Standardized method for solubility and storage of capsaicin-based solutions for cough induction

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Standardized method for solubility and storage of capsaicin-based solutions for cough induction
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Costanzo, Michael T.
Yost, Richard A.
Davenport, Paul W.
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Bio-Med Central (Cough)
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Background: Preparation of inhaled capsaicin solutions for cough induction varies greatly from one lab to another, which creates inconsistencies between tussigenic challenge results. The addition of Tween to these capsaicin solutions provides increased solubility and stability; however, the foul taste of Tween makes inhaling the solution for any prolonged period of time unpleasant. We sought to create a standard method for preparing soluble and stable capsaicin-based solutions (in 10% ethanol/water), without the addition of Tween. Methods: Capsaicin solutions were created at concentrations ranging from 0 to 500 μM in a variety of solvent systems, with and without Tween. Samples were stored in four different environments (−20°C, 3°C, and room temperature, protected from light; and room temperature, exposed to light) to test stability. Detection of capsaicin was carried out by UV absorption. A Grubb’s test was performed on all data to remove statistical outliers. Results: Similar capsaicin concentrations were seen for solutions prepared with or without Tween (Tween provided a slight increase in solubility), with neither solvent system providing complete solubility. Of the four environments tested, storing capsaicin solutions at 3°C while protected from light afforded the greatest stability, for a minimum of 30 weeks. Conclusion: We recommend the use of a 10% ethanol/water solvent system without Tween in the preparation of capsaicin solutions for tussigenic challenges. While this solvent system does not provide complete solubility, we have detailed a method for capsaicin solution preparation that will account for this loss of solubility, while maintaining a solution that is Tween-free and safe for human inhalation. Keywords: Capsaicin, Solubility, Stability, Tussigenic challenge, UV detection
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Costanzo et al. Cough 2014, 10:6 http://www.coughjournal.com/content/10/1/6; Pages
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doi:10.1186/1745-9974-10-6 Cite this article as: Costanzo et al.: Standardized method for solubility and storage of capsaicin-based solutions for cough induction. Cough 2014 10:6.

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© 2014 Costanzo et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHOpenAccessStandardizedmethodforsolubilityandstorageof capsaicin-basedsolutionsforcoughinductionMichaelTCostanzo1,RichardAYost1andPaulWDavenport2*AbstractBackground: Preparationofinhaledcapsaicinsolutionsforcoughinductionvariesgreatlyfromonelabtoanother, whichcreatesinconsistenciesbetweentussigenicchallengeresults.TheadditionofTweentothesecapsaicin solutionsprovidesincreasedsolubilityandstability;however,thefoultasteofTweenmakesinhalingthesolution foranyprolongedperiodoftimeunpleasant.Wesoughttocreateastandardmethodforpreparingsolubleand stablecapsaicin-basedsolutions(in10%ethanol/water),withouttheadditionofTween. Methods: Capsaicinsolutionswerecreatedatconcentrationsrangingfrom0to500 Minavarietyofsolvent systems,withandwithoutTween.Sampleswerestoredinfourdifferentenvironments( 20C,3C,androom