Electron ionization in LC-MS: recent developments and applications of the direct-EI LC-MS interface

REVIEW

Electron ionization in LC-MS: recent developmentsand applications of the direct-EI LC-MS interface

Pierangela Palma & Giorgio Famiglini & Helga Trufelli &Elisabetta Pierini & Veronica Termopoli &Achille Cappiello

Received: 1 October 2010 /Revised: 13 December 2010 /Accepted: 21 December 2010 /Published online: 8 January 2011# Springer-Verlag 2011

Abstract The purpose of this article is to underline thepossibility of efficiently using electron ionization (EI) inliquid chromatography (LC) and mass spectrometry (MS).From a historical perspective, EI accompanied the firstattempts in LC-MS but, owing to several technical short-comings, it was soon outshined by soft, atmosphericpressure ionization (API) techniques. Nowadays, twomodern approaches, supersonic molecular beam LC-MSand direct-EI LC-MS, offer a valid alterative to API, andpreserve the advantages of EI also in LC-MS applications.These advantages can be summarized in three crucialaspects: automated library identification; identification ofunknown compounds, owing to EI extensive fragmentinformation; inertness to coeluted matrix interferencesowing to very unlikely ion–ion and ion–molecule inter-actions in the EI gas-phase environment. The direct-EI LC-MS interface is a simple and efficient solution able toproduce high-quality, interpretable EI spectra from a widerange of low molecular weight molecules of differentpolarity. Because of the low operative flow rates, thisinterface relies on a nano-LC technology that helps inreducing the impact of the mobile phase on the gas-phaseenvironment of EI. This review provides an extensivediscussion on the role of EI in LC-MS interfacing, andpresents in detail several performance aspects of the direct-EI LC-MS interface, especially in terms of response, mass-

spectral quality, and matrix effects. In addition, several keyapplications are also reported.

Keywords Nano liquid chromatography .Massspectrometry . Electron ionization

Introduction

In the last few decades, a long and winding road, made ofdifferent ingenious efforts, was needed to increase compat-ibility between liquid chromatography (LC) and massspectrometry (MS). The apparent incompatibility betweenthe two techniques required many attempts and adjustmentsprior to finding the right compromise between the oppositerequirements. In fact, whereas LC involves a high-pressureliquid phase, the MS analyzer must operate only underhigh-vacuum gas-phase conditions. Furthermore, LC sepa-rations often require gradient conditions, or rely on the useof modifiers that can severely influence the performance ofthe MS instrumentation. The physicochemical properties ofthe analytes can also play a role in the instrument response,and pose additional limitations. In spite of all thiscomplexity, the range of LC-MS instrumentation and itsrelated market are constantly growing, opening the way tomany new fields of research.

Nowadays, we can count on market-ready, high-sensitivity LC-MS interfaces that allow the identificationand quantification of organic and inorganic compounds atvery low concentration. Along with these well-establishedinterfaces, new ideas are forthcoming, based on differentoperating strategies in the attempt to expand the fields ofapplication, the sensitivity, and the ease-of-use of LC-MS.

The development of atmospheric pressure ionization(API) interfaces, approximately two decades ago, boosted

Published in the special issue Advances in Analytical MassSpectrometry with Guest Editor Maria Careri.

P. Palma :G. Famiglini :H. Trufelli : E. Pierini :V. Termopoli :A. Cappiello (*)Dipartimento GeoTeCA, Laboratorio LC-MS,Università di Urbino,Piazza Rinascimento 6,61029 Urbino, Italye-mail: [email protected]

Anal Bioanal Chem (2011) 399:2683–2693DOI 10.1007/s00216-010-4637-0

the use of LC-MS, opening the way to the widespreaddevelopment of this technique. In particular, the mostwidely used is electrospray ionization (ESI) [1], followedby atmospheric pressure chemical ionization (APCI) [2].The first of these is suitable for medium-polarity to high-polarity compounds, with a wide range of molecularweights, whereas the second comes into play for low tomedium molecular weights, and less polar analytes. Othermore recent solutions, such as atmospheric pressurephotoionization [3] and atmospheric pressure laser ioniza-tion [4], are available for nonpolar compounds that do notgive satisfactory results with the other two techniques. Thepossibility of achieving high sensitivity in a wide molecularweight range of analytes has expanded the scope ofbiochemical analysis, and this not only paved the way tothe development of proteomics, but also to the developmentof many other fields of analysis, such as fuel, doping,metabolomic, drug, environmental, bionalytical, and foodanalysis.

All API interfaces are based on low-energy chemicalprocesses (soft ionization) with the formation of protonatedor deprotonated molecular ions (with or without adducts).The fragmentation is either scarce or absent; therefore,tandem MS (MSMS), MSn, or high-resolution accurate MSis required for structure elucidation.

