Skip to main content
NCBI home page
ACS AuthorChoice logoLink to ACS AuthorChoice
. 2022 Aug 1;94(32):11329–11336. doi: 10.1021/acs.analchem.2c02074

Nanoflow Sheath Voltage-Free Interfacing of Capillary Electrophoresis and Mass Spectrometry for the Detection of Small Molecules

Yousef S Elshamy †, Timothy G Strein ‡, Lisa A Holland †,*, Chong Li †, Anthony DeBastiani †, Stephen J Valentine †, Peng Li †, John A Lucas †, Tyler A Shaffer †
PMCID: PMC9387528  PMID: 35913997

Abstract

graphic file with name ac2c02074_0007.jpg

Coupling capillary electrophoresis (CE) to mass spectrometry (MS) is a powerful strategy to leverage a high separation efficiency with structural identification. Traditional CE-MS interfacing relies upon voltage to drive this process. Additionally, sheathless interfacing requires that the electrophoresis generates a sufficient volumetric flow to sustain the ionization process. Vibrating sharp-edge spray ionization (VSSI) is a new method to interface capillary electrophoresis to mass analyzers. In contrast to traditional interfacing, VSSI is voltage-free, making it straightforward for CE and MS. New nanoflow sheath CE-VSSI-MS is introduced in this work to reduce the reliance on the separation flow rate to facilitate the transfer of analyte to the MS. The nanoflow sheath VSSI spray ionization functions from 400 to 900 nL/min. Using the new nanoflow sheath reported here, volumetric flow rate through the separation capillary is less critical, allowing the use of a small (i.e., 20 to 25 μm) inner diameter separation capillary and enabling the use of higher separation voltages and faster analysis. Moreover, the use of a nanoflow sheath enables greater flexibility in the separation conditions. The nanoflow sheath is operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. This includes the use of a wider pH range as well. The versatility of nanoflow sheath CE-VSSI-MS is demonstrated by separating cationic, anionic, and zwitterionic molecules under a variety of separation conditions. The detection sensitivity observed with nanoflow sheath CE-VSSI-MS is comparable to that obtained with sheathless CE-VSSI-MS as well as CE-MS separations with electrospray ionization interfacing. A bare fused silica capillary is used to separate cationic β-blockers with a near-neutral background electrolyte at concentrations ranging from 1.0 nM to 1.0 μM. Under acidic conditions, 13 amino acids are separated with normal polarity at a concentration ranging from 0.25 to 5 μM. Finally, separations of anionic compounds are demonstrated using reversed polarity under conditions of suppressed electroosmotic flow through the use of a semipermanent surface coating. With a near-neutral separation electrolyte, anionic nonsteroidal anti-inflammatory drugs are detected over a concentration range of 0.1 to 5.0 μM.

Introduction

Mass spectrometry (MS) provides critical information about sample composition and molecular structure in pharmaceutical and bioanalytical research, especially when combined with liquid chromatography separations to reduce the sample complexity prior to mass analysis.1 Although liquid chromatography is a prevalent separation method coupled to MS, capillary electrophoresis is an alternative liquid-based separation method that has been successfully interfaced to MS through electrospray ionization. Capillary electrophoresis offers the advantages of low volume sample requirements, automation, and fast runs.2 These features make an integrated capillary electrophoresis MS system a powerful technique for metabolomics,3−7 proteomics,8,9 glycomics,10 biomarkers,11−14 and affinity binding.15,16 Notable applications of capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) include analyses at the cellular levels4,7−9 as well as determinations ranging from pharmaceutical purity,17 degradation, and metabolite studies. Progress in the coupling of capillary electrophoresis and MS continues to advance applications that integrate these methods.18

Electrospray ionization is interfaced directly to the mass analyzer as a sheathless flow, or it includes an additional sheath flow to assist in the electrospray process. The incorporation of a sheath flow offers greater flexibility in the design of the capillary electrophoresis separation. This is particularly important under conditions of suppressed or reversed electroosmotic flow in which there is little or no bulk fluid flow exiting the separation capillary. Although the use of a sheath flow sustains the electrospray process, it also dilutes the analyte band exiting the separation capillary. This process of analyte dilution can be minimized by reducing the volumetric flow rate of the sheath to nanoliters per minute. In a recent comparison of sheathless and nanoflow sheath electrospray ionization to electrospray ionization, the nanoflow systems demonstrated a 10- to 100-fold improvement depending on the analyte.19 Different strategies are reported to achieve nanoliter/minute flow rates.18 The use of a sheath flow of 400 nL/min19 or lower20 was achieved by delivering fluid which was replaced by using a syringe pump operated from 4 to 8 μL/min. With a 3D printed plug-and-play design, the separation capillary and sheath capillary are positioned in a way that prevents back-flow into the separation capillary.20 A second design integrated in a commercial instrument21 delivers a nanoflow sheath reported to be 50 nL/min22 through electrokinetic pumping of fluid.

Vibrating sharp-edge spray ionization (VSSI) is an alternative method of interfacing liquid separations to MS. VSSI achieves sample nebulization and ionization through the generation and application of acoustic waves through a sharp edge coupled to a piezoelectric transducer. The introduction of acoustic waves to the liquid surface results in a plume of droplets that desolvate to produce gas phase ions suitable for MS analysis. Different designs couple the acoustic energy and liquid through the sharp edge, including the corner of a piece of glass for direct contact-based nebulization23,24 or using a capillary attached to the piece of glass25−29 with fluid through the capillary. Unlike traditional electrospray interfacing, VSSI is voltage-free ionization and does not utilize nebulizer gas. Organic additives, which are commonly used to assist electrospray ionization interfacing, are compatible with VSSI and decrease droplet size.23 VSSI is compatible with fluid flow rates from 1 to 1000 μL/min when the liquid passes through the probe in a design similar to what is observed with electrospray systems.25,27 Lower flow rates have been coupled to VSSI with voltage26 or when liquid is brought into contact with the tip of the glass VSSI probe as was previously reported in a sheathless capillary electrophoresis-VSSI-mass spectrometry (CE-VSSI-MS) design.30 In that report, for the first time, a hollow pulled probe was used on the corner of a glass slide attached to the piezoelectric transducer and functioned with flow rates ranging from 70 to 200 nL/min.