temperature,protectedfromlight;androomtemperature,exposedtolight)toteststability.Detectionofcapsaicin wascarriedoutbyUVabsorption.AGrubb ’ stestwasperformedonalldatatoremovestatisticaloutliers. Results: SimilarcapsaicinconcentrationswereseenforsolutionspreparedwithorwithoutTween(Tweenprovided aslightincreaseinsolubility),withneithersolventsystemprovidingcompletesolubility.Ofthefourenvironments tested,storingcapsaicinsolutionsat3Cwhileprotectedfromlightaffordedthegreateststability,foraminimumof 30weeks. Conclusion: Werecommendtheuseofa10%ethanol/watersolventsystemwithoutTweeninthepreparationof capsaicinsolutionsfortussigenicchallenges.Whilethissolventsystemdoesnotprovidecompletesolubility,we havedetailedamethodforcapsaicinsolutionpreparationthatwillaccountforthislossofsolubility,while maintainingasolutionthatisTween-freeandsafeforhumaninhalation. Keywords: Capsaicin,Solubility,Stability,Tussigenicchallenge,UVdetectionBackgroundCapsaicin(CAP)canbeutilizedtotestthecoughthreshold ofpatientsthoughttohavecertainrespiratorydiseases,as wellashealthyindividuals[1-3].Thegreatestchallengesin usingCAPforsuchtestsareitslowsolubilityinwaterand storageinstability.Therefore,whileapurewatersolvent systemwouldbeidealforsuc hsolutionsrequiringhuman consumption,wateralonecannotbeusedsincethe capsaicindoesnotdissolveinsufficientquantitiesto affordmeaningfultesting.Hence,allofthereported studiesusedsolutionsofcapsaicininwatermixedwith lowconcentrationsoforganicsolvents.Fortunately,CAP hasbeenshowntobecompletelysolubleinorganic solvents,withethanol(EtOH)providingthegreatest solubility[4,5].FurtherresearchhasrevealedthatdissolvingCAPinamixtureofEtOHandpolysorbate-80 (Tween-80)allowsforahighersolubilitythanEtOH withoutTween[6-9].OurownresearchsuggestsTween20isanequivalentreplacementsolventforTween-80; thus,Tween-20wasutilizedinthisresearch. CAPsolutionscontainingTween-20havebeenpreparedandtestedonsubjectswithwelldocumented dose – responserelationshipsforcoughreflextesting [10-13].Regrettably,therewasacommoncomplaintof abadtastefromthesolution,asreportedinseveral previousstudiesofTween ’ suseinflavordetectionand awarenessastheadversestimulus[14-16].Thistaste, oftendescribedassoapyandunpleasant,canbeattributed tothepresenceofthesurfactant,Tween-20orTween80, insolution.SincesubjectsarerequiredtoinhaleCAP solutionsforanextendedperiodoftime,thetasteofthe *Correspondence: pdavenpo@ufl.edu2DepartmentofPhysiologicalSciences,UniversityofFlorida,Gainesville,FL 32610,USA Fulllistofauthorinformationisavailableattheendofthearticle Coug h 2014Costanzoetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublicDomain Dedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthisarticle, unlessotherwisestated.Costanzo etal.Cough 2014, 10 :6 http://www.coughjournal.com/content/10/1/6

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solutionsbeginstocausediscomfort.Althoughprevious studiesreportedquantitativecomparisonsofCAPinsolutionswithTweenversussolutionswithoutTween[6,7], totheauthors ’ knowledge,therehasyettobeastudy thatdirectlycomparesthesolubilityofabroadrange ofconcentrationsofCAPin10%EtOHalonetoCAP inaTweensolution.Furthermore,acomparisonof CAPsolubilityinsolutionsofvaryingpercentagesof EtOHhasyettobereported.Thepurposeofsucha comparisonwouldbetoproduceCAPsolutionsinan EtOH-basedsolventsystem,equivalenttothosedeemed fitforuseintheaforementionedcoughthresholdtests [1,3],butwithoutTween. Inthisstudy,wesoughttodetermine(1)thepercentage ofEtOHyieldingCAPsolubilitycomparabletoadesired concentrationofCAPinTween-20solution,(2)theconcentrationofCAPdissolvedin10%EtOHrequiredto produceanalogoussolubilitytoadesiredconcentration