超高效合相色谱法快速测定保健食品中10种脂溶性维生素

脂溶性维生素作为保健食品重要的功效指标,现有的标准方法存在测定组分少、样品前处理过程复杂、对人员操作能力要求较高等问题,因此建立一种快速、简便、准确,且能同时检测多种常见脂溶性维生素的方法具有重要的现实意义。该研究采用超高效合相色谱(UPC²)建立了同时测定保健食品中维生素A棕榈酸酯、维生素A醋酸酯、维生素E醋酸酯、维生素K₁、α-生育酚、β-生育酚、γ-生育酚、δ-生育酚、维生素D₂、维生素D₃等10种常用脂溶性维生素的方法。样品经含75%二甲基亚砜(DMSO)的水溶液在45 ℃水浴超声15 min破乳,再加入正己烷振摇提取90 min, 3000 r/min离心10 min,取上清液过滤后进行分析。使用LC-Simulator软件对色谱条件进行模拟及优化,选用ACQUITY UPC² Viridis HSS C18 SB色谱柱进行分离,CO₂和乙腈-甲醇(85∶15, v/v)为流动相,梯度洗脱,流速1.9 mL/min,柱温30 ℃,选取10种脂溶性维生素各自的最大吸收波长检测,外标法进行定量。结果表明,10种脂溶性维生素在各自范围内线性关系良好,相关系数(r)均大于0.9997,检出限(LOD)和定量限(LOQ)分别为片剂:0.2~30 μg/g和0.8~75 μg/g,胶囊:0.4~60 μg/g和2~150 μg/g,样品平均加标回收率在96.5%~113.9%之间,RSD均小于4%,精密度、稳定性、重复性测定结果的RSD值也均小于2%。经比较,该方法测定结果与现有的国家食品安全标准基本一致,但该方法简单、快速、灵敏、准确,可满足保健食品中10种脂溶性维生素的检测要求,为保健食品中脂溶性维生素的快速同时检测奠定基础。

Rapid determination of 10 fat-soluble vitamins in health foods by ultra performance convergence chromatography

Fat-soluble vitamins are important efficacy indicators in health foods because they are essential for human physiological functions. The existing method for the simultaneous determination of fat-soluble vitamins has various problems, such as limited determination components, complex sample, pretreatment process, and high requirements for personnel operating ability. Therefore, establishing a fast, simple, and accurate method that can detect various common fat-soluble vitamins at the same time is necessary. In this study, a method for the simultaneous determination of 10 commonly used fat-soluble vitamins such as vitamin A acetate (VA acetate), vitamin A palmitate (VA palmitate), vitamin E acetate (VE acetate), vitamin K₁ (VK₁), α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, vitamin D₂(VD₂) and vitamin D₃ (VD₃) in health foods was established by ultra performance convergence chromatography (UPC²). First, the contents of about 1.0 g of capsule samples were accurately weighed. A grinder was used to grind tablet samples into powder. The powder mixture was then precisely weighed at 2.0 g. Both substances were placed in 50 mL brown stopper tubes. The test tube was then filled with 20 mL 75% dimethylsulfoxide (DMSO) aqueous solution for demulsification. The tubes were then sonicated before being extracted with n-hexane. The centrifuged supernatant was added to vials for detection. Viridis HSS C18 SB column (100 mm×3.0 mm, 1.8 μm) was applied and CO₂ was used as the mobile phase A. After comparing the influence of acetonitrile, methanol, and their mixture on chromatographic peak separation, acetonitrile-methanol (85∶15, v/v) was used as the mobile phase B. The injection volume was 1 μL. Using simulator software, the optimal chromatographic conditions were obtained after a set of three-factor orthogonal experiments of flow rate, gradient slope, and column temperature. The flow rate and column temperature were both set at 1.9 mL/min and 30 ℃. Furthermore, the maximum absorption wavelength of these 10 fat-soluble vitamins was selected for detection. Ten vitamins were baseline separated after 7 min of gradient elution. The limits of detection (LODs) and quantification (LOQs) of capsule samples were 0.4-60 μg/g and 2-150 μg/g, respectively, whereas the results for tablet samples were 0.2-30 μg/g and 0.8-75 μg/g. The linear ranges of the 10 fat-soluble vitamins were 0.1-100 μg/mL. The recoveries of spiked samples ranged from 96.5% to 113.9%, with RSD values less than 4%. Precision, stability, and repeatability RSD values were all less than 2%. By comparison, the determination results of this method were basically consistent with the existing national food safety standards. This method is simple, rapid, sensitive, and accurate, and it can meet the detection requirements of the 10 fat-soluble vitamins in health foods. Simultaneously, this method lays the foundation for the rapid and simultaneous detection of fat-soluble vitamins in existing health foods.