Abstract: A reversed-phase high performance liquid chromatography (RP-HPLC) method for the simultaneous quantitative determination of vanillin, methyl vanillin, ethyl vanillin and coumarin in milk and dairy products has been established. Vanillin compounds from samples were extracted with acetonitrile, blown to dryness under nitrogen, and then cleaned up with n-hexane. The chromatographic separation was achieved using a mixture of 20 mmoL/L ammonium acetate (pH 5.6), acetonitrile and methanol as the mobile phase with gradient elution on a C18 column. The analysis was carried out using a UV detector at a wavelength of 267 nm and the analytes were quantified by an external standard method. Good linear relationships in the range of 0.05–5.00 μg/mL were observed for all analytes. The limit of quantification was 0.2 mg/kg and the limit of detection was 0.06 mg/kg for the four analytes in liquid milk, milk powder, yogurt, lactic acid fermented beverage, ice cream, and cheese. The recoveries for vanillin, methyl vanillin, ethyl vanillin and coumarin in these samples at spiked concentration levels of 0.2–2.0 mg/kg were 80.6%–110.0%, 80.1%–109.6%, 80.4%–109.5% and 80.1%–105.9%, with relative standard deviations of 1.87%–7.70%, 1.45%–9.80%, 1.66%–9.52% and 1.16%–9.52%, respectively. This method was rapid, accurate, repeatable and simple and could be applied to determine vanillin compounds in milk and dairy products.

Key words: milk and dairy products, high performance liquid chromatography, vanillin, methyl vanillin, ethyl vanillin; coumarin

摘要： 建立乳与乳制品中香兰素、甲基香兰素、乙基香兰素和香豆素的反相高效液相色谱分析方法。用乙腈作为提取剂，氮吹浓缩，正己烷除脂净化，以C18反相色谱柱分离，20 mmol/L乙酸铵缓冲溶液（pH 5.6）-乙腈-甲醇为流动相，采用梯度方式洗脱，紫外检测波长267 nm，外标法定量。结果表明：4 种香兰素类化合物在0.05～5.00 μg/mL范围内线性良好，液态乳、乳粉、发酵乳、乳酸菌饮料、雪糕和干酪样品中香兰素、甲基香兰素、乙基香兰素和香豆素的定量限均为0.2 mg/kg，检出限均为0.06 mg/kg；液态乳、乳粉、发酵乳、乳酸菌饮料、雪糕和干酪中香兰素、甲基香兰素、乙基香兰素和香豆素添加量为0.2～2.0 mg/kg时，加标回收率分别为80.6%～110.0%、80.1%～109.6%、80.4%～109.5%、80.1%～105.9%，相对标准偏差分别为1.87%～7.70%、1.45%～9.80%、1.66%～9.52%、1.16%～9.52%。本方法快速、准确、重复性好、操作简便，适用于乳制品中香兰素、甲基香兰素、乙基香兰素和香豆素的含量测定。

关键词: 乳与乳制品, 高效液相色谱法, 香兰素, 甲基香兰素, 乙基香兰素, 香豆素