Generating Electrospray Ionization on Ballpoint Tips - ACS Publications

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Generating Electrospray Ionization on Ballpoint Tips Baocheng Ji, Bing Xia, Yuanji Gao, Fengwei Ma, Li-Sheng Ding, and Yan Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03990 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 26, 2016

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Analytical Chemistry

Generating Electrospray Ionization on Ballpoint Tips ‡

‡

Baocheng Ji , Bing Xia , Yuanji Gao, Fengwei Ma, Lisheng Ding, and Yan Zhou* Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, P.R. China.

ACS Paragon Plus Environment


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ABSTRACT In this study, we report a simple and economical ballpoint electrospray ionization mass spectrometry (BP-ESI-MS) technique. This combines a small ballpoint tip with a syringe pump for the direct loading and ionization of various samples in different phases (including solution, semi-solid and solid) and allows for additional applications in surface analysis. The tiny metal ball on the ballpoint tip exhibits a larger surface for ionization than that of a conventional sharp tip end, resulting in higher ionization efficiency and less sample consumption. The adamant properties of the ballpoint tip allow sampling by simply penetrating or scraping various surfaces, such as a fruit peel, paper or fabric. Complex samples, such as fine herbal powders and small solid samples could be stored in the hollow space in the ballpoint socket and subsequently extracted online, which greatly facilitated MS analysis with little to no sample preparation. Positive ion mode was attempted, and various compounds, including amino acids, carbohydrates, flavonoids and alkaloids, were detected from different types of samples. The results demonstrated that the special and excellent physical characteristics of ballpoint tips allowed for fast and convenient sampling and ionization for mass spectrometry analysis by the BP-ESI-MS method.


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INTRODUCTION Electrospray ionization (ESI) is a powerful mass spectrometric technique for the analysis of various compounds. In conventional ESI, intact ions are generated from large and complex species in solution upon application of a high voltage to the capillary. The development of nano-electrospray ionization (nano-ESI),1,2 which utilizes capillaries of ~5 µm inner diameter, allows for less consumption of samples. However, capillary-based ESI techniques are prone to clogging and thus usually require time-consuming sample preparation prior to ESI-MS analysis. Along with the emergence of ambient ionization techniques, e.g., desorption electrospray ionization (DESI),3 extractive electrospray ionization (EESI)4 and other successively invented techniques,5-7 the development of ESI on solid substrates (solid-substrate ESI) has significantly facilitated the direct analysis of complex samples with little to no sample preparation, extending the applications of ESI-MS. In the past two decades, various materials such as wick element,8 copper wire,9 metal needle,10-12 optical fiber wired with a metal coil,13,14 surface-modified glass rod15 and nanostructured tungsten oxide16 have been successfully developed as emitters for ESI, which have improved the sampling and ionization processes and facilitated the analysis of various types of samples. Recently, the analysis of samples on surfaces or in difficult-to-access corners was accomplished by paper spray17,18 or wooden tip19 ESI-MS. Currently, solid-substrate ESI techniques have been further extended to direct mass spectrometric analysis of biological tissue.20-23 Because the ionization conditions of noncapillary ESI, such as loading of sample



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solutions on the surface and the absence of desolvation gas, are different from those of capillary-based ESI, different features have been observed with noncapillary ESI. The sequential and exhaustive ionization of analytes was observed with a solid metal probe by Hiraoka.24,25 Hu and co-workers reported the separate ionization of proteins and peptides in a mixture sample with hydrophilic wooden tips.19 The study on paper spray ionization of polar analytes using nonpolar solvents indicated that solvents could transport insoluble analytes to the paper tip and field desorption and surface tension of solvents might play important roles in the ionization of analytes.26 Further investigation on the characteristics and emitters of noncapillary ESI is necessary for the development and applications of these types of techniques. In this study, we demonstrated a quick, easy, cheap, efficient and rugged ESI method that used a ballpoint tip for the loading and ionization of samples with little to no sample preparation. This new technique was developed for the analysis of various types of samples, including solution, semi-solid as well as solid, and allowed for new applications in surface analysis. A great variety of compounds, including amino acids, carbohydrates, flavonoids and alkaloids were detected in different samples using this method.

