mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

Journal of Chromatography B

Using 1.5 mm internal diameter columns for optimal compatibility with current liquid chromatographic systems

SzabolcsFeketeâ^,^b^,^∗,Amarande Murisierâ^,^b,Gioacchino LucaLosaccoâ^,^b,Jason Lawhorn^c, Justin M.Godinho^c, Harry Ritchie^c,Barry E. Boyes^c,Davy Guillarmeâ^,^b

aSchool of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

bInstitute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland

cAdvanced Materials Technology, 3521 Silverside road, Suite 1-K, DE 19810, Wilmington, United States

a r t i cl e i n f o

Article history:

Received 30 March 2021 Revised 11 May 2021 Accepted 14 May 2021 Available online 18 May 2021 Keywords:

Thisarticledescribestheuseofanewprototypecolumnhardwaremadewith1.5mminternaldiameter (i.d.)anddemonstratessomebenefitsoverthe1.0mmi.d.micro-borecolumn.Theperformanceof2.1,1.5 and1.0mmi.d.columnsweresystematicallycompared.Withthe1.5mmi.d.column,thelossofapparent columnefficiency canbesignificantlyreducedcomparedto1.0mm i.d.columnsin bothisocraticand gradientelutionmodes.Intheend,the1.5mmi.d.columnisalmostcomparableto2.1mmi.d.column fromapeakbroadeningpointofview.Theadvantagesofthe1.5mmi.d.hardwarevs2.1mmi.d. narrow-borecolumnsarethelowersampleandsolventconsumption,andreducedfrictionalheatingeffectsdue todecreasedoperatingflowrates.

ThisisanopenaccessarticleundertheCCBY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/)

1.Introduction

Analyticalscaleliquidchromatographic(LC)columns are com-mercially available in 4.6, 3.9, 3.0, 2.1, 2.0 and 1.0 mm inter-nal diameters (i.d.). Terms such as standard-bore (4.6, 3.9 and 3.0 mm i.d.), narrow-bore (2.1 and 2.0 mm i.d.) and micro-bore (1.0mm i.d.),are oftenusedto describeanddistinguishthe dif-ferentcolumnformatsandtherequiredoperatingflowrate[1,2,3].

Columnsbasedon3.0– 4.6mm havehistorically dominatedthe fieldof chromatographyhowever,therehasbeenasignificant in-creasein using2.1 mm i.d.columns,largelydue tothe adoption ofultra-highpressure liquidchromatographic (UHPLC)technology andUHPLC–MSsystems[3].Conversely,theuseof1.0mmi.d. col-umnformatisstillnotwidelyadopted.

As possible advantages, smaller column diameters result in lowersolventconsumption,thusreducingthecostofanalysisand offeringagreenersolution[4].Onsmalldiametercolumns,the op-timalflow rateis lower, therefore frictional heat effectsbecome lessimportant, andgiverisetomoreefficient desolvationforLC–

MSanalyses,resultinginhighersensitivity.Finally,thesample

con-∗Corresponding author at: Waters Corp., School of Pharmaceutical Sciences, CMU - Rue Michel Servet, 1, 1211 Geneva, Switzerland.

E-mail address: szabolcs.fekete@unige.ch (S. Fekete).

sumptionisalsoreducedwithsmallercolumns,sinceinjected vol-umehastobescaledindirectproportionwiththecolumnvolume, tomaintainthesamesensitivity.

On the other hand, the disadvantages of micro-bore columns may lie in limited loading capacity anddecreased apparent effi-ciencyduetoextra-columnbandbroadening.The limitedloading capacityisoftennotverycritical,butthelossofapparentcolumn efficiencycanbeserious.Lestremauandco-workerscomparedthe apparentefficiencyof1.0×100mm and2.1×100mmcolumns packedwiththesamematerial-usingamodernUHPLCsystem– andonlyabout67%oftheefficiencywasobtainedonthe1.0mm i.d.columncomparedtothe2.1mmi.d.one,inisocraticmode[4]. The apparentefficiencycould beimprovedbyincreasing the col-umn lengthand thereforethe ratioof column volumetosystem volume. In gradient mode, the contribution of the extra-column bandspreading was significantly reduced and a peak capacity of about80%oftheirequivalent2.1mmi.d.columnscanbeobtained on the 1.0 mm i.d. column [4]. Wu and Bradley reported about 60%dropinplatenumbers(N)whencomparing2.1×50mmand 1.0 ×50 mm columns packedwith1.8µm particlesand operat-ingthemunderisocraticconditions(N=9010platesvs.3580)[5]. Theyconcludedthattheefficiencylossduetoextra-columnband broadening increases asthe columndiameter andcolumn length decrease. Thiseffectwaseven morepronouncedforearlyeluting components. Another study also reported that the extra-column

