Chromatography Forum

Recently I am developing one LC/MS/MS (ESI) method to measure reduced glutathione (GSH) and oxidized glutathione (GSSG). Got some difficulty:

GSH is not stable, thus we prepare the stock solution in some acidic solution, like 0.1% formic acid, 0.1M HCl, etc. So does GSSG to be consistent. Found that GSSG standard solution always has GSH signal. That just puzzled me. (GSH standard solution did not see GSSG signal even without derivatization, though). I don't know where the GSH signal is coming from.

Literatures only mentioned protecting GSH to prevent GSH --> GSSG. Did not see anybody observed such GSSG --> GSH.

Great thanks for any post.

Recently I am developing one LC/MS/MS (ESI) method to measure reduced glutathione (GSH) and oxidized glutathione (GSSG). Got some difficulty:

GSH is not stable, thus we prepare the stock solution in some acidic solution, like 0.1% formic acid, 0.1M HCl, etc. So does GSSG to be consistent. Found that GSSG standard solution always has GSH signal. That just puzzled me. (GSH standard solution did not see GSSG signal even without derivatization, though). I don't know where the GSH signal is coming from.

Literatures only mentioned protecting GSH to prevent GSH --> GSSG. Did not see anybody observed such GSSG --> GSH.

Great thanks for any post.

Have you actually carried out an LC separation yet? If there is genuinely GSH in your GSSG standard you should see two chromatographic peaks.

On the other hand, if you see a GSH signal at the same retention time as GSSG, you could be getting in-source fragmentation of the GSSG bond, resulting in a precursor identical to the ion you measure for GSH, which will of course have the same product ions too. Something like:
[GSSG + 2H]2+ --> [GSH + H]+ + [GS]+

As long as GSSG and GSH are chromatographically resolved this should not be a problem. But if it is, try tweaking the parameter than can help reduce in-source fragmentation. On Agilent systems it's called fragmentor voltage, and on ABI systems it's called declustering potential. If you're lucky, you may find a certain voltage for GSH that is not strong enough to fragment GSSG, but is strong enough to deliver GSH ions to the MS.

Thanks, Camisotro. Yes, we used LC. Our instrument is from AB Sciex. Will check this with your comments and get feedback here later on.