Glutamate & GABA Analysis in Tissue Homogenate –
12 Minute Analysis
- A flexible analytical method with adaptability to microdialysate and tissue homogenate
- No UHPLC or two systems required.
Detection of glutamate (Glu) and γ-aminobutyric acid (GABA) are in high demand, but current methods depend on pre-column derivatization with o-phthaldialdehyde and β-mercaptoethanol (OPA-BME) can take as long as 40 minutes to 1 hour per sample to analyze. There are alternatives that use UHPLC or literally employ two systems connected with an online valve after the pre-column. However, both systems have a high initial cost and require more maintenance than the system we present here.
We have developed a concise method to analyze Glu and GABA in 12 minutes without requiring significant system upgrades. This application is available for use with the Eicom HTEC-510 or 700 Series after configuring with the low-cost ELS-500 Liquid Switching Valve. This switching valve enables the mobile phase to be changed on the low-pressure side of the pump. Peaks that elute long after the target peaks can be washed out of the column quickly with a second mobile phase. Using such a switching system is only possible with Eicom Pulse Quenching Pumps because the elimination of a pulse damper from the system minimizes the volume of fluid between the pump and column.
While this procedure is available for both tissue and microdialysis samples, there are some restrictions on the use of the microdialysis samples. Please see the end of this note for more.
If you are only interested in detecting Glu, Eicom recommends using our online enzyme column method. Please refer to the application note which specifically discusses it.
Analytical Conditions
| HPLC-ECD | Eicom HTEC-500 or 700 Series + ELS-500 + Autosampler AS-700 |
| Separation Column | Eicompak FA-3ODS (3.0 ID x 50 mm) |
| Precolumn | Eicompak CA-ODS |
| Flow Rate | 500 μl/min |
| Column Temperature | 40˚C |
| Applied Potential | +600 mV vs. Ag/AgCl |
| Working Electrode | Glassy Carbon Electrode, WE-GC with Gasket (GS-25P) |
| Column Wash | During 8 to 11 minutes |
| Total Analysis Time | 12 min |
Typical Chromatograms
GABA peak appears at 9 mins under these conditions. There is a very large peak at about 15 min (not shown) if the column wash process is not started. The Glu peak is close to the large front peak. If the Glu level is less than 100 nM, the peak may be hard to see. Thus, the lowest detection limit is about 100 nM for Glu and 10 nM for GABA with a 10 μL injection.
Analyzing Microdialysis Samples
If the sample contains over 1 μM glutamate and 10 nM GABA, as is usually the case in a microdialysis study, this application is sufficient. Work continues to further refine the method for optimal detection with both tissue and microdialysis samples. Please contact us for the latest method.
Fig. 2. Internal Standard (IS) is homocysteine.
Questions?