B American Society for Mass Spectrometry, 2013DOI: 10.1007/s13361-013-0697-7

J. Am. Soc. Mass Spectrom. (2013)

APPLICATION NOTE

Direct Electrospray Ionization Mass Spectrometric Profilingof Real-World Samples via a Solid Sampling Probe

Zhan Yu,1 Lee Chuin Chen,2 Mridul Kanti Mandal,3 Kentaro Yoshimura,4 Sen Takeda,4

Kenzo Hiraoka3

1College of Chemistry and Biology, Shenyang Normal University, Shenyang, Liaoning 110034, China2Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Kofu, Yamanashi 400-8511,Japan3Clean Energy Research Center, University of Yamanashi, Kofu, Yamanashi 400-8511, Japan4Department of Anatomy and Cell Biology, Interdisciplinary Graduate School of Medicine and Engineering, University ofYamanashi, Chuo-ku, Yamanashi 409-3898, Japan

Abstract. This study presents a novel direct analysis strategy for rapid massspectrometric profiling of biochemicals in real-world samples via a direct samplingprobe (DSP) without sample pretreatments. Chemical modification is applied to adisposable stainless steel acupuncture needle to enhance its surface area andhydrophilicity. After insertion into real-world samples, biofluid can be attached onthe DSP surface. With the presence of a high DC voltage and solvent vaporcondensing on the tip of the DSP, analyte can be dissolved and electrosprayed.The simplicity in design, versatility in application aspects, and other advantagessuch as low cost and disposability make this new method a competitive tool fordirect analysis of real-world samples.

Key words: Electrospray ionization, Direct sampling probe, Direct analysis

Received: 14 March 2013/Revised: 30 May 2013/Accepted: 20 June 2013

Introduction

Normally, conventional electrospray ionization massspectrometry (ESI-MS) only deals with solutions

delivered by capillaries, which restricts its application indirect analysis of real-world samples. In recent years, it hasbeen recognized that charged droplets from the electrosprayplume can interact with and transfer their charges to neutralmolecules. This realization promotes the appearance of somecontemporary ESI-based ionization methods, which can beused for direct analysis of real-world samples, includingdesorption electrospray ionization (DESI) [1], extractiveelectrospray ionization (EESI) [2], and other two-steptechniques of coupling laser desorption and ESI, such aselectrospray-assisted laser desorption/ionization (ELDI) [3],

laser-induced acoustic desorption/electrospray ionization(LIAD/ESI) [4], matrix-assisted laser desorption electrosprayionization (MALDESI) [5], laser ablation electrosprayionization (LAESI) [6], and laser electrospray mass spec-trometry (LEMS) [7].

Except the methods listed above, there exist otheralternative ionization methods for direct analysis by usingsolid ESI emitters. In 2007, Hiraoka et al. developed amodified version of ESI, probe electrospray ionization(PESI) [8], where a solid needle with a sharp tip wasutilized for both sampling and electrospraying. The intro-duction of a sharp solid probe worked as an ESI emitter canconvey some unique features, such as no sample preparationrequirements, low sample consumption, and high toleranceto salts. Other approaches of using solid ESI emitters likepaper [9], wooden tip [10], and biological tissues [11] havebeen reported recently as well.

Herein, we report a new method for direct ESI massspectrometric profiling of real-world samples by employinga disposable direct sampling probe (DSP). Both samplingand electrospraying can be done by using the same DSP.Biofluid can be transferred to the hydrophilic surface of the

Electronic supplementary material The online version of this article(doi:10.1007/s13361-013-0697-7) contains supplementary material, whichis available to authorized users.

Correspondence to: Zhan Yu; e-mail: yuzhan@synu.edu.cn; KenzoHiraoka; e-mail: hiraoka@yamanashi.ac.jp

DSP by direct insertion the DSP tip into real-world samples.With the presence of solvent vapor condensing on the DSPtip, analytes can be dissolved and then electrosprayed. Thislow cost and disposable DSP is designed to simplify thetedious repetitive pre-/post-measurement work of conven-tional chemical characterization of large-scale samples.

ExperimentalChemicals, Reagents, and Materials

Stainless steel (SUS304) disposable acupuncture needles(i.d. 0.3 mm) were purchased from Seirin (Shizuoka, Japan).Stainless steel (SUS304) sheet was obtained from Nilaco(Tokyo, Japan). All chemicals, regents, and solvents used inthis study were of analytical grade or higher.