The performance of soft ionization techniques is weak-ened by a few, critical restraints. Quantitative analysis inESI and, to a lesser degree, in APCI can be influenced bycoeluted compounds coming from the matrix. This phe-nomenon is commonly known as matrix effects, and it isresponsible for unwelcome ion suppression or enhancement[5–11] that affects the entire quantitative analysis [8]. Thepolarity of the analytes can also play a role in the signalresponse [7, 12]. The insufficient structural informationprovided by the scarce fragmentation needs to be compen-sated by complex and expensive MS instrumentation. Inaddition, it is extremely difficult to create reference MSMSelectronic libraries, because the processes involved in thecollision-induced dissociation are influenced by manyevents that make them rather unpredictable. Electronionization (EI) is considered a hard ionization technique,based on a gas-phase physical mechanism, and operates in ahigh-vacuum, high-temperature environment. The massspectra are generated after collisions with high-energyelectrons (70 eV), with the production of a positive radicalion and a consequent highly reproducible fragmentation.These spectra are highly informative; therefore, they can beused in structure elucidation, and in the analysis ofunknown compounds. The comparison of the experimentalspectra with those collected in electronic libraries (NationalInstitute of Standards and Technology, NIST; Wiley) is astrong point of EI, and can be used in legal debates [13].The algorithms developed by NIST and Automated Mass

Spectral Deconvolution and Identification System(AMDIS) can be used to extract the mass spectra in thecase of complex unresolved chromatographic peaks. Theionization conditions (gas phase, high temperature, lowpressure) are particularly indicated for gas chromatography(GC)-amenable compounds; however, a significant numberof LC-amenable low to medium molecular weight analytescan be ionized under EI conditions and provide very goodspectra. The operating conditions of LC and EI, inprinciple, are very divergent, and substantial efforts areneeded to find a compromise between the differentreciprocal requirements. When the first attempts appearedon the market, the typical flow rate of LC columns wasbetween 0.3 and 1.0 mL/min, too high for full introductionof the liquid phase into the MS ion source, and there wastoo much liquid phase to eliminate easily. The particlebeam (PB) interface solved this limitation by eliminatingmost of the mobile phase through a gas-phase momentumseparation [14]. PB represented a very brilliant solution tocouple LC and EI-MS but was affected by several draw-backs, in particular in reverse-phase mode, such as scarcesensitivity, signal instability, and limited linearity. PB is notwidely used anymore and only a few applications can nowbe found in the literature [15, 16].

Among the most significant efforts, it is worth mention-ing the attempt by Kientz et al. [17], who used an eluent-jetformation by means of inductive heating of the micro-LCeffluent, and momentum separation in a jet separator.Interesting results were obtained by Dijkstra et al. [18],who, following the approach by Kientz et al., proposed andimproved that technique, focusing their attention on its useunder chemical ionization (CI) MS conditions. Anotherinteresting approach was developed at the University of TelAviv by the group of Amirav. The rationale upon whichthey based their studies was the observation that approxi-mately 30% of the compounds listed in the electroniclibraries are characterized by a weak molecular ion, havingan effect on the identification of truly unknown com-pounds. Amirav and Granot [19, 20] exploited the use of asupersonic molecular beam (SMB) to produce EI spectra,characterized by an intense molecular ion from an LCcolumn. The peculiarity of this approach, called LC-(coldEI)-MS, is that the SMB represents a medium for EI ofvibrationally cold molecules. The result is a spectrum withan enhanced molecular ion, still providing library-searchable fragments.

Our research group has been very active in finding theright design for a simple and straightforward LC-MSinterface, EI-based, following the developments of LC,from microcolumns to nanocolumns. The first attempt wasbased on the use of micro-LC columns, and it could beconsidered as a microversion of the PB interface [21].Owing to the consistent reduction of the liquid effluent into

2684 P. Palma et al.

the ion source, it was possible to record EI spectra with anincrease in sensitivity, compared with classical PB. Itsevolution was the capillary EI system (CapEI, commercial-ized by Waters, Milford, MA, in 1999). The twoapproaches showed many similarities, in terms of conceptand design: they were made to work efficiently atmicroscale flow rates (1-5 μL/min), and they gave goodresults in many applications, under reverse-phase condi-tions [22–31]. For the first time, it was possible to introducenonvolatile buffers into the ion source without variation ofthe MS performance [32, 33]. We developed a completelynew design when nano-LC columns arrived on the market,taking advantage of their nanoliter per minute flow rate.Thanks to the extremely low liquid intake, the interfacedoes not require complicate stages to eliminate the liquidphase, which can be fully introduced into the ion source atflow rates lower than 500 nL/min. This new interface wasnamed direct-EI LC-MS to draw attention to the directcoupling of the nanocolumn into the mass spectrometer. Inmany ways this solution recalls the simplicity of GC-MScoupling.

