1、BSI Standards PublicationBS EN 16521:2014Cosmetics Analyticalmethods GC/MS method forthe identification and assayof 12 phthalates in cosmeticsamples ready for analyticalinjectionBS EN 16521:2014 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN 16521:2014.The UK p
2、articipation 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 of a contract. Users are responsible for its
3、 correctapplication. The British Standards Institution 2014. Published by BSI StandardsLimited 2014ISBN 978 0 580 81191 3ICS 71.100.70Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Str
4、ategy Committee on 31 May 2014.Amendments issued since publicationDate Text affectedBS EN 16521:2014EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN 16521 May 2014 ICS 71.100.70 English Version Cosmetics - Analytical methods - GC/MS method for the identification and assay of 12 phthalates in cos
5、metic samples ready for analytical injection Cosmtiques - Mthodes analytiques - Mthode CG-SM pour lidentification et lanalyse de 12 phtalates dans des chantillons de produits cosmtiques prts tre injects dans un systme analytique Kosmetische Mittel - Analysenmethoden - GC/MS-Methode fr die Identifizi
6、erung und die Quantifizierung von 12 Phthalaten in zur direkten Injektion geeigneten Proben kosmetischer Mittel This European Standard was approved by CEN on 10 April 2014. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this Europe
7、an Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions
8、(English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belg
9、ium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switze
10、rland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2014 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national
11、 Members. Ref. No. EN 16521:2014 EBS EN 16521:2014EN 16521:2014 (E) 2 Contents Page Foreword 3 Introduction .3 1 Scope 5 2 Reagents .5 3 Apparatus and equipment 6 4 Procedure .6 4.1 Standard purity 6 4.2 Sample preparation .7 4.3 Gas chromatography (GC) measurement conditions 7 4.4 Detection .8 4.4.
12、1 General 8 4.4.2 MS detection in Selected Ion Monitoring (SIM) mode 8 5 Evaluation .9 5.1 Identification and quantitative determination .9 5.2 Calculation . 10 6 Test report . 10 Annex A (informative) Example of Chromatograms . 11 Bibliography . 12 BS EN 16521:2014EN 16521:2014 (E) 3 Foreword This
13、document (EN 16521:2014) has been prepared by Technical Committee CEN/TC 392 “Cosmetics”, the secretariat of which is held by AFNOR. This European Standard shall be given the status of a national standard, either by publication of an identical text or by endorsement, at the latest by November 2014 a
14、nd conflicting national standards shall be withdrawn at the latest by November 2014. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent rights. A
15、ccording to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germ
16、any, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. BS EN 16521:2014EN 16521:2014 (E) 4 Introduction Phthalates are esters of phthalic acid
17、(Figure 1). About 80 % of all phthalates manufactured are used as “plasticizers” to make plastics flexible without sacrificing strength or durability. These compounds are present in cosmetic products like perfumes and toiletries. Some phthalates, particularly those of low molecular weight, are intro
18、duced into cosmetics as ingredients, for examples DEP and DMP are used as solvents and perfume fixatives 1-3 or DEP can be used as alcohol denaturing 2, 4. Their presence in such products may come from their use as ingredients during the manufacturing process or may come from the migration of phthal
19、ates from packaging when plastic is used. Their presence as contaminant could also be due to the manufacturing process or raw materials used. Some analytical methods are proposed in the literature for the determination of phthalates in cosmetic products 1, 4-12. Figure 1 Esters of phthalic acid This
20、 standard proposes a GC/MS method for a simultaneous assay in cosmetic samples ready for analytical injection of 12 phthalates listed in Table 1. These chromatographic conditions are not suitable for the quantification of di-isononyl phthalate (DiNP) or di-isodecyl phthalate (DiDP). According to SCC
