Drug discovery and development

Quantitation of Nucleotides and Nucleosides without
Derivatization using Capillary Electrophoresis Coupledto Mass Spectrometry (CESI-MS)
CESI-MS method for analysis of challenging small molecule metabolites and similar drug products
Jose-Luis Gallegos-PerezSCIEX Separations, Framingham, MAIntroduction
CESI-MS is the integration of capillary electrophoresis (CE) withelectrospray ionization (ESI) into a single dynamic process within
the same device (Figure 1). Operating at ultra-low flow rates, it
helps reduce ion suppression and increases assay sensitivity for
optimal results. Nucleotides are charged molecules, precursors
and breakdown products of DNA and RNA that also form thebasic structure of cofactors. They act as energy carriers andmediators of important cellular processes.1 Nucleosides areneutral and are also vital cellular components that inside the cellare metabolized by nucleo-kinases into the active nucleotide
mono-, di-, triphosphates.2 Nucleoside analogues are used astherapeutics for the treatment of viral infections, such as thehuman immunodeficiency virus (HIV).1 These polar compounds
are usually difficult to analyze by traditional chromatography
techniques, needing an extra derivatization step. This technicalnote describes an alternative workflow using CESI-MS for thequantitative analysis of selected nucleotides and nucleosides
without derivatization with reproducibility and detection atnM levels. Application of this CESI-MS method is promising forthe analysis of these types of molecules in the pharmaceuticaland biological (e.g., metabolomics, neurosciences) fields
among others.

Figure 1. CESI-MS: CE integrated with the QTRAP® 6500
LC-MS/MS System

Experimental procedureReagents:• Ammonium acetate (AA), formic acid (FA) and acetic acid
were purchased from Sigma-Aldrich and used without any
further purification. Ultrapure water (Type 1) was obtained
from a Milli-Q Direct Ultrapure Water System. Cytidine
5′-triphosphate (CTP), cytidine 5′- diphosphate (CDP), cytidine
5’-mono- phosphate (CMP), adenosine triphosphate (ATP),
guanosine triphosphate (GTP), uridine triphosphate (UTP),
thymidine triphosphate (TTP), uridine (Ur) and thymidine (Th)were purchased from Sigma-Aldrich.

Advantages of the CESI-MS method

Sample preparation:• No derivatization step

• Stock solutions of standard compounds were preparedin DDI water. Standard solutions containing a mixture of
the analytes—in the range of 0.25, 0.50, 1, 5, 10, 25 and50 nM—were also prepared in DDI water.

• Short analysis time
• Sensitive, quantitative methodp1

Drug discovery and development
Instrumentation:

Instrument conditions:• Capillary electrophoresis (CE): Separations were performed
using a CESI 8000 Plus System from SCIEX. The instrument
was controlled using 32 Karat™ Software v10.

• MS: The QTRAP® 6500 System was run with electrosprayionization in positive ion mode and acquisition was from100 to 600 m/z values. Conditions for multiple reaction
monitoring (MRM) were evaluated individually for eachcompound to select precursor and fragment ions. Table 1
provides the transitions that were used for the determination.

• Capillary: A bare fused-silica capillary was used for theseparation; 30 µm i.d. x 91 cm total length.
• Mass spectrometry (MS): A QTRAP® 6500 System (SCIEX,Framingham, MA) coupled to the CESI through the CESI
sprayer interface (see Figure 2) operating in positive ion modewith Analyst® Software 1.7.

Figure 2. CESI sprayer Interface.

Instrument conditions:• CESI: Several background electrolytes (BGE) are recommended in the literature.1 Ammonium acetate was selected
because it was demonstrated in positive ionization mode.
Several experimental conditions were tested to achieve thebest separation using adenosine triphosphate (ATP) as themodel analyte. Formic acid was used as the stacking agent.
The final experimental conditions were:
Background electrolyte: 12.5 mM ammonium acetate pH 9.7.
Stacking solution: 1% Formic acid
Separation voltage: 30 kV with 2 psi of assisted pressurem/zm/zMonoisotopic
precursor fragment
mass (Da)ionionLowest detectedconcentration(nM)

NameFormulaCytidine5’-triphosphate(CTP)