Recent approaches: direct-EI LC-MS and SMB-LC-MS

Despite initial success owing to the PB interface, the worldof LC-MS has rapidly forgotten EI to develop softionization techniques able to analyze biological macro-molecules. Looking at research papers and conferencepresentations in LC-MS, one finds a disproportionatenumber of API versus EI applications are presented, andEI is considered only a GC-MS ionization technique.Consequently, efforts to develop a more efficient EI-basedinterface were abandoned, although a respectable numberof low to medium molecular weight compounds canprovide very good results. Nowadays, there are two groupsworking constantly on the development of an efficient EI-based LC-MS interface: the group directed by Amirav atTel Aviv University (Israel), who developed the SMB-LC-MS interface, and our group at the University of Urbino(Italy), who developed the direct-EI LC-MS interface. Bothapproaches represent a good starting point to promote EI inLC-MS.

SMB-LC-MS interface

Amirav and coworkers started their work aware that LC-amenable compounds, generally larger and more thermallylabile than GC-amenable compounds, have weak or nomolecular ions in their NIST or Wiley EI mass spectra [19,20, 34]. These compounds are usually less volatile, andneed a higher ion source temperature to vaporize them,

which can cause degradation or weaker molecular ionproduction. In light of this consideration, the sampleidentification would be more effective if it were supportedby a higher molecular ion signal. Starting from pastapproaches, SMB-LC-MS has an initial step of completelythermally assisted spray formation, similar to thermosprayor to PB, that does not involve pneumatic gas assistance.The nebulization step is obtained at relatively high pressure(approximately 0.1 bar), as in APCI, and a complete sampleparticle vaporization of isolated molecules prior to SMB-LC-MS expansion. The supersonic free jet is then colli-mated and forms an SMB that contains “vibrationally cold”sample molecules that proceed axially along a fly-throughBrink-type EI ion source to record cold-EI mass spectra. Toobtain better results, a soft thermal vaporization nozzlechamber accepts the liquid flow coming from an LCsystem, and converts it into a beam of vibrationally coldundissociated sample compounds. This addition provides amajor improvement of the method, with an increase ofperformance and robustness of the sample vaporizationstep. The capillary flow restrictor allows one to obtain the0.1-bar pressure behind the nozzle needed to suppresscluster formation with the solvent vapor, and to have goodvibration cooling. The LC-MS with SMB (SMB-LC-MS)apparatus is schematically shown in Fig. 1 and it is basedon a modified GC-MS system with the SMB system thatwas previously described. The cold EI provides enhancedmolecular ion and improved isomer and structural mass-spectral information, together with all the characteristiclibrary-searchable fragments. No CI due to the solventvapors is involved, and no ion–molecule reactions caninterfere with the mass spectra obtained. Cold EI can ionizeall the sample compounds that were vaporized, regardlessof their polarity, and no nitrogen generator is required. Fourorders of magnitude was the observed linear response, withcurrent limits of detection (LODs) around a few picogramsin selected ion monitoring (SIM) mode for most of thecompounds analyzed. Combined SMB-LC-MS and GC-MSis also possible because the transfer line and nozzle canaccept two columns simultaneously. With this arrangement,GC-MS with an SMB and LC-MS with an SMB can bechanged by a simple "click of the mouse" method, after afew minutes of equilibration without any hardware change[35].

Direct-EI LC-MS interface

The direct-EI LC-MS interface, designed and totallyrealized at the University of Urbino, is a very simpledevice in which the liquid flow coming from an LC systementers directly into the EI ion source of a mass spectrom-eter. This new simple interface was conceived after a long

Electron ionization in LC-MS: developments and applications 2685

period of study on a previous PB-EI-based interface. Thefirst advantages over a PB system were obtained with asignificant reduction of the mobile phase flow rate. Themodifications adopted were a miniaturization of thenebulization process (microPB), and of the entire apparatusin the next version (CapEI). Both devices used flow rates aslow as 1–5 μL/min and they were successfully tested in theanalysis of several classes of compounds under differentreverse-phase LC conditions. Good tolerance to nonvolatilebuffers or chemically modified mobile phases was alsoobserved to be extremely useful in a lot of chromatographicseparations. This is a great advantage with respect to ESIsystems, where the presence of salts could decrease orcompletely cut out the ion production. It was soon clear thatthe direction in which to go to improve the performance ofan EI-based LC-MS interface involved a decrease in themobile phase flow rate.

