Electric Field-Modulated Electrospray Ionization Mass Spectrometry for Quantity Calibration and Mass Tracking

Analyses conducted by electrospray ionization (ESI) mass spectrometry (MS) typically entail performing a number of preparatory steps, which include quantity calibration and mass calibration. Quantity calibration can be affected by signal noise, while mass calibration can be affected by instrumental drift if analyses are performed over an extended period of time. Here, we present two methods for achieving these calibrations using modulation of electrospray plume by alternating electric fields and demodulating the resulting MS ion currents. For this purpose, we use an ESI source fitted with three ring electrodes between the electrospray emitter and the mass spectrometer’s inlet. One of these electrodes is supplied with a sine electric signal. Optionally, a nanoESI emitter is also placed between the ring electrodes and the mass spectrometer’s orifice to supply calibrant ions. The ion currents, recorded with this setup, present wave-like features. In the first variant, using a triple quadrupole mass analyzer, the ion currents are subjected to data treatment by fast Fourier transform (FFT), and the resulting FFT magnitudes are correlated with analyte concentrations to produce a calibration plot. In the second variant, using a quadrupole time-of-flight mass analyzer, the mass spectra recorded at the analyte ion current maxima are mass-checked using the m/z value of the internal standard (injected via nanoESI emitter), which appears predominantly in the time intervals corresponding to the analyte ion current minima. The setup has been characterized using simulation software and optimized. Overall, the method enables the preparation of quantity calibration plots and monitoring (minor) m/z drifts during prolonged analyses.


ADDITIONAL TABLE
. Calibration equations for various tested analytes (n = 3).The analytes were dissolved in 25% (v/v) methanol in water.Concentration unit: μM.The RE3 potential in the control experiment: 0 V.

IFigure S1 .
Figure S1.Vertical position of the ESI capillary from the center of MS inlet (A) ~ 0 mm; (B) higher ~ 1 mm.The sample was 5 μM of analyte dissolved in 25% (v/v) methanol in water.The flow rate was 40 μL min -1 .The QQQ-MS was operated in SIM mode (lysine m/z 147).

Figure S2 .
Figure S2.Schematic of the electronic circuit used to control electrospray plume with AC field.

Figure S4 .
Figure S4.Wave-like features are generated by modulating the electric field with 0.05 Hz frequency.Each color refers to a different concentration of acetaminophen.Sequence: t = 0.00-0.50min, turn on the ESI; t = 0.50-1.00min, turn on the RE3 voltage; t = 1.00-2.50min, turn on the RE2 AC voltage.

Figure S5 .Figure S6 .
Figure S5.Comparison of the results obtained with the RE3 with and without DC voltage.(A) The RE3 with DC 10 V; (B) the RE3 without DC 10 V.The sample was 5 μM of analyte dissolved in 25% (v/v) methanol in water.The flow rate was 40 μL min -1 .The QQQ-MS was operated in SIM mode (lysine m/z 147).The arrows in (B) show the instabilities.The standard deviations of the signals in the 0-1 min region are (A) 1104 a.u. and (B) 2296 a.u.

Figure S7 .
Figure S7.Bar plots representing (A) signal intensity and (B) FFT magnitude for the varied distance between the RE3 and the sampling cone of the QQQ-MS; (C) signal intensity and (D) FFT magnitude for the varied drying gas flow rate of the QQQ-MS; (E) signal intensity and (F) FFT magnitude for the varied ESI voltage of the QQQ-MS; (G) signal intensity and (H) FFT magnitude for the varied DL temperature of the QQQ-MS.The sample was 5 μM of analyte dissolved in 25% (v/v) methanol in water.The flow rate was 40 μL min -1 .The QQQ-MS was operated in SIM mode (acetaminophen m/z 152, alanine m/z 90, lysine m/z 147).

Figure S9 .
Figure S9.Optimization of the distance from the RE3 to the Q-TOF-MS inlet and the pressure applied to the vial with nanoESI electrolyte solution.The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM L-glutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The flow rate of ESI was 10 μL min -1 .The drying gas flow rate was 3.0 L min -1 .The voltage applied to ESI and nanoESI was 4.0 kV in both cases.The DL temperature was 200 °C.The Q-TOF-MS was operated in MS scan mode.

Figure S11 .
Figure S11.Optimization of the ESI flow rate: (A) 10 μL min -1 ; (B) 20 μL min -1 ; (C) 30 μL min -1 ; (D) 40 μL min 1 ; (E) 50 μL min -1 .The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM Lglutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The distance from the RE3 to the Q-TOF-MS inlet was ~ 10 mm.The pressure applied to the vial with nanoESI electrolyte solution was ~ 47 kPa.The drying gas flow rate was 3.0 L min -1 .The voltage applied to ESI and nanoESI both were 4.0 kV.The DL temperature was 200 °C.The Q-TOF-MS was operated in MS scan mode.

Figure S12 .
Figure S12.Optimization of the drying gas flow rate: (A) 3 L min -1 ; (B) 6 L min -1 ; (C) 9 L min -1 ; (D) 12 L min -1 ; (E) 15 L min -1 .The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM L-glutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The distance from the RE3 to the Q-TOF-MS inlet was ~ 10 mm.The pressure applied to the vial with nanoESI electrolyte solution was ~ 47 kPa.The flow rate of ESI was 10 μL min -1 .The voltage applied to ESI and nanoESI both were 4.0 kV.The DL temperature was 200 °C.The Q-TOF-MS was operated in MS scan mode.

Figure S13 .
Figure S13.Optimization of the nanoESI voltage: (A) 3.2 kV; (B) 3.4 kV; (C) 3.6 kV; (D) 3.8 kV; (E) 4.0 kV.The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM L-glutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The distance from the RE3 to the Q-TOF-MS inlet was ~ 10 mm.The pressure applied to the vial with nanoESI electrolyte solution was ~ 47 kPa.The flow rate of ESI was 10 μL min -1 .The drying gas flow rate was 3.0 L min -1 .The voltage applied to ESI was 4.0 kV.The DL temperature was 200 °C.The Q-TOF-MS was operated in MS scan mode.

Figure S14 .
Figure S14.Optimization of the ESI voltage: (A) 3.2 kV; (B) 3.4 kV; (C) 3.6 kV; (D) 3.8 kV; (E) 4.0 kV.The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM L-glutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The distance from the RE3 to the Q-TOF-MS inlet was ~ 10 mm.The pressure applied to the vial with nanoESI electrolyte solution was ~ 47 kPa.The flow rate of ESI was 10 μL min -1 .The drying gas flow rate was 3.0 L min -1 .The voltage applied to nanoESI was 4.0 kV.The DL temperature was 200 °C.The Q-TOF-MS was operated in MS scan mode.

Figure S15 .
Figure S15.Optimization of the DL temperature: (A) 150 °C; (B) 175 °C; (C) 200 °C; (D) 225 °C; (E) 250 °C.The black line refers to the ESI plume modulated with AC (50 μM adipic acid, m/z 147.0652) while the red line refers to the nanoESI plume of a continuously sampled calibrant (5 μM L-glutamine, m/z 147.0764).Both the sample and calibrant were dissolved in 25% (v/v) methanol in water.The distance from the RE3 to the Q-TOF-MS inlet was ~ 10 mm.The pressure applied to the vial with nanoESI electrolyte solution was ~ 47 kPa.The flow rate of ESI was 10 μL min -1 .The drying gas flow rate was 3.0 L min -1 .The voltage applied to ESI and nanoESI both were 4.0 kV.The Q-TOF-MS was operated in MS scan mode.