A sheathless CE-VSSI-MS system developed previously demonstrated a limit of detection of 2 nM,30 which was comparable to limits of detection of 0.7 nM for beta blockers obtained with a sheathless CE-ESI-MS system.31 Sheathless CE-ESI-MS interfacing is ideal for low detection limits because it avoids analyte dilution. For example, the introduction of a nanoflow sheath to CE-ESI-MS systems increased the limit of detection to 70 nM.32 While the sheathless CE-VSSI-MS system was capable of detecting nanomolar levels of analyte,30 separation conditions were limited to the use of a 50 μm inner diameter separation capillary and background electrolyte systems that maintained an electroosmotic flow sufficient to sustain the VSSI process.

These challenges are overcome using a sheath flow CE-VSSI-MS operated at submicroliter per minute flow rates (i.e., 400–900 nL/min) regardless of the separation flow rate. This present report describes the implementation of a nanoflow sheath CE-VSSI-MS device with a lab-built instrument. The sheath flow eliminates the requirements for an electroosmotic flow, and it enables the use of a smaller bore capillary. The use of a smaller inner diameter reduces the effects of siphoning and allows for the application of a higher separation voltage, which reduces the run time. Additionally, the method of aligning the VSSI probe is improved with the use of a micromanipulator and a hand-held digital microscope. This modular design makes the VSSI probe and capillary independent so that either is easily replaced. With the probe design, the interaction of the fluid and the sharp edge directs the nebulized plume in an angular fashion. Similar to the prior report,30 the nanoflow sheath design is capable of detecting 10 nM pindolol with a separation efficiency of 50 000 plates per meter.

The functionality of the nanoflow sheath CE-VSSI-MS system is evaluated under different conditions to demonstrate the flexibility of this technique. The nanoflow sheath is operated using aqueous solutions in the background electrolyte and in the sheath, demonstrating the separation can be performed under normal and reversed polarity in the presence or absence of electroosmotic flow. Using bare-fused silica, CE-VSSI-MS separations of β-blockers are achieved using an ammonium acetate background electrolyte at pH 6.3 that provides an electroosmotic flow. The capillary electrophoresis separations are compatible with electrokinetic injections and electrokinetic stacked injections to achieve a linear range of 10 to 1000 nM and 1 to 100 nM, respectively. Additionally, sheath flow CE-VSSI-MS can be performed where the electroosmotic flow is suppressed using a 2% formic acid background electrolyte to separate and detect amino acids within a concentration range of 0.25 to 5 μM. Moreover, the electroosmotic flow is also suppressed using a semipermanent coating to separate anionic nonsteroidal anti-inflammatory drugs (NSAIDs). The separation is achieved at near-neutral pH with reversed polarity. Under these operating conditions, NSAIDs are detectable within a concentration range of 0.1 to 0.5 μM using electrokinetic sample stacking. The detection of the anionic analytes with VSSI-MS is achieved with positive mode MS, which demonstrates a 3 order of magnitude enhancement in signal relative to that observed in negative mode MS.

Materials and Methods

Chemicals and Reagents

An amino acid kit (LAA-21), pindolol (P-0778), acebutolol (A-3669), atenolol (A-7655), timolol (T-6394), tolmetin (T-6779), ketoprofen (K-1751), suprofen (S-9894), indoprofen (I-3132), acetic acid (A6283), and mineral oil (M5904) were purchased from Millipore Sigma (Burlington, MA). Oxprenolol (156023) was purchased from ICN Biomedicals Inc. (Aurora, OH). Caffeine (C5-3) was purchased from Aldrich Chemical Co. LLC (Milwaukee, WI). Formic acid (A13285) and ammonium acetate (A16343) were purchased from Alfa Aesar (Heysham, England). Ammonium hydroxide (BDH3016) was purchased from VWR Analytical (Radnor, PA). The phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) were from Avanti Polar Lipids (Alabaster, AL). Deionized water (18 MΩcm) was obtained from an Elga Purelab and Veolia Chorus water system (Lowell, MA).

VSSI Probe Fabrication

The bare fused silica sheath capillary with an inner diameter of 200 μm and outer diameter of 363 ± 10 μm (TSP200350, Molex, Phoenix, AZ) is cut to 30 cm. The polyimide coating is removed on both ends of the capillary and examined with a microscope for a clean cut. To prepare the capillary electrophoresis electrode, a 25 μm platinum wire (PT005113, Goodfellow, Huntingdon, England) is trimmed to approximately 1 cm in length, bent at two 90° angles in a “U” shape, and fixed to the sheath using 1 minute epoxy (1366072, Henkel, Düsseldorf, Germany). To create the electric connection, the platinum is attached to a wire using conductive epoxy (8331-14G, MG Chemicals, Ontario, Canada) and then covered with a layer of 1 minute epoxy (1366072, Henkel). The hydrodynamic flow through the sheath capillary is driven by applying a pressure of 20 to 40 kPa (3 to 6 psi). The VSSI probe device is constructed using a solid glass rod (GR100-4 Precision Instruments 1 mm × 10 cm), pulled using a laser puller (Sutter Instrument Company, Novato, CA), trimmed to a final tip diameter of approximately 60 to 100 μm, and attached to the underside of a piezoelectric transducer (7BB-27-4 L0, Murata diameter = 27 mm) using 5 minute epoxy. The parameters used for pulling the glass rod to form the VSSI probe are as follows: HEAT = 750, FIL = 4, VEL = 60, DEL = 130, and PUL = 50.

VSSI Instrumentation

The piezoelectric transducer is connected to a function generator (DDS signal generator/counter Koolertron, Hong Kong Karstone Technology Co, Hong Kong) and amplifier (7500, Krohn-Hite, Brockton, MA), and a square wave is applied with frequencies ranging from 92 to 96 kHz and amplitudes ranging from 10 to 12 Vpp. The applied frequency and amplitude vary between fabricated devices, and optimal settings are evaluated prior to use through visual observation of a microdroplet plume via a moistened cotton swab tip. A micromanipulator is utilized to position the VSSI probe at a 90° angle relative to the capillary. The VSSI probe is parallel to the platinum electrode at the end of the capillary. The sheath flow VSSI device is placed 2 to 3 mm from the inlet of the mass spectrometer. The alignment is evaluated by direct infusion of a standard compound (e.g., pindolol).

