SIST ISO 15819:2015

Cosmetics - Analytical methods - Nitrosamines: Detection and determination of N-nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS

This International Standard describes a method for the detection and quantification of
N-nitrosodiethanolamine (NDELA) in cosmetics and raw materials used in cosmetics.
This method is not applicable to the detection and/or quantification of nitrosamines other than NDELA
nor to the detection and/or quantification of NDELA in products other than cosmetics or raw materials
used in cosmetics.
If a product has a possibility of either NDELA contamination from ingredients or NDELA formation by
the composition of ingredients, the method is intended to be applied for quantitative determination of
NDELA. Accordingly, the method does not apply to routine testing of cosmetic products. Because of the
large variety of cosmetic products within this field of application, this method might need to be adapted
for certain matrices (refer to ISO 12787).
Therefore, International Standards dedicated to alternative methods for testing nitrosamines in cosmetic
products are being developed separately. Other methods can be employed provided that they are verified
as to their detection of NDELA and validated in terms of recovery and quantification of the analyte.

Cosmétiques - Méthodes analytiques - Nitrosamines: Recherche et dosage de la N-nitrosodiéthanolamine (NDELA) dans les produits cosmétiques par CLHP-SM-SM

L'ISO 15819:2014 d�crit une m�thode de recherche et de quantification de la N‑nitrosodi�thanolamine (NDELA) dans les cosm�tiques et les mati�res premi�res utilis�es dans les cosm�tiques.
La pr�sente m�thode n'est applicable ni � la recherche et/ou � la quantification de nitrosamines autres que la NDELA, ni � la recherche et/ou � la quantification de la NDELA dans les produits autres que les cosm�tiques ou les mati�res premi�res utilis�es dans les cosm�tiques.
Si un produit peut �tre contamin� par la NDELA � partir de ses ingr�dients ou que la composition d'ingr�dients cosm�tiques peut entra�ner la formation de NDELA, alors la m�thode sera appliqu�e pour la d�termination de la quantit� de NDELA. Par cons�quent, cette m�thode ne s'applique pas aux essais de routine relatifs aux produits cosm�tiques. En raison du large �ventail de cosm�tiques consid�r� dans ce domaine d'application, la pr�sente m�thode pourrait n�cessiter d'�tre adapt�e � certaines matrices (se r�f�rer � l'ISO 12787).

Kozmetika - Analizne metode - Nitrozamini: odkrivanje in določevanje N-nitrozodietanolamina (NDELA) v kozmetičnih izdelkih s tekočinsko kromatografijo visoke ločljivosti z masno spektrometrijsko detekcijo (HPLC-MS-MS)

Ta mednarodni standard opisuje način odkrivanja in kvantifikacije N-nitrozodietanolamina (NDELA) v kozmetičnih izdelkih in surovinah, ki se uporabljajo v kozmetiki. Ta metoda se ne uporablja za odkrivanje in/ali kvantifikacijo nitrozaminov, razen NDELA, ali odkrivanje in/ali kvantifikacijo NDELA v izdelkih, razen v kozmetiki ali surovinah, ki se uporabljajo v kozmetiki. Če je mogoča kontaminacija izdelka z NDELA iz sestavin ali nastanek NDELA zaradi sestave sestavin, je metoda namenjena za izvajanje kvantitativnega določevanja
NDELA. V skladu s tem se metoda ne uporablja za rutinsko testiranje kozmetičnih proizvodov. Zaradi velike raznolikosti kozmetičnih proizvodov na tem področju uporabe bo to metodo morda treba prilagoditi določenim matricam (glej ISO 12787).
Zato se mednarodni standardi, ki so namenjeni alternativnim metodam za testiranje nitrozaminov v kozmetičnih
izdelkih, razvijajo posebej. Druge metode je mogoče uporabiti, če so preverjene
za odkrivanje NDELA in potrjene za obnovo ter kvantifikacijo analita.

General Information

• Status: Published
• Public Enquiry End Date: 30-Nov-2013
• Publication Date: 19-Feb-2015
• ICS: 71.040.50 - Physicochemical methods of analysis
• ICS: 71.100.70 - Cosmetics. Toiletries
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 27-Jan-2015
• Due Date: 03-Apr-2015
• Completion Date: 20-Feb-2015
• Ref Project: ISO 15819:2014 - Cosmetics — Analytical methods — Nitrosamines: Detection and determination of N-nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS

Relations

• Replaces: SIST ISO 15819:2009 - Cosmetics - Analytical methods - Nitrosamines: Detection and determination of N-nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS
• Effective Date: 01-Mar-2015

Standards Content (Sample)

SLOVENSKI STANDARD
SIST ISO 15819:2015
01-marec-2015
1DGRPHãþD
SIST ISO 15819:2009
.R]PHWLND$QDOL]QHPHWRGH1LWUR]DPLQLRGNULYDQMHLQGRORþHYDQMH1
QLWUR]RGLHWDQRODPLQD1'(/$YNR]PHWLþQLKL]GHONLKVWHNRþLQVNRNURPDWRJUDILMR
YLVRNHORþOMLYRVWL]PDVQRVSHNWURPHWULMVNRGHWHNFLMR+3/&0606
Cosmetics - Analytical methods - Nitrosamines: Detection and determination of N-
nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS
Cosmétiques - Méthodes analytiques - Nitrosamines: Recherche et dosage de la N-
nitrosodiéthanolamine (NDELA) dans les produits cosmétiques par CLHP-SM-SM
Ta slovenski standard je istoveten z: ISO 15819:2014
ICS:
71.040.50 Fizikalnokemijske analitske Physicochemical methods of
metode analysis
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
SIST ISO 15819:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST ISO 15819:2015