C9H16N3O14P3

482.9840

483.9

112.1

0.3

Cytidine5’-diphosphate(CDP)

C9H16N3O11P2

403.0182

404.0

112.1

0.3

Cytidine5’-monophosphate(CMP)

C9H14N3O8P1

323.0513

324.0

112.1

1.0

Adenosine
C10H16N5O13P3
triphosphate (ATP)506.9952

507.9

136.1

0.3

Guanosine
C10H16N5O14P3
triphosphate (GTP)

522.9901

523.9

152.10.5

Uridinetriphosphate (UTP)

C9H15N2O15P3

483.9680

484.9

97.0

0.5

Thymidine
C10H17N2O14P3
triphosphate (TTP)

481.9887

482.9

81.0

0.5

Uridine (Uri)

C9H16N2O6

244.0690

245.1

113.0

5.0

Thymidine (Thy)

C10H14N2O5

242.0897

243.1

127.1

5.0

Table 1. MRM transitions for the analyzed compounds and the lowest
detected concentrations (nM) for each one.

Data Analysis• Analyses of the mass spectra were carried out using
PeakView® Software (SCIEX). The quantitative data analysis
was performed using MultiQuant™ Software (SCIEX).
Method parameters: See Figure 3 and Figure 4.

Sample storage temperature: 10 °C
Capillary temperature: 20 °C

Important:• A separation current above 5 μA might cause permanent
damage to the separation capillary.
• Generally, please do not apply >2,000 V to generate theelectrospray as it may result in capillary damage.

Figure 3. Scheme of the inlet and outlet buffer vial tray setup for theCESI separation.

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Drug discovery and development

Figure 4. Separation method parameters. In step 10 and 12, use 2 psi of forward pressure. In steps 3, 5, 6 and 13 a low separation voltage is appliedwith 100 psi in both directions to rinse in parallel the capillary in the separation and conductive lines.

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Drug discovery and development
Results and discussion• Analytical parameters such as peak shape and migration times
were evaluated by injecting samples individually. A standard
mixture was also injected to check separation performance(Figure 5).
• All compounds showed migration times below 15 min with highreproducibility and sharp peak shapes (Figure 5).
• Linearity and other parameters were evaluated using analyte
sample mixtures from 0.25 to 50 nM for each compound.
• Figure 6 shows extracted electropherograms for the lowest
concentration detected for the individual compounds, ranging
from 0.25 to 1 nM for nucleotides and 5 nM for nucleosides(see Table 1).
• Calibration curves were obtained using MultiQuant™ Software(SCIEX, Framingham, MA). The analytical method exhibited aquantitative linear response with r > 0.9 (Figure 7).

Figure 6. Extracted ion electropherograms (XIEs) for each of theanalyzed compounds at the lowest concentration detected.

Figure 5. Extracted ion electropherograms (XIEs) of the analyzedcompounds. All injected in the same experiment at 10 nM (see Table 1).

Figure 7. Calibration curves for the analyzed compounds. All analyte
concentrations are ranging from 0 to 50 nM.

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Drug discovery and development
Conclusions

References• A method for separation of some nucleotides and nucleosides
was developed for CESI-MS with a short analysis time andminimum required sample volume. In addition, this separation
does not require derivatization of the analytes.

1. Willems, An V., Dieter L. Deforce, Carlos H. Van Peteghem,and Jan F. Van Bocxlaer. “Analysis of nucleic acid constituentsby on-line capillary electrophoresis-mass spectrometry.”
Electrophoresis 26, no. 7-8 (2005): 1221-1253.

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

2. Liu, Charles C., et al. “Capillary electrophoresis-electrospraymass spectrometry of nucleosides and nucleotides: Application
to phosphorylation studies of anti-human immunodeficiency
virus nucleosides in a human hepatoma cell line.”
Electrophoresis 26.7-8 (2005): 1424-1431.

• CESI-MS could be applied for quantitative analysis with low
detected concentrations (< 5 nM) for the analyzed compounds.
• Application of this CESI method is promising formetabolomics, biotransformation pharmacology and analyticalcharacterization studies where nucleotide/nucleoside-typemolecules are quantitatively analyzed, particularly whensamples are obtained with low volumes and/or concentrations.

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