12 Ftalati in Cosmetice

Journal of Chromatography A, 1253 (2012) 144 153

Contents lists available at SciVerse ScienceDirect

Journal of Chromatography A

j our na l ho me p ag e: www.elsev ier .com

Analyti saproduc tioCosme iterchroma

Pascal Gi et, Pierre-AnAgence nationa (DC),

a r t i c l e i n f o

Article history:Received 26 MReceived in reAccepted 28 JuAvailable onlin

Keywords:PhthalatesCosmeticsExtractionISO 12787Validation critGas chromatog

a b s t r a c t

Esters of phthalic acid, more commonly named phthalates, may be present in cosmetic products as

1. Introdu

Phthalatare often ptoiletries. Thtaminants ipackaging wdamage fertfertility or thregulation 1diethylhexy

CorresponE-mail add

(P. Gimeno).

0021-9673/$ http://dx.doi.oarch 2012vised form 26 June 2012ne 2012e 4 July 2012

eriaraphymass spectrometry

ingredients or contaminants. Their presence as contaminant can be due to the manufacturing process,to raw materials used or to the migration of phthalates from packaging when plastic (polyvinyl chlo-ride PVC) is used. 8 phthalates (DBP, DEHP, BBP, DMEP, DnPP, DiPP, DPP, and DiBP), classied H360or H361, are forbidden in cosmetics according to the European regulation on cosmetics 1223/2009. AGC/MS method was developed for the assay of 12 phthalates in cosmetics, including the 8 phthalatesregulated. Analyses are carried out on a GC/MS system with electron impact ionization mode (EI). Theseparation of phthalates is obtained on a cross-linked 5%-phenyl/95%-dimethylpolysiloxane capillarycolumn 30 m 0.25 mm (i.d.) 0.25 mm lm thickness using a temperature gradient. Phthalate quan-tication is performed by external calibration using an internal standard. Validation elements obtainedon standard solutions, highlight a satisfactory system conformity (resolution > 1.5), a common quanti-cation limit at 0.25 ng injected, an acceptable linearity between 0.5 g mL1 and 5.0 g mL1 as wellas a precision and an accuracy in agreement with in-house specications. Cosmetic samples ready foranalytical injection are analyzed after a dilution in ethanol whereas more complex cosmetic matrices,like milks and creams, are assayed after a liquid/liquid extraction using ter-butyl methyl ether (TBME).Depending on the type of cosmetics analyzed, the common limits of quantication for the 12 phtha-lates were set at 0.5 or 2.5 g g1. All samples were assayed using the analytical approach described inthe ISO 12787 international standard CosmeticsAnalytical methodsValidation criteria for analyticalresults using chromatographic techniques. This analytical protocol is particularly adapted when it is notpossible to make reconstituted sample matrices.

2012 Elsevier B.V. All rights reserved.

ction

es are esters of phthalic acid (Fig. 1). These compoundsresent in cosmetic products such as perfumes andeir presence can result of their use as ingredient, of con-

n raw materials or of the migration of phthalates fromhen plastic is used. 8 phthalates, classied H360 May

ility or the unborn child or H361 Suspected of damaginge unborn child, are regulated by the European cosmetic223/2009 (Annex II) [1ac] (dibutyl phthalate (DBP);l phthalate (DEHP); butylbenzyl phthalate (BBP);

ding author. Tel.: +33 467913951; fax: +33 467913983.resses: [email protected], [email protected]

di(2-methoxyethyl) phthalate (DMEP); di-n-pentyl phthalate(DnPP); diisopentyl phthalate (DiPP), n-pentyl isopentyl phthalate(DPP) and diisobutyl phthalate (DiBP)).

Some phthalates, particularly those with a low molecularweight, can be introduced into cosmetics and perfume as ingre-dients, for example DEP and DMP are used as solvents or perfumexatives [24], DEP can also be used as alcohol denaturing [3,5].Phthalates can also come from plastic containers. Indeed, phtha-lates are commonly used as plasticizers of polyvinyl chloride (PVC)and are not chemically bound to it. As a consequence, these com-pounds can be extracted from containers by cosmetic matrix. Thiscontamination can either come from the cosmetic packaging orfrom raw materials containers.

In 2012, no ofcial or standard method exists for the deter-mination of phthalates in cosmetic products and only fewanalytical methods are proposed in the literature [2,514].

see front matter 2012 Elsevier B.V. All rights reserved.rg/10.1016/j.chroma.2012.06.090cal method for the identication and asts: Application of the ISO 12787 internaticsAnalytical methodsValidation crtographic techniques

meno , Annie-Franc oise Maggio, Claudine Bousqutoine Bonnet

le de scurit du mdicament et des produits de sant (ANSM), Direction des Contrles/ locate /chroma

y of 12 phthalates in cosmeticnal standardia for analytical results using

Audrey Quoirez, Corinne Civade,

Unit Physico Chimie, 635 rue de la Garenne, 34740 Vendargues, France

P. Gimeno et al. / J. Chromatogr. A 1253 (2012) 144 153 145

OR1

O

HPLC/UV iseparation,cosmetic pC18 colum150 mm 4using eithertion [8,11,1[10], MeOHto pH 2.8 [8elution gra[2,6]. If mostication of(DEP, DBP, less used phphthalate (ber of phthIndeed, motion of 4 or(BBP, DEHPseparation and DEHP) regulation.

