Electrospray ionization (ESI) is a routinely used ionization method for mass spectrometry (MS), which is widely used in various disciplines, e.g., proteomics [1], metabolomics [2], and lipidomics [3], due to its sensitivity and simplicity. However, the disadvantages for conventional ESI are also obvious, for instance, analyte suppression, matrix effects, and salt contamination [4]. As an improved ESI technique, nanoelectrospray ionization (nESI) allows much higher ionization efficiency with minimized sample flow rates (< 100 nL/min) [5]. The architecture of nESI emitter plays a key role in ensuring a successful nanoelectrospray, which can greatly influence the reproducibility and stability of analyte signals [5]. Normally, pulled glass capillary was employed as nESI emitter. However, its vulnerability to sample clogging and high spray voltages, as well as the high cost per unit [6], hindered its practical application.

In the past two decades, many alternative non-capillary-based ESI emitters have been developed in order to alleviate these problems, including metal wire [7], metal needle [8], optical fiber wired with a metal coil [9], surface-modified glass rod [10], nanostructured tungsten oxide [11], paper [12], thin film polyimide tape [6], leaf [13, 14], wooden tooth pick [15, 16], plastic tip [17,18,19], polyimide microfluidic chip [20, 21], pointed carbon fiber [22, 23], aluminum foil [24], ball point [25], biochip [26], coated blade [27], cotton swabs [28], gel loading tip [29, 30], sponge [31], and glass slip [32]. However, each of the above method had its own analytical advantages and disadvantages. Until now, none of them could outdo capillary ESI/nESI in terms of net performance. One of the common and critical problems with non-capillary emitters was the occurrence of arcing and atmospheric corona discharge, particularly during the analysis of aqueous solutions, which greatly affected the stability and durability of MS signals. This problem could also occur in capillary-based ESI/nESI, though the small inner diameters and high electrical resistance of ESI solution in capillary ESI already minimized the phenomenon [33]. Various approaches were already investigated to quench the atmospheric discharge in ESI from aqueous solutions, including the use of high gas pressure in ionization area [34,35,36,37,38], trace amount of trifluoroethanol [39], pneumatic-assisted ESI spray [40], electrosonic spray [41], strongly dielectric nebulizing gases [42, 43], and high-ohmic resistor in series with high voltage power supply [33, 44]. However, the analysis of aqueous solutions with nESI-MS was still problematic. In addition, though some highly efficient ionization methods which can tolerate complex matrices and enable direct analysis of raw samples have been developed, e.g., extractive electrospray ionization (EESI) [45,46,47,48,49,50,51], novel low-cost methods are still needed to meet specific requirements, such as direct analysis of various samples (aqueous, viscous, or solid) with lowest sample consumption.

Herein, we report a simple yet reliable current-limited nESI method for analysis of aqueous solutions and other untreated samples (viscous, solid, and untreated bulk samples) by using a commercially available disposable gel-loading plastic tip in series with a high-ohmic resistor (10 GΩ) (tip-ESI). Basically, the analyte solution was loaded in a plastic tip, and the ionizing voltage for analytes was supplied through a platinum (Pt) wire, which was connected to the power supply in series with the resistor. Though plastic tip-based ESI has been previously demonstrated, the analysis of various aqueous solution was still a big challenge because the aqueous solutions were normally difficult to be sprayed out of the tip due to their high surface tension. Aksyonov and Williams used a pipette tip with larger diameter (i.d. ~ 0.3 mm) as emitter; however, the flow rate was also relatively high (~ 300 nL/min) [52]. Application of high voltage could facilitate the formation of electrospray of aqueous solution; however, corona discharge was often observed [29]. Therefore, special setups were needed, e.g., under super atmospheric pressure [29] or using high proportion of organic components as spray solvents (e.g., 30% of methanol) [30]. In this study, the use of a high-ohmic resistor, however, effectively avoided electrical discharge and allowed the formation of stable electrospray of ionization solvents with significantly low contents of organic additives (e.g., 1% methanol or 0.1% formic (acetic) acid) at ambient condition. Therefore, quantification of analytes (vitamin B) in water using tip-ESI was successfully conducted. In addition, the analytical performances, including flow rates, MS signal intensities, reproducibility, stability, LODs, and linear response range, were investigated.

