Contents lists available atScienceDirect
Journal
of
Pharmaceutical
and
Biomedical
Analysis
j o u r n a l h o m e p a g e :w w w . e l s e v i e r . c o m / l o c a t e / j p b a
Development
of
a
fast
LC-MS/MS
protocol
for
combined
measurement
of
six
LSDs
on
dried
blood
spot
in
a
newborn
screening
program
Emanuela
Scolamiero
a,1,
Bruno
Casetta
a,1,
Sabrina
Malvagia
a,
Tetsuo
Tanigawa
b,
Giulia
Forni
a,
Silvia
Funghini
a,
Massimo
Mura
a,
Francesca
Raspini
a,
Sara
Poggiali
a,
Giancarlo
la
Marca
a,c,∗aNewbornScreening,BiochemistryandPharmacologyLaboratory,PediatricNeurology,UnitandLaboratories,MeyerChildren’sUniversityHospital,
Florence,Italy
bAnalytical&MeasuringInstrumentsDivisionShimadzuCorporation,Kyoto,Japan
cDepartmentofExperimentalandClinicalBiomedicalSciences,UniversityofFlorence,Florence,Italy
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received30August2018
Receivedinrevisedform9November2018 Accepted2December2018
Availableonline3December2018 Keywords:
Lysosomalstoragedisorders Newbornscreening Driedbloodspot Massspectrometry
a
b
s
t
r
a
c
t
NewtreatmentoptionsandimprovedstrategiesforLysosomalStorageDisorders(LSDs)diagnosison driedbloodspot(DBS)haveledtothedevelopmentofseveralpilotnewbornscreeningprograms.
Buildingonapreviouslypublishedprotocol,wedevisedanew6-plexassaybasedonasingleDBSpunch incubatedintoabuffercontainingacombinationofsubstrates(GAA,GLA,ASM,GALC,ABGandIDUA). Thisnewprotocolincorporatesanewtrappingandclean-upprocedureusingperfusionchromatography connectedon-linewithananalyticalcolumnforanalyteseparation,afterenzymaticreaction.Resultsare availableafter4.5min.
Severalincubationtimesweretestedinordertoreducesamplepreparationtimesandtoimprove accuracyandreproducibility,alsoregardingthequenchingofthereactionwithinthetimewindowof linearproductaccumulation.Thecollecteddatademonstratethatanincubationtimeof4hisenoughto achievegoodreactionefficiencywithoutanyimpactonsensitivity.
Themethodprovedversatileandrobustforvariousinstrumentconfigurations.Thefastsample prepara-tionandrunningtimesallowahighsamplethroughput;anadvantageinnewbornscreeningprocedures. Thismethodcanalsobeusedfordiagnosticpurposes,allowingarapiddiagnosisinafewhours.
©2018ElsevierB.V.Allrightsreserved.
1. Introduction
LysosomalStorageDisorders(LSDs)areagroupofmorethan50 diseasessharinganimpairmentoflysosomalfunction.Overrecent years,manyoftheseconditions,whichcanbemoresuccessfully
Abbreviations: ABG,ß-Glucocerebrosidase;ASM,Acidsphingomyelinase;CDC, Centre forDisease Controland Prevention; CV, Coefficient ofvariation;DBS, DriedBloodSpot;DL,DesolvationLine;ESI,Electrosprayionization;GAA,Acid ␣-glucosidase;GALC,Galactocerebrosidase;GLA,␣-galactosidaseA;HPLC,High PerformanceLiquidChromatography;IDUA,␣-L-iduronidase;IS,Internalstandard; LC–MS/MS,Tandemmassspectrometrycoupledwithliquidchromatography;LSDs, LysosomalStorageDisorders;MPS-I,Mucopolysaccharidosis-I;MRMMultiple, reac-tionmonitoring;P,Product;Q1,FirstQuadrupole;Q3,ThirdQuadrupole;QC,Quality Control;RF,ResponseFactor;S,Substrate.