---------------------- Page: 2 ----------------------

SIST ISO 15819:2015
INTERNATIONAL ISO
STANDARD 15819
Second edition
2014-10-15
Cosmetics — Analytical
methods — Nitrosamines:
Detection and determination of
N-nitrosodiethanolamine (NDELA) in
cosmetics by HPLC-MS-MS
Cosmétiques — Méthodes analytiques — Nitrosamines: Recherche
et dosage de la N-nitrosodiéthanolamine (NDELA) dans les produits
cosmétiques par CLHP-SM-SM
Reference number
ISO 15819:2014(E)
©
ISO 2014

---------------------- Page: 3 ----------------------

SIST ISO 15819:2015
ISO 15819:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested 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 2014 – All rights reserved

---------------------- Page: 4 ----------------------

SIST ISO 15819:2015
ISO 15819:2014(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Principle . 1
4 Reagents . 2
5 Apparatus . 2
6 Sample preparation and conservation . 3
6.1 General . 3
6.2 Standard solutions preparation . 3
6.3 Sample preparation . 4
7 Procedure. 5
7.1 General . 5
7.2 Chromatographic conditions . 5
7.3 HPLC-MS-MS conditions . 6
8 Calculation of results . 7
8.1 Determination of the R value . 7
8.2 Calibration curve . 7
8.3 Validity check procedure and its criteria . 7
8.4 Calculation of concentrations . 7
9 Test report . 8
Annex A (informative) Examples of a calibration curve and of chromatograms .9
Bibliography .11
© ISO 2014 – All rights reserved iii

---------------------- Page: 5 ----------------------

SIST ISO 15819:2015
ISO 15819:2014(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 15819:2008), which has been
technically revised.
iv © ISO 2014 – All rights reserved

---------------------- Page: 6 ----------------------

SIST ISO 15819:2015
ISO 15819:2014(E)

Introduction
Human exposure to N-nitrosamines can occur through diverse sources such as environment, food, or
personal care products. As a result of their perceived carcinogenic potential on several animal species,
minimization of exposure to N-nitrosamines is recognized as important to the preservation of human
health. Among N-nitrosamines, N-nitrosodiethanolamine (NDELA) has been recognized as a potential
contaminant of cosmetics.
In this context, several analytical methods have been developed to detect and determine its presence
in cosmetics, such as gas chromatography/thermal energy analysis, high performance liquid
chromatography (HPLC) coupled either with photolysis and colourimetric quantification or with mass
spectrometry (MS) determination. This latter method uses advanced technology to ensure the maximum
specificity towards NDELA, to minimize the risk of artefactual formation of the analyte of interest and
to allow precise quantification.
This analytical method uses high performance liquid chromatography coupled with mass spectrometry
to separate and detect trace levels of NDELA from a cosmetic ingredient or product matrix with
maximum specificity for NDELA.
© ISO 2014 – All rights reserved v

---------------------- Page: 7 ----------------------

SIST ISO 15819:2015

---------------------- Page: 8 ----------------------

SIST ISO 15819:2015
INTERNATIONAL STANDARD ISO 15819:2014(E)
Cosmetics — Analytical methods — Nitrosamines:
Detection and determination of N-nitrosodiethanolamine
(NDELA) in cosmetics by HPLC-MS-MS
1 Scope
This International Standard describes a method for the detection and quantification of
N-nitrosodiethanolamine (NDELA) in cosmetics and raw materials used in cosmetics.
This method is not applicable to the detection and/or quantification of nitrosamines other than NDELA
nor to the detection and/or quantification of NDELA in products other than cosmetics or raw materials
used in cosmetics.
If a product has a possibility of either NDELA contamination from ingredients or NDELA formation by
the composition of ingredients, the method is intended to be applied for quantitative determination of
NDELA. Accordingly, the method does not apply to routine testing of cosmetic products. Because of the
large variety of cosmetic products within this field of application, this method might need to be adapted
for certain matrices (refer to ISO 12787).
Therefore, International Standards dedicated to alternative methods for testing nitrosamines in cosmetic
products are being developed separately. Other methods can be employed provided that they are verified
as to their detection of NDELA and validated in terms of recovery and quantification of the analyte.
2 Normative references
The following documents, 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 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 12787:2011, Cosmetics — Analytical methods — Validation criteria for analytical results using
chromatographic techniques
3 Principle
Extraction of NDELA in cosmetics samples is carried out with water in the presence of deuterated d8-
NDELA used as internal standard (IS). Clean-up is performed either using solid phase extraction (SPE
clean-up, see 6.3.1) with a C18 cartridge or liquid–liquid extraction using dichloromethane (DCM clean-
up, see 6.3.2) when the samples are not dispersible in water. The extracts are analysed by HPLC-MS-MS
(high performance liquid chromatography coupled with tandem mass spectrometric detection).
Identification of NDELA is carried out by using the molecular ion and two diagnostic ions. NDELA
quantification is done by comparing the ratio of the major fragment ions of NDELA and d8-NDELA with
the calibration curve.
In accordance with the ISO 12787, the absence of NDELA in the sample could be confirmed with a second
analysis. A spiked preparation at a target value could be performed to evaluate the limit of detection of
NDELA in the sample.
If matrix effect is observed with significant impact on the performance of the method (sensitivity,
accuracy, etc.) for specific cosmetic product, standard addition calibration procedures could be utilized
(refer to ISO 12787).
© ISO 2014 – All rights reserved 1

---------------------- Page: 9 ----------------------

SIST ISO 15819:2015
ISO 15819:2014(E)

