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Validation of an analytical procedure for the determination of

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j. Cosmet.
Sci.,53, 43-58 (January/February
2002)

Validationof an analyticalprocedurefor the determination

of oxidativehair dyesin cosmeticformulations

URSULA VINCENT, GUY BORDIN, and

ADELA
[Link],
European
Commission,
JointResearch
Centre,Institutefor Red•rence
Materialsand Measurements,
Retieseweg,
B-2240 Geel,Belgium.

Accepted
for publication
November
15, 2001.

Synopsis
A high rangeand variety of cosmeticformulationsthat containoxidativehair dyesand matrix-forming
compounds havebeenindustriallydevelopedoverrecentyearsand are now availableon the international
market.

Memberstatesof the EuropeanUnion are responsiblefor conductinganalysesof cosmeticproductsas

deemednecessary
by law and Europeanregulationenforcement.
Therefore,inspectionauthoritiesaswell as
the cosmetics trade and industryneedvalidatedanalyticalmethodsfor the identification,characterization,
and/or quality controlof specificactive ingredientsor formulationswith the aim of implementingthe
EuropeanUnion CosmeticDirectives(76/768/ECC, 95/17/EC). In this frame,we validateda candidate
reference methodthat enablesthe identificationand quantificationof hair dye-formingcompounds. This
methodconsists of a separation
by RP-HPLC coupledwith a DAD aftera liquid-liquid extractionprocedure
for separating matrix components from the dye-formingcompounds. The validationof the methodincludes
commoncriteriasuchasthe repeatabilityof the analysisand the establishment of figuresof merit, aswell
as statisticalevaluationsand quality assurance in order to follow the recommendations of the Eurachem
guide for analyticalmeasurements.

INTRODUCTION

A cosmeticformulationfor permanenthair dyeingcan be describedasa mixture of two

kinds of products:oxidativehair dyes, the so-calledactive compounds,and matrix
compoundsin which the dyesare [Link] hair dyesare aromaticorganic
compounds, e.g., amines,phenols,and derivatives,while a broadrangeof productscan
be usedasmatrix [Link] differentrolesin hair formulations.
For example,they act as surfactants,pH adjusters,consistency providers,antioxidants,
emulsifiers,film formers,preservatives,
or [Link] biggestanalyticalchallengeis
thusto identifyandquantifythe activeproductsinto a complexmatrix mixture in order
to proposea reference methodfor the analysisof this type of cosmeticproductwith the
aim of implementingthe EuropeanUnion CosmeticDirectives76/768/ECC and 95/
17/EC (1).

43
44 JOURNAL OF COSMETIC SCIENCE

In our previouswork, an analyticalprocedurewasdevelopedthat enablesthe separation

and identificationof 52 different dye-formingproducts,accordingto their retention
timesand UV spectra,in standardpure solutions(2,3). Theseoxidativehair dyes,listed
in the C.T.F.A. International
CosmeticIngredient
DictionaryandHandbook (4), wereselected
according
to COLIPA• data(5)andto howrepresentative
theyareofdifferent
groups
of
dye-formingcompoundsin the [Link], the investigationof possibleeffects
of the variousmatrix componentson the separationof the active substances
was con-
cludedin the settingup of an extractionprocedureof thesematrix components
from the
dye-formingcompounds(3).
Beforeapplyingsuchmethodologyto commercialformulationsfrom which qualitative
and quantitativedata are expected,a prior validationof the wholeprocedurehasto be
performedto assureconfidencein the final [Link],in the presentcontribu-
tion, a validation processand its resultsare [Link] was carried out using (i)
severalrepresentative syntheticformulationspreparedwith cosmeticgraderaw materials
and(ii) commercialshampoos availableon the localmarket,spikedwith four commonly
usedhair dye [Link] quantitativedeterminationof eachdyewas
successfullyachievedin the two typesof formulations,andfiguresof merit of the method
have been established.

EXPERIMENTAL

INSTRUMENTATION

HPLC was performedusing a two-pistonHPLC pump with a low-pressureternary

gradientsystemmodule(System325 from Kontron InstrumentsS.P.A., Milan, Italy), an
Autosampler360 with a loop of 20 1-fi(KontronInstrumentsS.P.A.),a Diode Array
Detector440 (KontronInstrumentsS.P.A.),and a vacuumdegassing system(Degasys
DG 1300, Uniflows,Japan).The columntemperaturewaskept constantby meansof the
thermostatof an electrochemical
detector(Decade,Antec Leyden,Leiden,The Nether-
lands).Data processing
wasdonewith the Data System450-MT2/DAD Series(Kontron
InstrumentsS.P.A.).The columnwasa Merck LichrospherRP 60 SelectB (C8), 250 x
4 mm, 5-1•mparticlesize.

