Research article

Received: 27 May 2012 Accepted: 1 August 2012 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/jms.3077

Study of the electrical connection mechanism ofsheathless interface for capillary electrophoresis-electrospray ionization-mass spectrometryCai Tie,a De-Wen Zhang,a Hong-Xu Chen,b Sen-Lin Songc andXin-Xiang Zhanga*

With the combination of high separation ability of capillary electrophoresis (CE) and strong identification ability of massspectrometry (MS), CE/MS is becoming a powerful tool for polar and ionic analytes analysis. Different interfaces have beendeveloped to enhance the sensitivity and reliability since the first introduction of CE/MS in 1987. A sheathless porous interfacebased on a new ions transferring electric connection technique was reported to be with high sensitivity and reliability. In thiswork, a series of optical and electrochemical experiments were designed to study the electric connection process. The resultsindicated that closing CE electrical circuit and applying MS spray voltage were achieved by the small ions transferring throughthe interface porous wall. The new electric connection method significantly enhanced the sensitivity, resolution and stabilityof the CE/MS analysis. The interface was applied in CE/MS detection of morphine and 6-monoacetylmorphine in urine sampleand showed an equal sensitivity to LC/MS. With the significant improvement of sensitivity and stability, the CE/MS withthe new interface showed strong potential for the determination of low abundance analytes. Copyright © 2012 John Wiley& Sons, Ltd.

Keywords: porous sheathless interface; CE/MS; electrical connection mechanism; high sensitivity; abused drug control

* Correspondence to: Xin-Xiang Zhang, 1Beijing National Laboratory for MolecularSciences (BNLMS), Key Laboratory of Biochemistry and Molecular Engineering ofMinistry of Education, Institute of Analytical Chemistry, College of Chemistry,Peking University, Beijing, 100871, China. E-mail: zxx@pku.edu.cn

a Beijing National Laboratory for Molecular Sciences (BNLMS), Key Laboratory ofBiochemistry and Molecular Engineering of Ministry of Education, Institute ofAnalytical Chemistry, College of Chemistry, Peking University, Beijing, 100871,China

b Beckman Coulter Commercial Enterprise (China) Co., Ltd, 12/F., East OceanCenter, 24A Jian Guo Men Wai Avenue, Beijing, 100004, China

c Beijing Center for Drug Abuser, Chinese Association of Drug Abuse Preventionand Treatment (CADAPT), Psychiatric Hospital Huang Liang Rd. Daxing DistrictBeijing, 102600, China

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Introduction

The combination of capillary electrophoresis and mass spectrom-etry (CE/MS) can achieve high sensitivity and high resolution forcomplex separations. The high separation efficiency and lowsample consumption of CE and the structural identification capa-bility of MS for both qualitative and quantitative analysis can beachieved by the combination of these two techniques. Differentkinds of interfaces, such as coaxial sheath-flow interface[1] andliquid junction interface[2] were reported since the first sheath-liquid interface introduced in 1980s.[3] All these interfaces reliedon the presence of different kinds of sheath flows to close theCE electrical circuits and apply ESI spray voltage.

Due to the delivery of an additional liquid at a relative highflow rate than that presented in CE, these interfaces had beenfound to cause loss of sensitivity and resolution, which certainlylimited a wide acceptance of the CE/MS platform.[4] A sheathlessinterface[5] was first introduced by Wahl group in 1994. Theelectric connection was established through the conductive coat-ing at the spray outer surface. The conductive outer surface wasobtained by coating conductive material such as gold, silver,copper, nickel or graphite materials.[6–17] Despite the improvedsensitivity afforded by the sheathless design, the robustnesswas still challenge because of the poor stability of the metalcoating and the electrolytic reaction within the tip.

A new sheathless CE/MS interface was developed by Moinigroup.[18] The authors speculated that redox reactions of waterat the ESI needle and ion transporting through the porous tipinto the capillary provided the electrical connection for theESI and CE outlet electrode. The improvement of the CE/MSsensitivity, stability and resolution were achieved.[19] This technique

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was applied to protein–protein and protein–metal complexanalysis[20,21] with enhanced sensitivity recently.[22] Based on theseworks, a high sensitivity porous sprayer interface now called as CESIfor CE/MS had been developed by Beckman Coulter. This interfaceimproved the performance of CE/MS by closing the CE electricalcircuit and applying spray voltage with porous capillary. It isnecessary to find more direct evidences to support the proposedelectrical connection mechanism.

