogram.

6.5.2 Multiple substance stock solution

Dilute a sufficient volume, e.g. 5  ml, of the single substance stock solutions (6.5.1) in a volumetric flask
(e.g. 100 ml) with an appropriate solvent, e.g. acetonitrile (6.2.2) or hexane (6.2.1), to prepare a solution with a
mass concentration, ρ ≈ 10 µg/ml.

Alternatively, commercially available (certified) combined/mixed solutions containing one or a few of the
reference substances (see Table 2) at an appropriate mass concentration of the respective individual substance,
e.g. 10 µg/ml in an appropriate solvent, e.g. acetonitrile (6.2.2) or hexane (6.2.1), may be used.

Solutions 6.4.2, 6.5.1 and 6.5.2 are stable for at least 1 year when stored in the dark at room temperature and
protected from evaporation. The stability of the standard solution shall be checked regularly. For that purpose,
independent solutions for quality control shall be available within a laboratory.

6.5.3 Calibration solutions

Prepare at least five calibration solutions (CS1 to CS5) by appropriate dilution of the multiple substance stock
solution (6.5.2), using hexane (6.2.1) or acetonitrile (6.2.2) as solvent. Add to each solution the same amount
of the stock solution of the injection standard to a final concentration, ρ ≈ 100 ng/ml.

It is recommended that the solvent for the calibration solutions be the same as the solution of the final extract.

Transfer, for example, 50 µl of the multiple stock solution into a 5 ml one-mark volumetric flask and make up
to the mark with an appropriate solvent. A volume of 1  µl of this reference solution contains 100  pg of the
individual substances concerned (ρ ≈ 100 ng/ml).

The mass concentration of the non-polar substances in the multiple substance stock solution shall be checked
by comparison with an independent, preferably certified, standard solution. All individual substances shall
agree within ±10 %.

These solutions shall be used for the calibration of the GC system [mixture in hexane (6.2.1)] as well as for the
investigation of recovery rates [mixture in acetone (6.2.3)].

Store the solutions at (3 ± 2) °C in the dark. These solutions are stable for at least 1 month.

7 Apparatus

7.1 General requirements

Standard laboratory glassware and stirring bars cleaned to eliminate all interferences.

NOTE All glassware and stirring bars can be cleaned, for example by rinsing with detergent and hot water and drying
for about 15 min to 30 min at about 120 °C. After cooling, the glassware can be rinsed with acetone and sealed and stored
in a clean environment.

Do not re-use glassware and stirring bars that have been in contact with waste-water samples or samples with
high concentrations for drinking water analysis. This applies especially for PAH, PCB and HCH.

7.2 Coloured glass bottles, narrow-necked, flat-bottomed, 1 000 ml, with aluminium-lined cap.

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7.3 Magnetic stirrer, with stirring bars (size approximately 2  cm), glass or PTFE-coated, kept under the
solvent used for extraction.

7.4 Separating funnel, nominal capacity 1 000 ml, with PTFE stopcock and glass stopper.

7.5 Conical flask, nominal capacity 250 ml, with glass stopper.

7.6 Equipment for concentrating the eluates by evaporation, e.g. a rotary evaporator, regulatable for
constant vacuum and with a temperature-controlled water bath, or stripping equipment using nitrogen gas.

7.7 Vacuum device for solid-phase extraction, e.g. vacubox, extraction box.

7.8 Microlitre syringes, e.g. 500 µl and 1 000 µl.

7.9 Reduction flask, 100 ml (e.g. as shown in Figure B.3).

7.10 Centrifuge with rotor, with centrifuge tubes (e.g. as shown in Figure B.2) with tapered bottom, 50 ml.

7.11 Shaking apparatus, with adjustable rotational speed.

7.12 Glass autosampler vials, capacity e.g. 2 ml, with inert cap and PTFE-coated septum.

7.13 Glass vials, e.g. centrifuge tubes, graduated (scale division 0,1  ml), nominal capacity 10  ml, with
glass stoppers.

