Gotti R, Andrisano V, Gatti R, Cavrini V, Candeletti S
Department of Pharmaceutical Sciences, Bologna, Italy.
Biomed Chromatogr. 1994 Nov-Dec;8(6):306-8. doi: 10.1002/bmc.1130080612.
A selective and sensitive high performance liquid chromatographic (HPLC) method has been developed for the determination of reduced glutathione (GSH) in biological samples (rat liver, spleen and plasma). The method involved a prechromatographic thiol derivatization with methyl 4-(6-methoxynaphthalen-2-yl)-4-oxo-2-butenoate; the reaction was rapid (5 min) under mild conditions (pH 7.5 and ambient temperature) and selective for the sulphydryl group. The thiol adducts were separated on a reversed-phase C-18 column using acetonitrile: 0.05 M triethylammonium (TEA) phosphate (pH 4) solution 32:68 (v/v) as the mobile phase. Fluorescence detection (lambda em = 450 nm; lambda exc = 310 nm) was used and the detection limit (S/N = 3) was about 0.5 pmole of the injected GSH adduct. The method was also applied to the determination of total glutathione in rat plasma after a preliminary reduction with dithiothreitol.
已开发出一种选择性和灵敏的高效液相色谱（HPLC）方法，用于测定生物样品（大鼠肝脏、脾脏和血浆）中的还原型谷胱甘肽（GSH）。该方法涉及用4-(6-甲氧基萘-2-基)-4-氧代-2-丁烯酸甲酯进行色谱前硫醇衍生化；反应在温和条件（pH 7.5和室温）下快速（5分钟），且对巯基具有选择性。硫醇加合物在反相C-18柱上分离，使用乙腈：0.05 M三乙铵（TEA）磷酸盐（pH 4）溶液32:68（v/v）作为流动相。采用荧光检测（发射波长λem = 450 nm；激发波长λexc = 310 nm），检测限（S/N = 3）约为注入的GSH加合物的0.5皮摩尔。该方法还应用于用二硫苏糖醇初步还原后大鼠血浆中总谷胱甘肽的测定。
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