CHEMICALS

L-ascorbicacid sodiumsalt (NaAsc)wasobtainedfrom Fluka, andp-phenylenediamine

(1,4-pd), m-phenylenediamine sulfate(1,3-pds),m-aminophenol (3-ap), and resorcinol
(res)werekindly providedby "LesColorantsWackkerr"(Saint-Ouenl'Aum6ne,France).
Sodium tetraboratedecahydrate(p.a.), acetic acid 95% (suprapure),ammonia 25%
(suprapure),hydrochloricacid (0.1 M) and n-heptane(p.a.) were obtainedfrom Merck.
Methanol(HPLC quality)[Link]
©, sodiumacetate,erythor-
bic acid(AFB), sodiummetabisulfite(MBS), and tetraethylenediamineacetate
(ED) were
kindly providedby "LesColorantsWackherr" for the preparationof the synthetic
formulations. Triethanolamine (TEA) was obtained from Mobi-Lab bvba (Zutendaal,

• Comit•deLiaison
Europ6en
del'Industrie
dela Parfurnerie,
deProduits
Cosm6tiques
et deToilette.
 OXIDATIVE HAIR DYES 45

Belgium).Purewater(18.2 Mll/cm quality)usedfor thepreparationof solutions

was
obtainedfrom a MilliQ Plus185 system(Millipore,Molsheim,France).
PREPARATION OF REAGENTS

Solvents
andchromatographic
mobile
phase.
The solvents
weremixturesof methanol(MeOH)
andSoerensen
buffer(40%).The Soerensen
buffer(pH 8.1) wasprepared
asfollows:88
ml hydrochloricacid (0.1 N) and 2 g/1 L-ascorbicacid sodium salt (NaAcs) as an
anti-oxidant agent were addedto 112 ml sodium tetraboratesolution.
Mobilephase(aqueous
phaseB): a 0.05 M aceticacidsolutionwasadjusted
to a pH of
5.9 with a 10% ammoniasolutionand filtered througha 0.45-1amfilter. When not in
use,the eluentwasstoredat a temperature
of 4øCto preventmicrobiological
growth.
Preparationofthesynthetic
formulations.
Fivesynthetic formulations,eachcontainingfour
hair dye intermediates(1,4-pal,1,3-palS,3-ap, and res)at variousconcentrations,
were
preparedaccording to "LesColorants Wackherr"data(6) to reproduce the following
formulations: darkbrown,middlebrown,light brown,darkblonde,andlight blonde.
They werepreparedusingthe followingsubstances:
1. Dark brown
ß Matrix:Oxocap
© (600.00g/l), sodiumacetate
(10.00g/l), TEA (30.00g/l), AEB
(3.00 g/l), MBS (4.65 g/l), ED (9.60 g/l)
ß Hair dyes:1,4-pd(12.50g/l), 3-ap(5.00 g/l), res(36.50g/l), 1,3-pdS(1.62 g/l)
2. Middle brown
ß Matrix:Oxocap
© (600.00g/l), sodium
acetate
(10.00g/l), TEA (30.00g/l), AEB
(3.00 g/l), MBS (4.65 g/l), ED (9.60 g/l)
ß Hair dyes:1,4-pd(7.90 g/l), 3-ap(2.80 g/l), res(21.00g/l), 1,3 pdS(0.86 g/l)
3. Light brown
ß Matrix:Oxocap
© (600.00g/l), sodiumacetate
(10.00g/l), TEA (30.00g/l), AEB
(1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l)
ß Hair dyes:1,4-pd(5.00 g/l), 3-ap(1.58 g/l), res(12.10g/l), 1,3-pdS(0.48 g/l)
4. Dark blonde
ß Matrix:Oxocap
© (600.00g/l), sodiumacetate
(10.00g/l), TEA (30.00g/l), AEB
(1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l)
ß Hair dyes:1,4-pd(3.16 g/l), 3-ap(0.89 g/l), res(6.95 g/l), 1,3-pdS(0.27 g/l)
5. Light blonde
ß Matrix:Oxocap
© (600.00g/l), sodiumacetate
(10.00g/l), TEA (30.00g/l), AEB
(1.80 g/l), MBS (4.65 g/l), ED (9.60 g/l)
ß Hair dyes:1,4-pd(2.00 g/l), 3-ap(0.50 g/l), res(4.00 g/l), 1,3-pdS(0.14 g/l)
Eachsynthetic formulation wasextractedandinjectedintothechromatographicsystem,
afterappropriatedilutionin a mixtureof MeOH andSoerensen buffer,pH 8.1 (40%).
Preparation
of thespiked shampoos.
Three commercialshampoos availableon the local
marketweredilutedtentimesw/win a mixtureofMeOH andmobilephaseB, pH 5.9
(40%).Thesedilutedmatrixsolutions werethenspikedwith 4 g/kgof eachof thefour
hairdyeintermediates (1,4-pd,1,3-pdS,3-ap,andres).The spikedshampoos obtained
werealsodilutedten timeseachw/w in a mixtureof MeOH andmobilephaseB, pH
5.9 (40%),beforeextraction andinjectioninto thechromatographic
system.
46 JOURNAL OF COSMETIC SCIENCE

