- No precolumn derivatization required
- Flexible analytical method with adaptability to microdialysate, blood and tissue homogenate
- Microdialysis sample run time of 10 minutes
- 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. This is achieved by HPLC-ECD (DC mode) without sample derivatization.
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 methodology is sample medium. Figure 1 illustrates the detection from brain tissue homogenate. One chromatogram can be completed in 10 min. The enzyme column life is more than 6 months for microdialysis samples.
|Column||Eicom GU-GEL (4.6 ID x 150 mm)|
|Reactor||E-ENZ (3.0 ID x 40 mm)|
|M.P. flow rate||400 μl/min|
|Applied potential||+500 mV vs. Ag/AgCl|
|Working electrode||Eicom WE-PT|
|Time constant filter||3.0 sec|
- Microdialysis Samples
- Blood Plasma
- Tissue Homogenate
- Culture Media etc.
The following is a representative example of an assay we have performed to demonstrate how our new methodology works. By using two online auto-injectors (Eicom EAS-20), the microdialysate was automatically injected followed by a standard glutamate solution. This standard was used as an internal standard (IS) in order 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
A step-by-step protocol for this application is available for Eicom