2-(p-iodoacetylaminophenyl)benzothiazole: A pre-column fluorescent derivatizing reagent for thiol in liquid chromatography

A new derivatizing reagent with iodoacetylamine as a reaction group toward thiol, 2-(p-iodoacetylaminophenyl) benzothiazole (IPBT) has been developed. Its reactivity to thiol groups and detectability in analysis of thiol-containing compounds were investigated by high performance liquid chromatography ( HPLC) analysis of cysteine (Cys), glutathione (GSH), N-acetylcysteine (Nac), penicillamine(PA), and 2- mercaptoethanol ( 2- ME) with fluorescence detection. It was found that the reactions between IPBT and investigated thiols were finished after 30 min at 40 degrees C in pH 8.8H(3)BO(3)-KCl-Na2CO3 buffer to produce the stable derivatives. The derivatives as well as the decomposed IPBT could be well separated on a C-18 column within 21 min by using 59% aqueous methanol containing 135 mmol center dot l(-1) of pH 4.7 acetic acid (Hac)-triethanolamine(TEA) buffer as eluent, and then detected at emission wavelength of 383 nm with the excitation wavelength of 330 nm. The detection limits were of 5.0 - 20.0 fmol for an injection volume of 20 mu l. The proposed method had been applied in the determination of GSH and Cys in human blood with recoveries of 96.7 - 102.1%.