1、BSI Standards PublicationBS ISO 15819:2014Cosmetics Analyticalmethods Nitrosamines:Detection and determinationof N-nitrosodiethanolamine(NDELA) in cosmetics by HPLC-MS-MSBS ISO 15819:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 15819:2014. Itsupersedes
2、BS ISO 15819:2008 which is withdrawn.The UK participation in its preparation was entrusted to TechnicalCommittee CW/217, Cosmetics.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purport to include all the necessaryprovisions
3、 of a contract. Users are responsible for its correctapplication. The British Standards Institution 2014. Published by BSI StandardsLimited 2014ISBN 978 0 580 82029 8ICS 71.100.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under
4、 the authority of theStandards Policy and Strategy Committee on 30 November 2014.Amendments issued since publicationDate Text affectedBS ISO 15819:2014 ISO 2014Cosmetics Analytical methods Nitrosamines: Detection and determination of N-nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MSCosmtiqu
5、es Mthodes analytiques Nitrosamines: Recherche et dosage de la N-nitrosodithanolamine (NDELA) dans les produits cosmtiques par CLHP-SM-SMINTERNATIONAL STANDARDISO15819Second edition2014-10-15Reference numberISO 15819:2014(E)BS ISO 15819:2014ISO 15819:2014(E)ii ISO 2014 All rights reservedCOPYRIGHT P
6、ROTECTED DOCUMENT ISO 2014All 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. P
7、ermission can be requested from either ISO at the address below or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 15819:2014ISO 15819
8、:2014(E)Contents PageForeword vIntroduction vi1 Scope . 12 Normative references 13 Principle 14 Reagents 25 Apparatus . 26 Sample preparation and conservation 36.1 General . 36.2 Standard solutions preparation . 36.3 Sample preparation 47 Procedure. 57.1 General . 57.2 Chromatographic conditions 57.
9、3 HPLC-MS-MS conditions . 68 Calculation of results . 78.1 Determination of the R value 78.2 Calibration curve . 78.3 Validity check procedure and its criteria . 78.4 Calculation of concentrations . 79 Test report . 8Annex A (informative) Examples of a calibration curve and of chromatograms 9Bibliog
10、raphy .11 ISO 2014 All rights reserved iiiBS ISO 15819:2014ISO 15819:2014(E)ForewordISO (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 t
11、echnical 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 clos
12、ely 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 n
13、eeded 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 pate
14、nt 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
15、 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 ISOs adherence to the WTO principles in the Technical Barr
16、iers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe 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 reservedBS ISO 15
17、819:2014ISO 15819:2014(E)IntroductionHuman 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 i
18、mportant 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 chromatograph
19、y/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
20、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 fo
21、r NDELA. ISO 2014 All rights reserved vBS ISO 15819:2014BS ISO 15819:2014Cosmetics Analytical methods Nitrosamines: Detection and determination of N-nitrosodiethanolamine (NDELA) in cosmetics by HPLC-MS-MS1 ScopeThis International Standard describes a method for the detection and quantification of N
22、-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 cosmetic
23、s.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
24、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.
25、 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 referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for
26、 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 methodsISO 12787:2011, Cosmetics Analytical metho
27、ds Validation criteria for analytical results using chromatographic techniques3 PrincipleExtraction 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,
28、see 6.3.1) with a C18 cartridge or liquidliquid 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 o
29、f 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 se
30、cond 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 cali
31、bration procedures could be utilized (refer to ISO 12787).INTERNATIONAL STANDARD ISO 15819:2014(E) ISO 2014 All rights reserved 1BS ISO 15819:2014ISO 15819:2014(E)4 ReagentsDuring the analysis, unless otherwise stated, use only reagents of recognized analytical grade and only distilled water or wate
32、r 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-nitrosodiethanola
33、mine, with known purity greater than 95 %.4.6 Ammonium acetate (NH4Ac), analytical grade (suitable for HPLC-MS).4.7 1 mol/l ammonium acetate solution, for the preparation of 1,0 l, dissolve 77,08 g of NH4Ac in 1,0 l of water.4.8 Eluent A: 2 mmol NH4Ac in water, for the preparation of 1,0 l, by takin
34、g 2 ml of 1 mol/l NH4Ac (4.7) and making up to 1 l with water.4.9 Eluent B: 2 mmol NH4Ac in 90 / 10 MeOH / water v/v, formed, for the preparation of 1,0 l, by taking 2 ml of 1 mol/l NH4Ac (4.7) and making up to 1 l with mixture of 90/10 MeOH / water v/v.5 ApparatusUse standard laboratory glassware a
35、nd equipment, with the addition of:5.1 Vortex mixer.5.2 Sample processing station, in SPE application such as Vacmaster1) 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
36、clean-up process. If necessary, additional filtration step with 0,2 m pore size membrane filter could be added.5.4 Solid phase extraction columns, e.g. Bakerbond1)C18 6 ml, 500 mg reversed phase octadecylsilane bonded to silica gel, 40 APD (Average Particle Diameter), 60 .5.5 HPLC-MS-MS equipment.5.
