Alves de Almeida, Eduardo ; Di Mascio, Paolo ; Harumi,

Tatsuo ; Moscovitch, Adam ; Hardeland, Rdiger ; Cardinali,

Daniel P. ; Brown, Gregory M. ; Pandi-Perumal, S.R.

Measurement of melatonin in body fluids : standards,

protocols, and procedures

Posprint Publicado en Child's Nervous System Vol.27, N 6, 2011

Este documento est disponible en la Biblioteca Digital de la Universidad Catlica Argentina, repositorio institucional
desarrollado por la Biblioteca Central San Benito Abad. Su objetivo es difundir y preservar la produccin intelectual
de la institucin.
La Biblioteca posee la autorizacin del autor para su divulgacin en lnea.

Cmo citar el documento:

Alves de Almeida, E, Di Mascio, P, Harumi, T, et al. Measurement of melatonin in body fluids : standards, protocols,
and procedures [en lnea]. Child's nervous system. 2011;27(6):879-891.
Disponible en: http://bibliotecadigital.uca.edu.ar/repositorio/investigacion/measurement-melatonin-body-fluidsstandards.pdf
(Se recomienda indicar fecha de consulta al final de la cita. Ej: [Fecha de consulta: 19 de agosto de 2010]).

Publicadoen:ChildsNervousSystem27(6):879891;2011

MeasurementofMelatonininBodyFluids:Standards,Protocols,andProcedures

EduardoAlvesdeAlmeida1,PaoloDiMascio2,TatsuoHarumi3,AdamMoscovitch4,Rdiger
Hardeland5,DanielP.Cardinali6,GregoryM.Brown7,S.R.PandiPerumal8,CA

DepartamentodeQumicaeCinciasAmbientais,IBILCE,UNESP,RuaCristvoColombo

2265,CEP15054000,SoJosdoRioPreto,SP.BRAZIL.

DepartamentodeBioqumica,InstitutodeQumica,USP.Av.Prof.LineuPrestes,748,CEP

05513970,SoPaulo,SP.BRAZIL.

DepartmentofAnatomy,AsahikawaMedicalCollege,JAPAN.

CanadianSleepInstitute,Toronto,ON,CANADA.

JohannFriedrichBlumenbachInstituteofZoologyandAnthropology,UniversityofGttingen,

Gttingen,GERMANY.

DepartamentodeDocenciaeInvestigacin,FacultaddeCienciasMdicas,Pontificia

UniversidadCatlicaArgentina,1107BuenosAires,ARGENTINA.

DepartmentofPsychiatry,UniversityofToronto,100BronteRd.Oakville,ON,L6L6L5,

CANADA.

SomnogenInc,8790112thStreet,NewYork,NY11418,USA.

CA

S.R.PandiPerumal

200LansdowneAvenue
Toronto,
ONM6K2V9
CANADA
sleepresearch@gmail.com
1

Abstract
Thecircadianrhythmofmelatonininsalivaorplasma,orofthemelatoninmetabolite6
sulphatoxymelatonininurine,isadefiningfeatureofsuprachiasmaticnucleusfunction,the
endogenousoscillatorypacemaker.Thesemeasurementsareusefultoevaluateproblems
relatedtotheonsetoroffsetofsleepandforassessingphasedelaysoradvancesofrhythmsin
entrainedindividuals.Additionally,theyhavebecomeanimportanttoolforpsychiatric
diagnosis,itsusebeingrecommendedforphasetypinginpatientssufferingfromsleepand
mooddisorders.Thus,thedevelopmentofsensitiveandselectivemethodsfortheprecise
detectionofmelatoninintissuesandfluidsofanimalsemergesasnecessary.Duetoitslow
concentrationandthecoexistenceofmanyotherendogenouscompoundsinblood,the
determinationofmelatoninhasbeenananalyticalchallenge.Thisreviewdiscussescurrent
methodologiesemployedfordetectionandquantificationofmelatonininbiologicalfluidsand
tissues.

Keywords:Melatonin;Circadianrhythms;Radioimmunoassay;EnzymeLinkedImmunoassay
Highperformanceliquidchromatography;Massspectrometry;Capillaryelectrophoresis

1.Introduction
Melatonin(Nacetyl5methoxytryptamine)isacompoundsecretedmainlybythepineal
gland,butsynthesizedalsoinmanyothertissuesandcellslikeretina(CardinaliandRosner,
1971;TosiniandMenaker,1998;Liuetal.,2004),humanandmurinebonemarrowcells(Conti
etal.,2000),platelets(Champieretal.,1997),gastrointestinaltract(Bubenik,2002),skin
(Slominskietal.,2005)orlymphocytes(CarrilloVicoetal.,2004).Withregardtothe
multiplicityofsitesofformationandthepresenceofmelatoninreceptorsinvariousdifferent
places,melatoninisaversatilephysiologicalsignalthathasbeenrelatedtothecontrolof
numerousphysiologicprocesses(DubocovichandMarkowska,2005;PandiPerumaletal.,
2008).Inmammals,photoperiodicinformationisrelayedthroughthesecretionofthis
hormonebythepinealgland,whichthenactsonthebrainandtheneuroendocrinesystemto
produceadaptivechangesinendocrinology,anatomyandphysiology,thusaffectingsleep,
reproduction,moulting,immuneresponses,energybalanceandbehavior,amongothers(Lewy
etal.,2006;CarrilloVicoetal.,2006;Arendt,2006;Reiteretal.,2009b).Moreover,melatonin
exhibitsdirectandindirectantioxidantproperties,andthereisstrongevidencethatthis
compoundcancounteractthedeleteriouseffectsofreactiveoxygenandnitrogenspeciesin
differentsystems(deAlmeidaetal.,2003;Onukietal.,2005;Reiteretal.,2007;Hardelandet
al.,2009;Reiteretal.,2009a).
Althoughnumerousphysiologicalfunctionshavebeenattributedtomelatonin,
mechanismsinvolvedinsuchfunctionsarefrequentlyunclear,especiallywhenparallel
signalingpathwaysareinitiatedviathemembranereceptorsMT1orMT2,orwhenother
melatoninbindingsitesareinvolved(Hardeland,2009).Therefore,furtherinvestigationat
cellularandmolecularlevelsisneededtoelucidatehowthiscompoundreallyactsasa
relevantphysiologicalregulatorysignal.
Althoughinvitrostudiescanfurnishimportantinformationontheeffectsofmelatonin
incellcultureorperfusedtissues,studiesonthefluctuationsofmelatoninconcentrationin
bodyfluidsandtissuescanbethemostrelevantissuetocomprehenditsfunctionin
organisms.Thus,thedevelopmentofsensitiveandselectivemethodsfortheprecisedetection
ofthesecompoundsintissuesandfluidsofanimalsemergesasnecessary.However,duetoits
lowconcentrationandthecoexistenceofmanyotherendogenouscompoundsinblood,the
determinationofmelatoninhasbeenananalyticalchallenge.Thisreviewdiscussescurrent
methodologiesemployedfordetectionandquantificationofmelatonininbiologicalfluidsand
tissues.

2.Melatoninfluctuationsinorganisms
3

 Invertebrates,thechronobiologicallyrelevantfractionofmelatoninismainly
producedandreleasedintothecirculationbyeitherthepinealgland,especiallyinmammals,
orpinealglandplusretina,e.g.insomebirdsandamphibians(Bentley,2001;Doyleand
Menaker,2007).Nonmammalianpinealsaredirectlyphotosensitivewhilepinealglandsof
mammalsarecontrolledbyneuronalphototransductionpathwaysoriginatingintheretinaand
processedbythehypothalamiccircadianpacemaker,thesuprachiasmaticnucleus(SCN)(Doyle
andMenaker,2007)(Goldman,2001;Moore,2007).Invariousbirds,lightinfluencescircadian
oscillatorspresentinthepinealglandsandactadditionallyonneuronalpathwaysofretinalor
hypothalamicoriginmodulatingthegland(Zawilskaetal.,2004;Csernus,2006;Cassoneetal.,
2009).Thecontributionofextrapineal/extraretinalmelatonintobloodplasmaconcentrations
ofthehormoneareeitherverylowor,inthecaseofgastrointestinalorigin,onlyepisodicand
withoutprofoundchronobiologicalsignificance(Bubenik,2002).
Inhumans,nocturnallypeakinghighamplitudeoscillationsofmelatoninintheplasma
areparalleledbycorrespondingvariationsinsaliva(Benloucifetal.,2008).Althoughplasma
levelsaregenerallyabout10timeshigherthanthosefoundinsaliva,determinationsof
salivarymelatonincanbeofadvantage,especiallywheninvasiveprocedureshavetobe
avoided.Theprimarymelatoninmetaboliteintheurine,6sulphatoxymelatonin,alsooscillates
consistentlywithmelatoninconcentrationinplasmaandsaliva(Benloucifetal.,2008).
Interestingly,melatoninrhythmsinfishblood,whichlikewiseexhibitpeaksduringnighttime,
canbefollowedinthewaterofanaquarium,towhichthehormoneisreleased(Jamesetal.,
2004).
Melatoninlevelsinhumanplasmausuallybegintoincreasebetween18:00hand20:00
h,andpeakbetween00:00hand05:00h(Lewyetal.,1985;Laganaetal.,1995;Pandi
Perumaletal.,2007;Benloucifetal.,2008;Machidaetal.,2009),beingfollowedbyarapid
decrease.Thedurationofthenocturnalmelatoninpeakhasbeenshowntobethecrucial
signalforencodingseason(BartnessandGoldman,1989).Howeverchangesintheduration
alsooccurinnonseasonalanimals.Forexample,intheratthedurationofthemelatonin
increaseiscloselytiedtothephotoperiod(Hoetal.,1984).Seasonalchangesinthenocturnal
peakofmelatoninhavealsobeenwidelyreportedwiththeamountofchangevaryingbetween
species.ThenocturnalpeakofpinealmelatoninsecretionintheSiberianhamsterduring
winteris2timesgreaterthanduringsummer(Ribelaygaetal.,2000),whilethatofthe
Europeanhamstershowsa10foldincrease(VivienRoelsetal.,1992).Thusbothdurationand
magnitudeofmelatoninsecretionarestronglycorrelatedwiththechangingphotoperiodsof
theseason(Garidouetal.,2003;Srinivasanetal.,2008).Seasonalvariationsinmelatonin
durationandlevelsarealsofoundinhumans,butlessprominent.Again,thedurationand
4