In the new device, the flow rate of the mobile phaseintroduced should not exceed 0.75 μL/min, mainly imposedby the column backpressure. This flow rate generates lessthan 1 mL/min of vapor, which is within the pumpingcapacity of most MS systems, and no adverse effects on theMS hardware were detected. Nano-LC column technologyis extremely useful when working under these conditions,because flow at a low rate (100-500 nL/min) can be directlyintroduced into the ion source without the negative effectsof mobile phase vapors. To achieve this goal, a completelynew interface was designed, with the hardware reduced to aminimum to enhance robustness and simplicity. Theinterfacing process is carried out through aerosol produc-tion obtained at a nanoscale flow rate, without the help of amakeup gas, and taking into account that the transition ofthe solute into the gas phase has to be as smooth as possibleto avoid peak broadening and thermal decomposition. The

system is called direct-EI LC-MS because the LC effluent isdirectly coupled to the ion source, as well as in capillaryGC, without any other interface device.

The operating principle is simple and it is based on thegas-phase ionization of the sample, as long as it canwithstand the typical EI conditions (high vacuum andtemperature). The absence of ion–molecule or ion–ionreactions decreases the effect of the mobile phase on theionization process, reducing matrix effects, and avoidingany postcolumn adjustments. High-vacuum conditions andthe low flow rate allow one to quickly remove solventvapor from the aerosol that remains composed of smalldroplets of analyte particles. The increase in the surface-to-mass ratio promotes better solute exposure to the sourceheat, limiting thermal decomposition, and accelerating theconversion into the gas phase. Once the transition iscomplete, the original liquid sample is transformed in amix of vapors homogeneously distributed inside the ionsource volume, ready for EI. The interfacing apparatus isentirely contained in the EI source, as shown in Fig. 2. Theprocess is fast and requires a path length of less than 8 mm.The ion source temperature can be varied from 200 to350 °C for most high-boiling compounds. This temperaturecompensates for the latent heat of vaporization of dropletdesolvation, and converts the analytes into the gas phase.The hardware consists of a capillary tubing that protrudes afew millimeters inside the ion source, and a suitablevaporization surface incorporated into the ion source, aswell. A perfect nebulization process can be obtained byadjusting the capillary internal diameter accordingly to theflow rate. The lower the flow rate, the smaller the internaldiameter should be. Different diameters were tested, and thebest solution was a 25 μm internal diameter capillarytubing, with a flow rate of 200-400 nL/min, compatible

Fig. 1 The supersonic molecu-lar beam (SMB)–liquid chroma-tography–mass spectrometryapparatus. LC liquid chromato-graph, GC gas chromatograph,MS mass spectrometer


with nano-LC columns. Conversely, the lowest flow ratecapable of generating a fine and homogeneous spray isapproximately 100 nL/min [36]. It is mandatory that theeluate reaches the ion source as a liquid phase to preventpremature in-tube solvent evaporation, to obtain a good-quality aerosol, and to avoid solute precipitation that mayclog the capillary. Fused-silica capillary tubing could notguarantee sufficient thermal insulation to prevent thisphenomenon and, after a series of attempts, fused-silica/PEEK tubing was selected for thermal insulation. The finalversion of the direct-EI LC-MS interface has 25 μminternal diameter fused-silica/PEEK tubing. Neither theelectron path nor the electric fields are influenced by theinterface inside the ion volume, which protrudes 1 mm, andhigh-quality mass spectra are thus produced.

Compactness and simplicity without the need foradditional complex devices are strong points of the direct-EI LC-MS interface. The lack of any particular transportmechanism involved in the interfacing process reducessample losses, enhances sensitivity, and extends the rangeof possible applications. The strict dependence of thedirect-EI LC-MS interface on nano-LC columns could beconsidered a limitation; however, it is well known that in allconcentration-sensitive LC-MS systems nanocolumns arecommonly employed. The limitations mostly attributed tosmall injection volumes could be overcome by large-

volume injection techniques or by the use of an enrichmentapparatus. In fact, only approximately 1/10,000th of thegas-phase sample molecules are ionized, and this impliesthat EI-based LC-MS interfaces cannot compete with ESI interms of LODs. LODs at the picogram level are normallyobserved in SIM mode for most analytes, whereas hundredof picograms are required to record an interpretable massspectrum. Nevertheless, the impressive sensitivity of ESIhas the drawback of scarce structural information. Costlydouble-stage instrumentation is therefore necessary forstructural elucidation in ESI, whereas with EI, thanks tothe extensive and reproducible fragmentation, a single-stagemass spectrometer is sufficient for analyte characterizationor identification. An MSMS analyzer would probablyincrease the sensitivity, although at the moment there areno experimental data to support this hypothesis.

