HPLC WITH O-PHTHALALDEHYDE PRECOLUMN DERIVATIZATION TO MEASURE TOTAL,OXIDIZED, AND PROTEIN-BOUND GLUTATHIONE IN BLOOD, PLASMA, AND TISSUE

This HPLC assay with o-phthalaldehyde precolumn derivatization is used to measure the total, oxidized, and protein-bound forms of glutathion e in human blood, plasma, and rat tissue. Total glutathione (i.e., sum of reduced, oxidized, and protein-bound fractions) was determined aft er reduction with dithiothreitol and protein precipitation with perchl oric acid (PCA). A preliminary selective blockage of free sulfhydryl g roups with N-ethylmaleimide was necessary to evaluate the different ox idized forms. The assay showed high sensitivity (<0.05 pmol injected) and good precision (within-day CVs of 5.5% to 6.4%), recovery (101% +/ - 4%), and linearity (r >0.999). Samples, after PCA acidification, wer e stable at room temperature and 4 degrees C for 3 days, and at -20 de grees C and -80 degrees C for >1 month. The method (involving automate d derivatization) not only is very rapid and simple but also allows im mediate processing of many different biological samples.