Influence of Matrix Compounds on Oxidative Hair Dyes by HPLC

7/22/2019 Influence of Matrix Compounds on Oxidative Hair Dyes by HPLC

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j. Cosmet.ci., 50, 231-248 (July/August1999)

Influenceof matrix compounds n the analysisof oxidalivehair dyesby HPLCURSULA VINCENT, GUY BORDIN, andADELA .RODRIGUEZ,uropeanommission,oint esearchCentre,nstituteor RerenceMaterialsand Measurements,etieseweg,B-2440 Geel,Belgium.Acceptedor publicationune30, 1999.

SynopsisOxidativehair dyeingproducts onsist f a mixtureof a broadspectrum f organiccompoundsncludingthe hair dye'sso-called ctivecompoundsnd the matrix-formingcompounds. orty-seven ye intermedi-atescommonly sed n cosmeticormulations ave hus beenanalyzed y RP-HPLC, and their chromato-graphic haracteristicsavebeen ecorded. ince he matrix compoundsould nterferewith the quantitativeanalysis f the activecompounds,ighteenmatrix compoundsommonly sed n cosmeticormulations avebeen testedfor their influenceon dye intermediatedetermination.Since some of them do affect thechromatographicehaviorof the dye intermediates, n isolationprocedure,or separatingmatrix compo-nents rom the dye-forming ompounds,onsisting f a liquid-liquid extraction y n-heptane, asbeensetup. In mostcases,his procedures effectiveor the extraction f the matrix products.Moreover, he dyeintermediates re not extracted y n-heptane, nd their chromatographicehavior s not alteredby theextraction rocedure.n summary,his studyhasshown hat a reference ethod or the analysis f oxidativehair dyesshould ncludea compulsory xtraction tep beforesubmission f sampleso RP-HPLC.

INTRODUCTION

Among the differentmethodsof changing he colorof humanhair, oxidativehair dyeingplaysan important role. Formulations onsistof a wide rangeof organiccompounds ftwo distinct types, .e., the hair dye intermediates nd the matrix-formingcompounds.Some of the hair dyes, which are available or use in hair dye formulations,have atoxicological r sensitizing otentialand areprohibitedor restrictedn concentration ythe 6th Amendmentof the EuropeanUnion CouncilDirective 93/35/EEC.To identify and quantify substancessed in hair dye formulations,with the aim ofimplementing the EuropeanUnion CosmeticDirective, there is need for a reliableanalyticalmethod. Initially, a high-performanceiquid chromatographymethod wasdevelopedhat enableshe identification f a broad angeof dye-forming ompoundsnstandardsolutions 1,2). The next step for the setup of a reliable analyticalmethod,besides dditionaloptimizationsteps, nvolves he investigation f possible ffectsofmatrix components.

231


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232 JOURNAL OF COSMETIC SCIENCE

Due to the different and specific unctionsof matrix compoundsn hair dye formula-tions, the chemicalcomposition f matrix compounds overs broad range, ncludingsurfactants, H adjusters,consistency roviders,antioxidants,emulsifiers,perfumes,perfumesolubilizers, nd preservatives. achof theseadditivesplaysa clearrole in theformulation. For instance,preservatives uchas methylparabenor DEDM-hydantoin andantioxidants such as BHT, sodium sulfite, and L-ascorbic acid sodium salt have to beadded to keep the product stable.Literature on the influence of the various matrix products on the chromatographicseparation f oxidativehair dyes s very rare, although t is of tremendousmportance.Someauthorssimply do not consider heir influenceon the analysis3-5), while otherstry to reducematrix interference y optimizing the measurement avelength 6).From the point of view of the chromatographic eparationof the dyes, two mainsituations can occur: a given matrix componentshowsa specific etentive behaviorand can be detectedwith the UV detector. the matrix component orms a complexwith a dye intermediate hat will change heretentive behaviorof the dye intermediate.Furthermore, t must be noted that somematrix components an also nterferewith thecolumn itself, i.e., adsorbonto the stationary eversed hase, hereforechanging heseparation ropertiesof the oxidativehair dyes.Therefore, the aims of the work described n this article were to investigatepossiblematrix influences n the chromatographic eparation f the dye-formingcompounds ndto setup an effectivemethodof separatinghe matrix componentsrom the dye-formingcompounds. his method shouldnot, of course, ffect he chromatographic ehaviorofthe dyes.Due to the high numberof dyesand matrix compounds, selection f productshad to be made according o lists of frequently used dye intermediates nd matrixproductswith their concentrationsn formulationsprovided by COLIPA (Comit deLiaison Europen de l'Industrie de la Parfumerie,de Produits Cosmtiqueset de Toi-lette; seeAppendix).The forty-seven yesand eighteenmatrix components electedortheseexperiments re representativef four groupsof dye-formingcompounds, lassifiedaccording o their chemical haracteristics,nd of five classesf additivesusedasmatrixproducts n the formulations.The influenceof matrix components n the dye determi-nation has beenperformedon selecteddyes rom eachgroup.