In Table 1 the performance of the interface is reportedfor four test compounds belonging to different chemicalclasses: dimethyl phthalate, atrazine, lindane, and mestra-nol. It is noteworthy that lindane cannot be detected withthe other LC-MS interfaces. The linear regression equationswere calculated in flow injection analysis (FIA) approxi-mately from the limit of quantitation up to 3 orders ofmagnitude using a mobile phase of 1:1 v/v water andacetonitrile at 300 nL/min flow rate, and injecting 20 nL ofsolutions ranging from 1 to 1,000 ng/L with five data

Fig. 2 The direct electron ioni-zation (EI) liquid chromatogra-phy–mass spectrometryinterface. nano-LC nano liquidchromatography

Dimethyl phthalate Atrazine Lindane Mestranol

Linear regression equation y=2772x-166 y=1274x+1606 y=1592x-7595 y=2114x+2522

R2 0.9999 1 0.9984 0.9988

LOD (SIM) (pg) 2 10 10 20

LOD (scan) (ng) 0.2 0.5 0.5 1

RSD (%) 1.1 1.5 3.7 4.3

Table 1 Performance of theinterface evaluated for fourselected compounds

LOD limit of detection, SIMselected ion monitoring, RSDrelative standard deviation


points. Correlation coefficients, LODs, relative standarddeviations, and R2 calculated for ten replicates are alsoreported.

The performance of the direct-EI LC-MS interface

The effects of the mobile phase composition and flow rate

It is clear from the previous sections that the introduction ofnano-LC columns has represented a significant step forwardin the direct EI LC-MS interface structural design. Thelimited mobile phase flow rate typical of nanocolumnsfacilitates the elimination of the solvent, and this preventschemical reactions with the residual vapors in the ionsource. These circumstances are very similar to those of acapillary GC column, whose gas-phase flow is analogous tothat of a 75 μm internal diameter nano-LC column workingat a flow rate as low as 200-400 nL/min. The experimentalresults are classic EI spectra, with no trace of CI, with onlya possible increase in noise at the lower m/z values for theionization, and fragmentation of the solvent molecules. Apossible negative effect of the presence of solvent vapors inthe ion source is a reduction in the ionization efficiency.Although neither the chemical composition nor the flowrate of the mobile phase has an influence on the quality ofthe experimental mass spectrum, the mobile phase can actas a shield, limiting the electron flow. This limitation has itseffect in terms of sensitivity, which adds up to the fact thatEI is a low-efficiency ionization technique.

Our data indicate that the direct-EI LC-MS interface isconcentration-sensitive because the response increases asthe mobile phase flow rate decreases. This observation, atfirst sight, may seem nonsense, given that the mobile phaseis not a key factor in the solute ionization. Hence, onewould expect that the flow rate would not influence thesignal intensity. As can be seen in Fig. 3, it is clear that atlow flow rates the signal is more intense than at higher flowrates. This leads to an improved signal-to-noise ratio, butalso a higher ionization efficiency, owing to a limitedamount of solvent molecules present in the gas phase,

which may compete with the analyte for the availableelectrons. At high flow rates (more than 1 μL/min), CIinterferences can also be observed, indicating that the mass-spectral quality is getting worse, although this may notprevent an electronic matching of the spectrum.

The direct-EI LC-MS interface, in reverse-phase mode,is rather indifferent to the organic modifiers in the mobilephase, as in the API interfaces; however, the differentphysicochemical properties of methanol and acetonitrilemay have an influence on the evaporation and the quality ofthe aerosol [6]. This effect can seriously limit the success ofa chromatographic separation; in fact, many applicationsare based on pH modifiers in the mobile phase.

Particular emphasis should also be given to the fact thatthe operative reduced flow rates of nanocolumns allow theintroduction into the ion source of nonvolatile buffers, asmodifiers, improving the tolerance toward chemicallymodified mobile phases. The presence of such modifiersis particularly unwelcome in ESI, because they can causesalt depositions that can suppress the ion transmission tothe point that a very low signal is obtained. To overcomethis limitation, volatile buffers are commonly used in allthose chromatographic separations that require a modifierin the mobile phase [37]. The use of ion-pairing agents,such as trifluoroacetic acid, heptafluorobutyric acid, andhydrochloric acid, has the tendency to mask the analytesignal [38–40]. Thanks to the physical-based gas-phaseionization, together with the limited liquid intake of thenano-LC columns, the functioning of the direct-EI LC-MSinterface is not affected by the presence of modifiers in themobile phase, not even by the presence of nonvolatilebuffers. The limited salt deposition can be easily removedby routine cleaning of the ion source. No limitation in theselection of solvents is imposed by the interface mechanism[36–41].

The performance of the interface was tested by intro-ducing into the ion source for 6 days (8 h/day) a mobilephase composed of a 1:1 v/v mixture of two buffers (bufferA, H2O–10 mM KH2PO4; buffer B, 30% buffer A–70%CH3CN). Crucial parameters, such as repeller potential andother voltages, were monitored for 40 hours of total

Fig. 3 Signal intensities of sev-eral model compounds versusmobile phase flow rate


acquisition time, and the mass-spectral quality of a 10 ng/μL standard solution of caffeine was acquired daily in FIA.The probability values from the NIST library also indicatethat the presence of the buffer does not affect the interfacebehavior. A comparison between the recorded peak areas inSIM mode indicates that the sensitivity was not affected.The absolute amount of salt introduced into the system wasso low that it did not influence any of the parametersmonitored. The residue of the buffer introduced was easilyremoved during routine cleaning procedures.