7.14 Gas chromatograph, with MS detector (EI).

7.15 High resolution, low-bleeding capillary column for GC (see Annex A).

7.16 Microfilter, with solvent-resistant hydrophilic membrane, pore size 0,45 µm.

7.17 Pasteur pipettes

7.18 Glass cartridges, filled with at least 0,5 g silica (see 7.19).

NOTE These cartridges are commercially available.

7.19 Silica, average particle size approximately 40 µm, heated at 450 °C for 3 h and stored in a desiccator to
ensure maximum activity.

NOTE Pre-packed silica cartridges are commercially available.

7.20 Molecular sieve beads, pore diameter 0,4 nm.

7.21 Glass wool

8 Sampling
Collect the sample in a coloured glass bottle with a volume of 1 000 ml (7.2).

When sampling drinking water from a mains tap, collect the sample before the tap is sterilized by flame
treatment for bacteriological sampling.

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Fill the bottle to the shoulder (approximately 950 ml). Determine the volume of the sample to be extracted by
weighing, before extraction and after emptying, with an accuracy of ±5 g. Store the sample at (3 ± 2) °C and
protect it from light until the extraction is carried out (see also ISO 5667-3).

Ensure that the extraction is carried out within the maximum preservation time, as specified in ISO 5667-3, to
avoid losses.

It is generally recommended that the extraction be carried out as soon as practicable to minimize potential
adherence to glass which could be an issue.

9 Procedure

9.1 General considerations

The liquid-liquid extraction method shall not be used with samples containing more that 150  mg/l of
suspended matter.

NOTE Volatile solvents other than hexane can be used if it is proven that there is equal or better recovery (recovery
between 70 % and 110 %).

9.2 Extraction

9.2.1 Sample preparation and extraction

Add a precisely defined amount of the internal standard (e.g a volume containing 50 ng), dissolved in a water-
soluble solution (6.4.2). Add 25 ml of hexane (6.2.1) and a stirring bar, then close the flask with a PTFE cap
liner or close the conical flask (7.5) with a ground stopper. Thoroughly mix the sample using the magnetic
stirrer (7.3) at maximum setting for 60 min. Transfer the sample to a separating funnel and allow the phases to
separate for at least 5 min. If an emulsion is formed during the extraction process, collect it in a centrifuge tube
and centrifuge (7.10), e.g. for 10 min at about 3 000 r/min. Remove the separated water with a Pasteur pipette.
Transfer the extract to a conical flask (7.5) and dry it according to 9.2.2.

For waste and surface waters, repeat the extraction procedure twice. Transfer the sample from the separating
funnel back into the sample container, add 25 ml of hexane (6.2.1), and proceed as described above.

The extraction procedure can also be carried out in a separating funnel (7.4) using a shaking apparatus
(7.11) and a micro-separator (see Annex B). Rinse the bottle thoroughly with extraction solvent to extract any
adsorbed components.

NOTE 1 Other volatile solvents can also be used if it is proven that there is equal or better recovery (recovery between
70 % and 110 %).

NOTE 2 For the extraction of waste water and other water samples with expected high concentrations of PAH, only
10 ml to 100 ml of the homogenous sample can be transferred to a 250 ml conical flask (7.5) with a pipette and diluted with
water to 200 ml. After adding 25 ml of hexane (6.2.1), proceed as described above.

9.2.2 Drying of the extract

Transfer the hexane layer obtained according to 9.2.1 into a 100 ml conical flask. Rinse the funnel or centrifuge
tube with 5 ml of hexane and add it to the total extract.

Dry the extract with approximately 1,0 g sodium sulfate (6.1.1) for at least 15 min, swirl the vessel frequently.
The extract can also be dried by filtering through anhydrous sodium sulfate.

Decant the dry extract into a reduction flask (7.9). Rinse the conical flask twice with 5 ml of hexane and decant
this also into the reduction flask.