https://doi.org/10.1016/j.chroma.2021.462258

S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258

dispersionofagivenLCsystemcandramaticallydecreasethe ap-parentperformanceofhighlyefficientnarrow-borecolumns[6].To properlyoperate2.1 ×50mm columns,an LCsystempossessing systemdispersionaslowasσ²_≤10µL²isrequiredtomaintainat least55%ofintrinsiccolumnefficiency.Whencouplingmicro-bore columnstoMS,thetubingusedtointerfacetheUHPLCsystemto the MSdevice is particularly critical in both isocratic and gradi-ent modes, because this tubingis locatedafter the column out-let,wherethebandcompressioneffectsthatcompensateforband broadeningdo notoccur[7].Standard commercialUHPLC–MS in-struments(unmodified)exhibitσ²valuesrangingfrom20tomore than100µL².However,byminimizingthevolumeofthe interfac-ingtube, the extra-column variancecan be reduced toσ² _≤ 20 µL² for anytypeof MSdetector(please notethatroutinely,very longinterfacingtubesareusedin practice,such as50– 100cm).

WithanoptimizedUHPLC–MSconfiguration,thelossinefficiency witha 2.1mmI.D. columnwasnegligibleatretention factors(k) higher than7, while the 1mm I.D. columnwashardly compati-blewith currentinstrumentation, even at k> 20.The impact of theextra-columnbandbroadening onthe chromatographicpeaks ingradientmodewassubtle,thoughstillunacceptablewith micro-borecolumns[7].

The lowerefficiency of1.0 mmi.d.columns isnotexclusively duetoextra-columndispersionbutcan alsobecaused bypoorly packedbeds,fritdispersionandaxialbedheterogeneity.Grittiand Wahabreportedthatthepackedbednearthewall ofthecolumn isdenserthanthatofthebulkpacking,whichresultsindifferences inbothsoluteandmobilephasevelocitythroughthecolumn[8]. Fora1.0mmi.d.column,thewallregionvolume(denser)tobulk regionvolume(lessdense) ratioiscloseto1whichisthe worst case.Forlarger-diametercolumns,thewallregionvolumebecomes lesssignificant and -for smaller column internaldiameter - the bulkregionbecomeslesssignificant.Thus,the1.0mmi.d.isoften consideredtobe theworst-case frombedheterogeneitypointof view.

Despite the expectedly high efficiency loss with 1.0 mm i.d.

columns, these columns can be used by utilizing carefully opti-mizedsystems,optimalcolumnhardwaredesign,verylowinjected volumeandbybenefiting fromband focusingeffects usingweak injectionsolvent[9,10].Schoors andco-workersreportedthe suc-cessfulusageof1.0mmi.d.columnsfortheanalysisofmonoamine neurotransmitters[11].Theydemonstratedasignificantincreasein sensitivityusing1.0mmi.d.columnascomparedtoa2.1mmi.d.

column. In addition, weak solvent injection helped focusingthe sampleatthecolumninlet.

It seems today that despite the sensitivity increase and re-duced solvent consumption, the adoption of micro-bore columns isstilllimited.Theeffortsneededtocompensateforsystemband broadening seemto be astrong barrier for mostusers. The aim of this study was to evaluate a compromise between 1.0 and 2.1mm columns.Thus,aprototype 1.5mmi.d.column was pre-paredandsystematicallycomparedtocommercial2.1and1.0mm i.d.columns packedwithahighlyefficientcolumnpacking mate-rial(2.7µm superficiallyporous90 ˚AC18).Columnefficiency ob-servedinbothisocraticandgradientmodeswerecomparedusing averylowdispersion(AcquityI-Class)andastandardUHPLC (Ac-quityH–Class)systems.Inaddition,thesensitivityofMSdetection wasalsostudied.