37、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) Vacmasterand Bakerbond are examples of suitable products ava
38、ilable 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 reservedBS ISO 15819:2014ISO 15819:2014(E)5.5.2 Analytical reversed phase HPLC separating column, C18, e.g. Spherisorb2)O
39、DS II protected with a guard column, the dimensions of which are: separating column length: 100 mm; internal diameter: 2,1 mm; size of spherical particles: 5 m; guard column length: 10 mm; internal diameter: 2,1 3,0 mm; size of spherical particles: 5 m.The use of a guard column is optional. If used,
40、 its ID is preferably same as that of separating column. The condition should be adjusted based on the brand that is used for analytical reversed phase HPLC separating column.6 Sample preparation and conservation6.1 GeneralWARNING Most N-nitrosamines are potent carcinogens and every possible precaut
41、ion 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.Rubber surgical gloves, which are frequently employed, do not provide complete protection. They should be removed and
42、 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.UV
43、 degrades N-nitrosamines, so all solutions (standards/extracts) shall be stored in such a way that deterioration and change in composition are prevented.HPLC-MS-MS analysis should typically be carried out within 30 min of preparation of the sample extracts. If analysis is delayed, then stability sho
44、uld be verified.6.2 Standard solutions preparation6.2.1 Accurately prepare a stock solution (A) of NDELA containing approximately 1,0 mg/ml in ethanol and store in the absence of light at less than 18 C. Record the exact concentration.6.2.2 Accurately prepare a stock solution (d8A) of d8-NDELA conta
45、ining approximately 1,0 mg/ml in ethanol and store in the absence of light at less than 18 C. Record the exact concentration.6.2.3 Prepare working solutions (B, C, D, E, and F) by successive dilutions of the stock solution (A). All solutions shall be stored in the absence of light between 2 C and 8
46、C.2) Spherisorbis an example of a suitable product available commercially. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product. ISO 2014 All rights reserved 3BS ISO 15819:2014ISO 15819:2014(E)Working solutionsStock or
47、working solution volumeWater volumeFinal concen-trationStabilityWorking solution B 100 l of A 900 l 100,0 g/ml 1 dWorking solution C 100 l of B 900 l 10,0 g/ml 1 dWorking solution D 100 l of C 900 l 1,0 g/ml 1 dWorking solution E 100 l of D 900 l 100,0 ng/ml 1 dWorking solution F 100 l of E 900 l 10
48、,0 ng/ml 1 dNOTE Users can determine actual volume used for the preparation as far as final concentration is secured.6.2.4 Prepare d8 working solutions (d8B and d8C) by sequential dilutions of the stock solution (d8A). All solutions shall be stored in the absence of light between 2 C and 8 C.d8 work
49、ing solutionsStock or working solution volumeWater volumeFinal concen-trationStabilityWorking solution d8B 20 l of d8A 20 ml 1,0 g/ml 1 dWorking solution d8C 200 l of d8B 1 800 l 100,0 ng/ml 1 dNOTE Users can determine actual volume used for the preparation as far as final concentration is secured.6.2.5 Prepare standard solutions by dilutions of the working solutions. A standard curve from 1,0 ng/ml to 80,0 ng/ml is made, using at least five of the seven standard solutions given
