Concise Glutamate Analysis by Using an Online Enzymatic Reaction

  • No precolumn derivatization required
  • A flexible analytical method with adaptability to microdialysate, blood, and tissue homogenate
  • Microdialysis sample run time of 10 minutes
  • The robust detection limit (< 3 fmol). (Microdialysis samples have higher concentrations) > chromatogram

In the central nervous system, glutamate plays a key role as a neurotransmitter and can be a toxic factor for neurons. Here we introduce a concise procedure for the analysis of glutamate levels in biological samples such as tissue homogenate, tissue/cell culture media, blood serum/plasma, and microdialysate. HPLC-ECD (DC mode) without sample derivatization achieves this.

This new technique employs an online immobilized enzyme column (glutamate oxidase) positioned after the separation column. After the glutamate is separated from other biogenic compounds, it is oxidized by the enzyme column to hydrogen peroxide which can then be detected by the platinum working electrode when the electrochemical detector is set to the DC mode. The detection limit for this method is the sample medium. Figure 1 illustrates the detection of brain tissue homogenates. One chromatogram can be completed in 10 min. The enzyme column life is over 6 months for microdialysis samples.

Chromatographic Condition

HPLC-ECDEicom HTEC-500
ColumnEicom GU-GEL (4.6 ID x 150 mm)
ReactorE-ENZ (3.0 ID x 40 mm)
M.P. flow rate400 μl/min
Applied potential+500 mV vs. Ag/AgCl
Working electrodeEicom WE-PT
Time constant filter3.0 sec
System Temperature33°C

Available Applications

  • Microdialysis Samples
  • Blood Plasma
  • Tissue Homogenate
  • Culture Media etc.

The following is a representative example of an assay we have performed to show how our new method works. By using two online auto-injectors (Eicom EAS-20S), the microdialysate was automatically injected, followed by a standard glutamate solution. This standard was used as an internal standard (IS) to account for enzyme activity. However, the enzyme activity was stable enough to neglect the IS. Compared to the glutamate signal peak, the uric acid peak was relatively higher. By adjusting the injection time, the uric acid peak could be incorporated into the front-solvent peak and sequential 10 min. injections are achievable (Fig. 2).

Fig. 1 – The retention time of the glutamate peaks is at 8 min, samples can be injected every 10 min.

Fig. 2 – Sequential Microdialysis Sample Injections

Questions?