在线固相萃取/二维液相色谱法检测婴幼儿配方食品中视黄醇顺反异构体和α-生育酚手性异构体

Determination of Cis and Trans Isomers of Retinol andα-Tocopher⁃ol Chiral Isomers in Infant Formula by Online Solid Phase Extraction and Two-dimensional Liquid Chromatography

建立了在线固相萃取/二维液相色谱分离检测婴幼儿配方食品中全反式视黄醇、13-顺式视黄醇和α-生育酚手性异构体的方法。样品经皂化,定容、离心、过滤后进样,经二乙烯基苯与N-乙烯基吡咯烷酮大孔共聚物(HLB)在线固相萃取柱在线净化后,通过切换阀将视黄醇、4种生育酚洗脱至一维色谱柱五氟苯基填料柱(PFP),在一维色谱柱中实现总视黄醇和4种生育酚异构体的分离。再通过捕集柱与中心切换阀将总视黄醇和α-生育酚切割至二维色谱柱,在3,5-二甲基苯基氨基甲酸酯涂敷的手性色谱柱(CND)上实现了全反式与13-顺式视黄醇的异构体分离,以及α-生育酚的d、l手性异构体分离。采用紫外和荧光检测器检测,外标法定量。结果表明:全反式视黄醇、13-顺式视黄醇的线性范围为0.1~5.0 mg/L,d-α-生育酚、l-α-生育酚的线性范围为1.0~30.0 mg/L,相关系数均大于0.999,定量下限分别为53μg/100 g、56μg/100 g、290μg/100 g和340μg/100 g;低、中和高3个加标浓度下的回收率为90.9%~106%,相对标准偏差(RSD)不大于2.8%。该方法可用于乳基婴幼儿配方食品中视黄醇和α-生育酚的检测及其生物有效性评价。

A quantitative determination method for the separation of all-trans retinol,13-cis-retinol andα-tocopherol chiral isomers in infant formula was established by online solid phase extraction and two-dimensional liquid chromatography technique.After sample was purified by divinylbenzene and N-vinylpyrrolidone macroporous copolymer(HLB)online solid phase extraction column,retinol and four tocopherols were eluted to the first-dimensional high performance liquid chromatography with pentafluorophenyl packing column(PFP)by switching valve,and the total retinol and four tocopherol isomers were separated in the first-dimensional column.The total retinol andα-tocopherol were cut sequentially into the second-dimensional column by the trap column and the central switching valve,then the separation of all-trans and 13-cis-retinol isomers was realized in the chiral column(CND)coated with 3,5-dimethylphenylcarbamate,and the separation of d and l chiral isomers ofα-tocoph⁃erol was realized at the same time.All of the compounds are detected by ultraviolet and fluorescent detectors with external standard quantification.The linear range was 0.1-5.0 mg/L for all-trans reti⁃nol and 13-cis-retinol,1.0-30.0 mg/L for d-and l-α-tocopherol with correlation coefficients more than 0.999,and the quantitation limits of all-trans retinol,13-cis-retinol,d-and l-α-tocopherol were 53μg/100 g,56μg/100 g,290μg/100 g,and 340μg/100 g respectively.Recoveries at low,medium and high spiked concentrations were between 90.9%-106%,with relative standard deviations(RSD)not more than 2.8%.The method could meet the regular analysis of retinol andα-tocoph⁃erols in milk-based infant formula,and also the exact evaluation of their bioavailability after consider⁃ing relative bioactive factor for each.