Chromatography Forum

Hi,

I want to quantify Dopamine, DOPAC and HVA in mice brain striatum homogenates.

Previously I ran microdialysis samples (which is a lot cleaner than brain tissue homogenate) and I used this mobile phase (from esa Technical note 70-5094p): 90 mM sodium dihydrogen phosphate, 50 mM Citric acid monohydrate, 1.7 mM OSA, 50 uM EDTA, 10% Acetonitrile, pH 3.0

DA, DOPAC, HVA peaks look fine, and good retention time too.

Now, esa recommends slightly different recipe of mobile phase for running brain tissue: 75 mM sodium dihydrogen phosphate, no citric acid, 1.7 mM OSA, 100 ul/L Triethylamine (TEA), 25 uM EDTA, 10% Acetonitrile, pH 3.0

The TEA definitely helps eliminating shoulder on DA peak (that sometimes appear in some samples ran). However, suddenly there is a huge peak right before the DOPAC peak that overshadows the DOPAC peak and making quantitation difficult and possibly less accurate.

My questions are:
1. Is TEA really mandatory to be included in brain tissue analysis? (peaks of interest is DOPAC, dopamine, and HVA)
2. What is the benefit of adding TEA in mobile phase?
3. Do you think it might be better to switch to the recipe without TEA?

Thank you in advance for your advice and time!

1. Is TEA really mandatory to be included in brain tissue analysis? (peaks of interest is DOPAC, dopamine, and HVA)

Check with ESA and ask them *why* they recommend the TEA for the tissue analysis and not the dialysate. Electrochemical detectors have a reputation for being fussy beasts and there may be some detector issues involved.

2. What is the benefit of adding TEA in mobile phase?

Aside from possible detector issues, TEA is often used to suppress tailing of basic compounds when using older-technology "Type A" columns. It is uses much less frequently with newer "Type B" columns. Evidently with your samples it does retard the onset of your peak shoulder issues.

3. Do you think it might be better to switch to the recipe without TEA?

That one is your call. You're balancing a peak shoulder and a big interfering peak. You might want to try a couple of runs with varying (lower) TEA concentrations and see if you can find a happy medium.