CE-VSSI-MS Separation and Analysis

Unless otherwise noted, the dimensions of the separation capillary are 30 cm, 25 μm inner diameter, and 150 μm outer diameter (TSP050150, Molex, Arizona, USA). Injection conditions, electric field, and separation polarity are noted in the text and figure captions. The acoustic spray is off during extensive prerun flushes but remains active during shorter flushes in between runs. For the separations of beta blockers, prior to use, the capillary is flushed at 138 kPa (20 psi) with 0.1 N ammonium hydroxide for 60 min, water for 10 min, and the background electrolyte for 30 min. In between runs, the capillary is flushed for 1 min with background electrolyte. For the separations of amino acids, prior to use, the capillary is flushed at 138 kPa (20 psi) with 2% formic acid for 20 min. In between runs, the capillary is flushed for 1 min with background electrolyte. For the separations of NSAIDs, the capillary is treated with a semipermanent lipid coating. The procedure for coating the capillary involves a flush at 172 kPa (25 psi) with 0.1 N ammonium hydroxide for 30 min, deionized water for 15 min, methanol for 15 min, deionized water for 15 min, 50 mM ammonium acetate at pH 6.3 for 3 min, phospholipid coating for 20 min, and 50 mM ammonium acetate at pH 6.3 for 7 min. For all separations, the capillary used to split the flow for the sheath is 50 cm with an inner diameter of 200 μm (TSP050375, Molex, Arizona, USA), and the same background electrolyte solution is used. A Q-exactive mass spectrometer equipped with LTQ Tune Plus software (version 2.7) is used to collect the data (Thermo Fisher Scientific, San Jose, CA). Both the MS ion transfer tube and the capillary outlet are grounded, creating a field-free region. The instrument is operated with the VSSI source by overriding two interlocks for the standard HESI source and with the sweep gas cone removed. Data are processed using Thermo Fisher Scientific Xcalibur (version 4.1) and Microsoft Excel (2021, Microsoft, Redmond, WA).

Capillary Electrophoresis-UV (CE-UV) Absorbance Detection Separation and Analysis

All separations are conducted using a Beckman/Coulter P/ACE MDQ (Beckman Coulter, Fullerton, CA, USA). Capillaries have a total length of 40 cm, an effective length of 30 cm, an inner diameter of 25 μm, and an outer diameter of 360 μm. The capillary preparation and separation are the same as those used for the VSSI-MS analyses. To maintain the same electric field strength used on the CE–VSSI–MS system, the separation voltage is 21.3 kV, applied in normal polarity for the beta blocker and in reverse polarity for the NSAIDs, and 16 kV, applied in normal polarity for the amino acids. The analyses were performed using UV absorbance detection at a wavelength of 200 nm. The cartridge temperature is set to 25 °C for flushes and separations. Data collection and analysis are accomplished by the accompanying Beckman/Coulter P/ACE MDQ 32Karat Software (Beckman Coulter). It is worth noting that, in the CE-UV NSAID separations, capillaries are flushed in between runs for an additional 3 min with ammonium acetate, followed by 5 min of phospholipid, and then 3 min of background electrolyte at 172 kPa (25 psi).

Preparation of Phospholipid

The solutions of the phospholipids are prepared as previously described.33−35 Briefly, DMPC and DHPC are weighed and combined at a molar ratio of 0.5 DMPC/DHPC. The 5% phospholipid solution is prepared by adding 50 mM, pH 6.3 ammonium acetate to the phospholipid powder. The preparation is then vortexed until the solids were thoroughly dissolved. The solution was then subjected to three freeze–thaw cycles followed by centrifugation at 10 000 rpm for 10 min at 4 °C and stored at −20 °C. Before use, calcium chloride is added to the lipid to a final concentration of 1 mM.

Results and Discussion

VSSI Interface Design

A new VSSI interface design integrates a sheath flow that allows for stable VSSI nebulization and ionization. The sheath flow VSSI interface shown in Figure 1 demonstrates that the separation capillary is integrated inside of a sheath capillary. In this design, the VSSI interface operates independently of the capillary electrophoresis. The voltage applied to the separation capillary does not affect the ion intensity. The separation capillary is recessed less than 100 μm from the grounding electrode that is flush with the end of the sheath capillary. The use of the sheath overcomes the limitation of the previously reported sheathless design,30 which required flow rates greater than 70 nL/min in the separation capillary. The sheath provides a stable sheath flow rate of 400 to 900 nL/min. With the aid of a camera, the VSSI probe is aligned directly at the sheath with micromanipulators (Figure S1).

Figure 1.

Figure 1

Open in a new tab

Components of the capillary electrophoresis VSSI sheath flow design.

VSSI Droplet Formation

Capillary electrophoresis-VSSI spray is ejected at an angle and creates droplets that are approximately 8 μm in diameter. This angular direction of spray observed with sheath flow VSSI (Figure 2A) was also reported with sheathless VSSI.30 This spray orientation and the 1 mm size of the plume are similar to the results obtained through COMSOL modeling of the acoustic streaming shown in Figure 2B. The numerical simulation indicates that the maximum streaming velocity resides on the corner edge at the tip of the vibrating capillary, denoted with the yellow color scale in Figure 2B. This location is where the droplets are most probably ejected into to the air. The interaction of fluid with the vibrating probe likely forms streaming that, along with plume entrainment and the MS vacuum intake, leads to the angular appearance of the spray. Streaming was also visualized in a fluid system with fluorescent beads in which the directionality of the fluid flow is observed to move inward at the probe tip, exiting as a vortex and a right angle to the probe (Supporting Information Video). To further characterize the nebulization process, droplets ejected from the probe are captured in mineral oil (Figure 2C) and measured with imaging software. As shown in Figure 2D, the mean droplet size is 8 ± 3 μm. This measurement of droplet size is considered as an estimate as larger droplets formed due to merging, while smaller droplets are obtained at a larger collection distance as the desolvation process is a function of distance of droplet travel to oil.

Figure 2.

Figure 2

Open in a new tab

(A)Image of the angular VSSI spray direction as visualized in the presence of a scattered green laser light. The COMSOL simulation shown in (B) demonstrates the angular direction of the velocity field at the VSSI probe. In (C), droplets collected from the VSSI into mineral oil are sized with optical microscopy and ImageJ to obtain the histogram (D) depicting the distribution of droplet size.

