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J.Health Sci., 49(6), 527-530, 2003
Optimum Conditions for Derivatization of Glutathione, Cysteine and Cysteinylglycine in Human Plasma with Ammonium 7-Fluorobenzo-2-Oxa-1,3-Diazole-4-Sulfonate for Accurate Quantitation by High-Performance Liquid Chromatography
Yoshikazu Yoshida,a, b Yasuharu Ohiwa,b Masao Shimamura,e Toru Izumi,c Shozo Yoshida,c Katsuyuki Takahashi,d Shinichi Miyairi,a, f Mizue Makimura,b
and Akira Naganuma*, a
aLaboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-ku, Sendai 980-8578, Japan, bDepartment of Pharmacy, cDepartment of Emergency and Critical Care Medicine, and dDepartment of Clinical Chemistry, University of Nihon Itabashi Hospital, Itabashi-ku, Tokyo 173-8610, Japan, eCommon-Use Facilities for Medical Research, Nihon University School of Medicine, Itabashi-ku, Tokyo 173-8610, Japan, and fCollege of Pharmacy, Nihon University, Funabashi, Chiba 274-8555, Japan
For the accurate quantitation of glutathione (GSH), cysteine (Cys) and cysteinylglycine (CysGly) in human plasma by detection of fluorescence after derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate (SBD-F), a thiol-specific fluorogenic agent and high-performance liquid chromatography (HPLC) , we attempted to identify optimum conditions for the derivatization and treatment of samples. The extent of derivatization with SBD-F of endogenous thiol compounds in human plasma was affected by several factors, such as the concentration of SBD-F and the reaction time. Acidification and deproteinization of plasma immediately after the separation of plasma from whole blood were essential and prevented the determination of inappropriately low values. The method developed in the present study allowed recovery that was closed to 100% for each compound. The mean values determined for 61 healthy individuals were as follows: Cys, 50.1 mu M; CysGly, 12.2 mu M; and GSH, 5.3 mu M.
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