4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696:1987. Solvents shall be of a suitable
quality for HPLC-MS.
4.1 Methanol (MeOH), HPLC-MS grade.
4.2 Ethanol (EtOH), HPLC-MS grade.
4.3 Dichloromethane (DCM), HPLC-MS grade.
4.4 N-nitrosodiethanolamine, with known purity greater than 95 %.
4.5 d8-N-nitrosodiethanolamine, with known purity greater than 95 %.
4.6 Ammonium acetate (NH Ac), analytical grade (suitable for HPLC-MS).
4
4.7 1 mol/l ammonium acetate solution, for the preparation of 1,0 l, dissolve 77,08 g of NH Ac in
4
1,0 l of water.
4.8 Eluent A: 2 mmol NH Ac in water, for the preparation of 1,0 l, by taking 2 ml of 1 mol/l NH Ac
4 4
(4.7) and making up to 1 l with water.
4.9 Eluent B: 2 mmol NH Ac in 90 / 10 MeOH / water v/v, formed, for the preparation of 1,0 l, by
4
taking 2 ml of 1 mol/l NH Ac (4.7) and making up to 1 l with mixture of 90/10 MeOH / water v/v.
4
5 Apparatus
Use standard laboratory glassware and equipment, with the addition of:
5.1 Vortex mixer.
®1)
5.2 Sample processing station, in SPE application [such as Vacmaster ] sample processing station.
5.3 High speed centrifuge (ideally 20 000 G).
NOTE If centrifuge speed below 20 000 G is used, pay attention to possible clogging problem during the SPE
clean-up process. If necessary, additional filtration step with 0,2 µm pore size membrane filter could be added.
®1)
5.4 Solid phase extraction columns, e.g. Bakerbond C18 — 6 ml, 500 mg reversed phase
octadecylsilane bonded to silica gel, 40
...

INTERNATIONAL ISO
STANDARD 15819
Second edition
2014-10-15
Cosmetics — Analytical
methods — Nitrosamines:
Detection and determination of
N-nitrosodiethanolamine (NDELA) in
cosmetics by HPLC-MS-MS
Cosmétiques — Méthodes analytiques — Nitrosamines: Recherche
et dosage de la N-nitrosodiéthanolamine (NDELA) dans les produits
cosmétiques par CLHP-SM-SM
Reference number
ISO 15819:2014(E)
©
ISO 2014

---------------------- Page: 1 ----------------------
ISO 15819:2014(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2014
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form
or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior
written permission. Permission can be requested 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 2014 – All rights reserved

---------------------- Page: 2 ----------------------
ISO 15819:2014(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Principle . 1
4 Reagents . 2
5 Apparatus . 2
6 Sample preparation and conservation . 3
6.1 General . 3
6.2 Standard solutions preparation . 3
6.3 Sample preparation . 4
7 Procedure. 5
7.1 General . 5
7.2 Chromatographic conditions . 5
7.3 HPLC-MS-MS conditions . 6
8 Calculation of results . 7
8.1 Determination of the R value . 7
8.2 Calibration curve . 7
8.3 Validity check procedure and its criteria . 7
8.4 Calculation of concentrations . 7
9 Test report . 8
Annex A (informative) Examples of a calibration curve and of chromatograms .9
Bibliography .11
© ISO 2014 – All rights reserved iii

---------------------- Page: 3 ----------------------
ISO 15819:2014(E)

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical Barriers
to Trade (TBT) see the following URL: Foreword - Supplementary information
The committee responsible for this document is ISO/TC 217, Cosmetics.
This second edition cancels and replaces the first edition (ISO 15819:2008), which has been
technically revised.
iv © ISO 2014 – All rights reserved

---------------------- Page: 4 ----------------------
ISO 15819:2014(E)

Introduction
Human exposure to N-nitrosamines can occur through diverse sources such as environment, food, or
personal care products. As a result of their perceived carcinogenic potential on several animal species,
minimization of exposure to N-nitrosamines is recognized as important to the preservation of human
health. Among N-nitrosamines, N-nitrosodiethanolamine (NDELA) has been recognized as a potential
contaminant of cosmetics.
In this context, several analytical methods have been developed to detect and determine its presence
in cosmetics, such as gas chromatography/thermal energy analysis, high performance liquid
chromatography (HPLC) coupled either with photolysis and colourimetric quantification or with mass
spectrometry (MS) determination. This latter method uses advanced technology to ensure the maximum
specificity towards NDELA, to minimize the risk of artefactual formation of the analyte of interest and
to allow precise quantification.
This analytical method uses high performance liquid chromatography coupled with mass spectrometry
to separate and detect trace levels of NDELA from a cosmetic ingredient or product matrix with
maximum specificity for NDELA.
© ISO 2014 – All rights reserved v

