filtre UV

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AnalyticaChimicaActa752 (2012) 11–29

ContentslistsavailableatSciVerseScienceDirect

Analytica

Chimica

Acta

jo u r n al h om ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / a c a

Review

An

overview

of

the

analytical

methods

for

the

determination

of

organic

ultraviolet

ﬁlters

in

biological

ﬂuids

and

tissues

Alberto

Chisvert

a,∗

_,

_Zacarías

_{León-González}

b

_,

_Isuha

_Tarazona

a

_,

_Amparo

_Salvador

a

_,

_Dimosthenis

_Giokas

c

a_Departamento_de_Química_Analítica,_Facultad_de_Química,_Universitat_de_València,_Doctor_Moliner_St._50,₄₆₁₀₀_Burjassot,_Valencia,_Spain b_Unidad_Analítica,_Instituto_de_{Investigación}_Sanitaria_Fundación_Hospital_La_Fe,₄₆₀₀₉_Valencia,_Spain

c_Laboratory_of_Analytical_Chemistry,_Department_of_Chemistry,_University_of_Ioannina,₄₅₁₁₀_Ioannina,_Greece

h

i

g

h

l

i

g

h

t

s

Papersdescribingthedetermination ofUVﬁltersinﬂuidsandtissuesare reviewed.

Matrixcomplexityandlowamounts ofanalytesrequireeffectivesample treatments.

Thepublishedpapersdonotcover the study of all the substances allowedasUVﬁlters.

NewanalyticalmethodsforUVﬁlters determinationinthesematricesare encouraged.

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Articlehistory: Received3May2012

Receivedinrevisedform10August2012 Accepted29August2012

Available online 6 September 2012 Keywords: Biologicalﬂuids Biologicaltissues Bodydisposition Percutaneousabsorption Sunscreen Cosmeticproducts Ultravioletﬁlters

a

b

s

t

r

a

c

t

OrganicUVﬁltersarechemicalcompoundsaddedtocosmeticsunscreenproductsinordertoprotect usersfromUVsolarradiation.Theneedofbroad-spectrumprotectiontoavoidthedeleteriouseffectsof solarradiationhastriggeredatrendinthecosmeticmarketofincludingthesecompoundsnotonlyin thoseexclusivelydesignedforsunprotectionbutalsoinalltypesofcosmeticproducts.

DifferentstudieshaveshownthatorganicUVﬁlterscanbeabsorbedthroughtheskinaftertopical appli-cation,furthermetabolizedinthebodyandeventuallyexcretedorbioaccumulated.Thesepercutaneous absorptionprocessesmayresultinvariousadversehealtheffects,suchasgenotoxicitycausedbythe generationoffreeradicals,whichcanevenleadtomutagenicorcarcinogeniceffects,andestrogenicity, whichisassociatedwiththeendocrinedisruptionactivitycausedbysomeofthesecompounds.

Duetotheabsenceofofficialmonitoringprotocols,thereisademandforanalyticalmethodsthat enablethedeterminationofUVfiltersinbiologicalfluidsandtissuesinordertoretrievemore informa-tionregardingtheirbehaviorinthehumanbodyandthusencouragethedevelopmentofsafercosmetic formulations.Inviewofthisdemand,therehasrecentlybeenanoticeableincreaseinthedevelopment ofsensitiveandselectiveanalyticalmethodsforthedeterminationofUVfiltersandtheirmetabolitesin biologicalfluids(i.e.,urine,plasma,breastmilkandsemen)andtissues.Thecomplexityofthebiological matrixandthelowconcentrationlevelsofthesecompoundsinevitablyimposesampletreatment pro-cessesthataffordbothsampleclean-uptoremovepotentiallyinterferingmatrixcomponentsaswellas theenrichmentofanalytesinordertoachievetheirdeterminationatverylowconcentrationlevels.

Theaimofthisreviewistoprovideacomprehensiveoverviewoftherecentdevelopmentsinthe determinationofUVﬁltersinbiologicalﬂuidsandtissues,withspecialemphasisontheelucidationof newmetabolites,samplepreparationandanalyticaltechniquesaswellasoccurrencelevels.

∗ Correspondingauthor.Tel.:+34963544900;fax:+34963544436. E-mailaddress:[email protected](A.Chisvert).

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12 A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29 Contents

1. Introduction... 12

2. MetabolicprocessesrelatedtoorganicUVﬁlters... 13

3. AnalyticalmethodsforUVﬁltersdeterminationinbiologicalﬂuidsandtissues... 17

3.1. Biologicalmatricesstudied... 17

3.1.1. Urineandfaeces... 17

3.1.2. Plasmaandserum... 17

3.1.3. Otherbiologicalmatrices... 17

3.2. Samplepreparation... 17

3.3. Analyticaltechniques ... 21

3.4. Accuracyandsensitivity ... 24

4. OccurrencelevelsoforganicUVﬁltersinbiologicalﬂuidsandtissues... 24

5. Conclusionsandfuturetrends... 28

Acknowledgements... 28

References... 28

AlbertoChisvertreceivedhisB.Sc.fromUniversityof Valenciain1999andreceivedhisPh.D.inDepartment ofAnalyticalChemistryfromUniversityofValenciain 2003.HewasassistantprofessorattheDepartment ofFoodSciencesoftheUniversityofValencia(2005), assistantprofessorattheDepartmentofAnalytical ChemistryofUniversityofAlicante(2006–2008),and lecturerinDepartment ofAnalyticalChemistryof UniversityofValencia(2008–2011).Nowadays,he isassociateprofessorinthislastdepartmentsince 2011.Hehasactedasco-editorofthebookAnalysis ofCosmeticProducts(Elsevier,2007),andheis edito-rialboardmemberofAdvancesinAnalyticalChemistry, AmericanJournalofAnalyticalChemistry,ChromatographyResearchInternational, InternationalJournalofAnalyticalChemistryandJournalofTraceAnalysisinFood andDrugs.Hisresearchareasarefocusedonbothliquidandgaschromatography coupledtomass-spectrometry,liquid-phaseandsolid-phasemicroextraction, bioanalysisandcosmetic,pharmaceuticalandenvironmentalanalysis.

ZacaríasLeón-Gonzálezobtained his B.Sc.degree

inchemistryin2006(UniversityofValencia),and received his Ph.D. in 2012 from the Department of Analytical Chemistry (University of Valencia). Hisresearchhasfocusedonbioanalyticalchemistry (i.e.,cosmeticingredientsbodydisposition),using mainlyliquidchromatographyandmass spectrome-trytechniques.HecurrentlyworksasAnalyticalUnit coordinatorintheInstitutode Investigación Sani-tariaHospitalLaFe(Valencia).Hiscurrentresearch interests are both target and untarget bioanaly-sis,includingstructuralelucidationchemistry,drug metabolismandbiomarkerdiscovery.

IsuhaTarazonaobtainedherB.Sc.degreein chem-istryatthe University ofValencia in2007. From June2007toJuly2008,sheenjoyedagrantinthe DepartmentofExperimentalHepatologyatthe ‘Uni-versityHospitalLaFe’ofValencia.Latershestarted hisdoctorateandsheobtainedherMScinAnalytical Chemistryin2010.CurrentlysheisaPh.D.studentin theDepartmentofAnalyticalChemistryatthe Uni-versityofValencia.Herresearchareasarefocused on both liquid and gas chromatography coupled tomass-spectrometry,liquid-phaseandsolid-phase microextraction,bioanalysisandcosmeticand envi-ronmentalanalysis.

AmparoSalvadorobtainedherPh.D.inchemistryin 1982attheUniversityofValenciaandsheis profes-sorinthisuniversitysincethen,beingfullprofessor ofanalyticalchemistrysince2005.Herresearchis focusedondevelopingnewanalyticalmethods,with specialemphasisonthestudyofthequality,efﬁcacy andsafetyofcosmeticproductsinwhichshehas spe-cializedsince1999,carryingoutdifferentresearch projectsfundedbytheSpanishScienceMinistryand byseveralcosmeticenterprises.Sheistheresponsible personoftheResearchGroupforAnalyticalControl ofCosmeticProducts(GICAPC).Amongtheir publi-cationsisincludedthebook“AnalysisofCosmetic Products(Elsevier,2007)inwhichsheistheEditorandalsoauthorofseveral chapters.Shehasbeenappointedmemberofvariouscommitteesintheﬁeldof cosmeticproducts,suchastheSpanishWorkingGroupofAnalyticalMethods fromtheSubcommitteeSC2ofCosmeticProducts(AEN/CTN),fordeveloping andreviewingEUrulesoncosmetics.

DimosthenisL.GiokasobtainedaBachelor’sdegree onEnvironmentalSciencefromtheDepartmentof EnvironmentalStudiesoftheUniversityoftheAegean (1998),aMaster’sDegreeonChemicalTechnology (2001)andaPh.D.fromtheDepartmentof Chem-istryoftheUniversityofIoannina(2003).Since2004 heisworkingasapost-docresearcherandresearch associateintheLaboratoryofAnalyticalChemistry. In2007hewasappointedintheLaboratoryof Chro-matographyof theDepartment of Chemistry. His researchfocusesonsupramolecularassemblies,ﬂow analysis(includingliquidchromatography), molecu-larandatomicspectroscopy,appliedchemometrics andmorerecentlynanomaterialsinanalyticalscience.

1. Introduction

Moderateexposuretosolarradiationhasbothphysicaland

psy-chologicalbeneﬁcialeffects,suchasthestimulationofvitaminD

synthesis[1],thepromotionofbloodcirculation[2]andthesense

ofwellnessproducedbytheactionofthebrainneurotransmitters

[3].However,excessivesunexposurecancauseerythemaat

short-termandthedevelopmentofskincarcinomas,amongotherfatal

consequences[4].Thisfactisrelatedtotheincrease,overtheyears,

ofthedeleteriousUVradiationreachingtheEarth,asaresultof

theprogressiveandcontinuousdamageofthestratosphericozone

layer.