EXPERIMENTAL SECTION Materials. Ballpoint pens (0.5 mm) from True Color brand were purchased from a local supermarket. Reserpine and tangeretin (5, 6, 7, 8, 4′-pentamethoxyflavone) were purchased from Sigma. Amoxicillin capsules, norfloxacin capsules, ketoconazole cream, erythromycin ointment, compound scopolamine hydrobromide adhesive paste


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and Fritillaria cirrhosa Don. were purchased from licensed pharmacy stories in Chengdu. Sweet oranges and blue gel ink pens were purchased from a local supermarket. Water was distilled water prepared using a Milli-Q system (Millipore Laboratory, USA). Methanol was of HPLC grade and purchased from Tedia (Fairfeild, OH, USA). Nanospray emitters (360 µm o.d., 50 µm i.d.) were purchased from New Objective (Woburn, MA). A stereomicroscope (Zeiss Stemi 2000-C, Carl Zeiss Jena, Germany) was used to observed the ballpoint-tip electrospray which was illuminated by a red laser pointer. BP-ESI Setup. The developed BP-ESI technique combines a ballpoint tip with a syringe pump (Figure 1). The small o.d. of the BP tip and good electrical conductivity of metal enable the tip to be conveniently fixed to inlets of different types of mass spectrometers with only minor hardware modification. Commercial ballpoint pens typically consist of a metal socket embedded with a metal ball (0.5 mm o.d.) and a plastic pipe. The BP tips could be readily dismantled and first washed with tap water to eliminate most of the ink inside, followed by washing with MeOH/H2O 1/1 in an ultrasonic washer for 10 min (repeated for three times) prior to use. An HPLC peek pipe (0.3 mm i.d., 1.3 mm o.d., ~500 mm long) was inserted in the metal socket for solution delivery. The fluid could be delivered through the small gap between the metal ball and the socket, which made BP-ESI different from other ESI techniques on feeding solvent. Sampling with BP Tips. Sample solution could be easily applied to the BP tips simply by pumping through the HPLC peek pipe. Just a gentle touching on semi-solid



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samples would make the BP tip coated with a thin film of samples. For analysis of solid samples (eg. powder samples), the tiny metal ball in socket was first prewetted with MeOH/H2O/FA 50/50/0.5 directed by syringe pump, and then touched on the sample surface with the tip-end for several seconds until the constituents of the solid sample were extracted. Moreover, fine ground powder (such as herbal medicines) could be added into the BP socket, followed by a subsequent auxiliary solvent (MeOH/H2O/FA 50/50/0.5) to actualize online extraction and electrospray ionization. For surface analysis, analytes could be easily picked up by simply penetrating in a depth of no more than half millimeter into soft surfaces or scraping on solid surfaces. The sturdy and durable BP tips support multiple experiments. Purge the pipe and ballpoint tip with 500-µL wash solvent (MeOH/H2O 1:1) or a five-minute ultrasonic cleaning step was enough for clean-up, and no residual signals were observed for normal tests. Mass Spectrometry. Mass spectra were acquired on a triple-quadrupole mass spectrometer (Waters, Manchester, UK). The BP tip was held and positioned parallel to the MS inlet (extended with a self-made 90-degree bend stainless steel pipe (1 mm i.d, 2 mm o.d., 50 mm long)) via a copper clip, which was connected to an external high voltage supply (0-4 kV). BP-ESI method generated ions without gas assistance and thus a close interface distance (0.5-3 mm) between the tip end and the MS inlet was chosen to optimize signal intensity and lower its fluctuations. The accumulation time for the ion signal to acquire a single mass spectrum record was 0.2 s. Desolvation and nebulization gas was turned off, and the ion source temperature was


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set at 150 °C. For data acquisition, the ionization voltage was adjusted until optimal ion signals were obtained. The cone voltage of mass spectrometer was set at 30 V. Tandem mass spectra were obtained by collision-induced dissociation (CID) of selected precursor ion with argon as collision gas. Data acquisition and processing were performed using the Masslynx 4.1 software.