EXPERIMENTALThe chromatographic rocedurehas been described n detail in a previousarticle (1).INSTRUMENTATION

All chromatographic eparations ere carried out using the following equipment:atwo-pistonHPLC pump with a low-pressureernarygradient systemmodule (System325 from Kontron nstrumentsS.P.A., Milan, Italy), an autosampler 60 with a loop of20 ml (Kontron InstrumentsS.P.A.), a diode array detector440 (Kontron InstrumentsS.P.A.), and a vacuum degassingsystem,DegasysDG 1300 (Unifiows, Japan). The


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MATRIX COMPOUNDS AND OXIDATIVE HAIR DYES 233

column emperature askept constant y means f the thermostat f an electrochemicaldetector Decade,Antec Leyden,Leiden,The Netherlands).Data processing as donewith the Data System 50-MT2/DAD seriesKontron nstruments .P.A.).The columnwasa Merck Lichrospher P 60 SelectB, 250 x 4 mm, 5-1am articlesize. n somecases,UV-Vis spectrophotometerLambda fromPerkinElmer)wasused dditionallywith the aboveequipment.CHEMICALS

L-Ascorbic acid sodium salt (NaAsc), 3,4-diaminobenzoic acid (3,4-daba), p-aminophenol4-ap),m-aminophenol3-ap),0-aminophenol2-ap),2,4-diaminophenol(2,4-dap), ,4-diaminophenolC1 (2,4-daph), -amino-m-cresol4-a-3-mp),2-amino-p-cresol 2-a-4-mp), 6-amino-m-cresol2-a-5-mp), 5-amino-0-cresol4-a-2-ht),2-amino-5-nitrophenol2-a-5-np),2-amino-4-nitrophenol2-a-4-np),N,N-diethyl-m-aminophenol3-deap), -phenylenediamine1,4-pd),m-phenylenediamine1,3-pd),0-phenylenediamine1,2-pd), 2-nitro-p-phenylenediamine2-n-l,4-pd), 4-nitro-0-phenylenediamine4-n-l,2-pd),2-chloro-p-phenylenediamineulfate2-cl-l,4-pds), -phenyl-p-phenylenediamine4-adp), ,4'-diaminodiphenylamineulfate 4,4'-dadps),resorcinolres),4-chlororesorcinolchlres), -hexylresorcinolhres), -anisidine1,4-ad),2,6-diaminopyridine2,6-dap),2-amino-3-hydroxypyridine2-a-3-hp),2-methylresor-cinol (2,6-dht), toluene-2,4-diamine2,4-dat), toluene-3,4-diamine3,4-dat), toluene-2,5-diamine ulfate2,5-dats), -naphtol1-nap),2-naphtol 2-nap),1,6-naphtalenediol(1,6-dhnap),2,3-naphtalenediol2,3-dhnap),2,7-naphtalenediol2,7-dhnap),phloro-glucinol (phlg), pyrogallol (pg), hydroquinone (hq), pyrocatechol (pc), p-methylaminophenolulfate met), 2-methoxy-p-phenylenediamineulfate 2,5-das),3-tert-butyl-p-hydroxyanisole3-tb-4-ha),4-chloroaniline4-cla),3-methyl-l-phenyl-2-pyrazoline-5-one3-m-l-p-2-p-5-o)wereobtainedromFluka.p-Phenylenediamineul-fate 1,4-pds), ndm-phenylenediamineulfate1,3-pds)werekindlyprovided y "LesColorantsWackherr," Saint-Ouen l'Aum6ne, France. Sodium tetraboratedecahydrate(p.a.),acetic cid95% (suprapure),mmonia 5% (suprapure),ydrochloriccid 0.1M), oleicacid OA) (p.a.),n-heptanep.a.), sopropanolp.a.),sodium ulfite p.a.) SS),andpolyvinylpyrrolidonPVP) wereobtainedrom Merck. BHT (butylated ydroxy-toluene),methylparabenMP), and aurylsulfateLS)wereobtainedromSigma.LauricdiethanolamideLDA), TEA-dodecylbenzenesulfonateTDS), and Syntopon D1(ethoxylatedctylphenol-EOP)wereprovided y Witco S.A. TriethanolamineTEA)was obtained rom Mobi-Lab bvba, Zutendaal,Belgium. DEDM-hydantoin (DMDM),dimethicone opolyol DC), nonoxynol-12NOL), and polyquaternium-11PQ) werekindly providedby "Keuringsdienstan Waren," Enschede, he Netherlands.n-Nonylamine8% (NNO) was btainedrom anssenhimica, ndOranex T (ORA)wasprovided y SpinnradGmbH, Gelsenkirchen,ermany.Cetrimonium hloride(CC)andmethanolHPLCquality)wereobtainedromFluka.Purewater 18.2Ml/cmquality)used or the preparation f solutionswasobtained rom a MilliQ Plus 185system Millipore, Molsheim, France).PREPARATION OF REAGENTS

Solventsnd chromatographicobilehaseBuffersolution H 8 (Soerensenuffer):440 ml hydrochloriccid (0.1 N) and 2 g/1


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234 JOURNAL OF COSMETIC SCIENCEL-ascorbic cid sodiumsalt (NaAsc) as an antioxidantagent were added o a 560-mlsodium teraboratesolution. The solventswere mixtures of MeOH and Soerensen uffer,pH 8 in variousproportions.Mobile phase aqueous haseB): a 0.05 M aceticacid solutionwasadjusted o a pH of5.9 with a 10% ammonia olutionand filteredthrougha 0.45-lm filter. When not inuse, he eluentwasstoredat a temperature f 4C to preventmicrobiologicalrowth.Samples.he matrix productsand the dye intermediatesused in the experiments regiven in Tables I and II, respectively.All dye intermediates nd matrix componentconcentrations ere selected ccording o COLIPA data.Stocksolutions f dye intermediates t a concentration f 0.1 g/100 g werepreparednmixturesof various roportions f MeOH and Soerensenuffer containingNaAscasanantioxidant gent), anging rom40% to 90% Soerensenuffer.Sample olutions f dyeintermediates t a concentration f 0.025 g/100 g were preparedby dilution from thestocksolutionsn mixturesof Soerensenuffer and MeOH ranging rom 30% to 60%Soerensen buffer.Stock solutionsof matrix componentswere prepared n MeOH- or isopropanol-Soerensenuffermixturesof differentproportions. ample olutions f the matrix com-ponentsat variousconcentrationsTable I) were preparedby dilution from the stocksolutionsn MeOH- or isopropanol-Soerensenuffermixtures abbreviationsregivenin the Chemicals section).

