Simultaneous determination of creatine and guanidinoacetate in plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS).

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ContentslistsavailableatScienceDirect

Journal

of

Pharmaceutical

and

Biomedical

Analysis

jo u r n al h om epag 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

Short

communication

Simultaneous

determination

of

creatine

and

guanidinoacetate

in

plasma

by

liquid

chromatography–tandem

mass

spectrometry

(LC–MS/MS)

Sara

Boenzi

a,∗

_,

_Cristiano

_Rizzo

b

_,

_Vincenzo

_Maria

_Di

_Ciommo

c

_,

_Diego

_Martinelli

a

_,

Bianca

Maria

Goffredo

b

_,

_Giancarlo

_la

_Marca

d

_,

_Carlo

_Dionisi-Vici

a

a_Division_of_Metabolism,_Bambino_Gesù_Pediatric_Hospital,_Rome,_Italy

b_Laboratory_of_Metabolic_{Biochemistry,}_Bambino_Gesù_Pediatric_Hospital,_Rome,_Italy c_Unit_of_Epidemiology_and_{Biostatistics,}_Bambino_Gesù_Pediatric_Hospital,_Rome,_Italy d_Department_of_{Pharmacology,}_University_of_Florence,_Florence,_Italy

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received7April2011

Receivedinrevisedform31May2011 Accepted9June2011

Available online 16 June 2011 Keywords:

Guanidinoacetate Creatine LC–MS/MS

a

b

s

t

r

a

c

t

Background:Guanidinoacetate(GAA)andcreatinearereliablebiochemicalmarkersforprimaryand sec-ondarycreatinedefects.Wedescribeamethodbyliquidchromatographycoupledwithtandemmass spectrometry(LC–MS/MS)forsimultaneousdeterminationofplasmaGAAandcreatine.Weanalyzed 283healthysubjectsfrom0to63yearsoldtoobtainage-relatedcontrolvalues.

Methods:Plasmasampleswereextractedwithacetonitrilecontaining13C2-GAAandd3-creatine. Sam-pleswereanalyzedbyLC–MS/MSinpositiveionisationmode,afterderivatizationtobutyl-esters.Optimal chromatographicseparationwasachievedusingacolumnSupelcosilTM_LC-4.6_mm_with_isocratic_elution

in5min.

Results:Runtimewas5min.Standardcurveswerelinearfrom0.05to200␮mol/Lforcreatineandfrom 0.02to40␮mol/LforGAA.Limitofdetection(LOD)andlimitofquantitation(LOQ)wererespectively 0.005and0.05␮mol/Lforcreatine;LODandLOQwere0.002and0.02␮mol/LrespectivelyforGAA.Intra andinter-assayCVsforcreatineandGAAwere<8%.Recoveryexperimentsadding50and100␮mol/L creatineand10and20␮mol/LGAAwere102.1%and101.2%,forcreatine;102.95%and96.45%forGAA. Themethodwasappliedto283plasmacontrolsfromhealthysubjectstoobtaincontrolvaluesinthree speciﬁcageranges:0–12,13–20,>20yearsold.

Conclusion:ArapidandhighsensitiveLC–MS/MSmethodwasdevelopedandvalidatedfordetermination ofcreatineandGAAinplasmaanditcouldalsobeappliedtootherbiologicalmaterials,suchasCFSand urines.Thismethodisusefulfordiagnosesofprimaryandalsoforsecondarycreatinedefectsthatmay occurininheritedmetabolicdiseasesinwhichprecursorsofcreatinebiosynthesisareinvolved.

1. Introduction

Creatineandphosphocreatineplayanessentialroleinenergy

storage and transmissionin severaltissues. Creatine is

synthe-sizedmainlyintheliverandpancreasbytworeactioninvolving

twoenzymes:theﬁrstarginine:glicineamidinotransferase(AGAT;

EC2.1.4.1)transferstheamidino-groupfromargininetoglycine,

producing ornithine and guanidinoacetate (GAA); the second

enzymeisS-adenosyl-l-methionine:N-guanidinoacetate

methyl-transferase(GAMT; EC2.1.1.2), which transfersa methylgroup

toGAAproducing creatine.Creatineistransportedtotissuesby

thecreatinetransporter(CrT)andisnon-enzymaticallyconverted

∗ Correspondingauthorat:DivisionofMetabolism,BambinoGesùChildren’s Hos-pital,PiazzaS.Onofrio4,00165Rome,Italy.Tel.:+390668592225;

fax:+390668592791.