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9.2.3 Enrichment

Evaporate the dried hexane extract obtained according to 9.2.2 until it fills only the tapered tip of the reduction
flask (approximately 2 ml), with, for example, a rotary evaporator, at a temperature of 30 °C, slowly lowering
the pressure to 20 kPa.

Do not evaporate the extracts to dryness, as losses of 2-ring or 3-ring compounds, for example, and
1,2,4-trichlorobenzene can occur. Adding a few drops of decane (6.2.4) or isooctane (6.2.5) reduces the loss
of the most volatile compounds.

Dissolve the extract into a known volume, e.g. 2 ml. Be sure that any residues that may be deposited on the
glass wall are dissolved by shaking the extract using the shaking apparatus.

Clean the extracts of waste-water samples and other samples of unknown origin by silica clean-up according
to 9.2.4, if the chromatogram shows interferences that hamper the quantification.

Transfer the enriched sample, if necessary after filtration through a filter (7.16), into a glass sample vial. Keep
the sample in a cool and dark place until the analysis is carried out.

Proceed as described in 9.4.

NOTE Alternative enrichment methods can also be used. If a large volume injection is used or if higher concentrations
of the target compounds are expected, a lower enrichment factor can be used.

9.2.4 Clean-up

Applying the procedure described in 9.2 can lead to co-extraction of relatively polar and/or other undesired
substances, which can interfere by the appearance of unknown peaks overlapping the target compounds.
When the target compounds are PAH, the silica clean-up procedure described in Annex C can be used. Use
the clean-up procedures for other non-polar substances specified in ISO 6468.

9.3 Gas chromatography

Operate the gas chromatograph according to the manufacturer’s instructions.

Select a capillary GC column and chromatographic conditions that will lead to efficient separation (see Annex A).

When using an injection standard, add a precisely known amount of the injection standard (6.4.2) to the sample
extract, mix thoroughly and inject immediately into the GC.

9.4 Blank measurement

Perform blank determinations at least once per batch using water prior to and during a series of analyses.
This water should be free of detectable target compounds. Blank measurements shall include all steps of the
analytical procedure from the arrival of the sample in the laboratory to the evaluation of the gas chromatogram.
If blank values are unusually high (over 50 % of the lowest reporting level), every step in the procedure shall be
checked in order to find the reason for these high blanks. Ensure that blanks are reduced as much as possible
by various procedures, e.g. elimination of contamination of the sample by ambient air and solvents and checks
of analytical instrumentation.

If sample concentrations are close to the limit of detection, however, blank values higher than 50  % of the
lowest reported value can be tolerated. If this occurs, it is recommended that the sample be concentrated and
retested to provide confirmation.

9.5 Mass spectrometric conditions

Adjust the mass spectrometer in accordance with the manufacturer’s instructions. Chromatograms are recorded
in full scan (50 amu to 420 amu) or selected ion monitoring/recording mode (SIM/SIR).

Adjust the scan rate of the mass spectrometer to a velocity allowing one GC peak to be described by at least
seven data points.

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Diagnostic ions with relative intensities with reference to ISO 22892[8] are listed in Table 4.

NOTE Each of the non-polar target analytes can be quantified by using the deuterated internal standard stated above.

10 Calibration

10.1 General
A calibration curve encompassing the concentration range is prepared for each compound to be determined.
The relative response [Rrel or FR depending on whether labelled internal standards or other native (non-labelled)
standards were used] versus concentration in standard solutions is plotted or computed using a regression
function. Relative response is determined according to the procedures described below. At least five calibration
points are employed. Also consult ISO 8466-1.

10.2 Calibration by labelled internal standards

Labelled internal standard calibration is used for the non-polar substances for which labelled compounds are
added to samples.

Prepare a calibration curve encompassing the concentration range for each compound to be determined. Plot
the relative response, Rrel (labelled to native), versus concentration in standard solutions or compute using
a linear regression. Determine the relative response factor for each non-polar substance according to the
procedures described below. Employ at least five calibration points.