Theuseofsunscreencosmeticproductscouldhelptoprevent

orminimizetheharmfuleffectsassociatedwiththeexcessivesun

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A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29 13

UVﬁlters,eitherorganicorinorganic[6],whichareabletoabsorb

and/orreﬂect,respectively,theincidentUVradiation,thus

pro-tectinghumanskin.Althoughtheywereinitiallydesignedtobe

usedinsunscreencosmeticformulations,theyarenowadaysadded

toother daily cosmetic products, suchas moisturizing creams,

lipsticks, makeup formulations, after-shave lotions or hair-care

products,in ordertopreventfromtheharmfuleffects notonly

whensunbathingbutalsoinourdailylife.

However,despiteUVﬁltersaredesignedforexternalapplication

ontheoutermostlayersoftheskin,differentstudieshaveshown

thatsomeorganicUVﬁlterscanbeabsorbedthroughtheskin,

fur-thermetabolizedandeventuallybioaccumulatedand/orexcreted

[7].Thesepercutaneousabsorptionprocessesmayresultinvarious adversehealtheffects(i.e.,allergiccontactdermatitis[8])andother

moreserioussystemiceffects,suchasmutagenicorcarcinogenic

[9,10]andestrogenic[11–13]activity.Thereby,toachievean

opti-mumcompromisebetweenadequateprotectionandminimalside

effectstocosmeticusers,thecompoundsthatcanbeusedasUV

ﬁltersincosmeticsaswellastheirmaximumallowed

concentra-tionshavebeenregulatedbythelegislationinforceineachcountry

[14].ThemolecularstructuresofsomeorganicUVﬁltersarelisted inFig.1.Foramoreexpandedlist,readersarereferredto[14].

Inthissense,thebodydispositionoforganicUVﬁlters

asso-ciatedtothepercutaneousabsorptionprocesseshasbecomean

interestingﬁeldofresearchaimingtoretrievemoreinformation

aboutthe behavior ofthese chemicalsin the humanbody and

thusencouragethedevelopmentofsafercosmeticformulations.

Moreover,it hasbeenshown that theskinpenetration of

sun-screenagentscandiffersigniﬁcantlydependingonthenatureof

thecosmeticformulation[15].Severalstudieshavebeencarried

outtoevaluatethevehicleeffects,beingtheﬁnalaimtooptimize

thecosmeticformulationinordertominimizethepercutaneous

absorptionoftheactiveingredients.

Invitromethodologiesbasedontheuseofdiffusioncellsand

tape-strippingtechniques[16,17]havebeenemployedtoestimate

thepercutaneousabsorptionoftheseUVﬁlters.Regardingtheuse

ofdiffusioncells(i.e.,Franztypecells[18]),acosmeticformulation (orothervehicle)containingthetargetUVﬁltersisappliedonthe

upperfaceofapieceofexcisedhuman,animalorsyntheticskin,

whichinnerfaceisincontactwithastirredreceptorﬂuid

emu-latingthebloodstream.Oncethepercutaneousabsorptionprocess

isaccomplished,UVﬁlters aredeterminedinthereceptorﬂuid.

Regardingthetape-strippingtechnique,italsoinvolvesthe appli-cationofthevehiclecontainingthetargetUVﬁltersonapieceof

excisedhuman,animalorsyntheticskin.However,afteradeﬁned

periodoftime,theformulationismechanicallyremovedandan

adhesivetapeispastedonthetreatedskinarea.Then,thetapeis

takenoffinsuchawaythatitremovestheﬁrstlayerofstratum

corneum,wherethecontentofthetargetUVﬁltersisdetermined

afterpropertreatmentofthetape.Then,theestimationofUVﬁlters

percutaneousabsorptioncanbeperformedafterrepetitionofthe

processwithnewpiecesofadhesivetape,whichremovethe

sub-sequentlayersofstratumcorneum[19].Thislattertechniquecan

alsobeappliedinvivo,byusinghumanvolunteersorlaboratory

animals.

Ontheotherhand,takingintoaccountthatthebody

disposi-tionoftheorganicUVﬁltersnotonlyconsidersthepercutaneous

absorptionbutalsothedistribution,metabolismandexcretion

pro-cesses,invivomethodologiesbasedontheanalysisofbiological

ﬂuidsand/or tissues collectedafterthe applicationof cosmetic

productscontainingUVﬁltersareofgreatinterest.

Forallthis,analyticalmethodsarerequiredtodeterminethe

targetUVﬁltersinboththereceptorﬂuidsfromdiffusioncellsand

theadhesivetapesfromthetape-strippingtechniqueandalsoto

determinenotonlytheparentorganicUVﬁltersbutalsotheir

pos-siblemetabolitesinbiologicalﬂuidsand/ortissues.However,no

ofﬁcialanalyticalmethodsareavailable.Inviewofthisdemand,

therehasbeena noticeableincreaseofanalytical methods

con-cerningthedeterminationofUVﬁltersandtheirmetabolitesin

biological ﬂuids (i.e., urine, plasma, and even breast milk and

semen)andtissuesinthelastyears.Theevolutionofthenumberof publicationsrelatedtothissubjectoverthetimeisshowninFig.2. Theincrementofpublishedarticlesinthelast5yearsisan unequiv-ocalreﬂectionofthesocialconcernabouttheuseofsafecosmetic

productscontainingorganicUVﬁltersintheircomposition.

Fromthesepublications,around71%arebasedonthe

applica-tionofavehiclecontainingthetargetUVﬁlters(mainlyasunscreen

cosmeticproduct)overhumanvolunteers.Theresidualpercentage

correspondstostudieswithrats(24%)andpiglets(5%).Thistrend

agreeswiththefactthatmethodologiesbasedoninvivoassaysof

cosmeticingredientswithanimalsarebeingsuccessively

forbid-denbytheEuropeanUnionlegislation,whichsetMarchof2013as

deadlineforcompleteprohibitionofanimaltesting[20,21].

Concerning experimentalissues, the complexity of the

bio-logical matrix and the low concentration levels of the target

compounds(i.e.,ngmL−1,ngg−1orbelow)[7]certainlyimposethe

needofsampletreatmentprocesses.Inthatway,asampleclean-up

toremovematrixcomponentsthatmayinterfereintheanalysis,

and a targetcompounds enrichmentstep, areboth required to

achievetheselectiveandsensitivedeterminationofUVﬁltersas

wellastheirmetabolites.

Takingalltheaboveintoconsideration,theaimofthisreviewis

toprovideacomprehensiveoverviewoftherecentdevelopments

relatedtothedeterminationoforganicUVﬁltersinboth

biologi-calﬂuidsandtissues,withspecialemphasisontheelucidationof

newmetabolitesandtheemployedsamplepreparationand

ana-lyticaltechniques.Moreover,thereportedamountsofUVﬁltersin

biologicalhumansampleshavebeencompiledinordertoshow

theoccurrencelevelsofthesecompounds.Finally,itshouldbesaid

thatduetotheexcessiveuseofUVﬁlters,theyhavereachedthe

aquaticenvironmentbydirectandindirectsourcesandarebeing

accumulatedinit[7].However,theanalysisoftissuesofaquatic

animalsfromanenvironmentalpointofviewisoutsidethescope

ofthisreview,andthereforehasbeenexcluded.

2. MetabolicprocessesrelatedtoorganicUVﬁlters

Although thedetermination of organicUV ﬁlters in

biologi-calﬂuidsandtissuesdoesnotprovidepreciseinformationabout

thelevelofpercutaneousabsorptionofthesecompounds,itdoes

theevidenceofpenetrationacrosstheskin,asapartofthe

over-allprocessthatconstitutesthebodydispositionoftheUVﬁlters.

Additionally,thisinvivomethodologyallowstheresearchonthe

systemicbiotransformationprocessesoftheUVﬁlters.

Biotransformationcanbedeﬁnedastheprocesswherebya

for-eigncompound(i.e.,axenobiotic)ischemicallytransformedinthe

bodytoformametabolite.Thisistheprocessthatmostinﬂuences

thetransportofaxenobioticintothebody,itstoxicityanditsroute ofelimination.Inspitethefactthatallcellsandtissuesare

capa-bleofcarryingoutbiotransformationofexternalcompounds,itis

primarilycarriedoutinliver,lung,intestineandskin[22].

Biotransformation is usually divided into two main phases,

commonlyknownasphase Iand phaseII.PhaseIisusuallyan

oxidative and/or hydrolytic process whereby lipophilic

xenobi-oticsare turnedintomore polarspecies, andthus, more easily

excretables. Next, in phase II, if these phase I metabolites or

eventheparentcompoundhavenotyetbeenexcreted,theycan

reactwithhighlypolarspecies suchasglucuronicacid, sulfate,

methionine,cysteineorglutathione,resultingintheso-called

con-jugates. While phase Ioxidativereactionsare mainlycatalyzed

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14 A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29

Fig.1. SomeexamplesoforganicUVﬁltersusedincosmeticproducts,groupedaccordingtotheirchemicalstructureandnamedaccordingtoINCI(InternationalNomenclature forCosmeticIngredients).

ofenzymes,thehydrolyticreactionsarecatalyzedbyestearases.

The phase II conjugationis catalyzed by a variety of isozymes

ofN-acetyltransferase(NAT),

uridinediphosphoglucuronosyltrans-ferase(UGT),sulfotransferase(ST),andglutathioneS-transferase

(GST).Atleast50majorenzymesparticipateinthe

biotransforma-tionprocess[23].

Althoughscarcestudiescanbefoundregardingthemetabolic

pathwayoforganicUVﬁlters,ithasbeenelucidatedinthecase

ofBZ3,MBCandEDPbymeansofinvitroandinvivo

methodolo-gies.Moreover,itshouldbesaidthatthebiotransformationpattern

ofPABA,whichisacontroversialUVﬁltercurrentlyprohibitedin

theEuropeanUnionbut stillallowedinothercountries[14],is

knownformanyyears,sincethiscompoundhasbeentraditionally

includedinnutritionalsupplementsofvitaminBfororalintake.