E-mailaddress:sara.boenzi@opbg.net(S.Boenzi).

tocreatinine[1].Threeinheriteddefectsinthebiosynthesisand

transportofcreatinehavebeendescribed:AGAT[2]andGAMT[3]

deﬁcienciesandthefunctionaldefectsinthecreatinetransporter

(SLC6A8)[4].Acommonfeatureinallthesedisorders,called

Cre-atineDeﬁciencySyndromes(CDS),isthecompletelackofCr/PCr

inthebrainmeasuredbyinvivomagneticresonancespectroscopy

(MRS).InAGATandGAMTdeﬁcienciesthecreatinepoolcanbe

par-tiallyrevertedbycreatinesupplementation,whereasmalepatients

withtheX-linkedcreatinetransporterdefectareunresponsiveto

thissupplementation[5].BiochemicaldetectionofCDSrelieson

thedetermination of twomain metabolitesinbiological ﬂuids:

creatineandGAA[6,7].TheaccumulationofGAAinbiological

ﬂu-idsinGAMTdeﬁciencywasdemonstratedtobereliablediagnostic

markerofthisdisease.Incontrast,fordiagnosticinvestigationfor

AGATdeﬁciencyisrecommendedtoanalysecreatineandGAAin

bothurineandplasmainwhichthelevelsofthesetwometabolites

aredecreased.X-linkedcreatinetransporterdeﬁciencypatientsare

characterizedbyincreasedurinary creatine/creatinineratioand

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plasmacreatineconcentrationusuallywithinthereferencerange

orslightlyincreased[8].

Thereare also a few secondary creatine defects, in

particu-lar, two inherited conditions causing hyperornithinemia have

been associated to secondary creatine deﬁciency: deﬁciency

of ornithine delta-aminotransferase activity (OAT) [9], and

hyperornithinemia–hyperammonemia–homocitrullinuria

syn-drome(HHH)[10,11].Inthesecases,treatmentwithcreatinecan

improvesomeclinicalaspectsofthedisease.

CreatineandGAAconcentrationsinphysiologicalﬂuidsare

reli-ablediagnosticmarkersforprimarycreatinedeﬁciencysyndromes

(AGAT,GAMT),butalsotorecognizesecondarycreatine

deﬁcien-ciesthatmaypossiblyoccurinothermetabolicdiseasesinwhich

precursorsofcreatinebiosynthesis areinvolved. Therefore,it is

importanttodevelophighlyselectiveandsensitiveanalytical

tech-niquesforthedeterminationofcreatineanditsprecursorGAA.We

developedandvalidatedareliableandsensitivemethodby

liq-uidchromatographycoupledwithtandemmassspectrometryfor

quantitativedeterminationofcreatineandGAAasbuthyl-estersin

plasma.Weanalyzedalargenumberofcontrolsamples(283)by

thismethodfortheidentiﬁcationofage-relatedreferenceranges,

asadiagnostictoolforearlydiagnosisofprimaryandsecondary

creatinedeﬁciencies. Theimportance todetectwithreasonable

precisionanycreatineandGAAalterationsisbecause,inaddition

totheclassicprimarycreatinedeﬁciencies,increasingnumberof

inheritedmetabolicdiseasesoccurwithslightchangesofplasma

creatine.Moreover,inthosecasesinwhichtheconcentrationsof

GAAandcreatineareverylow,asforAGATdeﬁciency,is

neces-saryahighsensitivemethod,enoughtosafelydetectvaluesbelow

thenormalrange.Previousmethods[12–16]areusefulforgreat

increasingbutnotforadecreaseoftwometabolites.Bodameretal.

[14]usingthesamemetabolitesderivatizationproceduresdidnot

reportLLOQforcreatineneitherforGAAbutonlyhighlimitsof

detection,especiallyfor GAA,1.2␮mol/L.Our datademonstrate

thatthisvalueisatypicalconcentrationdetectableina normal

plasmasample.