Manufacture of the DSP

Owing to the considerations of safety, surface modification ofthe acupuncture needles was performed in a well-ventilatedfume hood. First, disposable acupuncture needles were sonicat-ed in a 30%HNO3 solution for 20 min to remove the inert layerof stainless steel. Second, these needles were put into a 30 %(wt/wt) H2SO4 solution containing KMnO4 (30 mg/mL) andK2Cr2O7 (15 mg/mL) and were heated at 80–100 °C for about1 h. Third, needles were sonicated sequentially by a 20% oxalicacid solution and deionized water to remove the attachedreduction products of the oxidants. Finally, needles were heatedat 120 °C for 1 h for activation.

Scanning electron microscopic (JSM-6500 F; JOEL, Tokyo,Japan) images of the original and surface modified acupunctureneedles are shown in Figure 1a and b, respectively.

Contact Angle Measurements

A SUS304 stainless steel (the same material as the acupunctureneedles) sheet was first cut into 1×1 cm pieces. After carefulcleaning with ethanol, pieces were treated by following theabove surface modification procedures. To demonstrate howthe modification procedures change the surface of SUS304,comparative contact angle measurements were done byphotographing 1.0 μL deionized water on the surfaces ofunmodified and modified SUS304 pieces with a digital long-focus microscope (VH-5500; Keyence CO. Ltd., Osaka,Japan), respectively. All measurements were taken at a fixedoptical magnification. Contact angle data were directlymeasured from the photographs by using the Drop-ShapeAnalysis plug-in for ImageJ [12].

Setup of the Ion Source

In this study, for a typical sampling, a DSP will penetrate intothe sample surface for 2 mm and last for 10 s. After sampling,this probe was fixed coaxially in the center of a plastic rod,which was directly connected to a DCmotor. When an input of

DC 1.5 V was applied to this motor, the probe would rotate at aspeed of about 300 rpm measured by a digital tachometer (HT-5500; Ono Sokki Co. Ltd., Yokohama, Japan). This probe wasaligned horizontally towards the ion sampling orifice of a massspectrometer. The distance between the probe tip and the apexof the ion sampling orifice was 1.5 mm.

A home-made auxiliary vapor generator was adopted in thiswork. A syringe pump (Pump 11; Harvard Apparatus,Holliston, MA, USA), an automatic temperature controlledheater, a piece of fused-silica capillary (i.d. 75 μm), and someconnectors formed the main body of the vapor generator.Typically, the mixture of acetonitrile and pure water (50:50,vol/vol) was pumped at a flow rate of 7 μL/min. When theheater was kept at 150 °C, stable and gentle solvent vaporwould be generated. The heater was placed perpendicularly tothe DSP. The distance from the DSP tip to the vapor generatortip is critical to the performance of DSP-MS. In this study, itwas optimized as 2.5 mm. When the solvent vapor condenseson the DSP tip, the analyte on the DSP surface will be dissolvedand electrosprayed during the rotation process after applying atypical high voltage of 1.7 kV.

Results and DiscussionsNormally, stainless steel is covered by a thin chromium oxidelayer, which protects the inner part from further oxidation.Owing to its compact structure and chemical composition, thispassive layer can also enhance the hydrophobicity of thestainless steel surface. In this work, heavy oxidation is adoptedto remove the passive layer of the DSP and increase its surfacehydrophilicity.

Scanning electron microscopic images of an unmodifiedacupuncture needle and a surface modified one are shown inFigure 1a and b, respectively. It can be clearly seen that due tothe oxidation treatment, the surface roughness is enhanced byforming somemicro holes andwells. This microporous structureincreases the surface area [13] and surface hydrophilicity of theDSP. Comparative side-view images of 1.0 μL water dropletsimpacted on the unmodified and modified SUS304 plates areshown in Figure 1c and d, respectively. After modification, thecontact angle increases from 19.734° to 56.287°.

