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

建立了一种简单、灵敏的测定维生素E氧化产物的高效液相色谱（HPLC）柱后还原-电化学检测（ECD）方法（α-生育酚，α-TH），包括8a-（磷脂酰胆碱-二氧基）-α-生育酮（TOO-PC）、8 α-（胆固醇酯-二氧基）-α-生育酮（TOO-ChE）、α-生育醌（TQ）和环氧-α-生育醌（epoxyTQs）。从生物样品中提取的脂质经硅胶-NH_2固相萃取柱处理后，用氯仿洗脱THs、TQ、epoxyTQs和TOO-ChE，用甲醇洗脱TOO-PC，进行HPLC分析。氯仿级分中的TH、TQ和环氧TQ通过J ′ sphere ODS-H80柱用含有10 mM乙酸锂和5 mM三氟乙酸的95%甲醇分离，并在在线锌催化还原后通过ECD定量。甲醇中的TOO-PC ...更多信息 通过J ′ sphere ODS-L 80柱，使用含有20 mM氯化胆碱的97.5%甲醇分离，通过还原柱（氧化铝上的5%铂），并通过ECD（+700 mV）定量还原形式;氯仿级分中的TOO-ChE与含有10 mM乙酸锂的98.5%甲醇作为溶剂的条件相同。通过该方法对每种化合物的检测限为约0.2pmol。方法用于过氧化人血浆中α-TH反应产物的测定。在37℃条件下，内源性α-TH能清除胆固醇酯（ChE）和磷脂酰胆碱（PC）的过氧化自由基，抑制ChE和PC过氧化氢的形成。反相高效液相色谱法检测到的主要产物为TH与ChE-过氧自由基的加成产物（TOO-ChE）、TQ和epoxyTQ，TOO-ChE的结构为胆甾醇（8 α-二氧-α-生育酮）-环氧十八烯酸酯。结果表明，人血浆中的内源性α-TH可与脂质过氧自由基反应生成多种反应产物，因此，本研究建立的HPLC技术可用于生物样品中维生素E自由基清除产物的灵敏、特异的测定。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