Nanoflow Sheath Enables Low Flow Separations

Nanoflow sheath CE-VSSI-MS allows for flexibility in interfacing capillary electrophoresis separations performed over a broad range of pH values and separation flow rates. Prior work with sheathless CE-VSSI-MS was performed in 50 μm inner diameter capillaries with stable VSSI spray only at flow rates above 70 nL/min.30 The use of smaller inner diameter separation capillaries is desirable as higher electric fields and faster separations are possible. As shown in Figure 3, a separation of pindolol and acebutolol is performed using a sheath flow CE-VSSI-MS system with sheath and separation background electrolyte simultaneously maintained at a pH of 4.7, 6.3, or 9.1. Pindolol and acebutolol, which are cationic beta blockers, are resolved by utilizing ammonium acetate as the background electrolyte at acidic, near-neutral, and basic pH using sheath flow CE-VSSI-MS (Figure 3). These data are obtained with a 20 μm inner diameter separation capillary for which the calculated volumetric flow rate of the separation ranges from 11 to 27 nL/min. This is determined using caffeine as a neutral flow marker (Figure S2). These data demonstrate stable VSSI spray with capillary electrophoresis separations with sub-70 nL/min flow rates over a wide pH range. While resolution is higher at low pH where the electroosmotic flow is slower, faster analyses are possible at higher pH. Although it appears from Figure 3 that the signal intensity increases with pH, it is important to note that greater mass loading is obtained with electrokinetic injections performed with increasing pH. For example, the mass loading calculated at pH 4.7, 6.3, and 9.1 for an electrokinetic injection36 of 1 μM pindolol is 81, 104, and 141fg, respectively, and for acebutolol, it is 99, 130, and 181 fg, respectively. This increase in mass loading corresponds directly with the increase in peak areas obtained at pH 4.7, 6.3, and 9.1.

Figure 3.

Figure 3

Open in a new tab

(A) CE-VSSI-MS separation of 1 μM beta blockers. Separation was achieved with a 49 cm (total and effective length), 20 μm i.d. capillary at an applied voltage of 12 kV with a current of 3.2 μA and injection voltage of 3 kV 4 s. The trace is obtained using masses of 249.1595 and 337.1118 for pindolol and acebutolol, respectively, with a mass tolerance of 50 ppm. For all separations, the sheath fluid is the same as the background electrolyte, which is 50 mM acetic acid adjusted to pH 4.7 with ammonium hydroxide, 50 mM ammonium acetate with a pH of 6.3, or 50 mM ammonium acetate adjusted to a pH of 9.1 with ammonium hydroxide. The sheath flow rates are 650 nL/min (pH 6.3) and 733 nL/min (pH 4.7 and 9.1).

Separation Performance

Thorough characterization of the nanoflow sheath system with beta blockers is performed using electrokinetically stacked injections and the near-neutral pH background electrolyte, which has an intermediate electroosmotic flow rate. The traces for both the CE-UV and CE-VSSI-MS, shown in Figure 4, are each 0.2 min wide with the trace for the CE-VSSI-MS being from a single run as compared to multiple runs superimposed onto each other with CE-UV. The CE-VSSI-MS distinguishes oxprenolol, atenolol, and timolol that comigrate in the capillary electrophoresis separation (Figure 4). Migration times obtained with CE-UV and CE-VSSI-MS are significantly different as determined with a Student’s t test (ρ = 0.05). However, this is attributed to the use of a slightly higher electric field strength and the absence of temperature control on the lab-built CE-VSSI-MS instrument as separations performed on the CE-UV instrument had migration times as much as 8 s faster at 31 °C than runs obtained at 25 °C (Table S2). Additionally, the lab-built CE-VSSI-MS instrument had higher variance in migration time as the runs are manually controlled. The theoretical plate counts achievable with the capillary separation do not decrease with the introduction of the CE-VSSI-MS nanoflow sheath. The linear response for CE-VSSI-MS is 1–100 nM compared to 10–1000 μM for CE-UV (Table S3).

Figure 4.

Figure 4

Open in a new tab

(A) CE–UV separation of 200 μM pindolol, oxprenolol, atenolol, timolol, and acebutolol. Separation was achieved with a 40 cm (total length), 30 cm (effective length), 25 μm i.d. capillary at an applied voltage of 21.3 kV with a current of 8.8 μA and injection voltage of 10 kV for 2 s. (B) CE-VSSI-MS separation of 10 nM beta blockers. Separation was achieved with a 27 cm (total and effective length), 20 μm i.d. capillary at an applied voltage of 16 kV with a current of 11 μA and injection voltage of 20 kV for 2 s. The trace is obtained using masses of 249.1594, 266.1746, 267.1683, 317.1639, and 337.1212 for pindolol, oxprenolol, atenolol, timolol, and acebutolol, respectively, with a mass tolerance of 10 ppm. The sample is injected with electrokinetic stacking achieved by diluting the sample in 1 mM ammonium acetate. All separations are performed using a background electrolyte of 50 mM ammonium acetate with a pH of 6.3. The sheath flow rate is 900 nL/min.

Stacking Enhancement

Nanoflow sheath is compatible with both electrokinetic injections (Figure S3) and stacked electrokinetic injections (Figure 4). The linear range for EK injections of beta blockers begins at a lower concentration for CE-MS compared to CE-UV (25–2500 μM for CE-UV and 0.010–1.0 μM for CE-VSSI-MS; see Tables S4 and S5). This is also observed for EK stacked injections with a CE-UV linear range of 10.00–500/1000 μM and a CE-VSSI-MS linear range of 0.0010–0.10 μM (Tables S3 and S5). For CE-VSSI-MS, an injection voltage of 20 kV is applied for 2 s, and for CE-UV, an injection voltage of 10 kV for 2 s is used. A lower injection voltage is used in CE-UV analysis because 10 kV is the maximum injection voltage of the commercial instrument. To compare the stacking enhancement, a ratio of the slope of the curves generated from peak areas obtained with stacking electrokinetic injection to nonstacking electrokinetic injections is taken (Table S6). For the CE-VSSI-MS stacked injections, the signal intensity is increased by 3–11-fold depending on the analyte, as compared to CE-UV for which stacking enhancement is 3–6-fold.

CE-VSSI-MS Separations of Amino Acids with Suppressed Electroosmotic Flow

CE-VSSI-MS is not only useful for the separation of cationic beta blockers but also applicable to compounds that require an acidic background electrolyte to remain cationic. The separation of amino acids is performed using a background electrolyte composed of 2% formic acid, similar to CE-ESI-MS separations of amino acids reported in the literature.37 Under these conditions, the electroosmotic flow is suppressed; therefore, the analyte migration is predominantly due to electrophoretic mobility. The sheath design enables MS detection of analytes regardless of the electroosmotic flow rate. With CE-VSSI-MS, 13 amino acids are separated and detected in under 5 min. The separation of amino acids shown in Figure 5A is performed using a stacked electrokinetic injection of 20 kV for 3 s in a single analysis of a sample containing a mixture of 1 μM lysine, arginine, and histidine as well as 5 μM valine, leucine, asparagine, threonine, glutamine, tryptophan, glutamic acid, phenylalanine, proline, and tyrosine all dissolved in 0.004% formic acid. These amino acids are detectable at concentrations ranging from 0.25 to 5.0 μM (Tables S7 and S8). These concentrations are comparable to those recently reported for CE-ESI-MS systems with microliter38,39 or submicroliter40,41 sheath flows. While these reported systems utilized organic solvents38−41 in the sheath fluid, no organic solvents are needed with VSSI. The low molecular weight amino acids alanine and glycine are poorly detected with the MS and therefore not included. Additionally, isoleucine comigrates with its isomer leucine and is indistinguishable by MS.

