A new approach to the analysis of anionic metabolites byCESI-MS with negative ion electrospray ionization
Dr. Stephen Lock1 and Dr. Rawi Ramautar2
1SCIEX, United Kingdom and 2Division of Analytical Biosciences, Leiden Academic Center for Drug Research,
Leiden University, The Netherlands
Introduction
Despite recent developments in hyphenated techniques and column
technology, the analysis of relatively small and highly polar analytes(e.g., small organic acids, phosphorylated sugars, and underivatizedamino acids) remains challenging. These small charged metabolites are
difficult to retain on standard reverse-phase liquid chromatography(RPLC) columns and usually require HILIC/Hypercarb columns or ion
chromatography. Ion chromatography run times are usually long,Hypercarb columns usually need extensive conditioning before samples
are run and to maintain peak shape and HILIC chromatography often
requires that extracts contain organic solvents such as acetonitrile which
may not be the optimal solvent for highly polar compounds. Moreover,different mobile phases or LC columns are needed for the analysis ofcationic and anionic metabolites, respectively.
Capillary Electrospray Ionization (CESI) is the integration of capillaryelectrophoresis (CE) and electrospray ionization (ESI) into a single
process in a single device (Figure 1). CESI-MS operates at low nL/minflow rates and offers several advantages. These include increased
ionization efficiency and a reduction in ion suppression. CESI-MS
separates analytes by their charge and size and is, therefore, acomplementary separation mechanism to more traditional techniques,
such as RPLC. The purpose of this study was to investigate the use ofCESI-MS to analyze highly polar and charged metabolites at
physiological levels without the need for derivatization. This work
evaluates how this technique can be used to separate isobaric andstructurally similar metabolites, including phosphorylated sugars, andshows 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.

CESI-MS method: Sample was injected by pressure (5 psi, 5 s) onto an
OptiMS CESI cartridge (30 μm ID x 91 cm bare-fused-silica capillary)which was thermostatted using recirculating liquid coolant regulated at25oC.
For this analysis, the SCIEX TripleTOF® 5600+ mass
spectrometer was fitted with the NanoSpray® III 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 ionspray voltage set at 2000 V. The curtain was set very low at 5psi(set automatically in the CESI software). 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 one TOF-MS
scan was acquired over the mass range 600 – 2000 amu together with
50 product ion TOF scans for potential 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 lessthan 1.5 seconds and was optimized to give greater than 8 points acrossan analyte peak.
Action
Rinse
Rinse
Rinse
Rinse
Injection
Injection
Separation
VoltageTime(mins)
312
25s
10s
30
3

Pressure(psi)
75
50
100
7550.525Direction
Forward
Forward
Forward
ReverseForward
Forward
Forward
ForwardVoltage(kV)
000000301

Solution
Methanol
Water
10% Acetic acid
10% Acetic acid
Sample Vial
10% Acetic acid
10% Acetic acid
10% Acetic acid

Table 1:- CESI separation method used for the detection of anionicmetabolites

Results

Figure 1: OptiMS® - Ultra low flow ESI Interface.

In CE, compounds are separated according to differences in theirintrinsic electrophoretic mobilities (i.e., on their charge-to-size ratios). A
way to speed up the separation in CE is to apply a pressure to theelectrophoretic analysis. In this context, it is important to assess whether
the use of such a pressure does not compromise the high separation
efficiency of CE. Therefore, one of the first experiments used in thedevelopment of this method was the evaluation of pressure on themigration times and resolution. For this test two closely related andisobaric phosphorylated disaccharides were used. Figure 2 shows theeffect of pressure on the separation of Sucrose-6-Phosphate (S-6-P) andTrehalose-6-Phosphate (T-6-P).

Materials and Methods
Chemicals: All chemicals were Reagent Grade and were purchasedfrom Sigma Aldrich.
Sample Preparation: Standards were prepared by dissolving the puresolids into water and serial diluting into water. The plant sample wasprovided by Inst. F. Pflanzengenetik & Kulturpflanzenforschun (IPK) in
Germany and resulted from an extraction of wheat using 50% aqueousmethanol.

p1

0.5 psi pressure
Wheat Extract

Standard

F-6-P

G-1-P
S-6-P 10.55 mins

G-6-P

G-1-P

T-6-P 10.68 mins

2 psi pressure

F-6-P

Figure 2: The effect of pressure on the separation of Sucrose-6Phosphate (S-6-P) and Trehalose-6-Phosphate (T-6-P)
In this experiment increasing the pressure during the separation from 0.5
to 2 psi speeded up the analysis without a significant effect on theresolution. The two disaccharides are isobaric with very similarfragmentation patterns and their major fragment ion, resulting from theloss of the phosphate group, is the same for both compounds. Therefore,
separation is essential for the selective identification of these analytes byMS. In this method the polarity used for separation is reversed and wasneeded to push the metabolites to the MS detector. Some pressure isrequired to help counteract the residual electro-osmotic flow on the barefused silica but due to the use of a low- pH separation buffer (10% aceticacid), only a low pressure was required.
When glucose enters an organism it is either broken down to releaseenergy or stored as fat via the intermediate, dihydroxyacetone
phosphate. In this process of cellular Glycolysis phosphorylated forms ofmonosaccharides are very important intermediaries in the pathway. Oneof these major intermediate products in this cycle is Glucose-6phosphate.
Isobaric to Fructose-1- and Glucose-1-phosphate theseparation of this intermediate from these other structural isomers isoften difficult to achieve. Using the same method, separation of thesethree isobaric metabolites is possible as illustrated in Figure 3. As withthe phosphorylated disaccharides the product ion spectra for each ofthese compounds are very similar and therefore separation is critical fora confident identification.

