Abstract: A reversed-phase high performance liquid chromatography (RP-HPLC) method was established for the determination of 6S-5-methyltetrahydrofolate calcium in milk powder. After protein precipitation with trichloroacetic acid solution, samples were extracted with ascorbic acid solution in a hot water bath, and the extract was separated on a C18 reversed-phase column by gradient elution using a mobile phase composed of 0.1% trifluoroacetic acid solution and methanol, and the detection wavelength was set at 280 nm. Quantitative analysis was carried out by the single standard method. The results showed that the limit of quantification and detection of 6S-5-methyltetrahydrofolate calcium in milk powder samples were 150 and 50 μg/100 g, respectively. The recoveries for samples spiked at levels ranging from 150 to 600 μg/100 g were 95.0%–109.1% with relative standard deviations (RSDs) between 1.26% and 4.04%. This method is simple, accurate, with high recovery and reproducibility, and suitable for the determination of 6S-5-methyltetrahydrofolate calcium in milk powder.

Key words: milk powder; reversed-phase high performance liquid chromatography; 6S-5-methyltetrahydrofolate calcium; trichloroacetic acid; ascorbic acid

摘要： 建立乳粉中6S-5-甲基四氢叶酸钙的反相高效液相色谱分析方法。使用三氯乙酸溶液沉淀蛋白，抗坏血酸溶液加热提取后，以C18反相色谱柱分离，三氟乙酸溶液（0.1%）-甲醇为流动相，采用梯度方式洗脱，检测波长280 nm，单标法定量。结果表明：乳粉样品中6S-5-甲基四氢叶酸钙的定量限为150 μg/100 g，检出限为50 μg/100 g；乳粉中6S-5-甲基四氢叶酸钙的添加量为150～600 μg/100 g时，加标回收率为95.0%～109.1%，相对标准偏差为1.26%～4.04%。本方法操作简便、准确、回收率高、重复性好，适用于乳粉中6S-5-甲基四氢叶酸钙的含量测定。

关键词: 乳粉；反相高效液相色谱法；6S-5-甲基四氢叶酸钙；三氯乙酸；抗坏血酸