Althoug(GC) is alsoproducts [7separation DCHP, DEP,one paper [tion detectifor the idenusing GC/Mspectrometcolumn of ization is pobtained usin splitless mgas. Regardstudy dealsphthalates

Among ation of pht[11,12] propmonolith ma new liqui(ultrasoundsolidicatiobased on aextraction pbased on thon a celite sibility of dextract phthdescribes thafter the exformed usinsolvent, of t

also discussed [7]. It is noteworthy that this method requires largeamounts of solvent during the purication step. Extraction withethanol/water is also reported [10] but only for the assay of signif-icant phthalates levels in the sample (phthalate amount > 0.1%) so

ethodfor csionntrifxtracselecto thnd entermre exxanealate

122or thes usuid mts adalysient, s andhalatting eredting per byr a phthaes to

ISO dsVc tectic pexityical men teheneticr to ard ded fro gival rec anaion e

gromilaan bion s assntied. Tpossise pr

erimOR2

O

R1, R2 : Alkyl group

Fig. 1. Esters of phthalic acid.

s the most commonly described method for the identication and quantication of phthalates inroducts [2,68,1012]. All of these methods usens, either 250 mm 4.6 mm 5 m [2,6,8,11,12] or.6 mm 3.5 m [7,10]. Phthalate separation is obtained

a gradient elution mode [2,6,7,10] or an isocratic condi-2]. The most commonly used elution solvents are EtOH

[7,11,12] or ACN [8] with an aqueous buffer adjusted] or water [7,1012]. Two papers use a more complex

dient with 4 solvents (H2O/CH3CN/2-propanol/MeOH)t of these papers focus on the identication and quan-

the phthalates the most commonly found in cosmeticsDEHP and BBP), some papers also discuss the assay ofthalates like di-n-heptyl phthalate (DnHP) [8] or di-allylDAP) [12]. Attention has to be payed to the few num-alates simultaneously assayed by these HPLC methods.st papers propose analytical methods for the separa-

5 phthalates among which only 2 or 3 are regulated and DBP). Only one HPLC/UV method [10] proposes theof up to 7 phthalates (DMP, DEP, DPP, DiBP, BBP, DBPamong which 5 are regulated by the European cosmetic

h less described for cosmetics, gas chromatography mentioned for the analysis of phthalates in cosmetic,9]. A 5% dimethylpolysiloxane column is used for theof up to 7 phthalates (di-n-octyl phthalate (DnOP), DPP,

BBP, DBP, DEHP) among which only 4 are regulated. If9] describes a gas chromatography (GC)/ame ioniza-on (FID) for the quantication of phthalates and GC/MStication, another paper [7] compares results obtainedS and HPLC/UV for the assay of 7 phthalates. When massry is employed for the analysis of phthalates, a capillary30 m 0.25 mm 0.25 m is used, the phthalate ion-erformed by electronic impact (EI) and the detectioning SIM mode (m/z = 149). The sample is injected eitherode [7] or in one fth split mode [9] using He as carrier

less analytical method used (HPLC or GC), no reported with the simultaneous quantication of the 8 regulatedin cosmetic products.nalytical methods found in the literature for the extrac-halates from cosmetic products [2,512], two papersose the use of innovative techniques, SMMEs (polymericroextraction) using a specic extraction tool [11] andd/liquid extraction (LLE) approach with USAEME SFO-assisted emulsication microextraction with dropletn of oating organic) [12]. Later extraction protocol isn optimized version of the conventional liquid/liquidrocess (LLE). A more conventional extraction processe extraction of several cosmetic samples with hexanecolumn [2,6] is mentioned. Papers discussing the pos-

this mAs

suspention/ce(LLE) eto the study food athe delates aand he5 phthulationgiven fmatricuid liqsolventhe andocumprocesof phtinteresconsidextracin wat

Afteof 12 pproposin themethographiCosmecomplAnalytare givcomprto cosmin ordestandaobtainorder tthe ngraphivalidatsamplevery siteria cvalidatsamplefor quaobtainis not in-hou

2. Exp

ispersing different cosmetic samples in methanol andalates by ultrasounds [79] are also available. A papere evaporation of the solvent under reduced pressuretraction step, the purication of the residue is per-g C18 SPE columns [7]. Optimization of the extractionhe extraction conditions and of the purication step are

2.1. Materi

2.1.1. Gas cAll anal

graph inter does not appears suitable for trace quantication.osmetics the sample treatment is often reduced to a

of the sample in an organic solvent followed by sonica-ugation/ltration prior to analysis, but as liquid/liquidtion process is also mentioned [12], it was decided priortion of the sample treatment to extend the literaturee assay of phthalates on other complexes matrices likevironment. A paper presents an extraction process forination of phthalates from milk products [15]. Phtha-tracted by a mixture of ter-buthyl methyl ether (TBME)

from liquid samples, before a LC/ESI/MSMS analysis.s, including 3 regulated by the European cosmetic reg-3/2009, are assayed. LLE extraction techniques are alsoe extraction of phthalates from different environmentaling single drop micro-extraction (SDME)/dispersive liq-icro-extraction (DLLME) [16] or water-soluble organicded with inorganic salts [17]. Another paper discussess of phthalates in landll leaching by GC/MS [18]. Thispublished in 2002, uses a conventional LLE extraction

compares different organic solvents for the extractiones from water. Ethyl acetate appears to be the mostsolvent (recoveries > 80% regardless of the phthalate