2. Materialsandmethods

2.1. Reagents

Thelabeledinternalstandardd3-creatinewassynthesizedby

Dr. H. Ten Brink (VU University Medical Center, Amsterdam,

The Netherlands) and 13_C

2-GAA were obtained from CDN

iso-topes(Quebec, Canada). Creatine and GAA wereobtainedfrom

Sigma–Aldrich (Steinheim, Germany). Ammonium acetate was

purchasedfromMerkKGaA(Damstadt,Germany).HPLCgrade

ace-tonitrileandwaterwerepurchasedfromRomilLtd.(TheSource

ConventDriveWaterbeachCambridge,UnitedKingdom).Formic

acid98/100%(FOA)wasobtainedfromBDH(VWRLtd.,Poole,

Eng-land).Butanolic-HClwasfromRegisTechnologies(MortonGrove,

IL,USA).

2.2. Preparationofstandardsolutions

Toinvestigate themolecularfragmentation a toobtain

cali-brationcurvesfor creatineandGAA,10mmol/L d3-creatineand

13_C

2-GAAstocksolutions(Stock-1)werepreparedinwater and

stored frozenat−20◦_C. ₅_mmol/L _d₃_-Cr _and ₁_mmol/L _of 13_C

2

-GAAwerepreparedbydilutioninacetonitrile–water(50:50,v/v)

inthesamesolution(Stock-2)andstoredfrozenat−20◦_C._“Daily

standardsolution”containing10␮mol/L13_C

2-GAAand50␮mol/L

d3-CrwaspreparedbydilutionofStock-2solution1:100(v/v)with

acetonitrile.

2.3. Sampletreatmentprocedure

Plasmasamplewasobtainedfrombloodsamplescollectedin

EDTAK2 tubesthat wereimmediatelycentrifuged,for 5minat

3000×g,plasmawasseparatedfromredcellsand immediately

storedfrozenat−20◦_C.

Fiftymicrolitersof plasmasample wasmixedwith25␮Lof

“Dailystandardsolution”.Fourhundredmicrolitersofacetonitrile

wasaddedtoprecipitateproteinsandextractthemetabolites.Each

vialwasmixedbyvortexfor2min,thencentrifugedfor5minat

10,000rpmandthesupernatantﬂuidwastransferredtoaclean

vialanddriedat40◦Cundernitrogensteam.Eighty microliters

ofbutanolic-HCl wasaddedfor derivatizationofmetabolitesas

buthyl-estersandheatedat65◦Cfor15min.Samplesweredried

againat40◦Cundernitrogensteamandﬁnallytheywere

recon-stitutedwith200␮Lofacetonitrile–water50:50(v/v)containing

0.05%FOAtoobtainpH4,theappropriatepHformetabolites

ion-ization.

2.4. Liquidchromatography

ChromatographywasperformedonaAgilentseries1200pump

andautosampler(AgilentTechnologiesInc.,Wilmington,DE,USA).

ThecolumnforchromatographicseparationwasaSupelcosilTM

LC-8,3␮m,7.5cm×4.6mm(Supelco,Bellefonte,PA,USA).Themobile

phase wassolution A(0.005mol/Lammoniumacetate

contain-ing0.03%FOA)andsolutionB(acetonitrilecontaining0.03%FOA).

Chromatographyseparationofmetaboliteswasobtainedwith

iso-craticelutionsolventA-solventB(30–70,v/v)attheﬂowrateof

1.0mL/min.5␮Lofsamplewasinjectedintothecolumn.Thetotal

runwas5min.

2.5. Massspectrometry

Tandem mass spectrometry experiments were carried out

on an API3200 triple quadrupole mass spectrometer (Applied

Biosystems-Sciex, Toronto,Canada), equippedwith aTurbo Ion

SpraySourceoperatinginpositiveionmodewithaneedlepotential

of5500V;thesourcetemperaturewas550◦C.Declustering

Poten-tial(DP),CollisionCellExitPotential(CXP)andCollisionEnergy(CE)

wereoptimizedintroducing5.0␮mol/Laqueousstandardsolution

of50%acetonitrilecontaining0.05%FOAofeachmetaboliteinto

spectrometerinfusionatﬂowrateof10␮L/min.TheresultingDP

was+41forcreatineand+36VforGAA.OptimalCEandCXPwere

foundat27and5Vforcreatine,and21and3VforGAA,

respec-tively.Thefollowingtransitionsweremonitored:m/z174.1>101.0

forGAA,176.1>103.0for13_C

2-GAA,188.1>90.0forcreatineand

191.1>93.0 ford3-creatine.Thedwelltimeswas60msandthe

pausebetweenthemasstransitionswas5ms.