21、P 13, the possible presence of DiNP or DiDP in cosmetics does not seem to be a problem for human health. A GC/MS method using positive chemical ionisation with ammonia as collision gas is proposed in literature for the determinations of those compounds in cosmetic products 14. Analyses are carried o
22、ut on a GC/MS system with electron impact ionization mode (Ei). The separation of phthalates is obtained on a cross-linked 5 %-phenyl/95 %-dimethylpolysiloxane capillary column 30 m 0,25 mm (i. d.) 0,25 m film thickness using a temperature gradient. Phthalate quantification is performed by external
23、calibration using an internal standard or by the standard addition. Cosmetic samples are analyzed directly or after a previous dilution in ethanol 15. BS EN 16521:2014EN 16521:2014 (E) 5 1 Scope This European Standard describes a GC/MS method for the assay of 12 phthalates, amongst which the 8 phtha
24、lates regulated by the European cosmetic regulation 1223/2009 16. This method is given for the analysis of samples ready for analytical injection from cosmetic products or raw materials used in cosmetic products. Samples should be compatible with GC analysis possibly after dilution. This method does
25、 not include requirements for the preparation of samples in cosmetic matrices for which direct injection in GC is not feasible. 2 Reagents If not otherwise specified, analytical-grade chemicals shall be used. 2.1 Phthalates considered Table 1 Phthalates considered Phthalates CAS ManufacturerbQuality
26、 DBP a(dibutyl phthalate) 84742 ALDRICH 97,0 % DEHP a(diethylhexyl phthalate) 117817 ALDRICH 99,8 % BBP a(butylbenzyl phthalate) 85687 ALDRICH 97,0 % DMEP a(di(2-methoxyethyl) phthalate) 117828 ALDRICH 97,0 % DnPP a(di-n-pentyl phthalate) 131180 CIL CLUZEAU 99,0 % DiPP a(diisopentyl phthalate) 60550
27、5 CIL CLUZEAU 95,0 % DPP a(n-pentyl isopentyl phthalate) 84777060 CIL CLUZEAU 95,0 %* DiBP a(diisobutyl phthalate) 84695 ACROS 98,0 % DCHP (dicyclohexyl phthalate) 84617 ALDRICH 98,0 % DEP (diethyl phthalate) 84662 ACROS 98,0 % DMP (dimethyl phthalate) 131113 ACROS 98,0 % DnOP (di-n-octyl phthalate)
28、 117840 ALDRICH 98,0 % aRegulated phthalates. bThis is 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 CEN of this product. * Mixed isomers (DiPP, DnPP and DPP) 2.2 Ethanol 2.3
29、Internal standard, 4,4-Dibromodiphenyl from Fluka 1)97,0 % was used as internal standard (ISTD). 2.4 Internal standard stock solution (SM-ISTD), c = 1 000 g/ml. 1) This is an example of a suitable product available commercially. This information is given for the convenience of users of this document
30、 and does not constitute an endorsement by CEN of this product. BS EN 16521:2014EN 16521:2014 (E) 6 Weigh approximately 10 mg of 4,4-Dibromodiphenyl (2.3) into a 10 ml volumetric flask. Firstly, dissolve in a small amount of ethanol (2.2) and then fill to the calibration mark with ethanol. This stoc
31、k solution is daily prepared. 2.5 Phthalates stock solution, c = 500 g/ml (SM). Weigh approximately 10 mg of each phthalate (2.1) into a 20 ml volumetric flask. Firstly, dissolve in a small amount of ethanol (2.2) and then fill to the calibration mark with ethanol. This stock solution has a shelf li
32、fe of at least 2 weeks if stored in a refrigerator. NOTE Due to the low amount of DIPP standard (sold in quantities of 10,0 mg), a stock solution of this phthalate is prepared independently from other phthalates. In this way, two intermediate stock solutions SM-1 and SM-2 at 1 000 g/ml are prepared.
33、 SM-1 is prepared weighting approximately 10 mg of each phthalate (2.1), except DiPP, into a 10,0 ml volumetric flask, whereas SM-2 is prepared weighting approximately 5,0 mg of DiPP (2.1) into a 5,0 ml volumetric flask. The final stock solution SM is obtained mixing equal volumes of both standard s
34、olutions (c = 500 g/ml). All these stock solutions have a shelf life of at least 2 weeks if stored in a refrigerator. 2.6 Calibration solutions (standard solutions) 1,0 ml of the phthalate stock solution SM (2.5) is transferred into a 10,0 ml volumetric flask and filled with ethanol (2.2) up to the
35、calibration mark (c = 50 g/ml). From this intermediate solution (S1), at least 5 calibration solutions are prepared by dilution in ethanol (2.2) after the addition of 100 l of the internal standard stock solution (SM-ISTD). Phthalates concentrations on these calibration solutions ranges from 0,25 g/
