High-Sensitive Analysis of the Free-Radical Scavenging Products of Vitamin E in Biological Samples by High-Performance Liquid Chromatography

建立了以铂或锌为催化剂，柱后还原，电化学检测（ECD）的高效液相色谱（HPLC）体系，定量测定维生素E （α-生育酚，α-TH）氧化产物8a-（磷脂酰胆碱-二氧基）-α-生育酚（TOO-PCs）、8a-（胆甾醇-二氧基）-α-生育酚（TOO-ChEs）、α-生育酚基醌（TQ）和环氧-α-生育酚基醌（epoxyTQs）的方法。生物样品中提取的脂质在HPLC分析前采用二氧化硅-氨筒固相萃取处理；THs、TQ、环氧TQ和TOO-ChEs分别用氯仿洗脱，TOO-PCs分别用甲醇洗脱。采用含10 mM乙酸锂和5 mM三氟乙酸的95%甲醇J'sphere ODS-H80色谱柱分离三氯甲烷馏分中的三氯甲烷、TQ和环氧TQ，锌催化在线还原后采用ECD定量。使用含20 mM氯化胆碱的97.5%甲醇，用J'sphere ODS-L80色谱柱分离，通过还原柱（氧化铝上5%铂），用ECD （+700 mV）定量还原形式；以含10mm乙酸锂的98.5%甲醇为溶剂，氯仿馏分中TOO-ChEs的反应条件相同。该方法对每种化合物的检出限约为0.2 pmol。本方法适用于人血浆过氧化a-TH反应产物的检测。37℃自由基引发过氧化反应时，血浆内源性α-TH可清除胆固醇酯（ChE）和磷脂酰胆碱（PC）的过氧自由基，抑制ChE和PC氢过氧化物的形成。反相高效液相色谱法检测的主要产物为TH与che -过氧基（TOO-ChE）、TQ和环氧TQ的加成产物。TOO-ChE的结构表征为胆固醇酯（8a-二氧基-α-生育激素）-环氧十八烯酸酯。结果表明，人血浆内源性α-TH可与脂质过氧化自由基发生反应，形成一定的反应产物。综上所述，高效液相色谱技术在生物样品中维生素E自由基清除产物的灵敏和特异性测定方面具有优势。少

A simple and sensitive method using a high-performance liquid chromatography (HPLC) system with post-column reduction using platinum or zinc as the catalyst followed by electrochemical detection (ECD) has been developed for the quantification of the oxidation products of vitamin E (α-tocopherol, α-TH), including 8a-(phosphatidylcholine-dioxy)-α-tocopherones (TOO-PCs), 8a-(cholestrylester-dioxy)-α-tocopherones (TOO-ChEs), α-tocopherylquinone (TQ), and epoxy-α-tocopherylquinones (epoxyTQs). The lipids extracted from biological samples were treated with solid-phase extraction of a silica-NH_2 cartridge before the HPLC analysis ; THs, TQ, epoxyTQs, and TOO-ChEs were eluted with chloroform, TOO-PCs being with methanol, respectively. THs, TQ, and epoxyTQs in the chloroform fraction were separated by a J'sphere ODS-H80 column with 95% methanol containing 10 mM lithium acetate and 5 mM trifluoroacetic acid and quantified by ECD after on-line zinc-catalyzed reduction. TOO-PCs in the methanol fr … More action were separated by a J'sphere ODS-L80 column using 97.5% methanol containing 20 mM choline chloride, passed through a reduction column (5% platinum on alumina), and the reduced forms were quantified by ECD (+700 mV) ; TOO-ChEs in the chloroform fraction being the same conditions with 98.5% methanol containing 10 mM lithium acetate as the solvent. The detection limit for each compound by this method was about 0.2 pmol. This method was applied to the detection of the reaction products of a-TH in the peroxidized human plasma. The endogenous α-TH in plasma could scavenge peroxyl radicals of cholesterylester (ChE) and phosphatidylcholine (PC) to suppress the formation of ChE and PC hydroperoxides when the peroxidation was initiated by a free radical initiator at 37℃. The main products detected by reversed-phase HPLC were the addition products of TH with ChE-peroxyl radicals (TOO-ChE), TQ, and epoxyTQ.The structures of TOO-ChE were characterized as cholestryl (8a-dioxy-α-tocopherone)-epoxyoctadecenoates. The results indicate that endogenous α-TH in human plasma can react with lipid peroxyl radicals to form some reaction products.In conclusion, the HPLC technique presented here is found to advantages for the sensitive and specific determination of the free-radical scavenging products of vitamin E in biological samples. Less