heightofthemelatoninpeakisnegativelycorrelatedwiththelengthofthephotoperiod
(MoreraandAbreu,2006;UenoTowatarietal.,2007).
Anotherimportantaspectofmelatoninfluctuationinhumansconcernsvariable
environmentallightintensities.Ithasbeenproposedthatmelatoninsecretionduringdark
phasesisgreatlyinfluencedbydimlight,whereasverybrightlightcanmaskmelatonin
production(Lewyetal.,1980).Dimlightcanbeparticularlyimportantinthecircadian
entrainmentoftherhythm.Moreover,thesuppressionofmelatoninformationandreleaseby
nocturnallightrepresentsawellknownphenomenon,ofparticularimportanceinshiftwork
(Stevensetal.,2007).Thephoticshutoffmechanismsdependontherespectiveorganismand
maybebasedeitherondephosphorylationofakeyenzymeinmelatoninbiosynthesis,
arylalkylamineNacetyltransferase(AANAT),leadingtotheincapabilityofinteractingwitha
1433proteinfollowedbyrapidproteasomaldegradation,and/orondownregulationof
AANATexpression(Schomerusetal.,2000;Gangulyetal.,2001).Consequently,melatonin
actionsarenotonlyinfluencedbythephaseofthelightdarkcycle,butcanbestrongly
affectedbyvariationsinlightanddimlightintensities.Moreover,bothnormalmelatonin
patternsandtheinfluencesbylightcanvaryconsiderablybetweenindividuals,eitherinterms
ofapersonalcharacteristic(Grofetal.,1985;WaldhauserandDietzel,1985;Kolleretal.,1994;
LerchlandPartsch,1994)orasaconsequenceofagingoraprogressingdiseases(Brownetal.,
1979;Girottietal.,2000;KarasekandReiter,2002;Girottietal.,2003;Yapraketal.,2003;
Peschke,2008).Studiesinsiblingshaveindicatedthatsomeofthisvariationhasagenetic
origin(Griefahnetal.,2003).Diseasedependentdecreasesofmelatoninwerealsodetectedin
Alzheimerpatients,notonlyintheblood(SkeneandSwaab,2003;WuandSwaab,2005)but
alsointhecerebrospinalfluid(Zhouetal.,2003),anotherbodyfluidofdiagnosticrelevance.
Thesechangesinnocturnalmelatonincouldberesponsibleforgreatvariationsinthe
pacemakerprocessescontrollingcircadianandannualrhythms.Thiscouldbealso
extrapolatedtomanyhealthdisorders.Inetiologicalterms,changesinmelatoninhavebeen
repeatedlysuspectedtobeinvolvedinnumerousdiseases,inthesusceptibilityto
inflammatoryprocessesoringeneticpredispositions.Thehealthrelatedrolesofmelatonin
seemtoreflectamixtureofhormonal,immunomodulatory,neuromodulatoryandvarious
typesofantioxidantactions,anditsefficacyinsafeguardingtheseunderlyingprocessesis
observedatverylowconcentrations(Hardeland,2009).Therefore,itseemsthatfluctuations
inmelatonindurationandlevelswhichmayappear,atfirstglance,tobeofonlyminorextent,
maycause,onthelongrun,importantpathophysiologicalchanges.Becausemelatoninlevels
arerelativelylowevenatnighttimehighlyspecificandsensitivemethodsformelatonin
measurementsinbiologicalsamplesareamatterofnecessity.
5


3.Determinationofmelatonininbiologicalsamples
3.1.Samplepreparation
Consideringitslowlevelsanimportantissueofmelatoninmeasurementsisitsadequate
extractionfrombiologicalsamples.Inserumsamples,melatonincanbeextractedbysimple
liquid/liquidprocedures,suchastheadditionofdichloromethane(1:1,v/v).Samplesarethen
vigorouslymixedandcentrifugedtoobtainaqueousandorganicphases.Withthisprocedure,
melatoninisretainedinthedichloromethanephasesthatarecollectedanddriedunder
nitrogenatmospheretoconcentratemelatonin.Thisyieldsasatisfactoryrecoveryrate
(generallymorethan70%)andcanbealsoappliedtobufferhomogenizedtissues.However,
Rizzoetal.(2002)reportedlowprecisionandaccuracywithsingleliquidliquidextractionsof
melatoninforhighperformanceliquidchromatography(HPLC)coupledtofluorescence
detector(Rizzoetal.,2002).Formultipleanalysesofmelatoninanditsprecursorsor
metabolites,moreprofoundliquidliquidextractionshavebeenalsodescribedusinga
combinationofdifferentsolvents(HarumiandMatsushima,2000).
Inolderinvestigations,chloroform(trichloromethane)wasmostlyusedformelatonin
extractionandisstillinusetoday.Althoughthismethodcanbeused,dichloromethaneis
preferredforreasonsoflowertoxicity.Generally,chlorinatedmethaneshouldbeofhighest
purityandprotectionfromlightandredoxactivecompoundsisofutmostimportancetoavoid
formationofreactiveintermediateswhichcandestroymelatonin.
Laganetal.(1995)describedanextractionprocedureforserumsamplesthroughan
LC18cartridgeplusaCarbographcartridgewitharecoveryrangingfrom86.3to91.7%for10
to200pgofmelatoninmL1.Briefly,2mLofserumsampleispassedthroughanLC18
cartridge,whichisthenwashedwith2mLofwaterand2mLofwatermethanol(90:10,v/v)
(Laganaetal.,1995).Thereafter,melatonincanbeelutedfromthecolumnwithpure
methanol,driedandresuspendedinanappropriatesolutionforanalysis(Sieghartetal.,1987)
orbefurtherpurifiedbyelutingwith2mLofwatermethanol(40:60,v/v)andloadingontoa
Carbographcartridge,asdescribedby(Laganaetal.,1995).Thecartridgeisthenwashedwith
10mLofmethanoland3mLofmethanoldichloromethane(80:20,v/v),andmelatoninis
finallyelutedwith1.5mLofmethanoldichloromethane(10:90,v/v).Theeluateisevaporated
todrynessunderN2atmosphereandresuspendedin100 Lofwatermethanol(75:25,v/v)for
analyses.
Samplepreparationwillalsodependonthemethodusedforanalysis,sincethe
presenceofothercompoundsinthesamplecaninterferewiththemelatoninsignal.The
extentofmelatoninprepurificationfrombiologicalsamplescan,insomecases,be
6

fundamentalforthesensitivityofthemethodused.Theproceduredescribedaboveallows
melatonindetectionwithhighsensitivityandwithoutinterferenceofothercomponentsinthe
sample(Laganaetal.,1995).Ithasbeenshownthathomogenizationin10volumesoficecold
0.1Mperchloricacidcanalsorepresentagoodmeansformelatonindeterminationintissues
byHPLCcoupledtoelectrochemicalorfluorescencedetection(Harumietal.,1996).Inthis
case,thehomogenateiscentrifugedat10000gfor20minat4Candtheresulting
supernatantcanbedirectlyinjectedintotheHPLCsystem.Itwasalsosuggestedtomix90 L
ofthesupernatantfractionwith10 Lof1Msodiumphosphate,pH4.3,forbetterresolution
ofpeaks.
Dependingonthemethodused,furthertreatmentofmelatoninextractsmaybe
needed.Gaschromatographycoupledtomassspectrometry(GCMS)detectionofmelatonin
requiressamplederivatizationformelatoninvolatilizationby,forexample
pentafluoropropionicanhydrideorheptafluorobutyrylimidazole(Degenetal.,1972;Covaciet
al.,1999).Inanothercase,humanplasmasamplesweredirectlyinjectedandevaluatedinan
HPLCsystemwithfluorescencedetectionwithoutpriorextractionorpurification,achievinga
detectionlimitof1ngpermlofhumanplasma(4pmol/ml)(Bechgaardetal.,1998).Also,it
hasbeenreportedthatderivatizationofmelatoninwithsodiumcarbonateandhydrogen
peroxideincreasessensitivityalmosttenfoldformeasurementinHPLCsystemscoupledto
fluorescencedetectors(Tomitaetal.,2003).
Roliketal.(2002)describedahighlyspecificmethodformelatoninisolationand
purificationfromcomplexbiologicalmatricesbyimmunoaffinitychromatography(Rolciketal.,
2002).PolyclonalantibodieshighlyspecificagainstmelatoninwereraisedbyMannich
synthesis(Grotaetal.,1981)andusedforpreparationofimmunoaffinitygel,witha95%
recoveryrateformelatoninextraction.Inthesesamples,melatoninconcentrationwas
determinedbyHPLCcoupledtomassspectrometrywithadetectionlimitof10fmol.
Regardingsamplepreparationforanalysisbymassspectrometry,theuseofadequate
isotopicallylabeledinternalstandardsrepresentsanimportantissue,improvingquantification
ofthehormoneandavoidingunderestimationofactuallevelsofmelatoninduetolosses
whichmighthaveoccurredinthesamplesduringextraction.
Finally,thecorrecthandlingandmaintenanceofsamplesisimportant,withsamples
keptconstantlyoniceandprotectedfromlightradiation,inordertoavoidmelatonin
degradation.Despiteitsrelativestability,melatoninoxidationcanoccurovertime,including
thereactionwithsingletoxygenthatcanbegenerateddependingonoxygenavailabilityand
lightincidence.Forsamplefreezing,itisrecommendedthatsamplesbedriedand
preferentiallykeptundervacuumornitrogenatmosphere.
7