2.6. Standardcurvesforquantiﬁcation

Aqueousstandardsolutionsof40,20,10,5,2,1,0.1,0.02␮mol/L

forGAAand200,100,50,25,10,5,0.5,0.05␮mol/Lforcreatine

wereanalyzedwiththesameprocedureofplasmasamples.The

acquireddatawereprocessedusingAnalystversion1.4.2software

(AppliedBiosystems-Sciex),includingoptionforchromatographic

andspectralinterpretationandforquantitativeinformation

gen-eration. Calibration curves were constructed with the Analyst

Quantitationprogramusingalinearleast-squareregression

non-weighted.

Thelimit ofdetection (LOD)wasdetermined byprogressive

dilutionsofcalibratorsolutionsofeachanalyteandwas

consid-eredasthelowestconcentrationforwhichthesignal-to-noiseratio

(S/N)wasindicatedbytheAnalystsoftwaretobeatleast3.The

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cali-Table1

PrecisiondatafortheLC–MS/MSassay.

Mean±SD(␮mol/L) CV(%) Intra-day(n=10) Creatine 53.42±1.81 3.39 GAA 2.66±0.16 6.01 Inter-assay(n=18) Creatine 54.71±2.73 4.99 GAA 2.87±0.22 7.66

bratorsolutionswithdecreasingconcentrationofeachanalyteand

wasconsideredasthelowestconcentrationforwhichthe

signal-to-noiseratio(S/N)wasindicatedbytheAnalystsoftwaretobeat

least10.

2.7. Samplecollectionofpatientsandcontrols

Children’splasma,forreferencevalues,wasobtainedfrom

hos-pitalizedneurologicallyhealthysubjectsandadults’plasmawas

obtainedfromanonymousblooddonor.Plasmafromtwopatients

previouslydiagnosedwithGAMTdeﬁciencywasstudied.All

sam-plesweretreatedasdescribedinSection2.3.

2.8. Statisticalanalysis

Apreliminarytest (Kolmogorov–Smirnov)wasperformedto

assessnormalityofdistributionsofcreatine,age,andGAA.

Differ-encesamongthethreeagegroupsweretestedusingANOVAfor

creatineandKruskal–WallistestforGAA.

3. Results

3.1. Chromatographyandmassspectra

Fig.1 shows anextract ion chromatogram froma 5_␮mol/L

GAAand20␮mol/Lcreatinestandardsolution.MultipleReaction

Monitoring(MRM)wasusedforthedetectionofthespeciﬁc

tran-sitionsinpositiveionmodeforthemonitoringofeachmolecule.

Under theconditions described above,thesystemwasefﬁcient

for the separation of GAA and creatine that were eluted with

theirrespectivelabeledinternalstandardattheretentiontimesof

3.96and4.18min,respectively.Moreover,inplasmasampleswere

separatedbytheseconditionstwoadditionalinterferingpeaks

pre-senting tothesame GAA MRM transition,m/z174.1>101.0,at

differentretentiontimes,1.20and4.76min,thatcouldinterfere

withtheGAAquantiﬁcation(Fig.4).

3.2. Linearity

Calibration curves were linear over concentrations of

0.05–200␮mol/Lforcreatineand0.02–40␮mol/LforGAAwhich

coversandexceedtheconcentrationrangesfoundinplasmafor

both metabolites. The coefﬁcient of linear correlation (r2₎_was

0.9997forcreatineand0.9998forGAA.

3.3. Limitofdetection,precisionandrecovery

The minimal detectableconcentrations, LOD of theanalytes

were0.005␮mol/Lfor creatine(S/Nratio7.5)and0.002␮mol/L

forGAA(S/Nratio4.6).LLOQwere0.05␮mol/Lforcreatine(S/N

ratio12.3)and0.02␮mol/LGAA(S/Nratio12.9).

Allvalidationexperimentswereperformedwithpooledplasma

fromhealthy controlsand thevalidation data of thepresented

methodarelistedinTables1and2.Theintra-assayvariationwas

assessedfrom10replicateswithinoneday(n=10)andinter-assay

Table2

AccuracydataforCreatineandGAA.