RESULTS AND DISCUSSION Analysis of Solution Samples. Upon application of 1 µM reserpine in MeOH/H2O/FA (50/50/0.1) on the BP tip at a flow rate of 200 nL/min and a high voltage of 2.5 kV, a desirable total ion chromatogram (TIC) and quality spectrum were obtained (data shown in Figure 2). The protonated molecules ([Reserpine + H]+, m/z 609) are clearly observed with no interfering background (background signals shown in Figure S1). The stable TIC indicates that there might be a linear relationship between the total ion signals and the consumption volume of the sample solution applied. The total ion signal was estimated by the integration of the ion intensity and corresponding duration time, and the consumption volume of the sample solution was acquired by the product of the flow rate and corresponding duration time. As expected, a strong linear relationship (R2= 0.9976) between the total ion signal and consumption volume of the sample solution is observed (see Figure 2c), which indicates a quite steady ionization efficiency at a rather low flow rate (200 nL/min). At a flow rate of 200 nL/min, useful ion signals with duration of ~5 s could be obtained by pumping as little as 17 nL of reserpine solution on the tip, indicating the high ionization efficiency of this BP-ESI method. On application of 0.1 µM reserpine in MeOH/H2O/FA



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(50/50/0.1) pumped at a flow rate of 200 nL/min, comparative experiments between nanospray emitters and BP tips were conducted. The nanospray emitter and the BP tip were both held and positioned parallel to the MS inlet. Both setups attained their highest operating efficiency under the optimum ionization voltages of 3 kV for nano-ESI and 2.5 kV for BP-ESI. The optimum interface distance between the emitter end and the MS inlet were ~0.5 mm for nano-ESI and ~2 mm for BP-ESI, respectively. The blunt BP tips have larger spray surface than sharp nanospray emitters, and typically this would put adverse effect on forming the Taylor cone and spray, resulting in lower sensitivity. However, BP-ESI presents higher response than that of nano-ESI under their optimum analytical conditions (data shown in Figure S2). Afterwards, the relationship between flow rates and ion signal intensities, corresponding to ionization efficiency, was investigated (data shown in Figure S3). Upon application of a high voltage of 2.5 kV, flow rates ranging from 50 to 400 nL/min were investigated. As the flow rate decreases, the ion signal intensity first climbs and then decreases. Upon reducing the flow rate to 50 nL/min, the TIC signal becomes unstable, and the ionization process is hindered by rapid evaporation of the sample solution applied on the metal ball. Accordingly upon application of a fixed voltage of 2.5 kV, the photos of BP electrospray at different flow rates were taken (see Figure 3). Apparently when the flow rate was set at 200 nL/min, a thin and stable liquid membrane formed on the metal ball surface and sustained to support the fine Taylor cone as well as the good ionization spray (see Figure 3c). The schematic diagrams of the Taylor cone formed on the BP tip and the nanospray emitter were


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shown in Figure 3(e-h). It is known that when the capillary radius increases, electric field (Ec) on the Taylor cone would decrease, thus making the formation of Taylor cone difficult (according to Equation 1). However, Taylor cone was clearly observed on the metal ball surface according to the photos taken. This may owe to the fact that a fairly thin liquid film formed on the metal ball surface, which possesses larger radii of curvature than that of the liquid drop on the tip of smaller pipe (such as capillaries and nano-ESI emitters). This means that the surface tension of the liquid on the ball surface is much weaker and thus could facilitate the formation of Taylor cone and the breakaway of small charged droplets. Furthermore, the electrical conductivity difference between the two emitters used (metal-made BP tips with good electrical conductivity

and

fused-silica

emitters

covered

with

a

thin

metal

layer of several micrometers in thickness) may as well affects the formation of Taylor cone and the efficiency of ionization. In the BP-ESI case, sample solution is closely contacted with the metal ball, making the conduction of charge easy. However, in the case of capillaries or nanospray emitters, the metal (as charge conductor) was only covered on the outer surface of the sharp tip. This means the charge conduction only occurred via the minimum contact between the solution and the metal layer, which would limit the ionization efficiency.