Accordingtothesestudies,thiscompound seemsnottoexhibit

phaseImetabolictransformation,butitisacetylatedand

conju-gatedwithglycinethroughoutthecorrespondingphaseIIreactions

to form p-aminohippuric acid (PAH), p-acetamidobenzoic acid

(PAcB)andp-acetamidohippuricacid(PAcH).However,thereare

veryfewstudiesregardingthetopicalapplicationofPABA.

Aran-cibiaetal.[24]andmorerecentlyWangetal.[25,26]determined

PABAanditsmetabolitesinhumanurineaftertopicalapplication

ofsunscreencosmetic.

RegardingBZ3,althoughsomedataconcerningtheformation

ofnon-deﬁnedhydroxylatedmetaboliteswereavailable[27],its

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A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29 15 Fig.1. (Continued). 0 10 20 30 40 50 60 1981 1986 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Year Number of publications

Fig.2. Evolution ofnumber of publications concerning the determinationof organicUVﬁltersinbiologicalﬂuidsandtissuessince1981(black=individual; white=accumulated).

[28] after oral administration of this UV ﬁlter to rats.Later, it

wasalsoconﬁrmedbythesameauthorsafterdermal

administra-tiontorats[29].Analysisofblood,urine,faecesandtissues(liver, kidney,intestine,testes,skin,spleenandothers)revealedthe

for-mationofthreephaseImetabolites(2,4-dihydroxybenzophenone

(DHB), 2,2-dihydroxy-4-methoxybenzophenone (DHMB) and

2,3,4-trihydroxybenzophenone(THB)).Accordingtotheseauthors,

BZ3undergoesO-dealkylationofthemethoxymoietyontheringA

toformDHB,whichishydroxylatedinthesameringtoformTHB,

whereasDHMBisformedbyhydroxylationofringBofBZ3(see

Fig.3).Inaddition,thephaseIIglucuronideconjugatesofBZ3and

itsmetaboliteswerealsofound.

In the case of MBC,its biotransformation pattern was

elu-cidated by means of proton nuclear magnetic resonance (1_H

NMR) and liquid chromatography tandem mass spectrometry

(LC–MS/MS)analysisofurine,faecesandplasmafromorallyMBC

administeredrats[30].Theformationof itsphase Imetabolites

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16 A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29 O OH O O OH HO O OH O OH O OH HO HO

BZ3

DHMB

THB

DHB

Fig.3. PhaseIbiotransformationpatternofBZ3.

CH3 C H3 C H3 O CH3

MBC

CBC

CBC-6OH

CH3 C H3 C H3 O OH O CH3 C H3 C H3 O OH O HO

Fig.4. PhaseIbiotransformationpatternofMBC.

of3-(4-carboxybenzylidene)hydroxycamphor(CBC-OH),being

3-(4-carboxybenzylidene)-6-hydroxycamphor(CBC-6OH)themajor

isomer) and the glucuronide forms of CBC and CBC-OH are

described.However,3-(4-hydroxybenzylidene)camphor,whichis

anintermediateencounteredbyinvitroexperimentscarriedout

byincubatingMBCwithratorhumanlivermicrosomes,wasnot

found.Inasubsequentpublication,thesameauthorsconﬁrmed

theirﬁndingsbyanalyzingurineandplasmafromhumanandrats

afterdermalapplicationofMBC[31].Theseauthorsconcludedthat

CBCisformedbyoxidationofthearomaticmethylmoietyofMBC,

andlater,CBCundergoesfurtheroxidationtoCBC-OH.Fig.4shows

thephaseIbiotransformationpatternforthisUVﬁlter.

Regarding EDP, its biotransformation pattern was recently

established. Firstly, in vitro experiments were carried out

with rat liver microsomes by incubating EDP in presence

of various cofactors. Under these conditions, two phase I

metabolites(i.e.,N,N-dimethyl-p-aminobenzoic acid(DMP) and

N-monomethyl-p-aminobenzoicacid(MMP))wereidentiﬁedby

gas chromatography–mass spectrometry (GC–MS) and liquid

chromatography–mass spectrometry (LC–MS) [32]. The results

showed that the ester group of EDP is hydrolyzed, losing the

2-ethylhexyl chain, thus forming DMP, which undergoes

N-demethylationtoformMMP,althoughthislastseemstobealso

directlyformedfromEDP(seeFig.5).PhaseIImetabolismwasalso

investigatedviaglucuronidationandacetylationbymeansof

incu-bationsofEDPwithhumanandratlivermicrosomesandcytosols

containingvariouscofactors,butnoinvitrophaseIImetabolites

werefound.Nevertheless,LC–MS/MSanalysisofurinefromhuman

volunteersaftertopicalapplicationofacosmeticformulation

con-tainingEDPshowedthepresenceofglucuronideconjugatesofDMP

andMMPtoagreaterextentincomparisontothenon-conjugated

forms[33].ThereasonwhyphaseIImetabolitesofEDPwereonly

detectedunderinvivoconditionsmightarisefromtheinherent

differencesonthebioavailabilityofchemicalsbetweeninvivoand invitrosystems[34].

TakingintoaccountthehighnumberoforganicUVﬁlters

avail-able,andthatthebiotransformationpatternhasbeenonlystudied

forfourofthem,newstudiesinthisﬁeldareencouraged.

N OH O CH3 C H₃

MMP

DMP

N O O CH3 C H3

EDP

N OH O CH3 H

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3. AnalyticalmethodsforUVﬁltersdeterminationin biologicalﬂuidsandtissues

WhenpublishedpapersdealingwiththedeterminationofUV

ﬁltersinbiologicalmatricesareclassiﬁedaccordingtothestudied

matrix(seeTables1–3),itcanbeobservedthatthemoststudied

biologicalmatrixisurine(57%),followedbyplasmaorserum(23%). Othermatrices,suchasfaeces(3%),breastmilk(3%)andsemen(3%), haveonlybeenanalyzedoccasionally.Theanalysisofbiological tis-sues(liver,kidney,intestine,spleen,brain,heart,testes,placental, skinoradiposetissue),hasbeenconsideredscarcely,andcomprises 10%oftheoverallpublications.

Todate,mostofthepublicationsarefocusedonthestudyof

BZ3anditsmetabolites,whichhavebeenwidelydeterminedinall

typesofbiologicalmatrices(i.e.,urineandfaeces,plasmaorserum,

breastmilk,semenandtissues).OtherUVﬁltersthathavebeen

studied,althoughscarcely,areBDM,BZ4,EDP,EMC,ES,HS,MBC,

PABA,PDT,P25,TDSand3BC,butarestillfarawayfromthetotal

numberofcompoundsconsideredasUVﬁlters[14].

Moreover,it shouldbeemphasized,thatsomeofthese

pub-lishedpapersareincompletefromananalyticalpointofview,since

theydealwithpharmacokineticstudies,butnon-validated

analyt-icalmethodsareused.Ontheotherhand,therearealsosomeof

themthatonlydealwiththeparentcompound,butnotwithits

metabolites. Allthis shows arealneedtodevelop efﬁcientand

reliableanalyticalmethodsthatallowstudyingthepercutaneous

absorption,biotransformation,bioaccumulationandexcretionof

allthesubstancesusedasUVﬁlters. 3.1. Biologicalmatricesstudied 3.1.1. Urineandfaeces

Achronologicalsummaryofthepublishedpapersdealingwith

thedeterminationofUVﬁltersinurineislistedinTable1.The anal-ysisoffaeceshasalsobeenincludedhere,sinceinbothcasesthey areroutesofexcretion.Differentstudiescarriedoutinratsshow

thaturineisthemainrouteofexcretionforBZ3andits

metabo-lites[28,29,36],whereasfaecesisthemainexcretionpathwayin

thecaseofMBCanditsmetabolites[30,31].Nodataarereported

forotherUVﬁlters.

ThetargetUVﬁlter,theaimofthestudy,thetypeofexperiment (e.g.,oraladministrationortopicalapplication,typesubject,etc.),

thesamplepreparation,theemployedanalyticaltechnique,aswell

assomecommentsregardingtheanalyticalperformance

(calibra-tionmodeused,limitofdetectioninthesample(SLD)andrecovery (R)),aredetailedinTable1.

Regardingtheaimofthestudy,publicationshavebeenclassiﬁed

dependingontheirinterestin(a)elucidatingthebiotransformation

pattern of the UV ﬁlter (BP), (b) showing evidences of the

percutaneous absorption (PA), (c) studying the changes of the

concentrationwithtime (i.e.,kineticproﬁles)(KP),(d)

develop-inganalyticalmethodsforsubsequentapplications(AM)and(e)

biomonitoringstudies(BM).Moreover,somerelevantcomments

concerningtheconsideration(ornot)ofbothphaseIandphaseII

metabolitesarealsopresented.

3.1.2. Plasmaandserum

Table2showsachronologicalsummaryofthepublishedpapers

dealingwiththedeterminationofUVﬁltersinplasmaandserum.

ThedescribedconceptsareanaloguetothoseexplainedinTable1.

Itcanbeconcludedthatplasmahasbeenlargelyanalyzedin

com-parisontoserum[68]. 3.1.3. Otherbiologicalmatrices

Achronologicalsummaryofthepublishedpapersdealingwith

thedeterminationofUVﬁltersinbiologicalmatricesdifferentfrom

urine,faeces,plasmaorserumislistedinTable3,includingthe

aforementionedconcepts. Ascanbe seen,UV ﬁlters have been

determinedinless commonbiological matrices,suchashuman

breastmilk[70–72],semen[63,73],andplacentaltissues[74,75].

Additionally, rat tissues have also been scanned for UV ﬁlters

[28,29,36,66,69].

