Abstract: By modification of our post-column derivatization system for thiols, an HPLC method has been developed for the simultaneous fluorometric determination of biogenic thiols and disulfides. They are separated by anion-exchange chromatography with gradient elution, reacted with sodium sulfite at pH 6-7 and o-phthalaldehyde (OPA) at pH 9.4-10.5 in the presence of taurine to produce fluorescent isoindole fluorophores, and monitored with a fluorescence detector (λex = 360 nm; λem > 405 nm). Under the optimized conditions for chromatography and the post-column derivatization, 250 pmol of L-cysteine (CySH), L-cystine (CySS), glutathione (GSH), and oxidized glutathione (GSSG) and 750 pmol of 3'-dephosphocoenzyme A (DP-CoASH), coenzyme A (CoASH), and their oxidized forms (DP-CoASS and CoASS) can be determined. The relative standard deviations (n = 5) of the method are 1.35%, 4.09%, and 6.87% for the analyzes of 5 nmol of CySS and GSSG and 8.5 nmol of CoASS, respectively.

Abstract: A flow-through cell is described for use in both flow injection analysis and HPLC. The carrier solution was 0.2 M NH4OH - NH4Cl buffer (pH 9.5) - 0.5% methanol - 0.5 mM CoCl2, and analysis by HPLC was on a column (25 cm x 4.6 mm) of C18 with a mobile phase similar to the carrier, but with 0.5 mM Na trichloroacetate instead of CoCl2; this system could separate several disulfides. The detection cell contained a dropping-Hg working electrode, a Pt auxiliary electrode and SCE. Differential-pulse polarography of four compounds showed that the catalytic (Brdicka) currents occurred at peak potentials from -1.5 to -1.65 V. With use of 10 µL samples, the detection limit in HPLC was 0.1 mM of dithiodiacetic acid.