Determine the response of each non-polar substance relative to its labelled analogue using Equation (1):

A1n ρ L
Rrel = (1)
A1L ρ n

where

A1n is the area of diagnostic ion 1 for the non-polar substance;

A1L is the area of diagnostic ion 1 for the labelled compound;

ρL is the concentration of the labelled compound in the calibration standard, in micrograms per litre;

ρn is the concentration of the native compound in the calibration standard, in micrograms per litre.

NOTE 1 If the relative response for any compound is constant (coefficient of variation of less than 20 %) over the five-
point calibration range, an averaged relative response can be used for that compound. Otherwise, the complete calibration
curve for that compound can be used over the five-point calibration range.

NOTE 2 It is also possible to use only one mass for calibration and quantification.

10.3 Calibration by internal standard

The internal standard method is applied to determination of other non-polar substances for which no labelled
standards have been added to the sample.

Calibration requires the determination of response factors, FR, defined by Equation (2):

A1s ρ is
FR = (2)
A1is ρ s

where

A1s is the area of diagnostic ion 1 for the non-polar substance;

A1is is the area of diagnostic ion 1 for the internal standard;

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ρis is the concentration of the internal standard, in micrograms per litre;

ρs is the concentration of the compound in the calibration standard, in micrograms per litre.

NOTE If the response factor, FR, for any compound is constant (coefficient of variation of less than 20  %) over
the five-point calibration range, an averaged response factor can be used for that compound. Otherwise, the complete
calibration curve for that compound over the five-point range can be used.

For the daily check of the calibration (recalibration), inject at least two calibration standards, e.g. concentrations
of (20 ± 10) % and (80 ± 10) % of the established linear range. Compare the calculated response factor with
those obtained in the previous batch of samples. They should not differ more than 20 %.

11 Measurement of samples
Equilibrate the measuring system before measuring samples and adjust the mass spectrometer according to
the manufacturer’s instructions.

The following measurement conditions shall apply.

Ionization method: electron impact

Mass range of the spectrum: 50 amu to 420 amu, at least 10 amu above the highest mass of the substances
to be determined

Cycle duration: <2 s so that five spectra can be taken per substance peak

If only single masses are registered in order to increase sensitivity, register the base peak and at least two
more ions, with the same cycle duration as above.

12 Identification
The quantification of a single substance requires a secure and non-ambiguous identification. Components that
have less fragmentation therefore require additional criteria for identification.

When taking whole spectra, the sample spectrum and the reference spectrum taken under the same working
conditions should be identical. The reference spectrum should be produced by each laboratory using
their equipment and should be stored in a reference spectrum database. These spectra are to be used for
identification purposes by MS.

The deviation of the non-basic peak (not 100 % mass peak) should be less than 10 %.

If there is a shift of retention time, confirmation of identity can be done by spiking. It is possible that the use of
isotopically labelled standards is the best way to confirm the identity.

A single substance is identified, if:

— the retention time of a substance in the total ion current chromatogram of the sample is congruent with that
of a reference standard in the latest acquired reference solution performed under identical chromatographic
conditions (limit: ±1 %, maximum ±6 s);

— the relative intensity of the diagnostic ions recorded in the mass spectrum of the sample acquired under
identical conditions does not differ by more than ±(0,1I  +  10)  % from those relative intensities of the
reference substance, where I is the relative intensity recorded from the characteristic ions in the mass
spectrum of the reference solution.

See Table 3.

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Table 3 — Degree of confirmation of the identity

Degree of
Technique Operating principle Additional criterion
identification
MS Possible Single mass monitoring Compliance of the mass ratio with that of
the standard compound within given limits
(SIM/SIR)
Confirmed Acquisition of total spectra Compliance of the spectrum with that of
the standard compound within given limits
(scan)

Critical peak couples can lead to incorrect automatic assignment. In such cases, a manual check is essential.
Critical peak pairs are: phenanthrene and anthracene; benzo[a]anthracene and chrysene; benzo[b]fluoranthene
and benzo[k]fluoranthene; benzo[a]pyrene and benzo[e]pyrene; and PCB28 and PCB31.