Mass-spectral quality

One of the advantages of the direct-EI LC-MS interface isobtaining mass spectra of the analytes comparable with thosepresent in electronic libraries. This characteristic is very usefulwhen an undoubted identification of unknowns is needed. Aswell as in GC-MS, the spectrum obtained is very informativebecause of a rich fragmentation. The identification isautomatic and it is legally defensible. Detailed and exhaustivecriteria of how mass-spectrometric data can be utilized inqualitative and quantitative analysis are reported in EuropeanCommission Decision 2002/657/EC. Algorithms developedby NIST and AMDIS can be applied to extract the realanalytes’ mass spectra when several overlapping peaks arepresent in the chromatogram of the sample. The presence ofsolvent vapors in the ion source was the main disadvantage inthe first attempts at direct coupling of LC and MS. As aconsequence, CI greatly influenced the mass-spectral results.With direct-EI LC-MS we never observed CI signals in thespectra, the EI spectra obtained are always reproducible, andaccurate isotope ion clusters can be recorded for molecularformula confirmation. The low rates of flow introduced intothe ion source produce only higher background noise in thelow-mass region of the spectrum, depending on the solventused. Low-mass noise rarely influences the matching qualityand excellent identification is generally achieved, as demon-strated by the spectrum of heptachlor in Fig. 4. Manycompounds that do not ionize with ESI produce a spectrumof very good quality with direct-EI LC-MS (matching of90% or more). To enhance the sensitivity and the quality ofthe spectrum, other strategies could be followed: it is realisticthat different surface materials and aerosol conditions can

optimize the liquid phase to gas phase transition. The firststudies on the surface process of vaporization indicated thatTeflon® polytetrafluoroethylene was able to enhance thesensitivity and mass-spectral quality with respect to astainless steel surface in the analysis of high-boilingcompounds [24]. Stainless steel and all the other materialsthat can be found in the standard EI ion sources wereselected to work with GC, where the analytes are eluted fromthe column in the gas phase, and they have very soft contactwith the source surface. When the analytes are eluted froman LC column, they are dissolved in a liquid phase that willbe nebulized and vaporized inside, and in strict contact withthe source walls. The hot surface of the ion source promotesthe vaporization of the less volatile molecules but, at thesame time, it could actively interact with these molecules,leading to thermal decomposition. Teflon® and other inertmaterials are notoriously less active than metals, and theycould represent a better solution to improve the sensitivityand the quality of the mass-spectral results, as demonstratedin previous preliminary studies.

Matrix effect evaluation

A serious limitation in the high selectivity of MSMS thataffects ESI- and APCI-based LC-MS methods is representedby matrix effects. Coeluted matrix components are responsi-ble for unexpectedly suppressing or enhancing the analyteresponse. The high selectivity ofMSMS does not seem to be asolution to reduce the sample preparation step, and to securethe quantification procedures, as reported by many authors [6,42]. The different interactions that are responsible for matrixeffects during the liquid-phase ionization are difficult topredict, and therefore to circumvent, and they appear to becompound-, matrix-, method-, and instrument-specific and,in many cases, sample- and lot-specific [5, 43]. Theminimization of matrix effects can consist of extensivecleanup procedures, improved chromatographic separations,and the use of stable-isotope-labeled internal standards [7, 8,44]. These solutions can help to reduce and control matrixeffects; nevertheless, they add additional steps to be added tothe method. It should not be understated that stable-isotope-labeled internal standards are not available for all analytes,and this limitation increases the complexity of the method

Fig. 4 The direct-EI mass spec-trum of heptachlor in tetrahy-drofuran (6 mg/L) is shown inthe upper trace, and the stan-dard NIST EI library spectrum isshown in the lower trace(matching factor 748, reversedmatching factor 778, probabilityfactor 95.8%)


development. The gas-phase EI of the direct-EI LC-MSinterface is less affected by matrix effects typical of APIinterfaces. The process of ionization is physical-based,involves odd numbers of electrons, and is highly energetic.It generates a spontaneous, highly reproducible fragmenta-tion, independent of the type of instrument. The coelutedcompounds are vaporized and ionized as a result of singlemolecule–electron interactions, independently of each other;as a consequence, the signal intensity is related only to theconcentration of each analyte.