Figure 5.

Figure 5

Open in a new tab

(A) CE-VSSI-MS separation of 1 μM lysine, arginine, histidine, respectively, and 5 μM valine, leucine, asparagine, threonine, glutamine, tryptophan, glutamic acid, phenylalanine, proline, and tyrosine. The extracted ion electropherograms were created using masses of 147.1127, 175.1188, 156.0767, 118.0864, 132.1019, 133.0607, 120.0602, 147.0762, 205.0974, 148.0602, 166.0861, 116.0708, and 182.0811 for lysine, arginine, histidine, valine, leucine, asparagine, threonine, glutamine, tryptophan, glutamic acid, phenylalanine, proline, and tyrosine, respectively, with a mass tolerance of 10 ppm. Separation was achieved with a 30 cm (total and effective length), 25 μm i.d. capillary at an applied voltage of 12 kV with a current of 8.2 μA and injection voltage of 20 kV 3 s. The sheath flow rate is 900 nL/min. (B) CE-UV separation of 100 μM arginine, histidine, tryptophan, phenylalanine, and tyrosine and 250 μM asparagine and glutamine. Separation was achieved with a 40 cm (total length), 30 cm (effective length), 25 μm i.d. capillary at an applied voltage of 16 kV with a current of 8.1 μA and injection voltage of 10 kV for 4 s. Stacking was achieved using 0.004% formic acid, and all separations were achieved using 2% formic acids as background electrolyte.

CE-VSSI-MS vs CE-UV

The separation performance with CE-VSSI-MS of amino acids can be compared using separations obtained with the same conditions but detected with UV absorbance detection. For CE-UV, only 7 of the 13 amino acids are detected with an electrokinetic stacking injection at 10 kV for 4 s. Also, because of peak overlap with CE-UV, full visualization of analyte peaks with UV requires two separate runs, which are then superimposed as shown in Figure 5B. The CE-UV traces are obtained at a concentration of 100 μM arginine, histidine, tryptophan, phenylalanine, and tyrosine and 250 μM asparagine and glutamine. While the migration times of the CE-VSSI-MS and CE-UV separations are statistically the same, the plate counts obtained with CE-UV are greater than those of CE-VSSI-MS (Table S9). This is due to injection overloading that occurs with the larger CE-VSSI-MS injections. As many of the amino acids are not detectable with UV absorbance, a simulation of the separation is used to predict the migration order for the amino acids that are undetectable with UV–visible absorbance (Figure S4). The simulation has comparable separations although tryptophan and proline migrate slower than predicted and arginine migrates faster than predicted by the simulation.

CE-VSSI-MS Separations with a Modified Capillary Surface and Reversed Polarity

The flexibility of the nanoflow sheath CE-VSSI-MS enables the use of reversed polarity with a suppressed electroosmotic flow to resolve the anionic NSAIDs. The passivation of the inner capillary wall surface is accomplished using a previously reported semipermanent capillary surface coating42−44 that masks the surface charge on the fused silica through the self-assembly of a phospholipid and is stable in solutions ranging in pH from 4 to 8. A benefit of the use of this reversed polarity separation is that it enables sample stacking of anions, which increases the peak height and area. The CE-VSSI-MS analysis of NSAIDs shown in Figure 6A is obtained in positive ion mode rather than with negative ion mode as a larger MS signal is observed. Experiments conducted with direct infusion reveal that this effect on the ionization efficiency of positive ions is more pronounced with higher pH (Figure S5). The increase in positive ionization efficiency is consistent with enhanced ionization associated with ammonium ions to either protonate the NSAID or form a transient adduct, which assists in ionization.45−47

Figure 6.

Figure 6

Open in a new tab

(A) CE-VSSI-MS separation of 1 μM NSAIDs. The extracted ion electropherograms are created using masses of 261.0577, 258.1123, 255.1013, and 282.1127 for suprofen, tolmetin, ketoprofen, and indoprofen, respectively, with a mass tolerance of 10 ppm. Separation is achieved with a 30 cm (total and effective length), 25 μm i.d. capillary at an applied voltage of −16 kV with a current of −11.2 to −14.9 μA and injection voltage of −20 kV for 2 s. (B) CE–UV separation of 20 μM suprofen, tolmetin, ketoprofen, and indoprofen. Separation is achieved with a 40 cm (total length), 30 cm (effective length), 25 μm i.d. capillary at an applied voltage of −21.3 kV with a current of −8.0 μA and injection voltage of −10 kV for 2 s stacking achieved using 1 mM ammonium acetate, and all separations were performed at pH 6.3, 50 mM ammonium acetate as background electrolyte. The sheath flow rate is 900 nL/min.

The CE-VSSI-MS separation of NSAIDs is obtained in a single run (Figure 6A), but the CE-UV analyses are completed in multiple runs, which are superimposed (Figure 6B). The migration times for CE-UV and CE-VSSI-MS are different (Table S10), which is attributed to a lack of temperature control in the lab-built CE-VSSI-MS system and the use of flushing and recoating of the programmable CE-UV capillary, which is not performed with the CE-VSSI-MS system. Moreover, the CE-VSSI-MS data display an increase in the separation current over time. This can potentially be attributed to a change in ion balance from the sheath. Although the migration times differ, the separation efficiency is the same for CE-VSSI-MS and CE-UV. The linear range of the CE-UV is 1 to 100 μM, whereas the CE-VSSI-MS linear range is from 0.1 or 0.5 to 5 μM (Tables S10 and S11). Previous CE-ESI-MS reports have a similar sensitivity. For example, a nanoflow sheath CE-ESI-MS analysis also based on positive mode MS and an aqueous background electrolyte had a detection limit of 4 μM for ketoprofen.48 In a different report,49 a detection limit of 0.4 μM was obtained for suprofen with negative mode MS using a sheath flow interface and an aqueous background electrolyte. The detection of the NSAID suprofen was improved when a nonaqueous background electrolyte49 was used with sheath flow and sheathless flow, generating 0.2 and 0.004 μM, respectively.