(1) where Ec represents the electric field, Vc is the applied potential, rc the capillary outer radius, and d the distance from capillary tip to the counter-electrode.27 Additionally, the influence of ionization voltage on ionization efficiency was



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also investigated. At a constant flow rate of 200 nL/min, ionization voltage was adjusted in the range of 1.5 to 3.5 kV with a step size of 0.5 kV and different ion intensities were recorded, as shown in Figure S4. As the voltage rises from 1.5 to 2.5 kV, the ion intensity is an increasing function of ionization voltage. A higher ionization voltage might lead to a more plenitudinous and sustainable charge supply, forming more charged microdroplets during off-spring droplet fission and producing more analyte ions. When ionization voltage was set to 3 kV and above, discharge phenomenon was observed and ion intensity reduced sharply. As a result, 2.5 kV was selected as the optimal ionization voltage. All experiments were carried out at least three times in an air conditioned room with constant temperature and humidity. Mass spectra were obtained by averaging the data acquired in a time window of 2 minutes.

Analysis of Semi-Solid Samples. Topical medications are applied to body surfaces to treat ailments along with minimal systemic absorption and acceptable safety via a large range of classes such as creams and ointments, which are less accessible to conventional capillary-based ESI. Analysis of these samples generally involves various time-consuming and labor-intensive experimental steps including extraction, separation and characterization. To investigate this application of BP-ESI, erythromycin ointment and ketoconazole cream were analyzed, and data acquired were shown in Figure 4. Due to the different bases used as the drug vehicle and unique preparation processes, the two above mentioned forms of semi-solid samples show definite differences in TIC, corresponding to different ionization stages. For the application of erythromycin ointment, protonated molecules ([Erythromycin + H]+)


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(m/z 734) are directly observed at the initial stage of ionization process. This phenomenon could be explained by its high oil content base in erythromycin ointment, which was not well ionized. Although effective ionization period for erythromycin lasts only for several seconds, high quality spectrum of predominant [M+ H]+ of erythromycin could be acquired, as shown in Figure 4b. As an emulsion of oil and water in approximately equal proportions, ketoconazole cream showed different ionization stages. Figure 4 shows its TIC (4c) and mass spectrum (4d) obtained for the period of T2 in part 4c, respectively. The [M + H]+ of ketoconazole (m/z 531) was only observed at T2. Mass spectra obtained for the period of T1 and T3 in part 4c were shown in Figure S5. The ketoconazole molecule remained in the liquid membrane of the sample attached to the BP tip at T1, and excess charges were continuously supplied to the liquid membrane until the total depletion of the sample. This observation indicated a sequential and exhaustive ionization process in BP-ESI, similar to that of the probe electrospray ionization method. 24 Both above mentioned experiments were completed within two minutes, indicating the strong applicability of the fast and convenient technique for analysis of semi-solid samples.

Analysis of Powder Samples. The slim and hard properties of BP tips also allow for the direct analysis of powder samples in an open environment. The pre-wetted ballpoint tip was first touched with the powder of amoxicillin, a commonly used β-lactam antibiotic, and then mounted in position. Subsequently, solvent ((MeOH/H2O/FA 50/50/0.1) was delivered to the metal ball deposited with sample powder for further dissolution and ionization. The high voltage of 2.5 kV was applied



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to the BP tip. As shown in Figure 5a, predominant [M + H]+ of amoxicillin (m/z 366) was observed. Similar results were acquired when applying BP-ESI method for the detection of norfloxacin in capsules (see Figure 5b). Figure S6 presents a plot of [Norfloxacin + H]+ intensity versus analysis time. It is evident that electrospray conditions are changing during the ionization period. At the initial ionization stage, massive protonated norfloxacin ions generated due to excessive norfloxacin molecules adhered to the metal ball surface. Following the exquisite consumption of norfloxacin in a short and unsteady state, a steady ionization state reached. Subsequently, a gentle decrease and total depletion of norfloxacin were acquired. This revealed the exhaustive ionization of BP-ESI once again. These results demonstrated the great advantages of BP-ESI in routine analysis of drug powders.

