Evaluation of a nanoflow interface based on the triple-tube coaxial sheath-flow sprayer for capillary electrophoresis-mass spectrometry coupling in metabolomics

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Article

Reference

Evaluation of a nanoflow interface based on the triple-tube coaxial sheath-flow sprayer for capillary electrophoresis-mass spectrometry

coupling in metabolomics

FERRE, Sabrina, et al .

Abstract

The performance of an original CE-MS interface that allows the in-axis positioning of the electrospray with respect to the MS inlet was evaluated. The variations in the geometrical alignment of this configu- ration in the absence of a nebulizing gas afforded a significant reduction in the sheath-liquid flow rate from 3 μL/min to as low as 300 nL/min. The sheath liquid and BGE were respectively composed of H2O—iPrOH–CH3COOH 50:50:1 (v/v/v) and 10% acetic acid (pH 2.2). A significant gain in sensitivity was ob- tained, and it was correlated to the effective mobility of the analytes. Compounds with low mobility val- ues showed a greater sensitivity gain. Special attention was paid to the detection of proteinogenic amino acids. Linear response functions were obtained from 15 ng/mL to 500 ng/mL. The limits of quantification, as low as 34.3 ng/mL, were improved by a factor of up to six compared to the conventional configu- ration. The in-axis setup was ultimately applied to the absolute quantification of four important amino acids, alanine, tyrosine, methionine and valine, in standard reference material (NIST plasma). The [...]

FERRE, Sabrina, et al . Evaluation of a nanoflow interface based on the triple-tube coaxial sheath-flow sprayer for capillary electrophoresis-mass spectrometry coupling in metabolomics.

Journal of Chromatography. A , 2021, vol. 1641, p. 461982

DOI : 10.1016/j.chroma.2021.461982 PMID : 33611118

Available at:

http://archive-ouverte.unige.ch/unige:150313

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ContentslistsavailableatScienceDirect

Journal of Chromatography A

journalhomepage:www.elsevier.com/locate/chroma

Evaluation of a nanoﬂow interface based on the triple-tube coaxial sheath-ﬂow sprayer for capillary electrophoresis-mass spectrometry coupling in metabolomics

Sabrina Ferré

^a^,^b

, Nicolas Drouin

^a^,^b

, Víctor González-Ruiz

^a^,^b^,^c

, Serge Rudaz

^a^,^b^,^c^,^∗

aSchool of Pharmaceutical Sciences, University of Geneva, Geneva, Switzerland

bInstitute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, Geneva, Switzerland

cSwiss Centre for Applied Human Toxicology (SCAHT), Switzerland

a rt i c l e i nf o

Article history:

Received 21 December 2020 Revised 3 February 2021 Accepted 7 February 2021 Available online 9 February 2021 Keywords:

Capillary electrophoresis CE-ESI-MS

Mass spectrometry Nanoﬂow interface Amino acids

a b s t r a c t

Theperformance ofanoriginalCE-MSinterfacethatallowsthein-axispositioningoftheelectrospray withrespecttotheMSinletwasevaluated.Thevariationsinthegeometricalalignmentofthisconfigu- rationintheabsenceofanebulizinggasaffordedasignificantreductioninthesheath-liquidflowrate from3μL/mintoaslowas300nL/min.ThesheathliquidandBGEwererespectivelycomposedofH₂O—

iPrOH–CH₃COOH50:50:1(v/v/v)and10% aceticacid (pH2.2). Asignificantgaininsensitivitywas obtained,anditwascorrelatedtotheeffectivemobilityoftheanalytes.Compoundswithlowmobilityval- uesshowedagreatersensitivitygain.Specialattentionwaspaidtothedetectionofproteinogenicamino acids.Linearresponsefunctionswereobtainedfrom15ng/mLto500ng/mL.Thelimitsofquantification, as lowas 34.3ng/mL,wereimprovedbyafactorofuptosixcomparedtothe conventionalconfigu- ration.Thein-axissetupwasultimatelyappliedtotheabsolutequantificationoffourimportantamino acids,alanine,tyrosine,methionineandvaline,instandardreferencematerial(NISTplasma).Theaccura- ciesrangedfrom78to113%,thusdemonstratingthepotentialofthisconfigurationformetabolomics.