36、ml to 5,0 g/ml with an ISTD concentration fixed at 10,0 g/ml. These calibration solutions are prepared extemporaneously and injected. If cosmetic samples (perfume) are directly prepared in a 1,5 ml GC vial, according to 4.2, the preparation of calibration solutions have to be adapted: Calibration so
37、lutions, ranging from 0,25 g/ml to 5,0 g/ml, are prepared without internal standard and 10 l of the internal standard stock solution (SM-ISTD) are added to 1,0 ml of each calibration solution directly in a 1,5 ml GC vial. These calibration solutions are prepared extemporaneously. Vials are shaken an
38、d the solution injected. 3 Apparatus and equipment 3.1 Standard laboratory equipment. 3.2 Gas chromatography/mass spectrometry apparatus. 3.3 Gas chromatography/FID (for standard purity). 3.4 Analytical separation column. The following parameters have proved useful: GC column, low bleeding phase: 5
39、%-Phenyl-95 %-dimethylpolysiloxane, 30 m 0,25 mm (i. d.) 0,25 m or equivalent material. 4 Procedure 4.1 Standard purity The purity of each standard and the internal standard and the respective percentages for geometrical isomers (n-pentyl isopentyl phthalate - DPP) shall be determined by GC-FID for
40、further calculations. BS EN 16521:2014EN 16521:2014 (E) 7 4.2 Sample preparation 1,0 g of the cosmetic sample ready for analytical injection is transferred into a 10,0 ml volumetric flask and filled with ethanol (2.2) up to the calibration mark after the addition of 100 l of the internal standard st
41、ock solution (SM-ISTD). This solution is injected. In case of excessive concentration of phthalates, an appropriate previous dilution of the sample is performed. The limit of quantification using this sample preparation was set at 5 ppm. An alternative sample preparation consists to prepare the cosm
42、etic sample directly in a 1,5 ml GC vial. 10 l of the internal standard stock solution (SM-ISTD) are added to 1,0 ml of cosmetic samples (perfume). Vials are shaken and the solution injected. The limit of quantification obtained using this sample preparation was set at 0,5 ppm. Calibration solutions
43、 are prepared according to 2.6. 4.3 Gas chromatography (GC) measurement conditions When using the apparatus (3.2) and column (3.4), the following conditions have shown to be useful: Table 2 GC/MS programme Apparatus Gas chromatography with mass selective detector (GC/MS) Autosampler Column GC column
44、, low bleeding phase: 5 %-Phenyl-95 %-dimethylpolysiloxane, 30 m 0,25 mm 0,25 m (or equivalent) Oven programme Ramp Temperature Time 100 C 0 min 30 C/min 200 C 0 min 3 C/min 260 C 0 min 30 C/min 320 C 5 min Injector T 300 C Interface T 250 C Source T 230 C Injection time 30 min Gas / flow rate He/1
45、ml/min Injection port split/splitless Injection parameters : 1 l/Constant pressure, Split 1/20 Detection mode Quadrupole Ionisation mode EI (70 eV) Mass detection (Full/SIM) Identification: full-scan (m/z 40 to 350) Quantification: SIM using 3 specific ions Internal standard 4,4-Dibromodiphenyl Cali
46、bration 0,25 g/ml to 5,0 g/ml Solvent used Ethanol (injection) BS EN 16521:2014EN 16521:2014 (E) 8 Examples of chromatogram, obtained using the GC/MS programme described in Table 2, are given in Annex A (Figure A.1). 4.4 Detection 4.4.1 General The detection and quantitative determination can be per
47、formed by evaluating the mass traces of each phthalate. Relative Retention (RR), m/z ions used for quantification and ions ratios obtained on a standard solution for each phthalate are given in Table 3. 4.4.2 MS detection in Selected Ion Monitoring (SIM) mode 1) from 2,5 min Ions 135 ;163 ; 194 (dwe
48、ll time 50 ms/ion); 2) from 4,0 min Ions 105 ; 149 ; 177 (dwell time 50 ms/ion); 3) from 5,0 min Ions 104 ; 149 ; 223 (dwell time 50 ms/ion); 4) from 7,0 min Ions 59; 104;149 ; 205; 223 (dwell time 30 ms/ion); 5) from 8,2 min Ions 104 ; 149 ; 219, 237, 310, 312 (dwell time 25 ms/ion); 6) from 10,5 m
49、in Ions 57; 71; 97 (dwell time 50 ms/ion); 7) from 13,0 min Ions 91 ; 149 ; 206 (dwell time 50 ms/ion); 8) from 16,5 min Ions 149 ; 167 ; 225; 226, 249; 279, (dwell time 25 ms/ion); 9) from 20,0 min Ions 149 ; 167 ; 279 (dwell time 50 ms/ion). Retention times of each group should be determined after the injection of a standard solution. NOTE 1 A dwell time at 50 msec per ion for a SIM window including three fragments is correct. If 6 ions are present, a 25 msec p