Conclusions and Future Directions

This report demonstrates a successful implementation of a nanoflow sheath CE-VSSI-MS design. Using this novel design, the separation can be performed using background electrolyte at different pH values regardless of flow rate in such a manner that high separation voltage and rapid separation can be achieved. This novel system is comparable to the previously reported sheathless CE-VSSI-MS, and analyte is detected without a significant dilution effect. The nanoflow sheath design offers the flexibility to select different volatile background electrolytes for the separation and analysis of small cationic, zwitterionic, and anionic compounds. Moreover, separation of these analytes via the nanoflow sheath is achieved with an untreated fused silica capillary as well as a capillary surface modified with a semipermanent lipid coating. Signal enhancement is realized by the stacked injection of the analytes. Further investigation into the phenomenon of anionic analytes demonstrating a greater signal in positive MS mode is necessary. Finally, future work will focus on the ability of this design to separate the larger compounds and to gain a better understanding about the ionization mechanism of VSSI on small molecules including pharmaceuticals and metabolites.

Acknowledgments

This material is based on the work supported by the National Science Foundation Grant No. CHE2004021.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.2c02074.

  • Additional electropherograms, a photograph of the system, data obtained with CE-UV and CE-VSSI-MS including migration time, plate count, resolution, area, and linear regression, and a summary of the ionization intensity of the compounds (PDF)

  • Streaming of the fluid system with fluorescent beads (AVI)

The authors declare no competing financial interest.

Supplementary Material

ac2c02074_si_001.pdf (588.3KB, pdf)
ac2c02074_si_002.avi (2.5MB, avi)