3.2.Immunoassay
3.2a.Preparationofantisera
Forthemonitoringofmelatonininbiologicalfluids,useofimmunologicalmethodsisthe
mostwidespreadmethod.Severalcommercialkitsbasedonthesemethodshavebeen
availableformelatonindetermination.Someofthesemethodsarehighlysensitiveandsimple
touse(lowerlimitofdetection:0.5pgmL1)butmaysufferfromapotentialriskofcross
reactivitytostructurallysimilarcompoundsifmelatoninisnotextracted(Lemaitreand
Hartmann,1980;Dietal.,1998).
Themostcrucialaspectofimmunoassaysisthepreparationoftheantiserum.Because
melatoninistoosmalltobecapableofproducingantiseraonitsownitmustbecoupledtoan
antigenicprotein.Insuchaconjugatethesmallmolecularweightsubstanceiscalledahapten.
Theresultingantiserumbindsboththeproteinandthehaptenplusaportionoftheadjacent
protein.Thehaptenhasfewantigenicdeterminantsrelativetotheprotein.Specificitystudies
ofantiseraproducedbysteroidproteinconjugatesshowedthatantiseraarenotableto
discriminatestructuraldifferencesinthehaptenthatareimmediatelyatorclosetothesiteof
coupling(Grotaetal.,1983).
Thechoiceofthehaptenandconjugationreactionshouldthereforebedeterminedby
thetypeofdiscriminationthatisrequired.Indolealkylamineshaveincommonaringnitrogen
(position1)andanadjacentcarbon(Position2).Thusformelatonin,couplingviatheposition1
orposition2shouldallowresultingantiseratodiscriminatedifferentindolesthatare
commonlyfoundintissues.
StudiesofantiseraresultingfromMannichcouplingofmelatonintobovineserum
albumin(BSA)revealthatthisapproachleadstoahighlyspecificmelatoninantiserumas
shownbycrossreactivitystudiesinradioimmunoassay(RIA)(Bubeniketal.,1976;Pangetal.,
1977;LemaitreandHartmann,1980;Yangetal.,2006).Todeterminethelocusofattachment
ofmelatonintoprotein,modelreactionswereconductedandresultingproductsanalyzedby
nuclearmagneticresonanceandinfraredspectroscopy(Grotaetal.,1983).Resultsindicated
thatcouplingwaslikelyatposition2.Furtherstudiesweredoneofcrossreactivityof
intermediatereactionproductsrevealingthathighestcrossreactivityoccurredwithC2
substitutedmelatoninderivatives.Thusitwasconcludedthatthemethylenebridge
conjugatingmelatonintoBSAoccurredatthe2positionoftheindolemolecule.Thisapproach
hasbeenusedwidelyformelatoninimmunoassays.Recentlytwodifferentgroupshaveused
thisapproachingeneratingmonoclonalantiseraagainstmelatonin(Yangetal.,2006;
Soukhtanlooetal.,2008).
8

Couplingattheringnitrogenusing1(pcarboxybenzyl)melatonin(deSilvaand
Snieckus,1978)coupledtoBSAasantigen(Grotaetal.,1981)resultsinantiserathatbind
melatoninspecifically.Melatonin1propionicacidcoupledtoBSAalsostimulatesproduction
ofhighlyspecificantisera.Asimilarapproachcoupling1(4carboybutyl)melatonintoprotein
resultedinahighlyspecificRIA(Kawashimaetal.,1984).Themelatoninderivative,3(3(2
acetamidoethyl)5methoxyindollyl)propionicacidcoupledtobovinegammaglobulin
producesspecificantiserumthathasbeenusedwidelyinRIA(BlairandSeaborn,1979;
Kennawayetal.,1982).YetanotherderivativeN[3(2aminoethyl)5methoxyindole]
hemisuccinamidehasbeenusedtogenerateantiserumasthebasisforaspecificRIA(Manzet
al.,1989).ThusmelatonincoupledattheNpositiongivesrisetoantiserathatarehighly
specificformelatoninascomparedtootherindoles.
Couplingatthesidechainhasalsosuccessfullyproduceduseablemelatoninantiserum.
MethodsusedincludeNacetyl5methoxytryptophancoupledusingcarbodiimide(Wilkinson
etal.,1977;Vakkurietal.,1984),succinyl5methoxytryptaminecoupledtoprotein(Rollag
andNiswender,1976)andindomethacincoupledtoprotein(LevineandRiceberg,1975).
Melatonincoupledviaadiazolinkagehasalsobeenreportedtoproduceareasonablyspecific
antiserum,howeverthesensitivityoftheresultingassaywaslow(Lynchetal.,1975;
Wurzburgeretal.,1976).
CouplingofNacetylserotoninusingformaldehydegeneratesantiserumthatbinds
melatoninandNacetylserotoninequally;crossreactivitystudiesandmodelreactionsshowed
thatcouplingoccursatthe4positionofthemolecule(GrotaandBrown,1974;Kennawayet
al.,1977).ResultingantiserumhasbeenusedasthebasisofaRIAthatrequiredprior
extractionandcolumnchromatographytoeliminatethecrossreactingindole(Kennawayetal.,
1977).
Thechiefmetaboliteofmelatonininurine,6sulphatoxymelatonin(6hydroxymelatonin
sulphate)hasalsobeenmeasuredbyimmunologicmeans.Theantiserumtypicallyusedfor
thisassayisgeneratedbyuseoftheMannichreactionandishighlyspecific(GrotaandBrown,
1974;Arendtetal.,1985).Antiseraproducedusingtheseapproacheshavebeenused
extensivelynotonlyforRIA,butalsoforimmunohistochemistryandforenzymelinked
immunoassays(ELISA).

3.2b.Radioimmunoassay(RIA)
TheprincipleofRIAmethodformelatoninmeasurementisthataknownamountof
radioactivemelatonin(2I125iodomelatoninor3Hmelatonin,forexample)ismixedwithafixed
amountofantibodyraisedagainstmelatonin.Increasingconcentrationsofunlabeled
9

melatoninareaddedtothemixture,whichwillcompetewithlabeledmelatonincausingits
displacementfromtheantibody.Freelabeledmelatoninisthenseparatedfromremaining
antibodyboundradioactivemelatoninandradioactivityismeasured.Astheconcentrationof
unlabeledmelatoninincreasesinthemixture,competitionfortheantibodiesalsoincreases
andboundlabeledmelatonindecreases.Acalibrationcurveconstructedfromknownamounts
oflabeledandunlabeledmelatoninallowsthedeterminationofunknownmelatonin
concentrationsinbiologicalsamples.
Fraseretal.(1983)describedaprotocolformelatoninmeasurementbyRIAinplasma
thathasbeenadoptedbyseveralresearchers,somewithslightmodifications(Fraseretal.,
1983).Briefly,200 Lof1000folddilutedantibodyisaddedto500 Lofsolutionscontaining
differentamountofmelatoninstandard(2.5to250.0pg).Thesolutionisvortexedandkeptat
roomtemperaturefor30min.3Hmelatoninisaddedtothetubes(100 L,4000cpm),mixed
andkeptat4Cfor18h.Then,0.5mLofDextrancoatedcharcoalsolution(0.1gofdextran75
plus10gofcharcoalper500mLofbuffer)isaddedandthesolutioniscentrifugedfor15min
at1500gand4C,inordertoseparatetheantibodyboundmelatoninfromthefree
fraction.Thesupernatantfractionisfinallydecantedinto10mLofscintillationfluidand
radioactivityiscountedonabetascintillatorcounter(Fraseretal.,1983).
SeveralvariationsinRIAmethodshavebeendescribed,byusingdifferentantibodies(as
notedabove),bychanging3Hmelatoninto2I125iodomelatonin,orbyalteringtheseparation
procedure.Ingeneral,becauseofitshigherspecificactivity2I125iodomelatoninallowsalower
detectionlimitthusallowingtheuseofasmalleramountofsample.Theconcentrationof
melatoninduringdaylightcanbeaslowas0.2to0.3fM(Rolciketal.,2002).Thiscouldbe
especiallyimportantifmeasurementsarenotprecededbymelatoninpurification.However,
125

