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

Abstract

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.

Figure 1

Components of the capillary electrophoresis VSSI sheath flow design.

Figure 2

(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.

Figure 3

(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).

Figure 4

(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.

Figure 5

(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.

Figure 6

(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.