References

  1. Beccaria M.; Cabooter D. Current Developments in LC-MS for Pharmaceutical Analysis. Analyst 2020, 145, 1129–1157. 10.1039/C9AN02145K. [DOI] [PubMed] [Google Scholar]
  2. Kristoff C. J.; Bwanali L.; Veltri L. M.; Gautam G. P.; Rutto P. K.; Newton E. O.; Holland L. A. Challenging Bioanalyses with Capillary Electrophoresis. Anal. Chem. 2020, 92, 49–66. 10.1021/acs.analchem.9b04718. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Huang L.; Wang Z.; Cupp-Sutton K. A.; Smith K.; Wu S. Spray-Capillary: An Electrospray-Assisted Device for Quantitative Ultralow-Volume Sample Handling. Anal. Chem. 2020, 92, 640–646. 10.1021/acs.analchem.9b04131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Kawai T.; Ota N.; Okada K.; Imasato A.; Owa Y.; Morita M.; Tada M.; Tanaka Y. Ultrasensitive Single Cell Metabolomics by Capillary Electrophoresis-Mass Spectrometry with a Thin-Walled Tapered Emitter and Large-Volume Dual Sample Preconcentration. Anal. Chem. 2019, 91, 10564–10572. 10.1021/acs.analchem.9b01578. [DOI] [PubMed] [Google Scholar]
  5. Saoi M.; Percival M.; Nemr C.; Li A.; Gibala M.; Britz-McKibbin P. Characterization of the Human Skeletal Muscle Metabolome for Elucidating the Mechanisms of Bicarbonate Ingestion on Strenuous Interval Exercise. Anal. Chem. 2019, 91, 4709–4718. 10.1021/acs.analchem.9b00149. [DOI] [PubMed] [Google Scholar]
  6. Sasaki K.; Sagawa H.; Suzuki M.; Yamamoto H.; Tomita M.; Soga T.; Ohashi Y. Metabolomics Platform with Capillary Electrophoresis Coupled with High-Resolution Mass Spectrometry for Plasma Analysis. Anal. Chem. 2019, 91, 1295–1301. 10.1021/acs.analchem.8b02994. [DOI] [PubMed] [Google Scholar]
  7. Zhang W.; Guled F.; Hankemeier T.; Ramautar R. Utility of Sheathless Capillary Electrophoresis-Mass Spectrometry for Metabolic Profiling of Limited Sample Amounts. J. Chromatogr. B 2019, 1105, 10–14. 10.1016/j.jchromb.2018.12.004. [DOI] [PubMed] [Google Scholar]
  8. Choi S. B.; Polter A. M.; Nemes P. Patch-Clamp Proteomics of Single Neurons in Tissue Using Electrophysiology and Subcellular Capillary Electrophoresis Mass Spectrometry. Anal. Chem. 2022, 94, 1637–1644. 10.1021/acs.analchem.1c03826. [DOI] [PubMed] [Google Scholar]
  9. Lombard-Banek C.; Moody S. A.; Manzini M. C.; Nemes P. Microsampling Capillary Electrophoresis Mass Spectrometry Enables Single-Cell Proteomics in Complex Tissues: Developing Cell Clones in Live Xenopus Laevis and Zebrafish Embryos. Anal. Chem. 2019, 91, 4797–4805. 10.1021/acs.analchem.9b00345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Szigeti M.; Guttman A. Sample Preparation Scale-up for Deep N-Glycomic Analysis of Human Serum by Capillary Electrophoresis and CE-ESI-MS*. Molecular & Cellular Proteomics 2019, 18, 2524–2531. 10.1074/mcp.TIR119.001669. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Salim H.; Pero-Gascon R.; Giménez E.; Benavente F. On-Line Coupling of Aptamer Affinity Solid-Phase Extraction and Immobilized Enzyme Microreactor Capillary Electrophoresis-Mass Spectrometry for the Sensitive Targeted Bottom-up Analysis of Protein Biomarkers. Anal. Chem. 2022, 94, 6948–6956. 10.1021/acs.analchem.1c03800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gill B.; Jobst K.; Britz-McKibbin P. Rapid Screening of Urinary 1-Hydroxypyrene Glucuronide by Multisegment Injection-Capillary Electrophoresis-Tandem Mass Spectrometry: A High-Throughput Method for Biomonitoring of Recent Smoke Exposures. Anal. Chem. 2020, 92, 13558–13564. 10.1021/acs.analchem.0c03212. [DOI] [PubMed] [Google Scholar]
  13. Pero-Gascon R.; Benavente F.; Minic Z.; Berezovski M. V.; Sanz-Nebot V. On-Line Aptamer Affinity Solid-Phase Extraction Capillary Electrophoresis-Mass Spectrometry for the Analysis of Blood Α-Synuclein. Anal. Chem. 2020, 92, 1525–1533. 10.1021/acs.analchem.9b04802. [DOI] [PubMed] [Google Scholar]
  14. Azab S.; Ly R.; Britz-McKibbin P. Robust Method for High-Throughput Screening of Fatty Acids by Multisegment Injection-Nonaqueous Capillary Electrophoresis-Mass Spectrometry with Stringent Quality Control. Anal. Chem. 2019, 91, 2329–2336. 10.1021/acs.analchem.8b05054. [DOI] [PubMed] [Google Scholar]
  15. Gstöttner C.; Hook M.; Christopeit T.; Knaupp A.; Schlothauer T.; Reusch D.; Haberger M.; Wuhrer M.; Domínguez-Vega E. Affinity Capillary Electrophoresis-Mass Spectrometry as a Tool to Unravel Proteoform-Specific Antibody-Receptor Interactions. Anal. Chem. 2021, 93, 15133–15141. 10.1021/acs.analchem.1c03560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Han M.; Wang Y.; Cook K.; Bala N.; Soto M.; Rock D. A.; Pearson J. T.; Rock B. M. Universal Automated Immunoaffinity Purification-CE-MS Platform for Accelerating Next Generation Biologic Design. Anal. Chem. 2021, 93, 5562–5569. 10.1021/acs.analchem.1c00149. [DOI] [PubMed] [Google Scholar]
  17. Řemínek R.; Foret F. Capillary Electrophoretic Methods for Quality Control Analyses of Pharmaceuticals: A Review. Electrophoresis 2021, 42, 19–37. 10.1002/elps.202000185. [DOI] [PubMed] [Google Scholar]
  18. Stolz A.; Jooß K.; Höcker O.; Römer J.; Schlecht J.; Neusüß C. Recent Advances in Capillary Electrophoresis-Mass Spectrometry: Instrumentation, Methodology and Applications. Electrophoresis 2019, 40, 79–112. 10.1002/elps.201800331. [DOI] [PubMed] [Google Scholar]
  19. Höcker O.; Montealegre C.; Neusüß C. Characterization of a Nanoflow Sheath Liquid Interface and Comparison to a Sheath Liquid and a Sheathless Porous-Tip Interface for Ce-Esi-Ms in Positive and Negative Ionization. Anal. Bioanal. Chem. 2018, 410, 5265–5275. 10.1007/s00216-018-1179-3. [DOI] [PubMed] [Google Scholar]
  20. Schlecht J.; Stolz A.; Hofmann A.; Gerstung L.; Neusüß C. Nanoceasy: An Easy, Flexible, and Robust Nanoflow Sheath Liquid Capillary Electrophoresis-Mass Spectrometry Interface Based on 3d Printed Parts. Anal. Chem. 2021, 93, 14593–14598. 10.1021/acs.analchem.1c03213. [DOI] [PubMed] [Google Scholar]
  21. Peuchen E. H.; Zhu G.; Sun L.; Dovichi N. J. Evaluation of a Commercial Electro-Kinetically Pumped Sheath-Flow Nanospray Interface Coupled to an Automated Capillary Zone Electrophoresis System. Anal. Bioanal. Chem. 2017, 409, 1789–1795. 10.1007/s00216-016-0122-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Sun L.; Zhu G.; Zhang Z.; Mou S.; Dovichi N. J. Third-Generation Electrokinetically Pumped Sheath-Flow Nanospray Interface with Improved Stability and Sensitivity for Automated Capillary Zone Electrophoresis-Mass Spectrometry Analysis of Complex Proteome Digests. J. Proteome Res. 2015, 14, 2312–2321. 10.1021/acs.jproteome.5b00100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li X.; Attanayake K.; Valentine S. J.; Li P. Vibrating Sharp-Edge Spray Ionization (VSSI) for Voltage-Free Direct Analysis of Samples Using Mass Spectrometry. Rapid Commun. Mass Spectrom. 2021, 35, e8232 10.1002/rcm.8232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ranganathan N.; Lozier A. M.; Rawson M. C.; Johnson M. B.; Li P. Direct Analysis of Surface Chemicals Using Vibrating Sharp-Edge Spray Ionization Mass Spectrometry. Rapid Commun. Mass Spectrom. 