PROCEDURES

Reversed-phaseHPLC separations.
A non-linearMeOH (A)/aqueous phase(B) gradientwas
usedas describedin our previouswork (3). The total flow was 1 ml/min. The column
wasequilibratedby 25 ml [Link]
[Link] columntemperature waskept at 48ø[Link] dataacquisition wascarried
out at the maximumUV absorbance wavelengths for eachdye intermediate(TableI) in
parallel with the UV spectraacquisition.
Extraction of thematrixcomponents
fromthefinal analytesolution.
This wascarriedout by a
three-stepliquid-liquid extractionby n-heptane,previouslyshownto be 100% efficient
for a large numberof matrix components while not affectingthe activecompounds (3).
Determinationofconcentration
[Link] quantitationof eachactivecompoundwasbased
on individualcalibrationlinesin pure standardsolutions[eachhair dye beingprepared
in a mixtureof Soerensen buffer(40%) andMeOH]. Eachsamplein a puresolutionwas
injectedin the HPLC systemat concentrations rangingfrom 0 g/1 to 0.9 g/l (1,4-pd),
0 g/1to 0.7 g/1(3-ap),and0 g/1to 0.4 g/l (resand 1,3-pdS).The analysis wasrepeated
three times in orderto test the repeatability.
Statisticalevaluations
and quality assurance.
For the chromatography,the quality of the
separations wasevaluatedaccordingto differentcriteriaclassicallyconsidered in HPLC.
The repeatabilityof the separationwascalculatedfor eachhair dye and wasbasedon
both the retentiontime and the [Link] account
to evaluatethe quality of the column:
© the resolutionR betweentwo consecutive peaks,whichcharacterizes the separating
powerof the columnfor two selectedsubstances,
© The peakasymmetryfactors, whichis the measurement of the deviationof peakform
from the theoreticallyexpectedGaussiancurve,and
© the capacityfactork', i.e., the ratioof the net retentiontime (retentiontime - dead
time) and the deadtime. This capacityfactorenablesa comparisonbetweencolumns
of the separationof selectedcompounds.

For the calibration,three regressiontypeswere tested(quadraticregression,linear re-

gression,and trendlinelinearregression with interceptsetat zero)in orderto determine
the best mathematicalmodel applicableto the experiment(data not reported).The
regressiontype finally chosenwas that providing the most accurateresultsfor the
concentrationof the maximum of selecteddyes,i.e., the "trendlinewith interceptsetat
zero"regression. The pipettesweretestedboth by gravimetryand colorimetry,and the
calculationof the uncertaintieswasperformedaccordingto internationalstandards(7).

Table I
Maximum Absorbance
Wavelengthsfor Four Hair Dye Intermediates

Maximum UV absorbance
wavelength(nm)

Dye intermediate Maximum 1 Maximum 2

p-Phenylenediamine 235 290

m-Phenylenediamine
sulfate 220 290
m-Aminophenol 235 280
Resorcinol 220 270
 OXIDATIVE HAIR DYES 47