Overlapping compounds with similar masses can be identified reliably only if the minimum between both peaks
is at least 25 % of the base peak; otherwise they are reported as a sum.

When using single masses, all three mass signals should be present. The signal-to-noise ratio for the smallest
peak of a mass should be over 3.

The ratio of the three masses in the spectrum should be determined from the peak heights at the peak maximum.
The two peaks corresponding to the masses below 100 % that are determined should, in relation to the 100 %
mass, be within 10 % of the value determined under the same conditions with the reference material.

The mass spectrum of the sample should include all ions that have a relative intensity of 10 % in the reference
spectrum. The ratio of the intensities of the different ions in the sample spectrum and the reference spectrum
should be within 20 %, tested on the three most important ions.

Single mass registration should be noted in the report.

For detection by MS, use the peak area of the base peak of substance i, after checking the identity by comparing
the spectra, or, with the SIM technique, the isotope and/or fragment ratios. If using an internal standard, the
reference is always the signal of the most intensive mass (main ion), after this signal has been checked for purity.

Table 4 — Recommended characteristic masses of non-polar substances, as specified in ISO 22892[8]

Diagnostic ion 1a Diagnostic ion 2a Diagnostic ion 3a

Compound
m/z m/z m/z
PAH
Naphthalene 128 (100) 102 (11) —
Acenaphthylene 152 (100) 150 (3) 76 (10)
Acenaphthene 153 (100) 154 (70) 76 (10)
Fluorene 165 (100) 166 (81) 139 (4)
Phenanthrene 178 (100) 152 (9) 76b (3)
Anthracene 178 (100) 152 (12) 76 (6)
Fluoranthene 202 (100) 200 (31) 100b (3)
Pyrene 202 (100) 200 (2) 101b (4)
Benzo[a]anthracene 228 (100) 226 (3) 114b (2)
Chrysene 228 (100) 226 (6) 113b (4)
Benzo[b]fluoranthene 252 (100) 250 (22) 126 (5)
Benzo[k]fluoranthen 252 (100) 250 (22) 126 (5)
Benzo[a]pyrene 252 (100) 250 (18) 113 (11)
Indeno[1,2,3-cd]pyrene 276 (100) 138 (12) 274b * (4)
Dibenzo[a,h]anthracene 278 (100) 139 (9) 276b * (5)

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Table 4 (continued)