2.Experimental

2.1. Chemicalsandsamples

Acetonitrile (AcN), methanol (MeOH), ethanol (EtOH), wa-ter and formic acid were purchased from Fisher Scientific (Reinach,Switzerland).Uracil,methylparaben,ethylparaben,

propy-lparaben, butyl–paraben, cannabidivarine (CBDV), cannabigerolic acid (CBGA), tetrahydrocannabivarin (THCV), cannabichromene (CBC), delta9-tetrahydrocannabinolic acid (THCA-A) and hu-man serum albumin (HSA), were purchased from Sigma–

Aldrich. Cannabidiolic acid(CBDA),cannabigerol (CBG), cannabid-iol (CBD), cannabinol (CBN), (-)-delta9-THC (d9-THC) and (-)-delta8-THC(d8-THC)werepurchasedfromLipomedAG(Arlesheim, Switzerland). Terbutaline,fenfluramine,norfentanyl,atenolol, ben-zoylecgonine, probenecid, hydrochlorothiazide, etacrynic acid, furosemide, chlorthalidone, bumetanide and bendroflumethiazide solution at1mg/mLinMeOHwerekindly providedbytheSwiss LaboratoryforDopingAnalyses(Epalinges,Switzerland).

2.2. Chromatographicsystem

ForUHPLC-UVmeasurements, twoUHPLCsystems were used.

One wasa very low-dispersion system,namely aWaters Acquity UPLC I-Class (Waters, Milford, MA, USA) equippedwith abinary solvent deliverypump,anautosampler andUVdetector.The sys-temincludedaflowthroughneedle(FTN)injectionsystemwith15 µL needleanda0.5µLUVflow-cell.The extra-columnvolumeof thesystemwasmeasuredasVec=7.5µL,whilethegradientdelay volumewasV_d =98 µL.The other systemwasa WatersAcquity UPLC H–Classequippedwithaquaternarysolvent deliverypump, an autosampler and UVdetector. The system included a FTN in-jection system with15µL needleanda 0.5µL UV flow-cell.The extra-column volumeof the system wasmeasured as Vec =11.5 µL,whilethegradientdelayvolumewasV_d=370µL.

ForUHPLC-MS/MSmeasurements,athirdWatersAcquityUPLC I-Class, composed of a binary solvent delivery pump andan au-tosampler(loop offline), washyphenated toaWaters TQD Triple Quadrupolemassspectrometer,fittedwithaZ-sprayESIsource.A capillaryvoltageof±1.5kV,sourcetemperatureof150°C, desolva-tiontemperatureat450°C,desolvationandconegassetat750L/h and0L/hwereappliedtoallanalyses. Nitrogen(N2)wasusedas both desolvationandconegas,whileargon(Ar)wasemployed as the collision gas.Multiple reaction monitoring(MRM) mode was usedduringUHPLC-MSanalyses.TheUHPLCsystemwasconnected totheMSvia65µmx50cmPEEKtube.

Data acquisition and instrument control for UHPLC-UV mea-surements were performed by EmpowerPro 3software (Waters, Milford, MA, USA),while for UHPLC-MS analyses, MassLynx v4.1 (Waters, Milford, MA, USA) was used. Data was treated in Excel (Microsoft)forUHPLC-UVanalyses,whileTargetLynxv4.1(Waters, Milford,MA,USA)wasusedforUHPLC-MSmeasurements.

2.3. Columns

Anewprototype1.5×100mmcolumnpackedwith2.7µm su-perficiallyporous90 ˚AC18particlesandcommercial1.0×100mm and 2.1 × 100 mm columns packed with the same material wereprovidedbyAdvancedMaterialsTechnology(Wilmington,DE, USA).

2.4. Sampleandmobilephasepreparation

Amixsolutioncontaining uracil,methylparaben,ethylparaben, propylparaben andbutylparaben was prepared in 10:90 v/v ace-tonitrile:waterat50µg/mL.Uracilandparabenswereelutedinthe mobilephase,namely35:65v/vacetonitrile:water.