Experimental Section

Instrumentation

LTQ-XL linear ion trap mass spectrometer (Finnigan, San Jose, CA) was used for all experiments. LTQ parameters were as follows: capillary temperature, 180 °C; capillary voltage, 5 V; cone voltage, 80 V; tube lens voltage, 5 V; multipole RF amplitude (Vp-p), 800 V. Mass range of detection was set from 50 to 2000 Th or from 50 to 4000 Th in both positive and negative ion modes. The maximum ion injection time was optimized and set to 0.7–1 ms. For collision-induced dissociation (CID) analysis, precursor ions were isolated within the window of 1.2 Da. The collision energy was 15–35%, and the collision time was 30 ms.

Tip-ESI Source

Figure 1 shows the schematic diagram of tip-ESI ion source. A home-made plastic union was used to fix a silver-coated Pt wire electrode (o.d. 50 μm; Ida Tianjin Co., Tianjin, China). The Pt wire was inserted into a commercially available gel-loading plastic tip (i.d. 120 μm or 410 μm; o.d. < 300 μm; L. 65 mm, epT.I.P.S, Eppendorf, Germany) which was originally designed for loading of samples onto polyacrylamide gels for DNA sequencing. The end of the tip was cut by ca. 10 mm in order to prevent tip vibration during analysis, which however, did not result in measurable change of the inner diameter of the tip. The distance between the end of the wire and the cut was typically 10 mm. The entire assembly was mounted on the x-y-z stage using a clamp. The pipette tip is 2 mm away from the inlet capillary of the mass spectrometer (Figure 1, S1). A 10-GΩ resistor was connected in series with power supply, and a voltage of 3.4–3.5 kV was applied. As for comparison, pulled glass capillary nESI analysis was also carried out. The capillary with the diameter (i.d.) of 1 μm was placed about 3 mm from the MS inlet. The applied capillary voltage was 2.1 kV.

Figure 1
figure 1

Schematic of tip-ESI with limited current by a high-ohmic resistor

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Chemicals

All analyte chemicals, as well as methanol, acetic acid, and formic acid, were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of horse heart myoglobin (purity, 90%), vitamin B (thiamine), and MRFA (Met-Arg-Phe-Ala) peptide (purity, 98%) were prepared in double deionized (DDI) water. Analyte solutions were prepared by appropriate dilution of stock solutions with water containing 0.1% acetic acid or 1.0% methanol (v/v). All biological samples including tomato, cherry tomato, potato, orange, zinger rhizome, small orange, chicken egg, yogurt, and soy milk were purchased from a local market of Nanchang, China, and analyzed without any sample pretreatment. Chinese herbal medicine samples of Coptis chinensis (dry root), Schisandra chinensis (dry fruit), and Schisandra sphenanthera (dry fruit) were purchased from a pharmacy shop in Nanchang, China. Catharanthus roseus (leaf) was collected locally in Nanchang, China.

Loading of Samples

One to 2 μL of aqueous sample was firstly loaded into the modified tip. Then, the Pt wire was immersed into the solution, and the tip was fixed on the x-y-z stage. Noteworthy, introduction of any air cushion should be avoided when loading aqueous sample into the tip with a pipette, which could greatly affect the accuracy of sample loading as well as the stability of electrospray. Therefore, it is necessary to disconnect the pipette when the tip is still immersed into the aqueous solution. To load juicy or viscous sample, the Pt wire was stabbed ~ 5 mm deep in the untreated sample and then inserted into a pipette tip pre-filled with 1 μL water/methanol mixture (v/v 99:1). Loading of solid samples (powder or bulk solid) could be conducted by pressing pre-wetted Pt wire on the surface of samples, or inserting a small portion of solid sample into the pipette tip directly. The pipette tip was pre-filled with 1–2 μL water/methanol mixture (v/v 99:1). Ionization of analytes was initiated by applying a high voltage to the Pt wire.

