快速皂化-气相色谱-串联质谱法同时测定乳粉中胆固醇和维生素E4种异构体

Determination of cholesterol and vitamin E isomers in milk powder by gas chromatography-tandem mass spectrometry coupled with rapid saponification

胆固醇和生育酚都是人体所必需的重要营养元素,是乳粉中的重要质量指标。现行国家食品安全标准对胆固醇和维生素E 4种异构体(α-生育酚、β-生育酚、γ-生育酚和δ-生育酚)的检测前处理复杂繁琐、耗时长,且不能同时测定,因此,开发简单、快速且可同时测定胆固醇和4种生育酚的检测方法具有重要的现实意义。该研究采用气相色谱-串联质谱(GC-MS/MS)建立了乳粉中胆固醇和4种生育酚的定性定量测定方法。样品经脂肪酶酶解,采用快速的碳酸钾-乙醇皂化法,同时对酶解时间、皂化温度、萃取溶剂的种类、萃取溶剂的体积、萃取时间等前处理进行优化,从而确定出最优的前处理方法。结果表明,样品在37℃下酶解4 h,然后室温(25℃)下振荡10 min皂化,最后使用5 mL正己烷萃取10 min,4000 r/min离心6 min,取上层正己烷过膜,经TG-5MS Sil色谱柱分离,GC-MS/MS多反应监测离子模式扫描分析,同时获得定性和定量结果。实验结果表明,胆固醇在0.5~50.0 mg/L、4种生育酚在0.25~25.0 mg/L范围内具有较好的线性关系,相关系数(r2)大于0.99,加标回收率为76.6%~93.1%,相对标准偏差为0.9%~3.3%,胆固醇定量限为10.0μg/100 g,4种生育酚的定量限均为5.0μg/100 g。随机抽取市面上出售的20种婴幼儿配方奶粉及4种低脂乳粉,对其胆固醇和4种生育酚含量进行了分析测试,结果显示,婴幼儿配方乳粉中胆固醇、4种生育酚的含量高于低脂乳粉,其中婴幼儿乳粉的4种生育酚中α-生育酚和β-生育酚含量较高。该方法简便、快速、灵敏、准确,可满足乳粉中胆固醇和4种生育酚生育酚的检测要求,为乳粉品质的快速检测奠定了理论基础。

Cholesterol and tocopherols,which are important quality indicators in milk powder,are essential nutrients for the human body.Current pretreatment methods for the detection of cholesterol and four isomers of vitamin E(α-tocopherol,β-tocopherol,γ-tocopherol,andδ-tocopherol)are based on national food safety standards,which are complicated,time-consuming,and unsuited for simultaneous measurements.Thus,developing a simple,fast,and simultaneous detection method for cholesterol and the four kinds of tocopherols is of practical significance.In this study,gas chromatography-tandem mass spectrometry(GC-MS/MS)was used to establish qualitative and quantitative methods for the determination of cholesterol and the above mentioned four isomers of vitamin E.The sample was digested with lipase and then saponified rapidly using a potassium carbonate-ethanol system.The optimal pretreatment method was established by optimizing the enzymolysis time,saponification temperature,type and volume of the extraction solvent,and extraction time.Then,cholesterol and the four tocopherols in milk power were simultaneously determined.The results revealed a good linear relationship for cholesterol and the tocopherols in the range of 0.5-50.0 mg/L and 0.25-25.0 mg/L,respectively.The correlation coefficients(r2)were greater than 0.99;the recoveries were 76.6%-93.1%;and the relative standard deviations were 0.9%-3.3%.The limits of quantification for cholesterol and the tocopherols were 10.0μg/100 g and 5.0μg/100 g,respectively.The recoveries of the added standards did not fully reflect the ability of the method to decompose and extract the actual sample,especially given that the five compounds considered in this study were fat-soluble.Thus,the added standard recovery could not verify the enzymatic hydrolysis effect.In order to investigate the effectiveness of this method for actual milk powder samples,the amounts of cholesterol and the four tocopherols in infant milk powder were determined according to the national standard methods(GB 5009.82-2016,GB 5009.128-2016)and the proposed method.For each method,six sets of measurements were carried out in parallel.The cholesterol content measured by this method was slightly lower than that measured by the national standard method,while the amounts of the four tocopherols were slightly higher.There was no significant difference(p>0.05)between the national standard method and our method on the amounts of cholesterol and the four tocopherols in milk powder.Twenty kinds of infant formula milk powder and four kinds of low-fat milk powder were randomly selected from the market,and the amounts of cholesterol and the four tocopherols were analyzed.The results showed that the amounts of cholesterol and the four tocopherols in the infant formula milk powder were higher than those in the low-fat milk powder.This method is simple,fast,sensitive,and accurate,thus meeting the detection requirements for cholesterol and tocopherols in milk powder.The findings of the study would provide a theoretical foundation for the rapid estimation of milk powder quality.