Amixsolutioncontainingelevencannabinoids(i.e.CBDV,CBGA, THCV, CBC,THCA-A, CBDA, CBG,CBD, CBN, d9-THCand d8-THC) was prepared from individual stock solutions diluted in solvent having the same composition as mobile phase “A” at 45 µg/mL.

The individual stock solutions were prepared in eithermethanol, acetonitrileorethanoldependingontheirsolubility.Cannabinoids

S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258

Fig. 1. Apparent plate numbers ( Napp) as function of solute retention factor ( k ) observed on Acquity I-Class system at high (A) and low flow rates (B) and on H-class system at high (C) and low (D) flow rates with 1.0, 1.5 and 2.1 mm i.d columns. Sample: methyl-, ethyl-, propyl- and butyl–paraben.

were separated in gradient mode. Mobile phase “A” was 0.1%

formicacidinwater,whilemobilephase“B” was0.1%formicacid inacetonitrile.Alineargradientof60– 95%Bwasappliedattwo different gradient steepness (corresponding to gradient times of t_G1 =10 min andt_G2 = 20 min), since peak width depends on gradientsteepness.

For UHPLC-MSanalyses, twomixtures of doping agents were used.Bothmixtures werepreparedin 5:95v/vmethanol:water at a finalconcentration of 200 pg/mL. Analyses were performed in gradient mode. Mobilephase “A” was 0.1% formicacid in water, whilemobilephase“B” was0.1%formicacidinacetonitrile.A lin-eargradient from5%to95%of mobile phase“B” was appliedat twodifferentgradient steepness (t_G1 =5 minfor allanalyses at high-flowratesoneachcolumn,t_G2 =3.3minforallanalysesat low-flowrates).

2.5. Comparisonofefficiency

The linear mobile phase velocity (u0) and the total column porosity(ε_T)weredeterminedfromthefollowingequation:

u₀= L t₀= 4F

ε_Td²_cπ ⁽¹⁾

whereListhenominalcolumnlength,t₀isthecolumndeadtime (correctedforsystem residencetime),Fisthemobilephase flow rate,anddcthenominalcolumndiameter.Thecolumndeadtime wasmeasuredbyinjectingnon-retainedcompound(uracil).

When comparing column efficiency, twomobile phase veloc-ities were set (low and high level). Linear velocities u0 = 15 and 25 cm/min were considered, corresponding to F ~ 0.3 and 0.5 mL/min on the 2.1 mm i.d., F ~ 0.15 and 0.26 mL/min on the 1.5 mm i.d. and F ~ 0.07 and 0.11 mL/min on the 1.0 mm

i.d. column.Mobile phasetemperature wasset to30 °C.Inboth isocratic andgradientmodes,threeinjectionvolumesweretested (V_inj=0.1,0.5and1µL)sinceinjectedvolumemightimpactboth system andcolumnband-broadening,especially forsmallvolume columns.

Inisocratic mode,the apparent platenumbers (Napp, not cor-rected for systemdispersion) obtained withthe parabenmixture werecompared.PlotsofNapp versusretentionfactor(k)were plot-tedandlogarithmictrendswerefitted.Theretentionfactorsofthe parabens were comprisedbetween k~ 1 and11, whichis repre-sentative of common practice. Since in isocratic mode, both the pre- andpostcolumndispersions impactthe totalband broaden-ing,twoUHPLCsystemswereusedtoseethedifferencesof appar-entefficiency.

Ingradientmode,thepeakwidthsofcannabinoidsmeasuredat halfheight(w_1/2)werecomparedandplottedasafunctionof ap-parent retention factor (kapp, based onobserved retention time).

The gradient measurements were performed only on the low-dispersion system.Pleasenotethatingradientmode,itismostly thepostcolumnvolumethatimpactstheoverallpeakbroadening, since the pre-column dispersion iscompensated by the gradient band focusingeffect. The postcolumnvolumeof thelow disper-sion(AcquityI-Class)andastandardUHPLC(AcquityH–Class) sys-tems were the same (0.5 µL UV cell andpost column connector tubingof65 µmx 30cm),thus whyonly onesystem wastested here.