Results and Discussion

Effect of High-Ohmic Resistor

To demonstrate the importance of high-ohmic resistor for the performance of tip-ESI, we compared the MS spectra of myoglobin in 0.1% acetic acid aqueous solution with and without the use of a 10-GΩ resistor. The current-voltage characteristic showed that the use of resistor greatly reduced ESI current, which could be easily detected by MS instrument (Figure S2). For instance, the ESI currents at an applied voltage of 3.4 kV with and without resistor were 0.12 μA and 4.71 μA, respectively (Figure S2). On the other hand, though the ESI current was also extremely low with a low applied voltage (e.g., 0–2 kV), the desired analytes could not be ionized efficiently. It can be seen from Figure 2c, d that myoglobin exhibited apo-form signals (the heme group was lost), with increased degree of charging. The yielding of positively charged ions (+ 18 and + 19) with m/z values around 1000 (Figure 2c, d) indicated a same ionization process with (Figure 2c) and without resistor (Figure 2d). However, unstable myoglobin signal was observed without the use of resistor (Figure 2b), probably due to the emergence of corona discharge resulted from the high ESI current at the end of the tip. A corona discharge is a process where a current flows from an electrode with a high potential into a neutral fluid (usually air), resulting in a bluish glow in the air adjacent to the electrode tip [29].

Figure 2
figure 2

Comparison of tip-ESI-MS analysis of myoglobin (167 μg/mL) in 0.1% acetic acid aqueous solution with (a, c) and without (b, d) a 10-GΩ resistor in series with the high voltage power supply

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Effect of Tip Diameter

A series of pipette tips with varied tip diameters (100–560 μm) were tested for tip-ESI-MS analysis. The results suggested that the spray efficiency deteriorated with the increase of tip diameters. Figure S3 compared the analyses of vitamin B sample using two pipette tips with the inner diameter of 120 μm and 410 μm, respectively. Obviously, stronger and reproducible MS signals were obtained with the tip diameter of 120 μm (Figure S3a, b). Similar result was also found for Catharanthus roseus leaf in 1% methanolic aqueous solution (Figure S4). Detected compounds from Catharanthus roseus leaf were identified as protonated [M+H]+ and potassiated [M+K]+ which were already confirmed by high-pressure liquid chromatography (HPLC)[53,54,55]. In comparison, both the signal stability and intensity decreased significantly with the tip diameter of 410 μm (Figure S4c, d), probably due to the poor stability of ESI process from the emitter with larger inner diameter. Replicate experiments confirmed that the smaller the tip i.d., the better the performance of tip-ESI.

Comparison with Pulled Glass Capillary nESI

First of all, the flow rates of tip-ESI, calculated based on the volume of solution consumed per unit time, were compared with capillary nESI. For analysis of 167 ng/mL of myoglobin in 0.1% formic acid solution, with the tip diameter of 120 μm, the applied voltage of 3.4 kV, and the resistor of 10 GΩ, 2.5 μL aqueous solution was consumed within ca. 50 min (Figure S5a, b). Therefore, the estimated flow rate of tip-ESI was ca. 50 nL/min. In another measurement of 10 μg/mL MRFA in 1% methanolic solution under the same experimental conditions, 1 μL mixture was taken within ca. 12 min (Figure S5c,d), yielding a flow rate of ca. 66 nL/min. Our study also showed that the flow rate of tip-ESI increased gradually with decreasing the surface tension of sample solution indicated by the content of methanol (Figure S6). Though the flow rate could be influenced by various parameters, e.g., magnitude of high voltage, inner diameter of the emitter tip, surface tension of the spraying solution, analyte concentration, and the rate of environmental evaporation [44], the obtained flow rate for tip-ESI was comparable to that of conventional nESI (~ 20–30 nL/min) [56], which was surprisingly low given that the diameter of the tip (ca. 120 μm) was significantly larger than that of pulled glass capillaries (~ 1–5 μm). This could be ascribed to the stable voltage (3.4–3.5 kV) applied, which had a significant influence on the rate of electroosmotic flow of aqueous solutions during ESI [57]. Also, the high hydrophobicity of plastic tips may help stabilizing the high surface tension of sample solution and thereby minimized the flow rates. On the other hand, analysis of various analytes (vitamin B and myoglobin) with different aqueous media (water or acetic acid solution) using the two different approaches showed great similarity on performances with respect to signal intensity, charge distribution, and other MS characteristics (Figure 3). The comparison experiment was repeated using three different capillaries and pipette tips. As discussed above, the flow rate in tip-ESI was only slightly higher than in capillary nESI. However, the much larger diameter of tip-ESI avoided tip clogging over the entire analysis run until the sample was finished. In contrast, tip clogging was observed for capillary nESI during the analysis of myoglobin sample (167 μg/mL), indicated by the disappearance of signal after a few minutes or even shorter time. The results suggested that tip-ESI is a simple but effective ionization technique with enhanced signal stability and durability for MS analysis.