Diagnostic ion 1a Diagnostic ion 2a Diagnostic ion 3a

Compound
m/z m/z m/z
Benzo[ghi]perylene 276 (100) 138 (12) 274 (4)
PCB
PCB 28 186 (100) 258 (74) 186 (82)
13C -PCB 28 268 270 —
12
PCB 52 292 (100) 294 (49) 220 (95)
13C -PCB 52 304 306 —
12
PCB101 326 (100) 328 (65) 256 (62)
13C -PCB101 338 340 —
12
PCB 118 326 (100) 328 (62) 254 (57)
13C -PCB 118 338 340 —
12
PCB 138 290 (100) 358 (42) 360 (94)
13C -PCB 138 372 374 —
12
PCB 153 360 (100) 362 (92) 290 (73)
13C -PCB 153 372 374 —
12
PCB 180 394 (100) 396 (96) 324 (84)
13C -PCB 180 406 408 —
12
OCP
Hexachlorobenzene (HCB) 284 (100) 142 (22) 249 (24)
α-Hexachlorocyclohexane (α-HCH) 181 (100) 219 (33) 109 (29)
β-Hexachlorocyclohexane (β-HCH) 181 (97) 219 (54) 109 (49)
γ-Hexachlorocyclohexane (γ-HCH) 181 (97) 219 (34) 109 (33)
δ-Hexachlorocyclohexane 109 (100) 219 (96) 183 (90)
ε-Hexachlorocyclohexane 109 (88) 219 (100) 183 (90)
Aldrin 66 (100) 263 (78) 293 (41)
Dieldrin 79 (100) 263 (70) 277 (18)
Endrin 81 (100) 263 (70) 277 (18)
Heptachlor 100 (100) 65 (65) 272 (89)
Heptachlor epoxide (cis-isomer) 253 (100) 183 (90) 289 (85)
Heptachlor epoxide (trans-isomer) 353 (100) 81 (67) 263 (26)
α-Endosulfan 195 (100) 159 (93) 265 (55)
β-Endosulfan 195 (100) 241 (80) 159 (56)
p,p′-DDE 246 (100) 318 (37) 176 (36)
o,p′-DDD 235 (100) 165 (66) 199 (29)
o,p′-DDT 235 (100) 165 (67) 199 (27)
p,p′-DDD 235 (100) 165 (66) 199 (20)
o,p′-DDE 246 (100) 318 (37) 176 (27)
p,p′-DDT 235 (100) 165 (68) 199 (20)
Methoxychlor 227 (100) 228 (18) 274 (5)
Chlorobenzenes
1,2,4-Trichlorobenzene 180 (100) 182 (97) 145 (45)
1,2,3-Trichlorobenzene 180 (100) 182 (92) 145 (38)
1,3,5-Trichlorobenzene 180 (100) 182 (95) 145 (32)
1,2,3,4-Tetrachlorobenzene 216 (100) 214 (74) 108 (24)

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Table 4 (continued)

Diagnostic ion 1a Diagnostic ion 2a Diagnostic ion 3a

Compound
m/z m/z m/z
1,2,3,5-Tetrachlorobenzene 216 (100) 214 (78) 108 (14)
1,2,4,5-Tetrachlorobenzene 216 (100) 214 (79) 108 (12)
Pentachlorobenzene 250 (100) 252 (63) 215 (25)
Pentachloronitrobenzene 237 (100) 295 (83) 142 (50)
Organophosphorus
Azinphos-ethyl 132 (100) 160 (80) 77 (68)
Bromofenvinphos 267 269 323
Chlorofenvinphos 267 (100) 323 (59) 81 (58)
Chloropyriphos-ethyl 97 (100) 197 (90) 314 (47)
Chloropyriphos-methyl 286 (100) 125 (95) 288 (78)
Heptenophos 124 (100) 89 (77) 215 (13)
a In brackets: relative intensity of the fragment ion.
b Often missing fragments.

13 Calculation

13.1 Quantification by internal standards

Compute the concentrations of those non-polar substances for which an internal standard is added using the
response factors determined from the initial calibration data (10.3) and Equation (3):

A1s mis
m ex = (3)
A1is FR

where

mex is the amount of the non-polar substance in the extract, in nanograms;

A1s is the area of diagnostic ion 1 for the non-polar substance;

mis is the amount of the internal standard, in nanograms;

A1is is the area of diagnostic ion 1 for the internal standard;

FR is the response factor as defined in 10.3.

Determine the response factor of the internal standards relative to the injection standard using the area
response of the diagnostic ion. Using the amount in the extract determined using Equation (3), compute the
percentage recovery, η, of the internal standards using Equation (4):

m ex
η= × 100 (4)
m spk

where

mex is the amount found, in nanograms;

mspk is the amount spiked, in nanograms.

For compounds for which no internal standard has been added, the recovery is determined as follows.

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Add, for example, 2 ml of reference solution prepared according to 6.4.1 to 1 000 ml water and proceed as
specified in Clause 9.

Determine the recovery rates for surface water samples by the method of standard additions.

Determine the mean recovery, η , of analyte i using Equations (5) and (6):

ρ i,N f
η i,N = (5)
ρ i,N e

n
∑ η i,N
N =1
ηi = (6)
n

where

hi,N is the recovery of analyte i at concentration level N;

ρ i,N f is the mass concentration of analyte i found at concentration level N, calculated with the calibration
function, in micrograms per litre;
ρ i,N e is the mass concentration of analyte i given at concentration level N, in micrograms per litre;

ηi is the mean recovery;

n is the number of concentration levels.