ThisisanopenaccessarticleundertheCCBYlicense(http://creativecommons.org/licenses/by/4.0/)

Introduction

CEhasthegreatadvantagesofhighresolution,highselectivity and shortanalysis time withlow samples andsolvent consump- tion.Whencombinedwithelectrosprayionizationmassspectrom- etry(ESI-MS),itisoneofthemostrelevanttoolsforthecharacter- izationofionizablepolarcompounds[1,2].Thetwotechniquescan becoupledviaseveralmodesbasedontheflowrateandthepres- enceofanassistingnebulizinggas[3].CE-ESI-MSinterfacescanbe classified as(i) nano-ESI interfaces operating atlow flow rate or withoutadditionalliquid(1–1000nL/min)withnonebulizinggas requiredor(ii)ESIinterfaceswithanadditionalsheathliquidsup- port operatingat1–1000μL/minandmostlyassistedwithaneb- ulizing gas. The sheathliquid provides electrical contact to close theCEcircuit,enablingmorerobustnessandflexibilitythroughits compositionincludingthepotentialforpostcapillaryderivatization.

However, it hasbeenreportedthat the signiﬁcantadditional vol-

∗Corresponding author. Institute of Pharmaceutical Sciences of Western Switzer- land (ISPSO), University of Geneva, Rue Michel Servet 1, 1211, Geneva 4, Switzerland.

E-mail address: serge.rudaz@unige.ch (S. Rudaz).

umecompared tothe flow rateforCE alonelimits thedetection sensitivitybecauseofthedilutioneffect[4].Thenebulizinggasas- sistssprayformationandimprovesESIrobustness,butthesuction effecthasbeendemonstratedtogenerateahydrodynamicflowin- side theseparationcapillary,thus restrictingseparationefficiency [5].

Sheathless interfaces introduced by the design ofMoini etal.

havebeenproposedtoavoidtheinfluenceofthesheathliquidand increase the detectioncapabilities[6].This kindofinterface pro- ducesapurenanosprayonthesolebasisoftheCEflowrate.Thus, althoughlessflexibleintermofthemethoddevelopmentandion- izationconditionsthan sheath-flowassistedinterfaces,sheathless interfacesofferthe opportunityto reduce thedropletsize andto upgrade ionization efficiency. A nanospray regime also makes it possibletoobtaina CEoutputclosertotheiontransfercapillary, havingapositiveeffectoniontransmission.

LiquidjunctioninterfacesaregenerallybasedontheuseofaT- junctionthatallowstheCEseparationcapillaryandanelectrospray tip tocome intocontact througha reservoir ofconductive liquid towhichavoltageisapplied,thusclosingtheelectricalcircuit[7–

9].Themaindiﬃcultieswiththistypeofinterfaceareduetothe

https://doi.org/10.1016/j.chroma.2021.461982

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S. Ferré, N. Drouin, V. González-Ruiz et al. Journal of Chromatography A 1641 (2021) 461982

variability of the electric ﬁeld over time and the formation of a deadvolume,causingalossofeﬃciency.

The triple tube coaxial sheath-flow sprayer works at μL/min withpneumaticassistanceandtheESIneedleisorthogonaltothe MS entranceinthemostconventional configuration.Variationsin the geometric alignment of the sheath-flow interface have been slightlyexploredinalimitednumberofapplications.

Although three types of interfaces including the sheath- liquidinterface,thesheathlessinterfaceandtheelectro-kinetically pumped sheath ﬂow interface [10,11]are commercially available, several alternative interfaces, including homemade setups, have beenproposedbyCE-MSuserstomodulatetheinterfacingparam- eters related to analysissensitivity [12–14].For a numberof developments,theobjectivewastoimprovedetectionwhilepreserv- ing both theﬂexibility androbustnessassociatedwiththeuse of an additionalliquid[15].Othergroupshaveexploredthepossibil- ity to operatecommercially-available interfacesusing customized conditions[16,17].

SinceCEisparticularlyadaptedforthedetectionofchargedand very polarcompounds [18] it constitutes a complementary tech- niquetomostLCapproachesusedinmetabolomics,withtheaim to broadenthecoverage ofthousandsofcompounds withawide range ofphysicochemicalproperties[2,19,20].Owing totheir im- plication in a large number of processes, amino acids (AAs) are of major interest in understanding metabolism functions. In fact, AAs are the main blocks of protein building, precursors of hor- mones and neurotransmitters,and regulators ofgene expression.