Figure 3
figure 3

Comparison between tip-ESI-MS (a, c, e) and conventional pulled glass capillary nESI-MS (b, d, f) under optimized experimental conditions for analysis of vitamin B and myoglobin. (a), (b) Vitamin B (1000 ng/mL) in water. (c), (d) Myoglobin (167 μg/mL) in water. (e), (f) Myoglobin (167 μg/mL) in 0.1% acetic acid aqueous solution

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Quantitative Analysis of Aqueous Solution by Tip-ESI-MS

Figure 4 shows the results of quantitative analysis for vitamin B in water by tip-ESI-MS. The vitamin B was prepared at a concentration of 1000 ng/mL, 500 ng/mL, 100 ng/mL, 50 ng/mL, 10 ng/mL, 1 ng/mL, 0.5 ng/mL, and 0.25 ng/mL, respectively. In order to avoid cross contamination of the ion transport tube, all measurements were run from low concentration to high concentration (0.25–1000 ng/mL). Stable and reproducible MS signals as well as good linearity (R ˃ 0.9983) were observed in the wide concentration range of 0.25–1000 ng/mL (Figure 4). The limit of detection for vitamin B, calculated based on the corresponding concentration of three times of the standard deviation for signal/noise (S/N), was 0.25 ng/mL. The inset in Figure 4 showed the MS spectrum of the standard of 0.25 ng/mL. Clearly, the characteristic protonated form of [M+H]+ with the m/z of 265 was detected. Noteworthy, the pipette tips were normally made of inert materials, and therefore, the same plastic tip might be washed and reused without leading to notable cross contamination.

Figure 4
figure 4

Quantitative analysis of vitamin B (thiamine) in aqueous solution containing 1% methanol. The analyte concentration ranged from 0.25 to1000 ng/mL. Each data point was the average of three independent experiments, and error bars represented the corresponding standard errors. The inset diagram showed the MS spectrum of a standard of 0.25 ng/mL

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Analysis of Untreated Biological Samples

Figure 5 shows the results of tip-ESI-MS analysis of onion, tomato, potato, orange, ginger rhizome, and cherry tomato. Most of the analytes such as flavonoids, glycoalkaloids, glucose, sucrose, choline, and gingerol were detected as protonated [M+H]+, sodiated [M+Na]+, and potassiated [M+K]+ in positive ion mode (Figure 5a–e). Some acids such as malic acid, citric acid, and glutamic acid were detected as deprotonated [M-H]− ions from cherry tomato in negative ion mode (Figure 5f). These results were consistent with earlier studies of these samples using in vivo nESI [58], leaf spray, or direct ionization [14]. Potentially, tip-ESI can be coupled with liquid extraction surface analysis (LESA) [59, 60], which also relies on tip sampling, for the direct analysis of biological samples (tissue analysis, biological fluids, and single-cell analysis) by a robotic system.

Figure 5
figure 5

Direct detection of bioactive ingredients in bulk biological samples using tip-ESI-MS. Onion (a), tomato (b), potato (c), orange (d), and ginger rhizome (e) were analyzed in positive ion mode; cherry tomato (f) was in negative ion mode

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Analysis of Viscous Sample

tip-ESI was also successfully applied to analysis of viscous samples including chicken egg white, yogurts, and soy milk. The samples were loaded in the same way as juicy sample analysis. Figure 6 shows the tip-ESI-MS analysis of above samples. It can be seen that different compounds (proteins, amino acids, lipids, and sugars) were identified from various viscous samples in the form of protonated [M+H]+, sodiated [M+Na]+, and potassiated [M+K]+ as well as calcium adduct species of [M+Ca]2+ (Figure 6a–c), which were in agreement with recent reports obtained by dipping probe electrospray ionization [61] and solid substrate electrospray ionization [62]. This indicated that tip-ESI could be an alternative to the expensive nESI approaches based on the metal coated/uncoated pulled glass capillaries. Owing to the relatively large inner diameter of the tip, no clogging problem was encountered in our study for all the tested samples, even without filtering treatment.