In this article, a series of experiments were carried out,and some direct evidences were found to support the ions trans-ferring electric connection mechanism. The interface was appliedto CE/MS analysis of morphine and 6-monoacetylmorphine(6-MAM) in urine sample and showed high sensitivity and separa-tion efficiency.

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Materials and methods

Materials

Sodium chloride, potassium chloride, lithium chloride, calciumchloride, morphine and ammonium acetate were purchased fromSigma-Aldrich (St. Louis, MO, USA); HPLC-grade methanol waspurchased from Fisher Scientific (Fair Lawn, NJ, USA). Calcium ionfluorescence probe Fluo-3, pentasodium salt was purchased fromFanbo Biochemicals Co. Ltd. (Beijing, China). 6-MAMwas purchasedfrom Isotec (Miamisburg, OH, USA). Water was purified by Milli-Qpure water system. Urine sample was provided by ChineseAssociation of Drug Abuse Prevention and Treatment BeijingCenter for Drug Abuser, and the volunteers have been informedbefore sampling.

Instrumentation

Inverted fluorescence microscope (type-Ti, Nikon, Japan) andZDMCI6-1 chip analysis platform (Jiangnan Optics and ElectronicsCo., Nanjing, China) were utilized to monitor ion transfer throughthe porous interface. The outer surface structure of the capillarywas obtained by atomic force microscopy (SPA-400, Canon,Japan). Autolab PG302N electrochemical workstation (ECOCHEMIE, Utrecht, Netherlands) was used to measure the imped-ance of the interface. Fused-silica capillary of 30 mm i.d. and150mm o.d. purchased from Polymicro (AZ, USA) was applied tothe interface as well. The CESI interface and associated consum-ables were provided by Beckman Coulter (Brea, CA, USA). TheCE/MS detection of morphine and 6-MAM was applied on thecoupling of PA800 plus CE system (Beckman Coulter, Brea, CA,USA) and Agilent 6320 ion trap Mass spectrometer (AgilentTechnologies, Palo Alto, CA, USA).

CESI interface surface morphology analyzed by AFM

CESI interface was immobilized on the platform with double-sidedtape. Scanning was carried out under tapping scanning modewith SI-DF3 silica tip (SII, Chiba, Japan). A 250nm� 250nm areawas scanned.

AC Impedance analysis of new interface

A part separation capillary, containing the new interface, was cutand further immobilized on a glass slidewith both ends surroundedby reservoirs filled with buffers as shown in Fig. 1A. Both reservoirsand the capillary were filled with the same buffer at a concentrationof 1mM and then rinsed with the solution to equilibrate theinterface. The working electrode and counter electrode werepositioned in the two reservoirs, respectively. The AC impedanceswere measured from 1Hz to 105Hz.

Ion transport through the CESI interface

Different buffers were used to fill the reservoirs as shown in Fig. 1A.For calcium ion transferring study, the capillary was rinsed with asolution containing 10mM Fluo-3 probes initially. Then bufferreservoirs A and B were filled with 1mM CaCl2 and 10mM Fluo-3probe solution, respectively.

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Application of new interface inmorphine and 6-MAMdetection

500ml urine samples was pretreated with C-18 SPE columnpurchased from Waters (Milford, MA, USA) and concentrated byten folds prior to be analyzed by CE/MS.