Table ISelectedMatrix Compounds, unctions, oncentrations,nd MeOH Proportions sed

ConcentrationCompound Function (g/100 g)

MeOHproportion(%)

Oleic acid (OA)Oranex HT (ORA)TEA-dodecylbenzenesulfonate(TDS)Cetrimonium chloride (CC)Lauric diethanolamide (LDA)BHTSyntopon D1 (EOP)Nonoxynol-12 (NOL)n-Nonylamine 98% (NNO)Sodium lauryl sulfate LS)Polyquaternium-11 PQ)MethylparabenMP)PolyvinylpyrrolidonPVP)Sodium sulfite (SS)Triethanolamine (TEA)DEDM-Hydantoin (DEDM)Dimethiconecopolyol DC)L-ascorbic acid sodium salt

(NaAsc)

Surfactant, leansing gent 5 100Unknown 3 100Surfactant, eansingagent 0.5 90Surfactant, mulsifying gent 0.5Surfactant, eansingagent 2Antioxidant 0.25Perfume solubilizer 6Surfactant, mulsifying gent 3Unknown 25Surfactant, leansing gent,denaturant 3Antistatic agent, ilm former,hair fixative 2Preservative 0.05Antistatic agent, ilm former,hair fixative 2Antioxidant,reducingagent 2pH adjuster 1.5Preservative 0.05Emollient, hair conditioning gent 2.5Antioxidant,pH adjuster **

907570707060606060604040

0

* DC waspreparedn an isopropanol-Soerensenuffermixturewith 67% isopropanol.** The Soerensenuffer containsNaAsc 2 g/l.


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Table IIRetention Times for Forty-SevenDye Intermediates,Each of Which Was Measuredas a SingleComponentSolution n = 3)

Retention time (rain)Dye Mean RSD (%)

3,4-Diaminobenzoicacid 2.75 1.542,4-Diaminophenol 3.95 0.172,4-DiaminophenolHC1 3.98 0.18p-Phenylenediamineulfate 5.21 0.54Phloroglucinol 5.23 12.56p-Phenylenediamine 5.60 9.34Pyrogallol 5.90 3.592-Amino-3-hydroxypyridine 5.91 0.84Hydroquinone 6.02 6.92p-Aminophenol 6.13 3.46m-Phenylenediamineulfate 7.00 3.23m-Phenylenediamine 7.15 6.422,6-Diaminopyridine 7.91 3.93m-Aminophenol 8.04 3.43Toluene-2,5-diamine sulfate 8.50 7.73Resorcinol 9.23 4.674-Amino-m-cresol 9.70 1.820-Phenylenediamine 9.86 3.872-Methoxy-p-phenylenediamineulfate 9.94 1.350-Aminophenol 10.51 5.98Pyrocatechol 11.23 18.06p-Methylaminophenolulfate 11.88 2.022-Methylresorcinol 11.89 6.842-Chloro-p-phenylenediamineulfate 12.33 2.01Toluene-2,4-diamine 12.78 8.852-Nitro-p-p enylenediamine 13.14 2.204-Nitro-0-phenylenediamine 15.10 2.532-Amino-4-nitrophenol 15.50 3.015-Amino-o-cresol 15.66 4.11Toluene-3,4-diamine 17.53 7.506-Amino-m-cresol 17.95 1.342-Amino-p-cresol 18.07 2.90p-Anisidine 18.47 4.212-Amino- nitrophenol 18.66 3.144-Chlororesorcinol 20.44 3.704,4'-Diaminodiphenylamineulfate 20.83 0.751,6-Naphtalenediol 25.23 2.242,7-Naphtalenediol 25.81 1.593-Methyl-1-phenyl-2-pyrazoline-5-one 26.01 6.394-Chloroaniline 26.82 2.502,3-Naphtalenediol 27.88 2.38N- Phenyl-p-phenylenediamine 29.56 3.642-Naphtol 29.71 0.93N, N-Diethyl-m-aminophenol 30.00 0.021-Naphtol 30.01 0.423 tert-butyl-4- ydroxyanisole 31.85 0.584-Hexylresorcinol 32.26 0.55


