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α-retinyl esters from retinyl esters
Hilary J. Goetz, Rachel Kopec, Ken M. Riedl, Jessica L. Cooperstone, Sureshbabu Narayanasamy, Robert W. Curley, Steven J. Schwartz
To cite this version:
Hilary J. Goetz, Rachel Kopec, Ken M. Riedl, Jessica L. Cooperstone, Sureshbabu Narayanasamy,
et al.. An HPLC–MS/MS method for the separation of α-retinyl esters from retinyl esters. Journal
of Chromatography B - Analytical Technologies in the Biomedical and Life Sciences, Elsevier, 2016,
1029-1030, pp.68-71. �10.1016/j.jchromb.2016.06.043�. �hal-02631924�
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Journal homepage : http://www.elsevier.com/locate/chromb
Short communication
An HPLC–MS/MS method for the separation of ␣ -retinyl esters from retinyl esters
Hilary J. Goetz
a, Rachel E. Kopec
a,b, Ken M. Riedl
a, Jessica L. Cooperstone
a, Sureshbabu Narayanasamy
c, Robert W. Curley Jr.
c, Steven J. Schwartz
a,∗aDepartmentofFoodScience&Technology,TheOhioStateUniversity,Columbus,OH,UnitedStates
bINRA,UMR408SécuritéetQualitédesProduitsd’OrigineVégétale,Avignon,France
cDepartmentofMedicinalChemistry,TheOhioStateUniversity,Columbus,OH,UnitedStates
Keywords:
␣-Retinylpalmitate
␣-Retinol Retinylesters
ProvitaminAcarotenoids Synthesis
HPLC–MS/MS
a b s t r a c t
EnzymaticcleavageofthenonsymmetricprovitaminAcarotenoid␣-caroteneresultsinonemoleculeof retinal(vitaminA),andonemoleculeof␣-retinal,abiologicallyinactiveanalogoftruevitaminA.Dueto structuralsimilarities,␣-retinylestersandvitaminAesterstypicallycoelute,resultingintheoverestima- tionofvitaminAoriginatingfrom␣-carotene.Herein,wepresentasetoftoolstoidentifyandseparate
␣-retinolproductsfromvitaminA.␣-Retinylpalmitate(␣RP)standardwassynthesizedfrom␣-ionone followingaWittig-Hornerapproach.Ahigh-performanceliquidchromatography–tandemmassspec- trometry(HPLC–MS/MS)methodemployingaC30columnwasthendevelopedtoseparatethespecies.
Authenticstandardsofretinylestersandthesynthesized␣-RPconfirmedrespectiveidentities,while other␣-retinylesters(i.e.myristate,linoleate,oleate,andstearate)wereevidencedbytheirpseudo- molecularionsobservedinelectrosprayionization(ESI)mode,fragmentation,andelutionorder.For quantitation,anatmosphericpressurechemicalionization(APCI)sourceoperatedinpositiveionmode wasused,andretinol,thepredominantin-sourceparentionwasselectedandfragmented.Theapplica- tionofthismethodtoachylomicron-richfractionofhumanplasmaisdemonstrated.Thismethodcan beusedtobetterdeterminethequantityofvitaminAderivedfromfoodscontaining␣-carotene.
©2016ElsevierB.V.Allrightsreserved.
1. Introduction
VitaminAdeficiencyremainsasignificantproblemworldwide, with deficient regions obtaininga majority of their vitamin A needsfromprovitaminAcarotenoidsfoundinfruitsandvegeta- bles[1,2].ThevitaminAcapacityofcarotenoidsisdictatedbythe presenceofanunsubstituted-iononeringinconjugationwitha polyenechain[3].Whilenumerousanalyticalmethodshavebeen developedforthedeterminationofvitaminAdeliveryfromaprovi- tamin A rich meal [4–6],most studiesfocus onthe provitamin Acarotenoid-carotene.Thesymmetricstructureof-carotene yieldstwomolecules ofretinalaftercentralcleavagemaking it
Abbreviations: ␣RP,␣-retinylpalmitate;APCI,atmosphericpressurechemical ionization;ESI,electrosprayionization;HPLC,highperformanceliquidchromatog- raphy;MeOH,methanol;MTBE,methyltert-butylether;NMR,nuclearmagnetic resonance;PDA,photodiodearray;RP,retinylpalmitate;MS/MS,tandemmass spectrometry.