Moreover, the functional groups ofAAs are candidates for modi- fication through enzymaticactivity, resultingin thegeneration of newspecies.Insomecases,theenzymaticreactionisnotspecific, andepimetabolitesofAAsaregenerated[21].Noncanonicalspecies can be implicatedin environmentaladaptation or produce toxic- ity.AAanalysiscanbeperformedinnormal(fromanodetocath- ode)orreversed mode,dependingontheir charge andthepH of thebackgroundelectrolyte [22–26].Manyeffortshavebeenmade intodevelopingCE-MSinterfacestoachievesensitiveAAsdetection inbiological matrices[26].Asan example,tenAAs wereresolved within 2 min withan electrokinetically pumpedlow-flow sheath liquid interface. The LODsfor histidine andserine were between 140 ng/mLand 1.58mg/mL,respectively[10].In another work,a flow-through microvial interface operating between100 and500 nL/minachieved5-foldimprovedLODsforAAscomparedtoacon- ventionalsheathliquidinterface[27].

Inthiswork,atriple-tubecoaxialsheath-flowsprayerwasem- ployedformetabolomicsinanoriginalin-axisconfigurationwitha greatlyreducedflowrateforthesheathliquidandwithoutnebu- lizinggas.Asfarasweknow,therehasbeennodemonstratedap- plication of such a configuration formetabolite detection.Herein specific attention was paid to the performance of the presented interfacefortheanalysisofAAs.Finally,quantificationbystandard additionhasbeenperformedincertifiedplasmaasaproofofcon- cept fortheapplicabilityofthenewsourcedesignforroutineap- plications.

Experimentalsection Samplesolutions

Standard compounds were dissolved with 5% acetonitrile and 0.1% formicacid(FA) at1000μg/mLandstoredat−80°C,except for neopterin andbiopterin, whichwere prepared inDMSO, and forguanine,dissolvedin1MHCl.Mixedstocksolutionswerepre- paredin5% acetonitrileand0.1% FAat10 μg/mL,then dilutedto 500ng/mLusing50mMFA.

The mix of 52 low molecular weight compounds con- tained the following standards: 3–hydroxy-DL-kynurenine,

3-methoxytyramine, 4-imidazoleacetic acid, acetylcholine, ade- nine, adenosine, agmatine, amphetamine, biopterin, choline, cis-4–hydroxy-l-proline, creatine, cytidine, cytosine, dopamine, epinephrine,

γ

-aminobutyric acid, guanidoacetic acid, gua- nine, histamine, isobutyrylcarnitine, l-arginine, l-carnitine, 3,4- dihydroxyphenylalanine, l-glutamine, l-histidine, l-kynurenine,l- lysine, l-phenylalanine, l-tryptophan, l-valine,lauroyl-l-carnitine, lidocaine, metanephrine, 3,4-methylenedioxyethylamphetamine, 3,4-methylenedioxymethamphetamine, neopterin, nicotinamide, nicotinic acid, normetanephrine, O-acetyl-l-carnitine, paraceta- mol, phenethylamine, procaine, pyridoxal, pyridoxine, serotonin, spermidine, thiamine, thiamine monophosphate, tryptamine and tyramine. Standard compounds were purchased from Sigma Aldrich (Buchs, Switzerland). The Mass Spectrometry Metabolite Library of Standards, containing 634 compounds distributed in seven 96-well plates according to their hydrophilicity, was also purchased from Sigma Aldrich. One hundred and twenty two compounds were combined into 25 mixtures as described in [28]. Brieﬂy, stock solutions were prepared at a concentration of 25 μg/mL according to the manufacturer’s instructions and dis- tributedintomixturesofupto12compoundswithdistinctmasses (8μg/mL).Themixturesweredilutedsuccessivelyto4μg/mLand 2 μg/mL with water, and eventually diluted to 1 μg/mL using a mixtureof500ng/mLparacetamolandprocaineinwater.

CE

The CEanalyses were carriedout with an Agilent7100 capil- laryelectrophoresissystemfromAgilentTechnologies(Waldbronn, Germany).The appliedvoltagewassetto30kVinpositivepolar- itymodeatroomtemperature(25°C).Fusedsilicacapillariesfrom BGBtechnologies(Boeckten,Switzerland)witha70cmlengthand 50μminternaldiameterwerepreconditionedbeforeﬁrstusewith MeOH,H₂O, 1MNaOH,H₂O,1MHCl,H₂O,0.1MHCl, H₂O,BGE (10%aceticacidv/vinH₂O,pH2.2)at5barforoneminuteeach.