). Those papers [12,1518] highlight the possibility ofhthalates from cosmetics after suspension/dissolution

an immiscible organic solvent.resentation of the analytical method used for the assaylates, including the 8 phthalates regulated, this paper

apply the approach for result validation described12787 international standard CosmeticsAnalyticalalidation criteria for analytical results using chromato-hniques [19] to the analysis of different cosmetics.roducts are various and complex, their diversity and

are due to the large extent of their formulation matrices.ethods developed to assess the quality of cosmetics

o be pertinent for their intended use, widely usable,sible and transferable. Nevertheless, their application

matrices requires the use of specic validation criteriassess the results obtained. The ISO 12787 internationalenes validation criteria to which analytical resultsom the analysis of cosmetic products should comply ine condence in performance, reliability and quality ofsult. It proposes an analytical approach for chromato-lyses in order to obtain an assay result and differentlements which can be determined for each sample (orups) submitted to the analysis. If the same matrix orr ones are used, validation results obtained on each cri-e extended to all other analyzed samples. Once thoseelements for analytical results are determined, otherays are carried out using an external calibration curvecation taking into account validation criteria previouslyhis analytical protocol is particularly adapted when itble to make reconstituted sample matrices (i.e. sold orepared blank sample matrix).

ental

als and methods

hromatography and mass spectrometryyses are carried out on a Varian 3800 gas chromato-faced with a Varian 1200 quadripole mass spectrometer

146 P. Gimeno et al. / J. Chromatogr. A 1253 (2012) 144 153

Table 1Selected ions for SIM mode acquisition.

SIM Time from Ions (m/z) Dwell time

1st group 2.5 min 105, 135, 149, 163, 177, 194 25 ms2nd group 5.0 min 59, 104, 149, 205, 223 3rd group 8.2 min 104, 149, 219, 237, 310,4th group 10.5 min 57, 71, 91, 97, 149, 206 5th group 16.5 min 149, 167, 225, 226, 249,6th group 20.0 min 149, 167, 279

(Palo Alto, USA). The column is an Agilent HP-5MS capillarycolumn (crosslinked poly 5% diphenyl/95% dimethylsiloxane);30 m 0.25 mm (i.d.) 0.25 mm lm thickness. The oven tem-perature is programmed as follows: 100 C ramped to 200 C at30 C min1, then to 260 C at 3 C min1, and then to 320 C at30 C min1 held for 5 min. One microliter of each extract is injectedin split mode one twentieth using a Varian 8400 autosampler.The injection port and transfer line temperatures are both xed at300 C. The ion source temperature is set at 230 C, and the heliumcarrier gas ow rate xed at 1 mL min1. The mass spectrometer istuned on electron impact ionization (EI) at 70 eV. The solvent delayis xed at 2.5 min and the run time at 30 min. The acquisition isperformed on full-scan (m/z = 40350) and on SIM mode accordingto Table 1. On SIM mode three different ions are monitored for eachphthalate ainjection usare determi

2.1.2. Refer12 phth

Table 2. Phulated by (99.0%), DEDEP (99.5%dibromobipQuentin Fa(99.5%) and(Sainte-Foyfrom Panre(more than(VWR, Fluk

Table 2Analyzed phth

Phthalates

DBP (dibutyDEHP (diethBBP (butylbDMEP (di(2-DnPP (di-n-DiPP (diisopDPP (n-penDiBPa (diisoDCHP (dicycDEP (diethylDMP (dimetDnOP (di-n-

[A1] European675, 677, 678,[A2] European[A3] European[B] Opinion onProducts (SCC

a DiBP is regDecember 201

2.1.3. StandThe pre

formed onphthalates inated befoan organic sularly injecare preparein a 20.0 mfor at least 3.2). A calibis preparedethanol. 4,4used as inte

bromtory arderens al staed fr

Cosmcosmes (se m

uantere sf one

g g afterto 4,d witfy thas peifferith 1f rep1 cxtra

Samp. Stainjecntiinjec

in according to literature (NIST library) and after a previousing the full-scan mode. Retention times of each groupned after the injection of a standard solution.

ence standardsalates are analyzed by the method and are listed inthalates marked in bold are the 8 phthalates reg-the European cosmetic regulation 1223/2009. DBPHP (99.0%), BBP (98.0%), DMEP (99.4%), DCHP (99.7%),), DMP (99.0%), DiBP (99.0%), DnOP (98.0%) and 4,4-henyle (ISTD) were purchased from SigmaAldrich (St.llavier, France), DPP (99.0% mixed of isomers), DiPP

DnPP (99.0%) were obtained from CIL Cluzeau Info Labo-La-Grande, France). Sodium sulphate anhydrous cameac Quimica Sau (Barcelona, Spain). All organic solvents

99%) were obtained from different industrial sourcesa, ChromaNorm).

alates.