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Samplesolutionsof dye intermediates t a concentration f 0.025 g/100 g in matrixmedia (at the differentconcentrationsiven above)were preparedby dilution from therespective tocksolutions t variousproportions f MeOH and Soerensen.It has o be noted hat all samples ontainedNaAsc asan antioxidantagentadded o theSoerensen uffer to ensure he stability of the dye samples.PROCEDURESReversed-phasePLC conditions. non-linearMeOH (A)/aqueous hase B) gradientwasusedas follows: 0-25% A for 19 rain, 25-80% A for 10 rain, 80% A for 5 rain, 80-95%A for 5 rain, 95% A for 10 rain, and 95-0% A for 3 min. The total flow was ml/min.Between he injections, he columnwas equilibratedby a 25-ml mobile phase.Eachanalysiswasrepeatedive times.The column emperaturewaskept at 48C. The dataacquisitionwascarriedout at two or threeselectedwavelengths:20 nm, 235 nm, and290 nm, in parallel with the spectra cquisition.Isolation f the matrix componentsrom the inal analytesolution.irst, experimentsusinganion exchange, olid phaseextraction, iquid cation exchange, nd liquid-liquid ex-traction were carried out. The results ed to the selectionand the optimization of aliquid-liquid extraction rocedure f the matrix componentsrom the sample olutionby n-heptane.Two milliliters of samplesolution (single-matrixproduct solutionor mixture of dyeintermediates nd a matrix compound, t the concentrationsiven in the Experimentalsection)were treatedwith n-heptane.Dependingon the matrix product, he extractioninvolvedone to three steps. n the first step, he extractionwasperformedon the 2-mlsample sing20 ml n-heptane, he two phases ereseparated,nd the resultingaqueousphase1 was hereafter ubmitted o HPLC or analyzed y UV-Vis spectrophotometry.For additionalextractionsteps,20 ml n-heptanewere added to the resultingaqueousphasen-l, separation f the two phases asperformed, nd the resultingaqueous hasen wassubmitted o HPLC or analyzedby UV-Vis spectrophotometry.

RESULTS AND DISCUSSIONA systematic tudywascarriedout on eachof the eighteen electedmatrix compoundsand forty-seven electedhair dyes (Tables and II). First, their individual retentivebehaviorwas determinedand their UV spectrum ecorded,using the DAD or theUV-Vis spectrophotometer.econd,he efficiency f the liquid-liquid extraction ro-tocolon the selectedmatrix compounds aschecked.When a matrix compound ouldnot be extractedrom the sample olution,ts influence n the retentive ehavior f thedye intermediateswas investigated.CHROMATOGRAPHIC STUDYSingle olutionsf dye-formingompounds.orty-sevenye-forming ompounds eresub-jected to chromatographynd their retention imes and UV spectrawere recorded.Resultsare presentedn Table II.It comes ut that 91% (43 dye ntermediates)f the dye ntermediatesestedhave heir


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retention time between 5 min and 30 min. Some of the tested dyes have very closeretention imes--4-n-l,2-pd, 2-a-4-np, and 4-a-2-ht for instance, heir retention imesbeing 15.10 min, 15.50 min, and 15.66 rain, respectively. owever,since heir respec-tive UV spectra how emarkable ifferences8), their identificationcanstill be carriedout easily.In order o test the repeatability, ive injectionsof eachsamplewere carriedout. Therelative standard deviation (RSD) for the retention time was between 0.2% and 1.1%,while that of the peak corrected reas definedas the peak areadividedby the retentiontime), recordedat 290 nm, was between 1.1% and 6.0% (9). The chromatographicbehaviorshowedgood repeatability.The injectionswere repeatedon three differentcolumns n order o test the reproduc-ibility (Table II). For thirty-six hair dye intermediates,he relativestandarddeviation(RSD) on the retention ime was ess han 5%; for nine dye ntermediates,his RSD wasbetween6% and 10% and more than 10% for only two dyes. The reproducibilitybetweencolumnswas considered atisfactory.Single olutionsf matrixcompounds.ight differentcompounds ere subjectedo chro-matographiceparation,nd heir retention imesandUV spectra ere ecorded. esultsarepresentedn Table III. It appearshat someof the testedcompounds avevery closeretention times--BHT, OA, and ORA, for instance.However, with their respectiveUVspectra lsoshowing emarkable ifferencesFigure 1), their identification an still becarriedout easily. t must alsobe noted hat most of the matrix compounds, ith theexceptionof DMDM, PVP and MP, have retention times greater than 30 minutes,meaning hat confusing ye intermediates ith matrix products houldnot occursincethe retention imesof the hair dyesgenerally ange rom 5 to 30 minutes 1). Concerningthe fourdye ntermediatesorwhich etentionimesareout of the rangeof 5 to 30 min,the discriminationrom the matrix compoundsanalsobe easilymadeaccordingo bothretention time and UV spectrum.In order o test the repeatability, ive injectionsof eachsamplewere carriedout. Therelative standard deviation (RSD) for the retention time was between 0.02% and 0.7%,while that of the peakcorrected reaswasbetween .3% and 3.7%, except or DMDM.The chromatographicehavior f the matrix compoundshowed oodrepeatability.

Table IIIRetentionTimes and CorrectedPeak Areas or Eight Matrix Compounds,Each n a SingleSolution (n = 5)

Retention time (min) Corrected eakarea AU)Matrix compound Mean RSD (%) Mean RSD (%) k of measurementrim)

DMDM 6.80 0.21 4.3 12.4 220PVP 7.10 0.24 1.1 0.2 220MP 26.87 0.06 1.0 1.8 290NNO 33.80 0.70 5.2 3.4 235EOP 35.85 0.08 5.8 0.3 290OA 37.97 0.30 20.6 2.9 235BHT 38.11 0.02 2.5 3.7 290ORA 38.35 0.09 12.2 0.5 235

AU: arbitrary unit.


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Butylated Hydroxytoluene

1000--500

-s22| I200 300

Figure 1. UV spectra f three matrix products.