Aportionofpolyimidecoatingwasremovedatbothextremitiesof thecapillaries(5mm).Betweeneachrun,thecapillarywasrinsed with BGE at 5 bar for one minute. Injections were performed hydrodynamically at 50 mbar for 12 s corresponding to 1.0% of the total capillary length as calculated by ZeeCalc v1.0b (http:

//www.unige.ch/sciences/pharm/fanal/lcap/zeecalc/zeecalc.zip).The autosampler was kept at approximately 10 °C using an external watercoolingsystemfromVWR(Nyon,Switzerland).

MS

The Agilent 7100 CE systemwas hyphenated with an Agilent 6490 triple quadrupole LC/MS from Agilent Technologies (Santa Clara,CA, USA) through a triple-tubecoaxialsheath-ﬂow sprayer (AgilentTechnologies,Waldbronn,Germany).Acquisitionswereop- eratedinESIpositivemodewithSRMmeasurements(seesupple- mentary data Table S1). Instrument control, data acquisition and datatreatmentwereperformedwithAgilentMassHuntersoftware versionB.08.00(Agilent,SantaClara,CA,USA).

Interfaces

Forthe conventionalinterface, the sprayerwasmounted on a commercial Agilent ESI source. The capillary voltage, drying gas flowrate,sheathliquidflowrateandcapillaryprotrusionwereset to 5500 V, 11 L/min, 3 μL/min and ~0.28 mm, respectively. The nebulizinggaswassetto 0psi.Thesprayerposition wasorthog- onal to the MS entrance as imposed by the design. For experi- mentswiththenanoflowinterfaceconfiguration,thecapillaryvolt- age,thesheathliquidflowrateandthe capillaryprotrusionwere 2

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A B C

Fig. 1. Scheme of (A) the conventional sheath liquid sprayer orthogonal conﬁguration. Scheme (B) and picture (C) of the new sheath liquid sprayer in-axis conﬁguration. A gas diverter was employed to shunt the drying gas. A 15 °angle to the horizontal was retained based on ESI current monitoring.

set to 4350 V, 300 nL/min and~0.28 mm, respectively. The drying gasflowwasdeviatedusinga diverter.Thetriple-tubecoaxial sheath-flow sprayer was assembled on a 3-axis support in front of the MS entrance withangle adjustments set at9.14, 5.33 and 12.45 mm for the x,y,and z-axes, respectively corresponding to 16.76,1.27and7.87mmfromthecenterofthehexaborecapillary, andaninclinationof15°(Fig.1).Forthefirstexperimentswith52 compounds, the capillaryvoltage andthe sheathliquid flow rate were set to 4750 V and500 nL/min, respectively. In both cases, thesheathliquidwasa50:50:1mixtureofH₂O—iPrOH–CH₃COOH (v/v/v).Acamerawasaddedwhenusingthenanoflowinterfaceto monitorthespray.

QuantitativedeterminationofaminoacidsinNISTplasma

Toperformabsolutequantificationusingstandard additions,l- alanine,l-valine,l-methionineandl-tyrosinewereaddedatdiffer- entdilutionratestoafixedamountofNISTSRM1950metabolites inhumanplasma(Sigma-Aldrich).Foreachquantifiedaminoacid, the corresponding internal standard, l-alanine-3,3,3-d₃ (Sigma- Aldrich), l-valine-d₈ (Cambridge Isotope Laboratories, Tewksbury, UnitedStates),l-methionine-(methyl-13C,d₃)(Sigma-Aldrich)orl- Tyrosine-(phenyl-d₄)(Sigma-Aldrich),respectively, wasaddedata fixed concentrationinall thesamples.Onehundredandfiftymi- croliters ofthe previously spiked NIST SRM 1950 were added to 300 μLofcoldacetonitrile forprotein precipitation(1:2 v/v).The samples were mixed for 15 min at 1200 rpm (4 °C), then cen- trifugedfor10minat15,000g(4°C).Thesupernatantswereevap- oratedto drynessunder anitrogenstream andstoredat−80 °C.

Before injections, 250 μL of water were added to all the samples. The sampleswere refrozen andunfrozen extemporaneously foranalysis,andanadditionaldilutionfactorof2wasappliedwith wateratthisstage.Three samplescorrespondingto twostandard additionswerepreparedandanalyzedintriplicate.

Computationalestimation

Thein-solutionionicdistributionswerecomputedbasedonpKa valuesusingChemAxon’sChemicalize.

