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

建立了一种简便、灵敏的测定维生素E(α-生育酚，α-TH)氧化产物的高效液相色谱-铂或锌催化柱后还原-电化学检测方法，其中包括8a-(磷脂酰胆碱-二氧基)-α-生育酚(TOO-PC)、8a-(胆固醇酯-二氧基)-α-生育酚(TOO-CHES)、α-生育酚(TQ)和环氧生育酚(α-TQs)。从生物样品中提取的脂质在高效液相分析前用硅胶-NH_2小柱固相萃取处理，TH、TQ、环氧TQS和TOCHES分别用氯仿洗脱，也用甲醇洗脱。氯仿馏分中的TH、TQ和环氧TQs采用J‘SphereODSH80色谱柱分离，95%甲醇含10 mM醋酸锂和5 mM三氟乙酸，在线锌催化还原后用电子捕获检测器进行定量。Too-甲醇fr…中的PC采用J‘SphereODSL80色谱柱，以97.5%甲醇(含20 mM氯化胆碱)为色谱柱，通过还原色谱柱(氧化铝上含5%铂)，用电子捕获检测器(+700 mV)对还原形式进行定量；以98.5%甲醇(含10 mM醋酸锂)为溶剂，在氯仿部分以98.5%甲醇和10 mM醋酸锂为溶剂，分离出更多的作用。该方法对每种化合物的检出限约为0.2pmol。将该方法应用于过氧化人血浆中α-TH反应产物的检测。血浆内源性α-TH可清除胆固醇酯(ChE)和卵磷脂(PC)的过氧化自由基，从而抑制ChE和PC氢过氧化的形成，在37℃时由自由基引发剂引发。反相高效液相色谱法检测到的主要产物为TH与CHE-过氧基的加成产物(TO-CHE)、TQ和环氧TQ。TO-CHE的结构为胆甾醇酯(8a-二氧基-α-生育酚)-环氧十八碳烯酸酯。结果表明，人体血浆中的内源性α-TH可与脂质过氧化自由基反应生成某些反应产物。因此，本研究建立的高效液相色谱技术可用于生物样品中维生素E自由基清除产物的灵敏、特异的检测。较少

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