CE/MS separations were carried out with two capillaries: one withCESI (30/150mm i.d./o.d. and 75 cm in length) was for separationand another (75/365mm i.d./o.d. and 80 cm in length) was used toprovide static conductive liquid for the electrical connection. TheCE background electrolyte was 10mM ammonium acetate atpH 5.2. Each new separation capillary was activated by rinsed under50psi with 0.1M NaOH for 2min, followed by water for 3min andthen running buffer for 5min, prior to the first use. Betweenanalyses, the capillary was rinsed with 0.1M NaOH under a 50psipressure for 0.5min, water for 1min, and running buffer for 3min.The sample was electro-kinetically injected at 10 kV for 10 s.Separation voltage of 20 kV was applied with an anode located atthe inlet part of the capillary. During CE separation, ion sprayvoltage was applied at �1.4 kV. MS parameters were: positivemode; nebulizer gas, 0 psi; dry gas, 3.0 L/min; cone temperature,325o C; scanning range from 150 to 500m/z.

Results and discussion

Interface surface morphology

The surfaces of the un-pretreated capillary without polyimidecoating and CESI interface were observed by AFM (Fig. 2).Comparing with CESI interface, the surface of un-pretreatedcapillary wasmuch smoother. 4–6nm radii cavities can be observedin the surface of CESI interface which was created by etchingprocess.[18] There were many pores formed in different radii duringthe etching process. The cavities observed on the capillary outersurface were the end of these pores. The electrical connection ofCE/MS could be achieved by the ions transferring throughthose pores.

Selective ion transfer based on the ion size

The impedances of CESI interface were measured to analyze thedifferent ions transferring in the CESI interface porous wall. ACimpedances measurements were carried out as mentioned in thework of Wainright to eliminate the capacitance influence in thesolution.[23] The impedance measured under relative highfrequency, its phase angle was almost 0�which indicated that thedata measured under high frequency was almost a resistancecharacter and barely effected by the capacitance. AC impedancewasmeasured in KCl, NaCl and LiCl solutions at 1mMconcentration.Under this salts concentration, the migration of positive andnegative ions was independent and only affected by their ionicmobility. The conductance of the solution was the summary ofpositive and negative ions conductance, as shown in Eqn (1). Theconductivity of positive ions could be calculated.

Λs¼lþ þ l� (1)

lþ ¼ Λs � l� ¼ Λs � l�Cl

lþ ¼ kþSL

Iþ ¼ S

L¼ lþ

kþ(2)

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Figure 1. The porous interface spray end was immobilized on a slide as (A) both for AC impedance measurement and ions transfer analysis. Thefluorescence intensity inner porous interface was increasing with the separation voltage applying as (B), the fluorescence image at0 min, and 2minseparation voltage applied(C/D).

Figure 2. The surface AFM image of a normal capillary without polymer coating and porous interface (A/B).

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Where Λs is the conductance of the salts solution, l+ and l-

were the conductance of positive and negative ions, к+ was theconductivity of positive ions, L was the positive migrationdistance and S was the section area.

The I+ value of different positive ions should be only related tothe value of S and L. If the porous interface showed no ions selectiv-ity, the I+ values would be constant because the measurementswere carried out with the same interface. Based on the measuredimpedance data, I+ values of different positive ions were calculatedas shown in Fig. 3. The I+ values decreased significantly fromK+ to Li+, which indicated the total section area for different ionsto transfer was decreased from K+ to Li+ with the constant

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migration length of the same interface. All the three positive ionswere in the same group and showed similar characters in thesolution but with different size. The hydrated ions radius ofK+ to Li+ increased from 3Å to 6Å. The transferring area of theinterface decreased with the increase of hydrated ions radius. Thetransferring section was made up of a huge number of pores inthe interface. Comparing with larger ions, there were more poresfor smaller ions to get through. The transferring section area forthe ions with smaller hydrated ions radius is larger. The decreasingof the I+ values became slight while the ions became larger. Itindicated that the amount of the pores between the size ofNa+ and Li+ was smaller than those between K+ and Na+. This result

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Figure 4. CE/MS analysis of drug abusers urine sample. Linear relation-ship of morphine and 6-MAM analyzed by CE/MS (A). Morphine and6-MAM tracking in urine sample (B).

Figure 3. I+ value of K+, Na+ and Li+.

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supported that the pores in most abundance was smaller than thesize of Na+. This pores size distribution could limited the larger ionssuch as analytes migration cross the interface effectually.