2.6. Comparisonofsensitivity

Peakintegrationandsignal-to-noise(S/N)measurements were performed via the TargetLynx tool available in MassLynx v4.1.

Smoothingprocess wasappliedtoallchromatograms prior toall

S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258

Fig. 2. Comparative chromatograms, obtained with 1.0 ×100 mm, 1.5 ×100 mm and 2.1 ×100 mm columns - packed with superficially porous C18 material - in isocratic mode (standard UHPLC, Acquity H –Class). Mobile phase 35:65 v/v acetonitrile:water, mobile phase velocity u0 = 25 cm/min. Sample: methyl-paraben (1), ethyl-paraben (2), propyl-paraben (3) and butyl–paraben (4).

calculations,usingaSavitzky-Golay methodwithasmoothing it-eration value of 3 and a smoothing width of 2. The “Peak-to-Peak” methodwasappliedforsignal-to-noisecalculation, measur-ingpeak signal level from the baselineand by fixing aconstant noisesignalwindowof1.0minforallchromatograms.

3.Resultsanddiscussion

Inthe first instance,the columnvolumes andporositieswere determined.Column volumes,V₀,were equalto173, 100and49 µL,while porositiesε_T were equal to0.50, 0.57 and0.63for the 2.1,1.5and1.0mmi.d.columns,respectively.

RegardingV0,itisimportanttokeep inmindthe general10%

rule of thumb for extra-columnband broadening and associated efficiencyloss[12]:theextra-columnvolumeshouldnotbemore than10%ofthe column’spackedbedvolume(tolimit the contri-butionofsystemdispersion).Theextra-columnvolumeofourvery lowdispersionUHPLC systemisVec =7.5µL, whichcorresponds to4, 7 and15% of the 2.1, 1.5and 1.0 mm i.d.columns volume (100mmlength),respectively.Ontheotherhand,thesystem vol-umeofourstandard UHPLCsystemisVec=11.5µL,which corre-spondsto7,11and23%ofthe2.1,1.5and1.0mmi.d.columns vol-ume(100mm length),respectively.Thesevaluessuggestthatthe 1.5×100mmcolumn canbeoperated onavery lowdispersion

systemwithoutsignificantefficiencyloss,whileonastandard UH-PLC,lowerapparentefficiencyisexpected,buttoamuchlesser ex-tentthanona1.0×100mmcolumn.Tooperatea2.1×100mm i.d. column (packed with superficially porous particles) without significant efficiency loss,a system volumeof Vec ≤17 µL is re-quired.Fora1.5and1.0×100mmcolumn,Vec≤10µL,andVec

≤5µL arerecommended,respectively.Thislattercriterion(Vec ≤ 5µL)isproblematic,sincecommercial UHPLCsystemsallpossess Vec >6–7µL(typicallybetween6and20µL)[6,7,12].

Itisalsoworthmentioningthattheobservedcolumnporosity increaseswhendecreasingthe columndiameter.Thisobservation islogicalandcanprobablybeexplainedbythefollowingreasons:

Theapparentporosityofsmallcolumnsincreaseswhendecreasing column diameter or length, dueto the higher ratio of extra-bed volume (V_eb) topackedbedvolumeanddue tosomedifferences in packing quality (density)too [13,14].The extra-bedvolumeof a column hardware was recently described as the total volume of column hardwareflow distributor,flow collector, frits,and in-let/outlet connections [13,14]. To have an idea about its value, a recentstudyreportedextra-bedvolumeofV_eb~ 4µLfora2.1mm i.d.columnhardware[14].Therefore,forverylowvolumecolumns (such asshortcolumns of 1.0,1.5and2.1 mm i.d.),notonlythe extra-column systemvolume, butalso theextra-bedcolumn vol-umeneedtobeconsideredaspossiblesourceofefficiencyloss.