∗Correspondingauthorat:235ParkerFoodScience&TechnologyBuilding,2015 FyffeCtColumbus,OH43210,UnitedStates.
E-mailaddress:schwartz.177@osu.edu(S.J.Schwartz).
themostpotentprovitaminAprecursor.Howeverinnature,- caroteneisoftenfoundconcurrentwithnonsymmetricprovitamin Acarotenoidslike␣-carotene.Analogousto-carotene,␣-carotene isenzymaticallycleavedatthecentraldoublebond,yetonlypro- ducesonemoleculeofretinal.Theremainingproduct,␣-retinal, containsan-ring,andpossessesonly2%ofthebioactivityofvita- minA in animal models[7–10]. Both␣-retinaland retinalare reducedto␣-retinoland retinol,respectively,then esterifiedto fattyacids,beforebeingpackagedandreleasedinbloodchylomi- crons.Thestructuralsimilaritybetweentheisobars␣-retinoland retinol,withonlyadifferenceintheplacementofasingledou- blebond,causesdifficultyresolving␣-retinol/␣-retinylestersfrom analogousretinol/retinylesters,respectively,usingtraditionalC18 reversed-phasechromatography.Duetothiscoelution,itislikely thatreportedlevelsofnewlyformedvitaminAaftertheconsump- tionofan␣-carotene-richmealhavebeenoverestimated.
Afewarticleshavereportedtentativeidentificationof␣-retinyl estersinanimaltissuesandbloodafterthefeedingof␣-carotene or␣-retinol[11–15].Collectively,thesestudiesidentified␣-retinyl esters based upon anticipated retention time, expected UV–vis spectra,anddisappearanceaftersamplesaponification[11–15].In
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onestudy,thepresumedresulting␣-retinolwasisolatedfromtis- suesandthen usedtoquantify␣-retinol fromthesametissues [11].Anotherstudyrequireddouble-analysisofbothasaponified andunsaponifiedsample,inadditiontoacalculation,toestimate
␣-retinylestercontribution[12].Saponificationofretinolspecies priortoanalysisrequiresmorepreparatorytime,increasespoten- tialfordegradationofthesesensitivecompounds,and doesnot allowforthedifferentiationofthecirculatingesters[12,14].Addi- tionally,withoutauthenticstandards,reportsofintact␣-retinyl esters in animal tissues are tentative [15]. Indeed, the lack of authenticstandards hasprevented unequivocalidentificationof
␣-retinylestersforthepast20years.
The objective of this work was to provide a tool to better measurethevitaminApotentialof␣-carotene-containingfoods.
␣-Retinolwassynthesizedandesterifiedtopalmiticacid(␣RP,the presumedpredominateacylatedformof␣-retinol[16]).Together with retinyl palmitate (RP), and other authentic retinyl ester standards,ahigh-performanceliquidchromatography-photodiode array-tandemmassspectrometry(HPLC-PDA-MS/MS)methodwas developed.Themethodwasthenappliedtodifferentiate␣-retinyl estersfromretinylestersfoundinthechylomicron-containingfrac- tionofhumanbloodplasma.
2. Materialsandmethods 2.1. Reagents
AmmoniumacetatewaspurchasedfromJ.T.Baker(Phillipsburg, NJ,USA).HPLCgrademethyltert-butylether(MTBE),Optimagrade water,methanol (MeOH)and formic acidwerepurchasedfrom FisherScientific(Pittsburgh,PA,USA).
2.2. Synthesisandpurificationofall-trans˛RPstandard
Adetaileddescriptionof␣RPsynthesisandpurificationispro- videdinthesupplementary materialand issummarized inFig.
S1.