Some standard peptide/protein samples were chosen todemonstrate the necessity of the surface modification of theDSP. Comparative mass spectrometric results obtained underthe same condition of two standard solutions, angiotensin II(10–5 M) and cytochrome c (10–4 M) by using surfaceunmodified and modified sampling probes are shown in FigureS1 of the Supplementary Materials. With the hydrophilic layer,chemically modified DSPs can convey stronger MS signalsthan the unmodified ones. The higher performance of thesurface modified DSPs can be hypothetically ascribed to itsmicroporous surface. Based on a previous work [14], a surface-modified DSP is assumed to load several tens of pL of bio-fluidduring a direct sampling process. One important factor to theperformance of DSP-MS is the quantity of solvent vaporcondensing on a DSP tip. At the present stage, frankly

Z. Yu et al.: DSP-MS Profiling of Real-World Samples

speaking, it is difficult for us to measure how much vapor willbe condensed during a DSP-MS measurement. Too muchvapor condensing on the DSP tip will decrease the concentra-tion of the formed sample solution and lead to worse s/n ration.

We are still searching for new techniques for a better controllingof the vapor condensation.

Another factor affecting the performance of this ion sourceis the rotation of the DSP. According to our observation, arotating probe will need less solvent for dissolving the sameamount of analytes than a stationary probe and will lead tostronger signal (shown in Supplementary Materials Figure S2).The time of detection can be shortened also, which is importantfor repetitive work of a large number of samples. Additionally,the rotation could accelerate the process of DSP tip wetness anddecrease the irregularities of the DSP tip, which is alwaysconsidered to be a major cause of unwanted corona dischargeduring the electrospray process.

Considering the most impressive characteristic of thismethod is direct analysis without sample preparation, it issuitable for rapid mass spectrometric profiling of real-worldsamples. Various samples ranging from biological tissues tomedicine tablets were chosen to demonstrate the applicabilityof DSP-MS. All mass spectra show similar patterns to thoseobtained by ESI-MS. Owing to the electrospray nature, onlynonvolatile compounds could be detected by this method. Evenafter 1 h of continuous heating, analyte attached on the DSPsurface such as saccharides and amino acids could still bedetected. Mass spectrometric results of various real-worldsamples could be found in the online supplementary materials.

Representative spectra of direct analysis of a normalkidney section and a kidney tumor section from the samepatient are shown in the Figure 2a and b, respectively. In thenormal kidney tissue, ions corresponding to the family ofphosphocholines (PCs) are detected predominantly. In thetumor tissue, although ions of PCs can also be observed; ionsof triacylglycerides (TAGs) appear with higher intensities. Itis well known that due to the decrease of the activity oflipoprotein lipase (LPL) [15], extremely high level of adiposedetected in tumor tissues could be used for identification. Themost interesting is the detection of ions at m/z 673.6 and

Figure 1. (a) Scanning electron microscopic (SEM) image of the tip of an original acupuncture needle; (b) SEM image of the tipof a DSP whose surface is chemically modified; (c) side view image of 1.0 μL water droplet impacted on the SUS304 plate; (d)side view image of 1.0 μL water droplet impacted on the surface modified SUS304 plate; (e) schematic drawing of the ionsource used in this work. The purple color around the sapling probe tip indicates dried analyte

Figure 2. Direct MS profiling of a normal kidney section (a)and a kidney cancer section (b) from the same patient. PC:phosphocholine; CE: cholesteryl ester; TAG: triacylglyceride

Z. Yu et al.: DSP-MS Profiling of Real-World Samples

689.6, corresponding to sodiated and potassiated cholesteryloleate. In a previous research [16], cholesteryl esters (CEs)content was found to be dozens of times higher in kidneycancer tissue than that in normal kidney tissue, which is seenas one biomarker for determination of renal cell carcinoma,the most common type of kidney cancer.

ConclusionsIn summary, we propose that a DSP can be readily used as a lowconsumption and direct analysis tool for rapid MS profiling ofreal-world samples. With the assistance of an external vaporgenerator, analytes can be dissolved and electrosprayed fromthe tip of the DSP. Considering the advantages of the currentmethod, such as low cost, disposability, and simplicity inconstruction and operation, DSP MS can be viewed as acompetitive candidate technique to be automated for the routineanalysis of large-scale samples.

AcknowledgmentThis work is financially supported by the Grants-in-Aid forScientific Research (S) and Development of System andTechnology for Advanced Measurement and Analysis Pro-gram (SENTAN) from Japan Science and Technology Agency(JST). Z.Y. thanks the financial support from the NationalNatural Science Foundation of China (21205080) and theScientific Research Fund of Liaoning Provincial EducationDepartment (L2012393).

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