Resultsanddiscussion

Newinterface,positioningandqualiﬁcation

Whencommerciallyavailable,ESIornano-ESIsourcesgenerally havedefaultsettingsthatmakeitquiteeasytogenerateionization andacquire data. Thecommercial CE-ESI-MS interface employs a sheathliquidinadditiontoabackgroundelectrolyte(BGE)topro- vide electrical contact anduses heated nitrogen as a nebulizing gastohelpdesolvation.Boththesheathliquidandnebulizinggas havebeendemonstratedto causedeleteriouseffectonsensitivity [4,5]; however,as shownin a previous work, the nebulizing gas canbe easily removedwhile operatingaconventional interfaceif theotherparametersareproperlyadjusted[18].

In this work, an original interface configuration for metabolomics based on the triple-tube coaxial sheath-flow sprayer has been used with the sprayer position modified from orthogonaltoin-axisinfrontofthedetector(Fig.1).Thisconfigu- ration,dedicatedtoworkintheabsenceofanebulizinggaswhile loweringthe sheath liquidflow rate, promotes ion samplingand improvessensitivity. Thepresence ofthe sheathliquidallows for flexibility regarding ionization conditions, while a reduced flow enables for the CE output to be in a closer position in front of the MS inlet. Reducing the flow rate was also expected to have positiveeffectonionizationefficiency[29].

The MS instrumentemployed benefitsfroma heatedcounter- flow drying gaswith a minimal flow rateof 11L/min. Thishigh flowrateofnitrogenhasbeenspecificallyadjustedfororthogonal nebulizationandwasexpectedtodrasticallyreducethestabilityof theMS signalwithin-axisnebulization.Therefore,thedryinggas wasshuntedby meansofadiverterasdescribedin[30].The ad- justablesettingswerethesheathliquidflow,theelectrosprayvolt- ageandthesprayerpositioning,includingtheangleadjustmentto horizontal.A camerawasaddedto thesetuptotrackTaylorcone generation and spray stability, and particular attention was paid tothe risk ofarcingorcorona dischargewhileincreasing theESI voltageandreducingthetip-to-samplingorificedistance.Thegen- erated ESI currentwas first systematically monitored to find the bestoperatingparameters andspatialtip position.Thex,y andz axes were respectively adjusted to 9.14,5.33 and 12.45 mm (see experimentalsection),anda15°angleofthesprayertohorizontal wasretainedasitwasobservedtoimprovestabilityofthespray.

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The nanosourcesettingswere employedonadailybasis,andthis conﬁgurationwasconﬁrmedbyseveraldaysofpreliminaryexper- iments.

The selectedsheathliquidandBGE compositionswere respec- tivelya50:50:1mixtureofH₂O—iPrOH–CH₃COOH(v/v/v)and10%

acetic acid (v/v) in H₂O (pH 2.2); the latter has been success- fullyemployedbothforcationicandanionicmetabolicprofilingby switching the CEpolarity or MS detectionmode [18,31,32].Elec- trophoretic mobility was chosen as a robust criterion for compound annotation andidentification[33–35], since itis aphysic- ochemical property ofa molecule for a definedBGE composition and temperature. Thus, the same BGE composition used in the mobility-based reference library has been selected to fit with a broadrangeofpotentialapplications.

Methoddevelopment

Aﬁrstsetofbasictestcompounds(N=52)fromdifferentbio- chemicalfamilies,includingAAs,nucleosides,nucleobasesandcat- echolamines,waschosen.Thesecompoundswereselectedtocover awiderangeofhydrophilicity(logPfrom−5.8to1.5),massesand pKavalues,resultinginabroadscaleofelectrophoreticmobilities

(from 633to 3910 mm² kV⁻¹ min^–1). Tocompare theperformance betweentheconventional orthogonal andin-axisconﬁgu- rations,thesheathliquidﬂowwasreducedfrom3μL/minto500 nL/min andtheESIvoltage wasset to4750 Vinsteadof5500 V.

The choice ofthenano flow rateandESIvoltagewasmadewith thehelpofESIcurrentmonitoring,wherestabilitywasconsidered as a crucial parameter asdescribed before.Samples were run in triplicate usingeach sourceconfiguration.A normalizationproce- dure was applied by dividingeach peak area by thecorrespond- ing migration time (MT)asrecommendedforquantitative CE-MS applications [36] and a relative quantification (ratio of normal- izedmeanarea)wasobtainedforeachcompound.Thecompounds wererankedaccordingtotheireffectivemobility,asthisvaluehas beendemonstratedtobe arobustcriterion forcompoundannota- tion inCEunderthe definedconditionsofBGE composition.Asa matteroffact,theuseofaneffectivemobilityscalehasbeenpart of the established strategies to address lack of reproducibility of migrationtimesinCE[18,37,38].