Figure 6
figure 6

Viscous sample analysis by tip-ESI-MS. (a) Chicken egg white. (b) Yogurt. (c) Soy milk. Hex, Lac, and LA indicated hexose, lactose, and lactic acid, respectively

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Analysis of Solid Sample

As is well known, conventional nESI can only be used for analysis of aqueous samples. Alternatively, the novel tip-ESI allowed direct analysis of solid sample without sample pretreatment. Figure 7 shows the MS spectra of erythromycin powder. After application of high voltage, reproducible, strong, and durable signal was observed (Figure 7a, b). The MS data obtained by tip-ESI was in agreement with that of ballpoint ESI analysis reported earlier [25]. In addition, the following different amino acid powders were also analyzed using this method. D-arginine, D-glutamic acid, and D-valine were detected as protonated monomer to pentamer (Figure 7c–f), and L-serine as protonated monomer to octamer. The formation of octamer in L-serine molecule was also evidenced by electrospray ionization (EI) [63]. The application of platinum wire enables direct sampling of powder samples and allows access to samples located in the areas where routine sampling procedure is difficult.

Figure 7
figure 7

Powder sample analysis by tip-ESI-MS. (a) Total ion chromatogram (TIC) of erythromycin. (b) Mass spectrum of erythromycin. (c) D-arginine. (d) L-serine. (e) D-glutamic acid. (f) D-valine

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Tip-ESI was also suitable for analysis of bulk solid sample. Two microliters of water/methanol mixture (99:1 v/v) was firstly loaded into a pipette tip, and then a small portion of solid sample was inserted into the pipette tip for online extraction and direct MS analysis. Undoubtedly, this sampling approach was not compatible with conventional capillary nESI due to capillary clogging. With tip-ESI, we experienced no any clogging, probably due to the large inner diameter of the tip and the high physical and chemical inertness of plastic surface. Figure 8 shows the tip-ESI-MS analysis of four popular Chinese herbal medicines: Coptis chinensis (dry root), Schisandra chinensis, and Schisandra sphenanthera (dry fruit). Alkaloids and lignans could be detected as protonated, sodiated, and potassiated species in the spectra (Figure 8). The obtained MS spectra exhibited strong similarity to earlier direct ionization method [64], and the presence of the detected analytes have been confirmed by other techniques such as EI [65], HPLC [66], and matrix-assisted laser desorption ionization (MALDI) [67]. Thus, tip-ESI was proven to be a good alternative to conventional approaches for direct analysis of solid samples with high analyte sensitivity.

Figure 8
figure 8

Analysis of four Chinese herbal medicines by tip-ESI using water-methanol mixture (99:1 v/v) as ionization reagent. (a) TIC of Schisandra chinensis. (b) Mass spectrum of Schisandra chinensis. (c) Coptis chinensis. (d) Schisandra sphenanthera

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Conclusions

nESI-MS analysis of various kinds of samples, particularly aqueous solutions, using a commercially available plastic tip combined with a high-ohmic resistor has been demonstrated. The tip-ESI performed equally well to the conventional capillary nESI but also added several new advantages, including high signal stability, durability, tolerance to high concentration and high voltage, and obviation of tip clogging and corona discharge. Generation of stable electrospray of solvents with low organic contents at ambient conditions enabled accurate quantification of analytes in aqueous solutions at trace level. Furthermore, tip-ESI facilitated sampling of untreated raw samples, viscous samples, and even solid samples for direct MS analysis. In perspective, tip-nESI could become a powerful approach for ambient point-of-care analysis or tissue imaging. Due to the low sample consumption and high sensitivity, tip-ESI is also potentially suitable for offline analysis of low-abundance metabolites in samples with limited volumes, e.g., at single-cell level or even at the subcellular level.