Mean±SD(␮mol/L) CV(%) Recovery(%) Creatineadded(n=5) 0␮mol/L 54.14±2.62 4.84 n.c. 50␮mol/L 106.31±3.40 3.20 102.08 100␮mol/L 155.92±3.91 2.51 101.15 GAAadded(n=5) 0␮mol/L 2.54±0.19 7.48 n.c. 10␮mol/L 12.91±0.46 3.56 102.95 20␮mol/L 21.74±0.97 4.46 96.45

fromthreetimesonsixdifferentdays(n=18);CVs forcreatine

andGAAinplasmawereinallcases<8%.Recoveryexperiments

wereperformedattwodifferentconcentrations:pooledplasma

wasspikedwithcreatine(50and100␮mol/L)andwithGAA(10

and 20␮mol/L).The creatine recoverywas 102.1% and 101.2%,

respectively,oftheexpectedamount;GAArecoverywas102.95%

and96.45%,respectively.

3.4. BiologicalvariationofcreatineandGAAinplasma

WemeasuredcreatineandGAAconcentrationsinplasma

sam-pleof283healthysubjectsfrom0to63yearsold.Ourresultsare

summarizedinTable3werewearbitrarilyindividuatedthreeage

groupranges:0–12years,13–20yearsand>20years.The

differ-encesamongthethreeagegroupsarehighlysigniﬁcantforcreatine

(F=40.920;p<0.001)andforplasmaGAAcontrolvalues(p<0.001)

(Fig.2).

Plasma GAA and creatine weresigniﬁcantly correlated with

age:creatineshowsaninversecorrelationwithage(rho=−0.450;

p<0.001) and GAA a direct correlation with age (rho=0.719;

p<0.001)(Fig.3).Forthisreason,itisnotpreferabletouseone

referenceagerangetodetectanyabnormalcreatineandGAA

con-centration.

Giventhebroaddistributionofvaluesespeciallyfortheyounger

agecohorts,weestimatereferencevaluesforbothcreatineandGAA

byuseofcontrolplasmasamplewithpercentiledistribution(2.5th

and97.5thpercentiles).

3.5. Patientssamplesanalysis

TheLC–MS/MSmethodwasusedtoanalysetwosamplesfrom

twodiagnosedGAMTpatientstotestifytheabilityofthismethod

todifferentiatethemfromcontrols.Plasmasamplefroma6years

oldpatientshowedanincreaseofGAAconcentration12.4␮mol/L

(range 0–12years:2.5th–97.5th percentile 0.8–1.6␮mol/L) and

reductionofcreatineconcentration7.4␮mol/L(range0–12years:

2.5th–97.5thpercentile16.2–97.0␮mol/L).Forthesecondpatient

11yearsoldplasmaGAAwas11.6␮mol/Landcreatine20.1␮mol/L

relativelytothesame0–12yearscontrolvalues.Theselevelsof

creatine and GAAare readilydistinguishable fromcontrolsand

Table3

CreatineandGAAcontrolvaluesin␮mol/L(n=283).

Agerange 0–12(n=93) 13–20(n=96) >20(n=94) Creatine 2.5thpercentile 16.2 14.1 13.2 Median 60.0 47.9 31.4 97.5thpercentile 97.0 92.0 56.8 GAA 2.5thpercentile 0.3 0.6 0.7 Median 0.8 1.1 2.2 97.5thpercentile 1.6 2.1 3.3

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. s p c 6 e 0 . 1 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I X 4.18 3.96 . s p c 6 e 0 . 1 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I X 4.18 . s p c 5 e 5 . 6 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 0 . 1 0 1 / 1 . 4 7 1 :) s ri a p 4 ( M R M + f o C I X Intensity, cps Intensity, cps Intensity, cps 3.96 . s p c 6 e 0 . 1 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I X 4.18 3.96 . s p c 6 e 0 . 1 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I X 4.18 Max.1.0e6cps. ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I XICof+MRM(4paris:)188.2/90.1amurfomSample14(CAL-6)ofCAL-28-MAR-2011.wiff(TurboSpray) Max.1.0e6cps. X 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Time, min 4.18 0.00 1.00e5 2.00e5 3.00e5 4.00e5 5.00e5 6.00e5 7.00e5 8.00e5 9.00e5 1.00e6 3.96 . s p c 6 e 0 . 1 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 1 . 0 9 / 2 . 8 8 1 :) s ri a p 4 ( M R M + f o C I X 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Time, min 0.00 1.00e5 2.00e5 3.00e5 4.00e5 5.00e5 6.00e5 7.00e5 8.00e5 9.00e5 1.00e6 _4.18 . s p c 5 e 5 . 6 . x a M ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 0 . 1 0 1 / 1 . 4 7 1 :) s ri a p 4 ( M R M + f o C I X 3.96 Max.6.5e5cps. ) y a r p S o b r u T ( ff i w . 1 1 0 2 -R A M -8 2 -L A C f o ) 6 -L A C ( 4 1 e l p m a S m o rf u m a 0 . 1 0 1 / 1 . 4 7 1 :) s ri a p 4 ( M R M + f o C I X 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 Time, min 0.00 5.00e4 1.50e5 2.50e5 3.50e5 4.50e5 5.50e5 6.50e5 3.96