ofCAPinsolutioncontainingTween-20,(3)iftheratio ofCAPpresentinEtOHsolutiontoCAPpresentin Tween-20solutionchangeswithvariationsinCAPconcentration,and(4)theshelflifeofCAPin10%EtOH. Basedupontheseresults,wearenowdevelopinga standardizedmethodforoptimalpreparationofCAP solutionsforuseintussigenicchallenges.MethodsChemicalsandreagentsCapsaicin,pharmaceuticalgrade,waspurchasedfrom FormosaLabs(Taoyean,Taiwan)andstoredat 20Cuntil use.190proofethylalcohol(95%EtOH)waspurchased fromDeconLabs(KingofPrussia,PA).HPLCgradewater waspurchasedfromFischer-Scientific(FairLawn,NJ). Tween-20waspurchasedfromMPBiomedicals(Solon, OH).Allsolutionswerestoredatroomtemperature, unlessotherwisestated. Priortoeachstudy,astocksolutionof25mMCAP wasprepared.Initially,theCAPpowderwasdissolvedin asolutionmade-upof80:10:10H2O:Tween-20:95% EtOH(hereafterreferredtoas “ Tweensolution ” ),but thismethoddidnotallowsufficientsolubilityofhigh concentrationsofCAP.Instead,thestocksolutionwas preparedbydissolving76mg(250 mol)ofsolidCAPin 10mLof95%EtOH.Preparingsuchahighconcentration ofCAPforthestocksolutionallowedanyerrorassociated withvariationofvolumetobeconsiderednegligible.In theresearchpresented,solutionswithEtOHlevelsat5, 10,15,20,25,35,50,65,75,and95%EtOHinH2Owere prepared.Additionally,solutionsofCAPwerepreparedat concentrationsof0,200,350,and500 MforallpercentagesofEtOHmentioned.Finally,amuchbroaderrangeof concentrations(0,50,100,150,200,250,300,350,400, 450,and500 M)werepreparedin10%EtOHformore in-depthanalysis.Allsamplesweredilutedtoatotal volumeof5mLintriplicate. TopreparetheTweensolutions,asolutionof80:10:10of H2O:Tween-20:95%EtOH(v/v/v)wasinitiallyprepared. TothisTweensolution,aspecifiedvolumeof25mM stockCAPwasaddedtoachievethedesiredconcentration ofCAP.SolutionsofCAPwerepreparedatconcentrations of0,50,150,250,350,and450 M,withatotalvolumeof 5mL.SimilartotheCAPsolutionsinEtOH,theTween solutionswerepreparedintriplicate. TostudythestabilityofCAPintheoptimizedsolvent system,solutionsatconcentrationsof0,200,350,and 500 MCAPin10%EtOH/H2O(withoutTween-20) wereprepared,andleftinoneoffourdifferentenvironments:1)roomtemperatureandexposedtolight,2)room temperatureandprotectedfromlight,3)approximately 3Candprotectedfromlight,and4)approximately 20C andprotectedfromlight.Allsolutionswerekeptinglass vials,andplacedinacardboardboxtoshelterthemfrom light,exceptonesetofvialswhichwasdirectlyexposedto roomlightforthepurposesofthestudy.Forsolutions storedbelowroomtemperature(3Cand 20C),the solutionswereallowedtowarmuptoroomtemperature fortwohoursandthenvortexed,beforeanalysis.Visual inspectionsofthesolutionspriortoanalysisindicatedno turbidity.Allsampleswerepreparedintriplicatetoafinal volumeof5mL.DeterminationofCAPconcentrationTheconcentrationofCAPinthesolutionswasmeasuredbyultraviolet(UV)absorptionspectrophotometry. AHewlett-Packard8450AUV/Visspectrophotometer wasutilizedtomonitortheabsorbanceat281nm[5]. Althoughtheinstrumentoffersautomaticblanksubtractionduetoitsdouble-beamgeometry,blanksoftheEtOH solventwereanalyzedseparatelyfromthesamples,and manualblank-subtractionwasperformedastheEtOH signalwasnegligible.However,asTween-20exhibited appreciableabsorbanceinthespectralregionofinterest, thedouble-beamcapabilitiesoftheinstrumentwere exploitedtoperformblank-subtractionautomatically. Toensureremovalofanomalousdata,aGrubb ’ stest withaconfidenceintervalof P =0.05wasperformedon anydatasuspectedtobeastatisticaloutlier.Alloutliers weresubsequentlyremovedfromthedataset.ResultsSolubilityofCAPindifferentsolventsystemsInitially,thesolubilityofCAPinvaryingconcentrations ofEtOHinH2Owasevaluated.Figure1displaysthe blank-correctedabsorbanceat281nmofCAPasafunctionofEtOHpercentagepresentinthesolvent.As expected,thesolubilityofCAPincreaseswithincreasing EtOHpercentage;rampingtheconcentrationofethanolCostanzo etal.Cough 2014, 10 :6 Page2of8 http://www.coughjournal.com/content/10/1/6