Iismorepronetononspecificbindingsothatsomedeterminationscanbefaulty.
Sieghartetal.(1987)reportedthatpriormelatoninpurificationfromplasmausing

reversedphasecolumnchromatographyreducesgreatlyproblemsofcrossreactivity(Sieghart
etal.,1987).MoreoverRoliketal.(2002)usedimmunoaffinitychromatographyemploying
specificantiseratoprocesssamplespriortoHPLCMSanalysis(Rolciketal.,2002).
Nonethelessitshouldberecognizedthatevenweakcrossreactivitycanbeaproblemifthe
crossreactingmoleculeispresentinlargequantities.Thusindependentvalidationofthe
procedureisessentialwhenadifferentmatrixisassayed.
OneexampleofsuchadifferentmatrixissalivaforwhichseveralRIAshavebeen
described(Milesetal.,1985;Vakkuri,1985;Milesetal.,1989;Englishetal.,1993).Toobtain
salivadifferentmethodshavebeenused,fromchewinggum,chewingoncottonswabsor
usingcommercialapparatus.Againextractionisusuallyessentialespeciallysincelevelsin
10

salivaareabout40%ofthoseinplasma.Salivaisparticularlyusefulifrepeatedsamplingis
required:forexampletocharacterizethefull24hrhythmofmelatoninortodeterminethe
dimlightmelatoninonset(DLMO),ameasurethathasbeenshowntobeveryusefulinstudies
ofavarietyofsleepdisorders(LewyandSack,1989;Leibenluftetal.,1996;PandiPerumalet
al.,2007).
Severalvariantsofthetimeconsumingcharcoalseparationprocedurehavebeen
developedandsuccessfullyapplied.Inthesocalledscintillationproximityassay,themelatonin
antibodyisboundtoasecondaryantibody(e.g.,antisheep)attachedtoscintillatorcontaining
microbeads(fluomicrospheres)(PoeggelerandHuether,1992).Thisrelativelyconvenient
proceduredepends,however,usuallyonthecommercialavailabilityofsuitable
fluomicrospheres,sincepreparationandstandardizationofsuchbeadsistootieconsuming
foranaveragelaboratory.Intheproximityassay,boundradioactivityisdetecteddirectlyby
thescintillatorsystemofthemicrospheres.Forphysicalandgeometricalreasons,sucha
systemhastohavealowerscintillationefficiencythanahomogeneousscintillationcocktail.
However,thisprocedurehasotheradvantages.Apartfrombeingmorerapid,thesystemis
lessaffectedbynonspecificbinding(valuesclosetobackground)suchasoccurinthecharcoal
procedure,hasabetterreproducibilityandshowsamuchlowerassaydriftuponrepetitive
measurements(proximityassay:about10%changewithin84h;charcoalmethod:about25%
overthesameperiodoftime)(PoeggelerandHuether,1992).Othervariantsinclude
separationusingadoubleantibodyprocedure(Dietal.,1998)andammoniumsulfate
precipitation(Hoetal.,1984).
Althoughnotmelatoninitself,therehasbeenconsiderableinterestinthemajorurinary
metaboliteofmelatonin,6sulphatoxymelatonin(Arendtetal.,1985).The24hpatternof
excretionofthemetaboliteaccuratelyreflectsthepatternofmelatonininblood(Markeyet
al.,1985;Nowaketal.,1987).RIAsforthissubstanceareavailableandhavebeenusefulin
assessingpinealfunctioninvariousconditions(Martinetal.,1984;Johnetal.,1992;Cavallo
andDolan,1996;Girottietal.,2000;Girottietal.,2003;Fideleffetal.,2006).

3.3EnzymeLinkedImmunoassay(ELISA)
AvarietyofELISAsformelatoninhavealsobeenreportedthatemployantisera
identicaltothoseusedintheRIAdescribedabove.Onesuchimmunoassayemployed
melatoninhemisuccinatehumanserumalbuminabsorbedonpolystyrenespheres,withthe
melatonincompetingforafixedamountofperoxidaselabeledIgGantibodytomelatonin
(FerruaandMasseyeff,1985).Thismethodhadadetectionlimitof22fmolpertubeand
thereforerequiredextraction.AcompetitivesolidphaseELISAforhumanandratserumand
11

ratpinealglandhasbeendescribedandvalidatedusingmicrotitreplatesthathasamuch
lowerdetectionlimit(1.0fmolperwell)aswellasprecisioncomparablewithothermethods
andthatcanbeappliedwithoutextractiontoratserum(Yieetal.,1993).Aimprovedversion
ofthisassaywithashorterincubationtimewassubsequentlyreported(Shavalietal.,1999).A
comparativestudyofanRIAandacommercialELISAreportedthattheELISArequireda
purificationsteptobevalidwhenappliedtohumanserum,astepthatwasnotpartofthe
procedurerecommendedbythemanufacturer(Cheginietal.,1995).Withtheextractionstep,
theassayhaddistinctadvantages,Enzymeassayshavemajoradvantagesinthattheenzyme
conjugateisstable,ismoreconvenientthan3Hor125Iandpresentnoproblemwithdisposalof
radioactivewaste.Furthermoreifmicrotitreplatesareusedcentrifugationisnotnecessary.
Althoughnotanenzymeimmunoassay,itisofinterestthatatimeresolvedfluoroimmunassy
hasalsobeendescribed(Yamadaetal.,2002).Anenzymeimmunoassayfor6
sulphatoxymelatoninhasbeenreported(PenistonBirdetal.,1996)andcommercialkitsare
available.

3.3.HPLCcoupledtoelectrochemicalandfluorescencedetection
Accordingto(Vitaleetal.,1996)RIAmethodologyhasbeenreplacedbyHPLCwith
electrochemicalandfluorescencedetectionformelatoninevaluationinbiologicalsamples,
duetoitsgreatsensitivityandspecificity.Howevertheyalsosuggestthatthisprocedureis
moreadequateformelatoninalone,andnotformixturesofseveralindoleslikeserotoninand
tryptamineamongothers,thatcancauseinterferencesintheassay.Indeed,theseauthors
reportedthatserotonin/melatoninratioishigherthan100inratpineal,whichcauses
disturbancesinchromatographicseparationsthatcandifficultmelatonindetection,so
requiringagoodprocedureformelatoninextraction.However,theavoidanceofpartial
coelutionwithotherindolesismostlyamatteroftheartofchromatography.Inourworkwe
havebeenabletodetectmelatoninwithgreataccuracyinbloodplasmaaftersimple
dichlorometaneextractionasdescribedabove,andusinganHPLCsystemconnectedto
electrochemicaldetection.Figure1showsachromatogramofmelatonindetectioninhuman
plasmabythisprocedure.GoodpeakseparationwasachievedbyusingaLC18columnand50
mMsodiumacetate100mMaceticacid(pH4.3),0.1mMNa2EDTA,andacetonitrile(75:25,
v/v)asmobilephasepumpedisocraticallyat1mL/min.
Harumietal.(1996)alsosuccessfullydeterminedmelatoninbyHPLCwith
electrochemicaldetection,withveryclearpeakseparationfordifferentindoleaminesamong
melatonin(Harumietal.,1996).However,thesensitivityofthisproceduredependsonthe
modelofelectrochemicalcell.Amperometricbasedelectrochemicalcellsaregenerallyless
12

sensitivethancoulometriccells,sothattheadequatepotentialshouldbepreviouslyoptimized
bytheconstructionofhydrodynamicvoltammograms.Withourcoulometricelectrochemical
system,thebestmelatoninsignalisobtainedat600mV.Sensitivitycanbealsogreaterwith
coulometricelectrochemicaldetectorslikeESAcoulochemIIImodel(ESA,Bedford,USA),
whichusesporouselectrochemicalcellsthatallowgreateraccuracyinmelatoninpeak
resolution.Harumietal.(1996)reportedtheuseofahigherpotential,900mV,forgood
melatoninsignalwiththeirgraphitecarbonworkingelectrode,andevensotheydetected
melatoninatverylowlevels(Harumietal.,1996).Rizzoetal.(2002)alsoused900mVfor
melatonindetectionwithanamperometricelectrochemicaldetector(Rizzoetal.,2002).
Withrespectoffluorescencedetection,somehighlysensitivemethodologieshavebeen
reportedformelatonindetectionatfmollevel(Simoninetal.,1999;Iinumaetal.,1999;Yang
etal.,2002;Luetal.,2002).MelatonincanbeseparatedonaC18columnbyusing75mM
sodiumacetatepH5.0andacetonitrile(72:28,v/v)asthemobilephasepumpedisocratically
at1.0mL/min,anddirectlydetectedbysettingupthefluorescencedetectoratanexcitation
wavelengthof275nmandanemissionwavelengthof345nm(Rizzoetal.,
2002).Nevertheless,insomecasesinwhichmelatoninconcentrationisverylow,derivatization
isrecommendabletoenhancethemelatoninsignal(Iinumaetal.,1999).Tomitaetal.(2003),
forexample,describesanoxidationprocedurethatcanenhancemelatoninfluorescenceby6.8
times,allowingitsdeterminationatamollevelsinbiologicalsamples(Tomitaetal.,2003).
Melatoninwasoxidizedtoanewfluorescentcompoundwithsodiumcarbonateandhydrogen
peroxide.However,precautionsshouldbetakenbyusingthiskindofapproach,because
otherscomponentsfromthebiologicalsamplemayleadforthegenerationoffluorophores
thatmayinterfereonthecorrectlevelevaluationcausinglackofmethodspecificity(Tomitaet
al.,2003).
Inanycase,carewithsamplepreparationcanimprovemelatoninsignal.Prepurification
ofmelatoninasdescribedbeforewilldecreasechromatogramnoisesandavoidthecoelution
ofmelatoninwithothercompoundsthatcaninterferewithmelatoninpeaks.Generally,the
useoffluorescencetechniquescanbeaffectednotonlybycoelutionwithotherfluorescent
compoundsinthesample,butalsobythepresenceofquenchers.Thisshouldnotbe
underratedsincethemajorityofaromatesabsorbaroundtheexcitationmaximumof
melatonin.Therefore,samplesshouldbetestedinadvanceforquenchingbyaddingknown
amountsofmelatonin.