Fig.1. Extractionchromatogram(upperpanel)withmultiplereactionmonitoringexperiments(lowerpanels)forplasmasample.GAAand13_C

2-GAA(RT:3.96min);creatine

andd3-creatine(RT:4.18min).

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Fig.3. ScatterplotofcreatineandGAAconcentration(␮mol/L)vsageexpressedinyears.Plasmacreatineconcentrationshowsaninversecorrelationwithage,whileGAA concentrationshowsadirectcorrelation.

areconsistentwithcharacteristicbiochemicalﬁndings ofGAMT

deﬁciency[3].

4. Discussion

Patientswithplasmacreatinereductioncouldbeeasilytreated

withcreatineoritsprecursorssupplementation,causing,insome

cases,arapidimprovementsofclinicalconditions.Earlydiagnosis

ofprimaryandsecondarydeﬁcienciescanimprovetheprognosisof

thesedisorders.Wehavedevelopedasensitiveandspeciﬁcmethod

fordeterminationofcreatineandGAAconcentrationsinplasma.In

recentyears,introductioninthelaboratoryofliquid

chromatogra-phycoupledwithmassspectrometry,havegreatlyincreasedthe

diagnosis of creatine primary defects that were not previously

easily diagnosed with traditional analytical techniques. Several

methodsforcreatineandGAAdeterminationhavebeendescribed,

includingstableisotopedilutiongaschromatography–mass

spec-Fig.4.Plasmasamplechromatogram.Upperpanel:extractionchromatogramwithmultiplereactionmonitoring(MRM)experimentm/z174.1>101.0ofGAA(RT:3.97min) andothertwounknownpeaks(RT:1.20,4.76min);Lowerpanel:extractionchromatogramwithMRMexperimentm/z176.1>103.0of13_C

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trometry(GC–MS)[12,13],andHPLC[6]andLC–MS/MS[14–16]

butthesemethodsreachlesssensitivityandspeciﬁcity.Allthese

GC–MSmethodsrequirealongtimeforseveralstepsofextraction

andderivatization.Newmethoddescribedinthispaperhasa

satis-factoryintra-dayprecision3.39%forcreatineand6.01%forGAAand

inter-dayprecision4.99%and7.66%respectively,similartothose

describedbyR.S.Carlingetal.[15]byun-derivatizedLC–MS/MS,

thatwere2.7%and6.4%forintra-dayprecisionbutnewdataare

slightbetterforinter-dayassayprecisionthatwere6.2%and9.5%

respectivelyforcreatineandGAA;LODandLLOQwere0.1␮mol/L

and0.4␮mol/LforbothcreatineandGAA,highercomparedwith

thenewmethod:LOD0.005␮mol/Lforcreatine,0.002␮mol/Lfor

GAA,LLOQ0.05␮mol/Lforcreatineand0.02␮mol/LGAA.

Proba-bly,newbetterdataareduetothechromatographicseparationof

twoadditionalinterferingpeakspresentingtothesameGAAMRM

transitionatdifferentretentiontimesthatcouldinterferewiththe

GAA(Fig.4).

Cognatetal.[16]publishedin2004amethodbyLC–MS/MS,

reachingLODof0.01␮mol/Lforcreatineand0.025␮mol/LforGAA,

and LLOQ 0.05␮mol/L and 0.1␮mol/L respectively for creatine

andGAA,slight higherthanournewdata.Theintraday

impre-cisionwere9%forGAAand2%forcreatine inplasmawhilethe

interdayimprecisionwere13%and9%respectively,higher

espe-ciallyforGAAcomparedwithourintra-dayandinterassayCVs.

Probably,differentdataobtainedmainlyforGAA,areduetothe

differentevaluationofinterferencesthatoccursinplasmasamples

analysis.

