A New Approach to the Analysis of Anionic Metabolites
by CESI-MS with Negative Ion Electrospray Ionization
Stephen Lock1 and Rawi Ramautar2
1 SCIEX, UK
2 Division of Analytical Biosciences, Leiden Academic Center for Drug Research,Leiden University, The Netherlands

Introduction
Despite recent developments in hyphenated techniques andcolumn technology, the analysis of relatively small and highlypolar analytes (e.g., small organic acids, phosphorylated sugars
and underivatized amino acids) remains challenging. Thesesmall charged metabolites are difficult to retain on standard
reversed-phase liquid chromatography (RPLC) columns andusually require HILIC/Hypercarb columns or ion chromatography.
Ion chromatography runtimes are usually long, and Hypercarb
columns usually need extensive conditioning before samples
are run and to maintain peak shape. HILIC chromatography
often requires that extracts contain organic solvents such asacetonitrile, which may not be the optimal solvent for highlypolar compounds. Moreover, different mobile phases or LC
columns are needed for the analysis of cationic and anionicmetabolites, respectively.
Capillary electrospray ionization (CESI) is the integration ofcapillary electrophoresis (CE) and electrospray ionization (ESI)
into a single process in a single device (Figure 1). CESI-MS
operates at low nL/min flow rates and offers several advantages.
These include increased ionization efficiency and a reduction
in ion suppression. CESI-MS separates analytes by theircharge and size and therefore is a complementary separation
mechanism to more traditional techniques, such as RPLC. The
purpose of this study was to investigate the use of CESI-MS toanalyze highly polar and charged metabolites at physiologicallevels without the need for derivatization. This work evaluates
how this technique can be used to separate isobaric andstructurally similar metabolites, including phosphorylated sugars,
and shows the utility of CESI-MS in negative ion mode for the
analysis of metabolites representing various chemical families,
such as sugar phosphates and organic acids.

Materials and methods

Figure 1. The OptiMS cartridge provides an ultra-low flow ESI interface.

Sample preparation: Standards were prepared by dissolving
the pure solids into water and serial diluting into water. The plant
sample was provided by the Leibniz-Institut für Pflanzengenetik
und Kulturpflanzenforschung (IPK) in Germany and resulted froman extraction of wheat using 50% aqueous methanol.
CESI-MS method: Sample was injected by pressure (5 psi, 5 s)
in a bare fused-silica OptiMS cartridge (30 μm ID x 91 cm), which
was thermostatted using recirculating liquid coolant regulated at
25 °C. For this analysis, the SCIEX TripleTOF® 5600+ LC-MS/MS System was fitted with the NanoSpray® III Ion Source. Gas
1, gas 2 and temperature were not applied (set to 0, 0 and 50,respectively) because ionization at these very low flow rates
occurs by simply applying the ion-spray voltage set at -2000
V. The curtain was set to 5 psi. The CE separation used theconditions shown in Table 1 with a background electrolyte of
10% acetic acid. Data was acquired by a looped experiment,
where a single TOF-MS scan was acquired over the mass range
600–2000 amu together with 50 product ion TOF scans forpotential metabolites. Where standards were available, the CE
voltage was optimized for the product ion scan for the individual
metabolite. The total cycle time for the analysis was less than
1.5 s and was optimized to give greater than 8 points across ananalyte peak.

Chemicals: All chemicals were reagent grade and werepurchased from Sigma Aldrich.
p1

ActionTime(min)PressureDirection
(psi)

Rinse

375

Rinse

1

Rinse

In this experiment, increasing the pressure during the separation

Voltage(kV)

Solution

Forward0

Methanol50

Forward0Water2100

Forward010% acetic acidfor both compounds. Therefore, separation is essential for theRinse

275

Reverse010% acetic acidselective identification of these analytes by MS. In this method,

Injection

5s5

Forward0Sample vial

Injection

10s

0.5

Forward010% acetic acid

Separation30

2Forward30

10% acetic acidthe bare fused-silica OptiMS cartridge, but due to the use of aVoltage

35

Forward110% acetic acid

low-pH separation buffer (10% acetic acid), only a low pressure

Table 1. CESI separation method used for the detection ofanionic metabolites.

Results

from 0.5 to 2 psi speeded the analysis without a significant
effect on the resolution. The 2 disaccharides are isobaric withvery similar fragmentation patterns and their major fragmention, resulting from the loss of the phosphate group, is the samethe polarity used for separation is reversed and was needed
to push the metabolites to the MS detector. Some pressure isrequired to help counteract the residual electroosmotic flow on

was required.
When glucose enters an organism, it is either broken down
to release energy or stored as fat via the intermediate,
dihydroxyacetone phosphate. In this process of cellular

In CE, compounds are separated according to differences inglycolysis, phosphorylated forms of monosaccharides are very

their intrinsic electrophoretic mobilities (i.e., on their charge-to-important intermediaries in the pathway. One of these major

size ratios). A way to speed up the separation in CE is to applyintermediate products in this cycle is glucose-6-phosphate.