∗ Correspondingauthorat:DepartmentofExperimentalandClinical Biomed-icalSciences,UniversityofFlorence,NewbornScreening,ClinicalChemistryand PharmacologyLab,MeyerChildren’sHospital,vialePieraccini24,Florence,Italy.
E-mailaddress:giancarlo.lamarca@meyer.it(G.laMarca).
1 Theseauthorscontributedequallytothiswork.
treatedifdiagnosedearly,havebeenaddedtonewbornscreening panelsaspilotprojects[1–3].
Since2001,approachesforidentifyingLSDpatientshavebeen developedinresearchsettings.Chamolesandcolleaguesdescribed a fluorometric assay for diagnosing LSDs based on the use of driedbloodspots(DBS)[4–7].Subsequently,Gelbandcolleagues providedappropriatesubstratesandisotopically-labelledinternal standardsforperformingmultipleenzymaticassaysbyLC–MS/MS onDBS[8].Currently,LC–MS/MSisthemostcommonlyused tech-niqueforidentifyingLSDsduringtheneonatalperiodbeforeclinical symptomsappear[2,3,9].
In2009,therewasasignificantbreakthroughwhenaspecificLC configurationwasdevised[10]forskippingthepurificationsteps thatwererequiredbeforeinstrumentalmeasurementinthe orig-inalprotocol.Thisconfigurationledtoamoreroutineapplication ofthemethodastherelativelyshortanalyticalmeasurementtime (around4min,injection-to-injection),meantthatitwaspossibleto achievehighersamplethroughputs.Moreover,theprotocolrelied ona singlehigh-pressurebinaryLCpumpand switchingvalve, https://doi.org/10.1016/j.jpba.2018.12.002
136 E.Scolamieroetal./JournalofPharmaceuticalandBiomedicalAnalysis165(2019)135–140
makingthehardwareconfigurationeasilyaffordableforany New-bornScreeningCenter.
Since this time, Mucopolysaccharidosis type I (MPS-I) [ ␣-L-iduronidase(IDUA)deficiency]hasalsobeenincludedinscreening protocols, alongside the five classical disorders, Pompe [acid ␣-glucosidase (GAA) deficiency], Fabry [acid ␣-galactosidase A (GLA) deficiency], Niemann-Pick [acid sphingomyelinase (ASM) deficiency],Krabbe[galactocerebrosidase(GALC)deficiency]and Gaucher[acidß-glucocerebrosidase(ABG)deficiency] [11]. This waspossiblethankstosomemodificationsonthechromatographic runwhichmadetherelatedIDUAISandIDUAenzymaticproduct measurable.
Manyprotocolsforlysosomalenzymeactivationand measure-menthavebeendevelopedtoallowthesimultaneousdetectionof severalenzymesononeortwoDBSpunches[12–15].
To ensure complete substrate digestion all the previously described protocols require long incubation times (at least 16–22hours).However,thisstudydemonstratesthatmore accu-rateproductquantificationcouldbeachievedwhentheenzymatic reactionisstillinitslinearitywindowwhereproductformation yieldisdirectlyproportionaltoincubationtime.
Shorteningincubationtimesmakeslargescalenewborn screen-ing more affordable and considerably reduces times for recall, diagnosisandtherapeuticinterventioninthecaseofsevere neona-talonsetformsforwhichveryearlytreatmentcontributestobetter outcomes[16].
2. Materialandmethods 2.1. Chemicalsandreagents
Substrate-Internalstandardmixtureswereobtainedfromthe CentreforDiseaseControlandPrevention(CDC,Atlanta,Georgia). Methanol,Isopropanol(LC–MSgrade)andFormicacid(purity 98–100%)were purchasedfromSigma-Aldrich(Riedel deHaën, Germany).Acetonitrile(LC–MSgrade)andwater (LC–MSgrade) were procured from Biosolve Chemicals, (Valkensward, The Netherlands).InvitrodiagnosticWhatman903®gradebloodspot
cardswerepurchasedfromWhatman(Dassel,Germany).Allthe chemicalsandreagentswereusedascommerciallysupplied with-outfurtherpurification.