Similarly,Bodameretal.[14]giveonlyfewinformationabout

chromatographyand nochromatogram withthepeaksortheir

retentiontimeshasshown,soitisunknownifthetwo

metabo-liteswerewellseparatedornot,orifbackgroundnoise orsuch

interferencewerepresentor not.Intra-day and inter-dayassay

aresimilartothoseobtainedbynewmethod,butlimitsof

detec-tionwereveryhigh:7.4and1.2␮mol/Lrespectivelyforcreatine

andGAA.Newmethodis2minlongerthantheothersLC–MS/MS

methodsbut it allowtoseparatethe unknownpeaksthat

cor-respondto unknowntwo substances withthe samemolecular

weight and that produce the same fragment m/z 101 of GAA

butyl-derivative.TheoreticallytheycouldinterferewiththeGAA

quantiﬁcation,if not wellseparated. Therefore, it is difﬁcultto

explain the reason for the increasedsensitivity of ourmethod

respecttheothersbyLC–MS/MS.Probably,itisdue toboththe

instrumentationandthechromatographicseparationbythe

col-umnweused,thatdiffersfrompreviouslystudypublished.The

columnweusedperformagoodseparationofcreatine,GAAand

someinterferencethatcouldoccur,asshowedinthereported

ﬁg-ureofchromatogram(Figs.1and4)withtwodistinctretention

times allowinghigh sensitivity for the quantitative

determina-tionofboth metabolites,conﬁrmedbyvery lowLODand LLOQ

values.ItcouldbesuitablefordetectinglowerGAAandcreatine

concentrations,suchasforAGATdeﬁciency,forseveralsecondary

creatinedeﬁciencies,andpossiblyalsotoexpandtheanalysisto

CFSinwhichpreliminarystudiesdemonstratethatGAA

concen-trationisalmost10timeslowerthaninplasma,howevermuch

lowerthandetectableconcentrationswithallmethodsprevious

published.

Moreover,Carling et al. [15] mentioned that the validity of

existingreferencerangesquotedinliteratureisquestionableby

therelativelysmallnumbersofsubjectsusedtodeterminethem

andnotalwaysspeciﬁedmodalitystorage.Speciﬁcationofstorage

modalityofsamplesanalyzedisveryimportantsinceitisknown

thatcreatine,overtimeandchangingpHofmedium,variesits

con-centrationtransformingitselfintocreatinine[15,17].Ourplasma

creatineandGAAreferencevaluesaredeterminedbylargegroup

ofcontrolsubjectallowingustoobtainthreeage-relatedranges,

clearlyindicatingstoragemodalityofsamples.Ourdataconﬁrm

thosealreadypublishedaboutthecorrelationofplasmacreatine

withtheageofsubjects[18]butinourstudy,thenumberof

con-trolvaluesareverylargerespectpreviouspublicationsinwhich

therewasanalyzedupto30samplesperagerange,withoutany

descriptionofsamplestorage[14–16].Thereforeusingourcontrol

values,itis possibletoestimateeven slightchanges ofcreatine

andGAAconcentrationinadeﬁnedagerange.Furthermore,the

applicabilityofthemethodisillustratedbythedeterminationof

GAAandcreatineinplasmaoftwopatientsaffectedwithGAMT,in

whichGAAwashighlyelevatedandcreatinewasdecreasedrespect

thecorrespondingagerelated control values.In summary, it is

knownthatitispossibletodetectlargealterationsofplasma

cre-atineandGAA,evenwithlowsensitivemethods.TheLC–MS/MS

methoddescribedinthispaperisarapidandusefultoolto

diag-noseprimarycreatinedeﬁcienciesandalsotorecognizetreatable

secondarycreatinedefectsthatmayoccurininheritedmetabolic

diseasesinwhichprecursorsofcreatinebiosynthesisareinvolved.

Thedevelopmentofthismethodisjusttodetectsmallchanges

comparedtocontrolsandtodetectgreatdecreasesoftwo

metabo-lites,suchasforAGATdeﬁciency,inwhichitisknownthatcreatine

andGAAconcentrations areverylow. Therefore,itwillbe

pos-sible to assess any variation of creatine and GAA in inherited

metabolicdiseasesandwillbepossibletoexploretherelationship

betweencirculating creatine levels and others metabolic

path-ways.

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