a pressure to the electrophoretic analysis. In this context, it is

Isobaric to fructose-1- and glucose-1-phosphate, the separation

important to assess whether the use of such a pressure doesof this intermediate from these other structural isomers is often

not compromise the high separation efficiency of CE. Therefore,difficult to achieve. Using the same method, separation of theseone of the first experiments used in the development of this

3 isobaric metabolites is possible, as illustrated in Figure 3. As

method was the evaluation of pressure on the migration times

with the phosphorylated disaccharides, the product ion spectraand resolution. For this test, 2 closely related and isobaric

for each of these compounds are very similar and therefore

phosphorylated disaccharides were used. Figure 2 shows theseparation is critical for a confident identification.

effect of pressure on the separation of sucrose-6-phosphate(S-6-P) and trehalose-6-phosphate (T-6-P).

Figure 3. CESI-MS Analysis of Phosphorylated Monosaccharides.

Figure 2. The effect of pressure on the separation of sucrose-6phosphate (S-6-P) and trehalose-6-phosphate (T-6-P).

p2

Other isobaric compounds included in this screen were citrate
and isocitrate, which form part of the citric acid cycle. Figure 6shows the separation of these structural isomers. In this example,
citrate and isocitrate differ by the position of the hydroxygroup, which affects their pKa and therefore their charge andmigration times.

Figure 4. Comparison of a standard with a wheat extract for theanalysis of phosphorylated monosaccharides.

Figure 4 shows how this method was applied to a wheat extract.
What can clearly be shown is the separation and detection of
glucose- and fructose-1-phosphate in the absence of glucose6-phosphate, even though the original sample that had beenpreviously prepared for HILIC chromatography had to be diluted
into water 8-fold. What can also be observed is the reproducibilityof the migration times of the standard when compared to theextracted sample.

Figure 6. Separation of isocitrate and citrate by CESI-MS.

Also included in this study were other intermediates of the citricIn order to assess migration time reproducibility, a mixed

acid cycle, such as succinate, malate, fumarate, oxaloacetatestandard containing over 15 compounds—including, among otherand pyruvate, and the analysis of 3 of these compounds is shown

compounds, phosphorylated mono and disaccharides, isocitrate,

in Figure 7.

citrate, succinate, malate, pyruvate and cyclic adenosine
monophosphate (cAMP)—was injected multiple times. In Figure
5, 100 μMolar standard of glucose-1-phosphate was injected
3 times, and it clearly shows that the migration times observed
are reproducible. This was repeated to give intraday RSD values≤1.1% for 10 repeated injections and an interday migration time
RSD ≤1.3% for 5 repeated injections using a series on anionicmetabolites with different migration times.2

Figure 7. Detection of fumarate, malate and succinate by CESI-MS.

Some of these citric acid cycle metabolites are very small andMS fragmentation leads to only a few fragments. This gives
limited options for selective detection by MS/MS, and therefore
Figure 5. 100 μMolar glucose-1-phosphate standard injected in triplicate
to study migration time stability taken from MultiQuant™ Software 3.0.

separation of these compounds is important.
To finally check the applicability of CESI-MS for quantificationof these metabolites calibration standards were prepared inwater. Standards were then injected multiple times and examples
are shown in Figures 8 and Figure 9 which indicated that theresponses are linear with R values ≥ 0.99 in all instances.

p3

So far, we have evaluated the sensitivity of this approach forover 20 different anionic metabolites, and their limits of detection
range from 100 nMolar for the phosphorylated monosaccharides
down to 10 nMolar for cAMP,2 an important secondary
messenger derived from adenosine triphosphate, which isinvolved in the energy transfer in cells. These limits of detection
mean that so far, the anionic metabolites tested can be detected
at physiologically relevant levels.

Conclusions
A fast, robust and reliable CESI-MS method for the highly
efficient and selective analysis of more than 20 negatively
charged polar metabolites has been developed. The
electrophoretic separation is assisted by the application ofa small pressure at the inlet and has been shown to provide
reproducible migration times and separation of isobaric species
and structural isomers of phosphorylated sugars, which is notpossible by reversed-phase HPLC techniques.
In the future, work is planned to evaluate electrophoretic preconcentration in order to increase the injection volume andtherefore the sensitivity for anionic metabolites. We also planto expand this analysis to include more metabolites for global
metabolic profiling studies.

References
Figure 8. Calibration curve for Fructose-6-phosphate (200 nMolar 200 μMolar).

1. Busnel, J.-M. et al. High Capacity Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry: Coupling aPorous Sheath less Interface with Transient-Isotachophoresis.
Anal. Chem. 2010, 82, 9476–9483.
2. Ramautar, R. Probing a new volume regime in metabolomics
with sheathless capillary electrophoresis-mass spectrometry.
Conference proceedings, HPLC 2015, Geneva, Switzerland,
June 21–25, 2015.

Acknowledgements
We would like to acknowledge Dr. N. Heinzel andDr. H. Rolletschek, IPK, for providing the plant extracts
Figure 8. Calibration curve for citrate (260 nMolar - 260 μMolar).

and some of the initial standards used in this project.

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