Transfer of ions

An optical experiment was designed to obtain the evidence forions transfer. As shown in Fig. 1A, reservoir B and the capillary werefilled with a Fluo-3 solution. Calcium chloride solution surroundedthe porous part of the interface in reservoir A. The fluorescenceintensity of Fluo-3 solution would be enhanced by formingcomplexes with calcium ions. Fluorescence images were obtainedwhile a separation voltage was applied. The results were shown inFig. 1C/D. The fluorescence inside the porous interface increasedwhile the voltage was applied and finally became constant in about50 s as showed in Fig. 1B. This indicated that there was enoughcalcium ions transferred through the porous interface when theelectric field was applied. At the given probe concentration of10mM, as predicated, the complex concentration would keepconstant when the calcium ions concentration achieved 102mMlevels. It indicated that the concentration of the calcium ionsshould be about 102mM when the fluorescence strength insidethe interface became constant. Based on these data, the totalcalcium ions transferred into the capillary, and the average currentcould be estimated.The current carried by these calcium ions would be at about pA

level. This current with such low salts concentration is too small tobe measured. The current with 1mM calcium chloride was measurealternatively under the same condition. It was at nA level. Itindicated that the current carried by the ions transferring took themajor part of the whole current. The results showed that ionstransferring through the interface closed the electrical circuit andapplied spray voltage while the CE/MS analysis. Based on thiselectrical connection mechanism, the redox was kept away fromthe spray tip which could improve the spraying stability.[5] Theelectric connection method also avoided the necessary of sheathliquid which would cause the significant sample dilution the signalsuppression and the peaks broadening. The stability, sensitivityand resolution of CE/MS were improved by the application ofCESI interface.On the other hand, the fluorescence intensity increasing out of

the porous interface was not observed during applying separationvoltage. It supported that the Fluo-3 ions cannot migrate cross theporous interface as calcium ions did. As mentioned above, most of

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the pores in the porous interface were smaller than the size ofhydrated sodium ions. The Fluo-3 ion was too big to get throughthe etched pores. The most analytes of CE/MS had similar size asFluo-3. The porous interface should keep them inside as Fluo-3.The porous interfaces showed a capability to minimize the analytesleakage during the CE/MS analysis.

The comparison among CESI CE/MS and othersanalysis methods

The CESI interface was applied in the detection of morphine and6-MAMdetection. Quantification was performed based on the peakareas in extracted ion chromatography. The results were showed inFig. 4A. The peaks widths at half height were no more than 0.2min.That supported that the peaks broadening was suppressedwith CESI interface, and the resolution of CE was maintained inCE/MS successfully. LODs of the two compounds were both aslow as 1 ng/ml. The results was compared with others analysismethods (Table 1). The sensitivity of CESI CE/MS was higher thansheath-liquid CE/MS and even comparable with LC/MS. The resultsemphasized that none sheath-liquid design of CESI interfaceenhanced the sensitivity of CE/MS analysis effectively. With theadditional consideration of the low sample consumption, CE/MSwith CESI interface could be a powerful tool for low abundancesample analysis.

CESI interface was further applied to tracking the major metabo-lites of heroin in drug abusers’ urine. Urine samples from different

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Table 1. Comparison of 6-MAM and morphine assays

Method 6-MAM Morphine References

LOD Linear Rang LOD Linear Rang

Sheath-flow CE/MS 100 ng/mL NA 100ng/mL NA Wey, A. B.[24]

CE/UV 100 ng/mL NA 100ng/mL NA Wernly, P.[25]

LC/MS 0.5 ng/mL 1–100 ng/mL 3 ng/mL 10–1000ng/mL Katagi, M.[26]

CESI CE/MS 1 ng/mL 5–500 ng/mL 1 ng/mL 5–500 ng/mL This work

NA: not available

Electro-connection of shealthless CE/MS interface

sampling time after the last intaking were analyzed by CE/MS.The concentration of morphine and 6-MAM decreased along withthe time extension as shown in Fig. 4B.