3.2. Samplepreparation

In order todetermine theconjugatedfractionof the parent

compoundand/orofthephaseImetabolites(i.e.,bounded

com-poundstoglucuronicacid,mainly,orsulfate)inurine,ahydrolysis

step is usually carried out todetermine the total content (i.e.,

free plus conjugated content). On theother hand, without the

hydrolysisstep,onlythefreecontentisdetermined.Thedifference

betweenbothcontentsresultsintheconjugatedcontent.Inthat

sense,intheﬁrststudies,itwasusualtoboiltheurinesample

with6Mhydrochloricacidtohydrolyzetheboundedcompounds

[28,29,36,39].However,nowadays,itisusualtoresorttoenzymatic

hydrolysisbyincubatingtheurinesamplewith␤-glucuronidase

(mainly fromHelixpomatia, since ithasalsosulfatase activity),

and then hydrolysis of the compounds bounded to glucuronic

acidandsulfateisachieved.Theenzymatichydrolysisiscarried

out under controlled conditions of pH, temperature and time

[28,29,31,33,36,37,42,44–47,51,52,56,59–61,63,68,71,72]. After

enzymatic hydrolysis,theenzyme is denaturedbytheaddition

of acetonitrile,coldmethanolor formic acid, thusstopping the

reaction and precipitating the enzyme, which is separated by

centrifugation.Then,thesupernatantissubmittedtothe

subse-quentsamplepreparationstep.Itshouldbeemphasized,however,

thatsomeofthepublishedpapersdo notcarry outthis

hydrol-ysis step, but theyonly considerthe phase I metabolites. This

mightcauseanerrorfromapharmacokineticstandpoint,sincea

fraction(i.e.,theconjugatedfraction)wouldnotbedetermined.

However,theaimofmostofthesepapersisthedevelopmentof

ananalytical methodtodeterminethefreecontentofboth the

parentcompound andphase Imetabolitesbypropervalidation

oftheanalyticalmethodology.Obviously,itshouldbetakeninto

accountthatahydrolysisstep(i.e.,atreatmentwithhydrochloric

acid or with ␤-glucuronidase) should be performed before in

casethedeterminationofthetotalcontentisofconcernfroma

toxicokineticpointofview.Anotherrecentapproach,thatenables

the determination of theconjugate forms withoutthe need to

performthehydrolysis step,is themeasurement byLC–MS/MS

bymeansoftheso-called‘constantneutralloss’mode[30,31,33].

Thisallowsthedetectionofanycompoundthatlosesafragment

ofgivenmassafterMS/MSprocess(inthiscaseafragmentof176.0

m/zwhichcorrespondstotheglucuronidemoiety[76]).

Ontheotherhand,duetocomplexityofthematrix,theusual

approachfollowedintheanalysisofurineistheuseofextraction

techniques in order to isolate the target compounds from the

restofthematrix,thusavoidinginterferencesinthesubsequent

measurement.Moreover,takingintoaccountthat UVﬁltersare

in thetrace levelor below [7], theseextractionmethodologies

havealsobeenusedwiththeaimtoconcentratethetarget

ana-lytes.AscanbeseeninTable1,themostpopulartechniquesare

liquid–liquid extraction(LLE)[26,28,29,36,60,61] orsolid-phase

extraction (SPE) either in manual mode [26,39,51,52,54,63,64]

or inan on-lineconﬁguration bymeansof sequential injection

manifolds[40,50,62],switchingchromatography[44–47,68,71,72]

orcommerciallyavailableautomatedworkstations[33,73].

Mem-brane assisted liquid–liquid extraction (MALLE) has also been

employed[57].Inorder toreducetheamountofsolventsused

andalsotoincreasetheenrichmentfactors,theso-called

microex-tractiontechniques,eitherinthesolid-orliquid-phase,havealso

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18 A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 Table1

PublishedpapersonUVﬁltersdeterminationinurineandfaeces(chronologicalorder).

UV ﬁltera

Aimb _Experiment _Sample_preparationc _Analytical

techniqued

Analytical performancee

Commentsa _Ref.

PABA PA Topicalsunscreenapplicationon humanvolunteers

Urineistreatedornotwith4MHClforhydrolysis,and thenitisderivatizedwithnitriteandN-naphthyl ethylendiamine

UV/Vis PABAanditsacetylatedphaseII metabolite(PAcB)areconsidered

[24]

BZ3 BP Oral,topicalandintravenous administrationonrats

DHBandotherhydroxylated undeﬁnedphaseImetabolitesare found.PhaseIIglucuronideforms arealsoidentiﬁed

[27]

BZ3 AM Oraladministration(incornoil)onrats LC-UV/Vis TheBZ3totalcontent

(free+conjugated)isdetermined. NophaseImetabolitesare considered

[35]

BZ3 BP KP

Oral(incornoil)ortopical(in petroleumjelly)administrationonrats

UrineissubjectedtoLLE,theextractisevaporated, redissolvedinMeOHandinjected.

Faecesaremechanicallyhomogenizedwithwaterand thensubjectedtoLLElikeurine

Forboundedcompounds,atreatmentwith ␤-glucuronidase(pH6,37◦_C,₂₄_h)_or_with₆_M_HCl_is

performedbeforeLLE

LC-UV/Vis PhaseImetabolites(DHB,DHMB andTHB)andphaseIIconjugated formsarealsoconsidered

[28,29,36]

BZ3 KP Topicalsunscreenapplicationon humanvolunteers

Urineistreatedwith␤-glucuronidase LC-UV/Vis TheBZ3totalcontent

[37]

BZ3 AM Topicalsunscreenapplicationona humanvolunteer

SPMEisperformedbydirectimmersiononurine (10–15min),andtheninjected

GC-MS (EI+₎

Standardadditioncalibration SLD=5ngmL−1

PhaseImetabolites(DHBand DHMB)arealsoconsidered,but THBisexcluded.NophaseII conjugatedformsareconsidered

[38]

BZ3 KP Topicalsunscreenapplicationon humanvolunteers

Urineisboiledwith6MHCl(30min),thenneutralized, subjectedtoSPEandinjected

LC-UV/Vis TheBZ3totalcontent

[39]

PBS AM PA

Topicalsunscreenapplicationon humanvolunteers

UrinesampleisﬁlteredandpHadjusted(5–7).Then,itis subjectedtoon-lineSPE

FL Standardadditioncalibration R=78–109%

SLD=12±5ngmL−1

NeitherphaseInorphaseII metabolitesareconsidered

[40]

TDS PA Topicalsunscreenapplicationon humanvolunteers

[41]

BZ3 AM KP

Urinesampleistreatedwith␤-glucuronidase(pH6.8, 37◦_C,₂₄_h)._Then,_it_is_mixed_with_MeCN_and_centrifuged.

ThesupernatantisevaporatedandredissolvedinMeOH, andﬁnallyinjected

LC-UV/Vis Externalcalibration R=90-95%

TheBZ3totalcontent

(free+conjugated)isdetermined. PhaseImetabolites(DHBandTHB) aredetectedbutnotdetermined

[42]

BZ3 EMC MBC

PA Topicalsunscreenapplicationon humanvolunteers

[43]

BZ3 AM BM

Urinecomingfromunknownhuman donors

Urineistreatedwith␤-glucuronidase/sulfataseorbuffer (pH5.0,37◦_C,₁₂_h)_for_enzymatic_hydrolysis_or_not._Then,

formicacidisaddedandcentrifuged,andthesupernatant submittedtoon-lineSPE-LC

LC-MS/MS (APCI−₎

Externalcalibration R=97-105% SLD=0.3-0.5ngmL−1

FreeandconjugatedformsofBZ3 aredetermined

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A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 19 BZ3 AM KP

Topicalsunscreenapplicationon piglets.Aftereuthanasia,urineis collectedfromthebladder

UrineismixedwithMeOH:MeCN,centrifugedandinjected LC-UV/Vis Matrix-matchedcalibration R=83-99%

SLD=50-70ngmL−1

PhaseImetabolites(DHB,DHMB andTHB)arealsoconsidered.No phaseIIconjugatedformsare considered

[48,49]

PDT AM PA KP

UrinesampleisﬁlteredandpHadjusted(4-7).Then,itis subjectedtoon-lineSPE

FL Standardadditioncalibration R=94-110%

SLD=10±4ngmL−1

[50]

MBC BP KP

Oraladministration(incornoil)onrats UrineismixedwithMeOH:water,centrifugedandinjected inLC-MS/MS

FaecesarelyophilizedandextractedbySoxhletextraction withMeOH

LCfractionsareisolated,evaporatedandredissolvedin D2OforNMR

LC-UV/Vis LC-MS/MS (APPI+_/ESI−₎ 1_H-NMR

Matrix-matchedcalibration PhaseImetabolites(CBCand CBC-OH)andphaseIIconjugated formsaredetermined

[30]

MBC BP PA KP

Urinesampleistreatedwith␤-glucuronidase(pH4.7, 37◦_C,₁₈_h),_then_mixed_with_cold_MeOH,_centrifuged_and

injected

LC-UV/Vis LC-MS/MS (APPI+_/ESI−₎

Matrix-matchedcalibration Total(free+conjugated)contentof MBC,CBCandCBC-OHare determined

[31]

BZ3 AM KP

Urinesample(pH6.5)iscentrifugedandsubjectedtoSPE. Theextractisdried,redissolvedinMeOHandinjected.For conjugatedcontent,itistreatedwith␤-glucuronidase (37◦C,16h)beforeSPE

LC-UV/Vis Externalcalibration Bothfreeandconjugatedcontent ofBZ3andDHBisdetermined

[51,52]

BZ3 AM KP

UrineisﬁlteredandpHadjusted(2.0),thenextractedby SDME(25min)andinjected

LC-UV/Vis Standardadditioncalibration R=80-93%

SLD=1.3ngmL−1

[53]

PABA AM PA KP

Urineiscentrifuged,mixedwithEtOH,andsubjectedto LLE.Theethylacetateextractisdried,reconstitutedwith MeOH:waterandsubjectedtoSPE.TheCH2Cl2extractis

dried,redissolvedinMeOHandinjected

LC-EC Externalcalibration R=96-99%

PhaseIImetabolites(PAH,PAcB andPAcH)aredetermined

[25]

P25 AM PA KP

UrineisﬁlteredandsubjectedtoSPE.TheDMFextractis injected

LC-FL Externalcalibration R=91-100% SLD=2.6ngmL−1

[54]

BZ3 EMC MBC

KP Topicalsunscreenapplicationon humanvolunteers

Urineislyophilized,suspendedinMeOH:water, centrifugedandinjected

LC−UV/VIS NeitherphaseInorphaseII metabolitesareconsidered

[55]

BZ3 AM Urinecomingfromsunscreenusers Urineistreatedwith␤-glucuronidase/sulfatase(pH6.8, 37◦C,3h).Then,waterisadded,subjectedtoSBSE (60min)andthermallydesorbedtoGC

GC-MS (EI+₎

Externalcalibration R=100-101% SLD=0.1ngmL−1

TheBZ3totalcontent

[56]

EDP AM PA

UrinesampleispHadjusted(7.2),extractedbydynamic MALLE(2h)andinjected

GC-MS (EI+₎

Standardadditioncalibration SLD=0.3ngmL−1

[57]

EDP AM PA

UrinesampleispHadjusted(7.2)extractedbyinvial MALLMEwithorbitalagitation(3h)andinjected

GC-MS (EI+₎

Standardadditioncalibration SLD=0.03ngmL−1

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20 A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 Table1(Continued) UV ﬁltera

techniqued

Commentsa _Ref.

