By alex on Tuesday, April 1, 2003 - 08:17 am:
I’m new to HPLC and I have a question: I have to quantify glutathione in plasma. I use a discovery C18 column, electrochemical detector and mobile phase NaH2PO4 25mM, octansulfonic acid 0.05M, ACN 5%, pH2.7. Glutathione peak is not weel resolved from another peak, so I decreased ACN to 2%, but nothing changed. Can I use ACN1% (which is the minum% of organic for a C18?) or is better to use MeOH? I have to increase the ion pair reagent?
Thanks.
By Ann on Wednesday, April 2, 2003 - 07:53 am:
When you say "nothing changed", do you mean that the glutathione peak was still not sufficiently resolved from the other peak or do you mean that there was no change in the retention time despite reducing the percentage of organic modifier
(If the latter, this would be rather surprising! How long did you allow for system equilibration after changing the mobile phase)??
What is the source of this other peak? Is it a contaminant of some sort (if so, can you eradicate it?)?
You may see quite different separation characteristics between MeOH and ACN mobile phases (combination of them both may suit).
How closely are the two peaks co-eluting? Might it be possible to use a longer column (i.e. use a 250mm column if you are currently using 150mm)? If the compounds are only co-eluting slightly, this might provide the extra resolution you need. Alternatively, are you able to try other C18 columns (different bonded phases)?
I am not familiar with the discovery C18 but C18 columns that are stable in 100% aqueous movile phases are available if necessary (such as Phenomenex's Synergi Hydro-RP).
Good luck!
By HW Mueller on Thursday, April 3, 2003 - 12:30 am:
There are sundry very specific HPLC methods for homocysteine with derivatization of the SH and fluorescence detection, which work quite well for glutathione. One can even buy kits now:
Glutathione: http://www.chromsystems.de/frame_e.html
Homocysteine: http://www.recipe.de/index_e.htm
If you do not have a fluorescence detector, a UV detctor might do.
I don´t remember the details of our trials, if you are desparate drop me an e-mail.
By Anonymous on Thursday, April 3, 2003 - 01:40 pm:
The site you provided does not show the details of derivatization and separtion conditions. Is it possible to find it in detail?
Thank you.
By HW Mueller on Friday, April 4, 2003 - 06:44 am:
Ubbink, et al, J Chrom Biomed Appl, 565, 441 (1991).
I am not certain that this is the method we used, have to get hold of a post-doc to find out. It was done a few years ago, so a lit search would be in oder anyway.