RESULTS AND DISCUSSION

The validationis obviouslya crucialstepin the development of an analyticalmethodand

hasthereforeto be basedon [Link] the presentwork, five syntheticformu-
lationsareusedto carryout this procedure,eachof them containingthe four activehair
dyes,p-phenylenediamine, m-phenylenediamine sulfate,m-aminophenol, and resorcinol,
chosenfor the following reasons:
ß They arerepresentative of threemajorclasses of oxidativehair dyes:aromaticamines,
aminophenols,and phenols.
ß They appearregularlyin the compositionof commercialformulations.
Theseactive compoundswere mixed with severalsubstances
classicallyused in the
matrices of cosmetic formulations:
ß Erythorbicacid (antioxidant)
ß EDTA (chelatant)
ß Sodiummetabisulfite(reducingagent)
ß Butyleneglycol (solvent,viscositydecreasingagent)
ß Laureth-8(surfactant,emulsifyingagent)
ß Laureth-3(surfactant,emulsifyingagent)
ß Cetrimoniumchloride(antistaticagent, cosmeticbiocide,surfactant,emulsifying
agent)
ß Trideceth-2carboxamide
MEA (surfactant,foam booster,viscosityincreasingagent,
aqueous)
ß Butoxyethanol(solvent,viscositydecreasing agent)
ß Oleyl alcohol(emollient, solvent,viscosityincreasingagent, non-aqueous)
Furtherseparations
with formulationscontainingup to 15 oxidativehair dyeswerealso
performedto confirmthe followingresults(data not reported).

CHROMATOGRAPHIC SEPARATION AND IDENTIFICATION OF THE PEAKS IN

SYNTHETIC FORMULATIONS

Each samplewas submitted to chromatographyafter [Link] analysiswas

repeatedthree times in order to test the repeatabilityof both the extractionand the
chromatographic [Link] an example,the chromatographic separationsobtained
for the "light brown"and the "light blonde"samplesaredepictedin Figure 1. Similar
chromatograms were obtainedfor the three other formulations.
Obviously,mostof the matrix compounds are 100% extracted,and the chromatograms
showonly six peaks,with an excellentresolution,calculatedaccordingto the following
formula:

(tR2- try) x 2
R1,
2= (/./21
-3-
w2
) (a)
whereR1,2 is theresolution
(determined
for twoconsecutive
peaks),tR1andt•2 arethe
retentiontimes of two consecutive
peaks,and w• and w2 are the peak widths at the
baseline (base width) (8).
All thecalculated
resolutions
rangedbetween
R4,5 = 1.77 andR3,4 = 4.76 (seepeak
labelingin Figure 1). The identificationof the hair dye intermediatescould then be
easilyperformedusing individual retention times and UV [Link], thesetwo
characteristics
havingbeenpreviouslyrecordedin a databasefor 68 hair dyesandmatrix
48 JOURNAL OF COSMETIC SCIENCE

+l • +l +1 +l +l

....

04

+l +1 +l +l +l +l
 OXIDATIVE HAIR DYES 49

2OOO

1500--

1000--

5
500 -- 6

-150
I I I
0 7.5 15.0 22.5 30.0

Retention time (min)

2000
1
3

1500_

1000 -

500 _
5

-150 I I I
0 7.5 15.0 22.5 30.0

Retention time (min)

Figure 1. Chromatographic separations
of two syntheticformulationsafter matrix extraction.a: "light
brown."b: "light blonde."Peaks:(1) NaAsc (intrinsicmatrix, antioxidant),(2) matrix compound,(3)
p-phenylenediamine,(4) m-phenylenediaminesulfate,(5) m-aminophenol,and (6) [Link] for
experimentaldata and acronyms.

compounds in pure standardsolutionscommonlyfoundin oxidativehair dye formula-

tions (9), the identificationcould then be performedby comparisonbetweenthe ex-
perimentaldataobtainedand the [Link] II and Figure2 showthe iden-
tificationobtainedby combiningthe two criteriaretentiontime andUV [Link]
3, 4, 5, and 6 are attributedto p-phenylenediamine,
m-phenylenediamine
sulfate,m-
aminophenol,and resorcinol,respectively.Peaks1 (attributedto sodiumascorbate)and
2, having retentiontimes shorterthan 5 min, are due to remainingmatrix compounds
not interferingwith the dye separation,in accordance
with previousobservations
(2,3).
50 JOURNAL OF COSMETIC SCIENCE
OXIDATIVE HAIR DYES 51
52 JOURNAL OF COSMETIC SCIENCE

4•0

1 ooc 001]

75C-

50C -

25C-

200 250 300

1ooo
I //
600 -I
/

o •o
Figure 2. UV spectracomparison of four hair dye intermediatesand a matrix compoundbetween200 and
400 nm. a: p-phenylenediamine reference.b: p-phenylendiamine in the analyte.c: m-phenylenediamine
sulfate reference.d: m-phenylenediaminesulfate in the analyte. e: m-aminophenolreference.f: m-
aminophenolin the analyte.g: resorcinolreference.h: resorcinol
in the analyte.i: sodiumascorbate
reference.
j: sodiumascorbatein the analyte.