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from5%to75%resultsina10to15%increasein solubility. Theeffectofamixedsolventsystemcontainingboth Tween-20andEtOHwasalsoinvestigated.Figure2 detailstheCAPabsorbanceresponsecurveforthree differentsolventsystems,10%EtOH,95%EtOH,and 80:10:10H2O:Tween-20:95%EtOH.CAPdissolvedinall threesolventsexhibitedsimilarabsorbance,suggesting thatthesolubilityofCAPissimilarineachsolventsystem tested.Despitethesimilarity,CAPappearedtohavethe Figure1 Theblank-correctedUVabsorbance( =281nm)of200,350,and500 MCAPforvaryingcompositionsofethanol. Errorbars correspondto1standarddeviation. Figure2 Calibrationcurvesforcapsaicininthreedifferentsolvents:10%EtOH,80:10:10H2O:EtOH:Tween-20(Tween),and95% ethanol. Errorbarscorrespondto1standarddeviation. Costanzo etal.Cough 2014, 10 :6 Page3of8 http://www.coughjournal.com/content/10/1/6

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greatestsolubilityin95%EtOH,asevidencedbythe greaterslopeoftheresponsecurve.AsCAPiscompletely solubleinpureEtOH[5],theabsorbancemeasurements obtainedfromCAPinthe95%EtOHsolventsystem wereusedtocalculatethecorrect(orpredicted)CAP concentrations.Theaverageratiooftheconcentration experimentallydeterminedforCAPin10%EtOHto theconcentrationofCAPin95%EtOH( E/P )isaround 91%(Table1).Theaverageratiooftheconcentrationsof CAPin10%EtOHtoTweensolution( E/T )isnearly99%.StabilityofCAPin10%EtOHsolutionsTodeterminethestabilityofCAPin10%EtOHunder differentenvironmentalconditions,atimecoursestudy wasconductedbymonitoringtheabsorbanceofCAP solutions.Thedataforeachofthethreeconcentrations monitored,200,350,and500 MCAP,areshownin Figures3,4,and5,respectively. Ofalltheenvironments,CAPstoredaround3Cand shelteredfromlightyieldedthemostconsistentabsorbancemeasurementsforallthreeconcentrationstested. Furthermore,allthreeconcentrationstestedyieldedabsorbancemeasurementswithinerrorrelativetotheinitial timepointfortheentiretyofthesevenmonthstudy. CAPsolutionsstoredatroomtemperature(~23C), whileprotectedfromlight,demonstratedaconsistency inabsorbancesimilartowhenstoredat3C.Withinerror, therewasnodecreaseinabsorbance,andthereforeno decreaseinCAPsolubilityfrom0to27weeks,forall threeoftheconcentrationstested.Theoneexceptionto thistrendwasthe12weekmeasurementforthe500 M concentration.Thisanomalousdatapointdemonstrated adecreaseinintensitythatisnotwithinerrorofthe observedtrend.Thus,bothvisualandstatisticalanalysis (byutilizationoftheGrubb ’ stest)resultinthedatapoint beingconsideredanoutlier( P =0.05). CAPsolutionsstoredatroomtemperature,andexposed tolight,exhibitnodecreaseinabsorbancethroughout theentiresevenmonthsforconcentrationsof200and 350 M.Conversely,forthehighestconcentrationof 500 M,thereisasignificantdecreaseinabsorbanceof CAPstartingafteronly2monthsofstorage.Asingle measurementforthe500 Msolutiontakenatthesixth weekofstorage(Figure5)showsadecreaseinabsorbance, beforereturningtoanabsorbancesimilartotheweek prior,andthengraduallydecreasingtolowerabsorbance asthefurtherweeksprogressed.AGrubb ’ stestwas againutilized,anddeterminedthatthisdatacouldbe consideredanoutlier( P =0.05). ThedatashownforCAPstoredat 20C,whileprotectedfromlight,provedtobetheleastconsistent,in termsofbothstabilityanderror.The200 Msolution demonstrateslittletonodecreaseinabsorbanceforthe firstfourmonthsofstoragebeforebeginningtoslowly diminishinintensity(Figure3).The350and500 M