BZ3 AM Urinecomingfromsunscreenusers Urineistreatedwith␤-glucuronidase/sulfatase(pH6.8, 37◦C,3h),andthensubjectedtodirectimmersion HF-LPME(15min)andinjected

GC-MS (EI+₎

Externalcalibration R=99%

SLD=0.005ngmL−1

TheBZ3totalcontent

[59]

PABA AM KP

Urineiscentrifugedandinjected LC-FL Externalcalibration R=99-101%

PhaseIImetabolites(PAH,PAcB andPAcH)aredetermined

[26]

BZ3 AM BM

Urinecomingfromunknownhuman donors

Urineistreatedwith␤-glucuronidase/sulfatase(37◦C, 12h),andthensubjectedtoLLE.Theorganicextractis dried,redissolvedinMeOH,centrifugedandinjected

LC-MS/MS (APCI−)

Externalcalibration R=85-99%

SLD=0.08-0.28ngmL−1

TheBZ3totalcontent

(free+conjugated)isdetermined. DHB(phaseImetabolite)isalso considered [60,61] BZ3 BZ4 AM KP

UrineisﬁlteredandpHadjusted(6.0).Then,itissubjected toon-lineSPEandtransferredtoLC

LC-UV/Vis Standardadditioncalibration R=92-109%

SLD=30-60ngmL−1

[62]

BZ3 AM Topicalsunscreenapplicationon humanvolunteers

Urineisﬁlteredandtreatedwith

␤-glucuronidase/sulfataseorwater(pH5-6,37◦C,12h)for enzymatichydrolysisornot.Then,formicacidisadded (pH3)andsubjectedtoSPE.Theacetoneextractisdried, reconstitutedwithMeOH:waterandinjected

LC-MS/MS (ESI+₎

Standardadditioncalibration R=98-115%

SLD=0.03-0.10ngmL−1

Bothfreeandconjugatedcontent ofBZ3andphaseImetabolites (DHB,DHMBandTHB)are considered

[63]

EDP BP AM

␤-glucuronidase/sulfataseorwater(pH5,37◦_C,₅₀_min

withultrasounds)forenzymatichydrolysisornot.Then,it issubjectedtoon-lineSPEandtransferredtoLC

LC-MS/MS (ESI+₎

Bothfreeandconjugatedcontent ofEDPandphaseImetabolites (DMPandMMP)areconsidered

[33]

MBC AM Topicalsunscreenapplicationon humanvolunteers

␤-glucuronidase/sulfataseorwater(pH5-6,37◦_C,₁₂_h)_for

enzymatichydrolysisornot.Then,formicacidisadded (pH3)andsubjectedtoSPE.Theacetoneextractisdried, reconstitutedwithwater:MeOH:MeCNandinjected

LC-MS/MS (ESI+_/ESI−₎

SLD=6±2ngmL−1

Bothfreeandconjugatedcontent ofMBCanditsphaseImetabolite CBCareconsidered

[64]

a_See_Fig.₁_for_key_{abbreviation.}_PAH₌_{p-aminohippuric}_acid;_PAcB₌_{p-acetamidobenzoic}_acid;_PAcH₌_{p-acetamidohippuric}_acid;_DHB₌_{2,4-dihydroxybenzophenone;}_DHMB₌_2,2_{-dihydroxy-4-methoxybenzophenone;}

THB=2,3,4-trihydroxybenzophenone;CBC=3-(4-carboxybenzylidene)camphor;CBC-OH=3-(4-carboxybenzylidene)hydroxycamphor;DMP=N,N-dimethyl-p-aminobenzoicacid;MMP=N-monomethyl-p-aminobenzoicacid.

b _BP₌_elucidation_of_the_{biotransformation}_pattern,_AM₌_development_of_the_analytical_method;_PA₌_showing_evidences_of_the_percutaneous_absorption;_KP₌_study_of_kinetic_proﬁles;_BM₌_{biomonitoring.}

c _APCI₌_atmosphere_pressure_chemical_ionization;_APPI₌_atmospheric_pressure_{photoionization;}_DMF₌_{dimethylformamide;}_EI₌_electronic_impact;_ESI₌_electrospray_ionization;_LLE₌_{liquid-liquid}_extraction;_HF-LPME₌

hollow-ﬁberliquid-phasemicroextraction;MALLE=membraneassistedliquid-liquidextraction;MALLME=membraneassistedliquid-liquidmicroextraction;MeCN=acetonitrile;MeOH=methanol;SBSE=stir-barsorptiveextraction; SDME=single-dropmicroextraction;SPE=solid-phaseextraction;SPME=solid-phasemicroextraction.

d _EC₌_{electrochemical}_detector;_FL₌_{ﬂuorescence;}_GC₌_gas_{chromatography;}1_H-NMR₌_proton_nuclear_magnetic_resonance;_LC₌_liquid_{chromatography;}_MS₌_mass _{spectrometry;}_MS/MS₌_tandem_mass_{spectrometry;}

UV/Vis=ultraviolet/visiblespectrometry.

(11)

stir-bar sorptive extraction (SBSE) [56], single-drop

microextraction (SDME) [53], hollow-ﬁber liquid-phase

microextraction (HF-LPME) [59] and in vial membrane

assisted liquid–liquid microextraction (MALLME) [58] have

been proposed for the determination of UV ﬁlters in urine.

Finally, lyophilization and subsequent redissolution in a

lit-tle amount of solvent has also been considered with urine

samples[55].

In the case of faeces, theyhave also been subjected to LLE

[28,29,36],ortoSoxhletextraction[30].

Regardingsampletreatmentinthecaseofplasmaorserum

anal-ysis,bloodhastobetreatedinordertoisolatetheplasmaorserum. Thus,incaseofplasma,itisnecessarytoremovethecellular com-ponentsoftheblood,suchasredandwhitecorpuscles,butavoiding theprecipitationoftheproteinsthatareinvolvedinthecoagulation process(i.e.,ﬁbrinogenandﬁbrin).Thisisachievedbycentrifuging bloodinheparinizedtubes.Inthecaseofserum,bloodisnormally

centrifugedinnon-heparinizedtubes,whichallowstheremoving

ofboththebloodcellularcomponentsandﬁbrinogenandﬁbrin.

Oncetheplasmaorserumisobtained,ahydrolysisstepisneeded

inorder todeterminetheconjugatecontent, eitherwithacidic

[28,29,35,36,67]orenzymatic[68]treatment,asmentionedabove

forurine.Moreover,itshouldbeemphasized,thattherecouldbe

targetcompoundsboundedtoproteins,whichcouldbereleasedby

acidichydrolysis[28].Ontheotherhand,despitefewexceptions,it

isverycommontoprecipitatetheproteinswhenanalyzingplasma

andserum,inordertoreducematrixinterferences.Thisisachieved

bymixingplasmaorserumwithorganicsolvents,suchas

acetoni-trile[42,55,65,67],acetonitrile/methanolmixtures[48,49,69],cold

methanolfollowedbycoldacetonitrile[30,31],mineralacidssuch

ashydrochloricacid[28,29,35],ororganicacidslikeformic acid

[68],thusachievingproteindenaturationandprecipitation,which arethenseparatedbycentrifugation.Insomecases,puriﬁcationis alsocarriedoutbyLLE[28,29,36,66]andbymanual[69]oron-line

[68]SPE.

Likeintheanalysisofplasmaorserum,theanalysisofbreast

milk,semenandtissuesalsorequirestheseparationofproteins.

The additionof isopropanol [71] or methanol[72] followed by

on-lineSPE,ortheuseofgelpermeationchromatography(GPC)

[70] has been reported in the case of breast milk. For semen,

theacidiﬁcationwithhydrochloricacid[63] orphosphoric acid

[73]facilitatesproteinprecipitation,and afterwardsSPEisused

toimproveclean-upandtoachieveaconcentration[63,73].Inthe

caseoftissues,theyarehomogenizedwithwaterandsubjected

toLLE[28,29,36,66,74,75]orhomogenizedinacetonitrileand sub-jectedtoSPE[69].Ontheotherhand,asabove-mentionedforurine, plasmaorserum,alsoincaseoftheanalysisofbreastmilk,semen ortissues,itismandatorytocarryoutahydrolysisstepifthetotal

amountofthetargetUVﬁltersisrequired.Enzymatichydrolysis

hasbeenusedincaseofbreastmilk[71,72]andsemen[63,73],

whereasbothacidicandenzymatichydrolysishavebeenusedin

tissues[28,29,36]. 3.3. Analyticaltechniques

Despite an exhaustive sample treatment is performed to

eliminatepotentialinterfering compoundsfromthematrix,the

complexityofthesampleencouragestoselectaproperanalytical

separationtechniquetoenhancethedeterminationofthetarget

compounds,sincesomeinterferingcompoundscanstillbepresent

inthematrix.Therefore,separationtechniquesarerequiredtocarry

outtheselectivedetermination ofUV ﬁlters,and theirpossible

metabolites,inbiologicalmatrices.