2.2. Assaycocktailpreparation
Each vial containing substrate-IS mixture was dissolved in methanolfollowingtheprotocoldevelopedbySpaciletal.[12].A 1mLaliquotfromeachvialwasmixedtogether,driedunder nitro-genflowandhydratedin10mLof6-plexreactionbuffer,prepared accordingtotheprocedurereportedbyElliottetal.[17].
2.3. Samplepreparation
A3.2mmdiameterdisc,equivalenttoapproximately3.4Land 2.8Lofwholebloodfornewbornsandadultsrespectively,was punchedoutdirectlyintoa96-wellplate.Sampleswereextracted andincubatedin30Lofcocktailbufferat37◦C;different incuba-tiontimesupto12hweretested.Foursampleswerepreparedand analysedateachtimepoint(2,4,6,8,10and12h).After incuba-tion,enzymaticreactionswerequenchedby250Lofmethanol +0.1% formic acidand the platewas centrifugedat 2500g for 5min.Thesupernatantwastransferredtoanew96-wellplateand a2LsamplewasinjectedintotheLC–MS/MSsystemthroughthe autosamplerkeptat8◦C.
2.4. MassspectrometryandHPLCparameters
AllmeasurementswereperformedusinganLCMS-8050 Triple-QuadMassSpectrometer(Shimadzu,Japan)equippedwithanESI sourceandworkinginMRMmode.Systemcontrolanddata anal-ysiswereperformedbyLabSolutionssoftware,version5.65.MRM transitionsforeachcompoundwereoptimizedbyflowinjection andarelistedinSupplementaryInformation(TableS1).
Otheroperatingparameters wereasfollows:NebulizingGas Flowof3.0L/min;HeatingGasFlowof5.0L/min;Interface Temper-atureof100◦C;DLTemperatureof100◦C;HeatBlockTemperature of100◦C;DryingFlowof15L/min.
TheHPLCsystem(Shimadzu,Japan)consistedofaDegassing UnitDGU-20A5R,controllerCBM-20A,twopumpmodulesNexera X2LC-30AD,autosamplerNexeraX2SIL-30ACandcolumnoven CTO-20ACequippedwithasix-portvalve.TheLCmoduleswere plumbedasshowninFig.1.
Trappingandelutionprocedureswereperformed,asa modifica-tionofthedescribedprotocol[12]withaperfusioncolumnPOROS R1/202×30mm(AppliedBiosystems,FosterCity,CA,USA).Forthe chromatographicelutiondifferentcolumnsweretested,including aSynergiFusionRP-802×50mm,4m(Phenomenex,Torrance USA),aShimPackXR-ODScolumn2×50mm,2.2m(Shimadzu, Japan),andaZorbaxSB-CN2,1×50mm,5m(Agilent,Italy).The ZorbaxSB-CNwaschosenforroutineuse.Thetemperatureofthe columnovenwassetat45◦C.
PumpAmodulewasfedbyanaqueoussolutionof0.05%formic acidcontaining10mmol/LofammoniumformatewhilepumpB modulewasfedbyamixtureofacetonitrile-methanol-isopropanol (50:25:25,v/v/v)containingformicacidat0.1%.
Theentireworkflowwasprogrammedinthreestepsmanaged bytheswitchingvalve:sampleloadingandclean-up(1min); ana-lyteelution(2.5min);forthefinalcolumnsreconditioning(1min) (Table1).
Inthefirststep,theinjectedsampleismovedtothetrapping col-umnwith1.6mL/minofeluentAforthecleanup.After1min,the valveisswitched,andboththeeluentsAandBflowsaremerged forproviding,aftera0.5min-delayat0%,agradientof50%eluent Bmovingto100%in2.0minatatotalflowrateof400L/minover thetrappingandelutingcolumnsnowconnectedinseries.Withthe analytesmovedfromthetrappingcolumntotheelutioncolumn, thevalveisswitchedbacktodisconnectthetwopumpsandenable boththecompletionofchromatographicseparationoftheanalytes withthefullsolventBat400L/minoverthechromatographic col-umn,andthesimultaneousreconditioningofthetrappingcolumn forthefollowinginjection.Thislaststeplasts1minforatotalrun of4.5min.