Plants are important natural resources for foods, supplementary products and herbal medicines, many of which are prepared in powder forms for convenient usage. The development of simple, rapid and efficient methods to analyze the metabolites and bioactive components in herbal powders is highly desirable for quality control and food safety. With BP-ESI method, only several milligrams of powder sample (or less in some cases) were needed to generate stable and durable MS signals, and representative metabolites could be quickly identified by MS and MS/MS experiments. Here, different driving solvents were used to obtain high quality metabolomics data of Fritillaria Cirrhosae Bulbus. Eventually, MeOH/H2O 1/1 containing 0.1% formic acid was selected as the online extraction solvent. As shown in Figure 6a, over ten constituents, including primary and secondary metabolites, are


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detected and further tentatively identified. The ion peak at m/z 175 was identified as the protonated arginine by MS/MS experiment (Figure S7a). The product ion spectrum of the selected ion at m/z 175 generates characteristic fragments of m/z 157, 158, 130 and 116 by the losses of H2O, NH3, [NH3+CO]28 and HN=C(NH2)2,29 respectively. As shown in Figure S7b, the ion peak at m/z 381 corresponded to the [Sucrose + K]+ ion, which produces ionic fragments of m/z 219 and 20130. The product ions of the precursor ion at m/z 219 are produced by the loss of glucoside or fructoside (C6H10O5, 162 Da), generating the characteristic product ion at m/z 201 by loss of H2O. The minor [M+ Na]+ of sucrose was also observed. Meanwhile, the potassium adduct dimer, trimer, tetramer and pentamer of sucrose were also detected at m/z 723 ([2Sucrose + K]+), m/z 1065 ([3Sucrose + K]+), m/z 1407 ([4Sucrose + K]+) and m/z 1749 ([5Sucrose + K]+), respectively. When subject to CID analysis, these ions presente the dominant product ion at m/z 381 under collision energy of 2 eV, as shown in Figure S8 (a-d). When the collision energy rises to 15 eV, the precursor ions at m/z 723 also generates characteristic fragments at m/z 219 and 201 (see Figure S8e), which further indicates that the above mentioned ions (m/z 723, 1065, 1407 and 1749) are potassium adduct ions of sucrose.

In addition to the typically primary metabolites (amino acids and saccharides), several typical steroid alkaloids are also profiled in the F. Cirrhosae sample by BP-ESI-MS. For example, the peaks at m/z 428, 430 and 432 are tentatively identified to be protonated peimisine, verticinone and peimine by comparing their MS/MS data (see Figure S9a-c) with published data.31 For peimisine, the fragment ion at m/z 410



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(C27H40NO2) is produced by a neutral loss of H2O, corresponding to a hydroxyl group at the C-3 position. The characteristic ion at m/z 114 (C6H12NO) is formed by the cleavage of the E-ring with the loss of C21H30O2. As shown in Figure S9d-j, other peaks are tentatively identified as protonated solanidine (m/z 398), ebeiedinone (m/z 414), ebeiedine (m/z 416), puqienine B (m/z 444), verticinone N-oxide (m/z 446), verticine N-oxide (m/z 448) and imperialine-3-β-D-glucoside (m/z 592) by comparing the MS/MS data with literatures.31,32 It cost less than ten minutes for simultaneous detection of over ten primary and secondary metabolites in F. Cirrhosae powder without any preparations, which demonstrated the efficiency and good application prospects of BP-ESI in direct analysis of powder samples.

Surface Analysis. Polymethoxyflavones (PMFs) exist exclusively from the citrus genus, particularly in the peel of sweet oranges. BP-ESI proposes a capability to directly analyze the constituents in orange matrices. The sharp BP tip was penetrated less than half millimeter into the orange peel, and the analytes were picked and adhered to the metal ball surface. Subsequently, with an auxiliary solvent (MeOH/H2O/FA 50/50/0.1), high quality mass spectrum was acquired (see Figure 6b). PMFs have regularity in elemental composition (chemical formula) and a basic aglycone structure with a maximum of seven substituents such as a methoxyl group (-OCH3) and/or hydroxyl group (-OH). The protonated molecules ([PMF + H]+) and potassium adduct ion ([PMF + K]+) are clearly observed in the mass range of m/z 250–500. PMFs have characteristic dissociation patterns, losing one or two methyl radicals (CH3·) to produce radicals [PMF+H−15]+ or [PMF+H−2 × 15]+ as the