CAS number Legislation

l phthalate) 84-74-2 [A1 ,2 ,3] [B]ylhexyl phthalate) 117-81-7 [A1 ,2 ,3] [B]enzyl phthalate) 85-68-7 [A1 ,2 ,3] [B]methoxyethyl) phthalate) 117-82-8 [A1 ,2 ,3]pentyl phthalate) 131-18-0 [A1 ,2 ,3]entyl phthalate) 605-50-5 [A1 ,2 ,3]tyl isopentyl phthalate) 84777-06-0 [A1 ,2 ,3]butyl phthalate) 84-69-5 [A1 ,2 ,3] [B]lohexyl phthalate) 84-61-7 [B]

phthalate) 84-66-2 [B]hyl phthalate) 131-11-3 [B]

4,4 -Dilaboraics. In o(allerginternaextract3.1).

2.1.4. 10

matricfrom thfor a qto 6 wence othan 5matrixCos-1 menteto verition wthe 4 dples wlimit o10 g gavoid e

2.1.5. 2.1.5.1lytical the qualytical dilutedoctyl phthalate) 117-84-0 [B]

regulation on cosmetics 1223/2009 30/11/2009 Annex-II (entrance 1151 and 1152) and article 15.

regulation 1272/2008 Annex VI. regulation 790/2009 Annexes II and V.

phthalates in cosmetic products. Scientic Committee on ConsumerP) 21/03/2007.ulated by the European cosmetic regulation 1223/2009 since the 1st0.

4,4-dibromcase of excedilution of

2.1.5.2. Staples Cos-1 tmetic matr30 ms 312 25 ms

25 ms 279; 25 ms

50 ms

ard solutionsparation and storage of standard solutions are per-ly with glass material to prevent the extraction offrom plastics. The glassware is appropriately decontam-re use [2,2022] by rinsing glassware several times witholvent (ethanol). During assay, ethyl acetate [23] is reg-ted to check and prevent carry over. Standard solutionsd in ethanol by weighing 20.0 mg of each phthalatesL volumetric ask. These stock solutions can be used7 days if they are stored in a refrigerator (see Sectionration curve ranging from 0.5 g mL1 to 5.0 g mL1

by diluting a 1000 g mL1 stock standard solution in-Dibromobiphenyl (retention time around 9.0 min) isrnal standard (ISTD) at a concentration of 10 g mL1.obiphenyl is already used as internal standard in ournd in literature [24] for the assay of allergens in cosmet-

to have a common ISTD for the assay of both compoundsnd phthalates), 4,4-Dibromobiphenyl was also used asndard for the assay of phthalates (phthalate may beom cosmetics in a similar way as allergens see Section

etic samplesetics samples, 6 perfumes and 4 complex cosmeticshampoo, cream, body milk and shower gel), all takenarket, were used to test the analytical method proposeditative analysis of phthalates. Samples identied Perf-1elected after a previous screening because of the pres-

of the 8 regulated phthalates at a concentration higher1 (limit of report xed for phthalates in this type of

a previous dilution 1/10 in ethanol). Samples identied initially blank of phthalates (blk), have been supple-h one or several phthalates (between 1 and 10 g g1)e accuracy and precision of the method. Supplementa-rformed randomly using the 8 regulated phthalates andent cosmetic matrices. The purpose was to obtain sam-3 phthalates at concentration ranging from 1 g g1,ort xed for phthalates in these complexes matrices toorresponding to the highest calibration level in order to

dilutions.

le preparationndard protocol used for cosmetic samples ready for ana-tion (samples Perf-1 to 6). This preparation is used forcation of phthalates in cosmetic samples ready for ana-tion such as perfumes or lotions. 1 mL of the sample is

10 mL volumetric ask with ethanol after addition of

obiphenyl as internal standard (ISTD) at 10 g mL1. Inssive concentration of phthalates, an appropriate extrathe sample in ethanol is performed.

ndard protocol used for complex cosmetic matrixes (sam-o 4). This extraction process is used for complex cos-ices like creams, milks, oils. 1 g of sample is suspended


in 10 mL of water and extracted with 10 mL of TBME. After extrac-tion, a centrifugation and a drying step with sodium sulphate isperformed. 5 mL of the TBME extraction phase are evaporated todryness under a gentle nitrogen ow. The residue after evapora-tion is dissoof excessivethanol areples 4,4-dixed nal c

2.2. ISO 127and cosmeti

2.2.1. GeneAll assa

described iinto three dusing standmethod ustion criteriaare: the antication (L(Rs: resolusignal and once at theperformed of the methchromatogrrange, etc.)uating the to adapt thscreening ranalyte in standard.