2.3. HPLC–MS/MSmethodforseparationof˛RPandRP
Separationof␣RPandRPstandardswasachievedusingaC30 column (4.6mm×250mm,3mparticlesize,YMC, Allentown, PA)).A1200SLseriesHPLCsystemwitha60mmpathlength1260 PDA(AgilentTechnologies,SantaClara,CA)wasemployed.Agra- dientofsolventA:90:10MeOH/H2Owith0.1%formicacid(v/v), and solventB:78:20:2MTBE/MeOH/H2O with0.1% formic acid
(v/v)wasasfollows:30%B,followedbyalineargradientto50%
Bover 18min,holdingat100%Bfor2min,andre-equilibrating at30%Bfor3.5min.Thecolumnwasheldat40◦C,withaflow rateof1.3mL/min,and 40Linjectionvolumes.TheHPLC-PDA wasinterfacedwithaQTRAP5500massspectrometer(ABSciex, FosterCity,CA)usinganAPCIsourceinpositiveionmodefor␣RP andRPquantitation.Thesourceparameterswereasfollows:cur- taingas:30psi,heatednebulizertemperature:450◦C,nebulizer gas:45psi,declusteringpotential:100V,entrancepotential:10V, andcollisioncellexitpotential:11V.Bothestersaffordedstrong in-sourceretinolfragmentsthatwereutilizedforMS/MSdetection.
Theparent-daughtertransitionswhichboth(1)displayeddistinctly differentMRMratiosfor␣-retinylandretinylestersand(2)pro- videdoptimalintensitiesforquantitationwerechosen(Table1).
PeakareaswereintegratedwithAnalyst1.5.1(ABSciex).
2.4. Biologicalsamplepreparation
Triglyceride-richlipoprotein(TRL)fractionsofhumanplasma containing newly formed chylomicronswere isolated [17] and extractedaspublishedpreviously[18].
2.5. HPLC-PDA-MS/MSmethodtoseparatetotal˛-retinylesters andretinylestersinbiologicalsamples
Todistinguishthenon-palmitateestersof␣-retinolandretinol foundinbiologicalsamples(i.e.myristate,linoleate,oleate,and stearate),theHPLCgradientdescribedabovewasextendedasfol- lows:beginningat30%B,followedbyalinearincreaseto55.6%B over23min,holdingat100%Bfor2min,andre-equilibratingat 30%Bfor3.5min.Todetecttheintactparent␣-retinylestersand retinylesters,theHPLCwasinterfacedwiththemassspectrometer viaanESIprobeoperatedinpositiveionmode.Sourceparameters, asdescribedinSection2.3,wereapplied,withtheexceptionofthe sourcetemperaturewhichwasincreasedto525◦Candcollision energies of17.5and 40Vfor the␣- andretinyl esters,respec- tively.Identificationof␣RPandretinylestersintheTRLsamplewas baseduponparentionmasses,UV–visspectra,andretentiontime coincidentwithauthenticstandards(Table1).Thenon-palmitate
␣-retinylestersweretentativelyidentifiedbyexpectedretention orderrelativetothecorrespondingretinylester,UV–visspectra, parentionmass,andexpecteddaughterratiosofthein-source␣- retinolparent.AnAPCIsource(withparametersprovidedinSection 2.3)wasusedforanalytequantitation.ForallMS/MSexperiments, MRMmodewasusedwithmaximized70msdwelltimespertransi- tion.All␣-retinylesterswerequantitatedusing␣RP,andallretinyl
Table1
MS/MSparametersusedfortheidentificationandquantitationof␣-retinylestersandretinylesters.