3.4.Massspectrometry

13

TheGCMStechniqueisverysensitiveandoffersmorespecificitythanHPLCwith
electrochemicalorfluorescencedetectors;however,aproblematicdifficultyistheneedof
derivatizationandthusthistechniquehavebeengraduallysubstitutedbyliquid
chromatographymassspectrometryprocedures.Thus,alternativeHPLCMSmethods
appropriateforuseinbiologicalissueshavebeendeveloped(Yangetal.,2002;Erikssonetal.,
2003;Motoyamaetal.,2004;Almeidaetal.,2004).However,thisapproachislimitedbythe
needofadequateinternalstandards.Yangetal.(2002)reportedamethodologybyusing
acetyltryptamineasinternalstandard(Yangetal.,2002);however,thisisnottheideal
situation.Themostappropriateistheemploymentofalabeledinternalstandardwhose
structureisthesameoftheanalyteunlessthemassdifference.Theadditionofanisotopically
labeledinternalstandardpriortoanalysisimprovesthemethodsconfidencelevel.
Anotheranalyticalmethodenabledthedeterminationofendogenousmelatoninin
humansaliva,byusingcolumnswitchingsemimicrocolumnliquidchromatography/mass
spectrometryandselectedionmonitoring(SIM).Melatoninwasmonitoredbasedonits
fragmentionatm/z174byinsourcedissociationandusingdeuteratedmelatoninasinternal
standard,andadetectionlimitof10fmolwasobtained(Rolciketal.,2002).Themain
limitationofthismethodologyistheuseofSIMmodetodetecttheionsgeneratedinthe
probe,whichdoesnotimplyanabsolutespecificity.Yet,Erikssonetal.(2003)reporteda
methodforthedeterminationofmelatonininhumansalivabyHPLCMS/MS,using7D
melatoninasinternalstandard(Erikssonetal.,2003).Thelimitofdetectionwas1.05pgmL1
andthelimitofquantificationwas3.0pgmL1.Oneofushasreportedthedevelopmentofa
newHPLCMS/MSassaywithelectrosprayionization(ESI)toquantitativelydetermine
melatoninandalsoitsdegradationproductN1acetylN2formyl5methoxykynuraminewith
highsensitivityandspecificity(Almeidaetal.,2004).Astableisotopicinternalstandard
melatoninD3(deuteratedmelatonin)waseasilysynthesizedbythereactionof5
methoxytryptaminewithdeuteratedacetylchloride(CD3COCl)(Figure2).
Thepredominantion[M+H]+inthefullscanmassspectraofmelatonin,andmelatonin
D3werelocated(Figure3AB).Thefragmentsgeneratedincollisioninduceddissociation
chamberrevealedapredominantfragmentatm/z=174formelatoninandmelatoninD3(loss
oftheNacetylgroup)(figure3CD).Them/ztransitionsfrom233to174(melatonin)andfrom
236to174(melatoninD3)werethereforechosenfortheMultipleReactionMonitoring(MRM)
detectionexperiments,whichensuredahigherspecificityandanaccuratequantificationof
melatonininhumanplasma(Figure3E).

3.4.Othertechniques
14

Somelaboratorieshavetakenanddevelopedcapillaryelectrophoresis(CE)fortheseparation
anddeterminationofmelstonininbloodplasma(Kimetal.,1999;Pobozyetal.,2005;
Musijowskietal.,2006)andpinealgland(Chenetal.,2001;Wuetal.,2006).Detectionof
analytewasperformedwithUVandfluoscencedetector(Kimetal.,1999;Pobozyetal.,2005;
Musijowskietal.,2006;Heviaetal.,2010)orelectrochemicaldetector(Chenetal.,2001;Wu
etal.,2006).DetectionlimitofmelatoninwithCEiscomparablewiththedataobtainedby
HPLCmethodsreportedpreviously.Recently,fortheseparationofmelatoninfromrelated
compounds,CEwithmicellarelectrokineticchromatographywasapplied(Pobozyetal.,2005;
Musijowskietal.,2006;Wuetal.,2006;Heviaetal.,2010).Thistechniqueallowedtoseparate
melatoninanditsprecursorsormetaboliteseffectively.Sodiumdodecylsulfateisusedto
produceapseudostationaryphase.

4.Conclusions
ThemostcommonmethodsfordeterminationofmelatonininbloodorsalivaareRIAs
andELISAs.Moreoverseveralcommercialkitsarenowavailablefortheseassays.Theyare
convenienttouse,especiallytheenzymebasedassays,butmustbeusedwithcarebecauseof
thepossibilityofcrossreactions,andnonspecificeffects.Thisisparticularlythecasebecause
oftheverylowlevelsofmelatoninthataretobemeasured.Thesepotentialproblemscanbe
reducedbydeterminingwhetherextractionisnecessaryandbycomparisonwithother
establishedmethods.Itistobeexpectedthatthesemethodswillcontinuetoimproveand
thatenzymeassayswillcontinuetogaingroundforroutinemeasurementsofmelatoninin
bloodandsaliva.

15


References
Almeida,E.A.,Klitzke,C.F.,Martinez,G.R.,Medeiros,M.H.,DiMascio,P.2004.Synthesisof
internallabeledstandardsofmelatoninanditsmetaboliteN1acetylN2formyl5
methoxykynuraminefortheirquantificationusinganonlineliquidchromatography
electrospraytandemmassspectrometrysystem.JPinealRes36,6471.
Arendt,J.2006.Melatoninandhumanrhythms.Chronobiol.Int23,2137.
Arendt,J.,Bojkowski,C.,Franey,C.,Wright,J.,Marks,V.1985.Immunoassayof6
hydroxymelatoninsulfateinhumanplasmaandurine:abolitionoftheurinary24hourrhythm
withatenolol.JClinEndocrinol.Metab60,11661173.
Bartness,T.J.,Goldman,B.D.1989.Mammalianpinealmelatonin:aclockforallseasons.
Experientia45,939945.
Bechgaard,E.,Lindhardt,K.,Martinsen,L.1998.Highperformanceliquidchromatographic
analysisofmelatonininhumanplasmaandrabbitserumwithonlinecolumnenrichment.J
Chromatogr.BBiomed.Sci.Appl.712,177181.
Benloucif,S.,Burgess,H.J.,Klerman,E.B.,Lewy,A.J.,Middleton,B.,Murphy,P.J.,Parry,B.L.,
Revell,V.L.2008.Measuringmelatonininhumans.JClinSleepMed4,6669.
Bentley,G.E.2001.Unravelingtheenigma:theroleofmelatonininseasonalprocessesin
birds.Microsc.ResTech.53,6371.
Blair,I.A.,Seaborn,C.J.1979.Thesynthesisofmelatoninantigens.AustJChem32,399403.
Brown,G.M.,Young,S.N.,Gauthier,S.,Tsui,H.,Grota,L.J.1979.Melatonininhuman
cerebrospinalfluidindaytime;itsoriginandvariationwithage.LifeSci.25,929936.
Bubenik,G.A.2002.Gastrointestinalmelatonin:localization,function,andclinicalrelevance.
Dig.Dis.Sci.47,23362348.
Bubenik,G.A.,Brown,G.M.,Grota,L.J.1976.Immunohistochemicallocalizationofmelatonin
intheratHarderiangland.JHistochem.Cytochem.24,11731177.
Cardinali,D.P.,Rosner,J.M.1971.Metabolismofserotoninbytheratretina"invitro".Journal
ofNeurochemistry18,17691770.
CarrilloVico,A.,Calvo,J.R.,Abreu,P.,Lardone,P.J.,GarciaMaurino,S.,Reiter,R.J.,Guerrero,
J.M.2004.Evidenceofmelatoninsynthesisbyhumanlymphocytesanditsphysiological
significance:possibleroleasintracrine,autocrine,and/orparacrinesubstance.FASEBJ18,
537539.
CarrilloVico,A.,Reiter,R.J.,Lardone,P.J.,Herrera,J.L.,FernandezMontesinos,R.,Guerrero,
J.M.,Pozo,D.2006.Themodulatoryroleofmelatoninonimmuneresponsiveness.Curr.Opin.
Investig.Drugs7,423431.