If the anthan 10, astions 2.2.4.1assays is toanalyte witdue to an apresence ofextraction othis analyteple. If the aSections 2.2formed usinand unspikanalyte conparametersracy, and thby performsample: 3 uof interest a1 sample tawith the anbefore, or vfrom PoEMvalidation crelative to matrix efferecoveries, yield of the relative to tRSD or a coa statistical

2.2.2. Validation on standard solutions for the 12 phthalatesanalyzed (1st part)2.2.2.1. Detection and quantication limits in solvent. As 8 of the12 analyzed phthalates are forbidden in cosmetics products and

rdinraph

ay bloweo estof deas pe787,

rang). Thbtainntictand

pea

. Anaeth

higher to utionion isthala

. Calalibr0, 1.5rent snt ca

rant calt. Thespomatetainlibrac sofutedrsionSA)) 4ed co

Sampore ae 2nounn cuined

jectently fibratn cueparion 2

Assay eachk thainat

rformtiblecosmparelved in 1 mL of ethanol (containing the ISTD). In casee concentration of phthalates, further dilutions with

performed. As for ready for analytical injection sam-bromobiphenyl is used as internal standard (ISTD) at aoncentration of 10 g mL1.

87 standard protocol: analyses on standard solutionsc samples

ralys were performed using the analytical approachn the ISO 12787 standard. This standard is dividedifferent parts. The rst part consists of determining,ard solutions, the main characteristics of the analyticaled before performing tests on samples. The valida-

on standard solutions checked during this rst stepalyte limit of detection (LoD) and/or limit of quan-oQ); the conformity of the chromatographic analysistion, As: assimetry); the linear range of the analytethe standard accuracy. This rst step is carried out

beginning of the analytical programme and should beagain or at least checked if any analytical parameterod is changed (calibration solvent, injection volume,aphic column type, separation conditions, calibration. The second part, called screening, consists of eval-quantity of analyte in the sample, if present, in ordere sample assay protocol. According to the sampleesult, two different protocols for the assay of thethe sample are proposed in the 3rd part of the ISO

alyte is not detected or detected with a S/N ratio lesssays are performed using spiked recoveries (see Sec-

and 2.2.4.2 for spiked preparations). The aim of these check that the non-detection, or the detection of theh a S/N ratio less than 10 in the analyzed sample, is notnalytical problem (suppression of the signal due to the

an interference compound in the cosmetic matrix, badf the analyte from the cosmetic) but to the absence of, at a known concentration limit, in the analyzed sam-nalyte is detected with a S/N ratio higher than 10 (see.4.1 and 2.2.4.2 for spiked preparations), assays are per-g a specic protocol based on the preparation of spikeded samples. The aim of these trials is to determine thecentration in the sample as well as several validation, like the matrix effect, the extraction yield, the accu-e condence interval. These parameters are determineding statistical analyses on six preparations of the samenspiked preparations, 2 samples spiked with the analytet the beginning of the analytical procedure (PrEMS) andken through the entire extraction procedure and spikedalyte of interest at the end of the extraction immediatelyery close to, detection (PoEMS). Recoveries obtainedS and/or PrEMS lead to the determination of differentriteria on the analytical results. The PoEMS recoverycalibration standards shows whether or not there is act. The difference between the PoEMS and the PrEMSrelative to calibration standards, gives the extractionanalytical process. The recovery obtained for the PrEMShe PoEMS gives the accuracy of the analytical result. Andence interval on the assay result can be obtained by

analysis of the replicates.

as acco(Paragmetic mat the order tlimits tion wISO 12system0.50 ngvalue oof quaing a sThe S/N

2.2.2.2of the mat the In ordethe dildetectthe ph

2.2.2.3dent c(0.5, 1.3 diffedifferebrationthe rssolvenment rbe estibias obThe castatistidistribrion veInc. (Uat a x

2.2.3. Bef

with ththe amibratiodetermand inpendethe calibratio4) is prin Sect

2.2.4. For

to checcontamare pecompain the are preg to the European regulation on cosmetics 1223/2009 7, Article 17 and Annex I) forbidden substance in cos-e present at trace levels, phthalates must be quantiedr possible amount (trace elements determination). Inimate this quantication amount, an evaluation of thetection (LoD) and quantication (LoQ) in standard solu-rformed. For the LoQ evaluation, in agreement with thesmall quantities of phthalates are injected in the GC/MSing from 0.025 ng to 0.5 ng (0.025, 0.050, 0.10, 0.25 ande limit of detection (LoD) is given by the y intercepted during the linearity assay (Section 2.2.2.3). The limitation (LoQ) is evaluated as 3 LoD and checked inject-ard solution at the estimated LoQ concentration value.k to peak at the LoQ must be >10.

lytical conformity/specicity. The analytical conformityod is checked by injecting a solvent calibration standardr expected level of the calibration curve (5.0 g mL1).ensure the absence of interference peak in the solvent,

solvent (ethanol) is also injected. The specicity of the checked using the MS spectra (m/z ions and ratio) andtes retention times.

ibration linearity standard accuracy. Three indepen-ation curves ranging from 0.5 g mL1 to 5.0 g mL1

, 2.0, 3.0, 4.0 and 5.0 g mL1) are prepared by dilutingtandard stock solutions in ethanol and are injected. Thelibration levels are uniformly distributed along the cali-ge. For the determination of low concentration analytes,ibration level corresponds to the quantication limit ine upper end is usually signied by a change in instru-nse. The method accuracy on standard solutions couldd, for each calibration level, as the mean concentrationed on this level (three values for each calibration level).tion linearity was checked on standard solutions usingtware (AVA Aide la Validation Analytique version 3.1.

