Chromatography Forum

Dear coleques,

i have a question if anybody has an experience with determination of glutathione in reduced and oxidized form in animal tissue by HPLC with fluorimetric detection. I try to use the method with OPA derivatization and reduction by DTT. There are a lot of signals after DTT using, and it is no quantitative relation between GSH and GSSG. Any suggestions will be helpfull.

With bestr wishes

maciek gawlik

I have done fluorescence detection after derivatizing the sulfhydryl group of glutathione, homocystein, etc. This was a somewhat tedious reaction which I don´t remember any more any way, but I have since seen some simpler -SH derivatizations. You should not have any trouble finding that. Note that the derivatization of the -SH is much more specific then -NH2 derivatization. The -SS- can be done after reduction, maybe there is even a direct method by now.

http://repository.tamu.edu/bitstream/ha ... sequence=1

http://apjcn.nhri.org.tw/server/APJCN/V ... /Bayor.pdf
http://eprints.ucl.ac.uk/18703/1/18703.pdf
http://scd.uhp-nancy.fr/docnum/SCD_T_20 ... GLOWKA.pdf

R Paroni, E De Vecchi, G Cighetti, C Arcelloni, I Fermo, A Grossi and P Bonini, HPLC with o-phthalaldehyde precolumn derivatization to measure total, oxidized, and protein-bound glutathione in blood, plasma, and tissue, Clinical Chemistry 41: 448-454, 1995;

Maura Floreani, Marcella Petrone, Patrizia Debetto, Pietro Palatini, A Comparison Between Different Methods for the Determination of Reduced and Oxidized Glutathione in Mammalian Tissues, Free radical research, 1997, Vol. 26, No. 5 , Pages 449-455