Automation of AOAC 988.13 for Increased Throughput in the ...€¦ · POSTER10-01 Step Solvent...

Automation of AOAC 988.13 for Increased Throughput in the Identification of FD&C Color Additives in Food

Megan York and Toni R. Hofhine, Gilson, Inc., Middleton, WI, USA and Judy Hadley, Ph.D. and Rick Laurell, Agilent Technologies, Santa Clara, CA, USA OVERVIEW

Purpose• Coloradditivesforfoodarecommonlyfoundinmanyplaces:candies,powdereddrinkmix,fruitskins.• TheadditionofsyntheticcoloradditivesisregulatedcloselybytheFDA.• Coloradditiveshavecomeunderfoodsafetyscrutinyrecentlybecauseoftheirpotentialadverse

physicalandmentalhealtheffectsthatmaybelinkedtoingestion,especiallyinchildren.• AOACOfficialMethod988.13providesamanualprocessforextraction,separation,

andidentificationofcoloradditivesfromvariousfoodproducts.• Automationofallthreesteps:samplepreparation,solidphaseextraction,andspectrophotometricanalysis

throughsinglesoftwarecontrolallowsforincreasedthroughputandconsistencyofsampletesting.• Automationofcommonmanualprocedurescandecreasesamplebacklogsbyproviding

addedefficiencyandincreaseddailythroughputforatypicallaboratory.

AGilsonGX-274ASPEC™System inconjunctionwithanAgilent8453UV-visibleSpectrophotometerwithMulticellTransportwasusedtoautomatethesolidphaseextraction(SPE),samplepreparation,andsampleanalysisofFD&Ccoloradditivesinfood.AOACOfficialMethod988.13wassuccessfullyautomatedandFD&Ccoloradditiveswereeffectivelyseparatedandqualitativelyidentifiedfrommultiplesamples.

EXPERIMENTAL

TheFD&Ccoloradditivescontainedwithinthreedifferentsampleswereseparatedviasolidphaseextractionandqualitatively identifiedagainstapreparedFD&Cstandard.TheFD&CcoloradditivesexaminedwereFD&CYellowNo.5(E102,Tartrazine),FD&CRedNo.40(E129,AlluraRedAC),andFD&CBlueNo.1(E133,Erioglaucine).Spectraofeachofthestandardscanbeseenbelow.

METHOD

Samples and SolventsAlluraRedAC(Sigma,P/N458848)Tartrazine(Sigma,P/NT0388)Erioglaucine(Sigma,P/N861146)Isopropanol(B&J,P/N10071758) 2.5,13,and20%solutionswerepreparedwithNanoPurewaterAceticAcid(Sigma,P/N320099) 1%solutionwaspreparedwithNanoPurewaterSodiumHydroxide(EMScience,P/NSX0600-1) 50%solutionwaspreparedwithNanoPurewaterHydrochloricAcid(Sigma,P/N258148)NanoPureWaterBlackFoodColoring(McCormick)Kool-Aid®(GrapeandOrange,powder) ProtocolsAlltestswereruninquadruplicate.Figure7belowshowsthecompleteprotocolfollowedfortheseparationandidentificationofblackfoodcoloring.

Sample Preparation

Reference Standard Solutions: (Stock)100mgofthereferencematerialwasdilutedto100mLwithNanoPurewater.(Standard)10mLofstocksolutionwasdilutedto100mLwiththeappropriateisopropanolsolution.

Black Food Coloring:A1:200dilutionofblackfoodcoloringwaspreparedwithNanoPurewater.

Kool-Aid:1gpowderwasdissolvedin100mLNanoPurewater.Filteredonbed.

Solid Phase Extraction

TheSPEschemefromAOAC988.13wasutilized,withsomevolumemodificationstoadjustforcartridgesize.ThisoriginalSPEschemecanbefoundbelowinFigure6.ThevolumesusedfortheapplicationcanbefoundinTables1,2,and3.

Sample Preparation and Analysis

ThefractionscollectedfromtheSPEprocesswerepreparedforidentificationusingtheGX-274ASPEC.

(1)Thefractionwastransferredtoacleantesttube;volumetransferredwas250µLlessthantheamountofsolventusedforelutioninthe Fractionatestep(2)Thefractionwasdilutedto6mLwithappropriateIPAsolutionandvolume(3)2mLwastransferredtoeachoftwosetsofcleantubes(4)1500µLwastransferredtotheflowcellsviathetransferports,andanabsorbancereadingwastakenontheneutraldilutedfraction(5)Adrop(23µL)ofconcentratedhydrochloricacidwasaddedtothesecondsetoftesttubesandthesolutionwasmixed(6)1500µLwastransferredtotheflowcellsviathetransferports,andanabsorbancereadingwastakenontheacidicfractionsolution(7)Adrop(23µL)of50%sodiumhydroxidesolutionwasaddedtothethirdsetoftesttubesandthesolutionwasmixed(8)1500µLwastransferredtotheflowcellsviathetransferports,andanabsorbancereadingwastakenonthebasicfractionsolution

AblankoftheappropriateIPAsolutionwastakenpriortoeachsetofabsorbancereadings.Thereadingsweretakenfrom190to1100nm,howeverthespectrawereonlyanalyzedfrom350to750nm,asspecifiedinAOAC988.13.Theflowcellsandlineswererinsedwith5mLNanoPurewateraftereachreadingtoeliminatecarryoverbetweensamples.