by Qualilab (45160 Olivet France) or Statgraphic Centu- 16.1.05 (32 bits) distributed by StatPoint Technologies,,4-dibromobiphenyl is used as internal standard (ISTD)ncentration of 10 g mL1.

le screening (2nd part)ssays, a screening of samples is performed, in agreementd step of the ISO 12787 standard, in order to estimatet of phthalates in analyzed samples if present. A cal-rve, in agreement with the linearity range previously

(ranging from 0.5 g mL1 to 5.0 g mL1), is preparedd. A control standard is performed at 2.0 g mL1 inde-rom the standard solutions used for the preparation ofion curve. This control standard is used to check the cal-rve preparation. Each sample (Perf-1 to 6 and Cos-1 toed according to the sample preparation protocol given.1.5 depending on the cosmetic matrix and injected.

of phthalates (3rd part) extraction batch, a whole reagent blank is carried outt the materials used for extraction and analysis had noted the samples. For each sample, spiked preparationsed using the minimum possible amount of a solvent

with the sample preparation to introduce the analyteetic. Depending on the sample type, spiked samplesd either mixing the analyte solution with the sample


Fig. 2. Examp(cream) and a

(Cos-1 to 46). SIM chro(cream) are

2.2.4.1. Assunspiked prat the estiming step) arthe absenceunspiked pPhthalate qusing an instandard adpreparationformed, usi(using 2 unscentration lusing the sithe ISO 127using the Finterval forthe multi-pmined. For was used. Pquanticatieries obtainpreparation(Section 2.1injection ssystematica

2.2.4.2. AssFor each assple and tw

1 g g1 of the overloaded samples before extraction are preparedaccording to Section 2.1.5.2. An additional spiked preparation at1 g g1 of the overload samples after extraction is performedaccording to Section 2.1.5.2. In order to check the absence of inter-

e peards ipara

tic sa by ei-poiiked

calcationts d

cy at of p

ults

ethod

o palatedpou

d wated. Ginat

chromtion an cassasumeticllow

DnPphthon ofle of chromatograms obtained for a blank reagent, the sample Cos-20.5 g L1 standard solution.

) or allowing dispersion into liquid samples (Perf-1 tomatograms obtained for a blank reagent and for Cos-2

given as example in Fig. 2.

ay for perfume samples (Perf-1 to 6). For each assay, threeeparations and two spiked preparations of the sample

ferencstandaple precosmeformeda mult3 unspies arepreparelemenaccuraamoun

3. Res

3.1. M

As n8 reguses commethoreguladetermof the separaEuropeto the on Conin cosmoped aDMEP,the 8 ticatiated concentration value (evaluated during the screen-e made according to Section 2.1.5.1. In order to check

of interference peak with the ISTD used, an additionalreparation without internal standard is performed.uantications are performed by external calibrationternal standard and using a multi-point/single levelsdition method (using the 3 unspiked and the 2 spikeds) [25,26]. An additional independent assay is per-ng a multi-point/multi-levels standard addition methodpiked preparations and 2 spiked preparations at 4 con-evels) [27]. Results were compared to those obtainedngle-point standard addition approach, as proposed in87 standard. For information, a prediction interval (PI)ieller theorem (allows the calculation of a condence

the ratio of two means) [28,29] based on data fromoint/multi-levels standard addition method is deter-this determination statistic software, like Statgraphichthalates recoveries are calculated by external standardon from spiked preparations. In order to check recov-ed for phthalates not detected, an additional spiked

at 5 g g1, corresponding after sample preparation.5.1) to the limit of report xed for ready for analyticalamples (Section 3.1.1), with all phthalates analyzed islly performed for all samples studied.

ay for cosmetic samples other than perfumes (Cos-1 to 4).ay, three unspiked preparations of the overloaded sam-o spiked preparation with all phthalates analyzed at

(DiDP). AccDiDP in cosA GC/MS mas collisionboth phthalble, to deveapplicable tion mode, considered

In agreepossible, inthe apparation/suspenTBME was tthe extractposed in liSuch extraclates becaube extracteing similarto have a cand phthalain landll ltion of phthacetate (EAextraction pendent exat 1 g g1k with the ISTD used, a preparation without internals also carried one. 1 g g1 corresponds, after sam-tion (Section 2.1.5.2), to the limit of report xed formples (Section 3.1.1). Phthalate quantications are per-xternal calibration using an internal standard and usingnt/single levels standard addition method (using the

and the 2 spiked preparations). Phthalates recover-ulated by external standard quantication from spikeds (before or after extraction) depending on validationetermined (matrix effect, extraction yield, etc.). Thend reliability of the assay are determined from thehthalate initially introduced in the blank matrix.