2.5. Calculationofenzymeactivity
Enzymeactivitywasreportedasmol/L/handcalculatedby usingthefollowingformula:
Enzymeactivity =RF×(Pa/ISa)x([ISc]xVa/Vdbs)/4h
Va=Assaycocktailvolume=30L
Pa=PeakareaProduct
ISa=PeakareaInternalStandard
ISc=InternalStandardconcentration
RF=Response Factor: ratio between the Internal Standard responseandtheProductresponse.
Vdbs=3.4 or 2.8L of wholeblood contained into a 3.2mm
punchfornewbornandadults,respectively 4h=incubationtime
Fig.1. Layoutofthenewplumbing.PositionofdepictedvalveisA.
Table1
LCparameters.
Step ValvePosition Time Pumps TotalFlow %B PumpAFlow PumpBFlow
1 A 0.0min Disconnected 1.6mL/min 0.4mL/min
2 B 1.0min Connected 0.4mL/min 0
1.5min 50
3.5min 100
3 A 3.5min Disconnected 1.6mL/min 0.4mL/min
End 4.5min
Table2
ReferencevalueofLSDenzymeactivityinnewbornandadultpopulation. CUTOFF(mol/L/h)
ENZYME NEWBORN (n=1000) ADULTS (n=200) Galactocerebrosidase(GALC) ≥0.4 ≥0.4 ␣-Glucosidase(GAA) ≥6.0 ≥6.7 ␣-L-Iduronidase(IDUA) ≥3.4 ≥6.6 ␣-Galactosidase(GLA) ≥1.9 ≥2.2 ß-Glucocerebrosidase(ABG) ≥2.9 ≥2.9 Sphingomyelinase(ASM) ≥2.6 ≥2.7
2.6. Interdayandintradayvariability
ImprecisionwasdeterminedinsixreplicatesoffourCDCcontrol
samplesonthesameday(intra-dayimprecision)andduplicates
for5timesoveraperiodofoneweek(inter-dayimprecision).The
imprecisionsoftheassaywereexpressedasapercentage
coeffi-cientofvariation(CV%).
2.7. Cutoffdetermination
Referencevalues forthesix lysosomal enzymeswere
deter-minedbothfor newbornandadultsby processingDBSof1000
randomlychosennewbornsand200adults.Qualitycontrols
pro-videdbyCDCandtruepositivesampleswereanalysedtotestthe
performanceofthemethodanditsabilitytodistinguishhealthy
individualsfromaffectedindividuals.
Thelowerlimitofnormalwassetatthelevelofthe0.2nd
per-centile(Table2)
3. Resultsanddiscussion 3.1. Enzymereactionparameters
Compared to similar studies, we totally revisedthe sample preparationprocessincludingtheincubationtimewhichis piv-otalforatimelydiagnosis.Longincubationtimesareoftenused toallowcompletesubstratedigestion.However,thismeansthat thecalculatedtime-basedenzymeactivitymaybeunderestimated afterthereactionplateauisreached.Ashortassaytimeshouldlead toamoreaccurateproductquantificationsincethereactionisstill inthelinearrange.
Severalincubationtimesofupto12hweretestedforthesix enzymaticreactions.Therelationshipbetweenproductyieldand timewasdifferentforthesixenzymesbutcommonlylinearupto 6h(Fig.2).
Withinthetimewindowoflinearproductaccumulation,we choseanincubationtimeof4hbecausethisprovedlongenoughto produceagoodsignalofproductsandisshortenoughtobe incorpo-ratedintoaroutinelabprocedure.Anincubationtimeoflessthan 4hwouldriskcompromisingaccuracyandanincubationtimeof morethan4hwouldbeincompatiblewiththetimedemandsand routinesofmanylaboratories.”