Ascan beseen inTables 1–3,LC is thetechnique ofchoice

todetermineUVﬁltersinallthestudiedbiologicalmatrices.The

mostusualdetectorcoupledtoLCisbasedontheultraviolet/visible

spectroscopy(UV/vis),sincewithonlyafewexceptions,UVﬁlters

do notexhibitneitherﬂuorescencenorelectrochemical

proper-ties,buttheydoexhibitahighabsorbanceintheUVrangeofthe

electromagneticspectrum.Thus,LCwithﬂuorimetric(FL)

detec-torwasjustusedforsensitiveandselectivedeterminationofP25

[54]andPABA[26]inurine,andalsoinjustonereport,LCwith

electrochemical(EC)detectorwasemployedforthesensitiveand

selectivedeterminationofPABAinthissamematrix[25].

Neverthe-less,whenhighselectivityandsensitivityarerequiredtoanalyze

thesecomplexmatrices, LCcoupledtomassspectrometry(MS)

detectorsintandem(MS/MS)isthebestoption.Inparticular,BZ3

hasbeendeterminedinhumanurine[44–47,60,61,63],serum[68],

breastmilk[71,72],semen[63]andplacentaltissue[74,75];3BCin

ratplasmaandtissues[66];MBCinhuman[31,64]andrat[30]

urine;andEDPinhumanurine[33]andsemen[73]byLC–MS/MS

employingdifferentionizationsources.InthecaseofBZ3,

atmo-sphericpressurechemicalionization(APCI)isthemostcommonly

used[44–47,60,61,71,74,75],althoughatmosphericpressure

pho-toionization(APPI)[68,72]andelectrosprayionization(ESI)[63]

have alsobeen usedin some cases.Regarding APCI, it is usual

toemploynegativemode(APCI−),butVela-Soriaandco-workers

[74]reportedthatabettersensitivityisobtainedinthepositive mode(APCI+_)._Ye_and_co-workers_[68]_also_stated_that_APPI−_slightly

improvedBZ3ionizationwithrespecttoAPCI−.However,intheESI mode,Leónetal.statedthatESI+_has_better_efﬁciency_than_ESI−_[63]_.

Incaseof3BC,ESI+_is_used_in_the_only_article_published_so_far_[66]_.

WithregardtoMBC,APPI+_[30,31]_and_ESI+_[64]_have_been_used

fortheparentcompound,butESI−ispreferredforitsmetabolites

[30,31,64].Finally,incaseofEDP,ESI+_is_used_for_its_better

efﬁ-ciencywithrespecttoESI−[33,73].Inallthesecases,determination

wasconductedbymultiplereactionmonitoring(MRM)modeofthe

mostintensetransition,butusinganotheroneforconﬁrmation.

UV/visspectrometrywithoutpreviouschromatographic

sepa-rationhasjustbeenusedinonecase,allowingthedetermination

ofPABAinaselectiveway,withtheuseofapreviousderivatization

withBratton–Marshallreaction[24].FLhasalsobeenused

with-out previouschromatographicseparation, but includingon-line

SPEbeforethemeasurement[40,50].Foridentiﬁcationpurposes,

nuclearmagneticresonance(NMR)hasalsobeenemployed,after

isolatingsamplefractionsbyLC[30].

Gaschromatography(GC)hasbeenusedlessfrequently,butin

anycasecoupledtoMS.Inparticular,GC–MSallowedthe

determi-nationofBZ3andEDPinurine[38,56–59],BZ3inplasma[67]and

BZ3andEMCinbreastmilk[70].Inallcases,ionizationwas

con-ductedbypositiveelectronicimpact(EI+_),_and_the_measurement

wasperformedbyselectedionmonitoring(SIM)modealthough

employingoneortwootherionsasqualiﬁers.

Finally,despitetheexhaustivesampletreatmentandtheuseof

selectiveanalyticaltechniques,resultsaresometimesaffectedby

thepresenceoftheso-called‘matrixeffect’.Thiseffectcausesthat

noquantitativerecoveriesare obtainedwhenemploying

exter-nalcalibration.Thiscouldbeduetoadifferenceinthebehavior

ofthetargetcompoundwiththepresenceofmatrixcompounds

that can enhance or mitigate the signal (i.e., quenching

phe-nomenonin FL, ionsuppressionin MS)or affecttheextraction

efﬁciencywhen anextractiontechnique isemployed.This

phe-nomenonhasbeen observedand reported bydifferent authors

in the determination of UV ﬁlters (see Tables 1–3) in urine

[30,31,33,38,40,48–50,53,57,58,62–64], plasma [30,31,48,49,67], semen[63,73],adiposetissue[66]andplacentaltissue[74,75].The

correctionofthisdeleteriouseffecthasbeensometimesachieved

by matrix-matched calibration, i.e.,theuse ofthesame matrix

(but free of analytes) toprepare thestandard calibration

solu-tions, such as in urine analysis [30,31,48,49], plasma analysis

[30,31,48,49,67] and placental tissue analysis [74,75]. In other

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22 A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 Table2

PublishedpapersonUVﬁltersdeterminationinplasmaandserum(chronologicalorder).

UV ﬁltera

techniqued

Commentsa _Ref.

BZ3 AM Oraladministration(incornoil)onrats AcidichydrolysiswithHCl LC-UV/Vis BothfreeandconjugatedformsofBZ3 aredetermined.PhaseImetabolitesare notconsidered

[35]

BZ3 BP KP

Oral(incornoil)antopical(in petrolatumjelly)administrationonrats

PlasmaissubjectedtoLLE.Theextractisevaporated, redissolvedinMeOHandinjected

Forboundedcompounds,acidichydrolysiswith6MHClis performedbeforeLLE

LC-UV/Vis PhaseImetabolites(DHB,DHMBand THB)andphaseIIconjugatedformsare alsoconsidered [28,29,36] BDM BZ3 EDP EMC ES

AM Spikedplasma PlasmaismixedwithMeCN,centrifugedandinjected LC-UV/Vis Externalcalibration R=89-10/%

NeitherphaseInorphaseIImetabolites areconsidered [65] BZ3 EMC ES HS

AM Topicalsunscreenapplicationson humanvolunteers

PlasmaispHadjusted(7.4)andthenmixedwithMeCN, centrifugedandinjected

LC-UV/Vis Externalcalibration R=91-104%

NeitherphaseInorphaseIImetabolites areconsidered

[42]

BZ3 EMC MBC

PA Topicalsunscreenapplicationson humanvolunteers

Plasmaisisolatedfrombloodandanalyzed NeitherphaseInorphaseIImetabolites areconsidered

[43]

BZ3 AM KP

Topicalsunscreenapplicationson piglets

PlasmaismixedwithMeOH:MeCN,centrifugedandinjected LC-UV/Vis Matrix-matched calibration R=79-99% SLD=50-70ngmL−1

BZ3phaseImetabolites(DHB,DHMB andTHB)arealsoconsidered.Nophase IIconjugatedformsareconsidered

[48,49]

3BC AM PA

Topicalalcoholicsolutionapplicationon rats

PlasmaissubjectedtoLLEwithheptane,dried,redissolved inMeOH:waterandinjected

LC-MS/MS (ESI+₎

Externalcalibration R=87-96% SLD=12.5ngmL−1

NeitherphaseInorphaseIImetabolites areconsidered

[66]

MBC BP PA KP

Oraladministration(incornoil)onrats Topicalsunscreenapplicationonhuman volunteersandrats

PlasmaismixedwithcoldMeOH,centrifuged,mixedwith coldMeCN,centrifugedagainandtheninjectedinto LC-MS/MS

LCfractionsareisolated,evaporatedanddissolvedinD2Ofor

NMR LC-UV/Vis LC-MS/MS (APPI+/_ESI−₎ 1H-NMR Matrix-matched calibration

PhaseImetabolites(CBCandCBC-OH) andphaseIIconjugatedformsare considered

[30,31]

BZ3 EMC MBC

KP Topicalsunscreenapplicationonhuman volunteers

PlasmaismixedwithMeCN,centrifugedandinjected LC− UV/VIS NeitherphaseInorphaseIImetabolites areconsidered

[55]

BZ3 BP AM KP

Oraladministration(incornoil)onrats PlasmaismixedwithMeCNandcentrifuged.The

supernatantishydrolyzed(6MHCl,100◦_C,₁_h),_pH_adjusted

(8.5)andsubjectedtoLLE.Theextractisevaporatedand derivatizedwithMSTFA

GC-MS (EI+₎ Matrix-matched calibration R=86-104% SLD=10ngmL−1

Total(free+boundedform)contentsof BZ3anditsphaseImetabolites(DHB, DHMB,THB)isconsidered

[67]

BZ3 AM Serumsamplescomingfromunknown humandonors

Serumismixedwithformicacid,centrifugedand transferredtoon-lineSPEandLC.Forboundedcompounds, serumistreatedwith␤-glucuronidase/sulfatase(pH5,37◦C, 4h)before LC-MS/MS (APPI−) Externalcalibration R=87-106% SLD=0.5ngmL−1

BothfreeandconjugatedformsofBZ3 aredetermined.NophaseImetabolites areconsidered

[68]

BZ3 KP Topicalalcoholicsolutionapplicationon rats

PlasmaismixedwithMeOH:MeCN,centrifuged,and subjectedtoSPE.TheMeOHextractisinjected

LC-UV/Vis NeitherphaseInorphaseIImetabolites areconsidered

[69]

a_See_Fig.₁ _for_key _{abbreviation.} _DHB₌_{2,4-dihydroxybenzophenone;} _DHMB₌_2,2_{-dihydroxy-4-methoxybenzophenone;}_THB₌_{2,3,4-trihydroxybenzophenone;} _CBC₌_{3-(4-carboxybenzylidene)camphor;}_CBC-OH₌

3-(4-carboxybenzylidene)hydroxycamphor.

b _BP₌_elucidation_of_the_{biotransformation}_pattern,_AM₌_development_of_the_analytical_method;_PA₌_showing_evidences_of_the_percutaneous_absorption;_KP₌_study_of_kinetic_proﬁles;_BM₌_{biomonitoring.}

c _APPI₌_atmospheric_pressure_{photoionization;}_EI₌_electronic_impact;_ESI₌_electrospray_{ionization;LLE}₌_{liquid-liquid}_extraction;_MeCN₌_{acetonitrile;}_MeOH₌_methanol;_MSTFA₌_{N-methy-N-(trimethylsilyl)}_{triﬂuoroacetamide.} d _GC₌_gas_{chromatography;}1_H-NMR₌_proton_nuclear_magnetic_resonance;_LC₌_liquid_{chromatography;}_MS₌_mass_{spectrometry;}_MS/MS₌_tandem_mass_{spectrometry;}_UV/Vis₌_{ultraviolet/visible}_{spectrometry.}

(13)

A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 23 Table3

PublishedpapersonUVﬁltersdeterminationinotherbiologicalmatrices(chronologicalorder).