As presented in Fig. 2, a significant gain in detection sensitivity was observed withan improvementfactor up to 9 (i.e., l- glutamine). Error bars were plotted to represent the 95% confi- denceintervalestimatedaccordingto[39].Interestingly,acorrela- tionbetweentheeffectivemobilityandthefactorofimprovement was observed. Analytes showing the lowest mobilities showed the best improvement in detection using the new configuration compared to the conventional interface (Fig. 3). Only six compounds were found to present an improvement factor <1, most of them corresponding to the high effective electrophoretic mo- bilityregion.In detail,theaveragearea wassimilar for17%ofall compounds (Mean of individual area/MT_in-axis/Mean of individual area/MT_orthogonal between0.5and1.5),andthereforeagaininde- tection was obtained for more than 80% of the tested analytes.

Theimprovementfactorwasbetween1.5and4.0for52%ofthese compounds,while31%ofthecompoundsshowedanimprovement greater than 4.5. Thistrend wasconfirmed in a second series of experiments with a larger numberof compounds. Approximately 100 additional analytes from a metabolomic commercial library were compared between both configurations (see supplementary dataTableS1). Forthissecondsetofexperiments,thesheathliquid flowandESIvoltagewerefound tobe suitablefornanospray generationwithslightlyloweredvaluescomparedtothefirstsetof experiments,at300nL/minand4350V,respectively,whereasthe three-dimensionalpositionofthesprayer,thatis,theangleadjust- ment,wasnotmodified.Similarresultswereobtainedonthisex-

tendedsetofcompounds,withnoimprovementmeasuredfor17%

of the compounds, 39% presenting an improvementbetween 1.5 and4.0, and 44%demonstrating a ratiogreater than 4.5 (Fig. 4).

Notethatthefactorofimprovementwashigherthan15forsome analytes(e.g.,approximately17for4-pyridoxate) withlow values ofeffectivemobility.Thelatterwasconﬁrmedtobeanimportant parameter in the improvement factor obtained betweenthe two conﬁgurations.

Effective mobility is affected by the degree ofionization of a molecule accordingto thepHofanalysis.Ata pHof2.2,mostof the compounds that exhibited a significant improvement in detection were found to be only partially ionized. As an example, the pKa of l-glutamine carboxylfunction is 2.1, meaning approximately half of the analyte molecules are not ionized. The mean area ofl-glutamineforthein-axisconfigurationcompared tothe conventionalconfigurationwasimprovedbyafactor9.5inthefirst setofexperimentsand9.4inthesecond set,suggestingahigher yieldofionizationwasobtainedintheprototypeconfigurationfor relativelylow ionizedanalytes.It isinteresting tonote thatsome compoundswithalreadyhighionizationefficiencieswiththeorig- inal set-up, as for instance carnitine derivatives or lidocaine, do not exhibit a significant gain in sensitivity (Fig. 2). In fact, low- eringtheflowratefromtheelectrospraytonanospray regimeal- lowsfor the generationof droplets withsmaller than submicron diameters, thus facilitating solvent evaporationand reducing fis- sionevents[40].Moreover,thegenerationofsmallerdropletsim- proves the surface to volume ratio,allowing fora larger propor- tion ofanalytesto betransfered in thegas phase.The generated ionsareeventually efficientlysampledwithatipclosertotheMS orifice.

AminoacidsandquantiﬁcationofNISTplasma

AAs are central components of metabolism. Comprehensive analysisofAAs incomplexsamples isofoutmost importancefor lifesciences. Hence,thisfamilywassignificantlyrepresenteddur- ing methoddevelopment. Approximately 40ofthe selectedcom- pounds were AAs with a median gain in detectionsensitivity of 4.5upto 12.7(i.e.,N-methylaspartate).Notably,10 ofthesecom- poundswere alreadyincluded inthefirst setof compounds,and thecorrespondingimprovementscouldbeconfirmed(i.e.,tyrosine, phenylalanine, tryptophan, cis-4-hydroxyproline, methionine, valine,alanine, guanidoacetate,histidine, arginine,

γ

-aminobutyrate, lysine).