16

Cassone,V.M.,Paulose,J.K.,WhitfieldRucker,M.G.,Peters,J.L.2009.Time'sarrowflieslike
abird:twoparadoxesforaviancircadianbiology.Gen.CompEndocrinol.163,109116.
Cavallo,A.,Dolan,L.M.1996.6Hydroxymelatoninsulfateexcretioninhumanpuberty.J
PinealRes21,225230.
Champier,J.,Claustrat,B.,Besancon,R.,Eymin,C.,Killer,C.,Jouvet,A.,Chamba,G.,Fevre
Montange,M.1997.EvidencefortryptophanhydroxylaseandhydroxyindolOmethyl
transferasemRNAsinhumanbloodplatelets.LifeSci.60,21912197.
Chegini,S.,EhrhartHofmann,B.,Kaider,A.,Waldhauser,F.1995.Directenzymelinked
immunosorbentassayandaradioimmunoassayformelatonincompared.ClinChem.41,381
386.
Chen,G.,Cheng,J.,Ye,J.2001.Applicationofanovelmicroinjectorinthedeterminationof
indolederivativesintheratpinealglandbycapillaryelectrophoresiswithelectrochemical
detection.Fresenius.JAnal.Chem.370,930934.
Conti,A.,Conconi,S.,Hertens,E.,SkwarloSonta,K.,Markowska,M.,Maestroni,J.M.2000.
Evidenceformelatoninsynthesisinmouseandhumanbonemarrowcells.JPinealRes28,193
202.
Covaci,A.,Doneanu,C.,AboulEnein,H.Y.,Schepens,P.1999.Determinationofmelatoninin
pharmaceuticalformulationsandhumanplasmabygaschromatographyelectronimpactmass
spectrometry.Biomed.Chromatogr.13,431436.
Csernus,V.J.2006.Theavianpinealgland.Chronobiol.Int23,329339.
deAlmeida,E.A.,Martinez,G.R.,Klitzke,C.F.,deMedeiros,M.H.,DiMascio,P.2003.
Oxidationofmelatoninbysingletmolecularoxygen(O2(1deltag))producesN1acetylN2
formyl5methoxykynurenine.JPinealRes35,131137.
deSilva,S.O.,Snieckus,V.1978.IndoleNalkylationoftryptaminesviadianionandphtalimido
intermediates.Newpotentialindolealkylaminehapten.CanJChem56,16211628.
Degen,P.H.,DoAmaral,J.R.,Barchas,J.D.1972.Agasliquidchromatographicassayof
melatoninandindoleaminesusingheptafluorobutyrylderivatives.Anal.Biochem.45,634644.
Di,W.L.,Kadva,A.,Djahanbakhch,O.,Silman,R.1998.Radioimmunoassayofboundandfree
melatonininplasma.ClinChem.44,304310.
Doyle,S.,Menaker,M.2007.Circadianphotoreceptioninvertebrates.ColdSpringHarb.Symp.
Quant.Biol72,499508.
Dubocovich,M.L.,Markowska,M.2005.FunctionalMT1andMT2melatoninreceptorsin
mammals.Endocrine.27,101110.
English,J.,Middleton,B.A.,Arendt,J.,WirzJustice,A.1993.Rapiddirectmeasurementof
melatonininsalivausinganiodinatedtracerandsolidphasesecondantibody.Ann.Clin
Biochem.30(Pt4),415416.

17

Eriksson,K.,Ostin,A.,Levin,J.O.2003.Quantificationofmelatonininhumansalivabyliquid
chromatographytandemmassspectrometryusingstableisotopedilution.JChromatogr.B
Analyt.Technol.Biomed.LifeSci.794,115123.
Ferrua,B.,Masseyeff,R.1985.Immunoassayofmelatoninwithenzymelabeledantibodies.J
Immunoassay6,7994.
Fideleff,H.L.,Boquete,H.,Fideleff,G.,Albornoz,L.,PrezLloret,S.,Suarez,M.,Esquifino,A.I.,
Honfi,M.,Cardinali,D.P.2006.Genderrelateddifferencesinurinary6sulfatoxymelatonin
levelsinobesepubertalindividuals.JournalofPinealResearch40,214218.
Fraser,S.,Cowen,P.,Franklin,M.,Franey,C.,Arendt,J.1983.Directradioimmunoassayfor
melatonininplasma.ClinChem.29,396397.
Ganguly,S.,Gastel,J.A.,Weller,J.L.,Schwartz,C.,Jaffe,H.,Namboodiri,M.A.,Coon,S.L.,
Hickman,A.B.,Rollag,M.,Obsil,T.,Beauverger,P.,Ferry,G.,Boutin,J.A.,Klein,D.C.2001.
RoleofapinealcAMPoperatedarylalkylamineNacetyltransferase/1433bindingswitchin
melatoninsynthesis.Proc.Natl.Acad.Sci.U.S.A98,80838088.
Garidou,M.L.,VivienRoels,B.,Pevet,P.,Miguez,J.,Simonneaux,V.2003.Mechanisms
regulatingthemarkedseasonalvariationinmelatoninsynthesisintheEuropeanhamster
pinealgland.AmJPhysiolRegulIntegrCompPhysiol284,R1043R1052.
Girotti,L.,Lago,M.,Ianovsky,O.,Elizari,M.,Dini,A.,PrezLloret,S.,Albornoz,L.E.,Cardinali,
D.P.2003.Lowurinary6sulphatoxymelatoninlevelsinpatientswithseverecongestiveheart
failure.Endocrine22,245248.
Girotti,L.,Lago,M.,Yanovsky,O.,Carbajales,J.,Elizari,M.,Brusco,L.I.,Cardinali,D.P.2000.
Lowurinary6sulphatoxymelatoninlevelsinpatientswithcoronaryarterydisease.JPineal
Research29,138142.
Goldman,B.D.2001.Mammalianphotoperiodicsystem:formalpropertiesand
neuroendocrinemechanismsofphotoperiodictimemeasurement.JBiolRhythms16,283301.
Griefahn,B.,Brode,P.,Remer,T.,Blaszkewicz,M.2003.Excretionof6hydroxymelatonin
sulfate(6OHMS)insiblingsduringchildhoodandadolescence.Neuroendocrinology78,241
243.
Grof,E.,Grof,P.,Brown,G.M.,Arato,M.,Lane,J.1985.Investigationsofmelatoninsecretion
inman.Prog.NeuropsychopharmacolBiolPsychiatry9,609612.
Grota,L.J.,Brown,G.M.1974.Antibodiestoindolealkylamines:serotoninandmelatonin.Can.
JBiochem.52,196202.
Grota,L.J.,Snieckus,V.,deSilva,S.O.,Brown,G.M.1983.AntibodiestoindolealkylaminesII:
siteofconjugationofmelatonintoproteinusingformaldehyde.Can.JBiochem.CellBiol61,
10961101.
Grota,L.J.,Snieckus,V.,deSilva,S.O.,Tsui,H.W.,Holloway,W.R.,Lewy,A.J.,Brown,G.M.
1981.Radioimmunoassayofmelatonininratserum.Prog.Neuropsychopharmacol5,523526.

18

Hardeland,R.2009.Melatonin:signalingmechanismsofapleiotropicagent.Biofactors35,
183192.
Hardeland,R.,Tan,D.X.,Reiter,R.J.2009.Kynuramines,metabolitesofmelatoninandother
indoles:theresurrectionofanalmostforgottenclassofbiogenicamines.JPinealRes47,109
116.
Harumi,T.,Akutsu,H.,Matsushima,S.1996.Simultaneousdeterminationofserotonin,N
acetylserotoninandmelatonininthepinealglandofthejuvenilegoldenhamsterbyhigh
performanceliquidchromatographywithelectrochemicaldetection.JChromatogr.BBiomed.
Appl.675,152156.
Harumi,T.,Matsushima,S.2000.Separationandassaymethodsformelatoninandits
precursors.JChromatogr.BBiomed.Sci.Appl.747,95110.
Hevia,D.,Botas,C.,Sainz,R.M.,Quiros,I.,Blanco,D.,Tan,D.X.,GomezCordoves,C.,Mayo,J.
C.2010.Developmentandvalidationofnewmethodsforthedeterminationofmelatoninand
itsoxidativemetabolitesbyhighperformanceliquidchromatographyandcapillary
electrophoresis,usingmultivariateoptimization.JChromatogr.A1217,13681374.
Ho,A.K.,Grota,L.J.,Brown,G.M.1984.RelationshipbetweenpinealNacetyltransferase
activity,pinealmelatoninandserummelatonininratsunderdifferentlightingconditions.
Neuroendocrinology39,465470.
Iinuma,F.,Hamase,K.,Matsubayashi,S.,Takahashi,M.,Watanabe,M.,Zaitsu,K.1999.
Sensitivedeterminationofmelatoninbyprecolumnderivatizationandreversedphasehigh
performanceliquidchromatography.JChromatogr.A835,6772.
James,J.D.,Ellis,T.,Scott,P.2004.Waterbasedmeasurementofrainbowtroutmelatonin.
JournalofFishBiology69,12981304.
John,T.M.,Brown,M.C.,Brown,G.M.1992.Anoralmelatoninreplacementregimenthatre
establishesthenormalcircadianlevelsofurinary6sulphatoxymelatonininfunctionally
pinealectomizedrats.JPinealRes13,145150.
Karasek,M.,Reiter,R.J.2002.Melatoninandaging.NeuroEndocrinol.Lett.23Suppl1,1416.
Kawashima,K.,Nagakura,A.,Wurzburger,R.J.,Spector,S.1984.Melatonininserumandthe
pinealofspontaneouslyhypertensiverats.ClinExp.Hypertens.A6,15171528.
Kennaway,D.J.,Frith,R.G.,Phillipou,G.,Matthews,C.D.,Seamark,R.F.1977.Aspecific
radioimmunoassayformelatonininbiologicaltissueandfluidsanditsvalidationbygas
chromatographymassspectrometry.Endocrinology101,119127.
Kennaway,D.J.,Gilmore,T.A.,Seamark,R.F.1982.Effectsofmelatoninimplantsonthe
circadianrhythmofplasmamelatoninandprolactininsheep.Endocrinology110,21862188.
Kim,Y.O.,Chung,H.J.,Chung,S.T.,Kim,J.H.,Park,J.H.,Han,S.Y.,Kil,K.S.,Cho,D.H.1999.
Determinationofmelatonininbiologicalsamplesbycapillaryelectrophoresis.JChromatogr.A
850,369374.