---------------------- Page: 5 ----------------------
INTERNATIONAL STANDARD ISO 15819:2014(E)
Cosmetics — Analytical methods — Nitrosamines:
Detection and determination of N-nitrosodiethanolamine
(NDELA) in cosmetics by HPLC-MS-MS
1 Scope
This International Standard describes a method for the detection and quantification of
N-nitrosodiethanolamine (NDELA) in cosmetics and raw materials used in cosmetics.
This method is not applicable to the detection and/or quantification of nitrosamines other than NDELA
nor to the detection and/or quantification of NDELA in products other than cosmetics or raw materials
used in cosmetics.
If a product has a possibility of either NDELA contamination from ingredients or NDELA formation by
the composition of ingredients, the method is intended to be applied for quantitative determination of
NDELA. Accordingly, the method does not apply to routine testing of cosmetic products. Because of the
large variety of cosmetic products within this field of application, this method might need to be adapted
for certain matrices (refer to ISO 12787).
Therefore, International Standards dedicated to alternative methods for testing nitrosamines in cosmetic
products are being developed separately. Other methods can be employed provided that they are verified
as to their detection of NDELA and validated in terms of recovery and quantification of the analyte.
2 Normative references
The following documents, 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 3696:1987, Water for analytical laboratory use — Specification and test methods
ISO 12787:2011, Cosmetics — Analytical methods — Validation criteria for analytical results using
chromatographic techniques
3 Principle
Extraction of NDELA in cosmetics samples is carried out with water in the presence of deuterated d8-
NDELA used as internal standard (IS). Clean-up is performed either using solid phase extraction (SPE
clean-up, see 6.3.1) with a C18 cartridge or liquid–liquid extraction using dichloromethane (DCM clean-
up, see 6.3.2) when the samples are not dispersible in water. The extracts are analysed by HPLC-MS-MS
(high performance liquid chromatography coupled with tandem mass spectrometric detection).
Identification of NDELA is carried out by using the molecular ion and two diagnostic ions. NDELA
quantification is done by comparing the ratio of the major fragment ions of NDELA and d8-NDELA with
the calibration curve.
In accordance with the ISO 12787, the absence of NDELA in the sample could be confirmed with a second
analysis. A spiked preparation at a target value could be performed to evaluate the limit of detection of
NDELA in the sample.
If matrix effect is observed with significant impact on the performance of the method (sensitivity,
accuracy, etc.) for specific cosmetic product, standard addition calibration procedures could be utilized
(refer to ISO 12787).
© ISO 2014 – All rights reserved 1

---------------------- Page: 6 ----------------------
ISO 15819:2014(E)

4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696:1987. Solvents shall be of a suitable
quality for HPLC-MS.
4.1 Methanol (MeOH), HPLC-MS grade.
4.2 Ethanol (EtOH), HPLC-MS grade.
4.3 Dichloromethane (DCM), HPLC-MS grade.
4.4 N-nitrosodiethanolamine, with known purity greater than 95 %.
4.5 d8-N-nitrosodiethanolamine, with known purity greater than 95 %.
4.6 Ammonium acetate (NH Ac), analytical grade (suitable for HPLC-MS).
4
4.7 1 mol/l ammonium acetate solution, for the preparation of 1,0 l, dissolve 77,08 g of NH Ac in
4
1,0 l of water.
4.8 Eluent A: 2 mmol NH Ac in water, for the preparation of 1,0 l, by taking 2 ml of 1 mol/l NH Ac
4 4
(4.7) and making up to 1 l with water.
4.9 Eluent B: 2 mmol NH Ac in 90 / 10 MeOH / water v/v, formed, for the preparation of 1,0 l, by
4
taking 2 ml of 1 mol/l NH Ac (4.7) and making up to 1 l with mixture of 90/10 MeOH / water v/v.
4
5 Apparatus
Use standard laboratory glassware and equipment, with the addition of:
5.1 Vortex mixer.
®1)
5.2 Sample processing station, in SPE application [such as Vacmaster ] sample processing station.
5.3 High speed centrifuge (ideally 20 000 G).
NOTE If centrifuge speed below 20 000 G is used, pay attention to possible clogging problem during the SPE
clean-up process. If necessary, additional filtration step with 0,2 µm pore size membrane filter could be added.
®1)
5.4 Solid phase extraction columns, e.g. Bakerbond C18 — 6 ml, 500 mg reversed phase
octadecylsilane bonded to silica gel, 40 APD (Average Particle Diameter), 60 Å.
5.5 HPLC-MS-MS equipment.
5.5.1 High performance liquid chromatography apparatus, consisting of an eluent reservoir, a
pump, an injection system, a data processor, e.g. an integrator with plotter, coupled with tandem mass
spectrometry using electrospray ionization.
® ®
1) Vacmaster and Bakerbond are examples of suitable products available commercially. This information
is given for the convenience of users of this document and does not constitute an endorsement by ISO of these
products.
2 © ISO 2014 – All rights reserved

---------------------- Page: 7 ----------------
...

NORME ISO
INTERNATIONALE 15819
Deuxième édition
2014-10-15
Cosmétiques — Méthodes analytiques
— Nitrosamines: Recherche et dosage
de la N-nitrosodiéthanolamine
(NDELA) dans les produits
cosmétiques par CLHP-SM-SM
Cosmetics — Analytical methods — Nitrosamines: Detection and
determination of N-nitrosodiethanolamine (NDELA) in cosmetics
by HPLC-MS-MS
Numéro de référence
ISO 15819:2014(F)
©
ISO 2014

---------------------- Page: 1 ----------------------
ISO 15819:2014(F)

DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2014
Droits de reproduction réservés. Sauf indication contraire, aucune partie de cette publication ne peut être reproduite ni utilisée
sous quelque forme que ce soit et par aucun procédé, électronique ou mécanique, y compris la photocopie, l’affichage sur
l’internet ou sur un Intranet, sans autorisation écrite préalable. Les demandes d’autorisation peuvent être adressées à l’ISO à
l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.
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
Publié en Suisse
ii © ISO 2014 – Tous droits réservés

---------------------- Page: 2 ----------------------
ISO 15819:2014(F)