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predominant fragment ions.33 The ions ([M + H]+) at m/z 343, 373 and 403 are tentatively identified as protonated tetramethoxyflavone, pentamethoxyflavone and hexamethoxyflavone, respectively, according to their MS/MS data (shown in Figure S10). The [PMF + H]+ and [PMF + K]+ of heptamethoxyflavone (m/z 433, 471) are also observed. Interestingly, in the high mass range, potassium adduct dimer and trimer ions ([PMF1 + PMF2 + K]+ at m/z 723, 753, 783, 813, 843, 873, 903 for potassium adduct dimers and [PMF1 + PMF2 + PMF3 + K]+ at m/z 1065, 1095, 1125, 1155, 1185, 1215, 1245, 1275, 1305, 1335 for potassium adduct trimers, where PMF1, PMF2 and PMF3 represent individual PMFs) are also observed. Among the potassium adduct dimer ions ([PMF1 + PMF2 + K]+at m/z 723, 753, 783, 813, 843, 873, 903), complex ion at m/z 813 reaches the maximum value of peak intensity and other ion peak intensities decrease gradually with the increase of m/z ratio difference (∆m/z). Apparently, peak intensity of adduct ions with more combination patterns tend to result in higher abundance. For example, the predominant ion at m/z 813 generates its fragments at m/z 471, 441, 411 and 381 under MS/MS experiment by the losses of tetramethoxyflavone (C19H18O6, 342 Da), pentamethoxyflavone (C20H20O7, 372 Da), hexamethoxyflavone (C21H22O8, 402 Da) and heptamethoxyflavone (C22H24O9, 432 Da) (MS/MS data shown in Figure S11c), respectively. This suggests that the predominant

ion

at

m/z

813

might

have

two

combination

patterns:

[Tetramethoxyflavone + Heptamethoxyflavone + K]+ and [Pentamethoxyflavone + Hexamethoxyflavone + K]+. Comparatively, the lower abundance peak at m/z 753 exhibits only one combination pattern, [Tetramethoxyflavone + Pentamethoxyflavone



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+ K]+, according to its MS/MS experiment (Figure S11a). Although the ion peak [PMF1

+

PMF2

+

K]+

(m/z

783)

exhibits

two

combination

patterns

[Tetramethoxyflavone + Hexamethoxyflavone + K] + and [2Pentamethoxyflavone + K]+ (MS/MS data shown in Figure S11b), its relative peak intensity was still lower than that of the ion peak [PMF1 + PMF2 + K]+ (m/z 813). This phenomenon might be related to the concentrations of individual PMFs in orange peel. This offers an attractive development direction for fast and trace in situ chemical analysis such as point-of-care diagnosis. Modern demands for specialized writing and printing instruments have resulted in an explosion of ink formulations, each of which may contain dozens of chemical components, such as coloring material, vehicle and other additives. The tiny and adamant properties of BP tips also allowed for convenient analysis of analytes on hard surfaces, such as ballpoint pen ink on paper and dried blood on ground, on which other solid-substrate ESI may fail to sample. Herein, an attempt was made to collect the analytes in ink mark for MS analysis. A pre-wetted BP tip was laid down onto the blue gel ink mark and carefully scraped in several circulars or liner motion for extraction and adhesion of the analytes on the metal ball surface. The tip was then mounted in position, and a driving solvent was delivered for ESI. As shown in Figure 7a, the detected ions with regular m/z could be classified into two classes: m/z 305, 349, 393, 437, 481, 525, 569, 613 for one class and m/z 409, 453, 497, 541, 585, 629 for another. In each class, the main peaks showed a fixed m/z difference (∆m/z 44), which indicated the presence of polymers. A direct ESI-MS analysis of the used ink


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was conducted to validate the detection of chemicals in ink by BP-ESI. The blue gel ink was spiked and dispersed in MeOH/H2O/FA (50/50/0.1) and filtered with 0.22 µm pore-size filter membrane before MS analysis. The direct ESI-MS (see Figure S12) agreed with that obtained by BP-ESI-MS, indicating good reliability of BP-ESI on detection of ink sample on paper. According to their fixed m/z ratio difference (∆m/z 44), the two classes of ions may generated from polyvinyl alcohol ([C2H4O]n).34-37 Scopolamine hydrobromide adhesive paste (used to treat motion sickness), a kind of external medicine usually combining three agents - oil, water, and powder, was also analyzed by BP-ESI. After a gentle rub on the drug layer followed by solvent-assisted BP-ESI, predominant [M + H]+ of scopolamine (m/z 304), the active ingredient in the paste, is clearly observed (see Figure 7b). These observations demonstrate the potential application of BP-ESI in surface analysis.

