超高效液相色谱法测定乳及乳制品中维生素D的含量

Determination of vitamin D in milk and dairy products by ultraperformance liquid chromatography

目的建立用正相高效液相色谱硅胶柱净化维生素D,通过反相超高效液相色谱测定乳及乳制品中维生素D的含量的一种快速检测方法。方法样品经氢氧化--钾皂化后,用石油醚萃取,萃取液经无水硫酸钠脱水、旋蒸,定容,用正向色谱净化,反相超高效液相色谱用以0.1mL/min流速的甲醇/水(97:3V:V)为流动相,采用WatersACQUITY UPLC BEH C18色谱柱分离,紫外检测器检测,内标法定量。结果维生素D3浓度在0.05–1.00μg/mL内标准曲线相关系数(r^2)均大于0.995。在质量分数为0.5、1.0、2.0μg/mL 3个加标水平下,加标回收率为65.0–115.0%,相对标准偏差为0.1%-1.4%。结论该方法简便、快速、实用、准确,各项技术指标满足标准的要求,可用于乳及乳制品中维生素D的定量检测。

Objective To establish a rapid method for the determination of vitamin D in milk and dairy products by reversed phase–ultra performance liquid chromatography(RP-UPLC)after normal phase–high performance liquid chromatography(NP-HPLC)with silica gel column purification.Methods Samples were saponified by potassium hydroxide and extracted with petroleum ether,then the extract liquor was dehydrated by anhydrous sodium sulfate,rotary evaporated and fixed volume.NP-HPLC was used to purify and for RP-UHPLC,methanol/water(97:3 V:V)at 0.1 mL/min flow rate served as mobile phase,WatersACQUITY UPLC C18 chromatographic column was used for separation,and UV detector and quantitative internal standard method were used for vitamin D determination.Results Relation coefficient(r^2)of the internal standard curve was greater than 0.995 in the range of vitamin D3 concentration of 0.05–1.00μg/mL.The recovery rate was 65.0%–115.0%and the relative standard deviation was 0.1%–1.4%when the mass fraction was 0.5,1.0 and 2.0μg/mL.Conclusion This method is simple,rapid,practical and accurate,and can be used for the quantitative determination of vitamin D in milk and dairy products.