400

Wavelength (nm)

Sampleolutionsontainingn additionalmatrixcompoundnd three ye ntermediates.romthe forty-seven ye ntermediatesested, ourwerechosenor further nvestigation asedon how representativehey wereof their differentclassesf hair dyesaswell ason theiruse in formulations.Three sample olutionswereprepared, achcontaining mixture of threeselected yeintermediates1,4-pd or 4-ap, res, and 2-n-l,4-pd), the intrinsic matrix compoundNaAsc asan antioxidant,and anothermajor matrix compound,BHT, DMDM, or EOP,respectively. HT is an importantantioxidant,DMDM a commonlyusedpreservative,and EOP a perfumesolubilizer. he retention ime of BHT and EOP is more han 30min, while DMDM is eluted within about 7 min. Three control solutions,mixturesofthe respective yeswithout the major matrix compound,were alsoprepared.All the components ere at the concentrationssed in formulations ccording oCOLIPA, and five injections f each ype of solutionwere made. For eachseries fmeasurementsof control and mixture, a new column was used, as it has been shown ina preliminary tudy hat after 50 injections,he efficiency f the column s dramaticallydecreaseddata not reported).Table IV gives he statistical esultsobtained or theretention imesand the corrected eakareas or eachof thesecompounds.Comparison f the data betweenmixture (BHT) and control 1, mixture 2 (DMDM)and control2, and finally mixture 3 (EOP) and control3 leads o several bservations.First of all, as shownby the results,DMDM or EOP do not interferewith the dyeintermediatesor with the column in a way that would change he chromatographic


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Table IVRetentionTimes and CorrectedPeak Areas or Three Matrix Compounds nd Four Dye IntermediatesnD,e-Matrix Mixtures and in Control Solutions (n = 5)

Retention ime (min) Corrected eak area AU)Column Mixture Compound Mean RSD (%) Mean RSD (%)

4-ap 5.59 0.85 11.5 7.2res 9.31 1.14 0.3 3.1

1 2-n-l,4-pd 13.75 0.53 9.1 21NaAsc 2.18 0.38 54.7 7.8

1 BHT 38.07 0.55 2.5 19.04-ap 5.57 0.40 10.6 5.9res 9.55 0.82 0.3 5.0Control 12-n-l,4-pd 13.89 0.50 10.0 6.5NaAsc 2.18 0.21 56.5 8.01,4-pd 5.36 0.39 14.6 1.6res 8.32 0.34 0.5 2.9

2 2-n-l,4-pd 12.91 0.55 14.4 1.1NaAsc 2.35 0.60 70.3 2.3

2 DMDM 6.72 0.42 5.4 15.1.......... 1,4-pd 5.33 0.19 15.0 2.0

res 8.28 0.53 0.5 1.3Control 22-n-l,4-pd 13.28 3.39 14.4 3.8NaAsc 2.35 0 80.7 1.54-ap 5.99 0.39 22.1 1.7res 9.53 0.79 0.6 1.5

3 2-n-l,4-pd 14.09 0.39 20.4 1.6NaAsc 2.27 0.25 35.7 5.5

3 EOP 36.05 0.71 6.4 0.64-ap 6.32 0.88 22.7 2.0res 10.26 1.99 0.6 5.9Control 32-n-l,4-pd 14.84 1.42 20.3 1.6NaAsc 2.24 0.24 53.2 6.3

behavior retention time, correctedpeak area) of the dye intermediates.Furthermore,they do not alter the repeatabilityof the separation. or instance,when DMDM is addedto the sample, he RSD on the retention ime of the varioushair dyesvaries rom 0.3%


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240 JOURNAL OF COSMETIC SCIENCEto 0.5% and that of the corrected eak areas taysbelow he limit of 5%. For DMDM,this RSD value is of the order of what was obtained when DMDM was analyzedseparately seeTable III). Nevertheless,DMDM has a retention time greater than 5minutes and could (potentially) be confusedwith a dye intermediate (such as p-phenylenediamine,or instance).However,DMDM, which shows wo chrornatographicpeaksat 220 nm with exactly he sameUV spectrum,doesnot show any absorbancesignalat any wavelengthgreater han 250 nm. Thus, a selective etectionof the dyeintermediatescan be cacriedout at a wavelengthgreater than 250 nm.For EOP, Tables II and IV show hat EOP has a retention time much greater than 30minutes,and thuscannotbe confused ith any dye intermediate. urthermore, he peakretention imesand the peakareas f the dye ntermediates renot significantly hangedby the matrix compound egardingeither the mean valuesor the RSD.Nevertheless, inceEOP doesnot interferewith the hair dye separationn thesecon-ditions,an additionalparameter, .e., the concentration, asbeenchecked.A broad angeof concentrations of EOP were tested around the concentration used in formulations.Five RP-HPLC analyses ere carriedout for eachconcentration f EOP in a three-dyeintermediatesolutioncontainingNaAsc asan antioxidant.Figure 2 shows he evolutionof the mean corrected reaof the peaksobtained or the three dye intermediateswithincreasingconcentrationof EOP in the sample. The graphs clearly show that thecorrected reasdo not vary significantly p -- 0.05) when EOP has been added to thesamplesolutionover a concentrationange of 2 g/100 g-10 g/100 g. The statisticalanalysis f the results datanot reported) hows ood epeatability f the analysisor eachselected concentration of EOP. The RSD is less than 5% for the retention time and thecorrected eak area.Concerning HT, the results how hat the presence f this matrix compound oesnot

25t *T-ap-, T 2-n-l,4-pd,,a.:0 't -- .. .10

0 2 4 6 8 10Concentrationof EOP (g/100g)

Figure 2. Influenceof the concentrationf EOP on the corrected eakareas f 4-ap, res,and 2-n-l,4-pd.(See ext for experimentaldata and abbreviations.)