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. Whatcan clearly be shown is the separation and detection of Glucose- andFructose-1-phosphate in the absence of Glucose-6-phosphate eventhough the original sample which had been previously prepared for HILIC
chromatography had to be diluted into water 8-fold. What can also beobserved is the reproducibility of the migration times of the standard
when compared to the extracted sample.
In order to assess migration time reproducibility a mixed standard
containing over 15 compounds including among other compounds
phosphorylated mono and disaccharides, isocitrate, citrate, succinate,malate, pyruvate, Cyclic adenosine monophosphate (cAMP) was injected
multiple times. In Figure 5 a 100 µMolar standard of Glucose-1phosphate 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 ten repeated injections and an intraday migration
time RSD ≤ 1.3% for five repeated injections using a series on anionicmetabolites with different migration times (2).

Most intense MS transition(phosphate fragment ion, 79amu)

Figure 5:- 100 µMolar Glucose 1 phosphate standard injected intriplicate to study migration time stability taken from MultiQuant™ 3.0software.

Gluctose-1-P Glucose-1-P

migrates at

migrates at

9.85 minutes 9.57 minutes

Fructose-1-P
Fructose-6-P

Glucose-6-P

migrates at
migrates at

migrates at

9.76 minutes
9.66 minutes

9.76 minutes

Other isobaric compounds included in this screen were citrate andisocitrate which form part of the citric acid cycle. Figure 6 shows theseparation of these structural isomers. In this example, citrate andisocitrate differ by the position of the hydroxy group which affects theirpKa and therefore charge and migration times.

Figure 3:- CESI-MS Analysis of Phosphorylated Monosaccharides.

p2

2Total Ion chromatogram forcitrate isomers

R = 0.99786

1

1 = Citrate

2 = Isocitrate

Figure 8:- Calibration for F-6-P ( 200 nMolar – 200 µMolar).

R = 0.99111
Figure 6:- Separation of isocitrate and citrate by CESI-MS.
Also included in this study were other intermediates of the citric acidcycle, such as succinate, malate, fumarate, oxaloacetate and pyruvate,
and the analysis of three of these compounds is shown in Figure 7.

Fumarate

MalateSuccinate

Figure 8:- Calibration for citrate (260 nMolar – 260 µ/Molar).

Conclusions

Figure 7:- Detection of fumarate, malate and succinate by CESI-MS.
Some of these citric acid cycle metabolites are very small and MS
fragmentation leads to only a few fragments. This gives limited options
for selective detection by MS/MS and, therefore, separation of thesecompounds is important.
To finally check the applicability of CESI-MS for quantitation of thesemetabolites calibration standards were prepared in water. Standards
were then injected multiple times and examples are shown in Figures 8and 9 which indicated that the responses are linear with R2 values ≥ 0.99
in all instances.
So far, we have evaluated the sensitivity of this approach for over 20
different anionic metabolites and their limits of detection range from 100
nMolar for the phosphorylated monosaccharides down to 10 nMolar forcAMP (2) an important secondary messenger derived from Adenosine
triphosphate which is involved in the energy transfer in cells. Theselimits of detection mean that so far the anionic metabolites tested can bedetected at physiological relevant levels.

A fast, robust, and reliable CESI-MS method for the highly efficient andselective analysis of more than 20 negatively charged polar plant
metabolites has been developed. The electrophoretic separation is
assisted by the application of a small pressure at the inlet and has beenshown to provide reproducible migration times and separation of isobaric
species and geometric isomers of phosphorylated sugars which is notpossible by reverse phase HPLC techniques.
In future, work is planned to evaluate electrophoretic pre-concentration inorder to increase the injection volume and therefore the sensitivity foranionic metabolites. We also plan to expand this analysis to include moremetabolites for global metabolic profiling studies.

References
1. Busnel, J-M., et. al., “High Capacity Capillary ElectrophoresisElectrospray Ionization Mass Spectrometry: Coupling a Porous
Sheath less Interface with Transient-Isotachophoresis”, Anal.
Chem. 82 9476-9483 2010.
2. Rawi Ramautar, “Probing a new volume regime in metabolomics
with sheathless capillary electrophoresis-mass spectrometry”.
Conference proceedings HPLC 2015 (Geneva).

Acknowledgements
We would like to acknowledge Dr. N. Heinzel and Dr. H. Rolletschek,Inst. F. Pflanzengenetik & Kulturpflanzenforschun for provided the plant
extracts and some of the initial standards used in this project.

Document number: RUO-MKT-02-2643-A© 2015 AB Sciex. For Research Use Only. Not for use in diagnostic procedures. AB Sciex doing business as SCIEX.
The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX™ is being used under license

p3