solutionsrevealedamuchsoonerdrop-offinintensity, astheabsorbancebegantosignificantlydecreaseafter onlyamonth(Figures4and5).Fortheconcentrations of200and350 M,thedropinabsorbanceintensity correlateswithadramaticincreaseinstandarddeviation ofthedata,renderingthedataquestionableatbest.The measurementstakenfor500 Mcontainmuchlessassociatederror,andcanbeconsideredmorereferential.DiscussionStudiesreportedbyKopecetal[6,7].providedthe groundworkforthisstudyofCAPsolutionsinvolvingthe useofTweenasasolvent.However,thisreportinvolvesa morecomprehensivecomparisonofTweenandEtOHbasedsolutions,intermsofbothsolubilityandstability. EachCAPsolutionwascomprisedofonlyafewcomponents(e.g.CAP,H2O,EtOH,andTween-20),andeach componenthasadifferentmaximumabsorbanceinthe UVspectrum.Duetoalackofsamplecomplexity,UV/Vis detectionwasperformedwithoutpriorHPLCseparation, unlikethepreviousstudy.Therefore,timeofanalysis wasgreatlyreduced.TheHPLCanalysisdescribedby Kopecetal.required12minutespersample[7],whereas anaveragesampleanalysistimewiththemethoddescribed inthisworkwassignificantlyfaster(~3minutes).Furthermore,therewereinitialconce rnsoverthesolubilityofthe capsaicin,asparticleslargeenoughtoscatterlightwere observedinsolutionsstoredbelowroomtemperature duringthetime-coursestudy;however,theseparticles allredissolvedoverthecourseofthe2-hourwarming periodandvortexingofsamplesbeforeanalysis.Visual inspectionsofthesolutionspriortoanalysisindicated noturbidity.Theuseofaspectroscopictechniquepermits non-destructiveandrapidanalysisofthesamples,but affordslessselectivitythanwouldbeachievedwitha supplementaryHPLCseparationstep[17].Futurestudies utilizingHPLCseparationorotheranalyticaltechniques couldprovideconfirmationoftheseresults. Fromtheresultsofourstudy,itcanbeconcludedthat CAPsolutionsthataresimilarinsolubilityandstability Table1CAPconcentrationinEtOH-vs.Tween-based solventsystemsConcentrations( M) Absat281nm1std.dev.(3replicates) 95%EtOH10%EtOHTweenRatio( E/P ) 50.02.345.13.343.12.70.900.08 150.00.1135.84.6137.70.80.910.03 250.00.1226.58.8232.34.40.910.05 350.00.1317.23.5326.94.40.910.01 450.00.1407.920.14218.90.910.05 Costanzo etal.Cough 2014, 10 :6 Page4of8 http://www.coughjournal.com/content/10/1/6

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tothosewithTween-20canbepreparedusingpurely EtOH-basedsolvents,yetlackthefoultasteassociated withTween.Kopecetal[6,7].determinedthatutilization ofasolventsystemofTween-80,ethanol,andwaterfor preparationofCAPsolutionsyieldsbettersolubilityand stabilitythanasolventsystemcomprisedofethanoland waterwithoutTween.However,theconcentrationofethanolintheirreportedsolutionswasonlyaround1%.The primarypurposeofourstudywasdetermineifpreparing capsaicinsolutionsinsimilarfashiontoKopecbenefitted Figure3 StabilityoftheUVabsorbancemeasurements( =281nm)of200 MCAPsolutionspreparedin10%EtOH,asafunctionoftime, forfourdifferentstorageenvironments: 20C(dark),3C(dark),23C(dark),23C(light). Errorbarscorrespondto1standarddeviation. Figure4 StabilityoftheUVabsorbancemeasurements( =281nm)of350 MCAPsolutionspreparedin10%EtOH,asafunctionoftime, forfourdifferentstorageenvironments: 20C(dark),3C(dark),23C(dark),23C(light) .Errorbarscorrespondto1standarddeviation. Costanzo etal.Cough 2014, 10 :6 Page5of8 http://www.coughjournal.com/content/10/1/6