2020, 34, e8902 10.1002/rcm.8902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Ranganathan N.; Li C.; Suder T.; Karanji A. K.; Li X.; He Z.; Valentine S. J.; Li P. Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI) for Voltage-Free Liquid Chromatography-Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2019, 30, 824–31. 10.1007/s13361-019-02147-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Li C.; Attanayake K.; Valentine S. J.; Li P. Facile Improvement of Negative Ion Mode Electrospray Ionization Using Capillary Vibrating Sharp-Edge Spray Ionization. Anal. Chem. 2020, 92, 2492–2502. 10.1021/acs.analchem.9b03983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Jayasundara K. U.; Li C.; DeBastiani A.; Sharif D.; Li P.; Valentine S. J. Physicochemical Property Correlations with Ionization Efficiency in Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI). J. Am. Soc. Mass Spectrom. 2021, 32, 84–94. 10.1021/jasms.0c00100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Majuta S. N.; DeBastiani A.; Li P.; Valentine S. J. Combining Field-Enabled Capillary Vibrating Sharp-Edge Spray Ionization with Microflow Liquid Chromatography and Mass Spectrometry to Enhance ‘Omics Analyses. J. Am. Soc. Mass Spectrom. 2021, 32, 473–485. 10.1021/jasms.0c00376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. DeBastiani A.; Majuta S. N.; Sharif D.; Attanayake K.; Li C.; Li P.; Valentine S. J. Characterizing Multidevice Capillary Vibrating Sharp-Edge Spray Ionization for in-Droplet Hydrogen/Deuterium Exchange to Enhance Compound Identification. ACS Omega 2021, 6, 18370–18382. 10.1021/acsomega.1c02362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kristoff C. J.; Li C.; Li P.; Holland L. A. Low Flow Voltage Free Interface for Capillary Electrophoresis and Mass Spectrometry Driven by Vibrating Sharp-Edge Spray Ionization. Anal. Chem. 2020, 92, 3006–3013. 10.1021/acs.analchem.9b03994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Bonvin G.; Veuthey J.-L.; Rudaz S.; Schappler J. Evaluation of a Sheathless Nanospray Interface Based on a Porous Tip Sprayer for CE-ESI-MS Coupling. Electrophoresis 2012, 33, 552–562. 10.1002/elps.201100461. [DOI] [PubMed] [Google Scholar]
  32. González-Ruiz V.; Codesido S.; Far J.; Rudaz S.; Schappler J. Evaluation of a New Low Sheath-Flow Interface for CE-MS. Electrophoresis 2016, 37, 936–946. 10.1002/elps.201500523. [DOI] [PubMed] [Google Scholar]
  33. Crihfield C. L.; Kristoff C. J.; Veltri L. M.; Penny W. M.; Holland L. A. Semi-Permanent Cationic Coating for Protein Separations. J. Chromatogr. A 2019, 1607, 460397. 10.1016/j.chroma.2019.460397. [DOI] [PubMed] [Google Scholar]
  34. Archer-Hartmann S. A.; Sargent L. M.; Lowry D. T.; Holland L. A. Microscale Exoglycosidase Processing and Lectin Capture of Glycans with Phospholipid Assisted Capillary Electrophoresis Separations. Anal. Chem. 2011, 83, 2740–7. 10.1021/ac103362r. [DOI] [PubMed] [Google Scholar]
  35. Luo R.; Archer-Hartmann S. A.; Holland L. A. Transformable Capillary Electrophoresis for Oligosaccharide Separations Using Phospholipid Additives. Anal. Chem. 2010, 82, 1228–33. 10.1021/ac902052m. [DOI] [PubMed] [Google Scholar]
  36. Landers J. P.Handbook of Capillary and Microchip Electrophoresis and Associated Microtechniques, second ed.; Taylor & Francis: Boca Raton, 1997; Appendix 1: Calculations of Practical Use, p 869. [Google Scholar]
  37. Soga T.; Heiger D. N. Amino Acid Analysis by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry. Anal. Chem. 2000, 72, 1236–1241. 10.1021/ac990976y. [DOI] [PubMed] [Google Scholar]
  38. DiBattista A.; McIntosh N.; Lamoureux M.; Al-Dirbashi O. Y.; Chakraborty P.; Britz-McKibbin P. Temporal Signal Pattern Recognition in Mass Spectrometry: A Method for Rapid Identification and Accurate Quantification of Biomarkers for Inborn Errors of Metabolism with Quality Assurance. Anal. Chem. 2017, 89, 8112–8121. 10.1021/acs.analchem.7b01727. [DOI] [PubMed] [Google Scholar]
  39. Cieslarova Z.; Lopes F. S.; do Lago C. L.; França M. C.; Colnaghi Simionato A. V. Capillary Electrophoresis Tandem Mass Spectrometry Determination of Glutamic Acid and Homocysteine’s Metabolites: Potential Biomarkers of Amyotrophic Lateral Sclerosis. Talanta 2017, 170, 63–68. 10.1016/j.talanta.2017.03.103. [DOI] [PubMed] [Google Scholar]
  40. Maxwell E. J.; Zhong X.; Zhang H.; van Zeijl N.; Chen D. D. Y. Decoupling CE and ESI for a More Robust Interface with Ms. Electrophoresis 2010, 31, 1130–1137. 10.1002/elps.200900517. [DOI] [PubMed] [Google Scholar]
  41. Schiavone N. M.; Sarver S. A.; Sun L.; Wojcik R.; Dovichi N. J. High Speed Capillary Zone Electrophoresis-Mass Spectrometry Via an Electrokinetically Pumped Sheath Flow Interface for Rapid Analysis of Amino Acids and a Protein Digest. J. Chromatogr. B 2015, 991, 53–58. 10.1016/j.jchromb.2015.04.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Cunliffe J. M.; Baryla N. E.; Lucy C. A. Phospholipid Bilayer Coatings for the Separation of Proteins in Capillary Electrophoresis. Anal. Chem. 2002, 74, 776–783. 10.1021/ac015627u. [DOI] [PubMed] [Google Scholar]
  43. White C. M.; Luo R.; Archer-Hartmann S. A.; Holland L. A. Electrophoretic Screening of Ligands under Suppressed EOF with an Inert Phospholipid Coating. Electrophoresis 2007, 28, 3049–55. 10.1002/elps.200600816. [DOI] [PubMed] [Google Scholar]
  44. Wells S. S.; De La Toba E.; Harrison C. R. Metal Cation Control of Electroosmotic Flow Magnitude in Phospholipid-Coated Capillaries. Electrophoresis 2016, 37, 1303–9. 10.1002/elps.201600012. [DOI] [PubMed] [Google Scholar]
  45. Draper W. M.; Xu D.; Perera S. K. Electrolyte-Induced Ionization Suppression and Microcystin Toxins: Ammonium Formate Suppresses Sodium Replacement Ions and Enhances Protiated and Ammoniated Ions for Improved Specificity in Quantitative LC-MS-MS. Anal. Chem. 2009, 81, 4153–4160. 10.1021/ac802735t. [DOI] [PubMed] [Google Scholar]
  46. Hua Y.; Jenke D. Increasing the Sensitivity of an LC-MS Method for Screening Material Extracts for Organic Extractables Via Mobile Phase Optimization. J. Chromatogr. Sci. 2012, 50, 213–227. 10.1093/chromsci/bmr049. [DOI] [PubMed] [Google Scholar]
  47. Zhou S.; Cook K. D. Protonation in Electrospray Mass Spectrometry: Wrong-Way-Round or Right-Way-Round?. J. Am. Soc. Mass Spectrom. 2000, 11, 961–966. 10.1016/S1044-0305(00)00174-4. [DOI] [PubMed] [Google Scholar]
  48. Tseng M.-C.; Chen Y.-R.; Her G.-R. A Beveled Tip Sheath Liquid Interface for Capillary Electrophoresis-Electrospray Ionization-Mass Spectrometry. Electrophoresis 2004, 25, 2084–2089. 10.1002/elps.200305907. [DOI] [PubMed] [Google Scholar]
  49. Bonvin G.; Schappler J.; Rudaz S. Non-Aqueous Capillary Electrophoresis for the Analysis of Acidic Compounds Using Negative Electrospray Ionization Mass Spectrometry. J. Chromatogr. A 2014, 1323, 163–173. 10.1016/j.chroma.2013.11.011. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ac2c02074_si_001.pdf (588.3KB, pdf)
ac2c02074_si_002.avi (2.5MB, avi)

Articles from Analytical Chemistry are provided here courtesy of American Chemical Society

RESOURCES