Thisshortenedsamplepreparationtimesatisfiestheneedfor amorerapidanalyticalstrategy whichleadstofasterdiagnoses meaningthatERTcanbeintroducedearlierandpatientoutcomes improved[16].
DBSfromhealthynewbornsandadultswereanalysedfor cut-offdeterminationandthemethodwasvalidatedbymeansofboth CDCcontrolsandtruepositivesamples.Although,itisoftenvery difficulttofindsufficientnumbersofDBSfromtruenewborn
posi-138 E.Scolamieroetal./JournalofPharmaceuticalandBiomedicalAnalysis165(2019)135–140
Fig.2. Thegraphaboveshowstheenzymaticreactionsstoppedatdifferenttimes.Theresultsareplottedasareaproductagainstincubationtimeinordertoidentifythe windowtimeoflinearity.Thecurvesappeartohavealinearportionupto6h.Theenzymaticactivitycanbeaccuratelyquantifiedifthereactionisstoppedinthelinear phasewhentheproductformationstillincreasesinagivenunitoftime.
tivepatients,wewereabletotesttheassayeffectivenesson8truly affectedpatientsofwhom4werenewborns(1Pompe,2Fabry, 1MPSI)and4adults(1Pompe,2Gaucherand1Krabbe).Since nopatientwithNiemann-PickA/Bdiseasewasavailable,theassay wastestedonCDCsamples.Allpositivesampleswerecorrectly identified.
3.2. MS/MSconditionsandLCconfiguration
In2009afirstapproachforafiveLSDenzymeassaybasedon anon-linecleanupfollowedbychromatographicseparationwas developedinordertosimplifyandspeedupsamplepreparation
[10].Subsequentlyamultiplexassaywasimplementedbyadding IDUAmeasurementtotheotherfiveenzymes[11].
In the new protocol described in this work, specific mass spectrometricparametershavebeenslightlymodified,whileLC configurationandoptimizationhavebeenprofoundlyrevised.This wasnecessarybecauseofdiscrepanciesconcerningtheaccuracy ofIDUAresultsafterrunningseveralthousandsofsamples.After checkingallthestepsoftheprotocol,concernsaroseaboutthe peak shape of both the IDUA IS and IDUA product.Their peak shapesdenotedasthesignalsweresignificantlydistortedby resid-ualmatrixcomponentssupposedlyelutingatthesameretention time(RT).IDUAISisahomologouscompoundoftheinvestigated IDUAproductandnotanisotopologue.Itislikelythatsomeminor
Fig.3.ExtractedionchromatogramsrelatedtothesixproductsandtheirrelativeinternalstandardsbyrunningaCDCcontrolsample.ChromatogramsofABGandGALC productresultintwopeaks.Thefirstpeak(ghost)isduetotheproductgeneratedbyin-sourcebreakdownofsubstrate.WhiletheRToftheghostpeakisthesameasthe substrate,theRTofthesecondpeakcorrespondstotheproductoftheenzymaticreaction.
matrixcomponentsreleasedbythetrappingcolumnatthevalve switchingweremakingtheion-suppressionimpactslightly differ-entonbothanalyteandinternalstandard.