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fromTweenadditionenoughthatitsusewouldberecommended.AsthereportedpreparationoftheTweensolutionsdidnotinvolveanyseparationstep,wewantedto analyzethesolutionsastheywouldbeprepared clinically.Inthisrespect,anyaspectofdissolutionthat mayresultinsuspension/dispersionofCAPwould accuratelyreflectclinicalim plicationswhenconducting thetest.Asdiscussedinmoredetailbelow,noturbidity wasobservedinsamplesreadyforanalysisfollowing theirwarmingtoroomtemperatureandvortexing.The presentworksuggeststhatTween-20yieldsalessthan 2%increaseinsolubilityoverasolventsystemofethanolandwater,wheretheethanolconcentrationis10% ratherthan1%byvolume.Furthermore,regardlessof theinclusionorexclusionofTween-20,solventsystems safeforhumanconsumption(i.e.,lessthan20%ethanol byvolume)generallydemonstrateCAPconcentrations belowthepredictedconcentration;therefore,neither allowscompletesolubility. AsnotedinFigure1,thereisamodestincreasein solubilityasthecompositionofEtOHisincreasedfrom 5to75%;however,thisdifferencecanbeeasilycorrected forbydissolvingadditionalCAPintothesolution,as describedinmoredetaillater.Thus,lowerpercentagesof EtOHarerecommendedtoallowsafehumaninhalation, approximately20%orlessEtOH.Therefore,wehave chosentouse10%EtOHsolutionstodecreasetheamount ofEtOHsolventintheinhaledvapor.Furthermore,the concentrationofCAPusedforthetussigenicchallenge doesnottypicallyexceed500 M;[12]therefore,anincreaseinvariationbetweenhighandlowEtOHcompositionsathigherconcentrationsofCAPdoesnotbecomea factor. Forsolutionsin10%EtOH,thereisa10%decreasein solubilityrelativetothepredictedsolubility(Table1); predictedconcentrationisdeterminedfromabsorbance measuredforCAPdissolvedinthe95%EtOHsolvent system.Thiscanbeovercomebyincreasing,by10%,the amountofCAPinitiallydissolvedinsolution.Accordingly,asuggestedstandard izedsamplepreparationfor CAPsolutionsinanEtOH-basedsolventsystemis detailedbelow.RecommendationsofpreparationofCAPsolutionsin EtOHsolventsystemInthisstudy,weused95%EtOHandaccountedforthe impurityinourdilutions;however,thefollowingguidelineuses100%EtOHtoallowforeasiercalculation. Note:Theethanolusedmustbesafeforhumanconsumptionandthecapsaicinpharmaceuticalgrade. Step1. DissolvesolidCAPin100%anhydrousEtOH tocreateaconcentratedCAPstocksolution.Dissolving 0.7635gofsolidCAP(2.5mmol)in100mLof100% EtOHwillproducea25mMstocksolutionofCAP. Step2. PreparethesolventfortheinhaledCAPsolutionsbydiluting100%EtOHwithH2O.Tocreate10% EtOHsolutions,combine100mLof100%EtOHwith 900mLofH2O. Figure5 StabilityoftheUVabsorbancemeasurements( =281nm)of500 MCAPsolutionspreparedin10%EtOH,asafunctionoftime, forfourdifferentstorageenvironments: 20C(dark),3C(dark),23C(dark),23C(light). Errorbarscorrespondto1standarddeviation. Costanzo etal.Cough 2014, 10 :6 Page6of8 http://www.coughjournal.com/content/10/1/6

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Step3. AddtheCAPstocktothe10%EtOHtoproducethedesiredconcentrationofCAPin10%EtOH, usingthefollowingequation: Vstock Ccap VcapCstock 1 : 1 1 Where Cstockand VstockaretheconcentrationandvolumeoftheCAPstocksolution,and Ccapand Vcaparethe concentrationandvolume,respectively,ofthedesired CAPsolutionin10%EtOH.Recallthattheinitialamount ofCAPaddedneedstobeincreasedby10%toaccount forthedecreaseinsolubilityassociatedwitha10%EtOH solvent.Equation1maybeutilizedtoprepareanydesired CAPsolution,orTable2mayprovideaquickreference forspecificconcentrations.StabilityofCAPin10%EtOHsolutionsThestabilityofsolutionsofCAPpreparedwithTween80havebeenpreviouslyreported[7],hence,wedidnot repeatasimilarinvestigation.Instead,wefocusedsolely onthestabilityofCAPsolutionspreparedin10%EtOH, whichweredilutedinHPLC-gradewaterratherthan salinesolution. Themostpertinentfindingsfromthisstudyshowthat thegreateststabilitymaybeachievedbystoringthe specifiedsolutionsofCAPinalight-freeenvironment, andatatemperatureofapproximately3C.Undersuch conditions,thesolutionsofCAPin10%EtOHremained stable,overarangeofconcentrations,foraminimumof 