and discussion

development

per deals with the simultaneous quantication of the phthalates in cosmetic products, and as most of the-nds can be considered as volatiles analytes, a GC/MSs developed for the assay of at least the 8 phthalatesC/MS is a commonly used analytical technique for the

ion of phthalates in the environment. The developmentatographic method proposed was rst focused on the

and identication of the 8 phthalates regulated by theosmetic regulation and then extended, when possible,y of phthalates discussed by the Scientic Committeeer Products (SCCP) in document Opinion on phthalates

products [13]. The chromatographic conditions devel- the determination of 12 phthalates (DBP, DEHP, BBP,P, DiPP, DPP, DiBP, DCHP, DEP, DMP, DnOP), includingalates regulated, but are not suitable for the quan-

di-isononylphthalate (DiNP) and di-isodecylphthalateording to SCCP [13], the possible presence of DiNP andmetics does not seem to be a problem for human health.ethod using positive chemical ionization with ammonia

gas is proposed in literature for the determinations ofates in cosmetics products [30]. In order, as far as possi-lop for cosmetic standardization an easy and currentlyanalytical method for control laboratories, this ioniza-which requires a specic equipment, was not preferably.ment with literature and in order to eliminate whenterfering compound and limit the contamination oftus, an extraction protocol based on the dissolu-sion of the cosmetic in water followed by a LLE usingested. This protocol, already used in our laboratory forion of volatile fragrance allergens in cosmetics, is pro-terature [31] for the extraction of allergens in foods.tion mode could be relevant for the assay of phtha-se, phthalates, which are esters of phthalic acid, mayd from cosmetics in a similar way as allergens contain-

organic function like esters of benzoic acid. In orderommon extraction process for the assay of allergenstes, TBME, not studied for the extraction of phthalates

eaching [18] but used with hexane for the determina-alates from milk products [15], was compared to ethyl), supposed to be the most interesting solvent for theof phthalates from water [18]. In this aim, three inde-tractions of the matrix Cos-1 (shampoo), supplemented(report limit) with phthalates classied H360, were


Table 3S/N ratio obtained for each phthalate on the quantier ion.

S/N ratio

Phthalates Quantier ion (m/z) Solvent 0.025 g mL1 0.050 g mL1 0.10 g mL1 0.25 g mL1 0.50 g mL1

DMP DEP DiBPDBPDMEPDiPP DPP DnPP BBPDCHPDEHPDnOP

S/N ratio are o

performed to the samppoint out siobtained arthe extractiative to thecompared tto an easierthe concent

3.2. Prelimi

In agree12787 stanperformed phthalates repeatabilittor calibratspectrometby injectingphthalates a


Table 4Specicity data.

Phthalates RT (min) Ion 1 (m/z) Ion 2 (m/z) Ion 3 (m/z) Ion ratio 2/1% Ion ratio 3/1%

DMP 3.8 163 194 135 6 4DEP 4.6 149 177 105 21 9DiBP 6.8 149 104 223 7 5DBP 7.9 149 205 223 3 3DMEP 8.4 59 149 104 13 10DiPP 9.4 149 237 104 7 5DPP 10.1 149 237 219 5 2DnPP 10.7 149 237 104 2 3BBP 14.4 149 91 206 73 19DCHP 17.9 149 167 249 31 3DEHP 18.5 149 167 279 31 2DnOP 22.5 149 279 167 2 1

Table 5Calibration lin

Validation p BBP

Determinati 0.993Slope 0.065y-Intercept 0.01Variance hom NS Aberrant val NO y-Intercept c SSignicant s HS Validity of c NS

Validation p DEP

Determinati 0.993Slope 0.156y-Intercept Variance homAberrant valy-Intercept cSignicant sValidity of c

NS: non signi

correlation used (NS), house speccorrelation (0.55.0 gthrough the(Students tacceptable a general sent calibratphthalate clevel 100)

Table 6Mean relative

Calibration l

0.5 g mL1

1.0 g mL1

1.5 g mL1

2.0 g mL1

3.0 g mL1

4.0 g mL1

5.0 g mL1

In-house valida Non retainb Calibrationearity.

aramaters DBP DEHP

on coefcient R2 0.9909 0.99150.2103 0.0898 0.0670 0.0422

ogeneity (Cochran) NS NS ues (Dixon) NO NO omparison with 0 (Student) S Slope existence (Fisher) HS HS alibration curve (Fisher) NS NS

aramaters DPP DCHP

on coefcient R2 0.9924 0.9909 0.0737 0.0905

0.0220 0.0155 0.04

ogeneity (Cochran) NS NS NSues (Dixon) NO NO NO omparison with 0 (Student) S S S lope existence (Fisher) HS HS HS alibration curve (Fisher) NS NS NS

cant; S: signicant; HS: highly signicant.

between experimental values and the regression modelas well as mean relative bias in agreement with in-ications (see Table 6) point out an acceptable linearfor all phthalates on the calibration range considered

mL1). For this study, the 0 value has not been forced origin and the comparison of y-intercept with zeroest) is signicant (S). The linearity may probably not beon a wider concentration range. Residual plots showymmetric repartition of bias obtained for the differ-ion levels using the linear regression model regardlessonsidered. Mean relative bias (absolute bias/calibration

are summarized in Table 6.

3.4. Sample

The aimlates in eacsamples asin the ISOthe determobtained focurve (


Table 7Assay of phthalates in samples Perf-1 to 06.