CONCLUSIONS BP-ESI has been demonstrated in this study to have multiple sampling capabilities for a wide variety sample phases in simple ways. The BP tips used are inexpensive and readily available, and they could be directly connected to ESI sources of various mass spectrometers. The tiny metal ball embedded in the BP tip supports a relatively large area of ionization surface, resulting in high ionization efficiency. An exhaustive ionization phenomenon was also observed in the experiments. BP-ESI method could be used for routine analysis of various samples in forms of solution, semi-solid and solid. The wear-resistant property of BP tips also permitted applications in surface analysis. The successful use of BP tips as sampling and ionization media simplifies



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ESI analysis to a large extent and provides a quick and convenient analysis method.


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AUTHOR INFORMATION Corresponding Author *Prof. Yan Zhou ([email protected]; Tel: 86-28-82890810, Fax: 86-28-82890825)

Author Contributions ‡

These authors contributed equally to this work.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (No. 21302180, 21572221), a scientific research equipment development project of CAS (No. YZ201204) granted to Dr. Y. Zhou.



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REFERENCE (1) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1-8. (2) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994,136, 167-180. (3) Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G. Science 2004, 306, 471-473. (4) Chen, H. W.; Venter, A.; Cooks, R. G. Chem. Commun. 2006, 2042-2044. (5) Venter, A.; Nefliu, M.; Cooks, R. G. TrAC, Trends Anal. Chem. 2008, 27, 284-290. (6) Chen, H. W.; Gamez, G.; Zenobi, R. J. Am. Soc. Mass Spectrom. 2009, 20, 1947-1963. (7) Huang, M. Z.; Yuan, C. H.; Cheng, S. C.; Cho, Y. T.; Shiea, J. Annu. Rev. Anal. Chem. 2010, 3, 43-65. (8) Fenn, J. B. US Patent 6297499 (1998). (9) Hong, C.M., Lee, C.T., Lee, Y.M., Kuo, C.P., Yuan, C.H., Shiea, J. Rapid Commun. Mass Spectrom. 1999, 13, 21-25. (10) Hiraoka, K.; Nishidate, K.; Mori, K.; Asakawa, D.; Suzuki, S. Rapid Commun. Mass Spectrom. 2007, 21, 3139-3144. (11) Mandal, M.K.; Chen, L.C.; Hashimoto, Y.; Yu, Z.; Hiraoka, K. Anal. Methods 2010, 2, 1905-1912. (12) Chen, L. C.; Nishidate, K.; Saito, Y.; Mori, K.; Asakawa, D.; Takeda, S.; Kubota, T.; Hori, H.; Hiraoka, K. J. Phys. Chem. B 2008, 112, 11164-11170. (13) Kuo, C.P.; Shiea, J. Anal. Chem. 1999, 71, 4413-4417. (14) Kuo, C.P.; Yuan, C.H.; Shiea, J. J. Am. Soc. Mass Spectrom. 2000, 11, 464-467. (15) Jeng, J.Y.; Shiea, J. Rapid Commun. Mass Spectrom. 2003, 17, 1709-1713. (16) Jeng, J.Y.; Lin, C.H.; Shiea, J. Anal. Chem. 2005, 77, 8170-8173.


