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

The application of Capillary Electrospray Ionization with negative ion electrospray ionization for the analysis of plant
metabolites
Stephen Lock1 and Edna Betgovargez2 , 1SCIEX, Warrington, United Kingdom and 2SCIEX, Brea, California, USA

RESULTSINTRODUCTION
Capillary Electrospray Ionization (CESI) is the integration of capillary electrophoresis (CE) and electrosprayionization (ESI) into a single process in a single device (Figure 1). CESI-MS operates at low nL/min flow rates
offering several advantages including increased ionization efficiency and a reduction in ion suppression. In thisstudy we describe the use of CESI-MS in negative ion mode for the analysis of plant metabolites including
phosphorylated sugars and low molecular weight organic acids. These are generally small polar compounds
typically not retained on traditional liquid chromatography columns making LC-separations difficult.
This study investigated both separation reproducibility and sensitivity for numerous plant metabolites difficult toanalyze by other means.

MATERIALS AND METHODSCE Conditions:
A method was set up for detection of over 40 plant metabolites using negative ion CESI-MS. This work usedreverse polarity with a pressure injection (5 sec. at 5 psi) and a background electrolyte of 10% acetic acid.
Electrophoretic separation was performed using a 90 cm bare fused silica capillary and a separation voltage of
30 kV creating a field strength of 333 V/cm. A pressure of 2 psi was applied to counter the reverse electro
osmotic flow from the bare fused silica capillary used for these separations.
Between each injection the capillary was conditioned by rinsing with 0.1 molar sodium hydroxide, water, 0.1
molar hydrochloric acid and finally the background electrolyte.

Initially both normal and reverse polarities were investigated. However, preliminary trials showed that reverse
polarity provided the best separation on the CESI-MS system in negative ion mode. Under these conditions a lower
ion spray voltage could be used which reduced the potential for discharge formation.

To check on the linearity of response for this method standards several compounds from Sigma Aldrich werepurchased and calibration standards were prepared in water. These standards were then injected in triplicate totest linearity of response (no internal standards were used in this test). Figures 5, 6 and 7 show the calibrationlines for 3 out of the 5 phosphorylated sugars. All lines were linear over the range tested (0.5- 50 µg/ml)
corresponds to concentrations in the µMolar range.

Using a bare fused silica OptiMS Cartridge and 10% acetic acid as a background electrolyte, low nMoles/10 µL
concentration of plant metabolites were detected. These metabolites included low molecular weight organic acidsincluding succinate and malate. In addition, separation of the isobaric phosphorylated disaccharides of Sucrose-6Phosphate and Trehalose-6-Phosphate was possible. Figure 2 shows the separation of these two plant metabolites.
The increase of pressure during the separation speeded up the analysis and increased the peak height but did nothave a negative effect on the overall resolution. Sucrose-6-Phosphate migrated first and both sucrose and trehalose
have nearly identical MS fragmentation patterns so separation of these metabolites is needed to clearly distinguishthese compounds.

Migration times are shorter for phosphorylated monosaccharides as these compounds are smaller. Baseline
separation of all three mono-phosphorylated sugars was not totally achieved (Figure 3) but Gluctose-1-phosphate(G-1-P), glucose -6-phosphate (G-1-P), fructose-1-phoshate (F-1-P) were distinguished by CESI. Baseline
resolution of G-1-P (migrating at 9.57 minutes) and G-6-P (migrating at 9.75 minutes) was possible but complicatedby F-1-P which eluted between these isomers at 9.66 minutes. Again fragmentation patterns were very similar butsome diagnostic ions were possible as soon in the bottom panes of Figure 3. Again the separation of these monophosphorylated sugars is not possible by standard reverse phase LC. When this method was then applied to a plant
extract two phosphorylated monosaccharides were identified (G-1-P and F-1-P) at low µMolar concentration in theextract (Figure 4).