Conclusions

In this work, a series of optical and electrochemistry experimentswere carried out to obtain direct evidences supporting theions transferring electric connection mechanism for CESI interface.These results indicated the ions migration through the sizeselective porous interface played an important role in the electricconnection and helped enhanced CE/MS performance. By thismechanism, CESI interface minimized the analytes leakage whilemaintaining the conductivity. Avoiding the dilution and broaden-ing of the sample bands caused by the sheath liquid, both sensitiv-ity and resolution of CE/MS were enhanced sequentially by CESIinterface. With the ions transferring, there was no redox reactionthat occurred around spray tip during the CE/MS analysis. Thishelped stabilize the ESI spraying. The CESI interface was appliedto trackmajormetabolites of heroin (morphine and 6-MAM) in drugabusers’ urine. With CESI interface, the resolution and sensitivity ofCE/MS methods were enhanced. The study on the urine samplesemphasized the optimization of CE/MS by CESI interface. Optimizedby CESI interface, CE/MS has a potential to be powerful analysistools for low abundance analytes.

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Acknowledgements

This work was supported by National Natural Science Foundation ofChina (No. 90713013, 30890142, 20975007) and the NationalScientific Support Project 2009CB320305 (MOST, China). The detailsuggestions from Dr. Jerald S. Feitelson and Dr. John C. Hudson ofBeckman-Coulter were highly appreciated.

References[1] M. A. Moseley, L. J. Deterding, K. B. Tomer, J. W. Jorgenson. Capillary-

zone electrophoresis/fast-atom bombardment mass spectrometry:design of an on-line coaxial continuous-flow interface. Rapid Commun.Mass Spectrom. 1989, 3, 87.

[2] S. Pleasance, P. Thibault, J. Kelly. Comparison of liquid-junction andcoaxial interfaces for capillary electrophoresis mass-spectrometry withapplication to compounds of concern to the aquaculture industry.J. Chromatogr. 1992, 591, 325.

[3] J. A. Olivares, N. T. Nguyen, C. R. Yonker, R. D. Smith. Onlinemass-spectrometric detection for capillary zone electrophoresis.Anal. Chem. 1987, 59, 1230.

[4] C. A. Nesbitt, H. X. Zhang, K. K. C. Yeung. Recent applications of capillaryelectrophoresis-mass spectrometry (CE-MS): CE performing functionsbeyond separation. Anal. Chim. Acta 2008, 627, 3.

J. Mass Spectrom. 2012, 47, 1429–1434 Copyright © 2012 John

[5] J. H. Wahl, D. C. Gale, R. D. Smith. Sheathless capillary electrophoresis-electrospray ionization mass spectrometry using 10 [mu]m I.D.capillaries: Analyses of tryptic digests of cytochrome c. J. Chromatogr.A 1994, 659, 217.

[6] A. P. Dahlin, M. Wetterhall, G. Liljegren, S. K. Bergstrom, P. Andren,L. Nyholm, K. E. Markides, J. Bergquist. Capillary electrophoresis coupledto mass spectrometry from a polymer modified poly(dimethylsiloxane)microchip with an integrated graphite electrospray tip. Analyst 2005,130, 193.

[7] X. F. Zhu, S. Thiam, B. C. Valle, I. M. Warner. A colloidal graphite-coatedemitter for sheathless capillary electrophoresis/nanoelectrosprayionization mass spectrometry. Anal. Chem. 2002, 74, 5405.

[8] Y. Z. Chang, Y. R. Chen, G. R. Her. Sheathless capillary electrophore-sis/electrospray mass spectrometry using a carbon-coated taperedfused-silica capillary with a beveled edge. Anal. Chem. 2001, 73,5083.

[9] Y. Z. Chang, G. R. Her. Sheathless capillary electrophoresis/electrospraymass spectrometry using a carbon-coated fused silica capillary. Anal.Chem. 2000, 72, 626.

[10] L. Bendahl, S. H. Hansen, J. Olsen. A new sheathless electrosprayinterface for coupling of capillary electrophoresis to ion-trap massspectrometry. Rapid Commun. Mass Spectrom. 2002, 16, 2333.

[11] A. D. Zamfir, N. Dinca, E. Sisu, J. Peter-Katalinic. Copper-coatedmicrosprayer interface for on-line sheathless capillary electrophoresiselectrospray mass spectrometry of carbohydrates. J. Sep. Sci. 2006,29, 414.