To estimate the limitsof detection (LOD) andlimits of quan- tiﬁcation (LOQ), the orthogonal andin-axisinterfaces were compared using 20 proteinogenic AAs standards. A calibration curve wasbuiltforeach AAwithbothinterfacesfrom15to500ng/mL, startingwithamixedsolutionwithaconcentrationof1000ng/mL that wasfurtherdiluted withwater. Foreach concentration level (k=6),dataacquisitionwasperformedintriplicate(n=3).Rela- tivestandard deviations(RSD) werecalculatedforthesecond (i.e.

30ng/mL)andfifth(i.e.250ng/mL) concentrationlevels foreach AA(see supplementarydata TableS2). TheLOD andLOQestima- tions were obtained based on the standard deviation of the response (Sy) and the slope of the curve (S) for a given analyte, asLOD =3.3(Sy/S)andLOQ =10(Sy/S),respectively (seesupple- mentarydataTableS3). S_y maybedefinedasthestandarddevia- tionofthey-interceptsoftheregressionlinesintriplicate.Forthis concentrationrange,thecorrelationcoefficientswereconfirmedto be greater than 0.98for all ofthe compounds except forglycine andcysteine, with respectivevalues of 0.96and0.88 using both configurations.Cysteineisasulfur-containingAAwhichmightun- dergooxidationintocystine.Serineshowedthebestimprovement, asthe LOQ was214.1 ng/mLwiththe conventional configuration and34.3ng/mLwiththe in-axisconfiguration.Leucine/isoleucine 4

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Fig. 2. The signal improvement factor with the in-axis configuration versus the orthogonal configuration for the first set of compounds ( N = 52). Compounds are ranked by decreasing μeff. The ratio corresponds to Mean of individual area/MT in-axis/Mean of individual area/MT orthogonal.

andvalinedemonstratedimprovementsinLOQfrom250.2ng/mL to 59.8ng/mL andfrom251.5ng/mL to 66.0ng/mL,respectively.

Noimprovementwasevidentforhistidine,glycineandcysteine,although theAAsdemonstratingLOQimprovementgreaterthan3.0 hadeffectivemobilitiesthatwere lowerthan50%ofthemaximal valuefortheset.Itshouldbenotedthattheparametersemployed fordetectionwere identicaltothoseused forthesecond method development set and no further adjustments were performed to speciﬁcallyenhancedAAdetection.

To evaluate the quantitative performances, four key proteinogenic AAs were selected with certified values in standard refer- encematerial(Metabolitesinhumanfrozenplasma,StandardRef- erence Material 1950, National Institute of Standards and Tech- nology) to investigate the applicability of the in-axis configuration forabsoluteestimationinrealsampleanalysis.The quantification of endogenousanalytesis essential forthe comprehensive knowledgeofmetabolicfunctionbutcanbechallengingduetothe lackofblankmatrices.Severalstrategieshavebeendeveloped,including standard addition, backgroundsubtraction, surrogate ma- trix and surrogate analyte methods [41]. The standard reference

materialemployed duringthisstudyhasbeen primarilydesigned to validate metabolitedetermination methods andcomparemea- surementtechnologiesin plasmaandsimilar matrices. The certified values intend to represent healthy human plasmaand have been measured with high confidence. In this case, the four selected AAs (i.e.,alanine, tyrosine,methionine, valine) withinter- mediate LOQ improvements from 1.9 to 3.8 were quantified us- ingthestandardadditionmethodologybybuildingthree-pointcal- ibrations in triplicate. Four internal standards, l-alanine-3,3,3-d₃, l-valine-d₈,l-methionine-(methyl-₁₃C,d₃)andl-tyrosine-(phenyl- d₄), were added at a constant concentration in all of the samples to allow for specific signal correction and decrease injec- tion and ionization variability. The samples were aliquoted and measured with both in-axis and orthogonal configurations. The acquisitions were performed in triplicate, thus the mean area of each point could be used to build three independent determinations resulting in a mean value of all determinations for eachAA.

The obtained concentrations of the endogenous species after calculationsforthein-axisandorthogonal setupsrespectivelyare

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4

e

Fig. 3. Illustrative examples of electropherograms obtained with the ﬁrst set of compounds ( N = 52) with the orthogonal setup (A) the in-axis setup (B).

Fig. 4. The signal improvement factor obtained with the in-axis conﬁguration with regard to the orthogonal conﬁguration versus 1/μefffor the second series of experiments (Pearson’s r = 0.7799). The compounds are highlighted according to their biochemical family.

presentedinTable1.Accuraciesrangedfrom78to113%andfrom 77 to 102% for the newand conventional conﬁgurations, respectively. Overall,this indicates that reliableresults can be obtained withthein-axisconﬁguration.Despitethein-axisspraysampling, noadditionalfoulingwasobservedinthetestedconditions.More-

over, thesimplicityof thedesign andimplementation donot re- quireadditionaltrainingcomparedtotheconventionaltriple-tube coaxial sheath-ﬂow sprayer, which makes the in-axis setup an interesting alternative while preserving the ﬂexibility of sheath liquid-supportedinterfaces.