13.2 Quantification by labelled internal standards

By adding a known amount of a labelled compound to every sample prior to extraction, correction for
recovery can be made because the non-polar substance and its labelled analogue exhibit similar effects upon
extraction, concentration, and GC. Use relative response, Rrel, values in conjunction with the initial calibration
data described in 10.2 to determine concentrations directly, as long as labelled compound spiking levels are
constant, using Equation (7):

A1n mL
m ex = (7)
A1L Rrel

where

mex is the amount of the non-polar substance in the extract, in nanograms;

A1n is the area of diagnostic ion 1 for the native compound;

A1L is the area of diagnostic ion 1 for the labelled compound;

mL is the amount of the labelled compound in the calibration standard, in nanograms;

Rrel is the relative response as defined in 10.2.

Determine the recovery rates for surface-water and waste-water samples by the method of standard additions.

Determine the mean recovery, η i , of analyte i using Equations (8) and (9):

ρ i,N f
η i,N = (8)
ρ i,N e

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n
∑ η i,N
N =1
ηi = (9)
n

where

hi,N is the recovery of analyte i at concentration level N;

ρ i,N f is the mass concentration of analyte i found at concentration level N, calculated with the calibration
function, in micrograms per litre;
ρ i,N e is the mass concentration of analyte i given at concentration level N, in micrograms per litre;

ηi is the mean recovery;

n is the number of concentration levels.

13.3 Recovery of internal standards

Recoveries of the internal standards for most samples are similar to those from reagent water. The recovery
limits are between 70 % and 110 %.

If the internal standard recovery is outside these ranges, a diluted sample shall be analysed.

If the recovery of any of the internal standards in the diluted sample is outside the normal range, the calibration
solution CS3 (6.5.3) shall be analysed and the calibration verified. For each compound, confirm that the result
of the verification analysis is within 20 % of the nominal concentration. If, however, any compound falls outside
its respective limit, the measurement system is not performing properly for that compound. In this event,
prepare a fresh calibration standard, or correct the problem causing the failure, and repeat the tuning of the
MS (9.5) and verification test, or recalibrate (10.2).

13.4 Concentration in the sample

Compute the concentration of a non-polar substance in the aqueous phase of the sample using the concentration
of the compound in the extract and the volume of water extracted, as follows:

m ex
ρ = (10)
Vs × 1 000

where

ρ is the concentration in aqueous phase, in micrograms per litre;

mex is the amount of the compound in the extract, in nanograms;

Vs is the sample volume, in litres.

14 Expression of results
Report the mass concentration of non-polar substance, in micrograms per litre, to not more than two
significant figures. Concentrations < 0,01 µg/l are rounded up to the nearest 0,001 µg/l. Rounding examples
are given in Table 5.

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Table 5 — Examples of rounding measurement results

Measured value Reported result

µg/l µg/l
13,54 14
1,354 1,4
0,135 4 0,14
0,013 5 0,014
0,008 6 0,009

15 Test report
The test report shall contain at least the following information:

a) the test method used, together with a reference to this Technical Specification (ISO/TS 28581:2012);

b) the data required for identification of the sample examined;

c) relevant information about sampling and sample preservation;

d) the concentration of each of the non-polar substances, expressed according to Clause 14;

e) if used, a note on single mass registration during MS analysis;

f) all operations not described in this Technical Specification which could have affected the results.