Toaddressthisproblemwithoutelongatingtherunningtime, plumbingsupportingthepreviousprotocolwasredesigned.The layoutshowninFig.1andtheLCoperatingparameters(Table1) providedagradientelutionatthetimethevalvewasswitchedfor ashiftedreleaseoftheanalytesfromthetrappingcolumn.With thisshifttheion-suppressionimpactmitigatedandwassimilarfor boththeIDUAISandtheIDUAproduct.Ontheotherside,eluting solventBcompositionwasreconsideredinorder,toruntheentire chromatographicprocessinlessthan4.5min,andtoguaranteethe cleanlinessofbothtrappingandelutingcolumns,injectionafter injection.Theelutingcolumnwaschosentopermitlarge-scaleuse. For the chromatographic separation differentcolumns were tested,includingaSynergiFusionRP-802×50mm,4m (Phe-nomenex,TorranceUSA),aShimPackXR-ODScolumn2×50mm, 2.2m(Shimadzu,Japan),andaZorbaxSB-CN2,1×50mm5m (Agilent,Italy).Besides,differentorganicsolventmixturesof ace-tonitrileandmethanol,withorwithoutisopropanol,weretested inordertoevaluatethemostappropriatecompositionofmobile phaseB.Experimentaldatashowedthataconcentrationof ace-tonitrilehigherthan50%wasnotsuitablesinceitextendedthe chromatographicrunuptosixminutes.Amixtureofacetonitrile/ methanol/isopropanolinthefollowingratio50:25:25(v/v/v) pro-vided thebest resultsin terms of chromatographic separation, robustnessandshortruntime.
Intermsofefficiencyandresolutionassociatedwithhigh long-termrobustnesstheZorbaxSB-CNcolumnperformedbetterthan theothertestedcolumns.Arepresentativechromatogramisshown inFig.3.
Thefinal choice of theZorbaxSB-CNcolumn instead ofthe previouslyusedSynergiFusioncolumn,togetherwiththe afore-mentioned organic mixture for mobile phase B and the new plumbingledtoanefficientandrapidchromatographicseparation whilemaintainingalowpressureinthechromatographic plumb-ing.
Thisnewconfigurationretainedtheadvantagesofafastsample injectionforadirectenzymeactivitymeasurementwithoutany otherpretreatment.
Table3
Intradayandinterdayimprecisions(expressedasCV%).
INTRADAY INTERADAY
QCCDC 0 Low Med High QCCDC 0 Low Med High
IDUA 9.3 3.5 6.9 9.3 IDUA 16.6 13.9 16.9 15.8 GLA 4.2 5.8 5.3 5.3 GLA 5.8 11.2 8.2 7.6 ABG 8.7 9.6 9.2 6.4 ABG 28.8 13.8 13.0 7.9 GALC 7.0 9.0 7.4 8.5 GALC 15.4 17.2 10.7 11.3 ASM 4.6 1.7 5.9 9.9 ASM 9.4 10.1 12.5 10.4 GAA 6.9 9.5 6.1 8.2 GAA 7.6 18.8 11.5 10.8 3.3. Methodvalidation
Theresultsforintra-dayand inter-dayimprecision(Table3)
wereacceptableconsideringmanyfactorscontributingto variabil-ity,includedmatrixinhomogeneity.Theintra-dayandinter-day imprecisionstudieswerelessthan20%CVsforall6enzyme activ-ities.
4. Conclusions
AtpresentenzymaticactivityassaysperformedonDBS,for new-bornscreeningpurposesorforconfirmingaclinicaldiagnosis,have anincubationtimeofatleast16h.Thisstudydemonstratesthatthis timecanbesafelyreducedtopermitgreaterworkloadsandfaster response times.Wedeveloped a protocolwitha4h-incubation time.
Themajorconcernregardinganenzymeactivityassaywitha shorttime isthat theproductformation maybeinsufficientto allowacleardistinctionbetweenhealthyandaffected individu-als.However,thisprotocolwasextensivelytestednotonlyonCDC butalsoontruepositivesamplestoconfirmthatthefourhourtime interval,thoughshort,issufficienttoconvertthesubstrateintoa quantifiableproductwhensupportedbythedescribedLC–MS/MS measurementsetting.
Afastdiagnosisofsevereneonatal-onsetformsofLSDsis essen-tialforeffectivepre-symptomaticintervention.Arapidscreening procedure can reduce recall times for newborns who stand to benefitfromtheprompt introductionoftreatment.Theshorter
140 E.Scolamieroetal./JournalofPharmaceuticalandBiomedicalAnalysis165(2019)135–140
incubationtimeadoptedinourprotocolreducesthetimeneeded tomakeadiagnosisandintroducetreatmentbytwodays.