MS/MS Conditions:
Beckman Coulter CESI 8000 (sold through SCIEX Separations) was coupled to a TripleTOF® 5600+ mass
spectrometer (SCIEX) operating in negative ion electrospray mode using looped dedicated product ion scan foreach of the plant metabolites (20 msec. accumulation time for each product ion with a mass range from 40 – 50
amu above the parent ion mass) together with a general TOF scan (100 -1000 amu for 100m sec) to screen forunknown targets. The total scan time for each cycle was approximately 1 second. The ionspray voltage used
was set to -1200V and the spray position and MS source were tuned using direct pressure injection at 10 psi.

Ascorbate, pyruvate, malate, fumarate and succinate were detected in addition to citrate using this approach.
This preliminary method has been set to detect over 30 negatively charged polar metabolites. An example ofthe separation of just three of these is shown in Figure 10 and an example of a calibration line for ascorbate
whose migration time 13.38 minutes is shown in Figure 11.

Fumarate

MalateSuccinate

Figure 10. Electropherograms for Fumarate (migration time of 13.4
minutes), malate (migration time of 14 minutes) and succinate (migration
time of 14.5 minutes).

Figure 6. Calibration line for Glucose-1-Phosphate.

Figure 7. Calibration line for Glucose-6-Phosphate.

Figure 11. Calibration for Ascorbate.

In order to test the versatility of this system theanalysis of underivitised amino acids as well asother positively charged plant metabolites wasattempted using the same capillary andbackground electrolyte system. In this test theMS was switched to positive mode and thepolarity for CE separation changed to normalmode. An example of the results is shown in
Figure 12. Using this approach, all underivitisedamino acids tested were detected in just a 20
minute window.

An important feature of this method is it’s ability to separate the isobaric species of citrate and isocitrate within
the same analysis. These compounds only differ by the position of the hydroxy-position and are highly polar
and again very difficult to separate by reverse phase LC analysis. CESI-MS produced a baseline separation ofthese two species (Figure 8).

0.5 psi(A)Figure 12. Electropherograms for several common amino acids acquired2 psi

Due to low flow rate used by the CESI, the curtain gas was lowered to around 4-5 psi and the temperature of
the nano-spray source interface was set at 50 ºC.

G-1-P

F-1-P

G-6-P

(B)

Figure 2. Separation of Sucrose-6-Phoshate and Trehalose-6-

Figure 3. Separation of phosphorylated monosaccharides

Phosphate by CESI and the effect of pressure on the separationby CESI at a pressure of 2 psi.

CONCLUSIONS
CitrateA

Iso-Citrate(C)

Figure 8. Baseline separation of citrate and isocitrate by CESI-MS.

Figure 9. Comparison on 3 repeat injection of a 25 µg/ml
standard (A=Glucose-1-Phosphate, B= Sucrose-6-Phosphate andC=Isocitrate)

G-1-P

To show reproducibility each standard was injected in triplicate. Figure 9 shows the electropherograms for 3compounds and show the high reproducibility of the migration times. Key to this was the conditioning of thecapillary between injections.
Figure 1. Schematic for CESI Sprayer which connects to MS systems via an adapter fitted to a nano spraysource4.

A fast, robust, and reliable method, for the detection over 30 negatively charged polar plant metabolites has
been developed. The method uses a simple pressure injection and has been shown to produce reproducible
migration times and separation of isobaric species and geometric isomers of phosphorylated sugars which is
not possible by reverse phase HPLC separation

BF-1-P

Figureon the same capillary and using the same background electrolyte but using
normal polarity and a different CE method.

Phosphorylated monosaccharides at uMolar
concentration (A) and adetected by CESI in a plant extractcontaining 6% methanol (B).

Figure 5. Calibration line for Sucrose-6-Phosphate.

In addition by simply switching the polarity of the CE and MS systems, CESI-MS can be used to detect
underivitised amino acids and positively charged plant metabolites in the same samples. This approach istherefore an ideal way for screening both positively and negatively charged polar metabolites in an automatedapproach which is very difficult to do using normal phase chromatography.

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For Research Use Only. Not for use in diagnostic procedures.
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© 2015 AB Sciex.