Contents Page
Avant-propos .v
Introduction .vi
1 Domaine d’application . 1
2 Références normatives . 1
3 Principe . 1
4 Réactifs . 2
5 Appareillage . 2
6 Préparation et conservation d’échantillons . 3
6.1 Généralités . 3
6.2 Préparation des solutions étalons . 4
6.3 Préparation de l’échantillon . 5
7 Mode opératoire. 6
7.1 Généralités . 6
7.2 Conditions chromatographiques . 6
7.3 Conditions CLHP-SM-SM . 7
8 Calcul des résultats . 7
8.1 Détermination de la valeur R . 7
8.2 Courbe d’étalonnage . 7
8.3 Mode opératoire de vérification de la validité et ses critères . 8
8.4 Calcul des concentrations . 8
9 Rapport d’essai . 8
Annexe A (informative) Exemples d’une courbe d’étalonnage et de chromatogrammes .10
Bibliographie .12
© ISO 2014 – Tous droits réservés iii

---------------------- Page: 3 ----------------------
ISO 15819:2014(F)

Avant-propos
L’ISO (Organisation internationale de normalisation) est une fédération mondiale d’organismes
nationaux de normalisation (comités membres de l’ISO). L’élaboration des Normes internationales est
en général confiée aux comités techniques de l’ISO. Chaque comité membre intéressé par une étude
a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,
gouvernementales et non gouvernementales, en liaison avec l’ISO participent également aux travaux.
L’ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui concerne
la normalisation électrotechnique.
Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont
décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier de prendre note des différents
critères d’approbation requis pour les différents types de documents ISO. Le présent document a été
rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir www.
iso.org/directives).
L’attention est appelée sur le fait que certains des éléments du présent document peuvent faire l’objet de
droits de propriété intellectuelle ou de droits analogues. L’ISO ne saurait être tenue pour responsable
de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant les
références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de l’élaboration
du document sont indiqués dans l’Introduction et/ou dans la liste des déclarations de brevets reçues par
l’ISO (voir www.iso.org/brevets).
Les appellations commerciales éventuellement mentionnées dans le présent document sont données pour
information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un engagement.
Pour une explication de la signification des termes et expressions spécifiques de l’ISO liés à l’évaluation de
la conformité, ou pour toute information au sujet de l’adhésion de l’ISO aux principes de l’OMC concernant
les obstacles techniques au commerce (OTC), voir le lien suivant: Avant-propos — Informations
supplémentaires.
Le comité chargé de l’élaboration du présent document est l’ISO/TC 217, Cosmétiques.
Cette deuxième édition annule et remplace la première édition (ISO 15819:2008), qui a fait l’objet d’une
révision technique.
iv © ISO 2014 – Tous droits réservés

---------------------- Page: 4 ----------------------
ISO 15819:2014(F)

Introduction
L’exposition humaine aux N-nitrosamines peut se produire au contact de sources diverses présentes
dans l’environnement, la nourriture ou les produits de soin personnels. Ces substances ayant montré un
effet cancérigène notoire sur plusieurs espèces animales, il est reconnu qu’une restriction d’exposition
aux N-nitrosamines est d’une importance primordiale pour la préservation de la santé humaine. Parmi
les N-nitrosamines, il a été établi que la N-nitrosodiéthanolamine (NDELA) est un contaminant potentiel
des cosmétiques.
Dans ce contexte, plusieurs méthodes analytiques ont été développées pour rechercher et doser sa
présence dans les cosmétiques, comme la chromatographie en phase gazeuse avec analyse d’énergie
thermique, la chromatographie en phase liquide à haute performance (CLHP) couplée soit à une photolyse
avec quantification colorimétrique soit à une détermination par spectrométrie de masse (SM). Cette
dernière méthode nécessite l’usage de techniques avancées pour garantir une spécificité maximale dans
la recherche de la NDELA, afin de limiter le risque de formation artefactuelle de l’analyte considéré et de
permettre une quantification précise de la substance.
La présente méthode analytique emploie la chromatographie en phase liquide à haute performance
couplée à la spectrométrie de masse afin de séparer et de rechercher la NDELA à l’état de trace dans un
ingrédient ou dans une matrice de produit cosmétique, avec une spécificité maximale pour la NDELA.
© ISO 2014 – Tous droits réservés v

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NORME INTERNATIONALE ISO 15819:2014(F)
Cosmétiques — Méthodes analytiques — Nitrosamines:
Recherche et dosage de la N-nitrosodiéthanolamine
(NDELA) dans les produits cosmétiques par CLHP-SM-SM
1 Domaine d’application
La présente Norme internationale décrit une méthode de recherche et de quantification de la
N-nitrosodiéthanolamine (NDELA) dans les cosmétiques et les matières premières utilisées dans
les cosmétiques.
La présente méthode n’est applicable ni à la recherche et/ou à la quantification de nitrosamines autres
que la NDELA, ni à la recherche et/ou à la quantification de la NDELA dans les produits autres que les
cosmétiques ou les matières premières utilisées dans les cosmétiques.
Si un produit peut être contaminé par la NDELA à partir de ses ingrédients ou que la composition
d’ingrédients cosmétiques peut entraîner la formation de NDELA, alors la méthode sera appliquée pour
la détermination de la quantité de NDELA. Par conséquent, cette méthode ne s’applique pas aux essais
de routine relatifs aux produits cosmétiques. En raison du large éventail de cosmétiques considéré dans
ce domaine d’application, la présente méthode pourrait nécessiter d’être adaptée à certaines matrices
(se référer à l’ISO 12787).
Par voie de conséquence, les Normes internationales consacrées à d’autres méthodes d’essai des
nitrosamines dans les cosmétiques sont élaborées séparément. D’autres méthodes peuvent être
employées à condition que la recherche de NDELA ait été vérifiée et que ces méthodes soient validées en
termes de récupération et de quantification de l’analyte.
2 Références normatives
Les documents ci-après, dans leur intégralité ou non, sont des références normatives indispensables à
l’application du présent document. Pour les références datées, seule l’édition citée s’applique. Pour les
références non datées, la dernière édition du document de référence s’applique (y compris les éventuels
amendements).
ISO 3696:1987, Eau pour laboratoire à usage analytique — Spécification et méthodes d’essai
ISO 12787:2011, Cosmétiques — Méthodes analytiques — Critères de validation pour les résultats
analytiques utilisant des techniques chromatographiques
3 Principe
L’extraction de la NDELA des échantillons cosmétiques est réalisée avec de l’eau en présence de d8-NDELA
deutérée utilisée en tant qu’étalon interne (IS). Une purification est effectuée soit par extraction en
phase solide (purification SPE, voir 6.3.1) à l’aide d’une cartouche C18, soit par extraction liquide-liquide
à l’aide de dichlorométhane (purification DCM, voir 6.3.2) lorsque les échantillons ne peuvent pas se
disperser dans l’eau. Les extraits sont analysés par CLHP-SM-SM (chromatographie en phase liquide à
haute performance couplée à une détection spectrométrique de masse).
L’identification de NDELA est obtenue à l’aide de l’ion moléculaire et de deux ions qualifiants. La
quantification de NDELA est réalisée par comparaison du ratio obtenu entre les ions fragments
majoritaires de la NDELA et du d8-NDELA avec une courbe d’étalonnage.
© ISO 2014 – Tous droits réservés 1