No. Retention time(min)
Compound identity
HPLC-PDA spectrum(nm)
HPLC-APCI(+)-MS/MS m/zparention>m/z daughterion
APCIcollision energies(volts)e
HPLC-ESI(+)-MS/MS m/zparention>m/z daughterion
ESIcollision energies(volts)e
1 12.8 ␣-Retinylmyristate 298,311a,325 269.2>123.1b 27.5 496.6>145.0 17.5
2 13.2 Retinylmyristate 325a 269.2>239.1c,d,145.1d 35.0,25.0 496.6>197.0 40
3 13.2 ␣-Retinyllinoleate 298,311a,325 269.2>123.1b 27.5 548.6>145.0 17.5
4 13.9 Retinyllinoleate 325a 269.2>239.1c,d,145.1d 35.0,25.0 548.6>197.0 40
5 15.4 ␣-Retinyloleate 298,311a,325 269.2>123.1b 27.5 550.6>145.0 17.5
6 15.9 Retinyloleate 325a 269.2>239.1c,d,145.1d 35.0,25.0 550.6>197.0 40
7 16.9 ␣-Retinylpalmitate 298,311a,325 269.2>123.1b 27.5 524.6>145.0 17.5
8 17.3 Retinylpalmitate 325a 269.2>239.1c,d,145.1d 35.0,25.0 524.6>197.0 40
9 21.0 ␣-Retinylstearate 298,311a,325 269.2>123.1b 27.5 552.6>145.0 17.5
10 21.6 Retinylstearate 325a 269.2>239.1c,d,145.1d 35.0,25.0 552.6>197.0 40
aDenotesmax.
bDaughterwiththestrongesttransitionfor␣-retinolderivatives,chosenforquantitation.
c Daughtersuniquetoretinolderivativesonly.
d Daughterschosenforquantitationofretinolderivatives.
eCollisionenergyusedforeachrespectivedaughterfollowstheorderinwhichtheyarelistedinthepreviouscolumn.
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esterswerequantitatedusingRP.Thefattyacidmoietyofeach esterislostin-sourceprovidingthesameparention(andthussame response)for␣-retinolandretinol,respectively(aspreviouslycon- firmedwiththeauthenticretinylestersusedforidentification).
3. Resultsanddiscussion
3.1. HPLC–MS/MSmethoddevelopment
Identificationand quantitation of␣-retinyl ester and retinyl ester pairs presents an analytical challenge due to theirstruc- turalsimilarityandisobaricnature.BycouplingHPLCandMS/MS, wewere abletochromatographand useunique fragmentation patternstodifferentiatethe␣-retinylproductsfromtheirretinyl counterparts.
Chromatographicseparation of␣RPandRPwasachievedby modifyingthegradient and increasing thepolarity ofthe non- elutingsolventofthemethodoutlinedbyKopecetal.[18].Asa result,␣RPelutedimmediatelybeforeRP,whilestillmaintaining peakresolution.Thiselutionpatternwasobservedforallesters of␣-retinolandretinol.C30columns,widelyusedforcarotenoid andretinoidseparation,weretestedatvaryinglengths(150mm, 250mm)andparticlesizes(3m,5m)toimproveresolution.A columnlengthof250mmwith3mparticlesizegave thebest separation.
UV–visspectralcharacteristicswerealsohelpfulindistinguish- ing␣-retinylestersfromretinylesters.Themovementofadouble bondoutofconjugationinthe-ringof␣RPcausesahypsochromic shift,resultinginamaxof311nm(ascomparedamaxof325nm forRP)(Fig.S2)[19].␣RPretainsthe3-prongedfine-structurechar- acteristicofcarotenoids,withshouldersclearlyvisibleat298and 325nm,while RPhasnofinestructure (ascommonly observed forshort-ringapo-carotenoidsandothervitaminAderivatives) [19].Furthermore,fattyacidacylationhasnoimpactonthemaxof
␣-retinolandretinol[20].␣-Retinolandretinolelutedat3.62and 3.76minrespectively.
Byextendingthegradient,weobservedandseparatedmyris- tate,linoleate, oleate,and stearateesters ofboth ␣-retinol and retinolinadditiontopalmitateintheTRLextracts(Fig.1).Iden- titieswereconfirmedinESIpositivemode(Fig.S3),whereeachof theestersformedaradicalcationwhichfragmentedtoyieldadehy- dratedretinolfragmentatm/z269.2.Thesamepseudomolecular ionhasbeendescribedpreviouslyforretinylesters[4].Transitions monitoredarelistedinTable1andamasschromatogramisshown inthesupplementarymaterial.