The repeatabilityof the analysiswasalsoinvestigated,in terms of retentiontimes and

peakareasfor the four hair dyesin the five [Link] aregatheredin Table III.
On the whole, the repeatabilityof the separationis high for both the retentiontimes
(0.10%<RSD<l.24%) in the five formulations and the peak responseintensity
(0.21%<RSD<9.46%) exceptfor this secondparameterin the "light blonde"formula-
tion: the RSD of peak areasin the "light blonde"formulation is somewhathigher
(5.84%<RSD<14.35%) becauseof the low concentration of the hair dyesin this for-
mulation.

The quality of the columnwasalsoevaluatedby calculatingthe peak asymmetryfactor

s and the capacityfactork' of the peaksattributedto the hair dyesaccordingto the
following formulas(b) and (c) respectively:
a5va+ b5v•
s- (b)
2 x a5v•
 OXIDATIVE HAIR DYES 53

whereas• andb5%denotethehorizontal
(time)intervalof theright andleftpeakflanks,
respectively,from the peakmaximumat 5% of [Link] optimumpeaks,s = 1,
but for real HPLC peaks,s is practicallyalwaysgreaterthan 1 (8), and
tR, - t o
k' - -- (c)
Zo

wheretRiis the retentiontime of the compoundi andto is the deadtime of the column
(8).

Resultsaregiven in Table IV. The peak asymmetryfactoris very closeto the optimum
valueof 1 for the peaksof m-phenylenediamine sulfate(peak4) and m-aminophenol
(peak 5) in all five [Link]-phenylenediamine (peak 3), the peak asymmetry
factor rangesbetween0.83 and 0.96, showinga slight fronting on the peak. For
resorcinol(peak 6), the s value is greaterthan 1, which commonlyoccursin HPLC.
Concerning
the k' factor,the valuescalculated
for eachhair dyepeakrangefrom 1.03
to 3.03 and were repeatableunder the experimentalconditionsused,the relative stan-
dard deviationranging from 0 to 2.0% on three consecutiveinjectionsin all formula-
tionsexceptfor m-phenylenediamine sulfatein "light blonde"(RSD = 5.9%). The range
of significance
of k' being1 to 5 (8), the capacityfactork' determined
is considered
to
be good.
Consideringall the criteria, the stationaryphaseand the chosenelution conditions,as
well as the samplepreparation,were found to be very appropriatefor the separationof
the selectedhair dyes.

QUANTITATION OF THE HAIR DYE INTERMEDIATES IN SYNTHETIC FORMULATIONS

As detailed in the Experimentalsection,the quantificationwas basedon individual

calibrationlines, after appropriatedilution of the [Link] 3 showsthe
calibrationlinesobtainedfor p-phenylenediamine and resorcinolas [Link] ana-
lytical data are given in Table V for the calibrationlines establishedfor the four hair
[Link] all cases,
excellent
linearity
wasobtained
(R2 > 0.987)forconcentration
ranges
commonlyfound in [Link] of detection,definedas the con-
centrationgiving a signal-to-noiseratio of 3 at the optimum wavelengths,resultedin
0.87 pg for p-phenylenediamine,1.10 pg for m-phenylenediamine sulfate,0.70 pg for
m-aminophenol, and0.52 pg for resorcinol,that is to say4 (m-phenylenediaminesulfate)
to 160 (resorcinol)timeslowerthan the lowesthair dye concentrationsto be determined
in the selected formulations.