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ISO 15819:2014(F)

Conformément à l’ISO 12787, l’absence de NDELA dans l’échantillon pourrait être confirmée par une
seconde analyse. Une préparation dopée à une valeur cible pourrait être effectuée pour évaluer la limite
de détection de la NDELA dans l’échantillon.
Si un effet matrice est observé avec un impact significatif sur la performance de la méthode (sensibilité,
exactitude, etc.) pour un produit cosmétique spécifique, il est possible d’utiliser une procédure
d’étalonnage par ajouts dosés (se référer à l’ISO 12787).
4 Réactifs
Sauf indication contraire, n’utiliser, au cours de l’analyse, que des réactifs de qualité analytique reconnue
et de l’eau distillée ou de l’eau de qualité 1, conformément à l’ISO 3696:1987. Les solvants doivent être de
qualité pour analyse CLHP-SM.
4.1 Méthanol (MeOH), qualité CLHP-SM.
4.2 Éthanol (EtOH), qualité CLHP-SM.
4.3 Dichlorométhane (DCM), qualité CLHP-SM.
4.4 N-nitrosodiéthanolamine, de pureté connue, supérieure à 95 %.
4.5 d8-N-nitrosodiéthanolamine, de pureté connue, supérieure à 95 %.
4.6 Acétate d’ammonium (NH Ac), qualité analytique (adapté à l’analyse par CLHP-SM).
4
4.7 Solution d’acétate d’ammonium 1 mol/l, pour la préparation de 1,0 l, dissoudre 77,08 g de NH Ac
4
dans 1,0 l d’eau.
4.8 Éluant A: 2 mmol de NH Ac dans l’eau, pour la préparation de 1,0 l, à partir de 2 ml de NH Ac à
4 4
1 mol/l (4.7), complété à 1 l avec de l’eau.
4.9 Éluant B: 2 mmol de NH Ac dans 90/10 MeOH/eau (v/v), réalisé, pour la préparation de 1,0 l, à
4
partir de 2 ml de NH Ac à 1 mol/l (4.7), complété à 1 l avec un mélange de 90/10 de MeOH/eau (v/v).
4
5 Appareillage
Utiliser une verrerie et un équipement de laboratoire courant, ainsi que:
5.1 Agitateur de type vortex.
5.2 Station de traitement d’échantillon, pour application SPE [telle que la station de traitement
1)
®
d’échantillon Vacmaster ].
5.3 Centrifugeuse à haute vitesse (idéalement 20 000 G).
NOTE Si une vitesse de centrifugeuse inférieure à 20 000 G est utilisée, faire attention à un possible problème
de colmatation pendant le processus de purification SPE. Si nécessaire, une étape de filtration supplémentaire
avec un filtre à membrane de 0,2 µm de porosité pourrait être utilisée.
® ®
1) Vacmaster et Bakerbond sont des exemples de produits appropriés disponibles sur le marché. Cette
information est donnée à l’intention des utilisateurs du présent document et ne signifie nullement que l’ISO approuve
ou recommande l’emploi exclusif des produits ainsi désignés.
2 © ISO 2014 – Tous droits réservés

---------------------- Page: 7 ----------------------
ISO 15819:2014(F)

®1)
5.4 Colonnes d’extraction en phase solide, par exemple Bakerbond C18 – 6 ml, avec 500 mg
d’octadécylsilane en phase inverse fixée sur gel de silice, 40 APD (diamètre moyen de particules), 60 Å.
5.5 Équipement CLHP-SM-SM
5.5.1 Chromatographe en phase liquide à haute performance, consistant en un réservoir d’éluant,
une pompe, un système d’injection, un système de traitement des données, par exemple un intégrateur
avec traceur, couplé à un spectromètre de masse en mode ionisation par électrospray.
®2)
5.5.2 Colonne analytique de séparation CLHP en phase inverse, C18, par exemple Spherisorb
ODS II protégée par une colonne de garde dont les dimensions sont les suivantes:
— colonne de séparation
— longueur: 100 mm;
— diamètre interne: 2,1 mm;
— taille des particules sphériques: 5 µm;
— colonne de garde
— longueur: 10 mm;
— diamètre interne: 2,1 – 3,0 mm;
— taille des particules sphériques: 5 µm.
L’utilisation d’une colonne de garde est facultative. En cas d’u
...