Anyanalyticalmethoddevisedforclinicalapplicationmust sat-isfyrequirementsofsensitivity,accuracyandprecisionaswellas takingintoconsiderationtherealityofroutinelabtesting,which demandsspeedandrobustness.Theconceptofrobustnessisoften alittle vague,butcanbedefined asthecapacity ofan analyti-calmethodtoprocessa significantnumber ofsampleswithout compromisingaccuracyorsensitivity.InanLC–MSorLC–MS/MS systemtherearetwopotentiallyweaklinks;theionizationsource andthechromatographicsystem.Ifchromatographiccolumnsare notproperlyflushed,residuesofthepreviousmatrixmayremain andcontaminatethenextsampleinjected.Residuesthat accumu-lateinanunexpectedwaygenerateunpredictableeffects ofion suppressionofthetargetedcomponents
Anoptimizedmethodmustbeconductedinatimeframe com-patiblewithalarge-scaleroutine.
Asoutlinedabove,testing in ourlaboratoryproved thatthe methodwedescribeinthispaperfulfilsrequirementsofaccuracy, sensitivity,precision,speedandrobustnessoveraverylargescale forthreedifferentinstrumentalsettings.
Declarationofinterest
TetsuoTanigawaisanemployerofShimadzucorporation.The otherauthorsdeclarenoconflictsofinterest.
Funding
Thisresearchdidnotreceiveanyspecificgrantfromfunding agenciesinthepublicorcommercialsphere.Theworkwaspartially supportedbyAMMeCOnlus(AssociazioneMalattieMetaboliche CongeniteONLUS).
AppendixA. Supplementarydata
Supplementarymaterialrelated tothis article canbefound, intheonlineversion,atdoi:https://doi.org/10.1016/j.jpba.2018.12. 002.
References
[1]P.C.J.I.Schielen,E.A.Kemper,M.H.Gelb,Newbornscreeningforlysosomal storagediseases:Aconcisereviewoftheliteratureonscreeningmethods, therapeuticpossibilitiesandregionalprograms,Int.J.NeonatalScreen.3 (2017)1–15,http://dx.doi.org/10.3390/ijns3020006.
[2]D.Matern,D.Gavrilov,D.Oglesbee,K.Raymond,P.Rinaldo,S.Tortorelli, Newbornscreeningforlysosomalstoragedisorders,Semin.Perinatol.39 (2015)206–216,http://dx.doi.org/10.1053/j.semperi.2015.03.005.
[3]M.H.Gelb,C.R.Scott,F.Turecek,Newbornscreeningforlysosomalstorage diseases,Clin.Chem.61(2015)335–346,http://dx.doi.org/10.1373/clinchem. 2014.225771.
[4]N.A.Chamoles,M.Blanco,D.Gaggioli,Fabrydisease:enzymaticdiagnosisin driedbloodspotsonfilterpaper,Clin.Chim.Acta.38(2001)195–196,http:// dx.doi.org/10.1016/S0009-8981(01)00478-8.
[5]N.A.Chamoles,M.B.Blanco,D.Gaggioli,C.Casentini,Hurler-likephenotype: enzymaticdiagnosisindriedbloodspotsonfilterpaper,Clin.Chem.47 (2001)2098–2102.
[6]N.A.Chamoles,M.Blanco,D.Gaggioli,Diagnosisofalpha-L-iduronidase deficiencyindriedbloodspotsonfilterpaper:thepossibilityofnewborn diagnosis,Clin.Chem.47(2001)780–781.
[7]N.A.Chamoles,M.Blanco,D.Gaggioli,C.Casentini,Gaucherand niemann-pickdiseases–enzymaticdiagnosisindriedbloodspotsonfilter paper:retrospectivediagnosesinnewborn-screeningcards,Clin.Chim.Acta 317(2002)191–197,http://dx.doi.org/10.1016/S0009-8981(01)00798-7. [8]Y.Li,C.R.Scott,N.A.Chamoles,A.Ghavami,B.M.Pinto,F.Turecek,M.H.Gelb,
Directmultiplexassayoflysosomalenzymesindriedbloodspotsfor newbornscreening,Clin.Chem.50(2004)1785–1796,http://dx.doi.org/10. 1373/clinchem.2004.035907.