30weeks(nearly8months,thedurationofthestorage study),asseeninFigures3 – 5.Additionally,thehighly concentratedstocksolutionsofCAPpreparedinpure EtOHremainedstableforatleastayear(themaximum timetested)underthesameconditions.Thisincreasein stabilityofnon-TweenCAPsolutioncomparedtothat showninthepreviousstudy[6,18]maybeattributedto eithertheuseofdistilledwaterasthedilutionsolvent ratherthansalinesolution,ortheincreaseinEtOH composition.UseofsalinemaydestabilizetheCAP solutionscausingthemtodegradequicker.Alternatively, thepresenceofahigherpercentageofEtOHinsolution mayprovidegreaterstabilizationofCAP.Thedrasticdrop insolubilityofsolutionsstoredat 20Cmightbedueto thelowtemperaturecausingsomeCAPtoprecipitateout ofsolutionandadsorbtothewallsofthecontainer,and thennotredissolveoncethesolutionisbroughtbackup toroomtemperatureandvortexed.ConclusionsInthisstudy,wedemonstratedthatCAPsolutionspreparedina10%EtOHsolventsystemarenearlyassoluble asCAPsolutionspreparedinaTween-incorporatedsolvent system.Althoughneithersolventsystemallowscomplete solubilityofCAP,theconcentrationsthatcanbeachieved arequiteadequatefortussigenicchallenges.Therefore,to avoidthefoultasteofTweenandstillefficientlyprepare solubleCAPsolutionsin10%EtOH,asimpleequation maybefollowed(Equation1).Inthiswork,wehaveprovidedthegroundworkforcreatingastandardizedapproach topreparingCAPsolutionsforuseintussigenicchallenges. Untilfurtherrefinedmethodsar eestablished,thepreceding preparationstepsshouldbetakentoachievemaximum solubilityofCAPinsolution.Similarly,foroptimalstorage, keepsolutionsshieldedfromlightandatatemperatureof around3C. OngoingstudieswereperformedutilizingtheTweenfreemethodsforpreparingcapsaicinsolutionstoelicit cough(unpublishedresults).Tween-free50 – 500 M capsaicinsolutionswerepreparedin90%physiological salineand10%ethanol.Thesesolutionswereusedto elicitcoughinnormalsubjects.Thesesolutionswere usedimmediatelyafterpreparationandafterstoragefor 1 – 4months,eitherrefrigeratedoratroomtemperature. Nodifferencesasafunctionofvehiclesolution(90% physiologicalsaline,10%ethanolvs80%physiological saline,10%Tween20,and10%ethanol),storagetime,or storagetemperaturewereobservedforcapsaicinelicited coughthreshold,coughnumber ,Urge-to-CoughThreshold, andUrge-to-Coughsensitivitycomparedtopriorstudies [13,19,20]forequalconcentrationsofcapsaicin.There appearstobefewervehicleelicitedcoughswith90% physiologicalsalineand10%ethanolsolutionscompared to80%physiologicalsaline,10%Tween20,and10% ethanolsolutions.Futurestudiesarerequiredtosystematicallycomparethecoughresponsetocapsaicinusing thesevehicle(90%physiologicalsaline,10%ethanolvs 80%physiologicalsaline,10%Tween20,and10%ethanol) solutions.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Table2Recommendedpreparationof1LCAPsolutions atarangeofuseful(tussigenic)concentrationsDesiredconcentrationof capsaicininsolution( M) Volumeof25mM capsaicinstock(mL) Volumeof10% ethanol(mL) 50.22999.78 100.44999.56 200.88999.12 401.76998.24 1004.40995.60 1506.60993.40 25011.00989.00 37516.50983.50 50022.00978.00 Costanzo etal.Cough 2014, 10 :6 Page7of8 http://www.coughjournal.com/content/10/1/6