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affect either the retention imes of the dyesor their RSD, nor the mean valuesof thecorrected eak area.Nevertheless,t is important o note that the repeatabilityof themeasurementsor one component2-n-l,4-pd) seems o be stronglyaffectedby thepresence f BHT, with the RSD increasingrom 6.4% to 21%. BHT itself hasa highRSD for its peak corrected rea.Regarding hese esults, t appears, uite clearly, that although the matrix compoundEOP doesnot interferewith the dye intermediatesor with the column in a way thatwould affect the chromatographicehaviorof the dye intermediates,t would be ofadvantageo removeBHT andDMDM from the sample olutionbeforeproceeding iththe separationn order to obtain an accurate eparation f the dye intermediates.OPTIMIZATION OF THE LIQUID-LIQUID EXTRACTION METHODSince severalmatrix compounds re present n a real formulation, t is of immenseinterest o determine he effectof the isolationmethod on eachof them. The optimi-zationof the extraction rotocoleads o a one-stepLDA, TDS, NNO, ORA), two-step(OA, BHT, DMDM, NaAsc), or three-step CC, DC) extractionby n-heptane,whileEOP, NOL, LS, SS,TEA, PQ, MP, and PVP arenot extracted. wo differentapproacheswere considered or testing the extractionmethod. BHT and EOP were submitted toextraction n a mixture containing he dye intermediates s well, whereas n a secondapproach,he extractionwasperformedon singlesolutions f matrix compounds.Extraction f sample olutionsontaining matrix compoundnd threedye ntermediates.heextraction rotocolhas o be efficient o extract he matrix compounds, f course, ut thetarget dyesshouldnot be extracted.A one- to three-stepextractionprotocolwas thusapplied o sample olutions ontaining hreeselected ye intermediates4-ap, res,and2-n-l,4-pd), the antioxidantNaAsc,and a major matrix product BHT or EOP). Theaqueous hases btainedwere submitted o HPLC, and the extraction ield wascalcu-lated according o the followingequation:( correctedreafterxtractionield%) 100k, orrecte-------re--efor-'xtractio----100 (1)Table V gives he extraction ield for eachcomponentn the solution,and the successivechromatograms btained or the solutioncontainingBHT before a) and after (b) theextractionprocedureare shown n Figure 3. First of all, it is clearly seen hat theextractionprocedure ffectsneither the chromatographic haracteristicsf the dye in-

Table VStatisticalExtractionYield of the Components f a Dye-EOP and a Dye-BHT Mixture

Compound One-stepextractionyield (%) Two-step extractionyield (%)4-ap 0 0Resorcinol 0 02-n- 1,4-pd 0 0NaAsc 19 50EOP -- 0BHT 89 100 (n.p.)n.p.: no peak.


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550,

250__

--

-60

4

250--

Retention ime (rain)550 4

I--

2

,

I-60 0.0 15.0 30.0 49.0Retention ime (rain)

Figure3. Successivehromatogramsf a dye ntermediate ixturewith BHT addedo the sample eforeandafterextraction.: Before xtraction.: Aftera two-step xtraction.eaks:1) NaAsc intrinsicmatrix,antioxidant);2) 4-ap; (3) res; 4) 2-n-l,4-pd; and (5) BHT (matrix,antioxidant). eaks , 3, and 4 areunchangedetween teps and b. (See ext for experimentalataand abbreviations.)termediatesretentionimes,correctedeakareas f peaks , 3, and4) nor he repeat-ability of the analysis. hen, after a two-step xtraction y n-heptane, 0% of theantioxidantNaAsc s extractedwhile 100% of BHT (peak 5) is extractedrom thesample.However, t appearshat EOP is not at all extracted y n-heptane. dditionalexperimentalorkhasshownhat EOP was100%extractabley other ested rganicsolventstrichloromethane,ichloromethane,r diethylicether, or instance) ut thattheyalso ead o the partialextraction f the phenolic yes datanot reported).This extraction rocedurey n-heptanes therefore eryeffectiveor the separationf


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BHT from the sampleand partially effective n the caseof NaAsc, before he HPLCanalysis.Moreover, t has o be stressedhat with this extractionprocedure y n-heptane,the non-extractability f the dye intermediatess ensured 9).Extractionfsingle-compoundatrixsolutions.ue to the fact that the dye ntermediates renot extractedby n-heptane, he extractionprotocolwas then performedon single-compoundmatrix solutions. he extractionprotocolwas applied to 16 individual so-lutions of matrix compounds,namely OA, NNO, ORA, DMDM, BHT, PVP, MP,LDA, TDS, DC, NOL, SS, LS, TEA, PQ, and CC (see heir function in Table I). Theresultingaqueous hases btainedwere submittedto HPLC or analyzedby UV-Visspectrophotometrywhen necessary.The first sevencompounds OA, NNO, ORA, DMDM, BHT, PVP, and MP) showchromatographiceaks or which the chromatographicharacteristicsregiven in TableIII. For theseproducts, he extractionyield wascalculated ccording o equation1. Forthe lastnine compounds,.e., LDA, TDS, DC, NOL, SS,LS, TEA, PQ, and CC, the finalanalysis ould not be performedby RP-HPLC for several easons: ecause DA, NOL,and TDS are surfactants,he submissionf thesecompoundso RP-HPLC leads o theiradsorptionn the system, hus provoking urther contamination, nd the compoundsCC, LS, SS, TEA, DC and PQ do not showany chromatographic eak when submittedto RP-HPLC. As an alternative,a UV-Vis spectrophotometer as then used o test theextractionprocedure n thesenine compounds, y recording he spectra btainedbeforeand after extraction. n order to determine f the UV-Vis spectrophotometeras sen-sitive enough for the extraction measurements,BHT, which has been shown to beextracted y n-heptaneseeabove),wasused or comparing esultsobtained singbothprocedures.Evaluation f the extraction ield usingRP-HPLC.Figure4 shows he successivehro-matograms btainedbeforeand after extractionof four compoundsOA, NNO, ORA,DMDM). Table VI gives the extractionyield obtained. It must be noted that theefficiency f the extraction rotocolvariesslightly, depending n the matrix product.OA needs two-stepextraction rocedureo be fully extracted Figure4, la-c), whilethe peaksof NNO (Figure4, 2a,b)and ORA (Figure4, 3a,b)disappear ftera one-stepextractionprocedure.Moreover,even a two-stepproceduredoesnot lead to a completeextraction f DMDM (Figure4, 4a-c) and NaAsc.Finally, PVP and MP are not at allextracted.The extraction rotocolhas herefore eenshown o be efficient or isolatingOA, NNO,and ORA from the sample.ConcerningDMDM, the extractionyield is about 80% andMP is not extracted.Nevertheless, y analogywith EOP (Figure 2), a similar studyinvolving the concentration s an additional parameter eads to the conclusion hatDMDM andMP do not interferewith the dye ntermediates r with the column n a waythat would affect he chromatographicehaviorof the dye intermediatesresultsnotshown).ConcerningPVP, specialattention shouldbe given to real samples ontainingthis matrixproduct,as t formsa film on the columnsurface uring he first injection,leading o a modification f the retention imes of the dye intermediates. he determi-nationof the dye ntermediates as hereforeo be carriedout mainly accordingo theirspectrum results not shown).Evaluationof the extraction ield usingUV-Vis spectrophotometry.he extractionyields