[9]D.Ombrone,E.Giocaliere,G.Forni,S.Malvagia,G.laMarca,Expanded newbornscreeningbymassspectrometry:Newtests,futureperspectives, MassSpectrom.Rev.35(2016)71–84,http://dx.doi.org/10.1002/mas.21463. [10]G.laMarca,B.Casetta,S.Malvagia,R.Guerrini,E.Zammarchi,Newstrategy
forthescreeningoflysosomalstoragedisorders:theuseoftheonline trapping-and-cleanupliquidchromatography/massspectrometry,Anal. Chem.81(2009)6113–6121,http://dx.doi.org/10.1021/ac900504s. [11]D.Ombrone,S.Malvagia,S.Funghini,E.Giocoliere,M.L.DellaBona,G.Forni,
A.DeLuca,F.Villanelli,B.Casetta,R.Guerrini,G.laMarca,Screeningof lysosomalstoragedisorders:applicationoftheonlinetrapping-and-cleanup liquidchromatography/massspectrometrymethodfor
mucopolysaccharidosisI,Eur.J.Mass.Spectrom.19(2013)497–503,http:// dx.doi.org/10.1255/ejms.1257.
[12]Z.Spacil,H.Tatipaka,M.Barcenas,C.R.Scott,F.Turecek,M.H.Gelb, High-throughputassayof9lysosomalenzymesfornewbornscreening,Clin. Chem.59(2013)502–511,http://dx.doi.org/10.1373/clinchem.2012.189936. [13]S.Tortorelli,C.T.Turgeon,D.K.Gavrilov,D.Oglesbee,K.M.Raymond,P.
Rinaldo,D.Matern,Simultaneoustestingfor6lysosomalstoragedisorders andX-adrenoleukodystrophyindriedbloodspotsbytandemmass spectrometry,Clin.Chem.62(2016)1248–1254,http://dx.doi.org/10.1373/ clinchem.2016.256255.
[14]R.Mashima,E.Sakai,M.Kosuga,T.Okuyama,Levelsofenzymeactivitiesin sixlysosomalstoragediseasesinJapaneseneonatesdeterminedbyliquid chromatography-tandemmassspectrometry,Mol.Genet.Metab.Rep.9 (2016)6–11,http://dx.doi.org/10.1016/j.ymgmr.2016.08.007.
[15]G.S.Ribas,J.F.DeMari,G.Civallero,H.M.deSouza,M.G.Burin,C.R.Vargas,R. Giugliani,Validationofamultiplextandemmassspectrometrymethodfor thedetectionofselectedlysosomalstoragediseasesindriedbloodspots, JIEMS5(2017)1–7,http://dx.doi.org/10.1177/2326409817692360. [16]C.F.Yang,C.C.Yang,H.C.Liao,L.Y.Huang,C.C.Chiang,H.C.Ho,C.J.Lai,T.H.
Chu,T.F.Yang,T.R.Hsu,W.J.Soong,D.M.Niu,Veryearlytreatmentfor infantile-onsetpompediseasecontributestobetteroutcomes,J.Pediatr.169 (2016)174–180,http://dx.doi.org/10.1016/j.jpeds.2015.10.078,e1. [17]S.Elliott,N.Buroker,J.J.Cournoyer,A.M.Potier,J.D.Trometer,C.Elbin,M.J.
Schermer,J.Kantola,A.Boyce,F.Turecek,M.H.Gelb,C.R.Scott,Datasetand standardoperatingprocedurefornewbornscreeningofsixlysosomalstorage diseases:Bytandemmassspectrometry,DataBrief.8(2016)915–924,http:// dx.doi.org/10.1016/j.dib.2016.06.052.