With this set of calibrations,the dye concentrations

weremeasuredin the five formu-
[Link] experimentalconcentrations determinedwerethen comparedto the initially
introducedconcentrations. Table VI expressesthis comparisonas a percentof recovery
Re, that is concentrationfound divided by known [Link]
resultsare given with an expandeduncertainty,with a coveragefactor of 2 giving
confidencelimits of 95% (Re e 2s) following the recommendationsincluded in the
Eurachemguide (7).
The outcomeis that in all casesthe recoveryis higher than 90% and often higher than
95%. The procedureapplied for the determinationof hair dyes in complexsynthetic
54 JOURNAL OF COSMETIC SCIENCE

p-phenylenediamine (235 nm)

Linear regression, confidence limits and error bars

...
•' 250
]
200 Slope = 223.65

.• 150
1
,
0.0 0,2 0.4 0.6 0,8 .0

Concentration of p-phenylenediamine (g/I)

Resorcinol (270 nm)

Linear regression, confidence limits and error bars

16

• 14
; 12
lO
8
'= 6

0
0.0 0.1 0.2 0.3 0.4 0.5

Concentration of resorcinol (g/I)

Figure 3. Calibrationline of the correctedpeak area vs concentrationfor p-phenylenediamine

(a) and
resorcinol(b).

formulationsmimicking real commercialformulationsis then provento be very appro-

priate in termsof identificationand accuracyand precisionof quantitation.

DETERMINATION OF THE FOUR HAIR DYE INTERMEDIATES IN THREE COMMERCIAL SHAMPOOS

To furthervalidatethe entireprocedure,threecommercialshampoos,
known to contain
 OXIDATIVE HAIR DYES 55

Table V
Figuresof Merit for the CalibrationLinesObtainedfor the Four Hair Dyes

Dye Slope(AU/g.I-•) R2 Detection

limit (g/l) Range(g/I) n
p-Phenylenediamine 223.650 + 2.065 0.9976 0.044 0-0.9 30
rn-Phenylenediamine
sulfate 28.274 + 0.945 0.9874 0.055 0-0.4 14
rn-Aminophenol 145.520 _+1.507 0.9976 0.035 0-0.7 24
Resorcinol 32.580 _+0.509 0.9969 0.026 0-0.4 15

Table VI
Recoveries
(%) of Four Hair Dye Intermediatesin Five SyntheticFormulations

Hair dye recoveryRe + 2s (%)

Dye Dark brown Middle brown Light brown Dark blonde Light blonde

p-Phenylenediamine 95.05 + 3.05 93.15 + 2.95 91.32 + 5.20 95.08 + 4.58 96.45 + 3.66
m-Phenylened
iamine
sulfate 99.56 -+4.17 96.93 + 8.31 96.06 + 9.29 93.50 + 9.65 90.58 + 9.95
m-Aminophenol 95.99 + 3.63 99.37 + 0.85 97.27 + 3.29 98.89 + 2.17 96.48 + 5.21
Resorcinol 93.76 -+2.66 92.14 + 2.76 98.24 _+1.77 98.58 + 1.36 97.51 + 2.49

Percentage
of recovery+ 2s,expanded
uncertainty
with a coverage
factorof 2, whichgivesconfidence
limits
of 95%.

no hair dye intermediates(this wascheckedby confirmingthe absenceof any peaksat

the hair dye intermediatesretentiontimes reportedin Table II), were spikedwith
weighedamountsofp-phenylenediamine, m-phenylenediamine sulfate,m-aminophenol,
andresorcinol. Figure4 showsthe chromatograms obtainedfor the threeshampoos after
appropriate dilution in a mixtureof methanolandmobilephase(40%) followedby the
[Link] matrix compoundsare 100% extractedand the chromato-
gramsshowfive peaks,with a satisfactory resolution,calculatedaccordingto formula(a)
andcomprisedbetween R3,4 = 1.03andR2,3 -- 4.12(shampoo
1),R3,4 -- 0.85andR2,3
= 3.01 (shampoo
2), andR3,4 = 1.03andR•,3 = 3.79 (shampoo 3) (seepeaklabeling
in Figure 4). The identificationof the hair dye intermediatescould also be easily
performedusingindividualretentiontimesand UV [Link] peaks1 and 2 had to
be attributed to p-phenylenediamine becauseof the presenceof both amphotericand
basicformsof thiscompound [experimentalacidityconstantsof 2.46 and6.04 (9)] at the
workingpH of 5.9. Both formshavethusto be takeninto accountfor the quantitation.
The concentrationof eachhair dye intermediatewascalculatedwith the sameprocedure
previouslyreportedand comparedto the [Link] shownin Table VII, the
recoveriesobtained,as well as the expandeduncertainties,were very good. Therefore,
this wholeprocedureappearsto be suitablefor a completeidentificationand quantifi-
cationof thesehair dye intermediates.