We compared the behavior of direct-EI LC-MS and ESI-MSMS with model pesticides and pharmaceutical compoundson matrices of different composition (human plasma, riverwater, and seawater) [36, 45–47]. We used liquid–liquidextraction and solid-phase extraction (SPE) to extract theanalytes from the matrix. Matrix effects were calculatedfollowing the methods already reported in the literature,performing postcolumn infusion and postextraction additionexperiments. The absolute matrix effect was calculated as theratio between the average peak area of the sample spiked afterextraction (n=3) and the average peak area of the neatstandard solution (n=3), multiplied by 100. In this case, aresult higher than 100% indicates ionization enhancement,whereas a result lower than 100% indicates ionizationsuppression. The results obtained on the selected compoundsare summarized in Table 2 and demonstrate that the ionizationprocess of direct-EI LC-MS is not affected by matrix effects.The signals of the same compounds ionized under LC-ESI-MS conditions are influenced by matrix effects, with the onlyexceptions being methomyl and atrazine in river water.

Effects of the ion source temperature on signal response

Temperature is a fundamental parameter in EI andconsequently in direct-EI LC-MS. When the LC effluent

is eluted from the capillary, the source heat promotesvaporization of the low-boiling compounds, whichbecome promptly available for EI. As already stated,the temperature has a double function: to compensatefor the latent heat of solvent evaporation, and to convertthe analytes to the gas phase. It is mandatory that thisconversion occurs as rapidly as possible to avoidthermal decomposition of the sensitive compounds. Thegeneration of a fine aerosol is recommended to obtaingood and fast heat distribution, together with correctheating of the system. This could be achieved by properthermal insulation of the interface capillary, and by thehigh temperature of the ion source.

During the development of the interface, differenttemperatures of the ion source were tested in the rangefrom 150 to 350 °C. Compounds with diverse physico-chemical characteristics were analyzed in FIA to verifytheir response. The results suggest that high temperaturepromotes better sensitivity, especially for high-boilingcompounds, as expected. High-boiling compounds gener-ally require the presence of a suitable vaporization surfaceas well, on which the unvaporized residues can be broughtinto the gas phase, before being removed by the pumpingsystem. From our experience, there is a discrepancybetween the set temperature and the actual one measuredat the top of the interface capillary. The real temperature atthe top of the interface capillary was measured by replacingthe interface capillary with a special thermocouple. Asetting of 350 °C resulted in a real temperature of 103 °C.The heat transfer is not particularly efficient in high-vacuum conditions, and this does not help the vaporizationof high-boiling compounds, with a consequent lowresponse. To maximize the sensitivity and, at the sametime, to expand the range of molecules that can be analyzedby direct-EI LC-MS, a more effective heat transfer system

Compound Summary of the experimental conditions ME±RSD (%)

Matrix investigated Extraction method ESI-MS Direct-EI LC-MS

Ibuprofen Human plasma LLE 64±22 101±5

Human plasma SPE 52±14 97±4

Phenacetin Human plasma LLE 135±14 99±2

Human plasma SPE 123±13 101±2

Aldicarb Artificial matrix Injected without extraction 53±7 115±6

River water SPE 74±8 100±10

Atrazine Artificial matrix Injected without extraction 48±6 99±4


Methomyl Artificial matrix Injected without extraction 60±6 101±3


Propazine Artificial matrix Injected without extraction 61±4 99±5


Table 2 Relative matrix effect(ME) evaluation in human plas-ma extract and water samplesusing liquid chromatography(LC)–electrospray ionization(ESI)–tandem mass spectrome-try (MS) and direct electronionization (EI) LC-MS

LLE liquid–liquid extraction,SPE solid-phase extraction


should be used, preferably directly on the top of thecapillary. Work is in progress on this fundamental topic.

Fields of application of the direct-EI LC-MS interface

The direct-EI LC-MS interface is particularly indicated forthe analysis of low to medium molecular weight com-pounds with a wide range of polarities. Interestingapplications involve all those compounds that are hardlyionized with the LC-API-MS techniques. The direct-EI LC-MS interface has demonstrated good performance in thepresence of complex mixtures with a large number ofanalytes having different physicochemical properties. Inparticular, differences in polarity are not an issue as they are

in ESI, and this is advantageous in environmental applica-tions. We presented a method for the determination of 29endocrine-disrupting compounds, belonging to the classesof phenols, steroids, polycyclic aromatic hydrocarbons,organochlorines, carbamates, triazines, phthalates, andalkaloids in seawater samples of the mid-western AdriaticCoast of Italy [48]. A preconcentration SPE step wasneeded before direct-EI LC-MS analysis. The simultaneousdetection was possible in a single analysis, and all theanalytes gave high-quality EI spectra. This represents anadvantage compared with other approaches, where, with asimilar mixture, ESI-MS requires double detection innegative and positive ion modes [49].