SLOVENSKI STANDARD
oSIST ISO/DIS 15819:2013
01-november-2013
.R]PHWLND$QDOL]QHPHWRGH1LWUR]DPLQLGHWHNFLMDLQGRORþHYDQMH1
QLWUR]RGLHWDQRODPLQD1'(/$YNR]PHWLþQLKL]GHONLKVWHNRþLQVNRNURPDWRJUDILMR
YLVRNHORþOMLYRVWL]PDVQRVSHNWURPHWULMVNRGHWHNFLMR+3/&0606
Cosmetics - Analytical methods - Nitrosamines: Detection and determination of N-
nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS
Cosmétiques - Méthodes analytiques - Nitrosamines: Recherche et dosage de la N-
nitrosodiéthanolamine (NDELA) dans les produits cosmétiques par CLHP-SM-SM
Ta slovenski standard je istoveten z: ISO/DIS 15819
ICS:
71.100.70 .R]PHWLND7RDOHWQL Cosmetics. Toiletries
SULSRPRþNL
oSIST ISO/DIS 15819:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST ISO/DIS 15819:2013

---------------------- Page: 2 ----------------------
oSIST ISO/DIS 15819:2013

DRAFT INTERNATIONAL STANDARD ISO/DIS 15819
ISO/TC 217 Secretariat: ISIRI
Voting begins on Voting terminates on

2013-07-25 2013-10-25
INTERNATIONAL ORGANIZATION FOR STANDARDIZATION  •  МЕЖДУНАРОДНАЯ ОРГАНИЗАЦИЯ ПО СТАНДАРТИЗАЦИИ  •  ORGANISATION INTERNATIONALE DE NORMALISATION


Cosmetics — Analytical methods — Nitrosamines: Detection
and determination of N-nitrosodiethanolamine (NDELA) in
cosmetics by HPLC-MS-MS
Cosmétiques — Méthodes analytiques — Nitrosamines: Recherche et dosage de la N-nitrosodiéthanolamine
(NDELA) dans les produits cosmétiques par CLHP-SM-SM
[Revision of first edition (ISO 15819:2008)]
ICS 71.100.70









To expedite distribution, this document is circulated as r eceived from the committee
secretariat. ISO Central Secretariat work of editing and text composition will be undertaken at
publication stage.
Pour accélérer la distribution, le présent document est distribué tel qu'il est parvenu du
secrétariat du comité. Le travail de rédaction et de composition de texte sera effectué au
Secrétariat central de l'ISO au stade de publication.



THIS DOCUMENT IS A DRAFT CIRCULATED FOR COMMENT AND APPROVAL. IT IS THEREFORE SUBJECT TO CHANGE AND MAY NOT BE
REFERRED TO AS AN INTERNATIONAL STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS BEING ACCEPTABLE FOR INDUSTRIAL, TECHNOLOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN NATIONAL REGULATIONS.
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT, WITH THEIR COMMENTS, NOTIFICATION OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPORTING DOCUMENTATION.
©  International Organization for Standardization, 2013

---------------------- Page: 3 ----------------------
oSIST ISO/DIS 15819:2013
ISO/DIS 15819

COPYRIGHT PROTECTED DOCUMENT


©  ISO 2013
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any
means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission.
Permission can be requested from either ISO at the address below or ISO’s member body in the country of the requester.
ISO copyright office
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Tel. + 41 22 749 01 11
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E-mail [e-mail]
Web www.iso.org
Published in Switzerland

ii © ISO 2013 – All rights reserved

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oSIST ISO/DIS 15819:2013
ISO/DIS 15819
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 15819 was prepared by Technical Committee ISO/TC 217, Cosmetics.

ii Error! Reference source not found.

---------------------- Page: 5 ----------------------
oSIST ISO/DIS 15819:2013
ISO/DIS 15819
Introduction
Human exposure to N-nitrosamines can occur through diverse sources such as environment, food or personal
care products. As a result of their perceived carcinogenic potential on several animal species, minimization of
exposure to N-nitrosamines is recognised as important to the preservation of human health. Among
N-nitrosamines, N-nitrosodiethanolamine (NDELA) has been recognised as a potential contaminant of
cosmetics.
In this context, several analytical methods have been developed to detect and determine its presence in
cosmetics – such as gas chromatography/thermal energy analysis, high performance liquid chromatography
(HPLC) coupled either with photolysis and colorimetric quantification or with mass spectrometry (MS)
determination. This latter method uses advanced technology to ensure the maximum specificity towards
NDELA, to minimize the risk of artifactual formation of the analyte of interest and to allow precise
quantification.
This analytical method uses high performance liquid chromatography coupled with mass spectrometry to
separate and detect trace levels of NDELA from a cosmetic ingredient or product matrix with maximum
specificity for NDELA.

Error! Reference source not found. iii

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oSIST ISO/DIS 15819:2013
ISO/DIS 15819
Revision of ISO 15819 Cosmetics-Analytical methods-Nitrosamines:
Detection and determination of N-nitrosodiethanolamine (NDELA) in
cosmetics by HPLC-MS-MS
1 Scope
This International Standard describes a method for the detection and quantification of N-
nitrosodiethanolamine (NDELA) in cosmetics and raw materials used in cosmetics.
This method is not applicable to the detection and/or quantification of nitrosamines other than NDELA nor to
the detection and/or quantification of NDELA in products other than cosmetics or raw materials used in
cosmetics.
If a product has a possibility of either NDELA contamination from ingredients or NDELA formation by the
composition of ingredients, the method shall be applied for quantitative determination of NDELA. Accordingly
the method would not be applied in routine testing of cosmetic products. Because of the large variety of
cosmetic products within this field of application, this method might need to be adapted for certain matrices.
Therefore, International Standards dedicated to alternative methods for testing nitrosamines in cosmetic
products are being developed separately. Other methods can be employed provided that they are verified as
to their detection of NDELA and validated in terms of recovery and quantification of the analyte.