CONCLUSIONS

The validationof the analyticalprocedurefor the determinationof oxidativehair dyesby

RP-HPLC, coupledwith DAD detectionafter a liquid-liquid extraction,has been
56 JOURNALOF COSMETICSCIENCE

+1 +1 +1 +1

....
 OXIDATIVE HAIR DYES 57

500

e 300 -- 5

8 2

• 150
--

-30 I I I
0 7.5 15.0 22.5 30.0

Retention time (min)

500

• 300 -
E
--- 2

o 150
- 3

-30 I I I
0 7.5 15.0 22.5 30.0

Retention time (min)

500

z• 5
• 300--

-3O I I I
0 7.5 15.0 22.5 30.0

Retention time (rain)

Figure 4. Chromatographicseparationsof three shampoosspikedwith four hair dye intermediates,after
matrixextraction.a: shampoo1. b: shampoo 2. c: shampoo3. Peaks:(1)p-phenylenediamine(amphoteric
form), (2)p-phenylenediamine
(basicform), (3) m-phenylenediamine sulfate,(4) m-aminophenol,
and (5)
resorcinol.
58 JOURNAL OF COSMETIC SCIENCE

performedon complexsyntheticformulations mimickingrealcommercial formulations.

Five syntheticformulations,eachcontainingfour hair dyesembeddedin a complex
matrix,havebeenanalyzed,aswell asthreecommercialshampoos spikedwith weighed
amountsof the four chosenhair [Link] eachformulation,the four hair dyescouldbe
clearlyidentifiedusingtheir individualretentiontimesandUV spectra,andthe analysis
alsoshowedvery goodrepeatabilityin termsof retentiontimesand peakareasfor the
four hair dyesin the eight [Link] a quantitativepoint of view, calibration
lineswereperformedfor eachof the fourhair dyes,andin all casesexcellentlinearitywas
obtainedfor concentrationrangescommonlyfound in commercialformulationswith
very low detectionlimits. The concentrations of eachof the hair dyesin the eight
formulationscouldbe determinedwith an excellentrecoveryrate in eachformulation,
in all caseshigher than 90%.
As a whole, the validatedprocedurethus appearsto be highly appropriatefor the
identificationandthe quantificationof oxidativehair dyesin realcommercial
permanent
hair [Link] a next step,the methodis currentlybeing appliedand
beingvalidatedfor actualcommercial permanenthair dyeingformulations availableon
the market.

ACKNOWLEDGMENTS

We would like to thank "LesColorantsWackherr"and COLIPA for providingraw

[Link] wouldalsolike to thankF. CordeiroRaposo forhisgreat
help in the statisticalcalculations.
The work wascarriedout in the frameof supportto
DG "Enterprise"of the EuropeanCommission.

REFERENCES

(1). European Union CosmeticDirectives76/768/ECC,0.], L262, 169-200 (September 1976);95/17/EC,

0.], L140, 26-29 (June1995).
(2). E. Pel, G. Bordin, and A. R. Rodr;guez,HPLC candidatereferencemethod for oxidativehair dye
analysis.I. Separationand stability testing,.]. Liq. [Link]. Technol.,
21,883-901 (1998).
(3). U. Vincent, G. Bordin, and A. R. Rodr;guez,Influenceof matrix compounds on the analysisof
oxidativehair dyesby HPLC,.]. Cosmet. Sci.,50, 231-248 (1999).
(4). J. Wenningerand G. N. McEwan,Eds.,International Cosmetic
Ingredient
Dictionary
andHandbook, 7th
ed., (Cosmetic,Toiletry and FragranceAssociation,Washington, DC, 1997), Vols. 1-3.
(5). COLIPA: Personalcommunication(1994).
(6). Les ColorantsWackherr: Personalcommunication(1998).
(7). S. L. Ellison,M. Rosslein,and A. Williams, Eds., QuantifyingUncertainty
in AnalyticalMeasurement,
Eurachem/CITACGuide, 2000, pp. 1-120.
(8). L.R. Snyder,J.J. Kirkland, andJ. L. Glajch,Praaical HPLC MethodDevelopment, 2nd ed. (Wiley-
Interscience,New York, 1997), pp. 24, 29, 208-210.
(9). U. Vincent,PracticalAspectsoftheDetermination
ofOxidative Hair DyesbyRP-HPLC--Development ofa
Database (AnnualReport,GE/R/ACH/04/99, 1999), pp. 1-24.

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