UV ﬁltera

techniqued

Commentsa _Ref.

BZ3 BP Oral(incornoil)andtopical(in petrolatumjelly)administrationon rats.Aftereuthanasia,liver,kidney, intestine,testes,skin,spleenandheart arecollected

Tissueiswashed,mechanicallyhomogenizedwithwater,and thensubjectedtoLLE.Theextractisevaporated,redissolvedin MeOHandinjected

Forboundedcompounds,atreatmentwith␤-glucuronidase (pH6,37◦_C,₂₄_h)_or_with₆_M_HCl_is_performed_before_LLE

LC-UV/Vis PhaseImetabolites(DHB,DHMB andTHB)aredetermined.PhaseII conjugatedformsarealso considered

[28,29,36]

BZ3 EMC

PA Humanbreastmilkfromunknown femaledonors

Milkiscleaned-upbyGPC,andtheextractisinjected GC-MS (EI+₎

R=87-116% NeitherphaseInorphaseII metabolitesareconsidered

[70]

3BC AM PA

Topicalalcoholicsolutionapplication onrats.Aftereuthanasiaadipose tissue,brain,liver,muscleandtestes arecollected

TissueismixedandmortaredwithanhydrousNa2SO4,and

extractedwithMeOH(adipose)orheptane(rest),dried, redissolvedinMeOH:water,centrifugedandinjected.

LC-MS/MS (ESI+₎ Externalcalibration R=78-107%,exceptfor adiposetissue(40%) SLD=5ngg−1

[66]

BZ3 AM Humanbreastmilkfromunknown femaledonors

Milkismixedwithi-PrOH,centrifuged,mixedwithformicacid andtransferredtoon-lineSPE-LC

Forboundedcompounds,milkistreatedwith ␤-glucuronidase/sulfatase(37◦_C,₁₂_h)_before

LC-MS/MS

(APCI−₎ _RExternal₌_95-112%calibration

SLD=0.5ngmL−1

Bothfreeandconjugatedformsof BZ3aredetermined.NophaseI metabolitesareconsidered

[71]

BZ3 AM Humanbreastmilkfromunknown femaledonors

MilkismixedwithMeOH,centrifuged,mixedwithformicacid andtransferredtoon-lineSPE-LC

Forboundedcompounds,milkistreatedwith ␤-glucuronidase/sulfatase(pH5,37◦_C,₄_h)_before LC-MS/MS (APPI−₎ Externalcalibration R=98-107% SLD=0.4ngmL−1

Bothfreeandconjugatedformsof BZ3aredetermined.NophaseI metabolitesareconsidered

[72]

BZ3 AM Topicalsunscreenapplicationon humanvolunteers

Semenismixedwith1MHClandcentrifuged.Thesupernatant istreatedwith␤-glucuronidase/sulfatase(pH6.5,37◦_C,₁₂_h)

orwaterforenzymatichydrolysisornot.Then,formicacidis addedandsubjectedtoSPE.Theextractisdried,redissolvedin MeOH:waterandinjected

LC-MS/MS (ESI+₎ Standardaddition calibration R=86-111% SLD=1-3ngmL−1

Thefreeandconjugatedformsof bothBZ3andphaseImetabolites (DHB,DHMBandTHB)are considered

[63]

BZ3 AM Topicalhydroethanolicsolution applicationonrats.Afthereuthanasia, liverandbrainarecollected

TissueismechanicallyhomogenizedwithMeCN,subjectedto SPEandtheMeOHextractinjected

LC-UV/Vis NeitherphaseInorphaseII metabolitesareconsidered

[69]

EDP AM Topicalsunscreenapplicationon humanvolunteers

Semenismixedwith1MH3PO4andcentrifuged.The

supernatantistreatedwith␤-glucuronidase/sulfataseor water(pH5,37◦C,50minwithultrasounds)forenzymatic hydrolysisornot.Then,itispHadjusted(3.0)andsubjectedto on-lineSPE-LC LC-MS/MS (ESI+₎ Standardaddition calibration R=95-109%

Total(free+conjugated)contentof bothEDPandphaseImetabolites (DMPandMMP)isconsidered

[73]

BZ3 AM BM

Humanplacentalcomingfrom unknownfemaledonors,collectedat thetimeofdelivery

Tissueismechanicallyhomogenizedwithwaterandsubjected toLLE.Aftercentrifugation,thesupernatantisevaporatedand redissolvedinMeOH:ammoniabuffer.Then,itiscentrifuged andinjected LC-MS/MS (APCI+₎ Matrix-matched calibration R=98-104% SLD=0.07.0.3ngg−1

PhaseImetabolites(DHBand DHMB)arealsoconsidered.Phase IIglucuronideformsarenot considered

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24 A.Chisvertetal./AnalyticaChimicaActa752 (2012) 11–29 Table 3 (Continued ) UV ﬁlter a Aim b Experiment Sample preparation c Analytical technique d Analytical performance e Comments a Ref. BZ3 AM BM Human placental coming from unknown female donors, collected at the time of delivery The placental tissue is mechanically homogenized with water and subjected to LLE. After centrifugation, the supernatant is evaporated and redissolved in MeOH:ammonia buffer. Then, it is centrifuged and injected LC-MS/MS (APCI −) Matrix-matched calibration R= 99-104% SLD = 0.1-.0.2 ng g − 1 Phase I metabolites (DHB and DHMB) are also considered. Phase II glucuronide forms are not considered [75] a See Fig. 1 for key abbreviation. DHB = 2,4-dihydroxybenzophenone; DHMB = 2,2 -dihydroxy-4-methoxybenzophenone; THB = 2,3,4-trihydroxybenzophenone; DMP = N,N-dimethyl-p-aminobenzoic acid; MMP = N-monomethyl-p-aminobenzoic acid. b BP = elucidation of the biotransformation pattern, AM = development of the analytical method; PA = showing evidences of the percutaneous absorption; KP = study of kinetic proﬁles; BM = biomonitoring. c APCI = atmosphere pressure chemical ionization; APPI = atmospheric pressure photoionization; EI = electronic impact; ESI = electrospray ionization; LLE = liquid-liquid extraction; MeCN = acetonitrile; MeOH = methanol; SPE = solid-phase extraction. d GC = gas chromatography; GPC = gel permeation chromatography; LC = liquid chromatography; MS = mass spectrometry; MS/MS = tandem mass spectrometry; UV/Vis = ultraviolet/visible spectrometry. e R = recovery; SLD = limit of detection in the sample.

becauseit wassample-dependent,and thus thestandard

addi-tioncalibrationmethod[77] wasemployed,asit isthecase of

urine[33,38,40,50,53,57,58,62–64],semen[63,73]andadipose tis-sue[66].Otherstrategies,astheuseofisotopicinternalstandards

ofthetargetcompoundsarebarelyemployed[68,72]because

stan-dardsarenotavailable. 3.4. Accuracyandsensitivity

As can be seen in Tables 1–3, there are not many articles

reportinginformationregardingrecoverystudies.Theanalytical

methodsdescribedthereachieve,achieverecoveriesthatenable

accuratequantiﬁcationofthetargetUVﬁltersandtheirmetabolites

in the different matrices, either withexternal or with

matrix-matchedor withstandardaddition calibrationapproaches. It is

especiallyworthytoconsiderthehighdifﬁcultyoftheconsidered

matrices.

Regardingsensitivity,exceptafew exceptions,thepublished

methods(seeTables1–3)allowsthedeterminationsofUVﬁlters

andtheirmetabolitesinthelowngmL−1range,whichallows deter-miningtheminbiologicalﬂuidsandtissues[7](asitwillbeshown inTable4).Ononehand,andassaidbefore,theuseofsensitive

ana-lyticaltechniques,suchasFLandMS,andontheotherhand,and

aswasabove-mentioned,theuseofenrichmenttechniques,such

asLLE,SPE,MALLE,SPME,SBSE,SDME,HF-LPME,MALLMEallow

reachingthelowlevelsinwhichtheyareencountered.

4. OccurrencelevelsoforganicUVﬁltersinbiologicalﬂuids andtissues

ThecontentoforganicUVﬁltersinbiologicalﬂuidsandtissues isamatterofcontroversy,sinceitdependsontheexpositionofthe

subjecttothetargetUVﬁlter,whichisveryvariable,and

more-overitisuncontrollableevenundernormalconditionsofuse,due

tothemotivesexposedbelow.Thus,inordertoreachthe

protec-tionlevelindicatedbythe‘SunProtectionFactor’markedinthe

label,sunscreenproductshavetobeappliedinsimilarquantities

totheonesusedfortesting,i.e.2mgcm−2ofskin[78]butpeople

unconsciouslytendtoapplyaloweramount.Thisisdueto

vari-ousreasons;ononehand,thereisalackofinformationaboutthe

recommendedamount(itisnotusuallyindicatedinthelabel),and

ontheotherhand,ifthisdatawasknow,takingintoaccountthe

bodyof anaverageadultpersonas amodel,therecommended

amountwouldequateto6teaspoonsofsunscreencosmetic(ca.

