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- Posts: 5
- Joined: Mon Nov 06, 2017 9:42 pm
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:
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!