Abstract
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°C in pH 8.8 H3BO3‐KCl‐Na2CO3 buffer to produce the stable derivatives. The derivatives as well as the decomposed IPBT could be well separated on a C18 column within 21 min by using 59% aqueous methanol containing 135 mmol · l−1of 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 µl. The proposed method had been applied in the determination of GSH and Cys in human blood with recoveries of 96.7–102.1%.
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