19

Koller,M.,Harma,M.,Laitinen,J.T.,Kundi,M.,Piegler,B.,Haider,M.1994.Differentpatterns
oflightexposureinrelationtomelatoninandcortisolrhythmsandsleepofnightworkers.J
PinealRes16,127135.
Lagana,A.,PardoMartinez,B.,Marino,A.,Fago,G.,Bizzarri,M.1995.Determinationofserum
totallipidandfreeNacetylneuraminicacidingenitourinarymalignanciesbyfluorimetrichigh
performanceliquidchromatography.RelevanceoffreeNacetylneuraminicacidastumour
marker.ClinChimActa243,165179.
Leibenluft,E.,FeldmanNaim,S.,Turner,E.H.,Schwartz,P.J.,Wehr,T.A.1996.Salivaryand
plasmameasuresofdimlightmelatoninonset(DLMO)inpatientswithrapidcyclingbipolar
disorder.BiolPsychiatry40,731735.
Lemaitre,B.J.,Hartmann,L.1980.Preparationofantimelatoninantibodiesandantigenic
propertiesofthemolecule.JImmunol.Methods32,339347.
Lerchl,A.,Partsch,C.J.1994.Reliableanalysisofindividualhumanmelatoninprofilesby
complexcosinoranalysis.JPinealRes16,8590.
Levine,L.,Riceberg,L.J.1975.Radioimmunoassayformelatonin.ResCommunChem.Pathol.
Pharmacol10,693702.
Lewy,A.J.,Emens,J.,Jackman,A.,Yuhas,K.2006.Circadianusesofmelatonininhumans.
Chronobiol.Int23,403412.
Lewy,A.J.,Sack,R.L.1989.Thedimlightmelatoninonsetasamarkerforcircadianphase
position.Chronobiol.Int6,93102.
Lewy,A.J.,Sack,R.L.,Singer,C.M.1985.Immediateanddelayedeffectsofbrightlighton
humanmelatoninproduction:shifting"dawn"and"dusk"shiftsthedimlightmelatoninonset
(DLMO).Ann.N.Y.Acad.Sci.453,253259.
Lewy,A.J.,Wehr,T.A.,Goodwin,F.K.,Newsome,D.A.,Markey,S.P.1980.Lightsuppresses
melatoninsecretioninhumans.Science210,12671269.
Liu,C.,Fukuhara,C.,Wessel,J.H.,III,Iuvone,P.M.,Tosini,G.2004.LocalizationofAanat
mRNAintheratretinabyfluorescenceinsituhybridizationandlasercapturemicrodissection.
CellTissueRes315,197201.
Lu,J.,Lau,C.,Morizono,M.,Ohta,K.,Kai,M.2002.Achemiluminescencereactionbetween
hydrogenperoxideandacetonitrileanditsapplications.Anal.Chim.Acta455,193.
Lynch,H.J.,Ozaki,Y.,Shakal,D.,Wurtman,R.J.1975.Melatoninexcretionofmanandrats:
effectoftimeofday,sleep,pinealectomyandfoodconsumption.IntJBiometeorol.19,267
279.
Machida,M.,Dubousset,J.,Yamada,T.,Kimura,J.2009.Serummelatoninlevelsinadolescent
idiopathicscoliosispredictionandpreventionforcurveprogressionaprospectivestudy.J
PinealRes46,344348.

20

Manz,B.,Seidel,A.,Alexander,H.,Vollrath,L.,Wagner,B.,Zimmermann,G.,Wiedemann,K.,
Pollow,K.1989.Developmentandvalidationofaradioimmunoassayforserummelatonin.J
ClinChem.ClinBiochem.27,797802.
Markey,S.P.,Higa,S.,Shih,M.,Danforth,D.N.,Tamarkin,L.1985.Thecorrelationbetween
humanplasmamelatoninlevelsandurinary6hydroxymelatoninexcretion.ClinChim.Acta
150,221225.
Martin,P.R.,Higa,S.,Burns,R.S.,Tamarkin,L.,Ebert,M.H.,Markey,S.P.1984.Decreased6
hydroxymelatoninexcretioninKorsakoff'spsychosis.Neurology34,966968.
Miles,A.,Philbrick,D.,Tidmarsh,S.F.,Shaw,D.M.1985.Directradioimmunoassayof
melatonininsaliva.ClinChem.31,14121413.
Miles,A.,Philbrick,D.R.,Grey,J.E.1989.Salivarymelatoninestimationinassessmentof
pinealglandfunction.ClinChem.35,514515.
Moore,R.Y.2007.Suprachiasmaticnucleusinsleepwakeregulation.SleepMed8Suppl3,27
33.
Morera,A.L.,Abreu,P.2006.Seasonalityofpsychopathologyandcircannualmelatonin
rhythm.JPinealRes41,279283.
Motoyama,A.,Kanda,T.,Namba,R.2004.Directdeterminationofendogenousmelatoninin
humansalivabycolumnswitchingsemimicrocolumnliquidchromatography/mass
spectrometrywithonlineanalyteenrichment.RapidCommunMassSpectrom.18,12501258.
Musijowski,J.,Pobozy,E.,Trojanowicz,M.2006.Onlinepreconcentrationtechniquesin
determinationofmelatoninanditsprecursors/metabolitesusingmicellarelectrokinetic
chromatography.JChromatogr.A1104,337345.
Nowak,R.,McMillen,I.C.,Redman,J.,Short,R.V.1987.Thecorrelationbetweenserumand
salivarymelatoninconcentrationsandurinary6hydroxymelatoninsulphateexcretionrates:
twononinvasivetechniquesformonitoringhumancircadianrhythmicity.ClinEndocrinol.
(Oxf)27,445452.
Onuki,J.,Almeida,E.A.,Medeiros,M.H.,DiMascio,P.2005.Inhibitionof5aminolevulinic
acidinducedDNAdamagebymelatonin,N1acetylN2formyl5methoxykynuramine,
quercetinorresveratrol.JPinealRes38,107115.
PandiPerumal,S.R.,Smits,M.,Spence,W.,Srinivasan,V.,Cardinali,D.P.,Lowe,A.D.,
Kayumov,L.2007.Dimlightmelatoninonset(DLMO):Atoolfortheanalysisofcircadianphase
inhumansleepandchronobiologicaldisorders.ProgressinNeuroPsychopharmacology&
BiologicalPsychiatry31,111.
PandiPerumal,S.R.,Trakht,I.,Srinivasan,V.,Spence,D.W.,Maestroni,G.J.M.,Zisapel,N.,
Cardinali,D.P.2008.Physiologicaleffectsofmelatonin:roleofmelatoninreceptorsandsignal
transductionpathways.ProgressinNeurobiology185,335353.

21

Pang,S.F.,Brown,G.M.,Grota,L.J.,Chambers,J.W.,Rodman,R.L.1977.DeterminationofN
acetylserotoninandmelatoninactivitiesinthepinealgland,retina,harderiangland,brainand
serumofratsandchickens.Neuroendocrinology23,113.
PenistonBird,J.F.,Di,W.L.,Street,C.A.,Edwards,R.,Little,J.A.,Silman,R.E.1996.An
enzymeimmunoassayfor6sulphatoxymelatonininhumanurine.JPinealRes20,5156.
Peschke,E.2008.Melatonin,endocrinepancreasanddiabetes.JPinealRes44,2640.
Pobozy,E.,Michalski,A.,SotowskaBrochocka,J.,Trojanowicz,M.2005.Determinationof
melatoninanditsprecursorsandmetabolitesusingcapillaryelectrophoresiswithUVand
fluorometricdetection.JSep.Sci.28,21652172.
Poeggeler,B.,Huether,G.1992.Versatileonetubescintillationproximityhomogeneous
radioimmunoassayofmelatonin.ClinChem38,314315.
Reiter,R.J.,Paredes,S.D.,Manchester,L.C.,Tan,D.X.2009a.Reducingoxidative/nitrosative
stress:anewlydiscoveredgenreformelatonin.CritRevBiochemMol.Biol44,175200.
Reiter,R.J.,Tan,D.X.,Manchester,L.C.,Paredes,S.D.,Mayo,J.C.,Sainz,R.M.2009b.
MelatoninandReproductionRevisited.BiolReprod.
Reiter,R.J.,Tan,D.X.,Manchester,L.C.,Tamura,H.2007.Melatonindefeatsneurallyderived
freeradicalsandreducestheassociatedneuromorphologicalandneurobehavioraldamage.J
PhysiolPharmacol.58Suppl6,522.
Ribelayga,C.,Pevet,P.,Simonneaux,V.2000.HIOMTdrivesthephotoperiodicchangesinthe
amplitudeofthemelatoninpeakoftheSiberianhamster.AmJPhysiolRegulIntegrComp
Physiol278,R1339R1345.
Rizzo,V.,Porta,C.,Moroni,M.,Scoglio,E.,Moratti,R.2002.Determinationoffreeandtotal
(freeplusproteinbound)melatonininplasmaandcerebrospinalfluidbyhighperformance
liquidchromatographywithfluorescencedetection.JChromatogr.BAnalyt.Technol.Biomed.
LifeSci.774,1724.
Rolcik,J.,Lenobel,R.,Siglerova,V.,Strnad,M.2002.Isolationofmelatoninbyimmunoaffinity
chromatography.JChromatogr.BAnalyt.Technol.Biomed.LifeSci.775,915.
Rollag,M.D.,Niswender,G.D.1976.Radioimmunoassayofserumconcentrationsof
melatonininsheepexposedtodifferentlightingregimens.Endocrinology98,482489.
Schomerus,C.,Korf,H.W.,Laedtke,E.,Weller,J.L.,Klein,D.C.2000.Selective
adrenergic/cyclicAMPdependentswitchoffofproteasomalproteolysisaloneswitcheson
neuralsignaltransduction:anexamplefromthepinealgland.JNeurochem.75,21232132.
Shavali,S.,Samejima,M.,Uchida,K.,Morita,Y.,Fukuda,A.1999.Improvedenzyme
immunoassaymethodformelatonin:applicationtothedeterminationofserummelatoninin
rats,sheep,andhumans.ClinChem.45,690692.
Sieghart,W.,Ronca,E.,Drexler,G.,Karall,S.1987.Improvedradioimmunoassayofmelatonin
inserum.ClinChem.33,604605.
22