The analysis of organochlorine pesticides in watersamples is another application considered particularly

Table 3 Method validation for boronic acids with direct-EI LC-MS

Compound LOD (pg) scan/SIM LOQ (pg) scan/SIM Linear regression equation R2

4-Propylphenylboronic acid 232/2.5 772/8.3 y=10698.0x -167579 0.9998

4-Ethylphenylboronic acid 387/2.5 1,289/8.4 y=7797.0x -133169 0.9999

Phenylboronic acid 386/3.6 1,285/12.1 y=7428.4x -108795 0.9998

2-(2′-Methoxybenzyloxy)-phenylboronic acid 337/3.3 1,124/10.9 y=8393.9x -156893 0.9986

cis-Propenylboronic acid 636/5.0 2,120/16.5 y=1271.2x -26940 0.9970

2-Thienylboronic acid 586/10.6 1,893/35.2 y=4019.7x -100102 0.9998

Methylboronic acid 4,275/726 14,249/2422 y=50.5x -3449.6 1

LOQ limit of quantitation

Fig. 5 Total ion chromatogram of a standard solution of nonesterifiedfree fatty acids (62.5 μg/mL). Column Agilent Zorbax SB C18,150 mm×75 μm internal diameter, 3.5-μm particle size; gradient from100% buffer A to 100% buffer B in 40 min (buffer A, water plus 0.1%trifluoroacetic acid; buffer B, acetonitrile plus 0.1% trifluoroacetic

acid); injection volume 500 μL; flow rate 400 nL/min. 1 caprylic acid,2 capric acid, 3 lauric acid, 4 lauric-1-13C acid, 5 arachidonic acid, 6linolenic acid, 7 myristic acid, 8 palmitoleic acid, 9 linoleic acid, 10palmitic-d31 acid, 11 palmitic acid, 12 oleic acid, 13 stearic-d35 acid,14 stearic acid, 15 arachidic acid


challenging, because these very persistent and nonbiode-gradable compounds do not ionize with the LC-API-MStechniques. Such compounds are commonly analyzed byGC-MS; however, in multiresidue determination high-polarity, poorly volatile compounds can be present in thesample. The presence of these compounds would requirethe development of multistep GC-MS and LC-API-MSmethods. Direct-EI LC-MS can overcome this restraint; infact, with use of this approach the simultaneous trace-leveldetection of organochlorine pesticides and phenoxy acidpesticides was possible [50]. This represents the first single-step method for the analysis of LC-amenable and GC-amenable compounds together, with the recording of high-quality EI spectra for 19 pesticides in river water samples.

Direct-EI LC-MS was successfully used for the analysisof boronic acids, another class of compounds scarcelyionizable with the LC-API-MS methods, and that requires aderivatization step prior to GC-MS analysis. These analytesare used as coupling reagents to produce drugs, agro-chemicals, or herbicides, and their degree of purity isdirectly related to the purity of the final product. Asreported in Table 3, the method shows good linearity andLODs [51].

Lipidomics and metabolomics can also take advantage ofthe use of direct-EI LC-MS analysis. The simultaneousprofiling of nonesterified free fatty acids (NEFAs) inbiological matrices is particularly important because oftheir role in metabolic pathways, and they can beconsidered biomarkers for several diseases. The profilingof NEFAs in complex samples involves GC-MS afterextraction and derivatization of the lipid material, which isa complex and time-consuming procedure, despite the goodsensitivity and precision [52]. In addition, owing to thecomplexity of plasma and tissue matrices, quantitativeresults can be affected by matrix effects. In the analysis ofNEFAs, thanks to the absence of matrix effects, direct-EILC-MS reduces the sample preparation procedure, leadingto an overall shorter analysis time, and allows theundoubted identification of the target compounds. Prelim-inary results for the analysis of several saturated andunsaturated NEFAs in plasma are reported in Fig. 5 [53].The compounds of interest did not need derivatization, andthe spectra recorded showed NIST probability factorshigher than 80%.

Conclusions

The direct-EI LC-MS interface has been constantly im-proved since the first design, presented in 2002. Nowadays,it represents a challenging approach for low to mediummolecular weight analytes involved in many fields ofapplication. The response does not depend on the polarity

of the analytes, making the interface extremely flexiblewhen compared with other interfaces available on themarket. The design is robust and straightforward, and itcan be installed on simple instruments equipped with asingle quadrupole, although more sophisticated analyzerscan be used as well. The strong points of the direct-EI LC-MS interface are the absence of matrix effects, the tolerancefor salts and other nonvolatile buffers, and overall simplic-ity of operation. These characteristics extend the number ofdetectable molecules and expand the fields of application ofthe interface. Further efforts will be devoted to optimizingthe design to boost the sensitivity.

Acknowledgement The authors acknowledge Agilent Technologiesfor supplying the instrumentation.

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Electron ionization in LC-MS: recent developments and applications of the direct-EI LC-MS interface