Page 20 of 32

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(17) Liu, J.J.; Wang, H.; Manicke, N.E.; Lin, J.M.; Cooks, R.G.; Ouyang, Z. Anal. Chem. 2010, 82, 2463-2471. (18) Wang, H.; Liu, J.J.; Cooks, R.G.; Ouyang, Z. Angew. Chem., Int. Ed. 2010, 122, 889-892. (19) Hu, B.; So, P.K.; Chen, H.W.; Yao, Z.P. Anal. Chem. 2011, 83, 8201-8207. (20) Hu, B.; Lai, Y.H.; So, P.K.; Chen, H.W.; Yao, Z.P. Analyst 2012, 137, 3613-3619. (21) Liu, J.J., Cooks, R.G., Ouyang, Z. Anal. Chem. 2011, 83, 9221-9225. (22) Liu, J.J.; Wang, H.; Cooks, R.G.; Ouyang, Z. Anal. Chem. 2011, 83, 7608-7613. (23) Chan, S.L.F.; Wong, M.Y.M.; Tang, H.W.; Che, C.M.; Ng, K.M. Rapid Commun. Mass Spectrom. 2011, 25, 2837-2843. (24) Mandal, M. K.; Chen, L. C.; Hiraoka, K. J. Am. Soc. Mass Spectrom. 2011, 22, 1493-1500. (25) Yue Ren; He Wang; Jiangjiang Liu; Zhiping Zhang; Morgan N. McLuckey; Zheng Ouyang. Chromatographia. 2013, 76, 1339-1346. (26) Li, A.Y., Wang, H., Ouyang, Z., Cooks, R.G. Chem. Commun. 2011, 47, 2811–2813. (27) L. B. Loeb; A. F. Kip;G. G. Hudson; W. H. Bennett. Phys. Rev. 1941, 60, 714-722. (28) Shek, P.Y.I.; Zhao, J.; Ke, Y.; Siu, K.W.M.; Hopkinson, A.C. J. Phys. Chem. A 2006, 110, 8282-8296. (29) Li, M.; Hu, B.; Li, J.; Chen, R.; Zhang, X.; Chen, H. Anal. Chem. 2009, 81, 7724-7731. (30) Overy, D.P.; Enot, D.P.; Tailliart, K.; Jenkins, H.; Parker, D.; Beckmann, M. ; Draper, J. Nat. Protoc. 2008, 3, 471-485. (31) Zhou, J.L.; Xin, G.Z.; Shi, Z.Q. ;Ren, M.T.; Qi, L.W.; Li, H.J.; Li, P. J. Chromatogr., A 2010, 1217, 7109–7122. (32) Giuliana B.; Philippe S.K.; Giuseppe D. B.; Antonius K.; Tommaso, R. I. C. Electrophoresis



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2002, 23, 2904-2912. (33) Zhou, D.Y.; Chen, D.L.; Xu, Q.; Xue, X.Y.; Zhang, F.F.; Liang, X.M. J. Pharm. Biomed. Anal. 2007, 43, 1692-1699. (34) Kondo M, M. N. US Patent 5466283 (1995). (35) Koyama T. US Patent 5977211 (1999). (36) Ohnishi J’Oshima K;Kudo M. US Patent 5928989(1999). (37) Sawa T, Yamamoto Y. US Patent 6616741(2003).


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Figure Captions Figure 1. Schematic diagram of the experimental setup of BP-ESI. Figure 2. (a) Total ion chromatogram of 1 µM reserpine in MeOH/H2O/FA (50/50/0.1) obtained by pumping at a flow rate of 200 nL/min and setting the voltage at 2.5 kV, (b) positive ion mass spectrum of 1 µM reserpine in MeOH/H2O/FA (50/50/0.1) and (c) total ion signals of reserpine solution showed a linear relationship with the volume of solution applied on the tip. Each data point was the average obtained from three independent experiments. Figure 3. Photos of the BP-ESI taken by a stereomicroscope at different flow rates of (a) 400 nL/min, (b) 300 nL/min, (c) 200 nL/min and (d) 50 nL/min (Ionization voltage: 2.5 kV). Schematic diagrams of the Taylor cone formed on (e-g) ballpoint tip and (h) nanospray emitter. The liquid flow rates are high (e), medium (f) and low (g), respectively. Figure 4. (a) TIC of erythromycin ointment, (b) mass spectrum of erythromycin ointment, (c) TIC of ketoconazole cream and (d) mass spectrum of ketoconazole cream at the period of T2 (0.05 min < T1

Generating Electrospray Ionization on Ballpoint Tips - ACS Publications

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