Each sample was
first diluted with distilled water to make the volume up to
250 ll, and then 750 ll of NPM (1 mM in acetonitrile) were added.
The resulting solution was mixed and incubated at room temperature
for 5 min. 10 ll of 2 N HCl were added to stop the reaction.
After filtration through a 0.2 lm acrodisc, the derivatised samples
were injected onto a 5 lmC18 column in a reverse phase HPLC system.
GSH and Cys were determined concurrently since all known
monothiols form fluorescent derivatives with NPM. All determinations
were performed in triplicate (n = 3).