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Authors ’ contribution MCparticipatedintheconception,design,analysis,interpretation,drafting themanuscript,criticallyreadingitandfinalapprovalofthesubmitted manuscript.RYparticipatedinthedesign,analysis,interpretation,critically readingthemanuscriptandfinalapprovalofthesubmittedmanuscript. PDparticipatedintheconception,design,analysis,interpretation,critically readingthemanuscriptandfinalapprovalofthesubmittedmanuscript. Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements TheauthorswouldliketothankDr.BenjaminKillianforaccesstothe spectrophotometerusedinthisresearch. Authordetails1DepartmentofChemistry,UniversityofFlorida,Gainesville,FL32611,USA.2DepartmentofPhysiologicalSciences,UniversityofFlorida,Gainesville,FL 32610,USA. Received:11April2013Accepted:24April2014 Published:25September2014 References1.O'ConnellF,ThomasVE,PrideNB,FullerRW: Capsaicincoughsensitivity decreaseswithsuccessfultreatmentofchroniccough. AmJRespirCrit CareMed 1994, 150: 374 – 380. 2.FullerRW: Pharmacologyofinhaledcapsaicininhumans. RespirMed 1991, 85: 31 – 34. 3.IrwinRS,CorraoWM,PratterMR: Chronicpersistentcoughintheadult: thespectrumandfrequencyofcausesandsuccessfuloutcomeof specifictherapy. AmRevRespirDis 1981, 123: 413 – 417. 4.TurgutC,NewbyB,CutrightTJ: Determinationofoptimalwatersolubility ofcapsaicinforitsusageasanon-toxicantifoulant. EnvironSciPollutRes Int 2004, 11: 7 – 10. 5. TheMerckIndex. In EncyclopediaofChemicals,Drugs,andBiologicals (14thedition). EditedbyO ’ NeilMJ.WhitehouseStation,NJ:Merck ResearchLaboratories;2006. 6.KopecSE,DeBellisRJ,IrwinRS: ChemicalAnalysisofFreshlyPrepared andStoredCapsaicinSolutions:ImplicationsforTussigenicChallenges. PulmPharmacolTher 2002, 15: 529 – 534. 7.KopecSE,IrwinRS,DebellisRJ,BohlkeMB,MaherTJ: TheeffectsofTween-80 ontheintegrityofsolutionsofcapsaicin:usefulinformationforperforming tussigenicchallenges. Cough 2008, 4: 3. 8.FujimuraM,KasaharaK,KamioY,NaruseM,HashimotoT,MatsudaT: Femalegenderasadeterminantofcoughthresholdtoinhaled capsaicin. EurRespirJ 1996, 9: 1624 – 1626. 9.FujimuraM,KasaharaK,YasuiM,MyouS,IshiuraY,KamioY,HashimotoT, MatsudaT: Atopyincoughsensitivitytocapsaicinandbronchial responsivenessinyoungfemales. EurRespirJ 1998, 11: 1060 – 1063. 10.DavenportPW,SapienzaCM,BolserDC: Psychophysicalassessmentofthe Urge-to-Cough. EurRespirRev 2002, 12: 249 – 253. 11.DicpinigaitisPV,AllussonVR,BaldantiA,NalamatiJR: Ethnicandgender differencesincoughreflexsensitivity. Respiration 2001, 68: 480 – 482. 12.DicpinigaitisPV,AlvaRV: Safetyofcapsaicincoughchallengetesting. Chest2005, 128: 196 – 202. 13.VovkA,BolserDC,HeyJA,DanzigM,VickroyT,BerryR,MartinAD,Davenport PW: Capsaicinexposureelicitscomplexairwaydefensivemotorpatternsin normalhumansinaconcentration-dependentmanner. PulmPharmacol Ther 2007, 20: 423 – 432. 14.BaeyensF,HendrickxH,CrombezG,HermansD: Neitherextended sequentialnorsimultaneousfeaturepositivetrainingresultin modulationofevaluativeflavor-flavorconditioninginhumans. Appetite 1998, 31: 185 – 204. 15.CoppensE,VansteenwegenD,BaeyensF,VandenbulckeM,VanPW, EelenP: Evaluativeconditioningisintactafterunilateralresectionofthe anteriortemporallobeinhumans. Neuropsychologia 2006, 44: 840 – 843. 16.DickinsonA,BrownKJ: Flavor-evaluativeconditioningisunaffectedby contingencyknowledgeduringtrainingwithcolor-flavorcompounds. LearnBehav 2007, 35: 36 – 42. 17.SkoogDA,HollerFJ,CrouchSR: PrinciplesofInstrumentalAnalysis. 6th edition.ThomsonBrooks/Cole:Belmont,CA;2007. 18.MidgrenB,HanssonL,KarlssonJA,SimonssonBG,PerssonCGA: Capsaicininducedcoughinhumans. AmRevRespirDis 1992, 146: 347 – 351. 19.HeglandKW,BolserDC,DavenportPW: Volitionalcontrolofreflexcough. JApplPhysiol 2012, 113: 39 – 46. 20.HeglandKW,TrocheMS,DavenportPW: Coughexpiredvolumeand airflowratesduringsequentialinducedcough. FrontPhysiol 2013, 4: 167.doi:10.1186/1745-9974-10-6 Citethisarticleas: Costanzo etal. : Standardizedmethodforsolubility andstorageofcapsaicin-basedsolutionsforcoughinduction. Cough 2014 10 :6. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Costanzo etal.Cough 2014, 10 :6 Page8of8 http://www.coughjournal.com/content/10/1/6