AnAPCIsourceoperatedinpositiveionmodeprovedtobemore sensitiveforthedetectionof␣RPandother␣-retinylestersascom- paredtoanESIsource,confirmingpreviousreportsforretinoids [5,18,21].Furthersignalenhancementwasachievedbyreplacing themobilephase additiveammoniumacetate withformic acid.
␣RPandretinylesterstandardsolutionsconfirmedAPCIin-source fragmentationtoproduceaparentionat269.2m/z(representing theneutral lossofthefattyacidmoiety and water,[M+H-fatty acid-H2O]+).Thisobservationisconsistentwithotherreportsof retinylpalmitateanalysisbyMS[5,18,21,22].Notably,thedehy- drated␣-retinolspecies(containingan-ring)andretinolspecies (containinga-ring)havedifferentfragmentationpatterns,which simplifiedtheidentificationofthe␣-analoguesintheTRLextracts.
Inaddition,thesefragmentationpatternsmirrorthefragmenta- tionoftheparentprovitaminAcarotenoids␣-carotene(containing an-ringanda-ring)and-carotene(containingtwo-rings) [19,23].Theparent-daughterpairm/z269.2>123.1,characteris- tic of-ringcarotenoids, wasthestrongest transitionobserved for␣-retinolspeciesand isthoughttocorrespondtothe-ring moietyitself[19,23].Thisdaughterwasalsoobservedforretinol, but atlower levels,and thuswasselected forquantification of
␣RP.Incontrast,m/z269.2>239.1showedexceptionalselectivity fortheretinylestersandwasnotproduced fromfragmentation of ␣-retinol. Thisfragment was summed with m/z 145.1 (also
Fig.1. HPLC–MS/MSchromatogramofaTRLextractfromarepresentativesubject6hpost-prandial,utilizingtheHPLCmethodseparatingallestersandtheMSequippedwith anAPCI+source.MRMtransitionsspecificfor␣-retinoland␣-retinylesters(269m/z>123m/z—)andretinolandretinylestersRP(269m/z>145m/z----,269m/z>239m/z
···)aredisplayed.PeaknumberscorrespondtothosedetailedinTable1.
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representative of retinol) for quantitation. Fig. 1 demonstrates a mass chromatogram of a TRL extract withthese characteris- tic transitionsselected. Usingthese aforementionedtransitions, thismethodwasdeterminedtohavealimitofquantitation(LOQ, definedassignaltonoise=10)of4.8and29.5nMfor␣RPandRP, respectively,and a limitof detection(LOD,definedassignalto noise=3)of1.4and8.9nMfor␣RPandRP,respectively.Intotal, the3MS/MStransitionschosenwerecontinuouslymonitoredto providemoreconfidenceintheidentitiesof␣-retinylandretinyl estersbycomparingratiointensities.
4. Conclusions
Withoutpropertools,thecontributionof ␣-retinylestersto thenewlycirculatingretinylesterpoolcanbeeasilyoverlooked.
Herein,wehaveprovidedadescriptivemethodof␣-retinoland
␣RPsynthesis,anddetailedHPLC–MS/MSmethodsthatallowfor thedirect characterizationand quantificationof␣-retinylesters (bothintactandfragmented)inbiologicalsamples.Weanticipate thatthesemethodswillaidintheaccuratequantificationofvita- minAderivedfrom␣-carotenesources,tobetterassessitsvitamin Apotential.
Authordisclosures
Theauthorshavenoconflictsofinteresttodisclose.
Acknowledgements
This work was supported by a seed grant from The Ohio StateUniversityFoodInnovationCenter,andbytheNutrientand PhytochemicalSharedResourceofTheOhioStateUniversityCom- prehensiveCancerCenter(NIHP30CA016058).Wewouldalsolike tothankDr.EarlH.Harrisonforhisgenerouscontributionofthe retinylesterstandards.
AppendixA. Supplementarydata
Supplementarydataassociatedwiththisarticlecanbefound, intheonlineversion,athttp://dx.doi.org/10.1016/j.jchromb.2016.
06.043.
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