6

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Table 1

Results of AA quantiﬁcation in standard reference material (Metabolites in human frozen plasma, Standard Reference Material 1950, National Institute of Standards and Technology) with both conﬁgurations.

AA

Expected concentration ±σ (g/L)

(μmol/L)

Concentration measured with the orthogonal conﬁguration ±σ (g/L)

(μmol/L)

Concentration measured with the in-axis

conﬁguration ±σ (g/L)

(μmol/L)

A 26.7 ±2.3

300.0 ±26.0

20.6 ±1.2 231.2 ±13.5

22.8 ±5.2 255.9 ±58.4

V 21.3 ±1.2

182.2 ±10.4

21.7 ±4.5 185.2 ±38.4

24.2 ±4.0 206.6 ±34.1

M 3.3 ±0.3

22.3 ±1.8

2.7 ±0.6 18.1 ±4.0

2.6 ±0.7 17.4 ±4.7

Y 10.4 ±0.5

57.3 ±3.0 9.3 ±1.1

51.3 ±6.1 9.0 ±2.0

49.7 ±11.0

Conclusion

The dilutionandsuction effectsdescribedwiththetriple-tube coaxial sheath-ﬂow sprayer limited the technique’s performance.

A simplealternative hasbeen proposed to improveCE-MS sensitivity withoutnebulizing gas andwitha drasticallyreducedﬂow of additionalliquid. Moreimportantly, thegeometrical alignment ofthesprayerhasbeenmodiﬁedfromorthogonal toin-axis. This setup hasbeenemployedtodetectdifferentbiochemicalfamilies.

Twosetsofexperimentswereperformedandanimprovementwas found for approximately 80% of the studied compounds in both cases witha broadrangeof effectivemobilities. Interestingly,we demonstrated a further gain in sensitivitywith thein-axis inter- faceforpoorlychargedcompoundsinsolution.Infact,thereduced ﬂow operating withthe in-axissetup wasfound tobe especially valuableforthosecompoundsasitfacilitatesionization.

CE-MS is the technique of choice for the detection of ioniz- able polar compounds in scarce samples. Improvements in sensitivity contribute to the attractiveness ofCE-MS for quantitative purposes. Inthe presentstudy,AAs were ofspecific interest.The generationofindividualcalibrationscurvesdemonstratedimprove- ments inLOQsofupto 6for20proteinogenicAAstandards.The quantificationofaselectedsubsetofAAswasachievedincertified plasma withan internal calibrationapproach (i.e.,standard addition)demonstratingthepotentialofthisapproachforthedetermi- nation ofAAs incomplex matricesandits likely applicability for othersmallmetabolites.

DeclarationofCompetingInterest

Theauthorsdeclarethattheyhavenoknowncompetingﬁnan- cialinterestsorpersonalrelationshipsthatcouldhaveappearedto inﬂuencetheworkreportedinthispaper.

CRediTauthorshipcontributionstatement

Sabrina Ferré: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing.NicolasDrouin: Conceptualiza- tion, Data curation, Formal analysis, Investigation, Methodology, Writing - review & editing.VíctorGonzález-Ruiz: Conceptualiza- tion, Formal analysis, Methodology, Supervision, Validation, Visu- alization, Writing-review&editing.SergeRudaz:Conceptualiza- tion,Formalanalysis,Fundingacquisition,Methodology,Projectad- ministration,Resources,Supervision,Validation,Writing-review&

editing.

Acknowledgements

TheauthorswouldliketothankChristianWenzandHans-Peter Zimmermann(AgilentTechnologies,Waldbronn,Germany)forpro- vidingthe in-axiscapillaryelectrophoresis–massspectrometryin- terface.JulienBoccardisalsothankedforhisvaluablediscussions.

SRdeeplythankshismentorProf.S.Fanaliforsteeringhimalong hisﬁrststepsonCE,chiralCEandCE-MS,duringhispost-doctoral stayattheCNRS(Roma),longtimeago.

Supplementarymaterials

Supplementary material associated with this article can be found,intheonlineversion,atdoi:10.1016/j.chroma.2021.461982. References

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