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Annex A
(informative)

Examples of GC-MS conditions

Table A.1 — Examples of chromatographic conditions

Column Dimensions Temperature

programme
Length: 30 m 40 °C, 8 min isothermal
95 % dimethylpolysiloxane Inner diameter: 5 °C/min to 310 °C
5 % diphenylpolysiloxane  0,25 mm
Film thickness: 25 µm 15 min isothermal
Length: 30 m 40 °C, 6 min isothermal
86 % dimethylpolysiloxane
Inner diameter: 5 °C/min to 220 °C
14 % cyanopropylene- 0,25 mm
polysiloxane 
Film thickness: 1,0 µm 4 min isothermal

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Annex B
(informative)

Examples for the construction of special apparatus

Dimensions in millimetres

Key
1 PTFE screw cock

Figure B.1 — Microseparator

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Dimensions in millimetres Dimensions in millimetres

Key Key
1 PTFE screw cap 1 ISO 383:[1] 14/23
2 total graduated volume, 2 ml; graduations of 0,1 ml

Figure B.2 — Centrifuge tube with tapered Figure B.3 — Reduction flask

bottom and screw cap

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Annex C
(informative)

Silica clean-up

For clean-up of the extract, use columns [Pasteur pipettes (7.17) with a glass wool plug] or cartridges (7.18)
containing at least 0,5 g of silica (7.19). Clean the silica in the column or in the cartridge by rinsing with five bed
volumes of a mixture of dichloromethane (6.2.6)/hexane (6.2.1) (1+1), followed by conditioning with the same
volume of hexane (6.2.1).

NOTE 1 The clean-up is not possible for solutions that contain acetone (6.2.3).

Dry the solvents used for cleaning the extract by applying a molecular sieve (7.20). The silica should have its
maximum activity.

Concentrate the enriched extract (9.2.3) by blowing with a gentle stream of nitrogen (6.3.1) so that a volume of
500 µl remains.

Transfer the concentrated extract using a Pasteur pipette (7.17) on to the hexane-covered silica and allow it to
soak almost completely into the silica. Collect the eluate in a glass vial (7.13).

Rinse the reduction flask with 500 µl of hexane (6.2.1), add this solution to the column and allow it to soak
almost completely into the silica.

Elute the PAH with a mixture of dichloromethane (6.2.6)/hexane (6.2.1) (1+1).

NOTE 2 Commercially available cartridges containing 0,5 g of silica require a volume of at least 3 ml of the mixture of
dichloromethane (6.2.6)/hexane (6.2.1) (1+1) for the elution of the PAH.

Add a few drops of decane (6.2.4) or isooctane (6.2.5) to the eluate, homogenize by shaking, and concentrate
(see 9.2.3) to between 200 µl and 250 µl, e.g. first with a rotary evaporator (7.6) to about 2 ml, then by a stream
of nitrogen (6.3.1).

Fill the extract up to a known volume (e.g. 2 ml) with the same solvent that has been used for the preparation
of the calibration solutions (6.5.3).

Proceed as described in 9.4. Use an aliquot for the GC-MS determination.

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Bibliography

[1] ISO 383, Laboratory glassware — Interchangeable conical ground joints

[2] ISO 7981 (all parts), Water quality — Determination of polycyclic aromatic hydrocarbons (PAH)

[3] ISO/TS  13530:2009, Water quality — Guidance on analytical quality control for chemical and
physicochemical water analysis

[4] ISO 15089:2000, Water quality — Guidelines for selective immunoassays for the determination of plant
treatment and pesticide agents

[5] ISO  17858:2007, Water quality — Determination of dioxin-like polychlorinated biphenyls — Method
using gas chromatography/mass spectrometry

[6] ISO 17993:2002, Water quality — Determination of 15 polycyclic aromatic hydrocarbons (PAH) in water
by HPLC with fluorescence detection after liquid-liquid extraction

[7] ISO  18073:2004, Water quality — Determination of tetra- to octa-chlorinated dioxins and furans —
Method using isotope dilution HRGC/HRMS

[8] ISO  22892:2006, Soil quality  — Guidelines for the identification of target compounds by gas
chromatography and mass spectrometry

[9] ISO  28540:2011, Water quality — Determination of 16 polycyclic aromatic hydrocarbons (PAH) in
water — Method using gas chromatography with mass spectrometric detection (GC-MS)

[10] Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption.
Off. J. 1998-12-05, L330, pp. 32–54

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