36g),thatisusuallyquiteahigheramountthanthecommondose

peopleapplythemselves.Moreover,theindividualapplicationdose

ofthetargetcompound(i.e.,anygivenorganicUVﬁlter)is

com-pletelyunknown,asitobviouslydependsontheconcentrationof

theUVﬁltercontainedinthesunscreencosmetic,thenumberof

dailysunscreenapplications(i.e.,singleversusrepeated)andthe

assiduityinwhichthesunscreencosmeticisapplied(i.e.,summer

versuswinterseasons).Apartfromthis,andasstatedbefore,the

percutaneousabsorptionmaysigniﬁcantlydifferdependingonthe

natureofthecosmeticvehicle,butalsoonthephysiologicalaspects

ofeach subject.Asanexample ofallthis, itis worthyto

men-tionthebiomonitoringstudycarriedoutbyCalafatandco-workers

[47],whodeterminedthetotalBZ3contentin2517urinesamples

collectedbetween2000and2004.Theirﬁndingsrevealedagreat

intersubjectvariability(rangingfrom0.4to21700ngmL−1,witha meanvalueof22.9ngmL−1).Moreover,theseauthorsfound

differ-encesbysexandrace,whichwasattributedtodifferencesinthe

useofpersonalcareproductscontainingBZ3.Later,Kunisueand

co-workers[61]determinedtotalBZ3inurinefrom625women,

and found values ranging from undetectable (<0.28ngmL−1)

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A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 25 Table4

OccurrencelevelsofUVﬁltersinhumanbiologicalﬂuidsandtissues(chronologicalorder).

UV ﬁltera

Numberof subjectsb

Exposuredataa _Individual_levels_founda,c _Remarksa,c _Ref.

Urine

PABA 6(M) Singleapplication(20g)overthewholebodyofahome-made sunscreencontaining5%PABA

Notreported Greatintersubjectvariability

PABAtotalamountexcretedfor48hwas15.8-96.3mg,which correspondsto1.6-9.6%oftheapplieddose

PAcBisthemajormetabolite

[24]

BZ3 9 Singleapplication(13g)intheforearms(1051cm2₎_(ca.

12.4mg/cm2₎_of_a_commercial_sunscreen_containing_6%_BZ3_for

12h

Notreported Between1-2%ofthetotalappliedamountwasexcretedinformof BZ3tovera10-hperiodaftertopicalapplication

[37]

BZ3 1 Singleapplication(8mL)ofacommercialsunscreenwith non-speciﬁedBZ3content

BZ3f=260ngmL−1at4h [38]

BZ3 11(7M/4F) Singleapplication(40g)overthewholebody(2m2₎_(ca.

2mg/cm2₎_of_a_commercial_sunscreen_containing_4%_BZ3_for₁₂_h

Notreported BZ3taveragetotalamountexcretedfor48hwas11mg,whichisca.

0.4%oftheapplieddose Greatintersubjectvariation

[39]

PBS 3(2M/1F) Singleapplication(10mL)ofahome-madesunscreencontaining 5%PBS

PBSf=18–141ngmL−1 PBSftotalamountexcretedfor12hwas11.2-32.3␮g,which

comprises0.0022-0.0065%oftheappliedamount Greatintersubjectvariation

[40]

BZ3 3(F) Singleapplication(1.7g)tothearmsandback(860cm2₎_(ca.

2mg/cm2₎_of_a_commercial_sunscreen_containing_6%_BZ3_for₈_h

Notreported 1%ofthetotalappliedamountwasexcretedinformofBZ3t,DHBt

(majormetabolite),DHMBtandTHBt(minormetabolite)

[42]

BZ3 EMC MBC

32(15M/17F) Repeatedapplications(40gperdayduring7days)overthe wholebody(2m2₎_(ca.₂_mg/cm2₎_of_a_sunscreen_containing_10%

ofBZ3,MBCandEMC

BZ3fmax.valuesof

140ngmL−1

EMCfmax.valuesof

8ngmL−1 MBCfmax.valuesof 7ngmL−1 [43] BZ3 30 Unknown BZ3t=437ngmL−1(mean) [44] BZ3 30 Unknown BZ3f=<0.5–50.1ngmL−1 BZ3c=<0.5–2961ngmL−1 BZ3t=<0.5–3000ngmL−1

BZ3ismainlyexcretedasitsconjugatedform [45]

PDT 5 Singleapplication(10mL)overthewholebodyofahome-made sunscreencontaining5%PDT

Notreported PDTtaveragetotalamountexcretedwas77␮g,whichisca.0.015%of theapplieddose

Greatintersubjectvariation

[50]

MBC 6(3M/3F) Singleapplication(ca.2mg/cm2₎_of_a_home-made_sunscreen

containing4%MBC

MBCtnotfound

CBCtmax.valuesofca.

425ngmL−1

CBC-OHfmax.valuesofca.

1500ngmL−1

MBCnotfound,whereasCBCandCBC-OHwereexcretedmainlyas glucuronideandfreeforms,respectively

[31]

BZ3 90(F) Unknown BZ3t=<0.5–26700ngmL−1 Differencesbyseasonareobserved [46]

BZ3 2517

(1229M/1288F)

Unknown BZ3t=<0.5–21700ngmL−1 Differencesbysexandrace/ethnicityareobserved [47]

25(9M/16F) Repeatedapplications(2applications/dayx5days)overthe wholebody(ca.2mg/cm2₎_of_a_commercially_available

sunscreencontaining4%BZ3

Notreported Greatintersubjectvariabilityintheexcretionpattern BZ3andDHBextensivelyconjugated:

BZ3c=94.1%versusBZ3f=5.9%

DHBc=91.2%versusDHBf=8.8%

Between1.2-8.7%ofthetotalappliedamountwasexcretedinformof BZ3t

[51,52]

BZ3 1(M) Singleapplication(15g)ofahome-madesunscreencontaining 5%BZ3for12h

BZ3f=<1.3–48ngmL−1 BZ3ftotalamountexcretedfor12hwas42.6␮g,whichisbelow0.01%

oftheapplieddose

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26 A. Chisvert et al. / Analytica Chimica Acta 752 (2012) 11– 29 Table4(Continued) UV ﬁltera Numberof subjectsb

Exposuredataa _Individual_levels_founda,c _Remarksa,c _Ref.

PABA 6(3M/3F) Singleapplicationofahome-madesunscreencontaining5% PABA PAH=<2–1360ngmL−1 PAH PAcB=<2– 31200ngmL−1 PacB PacH=<2– 503ngmL−1 PacH

Differentexcretionpatterns [25]

P25 3(1M/2F) Singleapplication(ca.2mg/cm2₎_of_a_home-made_sunscreen

containing8%P25

P25f=<2.6– 210ngmL−1 P25ftotalamountexcretedwas80-300␮g,whichrepresents

0.011-0.019%oftheapplieddose

[54]

BZ3 EMC MBC

32(15M/17F) Repeatedapplications(40gperdayduring4days)overthe wholebody(2m2₎_(ca.₂_mg/cm2₎_of_a_sunscreen_containing_10%

ofBZ3,EMCandMBC

BZ3f=<4–519ngmL−1

EMCf=<4–207ngmL−1

MBCf=<3– 189ngmL−1

Greatintersubjectvariability [55]

BZ3 6 Nodata BZ3t=<0.1– 1.2ngmL−1 [56]

EDP 2 Singleapplication(20g)ofahome-madesunscreencontaining 8%EDP

EDPf=1.2– 16ngmL-1 _[57]

EDP 6 Singleapplication(7g)ofahome-madesunscreencontaining8% EDP

EDPf=0.4–5.8ngmL−1 [58]

BZ3 10 Nodata BZ3t=0.43–5.17ngmL−1 [59]

PABA 6(3M/3F) Singleapplicationofahome-madesunscreencontaining5% PABAfor16h

PAH=1.97–6.59␮gmL−1 PAcH=0.91–8.92␮gmL−1 PAcB=10.17– 88.75␮gmL−1

EssentialoilsincreasedPABApercutaneousabsorption [26]

BZ3 55(35M/20F) Unknown BZ3t=<0.28–330ngmL−1

DHBt=<0.082–250ngmL−1

DHMBt=<0.13ngmL−1

Differencesbylocationandbytheuseofsunscreenareobserved [60]

BZ3 BZ4

3(2M/1F) Singleapplication(12.5g)ofahome-madesunscreencontaining 8%BZ3and4%BZ4

Notreported Greatintersubjectvariability

BZ3fandBZ4ftotalamountsexcretedwere55-180␮gand

190-540␮g,whichare0.006-0.018%and0.04-0.1%,respectively,ofthe totalapplieddose

[62]

BZ3 1(M) Singleapplication(13g)ofahome-madesunscreencontaining 10%BZ3

Notreported BZ3andDHBarepredominant: BZ3f=0.4-1.5␮g;BZ3c=0.5-1.4␮g

DHBf=0.2-0.8␮g;DHBc=0.4-3.6␮g

DHMBf=0.004-0.010␮g;DHMBc=0.004-0.006␮g

THBf=0.001-0.059␮g;THBc=0.002-0.170␮g

[63]

EDP 4(2M/2F) Singleapplication(13g)ofahome-madesunscreencontaining 8%EDP

Notreported EDPfnotfound

DMPf=46␮g;DMPt=3231␮g

MMPfnotfound;MMPt=968␮g

Thetotalexcretedamountcorrespondsto0.4-0.5%oftheapplieddose

[33]

BZ3 625(F) Unknown BZ3t=<0.28– 5900ngmL−1

DHBt=<0.082– 3200ngmL−1

DHMBt=<0.13ngmL−1