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244 JOURNAL OF COSMETIC SCIENCE1300

900600

3oo-

-750.0

1300

9oo_

E 600-

,. 300-

-75oo

1300

900-

g600-" 300-

OA la

I I I

16u

-2515.0 30.0 45.0 57.0

Retention time (min)0.0

NNO I 2a

I I15.0 30.0 45.0 57.0Retention time (min)

15.0 30.0 44.0Retention time (min)

160113 I 213

.1oo

I -25 I Io.o 15.o 30 o 44.0Retention time (rain)

lc

o.o 15.o 30.0 45.0 57 oRetention time (rain)

Figure 4. Successivehromatogramsf four matrix productsamples eforeand after extraction.1: OA;2: NNO; 3: ORA; 4: DMDM. a: Beforeextraction.b: After a one-step xtraction.c: After a two-stepextraction. See ext for experimentaldata and abbreviations.)obtained are reported n Table VI. The complete extractionof BHT in a two-stepextraction rocedurewasconfirmed, nd the extraction rotocolhasalsobeenshown obe efficient or isolatingLDA, TDS, CC, and DC from the sample.Nevertheless, nce


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1300ORA 3_a

6OO-

300_

-750.0 150 30.0 45.0 57 0

Retention time (min)1300

9OO-

600-

300-

-750.0

3b

I15.0 30.0 45 0 57.0

Retention ime (min)

-500.0

-500.0

100

500.0

Figure 4. Continued

DMDM 4a

I I I7.5 15 0 22.5 30 0

Retention time (mln)

4b

I I I7.5 150 22.5 30.0

Retention time (min)4_qc

7.5 15.0 22.5 30.0Retention ime (min)

more, t has o be noted hat the efficiencyf the extractionrotocol aries lightly,depending n the matrixproduct. DA and TDS needa one-step xtraction rocedureto be ullyextracted, hile hecompletextractionf CC andDC involves three-stepextractionprocedure.Moreover,NOL, LS, SS,TEA, and PQ are not at all extracted.


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246 JOURNAL OF COSMETIC SCIENCETable VI

StatisticalExtractionYield of EighteenMatrix CompoundsEvaluation

using Compound Extractionyield (%) Extractionstep number

HPLC

UV/

VIS

OA 100NNO 100ORA 100DMDM 80NaAsc 50EOP 0MP 0PVP 0BHT 100LDA 100TDS 100CC 100DC 100LS 0SS 0NOL 0PQ 0TEA 0

(n.p.)(n.p.)(n.p.)

(n.p.)

n.p.: no peak.UV/VIS: UV-Vis spectrophotometry.CONCLUSION

The chromatographic ethodpreviously et up for the determination f hair dye in-termediates asbeenshown o be extremelyeffective or the analysis f forty-seven airdye intermediates ommonlyused n cosmeticormulations. sing that method,a databaseof the retention ime andof the spectrum asbeenbuilt up for the forty-seven yes,which allows he determinationof the dyesand their discrimination. n most cases,heretention imes of the hair dyes range rom 5 to 30 minutes.Thus problemswithconfusing ye ntermediates ith matrix products,which generallyhave etention imesof less han 5 minutesor greater han 30 minutes,shouldnot be encountered. ncemore, the UV spectrumconstitutes powerful criterion of discriminationwhen theretention time of a particular dye or a particular matrix compound s outside heexpected ange.The influenceof eighteenmatrix products ommonlyused n hair dye formulations asalsobeen nvestigatedn relation o the determination f hair dye intermediates. ineof the eighteenmatrix compound howretentivebehaviorand UV-detectablepeaks.Their individualretention imes and UV spectra ave herefore lsobeenrecorded ndincluded n the databasewith the aim of providingascomplete pictureaspossible fthe separation f hair dyes n complexmatrix media.The nine matrix compoundseftovercannotbe identifiedunder thesechromatographiconditions, ut their UV spectrahavealsobeenrecordedn order o complete he data base.However,on the whole, he presence f somematrix compoundseemso affect he finalaccuracyof the quantitative measurements f some hair dyes (caseof 2-n-l,4-pd),leading to the conclusionhat a separation f the matrix compoundsrom the dye


