Electrochemical,fluorescence, and UV detection for HPLC analysis of various cysteine derivatives

Electrochemical (ECD), fluorescence (FLD), and UV spectrophotometric (UVD) detection were used to monitor various S-alk(en)yl-L -cysteines and their corresponding sulfoxide isomers following pre-column derivatization with o-phthaldialdehyde-tert-butylthiol and separation by reversed-phase HPLC. Recording of hydrodynamic voltammograms, FLD stop-flow scanning, and on-line captured UV spectra were methods used for establishing optimal detector settings which were defined as a compromise between favorable selectivity and high sensitivity. Optimal detector settings were found at: (A) 750 mV vs. Ag/AgCl for ECD; (B) excitation at 230 nm and emission at 420 nm for FLD; and (C) 337 nm for UVD. Various aspects of detector practicability such as selectivity, baseline disturbances due to excessive reagent, scanning possibilities, as well as detection limits were evaluated and compared. Minimal detectable amounts of the compounds were in the range of 130-160 fmol for ECD, and 2.5-3.5 pmol and 13-16 pmol for FLD and UVD. In addition, the possibilities and benefits of detector coupling were examined.