Simonin,G.,Bru,L.,Lelievre,E.,Jeanniot,J.P.,Bromet,N.,Walther,B.,BoursierNeyret,C.
1999.Determinationofmelatonininbiologicalfluidsinthepresenceofthemelatoninagonist
S20098:comparisonofimmunologicaltechniquesandGCMSmethods.JPharm.Biomed.
Anal.21,591601.
Skene,D.J.,Swaab,D.F.2003.Melatoninrhythmicity:effectofageandAlzheimer'sdisease.
Exp.Gerontol.38,199206.
Slominski,A.,Fischer,T.W.,Zmijewski,M.A.,Wortsman,J.,Semak,I.,Zbytek,B.,Slominski,R.
M.,Tobin,D.J.2005.Ontheroleofmelatonininskinphysiologyandpathology.Endocrine.27,
137148.
Soukhtanloo,M.,Ansari,M.,Paknejad,M.,Parizadeh,M.R.,Rasaee,M.J.2008.Preparation
andcharacterizationofmonoclonalantibodyagainstmelatonin.Hybridoma(Larchmt.)27,
205209.
Srinivasan,V.,Spence,D.W.,Trakht,I.,PandiPerumal,S.R.,Cardinali,D.P.,Maestroni,G.J.
M.2008.Immunomodulationbymelatonin:Itssignificanceforseasonallyoccurringdiseases.
Neuroimmunomodulation15,93101.
Stevens,R.G.,Blask,D.E.,Brainard,G.C.,Hansen,J.,Lockley,S.W.,Provencio,I.,Rea,M.S.,
Reinlib,L.2007.Meetingreport:theroleofenvironmentallightingandcircadiandisruptionin
cancerandotherdiseases.Environ.HealthPerspect.115,13571362.
Tomita,T.,Hamase,K.,Hayashi,H.,Fukuda,H.,Hirano,J.,Zaitsu,K.2003.Determinationof
endogenousmelatoninintheindividualpinealglandsofinbredmiceusingprecolumn
oxidationreversedphasemicrohighperformanceliquidchromatography.Anal.Biochem.316,
154161.
Tosini,G.,Menaker,M.1998.Theclockinthemouseretina:melatoninsynthesisand
photoreceptordegeneration.BrainRes789,221228.
UenoTowatari,T.,Norimatsu,K.,Blazejczyk,K.,Tokura,H.,Morita,T.2007.Seasonal
variationsofmelatoninsecretioninyoungfemalesundernaturalandartificiallightconditions
inFukuoka,Japan.JPhysiolAnthropol.26,209215.
Vakkuri,O.1985.Diurnalrhythmofmelatonininhumansaliva.ActaPhysiolScand.124,409
412.
Vakkuri,O.,Leppaluoto,J.,Vuolteenaho,O.1984.Developmentandvalidationofamelatonin
radioimmunoassayusingradioiodinatedmelatoninastracer.ActaEndocrinol.(Copenh)106,
152157.
Vitale,A.A.,Ferrari,C.C.,Aldana,H.,Affanni,J.M.1996.Highlysensitivemethodforthe
determinationofmelatoninbynormalphasehighperformanceliquidchromatographywith
fluorometricdetection.JChromatogr.BBiomed.Appl.681,381384.
VivienRoels,B.,Pevet,P.,MassonPevet,M.,Canguilhem,B.1992.Seasonalvariationsinthe
dailyrhythmofpinealglandand/orcirculatingmelatoninand5methoxytryptophol
concentrationsintheEuropeanhamster,Cricetuscricetus.Gen.CompEndocrinol.86,239
247.
23

Waldhauser,F.,Dietzel,M.1985.Dailyandannualrhythmsinhumanmelatoninsecretion:role
inpubertycontrol.Ann.N.Y.Acad.Sci.453,205214.
Wilkinson,M.,Arendt,J.,Bradtke,J.,deZiegler,D.1977.Determinationofadarkinduced
increaseofpinealNacetyltransferaseactivityandsimultaneousradioimmunoassayof
melatonininpineal,serumandpituitarytissueofthemalerat.JEndocrinol.72,243244.
Wu,X.,Wu,W.,Zhang,L.,Xie,Z.,Qiu,B.,Chen,G.2006.Micellarelectrokineticcapillary
chromatographyforfastseparationandsensitivedeterminationofmelatoninandrelated
indoleaminesusingendcolumnamperometricdetection.Electrophoresis27,42304239.
Wu,Y.H.,Swaab,D.F.2005.ThehumanpinealglandandmelatonininagingandAlzheimer's
disease.JPinealRes38,145152.
Wurzburger,R.J.,Kawashima,K.,Miller,R.L.,Spector,S.1976.Determinationofratpineal
glandmelatonincontentbyaradioimmunoassay.LifeSci.18,867877.
Yamada,H.,Chiba,H.,Amano,M.,Iigo,M.,Iwata,M.2002.Rainbowtrouteyedstageembryos
demonstratemelatoninrhythmsunderlightdarkconditionsasmeasuredbyanewly
developedtimeresolvedfluoroimmunoassay.Gen.CompEndocrinol.125,4146.
Yang,S.,Zheng,X.,Xu,Y.,Zhou,X.2002.RapiddeterminationofserummelatoninbyESIMS
MSwithdirectsampleinjection.JPharm.Biomed.Anal.30,781790.
Yang,T.,Wang,J.,Qu,L.,Zhong,P.,Yuan,Y.2006.Preparationandidentificationofanti
melatoninmonoclonalantibodies.JPinealRes40,350354.
Yaprak,M.,Altun,A.,Vardar,A.,Aktoz,M.,Ciftci,S.,Ozbay,G.2003.Decreasednocturnal
synthesisofmelatonininpatientswithcoronaryarterydisease.IntJCardiol.89,103107.
Yie,S.M.,Johansson,E.,Brown,G.M.1993.Competitivesolidphaseenzymeimmunoassayfor
melatonininhumanandratserumandratpinealgland.ClinChem.39,23222325.
Zawilska,J.B.,Berezinska,M.,Lorenc,A.,Skene,D.J.,Nowak,J.Z.2004.Retinalillumination
phaseshiftsthecircadianrhythmofserotoninNacetyltransferaseactivityinthechickenpineal
gland.NeurosciLett.360,153156.
Zhou,J.N.,Liu,R.Y.,Kamphorst,W.,Hofman,M.A.,Swaab,D.F.2003.Early
neuropathologicalAlzheimer'schangesinagedindividualsareaccompaniedbydecreased
cerebrospinalfluidmelatoninlevels.JPinealRes35,125130.

24


Figure1Chromatogramofmelatoninstandard(2pmol;A)andmelatoninextractedfrom2
mLofaplasmasample(B),byHPLCwithcoulometricelectrochemicaldetector.

25

Figure2SynthesisoflabeledD3melatoninforitsuseasinternalstandardduringmelatonin
measurementinplasmasamples.

26

Figure3FullmassscanspectraofmelatoninandD3melatoninintheESI+mode

174

100

Daughters of 233ES+

[(M N-acetil) +

H]+

1.16e6

m/z = 174
H3C

O
N
H

CH3

Relative abundance (%)

m/z = 233

125

145

165

185

205

225

174

100

245

265

Daughters of 236ES+

[(M N-acetil) + H]+

1.16e6

m/z = 174
H3C

O
N

N
H

CD3

m/z = 236

125

145

165

185
m/z

205

225

245

265

27

Figure4DaughtersofmelatoninandD3melatoninionsafterfragmentation inthecollision
cellofthemassspectrometer.

28

Figure5Standardcalibrationcurveofmelatonin.OntheYaxis,datawasplotedbydividing
theareaoflabeledstandard(D3melatonin)bynotlabeledmelatoninstandard.

29

Relative abundance (%)

100

MRM ES+
236.00 > 174.00

MELD3 (1 pmol)

0
MRM ES+
233.00 > 174.00

100

MEL

2.00

4.00

6.00

8.00

10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00

Time (min)

Figure6ChromatogramofmelatoninanddeuteratedmelatoninbyHPLCMS/MS.

30