Sample Phthalate Screening Assay Additionalindependent assay

rd

Perf-1 )

Perf-2)

Perf-3 )

Perf-4 )

Perf-5 )

Perf-6 )

a Predictiveb Each other

3.5. Assay o

3.5.1. ResulIn agree

realized forto 6 at 10 ing step (Border to chadditional slyzed wereResults obtples Perf-1each samplexternal caaddition mmulti-pointinterval (PIaddition mlated by extValidation show analyto those obfor n = 3 lesaround 10%spiked prepfrom 80% toare in agreeout a satisfation quantianalyte amScreening amountb External standaquantication

BBP 100 g g1 126 g g1(RSD = 5.6%. n = 3Mean recovery:100%(90%. 109%)

BBP 300 g g1 312 g g1(RSD = 3.9%. n = 3Mean recovery:91%(85%. 96%)

DEHP 100 g g1 103 g g1(RSD = 4.7%. n = 3Mean recovery:113%(109%. 118%)




interval (PI) calculated using the Fieller theorem approach [30,33].

phthalates


Table 8Assay of phthalates in samples Cos-1 to 4.

Sample Added phthalate Added amounta Assay (externalquantication)

Multi-point/single levelstandard addition method

Matrix effect (ME)Extraction yield (EY)

Cos-1(shampoo) = 3)

6%

= 3)8%

= 3)6%

Cos-2(cream) = 3)

7%

= 3)6%

Cos-3(body milk) = 3)

0%

n = 3)%

Cos-4 (show n = 3)1%

a Initially bl

to 4 are givknown amoand quantia multi-pointeria obtainmean recovdard by ext(previous ormined). Thethe determin the blanresults obtaation (RSD)initially intwith a RSDDPP in Cos-high RSD rewith other In additioneffect and 12787 stanple considecould be co(liquid/liqusignicant preparationThis last criobtained beaddition qupreparationare directlyvalues.

For phthrecoveries rthe performusing TBMEple preparais adequate

clus

safe Propmeal (ISpatedrd Ccal r

ana andhtha3/20d DDBP 4.4 g g1 4.7 g g1

(RSD = 1.5%. nAccuracy: 10

DiBP 2.2 g g1 2.8 g g1


DEHP 5.3 g g1 5.6 g g1


DMEP 5.9 g g1 6.3 g g1


DiPP 2.0 g g1 2.1 g g1


BBP 7.9 g g1 8.7 g g1


DnPP 5.2 g g1 4.2 g g1

(RSD = 16.5%.Accuracy: 80

er gel) DPP 1.0 g g1 1.0 g g1

(RSD = 11.3%.Accuracy: 10

ank samples supplemented with one or several phthalates.

en in Table 8. This table compares for each sample, theunt of phthalate initially introduced in the blank matrixcation results obtained by external calibration or usingt/single level standard addition method. Validation cri-ed for each sample (matrix effect, extraction yield andery) are calculated according to the ISO 12787 stan-ernal standard quantication from spiked preparations

after extraction depending on validation criteria deter- accuracy and reliability of the assay are checked fromination of the amount of phthalate initially introducedk matrix. Whatever phthalate or sample considered,ined highlight an accuracy and a relative standard devi-

of the assay, determined from the amount of phthalateroduced in the blank matrix, between 80% and 130%

4. Con

ForHealthdevelonationparticistandaanalyti

Thearationthe 8 pics 122DPP an for n = 3 usually


(perfumes, lotions), the sample treatment was limited for phtha-lates quantication to a dilution in ethanol and for phthalatesidentication to a direct injection of the cosmetic samples in theGC/MS system.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.chroma.2012.06.090.

References

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phtation of results (notied under document number C (2002) 3044).92 N73 NWI GC/MS method for the identication and assay of 12s in ready to inject cosmetic samples.

Analytical method for the identification and assay of 12 phthalates in cosmetic products: Application of the ISO 12787 int...1 Introduction2 Experimental2.1 Materials and methods2.1.1 Gas chromatography and mass spectrometry2.1.2 Reference standards2.1.3 Standard solutions2.1.4 Cosmetic samples2.1.5 Sample preparation2.1.5.1 Standard protocol used for cosmetic samples ready for analytical injection (samples Perf-1 to 6)2.1.5.2 Standard protocol used for complex cosmetic matrixes (samples Cos-1 to 4)

2.2 ISO 12787 standard protocol: analyses on standard solutions and cosmetic samples2.2.1 General2.2.2 Validation on standard solutions for the 12 phthalates analyzed (1st part)2.2.2.1 Detection and quantification limits in solvent2.2.2.2 Analytical conformity/specificity2.2.2.3 Calibration linearity standard accuracy

2.2.3 Sample screening (2nd part)2.2.4 Assay of phthalates (3rd part)2.2.4.1 Assay for perfume samples (Perf-1 to 6)2.2.4.2 Assay for cosmetic samples other than perfumes (Cos-1 to 4)

3 Results and discussion3.1 Method development3.2 Preliminary validation criteria using the ISO 12787 standard3.3 Validation data obtained on standard solutions (ISO 12787 1st part)3.3.1 Detection and quantification limits on the quantifier ion3.3.2 Analytical conformity/specificity3.3.3 Calibration linearity standard accuracy

3.4 Sample screening (ISO 12787 2nd part)3.5 Assay of phthalates in samples (ISO 12787 3rd part)3.5.1 Results obtained for the assays of perfumes3.5.2 Results obtained for the assay of Cos-1 to 4

4 ConclusionAppendix A Supplementary dataAppendix A Supplementary data

12 Ftalati in Cosmetice

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