S. Fekete, A. Murisier, G.L. Losacco et al. Journal of Chromatography A 1650 (2021) 462258

Fig. 3. Peak widths (w1/2) as function of solute apparent retention factor (k app) observed at b = 0.025 (A), 0.04 (B), 0.05 (C) and 0.08 (D) gradient steepnesses with 1.0, 1.5 and 2.1 mm i.d columns. Sample: cannabinoinds standard mixture. The widths of only the well-resolved peaks are considered. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.1. Apparentefficiencyinisocraticmode

Fig.1showsthe measuredplatenumbers(Napp)asafunction ofsoluteretentionfactor(k).Figs.1AandBcorrespondtothevery lowdispersion system.Athighflow rate,Napp =15,000– 19,000 plateswere obtainedonthe 2.1mm i.d.column.The 1.5mmi.d.

column resulted in Napp = 12,500 - 19,000 and the 1.0 mm i.d.

column provided Napp = 9500 – 16,000. In average, the 1.5 and 1.0 mm i.d. columns performed ~93% and ~76% efficiency com-pared to the 2.1 mm i.d.column, respectively. At lowflow rate, Napp = 19,500 – 21,000 (2.1 mm i.d.), Napp = 16,500 – 21,000 (1.5mm i.d.)and Napp =11,500 – 16,700 (1.0mm i.d.)were ob-served.Inaverage,weobserved~94%and69%efficiencywiththe 1.5and1.0mmi.d.columns,respectively,comparedtothe2.1mm i.d.column.

It can also beseen that the lessretained peaks aremore af-fectedbysystemdispersion.Thisisobviouslyduetothefactthat columnpeakvariance(σ_col²)-andthereforepeakwidth-depends onsoluteretention:

σ_col² ₌ ^V

N_col(¹+k )² ⁽²⁾

where N_col is the column intrinsic efficiency (plate number un-affected by system dispersion). Therefore, at low k, the ratio of system dispersionto columndispersion increases duetothe de-creaseofcolumndispersion.Whencomparingtheefficiency corre-spondingtopoorly(k~ 1)andhighlyretained(k~ 10)compounds, theefficiencyobtainedforthepoorlyretainedcompounddropsby about20,35and40%onthe2.1,1.5and1.0mmi.d.columns, re-spectively,at lowflow rate. Similarly,at highflow rate, this effi-ciencylosscorrespondsto7%(2.1mmi.d.),17%(1.5mmi.d.)and 31%(1.0mmi.d.).

Figs.1CandDshowtheapparentefficiencyobtainedona stan-dard UHPLC system. At high flow rate, Napp = 13,000 – 16,000 plateswereobservedwiththe2.1mmi.d.column.The1.5mmi.d.

columnprovidedinNapp=10,500– 16,000,whilethe1.0mmi.d.

column performedNapp =8500– 15,000.Inaverage,the1.5mm i.d. column performed ~92% while the 1.0 mm i.d. column per-formed ~88% efficiencycompared to the 2.1 mm i.d.column. At lowflowrate,Napp=17,500– 19,000(2.1mmi.d.),Napp=14,200 – 18,500 (1.5 mm i.d.) and Napp = 8200 – 16,000 (1.0 mm i.d.) were observed.In average, weobtained ~90%and 80% efficiency withthe1.5mmand1.0mmi.d.columns,respectively,compared tothe2.1mmi.d.column.Betweenpoorlyandhighlyretained so-lutes,athighflowrates,wesaw19,34and43%differenceinplate numbers onthe 2.1,1.5and1.0mmi.d.columns,respectively.At low flowrate, weobserved 8%(2.1 mm), 23%(1.5 mm)and49%

(1.0mm)differencesinplatenumbersbetweenk~ 1and10.

Asanexample,Fig.2showscorrespondingchromatograms ob-tained with the standard UHPLC system operating at high flow rate.Itisworthmentioningthatnotonlyplatenumbers,butpeak symmetryisalsoaffectedbythe columndiameter.Thepoorly re-tained compounds elutein moreasymmetrical peaks onsmaller borecolumns.

3.2. Apparentefficiencyingradientmode

Ingradientelutionmode,theapparentefficiencyisexpectedly less affected by system dispersion, since most of the dispersion occurring in the pre-column volumes are compensated by band focusing taking place at the top of the column. Therefore, pre-columndispersionisalmostnegligible,whileapparentefficiencyis

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