乳粉中4种生育酚的正相高效液相色谱荧光法含量测定

Simultaneous Determination of 4 Tocopherol in Milk Powder by Normal Phase High Performance Liquid Chromatography with Fluorometric Detection

建立了反相色谱法样品前处理与正相酰胺柱色谱分离相结合的正相高效液相色谱荧光(HPLC-FLU)法同时测定乳粉中α-、β-、γ-和δ-生育酚的含量,用于乳粉中维生素E含量的准确测定。考察了不同色谱柱类型、不同流动相比例和不同进样量对4种生育酚的色谱分离情况。2.5 g乳粉样品经氢氧化钾碱性溶液皂化后,用石油醚-乙醚混合液提取浓缩至近干,用甲醇定容至10 mL,经过滤膜后进样。以V(正己烷)∶V(叔丁基甲基醚)∶V(四氢呋喃)∶V(甲醇)=810.0∶85.3∶104.3∶0.4的混合溶液为流动相等度洗脱,α-、β-、γ-、δ-生育酚在酰胺柱上18.5 min内达到基线分离。激发波长λ_(x)=294 nm,发射波长λ_(m)=328 nm,进样体积为2μL。4种生育酚在2~80 mg/L范围内,相关系数R^(2)≥0.9991;4种生育酚的定量限均为0.6 mg/kg,3水平加标平均回收率范围为88.2%~103.0%,相对标准偏差范围为1.3%~4.7%。方法色谱分离效果好、重复性好、准确度高、定量限也满足标准限量要求,具有较好的实用性,并已用于实际样品的检测,可为乳粉维生素E含量的准确测定提供技术参考。

A normal phase high performance liquid chromatography fluorometric(HPLC-FLU)method,with the characteristics of sample pretreatment from a reversed phase chromatography combining with normal phase chromatographic separation on a amide column,was established to determine the contents ofα-,β-,γ-andδ-tocopherol in milk powder,and then to determinate the content of vitamin E.The different types of chromatography column,ratios of mobile phase components and injection volumes were investigated.The milk powder sample(about 2.5 g)was firstly saponificated with potassium hydroxide solution,then extracted with mixture of petroleum ether and diethyl ether.The extracted solution was concentrated to near dryness,and then 10 mL of methanol was added.The final sample solution was filtered with a filter before injection.With the mixture solution(V(hexane)∶V(tert-butyl methyl ether)∶V(tetrahydro furan)∶V(methanol)=810.0∶85.3∶104.3∶0.4)as mobile phase and isocratic elution,the base line separations ofα-,β-,γ-andδ-tocopherol could be achieved within 18.5 min on an amide column.The measuring wavelengths ofλx andλm were set at 294 nm and 328 nm,respectively.The injection volume was set at 2μL.The correlation coefficient R^(2) was greater than or equal 0.9991 in the range of 2~80 mg/L.The limits of quantification(LOQs)were 0.6 mg/kg.The average recoveries forα-,β-,γ-andδ-tocopherol were between 88.2%~103.0%with the relative standard deviation(RSD)between 1.3%~4.7%in the milk powder sample.The method has shown satisfactory chromatographic separation,good accuracy and repeatability.The LOQs ofα-,β-,γ-andδ-tocopherol are also met the requirements of relevant standards.The method has been applied to analyze milk powder samples,and can be used as useful technical reference for accurate determination of the content of vitamin E.