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solutionss desirable. liquid-liquid extraction rocedure y n-heptane as hereforebeen etup. Thismethodwas ested nall theeighteen elected atrixproducts ndwas100% effectiveor eightproductsLDA, TDS, CC, DC, NNO, ORA, OA, BHT) in aone- o three-stepxtraction rocedure. MDM andNaAscwereonlypartiallyextracted(80% and 50%, respectively),hile EOP, MP, PVP, LS, SS,NOL, PQ and TEA werenot extracted.Nevertheless, he non-interference f these compoundswith the dyeintermediates r with the column,hasbeenproved,except n the caseof PVP.A three-stepiquid-liquid extraction rocedure y n-heptane, hich s not toxic o theenvironment,s therefore uggesteds a compulsorytep or the separationf matrixproductsrom the dye-containingamplebefore nalysis y reversed-phasePLC toavoid ll problems f potentialnterferenceetweenhedyes nd hematrixcompoundsor between he matrix compoundsnd the column n a way that would affect thechromatographiceterminationf thedyes. hisprocedure ill nowbeapplied o actualcommercial formulations.As a generalconclusion,t has beenshown hat after applyinga matrix extractionprocedure singn-heptane,he RP-HPLC method,combinedwith diodearraydetec-tion, s verypromisingn termsof beingan efficient andidateeference ethod or theidentification nd quantitationof oxidativehair dyes.NOTE

A spectra' atabasef eighteenmatrix products asbeen ecordedhrough he diodearray etectornd heUV spectrophotometerswellasa spectra' atabasef forty-sevendye intermediates8). For further informationabout the database, mail contact:[email protected] would like to thank COLIPA for providing raw materialsand information.Wewould also ike to thank Dr. E. Pel for performing nitial studieson separation roce-dures. This work was carried out in the frame of support to DG XXlV "ConsumerPolicy"and DG III "Industry"of the EuropeanCommission.APPENDIX

List of Some Frequently Used Matrix ProductsMatrix component Usualconcentrationg/100 g)

Ammonia 6.00-9.00Ascorbic acid 0.20L-ascorbic acid sodium salt 0.20BHT 0.25Diethyleneglycolmonoether 5.00Citric acid 0.30Diethanolamine 2.00Triethanolamine 0.10-1.50TEA-dodecylbenzenesulfonate 0.50DEDM-hydantoin 0.10


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248 JOURNAL OF COSMETIC SCIENCEAppendix (continued)

Syntopon D1 6.00Ethoxylatednonylphenol 3.00Ethyl acetate 2.00Hydroxyethylcellulose 2.40Isopropanol 3.00-15.00Lauric diethanolamide 1.50-2.00Methyl paraben 0.05Propyleneglycol 5.00-9.00Sodium auryl sulfate 2.95Sulfated castor oil 4.00Polyvinylpyrrolidon 2.00Oleic acid 5.00n-Nonylamine 25.00Oranex HT 3.00Cetrimonium chloride 0.50

REFERENCES(1) E. Pel, G. Bordin, and A. R. Rodrfguez,HPLC candidate eferencemethod or oxidativehair dyeanalysis.. Separation nd stability testing,J. Liq. Chrom.Re/. TechnoL,1(6), 883-901 (1998).(2) E. Pel,ApplicationfHighPerformaneiquidChromatographyHPLC) to heStudy fOxidative air Dyes(Internal annual report, GE/R/ACH/03/95, 1995), pp. 1-19.(3) D. H. LiemandJ. Rooselaar,PLC of oxidation aircolours, itt. Gebieteebensm.yg.,72, 164-176(1981).(4) N. Goetz,J. Mavro, L. Bouleau, nd A. de Labbey, Analysis f OxidativeHair Dyes Using HighPerformanceiquid Chromatography,"n Cosmeticnalysis: electiveethodsnd Techniques,. Bord,

Ed. (MarcelDekker, New York, 1985), pp. 245-287.(5) C. Gennaro,P. L. Bertolo,and E. Marengo,Determinationof aromaticaminesat trace evelsby ioninteraction eagent eversed-phaseigh-performanceiquid chromatography--Analysisf hair dyesand otherwater-solubleyes, . Chromatogr.,18, 149-156 (1990).(6) V. Andrisano, . Gotti, A.M. di Pietra,and V. Cavrini,Analysis f basichair dyesby HPLC withon-line post-column hotochemical erivatisation,Chromatographia,9, 138-145 (1994).(7) C. Genova, A. Zatta, L. Deiana, F. Montesion, F. Buosi, and G. Gazzaniga,"EthoxylatedMono-Branched rimaryAlcoholsn Hair Dyes," n Cosmetics997 ConjUrericeroceedings:odern hallengesotheCosmeticormulation,997, pp. 359-377.(8) U. Vincent,Practitwl spectsf theDeterminationf Oxidative air DyesbyRP-HPLC--DevelopmentfaData Base Annualreport,GE/R/ACH/04/99, 1999), pp. 1-24.(9) U. Vincent,Evaluation f High PerformanceiquidChromatographyHPLC) in Relationo theStudyofOxidative air Dyes Internalannual eport,GE/R/ACH/13/97, 1997) pp. 1-26.

Influence of Matrix Compounds on Oxidative Hair Dyes by HPLC

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