2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 3696:1987, Water for analytical laboratory use — Specification and test methods.
ISO 12787:2011, Cosmetics — Analytical methods — Validation criteria for analytical results using
chromatographic techniques.
3 Principle
Extraction of NDELA in cosmetics samples is carried out with water in the presence of deuterated d8-NDELA
used as internal standard. Clean-up is performed either using solid phase extraction (SPE clean-up, see
6.3.1) with a C18 cartridge or liquid–liquid extraction using dichloromethane (DCM clean-up, see 6.3.2) when
the samples are not dispersible in water. The extracts are analysed by HPLC-MS-MS (high performance liquid
chromatography coupled with tandem mass spectrometric detection).
Identification of the presence of NDELA is carried out by using the molecular ion and two diagnostic ions.
NDELA quantification is done by comparing the ratio of the major fragment ions of NDELA and d8-NDELA
with the calibration curve.
In accordance with the ISO 12787, the absence of NDELA in the sample could be confirmed with a second
analysis. A spiked preparation at a target value (LOD) could be performed to evaluate the limit of detection of
NDELA in the sample.
4 © ISO 2002 – All rights reserved

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oSIST ISO/DIS 15819:2013
ISO/DIS 15819
If matrix effect is observed with significant impact on the performance of the method (sensitivity, accuracy,
etc.) for specific cosmetic product, standard addition calibration procedures could be utilized. See ISO 12787
for reference.
4 Reagents
During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only
distilled water or water of grade 1 in accordance with ISO 3696:1987. Solvents shall be of a suitable quality for
HPLC-MS.
4.1 Methanol (MeOH), HPLC-MS grade.
4.2 Ethanol (EtOH), HPLC-MS grade.
4.3 Dichloromethane (DCM),HPLC-MS grade.
4.4 N-nitrosodiethanolamine, with known purity greater than 95 %.
4.5 d8-N-nitrosodiethanolamine, with known purity greater than 95 %.
4.6 Ammonium acetate (NH Ac), analytical grade (suitable for HPLC-MS).
4
4.7 1 mol/l ammonium acetate solution. For the preparation of 1,0 l, dissolve 77,08 g of NH Ac in 1,0 l
4
water.
4.8 Eluent A: 2 mmol NH Ac in water, formed, for the preparation of 1,0 l, by taking 2 ml of 1 mol/l NH Ac
4 4
(4.7) and making up to 1 l with water.
4.9 Eluent B: 2 mmol NH Ac in 90 / 10 MeOH / water v/v,formed, for the preparation of 1,0 l, by taking
4
2 ml of 1 mol/l NH Ac (4.7) and making up to 1 l with mixture 90/10 MeOH / water v/v, formed by mixing 900
4
ml MeOH and 100 ml water.
5 Apparatus
Use standard laboratory glassware and equipment, with the addition of:
5.1 Vortex mixer.
1
5.2 Sample processing station, in SPE application (such as Vacmaster ) sample processing station,
IST).
5.3 High speed centrifuge (ideally 20 000 G).
NOTE If centrifuge speed below 20 000 G is used, pay attention to possible clogging problem during the
SPE clean-up process. If necessary, additional filtration step with 0,2 µm pore size membrane filter could be
added.



1
® ® ®
Vacmaster , Bakerbond and Spherisorb are examples of suitable products available commercially. This information is
given for the convenience of users of this International Standard and does not constitute an endorsement by ISO of these
products.
© ISO 2002 – All rights reserved 5

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oSIST ISO/DIS 15819:2013
ISO/DIS 15819
2
5.4 Solid phase extr action columns, e.g. Bakerbond C18– 6 ml, 500 mg reversed phase
octadecylsilane bonded to silica gel, 40 APD (Average Particle Diameter), 60 Å.
5.5 HPLC-MS-MS equipment
5.5.1 High performance liquid chromatography apparatus, consisting of an eluent reservoir, a pump, an
injection system, a data processor, e.g. an integrator with plotter, coupled with tandem mass spectrometry
using electrospray ionization.
® 2
5.5.2 Analytical reversed phase HPLC separating column, C18, e.g. Spherisorb ODS II protected with
a guard column, the dimensions of which are:
separating column
 length: 100mm
 internal diameter: 4,6 mm
 size of spherical particles: 5 µm
guard column
 length: 10 mm
 internal diameter: 2,1 – 3,0 mm
 size of spherical particles: 5 µm

NOTE : Condition of “the guard column” should be adjusted based on the brand that is used for analytical
reversed phase HPLC separating column
6 Sample preparation and conservation
6.1 General
WARNING — Most N-nitrosamines are potent carcinogens and every possible precaution shall be
taken to avoid human exposure.
All operations involving handling of N-nitrosamines or their solutions should take place in an adequately
ventilated fume hood or glove box.
NOTE Rubber surgical gloves, which are frequently employed, do not provide complete protection. They should be
removed and disposed of immediately after use and not worn for long periods.
Use safe disposal to discard any solution of material containing N-nitrosamines (such as, for example, tins or
buckets for hazardous chemical waste).
N-Nitrosodiethanolamine shall be stored in the absence of light between 2 °C and 8 °C.

2
® ® ®
Vacmaster , Bakerbond and Spherisor
...