For Research Use Only. Not for use in diagnostic procedures.

The Use of Capillary Electrophoresis Coupled to Mass Spectrometry (CESI-MS) for Quantitation ofNucleotides and Nucleosides
Jose-Luis Gallegos-Perez1, Bryan Fonslow2, SCIEX, 1Framingham, MA and 2Brea, CAOVERVIEWOBJECTIVE
Develop a simple methodology for separation and quantitation of nucleotides using CESI-MS.

••••

MATERIALS AND METHODSRESULTSRESULTS (continued)

Chemicals:

Establishment of experimental conditions: Background Buffer Electrolyte, pH, voltage and pressure

Quantitative Analysis

All chemicals were Reagent Grade from Sigma-Aldrich. The method was set up for detection of 7 nucleotides.

Several Background Electrolytes (BGE) are recommended in the literature1, ammonium acetate was selected
because it was demonstrated its use in positive ionization mode. Several experimental conditions were tested toachieve the best separation using Adenosine triphosphate (ATP) as model analyte. Figure 2 shows theExtracted Ion Electropherogram (XIE) for ATP at the final experimental conditions.

Linearity and limits of detection (LODs) were evaluated using analyte sample mixtures from 50 nM to 50 mM foreach compound. Calibration curves and integration parameters were adjusted using MultiQuant™ software(SCIEX, Framingham, MA). The analytical method exhibits a quantitative linear response (r > 0.99) and LODs
between 100 nM and 200 nM for all Results:analyzedcompounds.
(seeFigure 5).
Cytidine
series and othertriphosphates

Name
Cytidine 5′-triphosphate

FormulaC9H16N3O14P3[M+H]
483.99Standards are dissolved in water
Background electrolyte is a 12.5 mM ammonium acetate adjusted to pH 9.7
Separation voltage is 30 kV assisted with 5 psi forward pressure
Data acquisition was done using CESI 8000 interfaced to SCIEX TripleTOF® 5600 system. The
quantitative data analysis was performed using MultiQuant™ software (SCIEX, Framingham, MA).

Cytidine 5′-diphosphate

C9H15N3O11P2404.03

Cytidine monophosphate

C9H14N3O8P1324.06

Adenosine triphosphate

C10H16N5O13P3508.00

Guanosine triphosphate

C10H16N5O14P3524.00

Uridine triphosphate

C9H15N2O15P3484.98

Thymidine triphosphate

C10H17N2O14P3483.00

RESULTS•+METHODS

A method, that does not required derivatization, for separation of some nucleotides was developed forCESI with a short time of analysis (< 10 min), and minimum required sample volume.

GTP

30 µm ID x 90 cm total length
Ammonium acetate (12.5 mM, pH = 9.7)
30 kV, assisted with 5 psi of forward pressure

Nucleotides and nucleosides are charged particles that are vital compounds in the biologicalprocesses. These polar compounds are usually difficult to analyze by traditional chromatography
techniques, needing an extra derivatization step.

Figure 2. Extracted Ion Electropherogram (XIE) of ATP.
To evaluate analytical parameters as peak shape and migration times, samples were injected individually (seeFigure 3) , then a standard mixture was also injected to check
Separation performance (see Figure 4). All compounds showed migration times below 10 min, highreproducibility and good peak shapes.

Cyt-tri, 200nM

Capillary Electrophoresis (CE) is a separation method that is ideal for highly charged and polar
molecules, which can be notoriously challenging for liquid or gas chromatography. In addition, CE
offers advantages of nano- and picoliter sample-volume, high separation efficiency, and fast injectionto-injection times.

•CESI is a technology that integrates capillary electrophoresis (CE) and electrospray ionization (ESI)
into a single process in a single device (see Figure 1). With CESI, a stable ESI spray is generated atflow rates in the range of 5-30 nL/min. Operating at these ultra-low flow rates, it helps reduce ionsuppression and increases assay sensitivity for optimal results.
In this study, we present a workflow using CESI-MS for the quantitative analysis of selectednucleotides using minimal sample volume, demonstrating baseline separation, reproducibility anddetection at nanoMolar levels.

UTP, 200nM

ATP, 50 nM

TTP, 200nM

Cytidine 5′-triphosphate (Cyt-tri)

Figure 5. Top: Calibration curves for the nucleotides sample mixtures. Bottom: Graphs show the lowest
concentration where the analytes can be distinguished from noise.

Cytidine 5′-diphosphate (Cyt-di)

CONCLUSIONS

Adenosine triphosphate (ATP)Figure 1. CESI Sprayer Interface•A method for separation of some nucleotides was developed for CESI with a short analysis time (< 10min) and minimum required sample volume. In addition it does not require derivatization for theanalytes.

•Separation of challenging charged molecules: cytidine phosphates, ATP, GTP, UTP and, TTP has
been demonstrated using the CESI-MS system.

•CESI could be applied for quantitative analysis with low detection limits (~ 200 nM) for the analyzed
compounds.

•Application of this CESI-method is promising for metabolomics in biological areas (e.g. neurosciences,pharmaceutical industry) where almost all the samples are obtained at low volume and concentration.

Guanosine triphosphate (GTP)

Uridine triphosphate (UTP)MS Conditions:
CESI 8000 (sold through SCIEX Separations) was coupled to a TripleTOF® 5600+ mass spectrometer (SCIEX)
operating in positive ion mode with Analyst 1.7™ software

Thymidine triphosphate (TTP)

Figure 3. XIE of individual sample injections (~ 50 mM)
Mass Spectrometer:
ToF acquisition mode:
Ionization mode:

Cyt-mono, 200nM

Cyt-di, 200nM

GTP, 200nM

Cytidine monophosphate (Cyt-mono)•Cyt-mono

CE Conditions:

INTRODUCTION•

Cyt-diCyt-tri,mM
50.12
12.53
3.13
0.78
0.20
0.05
0.01

UTPTTPCESI-MS Silica Surface Cartridge:
Background Electrolyte (BGE):
Separation Voltage:•ATP

SCIEX TripleTOF® 5600+
200-600 m/z values
Positive, [M+H]+REFERENCES
1Liu, Charles C., et al Electrophoresis 26.7‐8 (2005): 1424-1431

ATP Image from: http://en.wikipedia.org/wiki/Adenosine_triphosphate
Data Analysis

TRADEMARKS/LICENSING

Analysis of the mass spectra were carried out using Peak View® software (SCIEX, Framingham, MA). The
quantitative data analysis was performed using MultiQuant™ software (SCIEX, Framingham, MA).

For Research Use Only. Not for use in diagnostic procedures.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners.
Figure 4. XIE of analytes standard mixture (~ 50 mM/each}© 2015 AB Sciex.