[12] Y. R. Chen, G. R. Her. A simple method for fabrication of silver-coatedsheathless electrospray emitters. Rapid Commun. Mass Spectrom.2003, 17, 437.

[13] Z. Kele, G. Ferenc, T. Klement, G. K. Toth, T. Janaky. Design andperformance of a sheathless capillary electrophoresis/massspectrometry interface by combining fused-silica capillaries withgold-coated nanoelectrospray tips. Rapid Commun. Mass Spectrom.2005, 19, 881.

[14] S. Nilsson, O. Klett, M. Svedberg, A. Amirkhani, L. Nyholm. Gold-coatedfused-silica sheathless electrospray emitters based on vapor-depositedtitanium adhesion layers. Rapid Commun. Mass Spectrom. 2003, 17,1535.

[15] R. S. Ramsey, S. A. McLuckey. Capillary electrophoresis electrospray-ionization ion-trap mass-spectrometry using a sheathless interface.J. Microcolumn Sep. 1995, 7, 461.

[16] S. Nilsson, M. Wetterhall, J. Bergquist, L. Nyholm, K. E. Markides. Asimple and robust conductive graphite coating for sheathless electro-spray emitters used in capillary electrophoresis/mass spectrometry.Rapid Commun. Mass Spectrom. 2001, 15, 1997.

[17] L. L. Fang, R. Zhang, E. R. Williams, R. N. Zare. Online time-of-flightmass-spectrometric analysis of peptides separated by capillaryelectrophoresis. Anal. Chem. 1994, 66, 3696.

[18] M. Moini. Simplifying CE-MS operation. 2. Interfacing low-flowseparation techniques to mass spectrometry using a porous tip.Anal. Chem. 2007, 79, 4241.

[19] J.-M. Busnel, B. Schoenmaker, R. Ramautar, A. Carrasco-Pancorbo,C. Ratnayake, J. S. Feitelson, J. D. Chapman, A. M. Deelder, O. A. Mayboroda.High Capacity Capillary Electrophoresis-Electrospray Ionization MassSpectrometry: Coupling a Porous Sheathless Interface with Transient-Isotachophoresis. Anal. Chem. 2010, 82, 9476.

[20] A. Nguyen, M. Moini. Analysis of major protein-protein and protein-metal complexes of erythrocytes directly from cell lysate utilizingcapillary electrophoresis mass spectrometry. Anal. Chem. 2008,80, 7169.

Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/jms

3

C. Tie et al.

1434

[21] M. Moini. Metal displacement and stoichiometry of protein-metal complexes under native conditions using capillary electro-phoresis/mass spectrometry. Rapid Commun. Mass Spectrom. 2010,24, 2730.

[22] R. Haselberg, C. K. Ratnayake, G. J. de Jong, G. W. Somsen.Performance of a sheathless porous tip sprayer for capillary electro-phoresis-electrospray ionization-mass spectrometry of intact proteins.J. Chromatogr. A 2010, 1217, 7605.

[23] D. Shental-Bechor, Y. Levy. Folding of glycoproteins: towardunderstanding the biophysics of the glycosylation code. Curr. Opin.Struct. Biol. 2009, 19, 524.

wileyonlinelibrary.com/journal/jms Copyright © 2012 Joh

[24] A. B. Wey, W. Thormann. Head-column field-amplified sample stackingin presence of siphoning - Application to capillary electrophoresis-electrospray ionization mass spectrometry of opioids in urine. J.Chromatogr. A 2001, 924, 507.

[25] P. Wernly, W. Thormann. Drug of abuse confirmation in human urineusing stepwise solid-phase extraction and micellar electrokineticcapillary chromatography. Anal. Chem. 1992, 64, 2155.

[26] M. Katagi, M. Nishikawa, M. Tatsuno, A. Miki, H. Tsuchihashi. Column-switching high-performance liquid chromatography-electrosprayionization mass spectrometry for identification of heroin metabo-lites in human urine. J. Chromatogr. B 2001, 751, 177.

n Wiley & Sons, Ltd. J. Mass Spectrom. 2012, 47, 1429–1434