RESULTSANDCONCLUSION

McCormickbrandblackfoodcoloringwasusedasaprimarytestsampleduetoitsrelativelycleanmatrix.TheproductingredientlabelingfortheblackfoodcoloringindicateditcontainedFD&CYellowNo.5,FD&CRedNo.40,andFD&CBlueNo.1.Theresultantspectraconfirmedthepresenceofthesecoloradditives.RepresentativespectrafromeachofthefractionscanbefoundbelowinFigures8,9,and10.AslightcarryoverofFD&CRedNo.40intotheFD&CBlueNo.1fractioncanbeobservedinFigure10.

GrapeandOrangeKool-Aidpowderwereusedas secondary test samples for thisapplication.Thedrinkmixesprovideda slightlymorecomplexsamplematrix,containingchemicalssuchascitricacid,calciumphosphateandnaturalflavorings.TheGrapeKool-AidpowderwaslabeledascontainingFD&CRedNo.40andFD&CBlueNo.1.TheOrangeKool-AidlistedbothFD&CYellowNo.5andFD&CRedNo.40,aswellasFD&CRedNo.40Lake.SomeoverlappingofcolorbandswasapparentinthespectraforbothKool-Aidflavors.

RepresentativespectrafortheGrapeKool-AidcanbefoundinFigures11and12.CrossoverbetweentheFD&CRedNo.40andFD&CBlueNo.1fractionsisapparentinbothspectra,butdoesnothindertheidentificationofthecoloradditive.

RepresentativespectrafortheOrangeKool-AidcanbefoundinFigures13and14.DespitelowconcentrationintheOrangeKool-aid,aswellastheobservanceofsomeFD&CYellowNo.5remaininginthefraction,theFD&CRedNo.40inFigure14isqualitativelyidentifiedthroughthecharacteristicspectradisplayedseenfrom475to575nm.

Findingviableautomationsolutionstotediousandmanualmethods,withtheaddedconcernforworkersafetyinhandlingstrongacidsandbases,createsefficiency,promotesasaferworkenvironment,andallowsforday-to-dayconsistency.Byautomatingstandardlabpracticessuchasspectrophotometerreadingsandsamplepreparation,atypicallaboratoryisabletoincreasesamplethroughput,eliminatepersonneltopersonnelvariationanduselaboratorypersonnelforothermoreimportantlaboratoryworksuchasdataanalysis.Thebasicprocessofhowthemanualmethodwasautomatedcanalsobeappliedtoothermanualmethodsrequiringbothqualitative,asinAOAC988.13,andquantitativespectrophotometricsamplereadings,expandingitsapplicationintomanydifferentsamplecategories.

Thisapplicationshowsequivalencybetweenestablishedmanualmethodologyanditsautomatedcounterpartacrossavarietyofdifferentsamplematrices.Throughadditionaldevelopmentandoptimizationtheoverlappingofcolorbandscouldeasilybeeliminated.Usingflowcellswithalongerpathlengthcouldalsoassistwithobtaininghigherabsorbancereadingsforlowconcentrationsamples.

POSTER10-01

Step Solvent Volume (µL) Air Push (µL)

Condition #1 IPA 2000 1250

Condition #2 1% Acetic Acid 2500 1750

Load Black Food Coloring 1500 600

Fractionate #1 2.5% IPA 2250 750

Fractionate #2 13% IPA 1500 1000

Table 1. SPE Parameters used for the Separation of Black Food Coloring

Load Kool-Aid 2000 1200

Wash 2.5% IPA 3000 1500

Table 2. SPE Parameters used for the Separation of Grape Kool-Aid

Load Kool-Aid 2000 1200

Fractionate #1 2.5% IPA 4000 2500

Table 3. SPE Parameters used for the Separation of Orange Kool-Aid

Figure 13. FD&C Yellow No. 5 from Orange Kool-Aid Figure 14. FD&C Red No. 40 from Orange Kool-Aid

Figure 7. Sample List for Black Food Coloring Separation and Identification

Figure 1. Gilson GX-274 ASPEC with 406 Dual Syringe Pumps

Figure 2. Agilent 8453 UV-Visible Spectrophotometer with Multicell Transport

Figure 3. Standard Spectra for FD&C Yellow No. 5 Figure 4. Standard Spectra for FD&C Red No. 40 Figure 5. Standard Spectra for FD&C Blue No. 1

Figure 6. AOAC Official Method 988.13 SPE Scheme

Sample

Solvent

Colors Eluted

Colors Retained

R2 – FD&C Red 2 R3 – FD&C Red 3 R40 – FD&C Red 40 B1 – FD&C Blue 1 B2 – FD&C Blue 2 Y5 – FD&C Yellow 5 Y6 – FD&C Yellow 6 G3 – FD&C Green 3

ApparatusGilsonGX-274ASPEC™withtwo406DualSyringePump (4)10mLsyringes GXTransferPortAssembly(Special1785) Code386rackfor6mLSPEcartridgesAgilent8453UV-visibleSpectrophotometer MulticellTransport(8-cell) (8)FlowCell(1mm,40µL)PhenomenexStrata®C18-ESPECartridge(6mL/1000mg), P/N8B-S001-JCHGraceAlltech®Extract-Clean™FilterColumns(8.0mL), P/N211108

Figure 8. FD&C Yellow No. 5 from Black Food Coloring Figure 9. FD&C Red No. 40 from Black Food Coloring Figure 10. FD&C Blue No. 1 from Black Food Coloring

Figure 11. FD&C Red No. 40 from Grape Kool-Aid Figure 12. FD&C Blue No. 1 from Grape Kool-Aid

AOAC Official Method 988.13 provides ascheme to follow for the separationof thecoloradditivesviasolidphaseextraction.Arepresentationof this scheme canbe seento the right in Figure 6. This scheme wasutilizedtoestablishproperSPEmethodsforthesamplestested.

Automation of AOAC 988.13 for Increased Throughput in the ...€¦ · POSTER10-01 Step Solvent...

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