Glutathione Detection [UPLC]

Discussions about HPLC, CE, TLC, SFC, and other "liquid phase" separation techniques.

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Hello!

So, I have been trying to detect glutathione in both its reduced (GSH) and oxidized (GSSG) forms. I have been trying to replicate the method in the paper entitled Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) Column Chemistries for the UPLC–MS–MS Analysis of Glutathione, Glutathione Disulfide, and Ophthalmic Acid in Mouse Liver and Human Plasma from the Journal of Chromatographic Science, Vol. 46, March 2008.

Here is my method:
System: Waters UPLC H-Class
Column: Acquity UPLC BEH C18 1.7 um, 2.1 x 50 mm
Column Temp: 60° C
Run Time: 6 minutes
Injection Volume: 3 uL
Mobile Phase solvents:
A: 0.1% Formic Acid in DI Water
B: 0.1% Formic Acid in Acetonitrile
Gradient: Image

I have prepared at least two standards of GSH and one of GSSG in [50/50] HPLC Grade Methanol/DI water. They were filtered through a 0.22 um PTFE filter and centrifuged at 13,000 rpm for 4 minutes. I have no worried about derivatization of GSH just yet as I'm just worrying about getting any peaks. Any GSH that is oxidized should become GSSG anyway.

My method differs from that described in the paper in the column, the one described is 2.1 x 100 mm rather than 2.1 x 50 mm. I would not expect this to have a great difference on the results, but I am fairly new to LC.

I have run this method multiple times and have gotten what amounts to nonsense (see below). I have also run several simpler gradients ranging from 1/99 to 99/1 with and without formic acid with no real luck.

I have run caffeine several times and get clean peaks consistently with and without the formic acid. However, I have been having difficulty with other standards with this specific column. If a standard such as caffeine is giving peaks, but other analytes aren't working could the column still be at fault? How can I diagnose whether my column is at fault?

If anyone sees any issues with my methods or has some suggestion for detecting either GSH or GSSG please let me know. Thank you in advance for your time!
What kind of detection are you using? MS as described in the original article? Or a different kind of detector?
I'm using a PDA detector set for 280 nm on 2D channel. It also records on a 3D channel as well. We don't have a MS in-line. I maybe misunderstood the paper as using a traditional PDA detector in the obtaining the peaks first and then using an MS later in their experiment.

Thanks for the response. Sorry for the delay in response! I had 0 notifications!

Edit: Subscribed properly now.
Take a look at the molecular structure of glutathione: there is no chromophore present that may absorb @280nm, so no wonder you see no peak. UV (or PDA) detector is not the first choice here. You may try changing to a lower wavelength (such as 210 nm) in order to get absorption from the carbonyl groups. Sensitivity probably will not be really good (at least much worse than with an MS) but depending on your purpose, you might be lucky.
Luckily, I ran these with the 3D detector. And I actually do get peaks at roughly 215 nm at a retention time of 0.557 min that seem to resemble the peaks from this other source I found here. Granted that is a HPLC method, but this is encouraging. I'm going to see if I can get a calibration curve from this and run with that for now.

I really appreciate the help. I have been stumbling around in the dark for a little while without much of a background in analytical chemistry. I'm off to look up what a chromophore is. Thank you very much.
jcpal15 wrote:
Luckily, I ran these with the 3D detector. And I actually do get peaks at roughly 215 nm at a retention time of 0.557 min that seem to resemble the peaks from this other source I found here. Granted that is a HPLC method, but this is encouraging.


...and unfortunately that HPLC method is a very good example of a very bad method. There is no retention of the analyte peak, it is eluting in the solvent front. There's no separation involved, so this is not even chromatography. This is asking for trouble. Forget to use a method like this for serious work.
I always wonder how articles like this can get published, as there a literally thousands of methods out there in the academic journals which scream "not enough retention!".
As a first step into learning HPLC, look up "retention factor" even before looking what "chromophore" means.
Glutathion is not an easy one by HPLC. As mentioned above, you better have an MS for detection. Then about chromatography: the BEH C18 is not a good choice here, GSH elutes almost in the void on this type of column. We tested many different columns (several of which supposed to provide acceptable retention according to the literature) but none worked properly for both GSH and GSSG. Finally we found that the HSS T3 (2.1mmx100, 1,7um) works fine provided you "condition" it, otherwise you get strong peak tailing for GSSG. Probably something to do with metal ions binding strongly with the disulfide bridge. Anyway, by injecting repeatedly a cabbage extract (we were suggested to use BSA or something similar but it did not work at all, so I thought of using something containing lots of sulfur atoms, i.e. glucosinolates in cabbage, and bingo!) before analyzing glutathion we could prevent peak tailing on GSSG and GSH showed very decent retention starting from 100% H2O + 0.05% formic acid. Details on the method can be found there:

https://www.frontiersin.org/articles/10 ... 00067/full
Wow, thanks! Many of the methods I have come across have had to 'condition' or derivatize both GSH and GSSG to ensure that they don't auto-oxidize or auto-reduce. Your method is attractive because I worry about derivatization being slightly inaccurate.

I am working on obtaining an in-line MS for the system I have, but may have access to another lab's in the meantime. I definitely have a HSS T3, maybe not the same size though. So, hopefully, I can give it a whirl.

I appreciate the help! I'm a lot better informed then I was previously.
A precision about stability: TCA prevents rapid oxidation to some extent, but we still observe oxidation from GSH into GSSG after a few hours. We thus prepare samples and inject them immediatley after preparation. Specificially we prepare max. 30 samples per batch and run maximum 2 batches per day.
The method used in the article you referred to (Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) Column Chemistries for the UPLC–MS–MS Analysis of Glutathione, Glutathione Disulfide, and Ophthalmic Acid in Mouse Liver and Human Plasma from the Journal of Chromatographic Science, Vol. 46, March 2008) was actually measuring derivatized GSH.

It was stated in the last paragraph of introduction: "To prevent the auto-oxidation of GSH to GSSG during the sample work-up, GSH in mouse liver and human plasma was rapidly derivatized with NEM to form the stable derivative, GSH-NEM. The NEM derivatization step is important to circumvent the overestimation of GSSG and the underestimation of GSH".
kiptx wrote:
The method used in the article you referred to (Evaluation of BEH C18, BEH HILIC, and HSS T3 (C18) Column Chemistries for the UPLC–MS–MS Analysis of Glutathione, Glutathione Disulfide, and Ophthalmic Acid in Mouse Liver and Human Plasma from the Journal of Chromatographic Science, Vol. 46, March 2008) was actually measuring derivatized GSH.

It was stated in the last paragraph of introduction: "To prevent the auto-oxidation of GSH to GSSG during the sample work-up, GSH in mouse liver and human plasma was rapidly derivatized with NEM to form the stable derivative, GSH-NEM. The NEM derivatization step is important to circumvent the overestimation of GSSG and the underestimation of GSH".


In retrospect, that was the second problem with what I was doing. I reasoned that I'd detect GSSG at the very least. However, I was attempting this method without a MS-MS. I don't believe GSH or GSH-NEM are detectable with a UV detector. But, I could be wrong.
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