61高效液相色谱在牛奶中雌三醇含量检测中的应用孙林超江苏食品职业技术学院 (淮安 223003)摘要对不同牛奶中雌三醇的含量进行分析，探索建立HPLC法快速测定牛奶中雌三醇含量的方法。样品经乙腈处理沉淀蛋白质，正已烷脱脂净化后，取上清液浓缩，采用反相高效液相色谱法，流动相为乙腈+甲醇+四氢呋喃+醋酸钠溶液(0.01 moL/L)=20+20+10+50(V/V)，Agilent-C l8 柱(4.6 mm×300 mm，5 um)，流速为1.0 mL·min -1，波长为230 nm，进样量20 μL，雌三醇浓度在(0.1×10-6~2.0×10-6) g/mL时，浓度与峰面积呈线性关系，加标回收率为92.50%~103.9%，最低检出限为0.08 ng/mL，定量限0.27 ng/mL，相对标准偏差为2.04%~7.76%。关键词牛奶；雌三醇；高效液相色谱法Application of the HPLC in the Determination of Estriol Concentration in MilkSun Lin-chaoJiangsu food science college (Huaian 223003)Abstract The concentrations of estriol in different milk were analyzed, in order to establish a rapid method by HPLC to determine the estriol concentrations in milk. The proteins of samples were deposited by acetonitrile and the fats were removed by hexane and then the supernatant liquor concentrations were detected by RP-HPLC method. The chromatographic conditions included Agilent-C l8 column (4.6 mm×300 mm, 5 um) and the mobile phase consisting of a mixture of acetonitrile, methanol, tetrahydrofuran and 0.01 mol/L of sodium acetate solution by 20:20:10:50 (V/V). The fl ow rate was 1.0 mL/min. Detection wavelength was at 230 nm. The sample dose was 20 μL. When the concentration of estriol was at 0.1×10-6 g/mL to 2.0×10-6 g/mL, the perfect linearity between concentration and peak area ratio was obtained. The recovery rate was 92.50% to 103.9%. The detection limit was 0.08 ng/mL and the quanti fi cational limit was 0.27 ng/mL. The relative standard deviation (RSD) was 2.04% to 7.76%.Keywords milk ；estriol ；HPLC method牛奶是人类重要食品之一[1]，富含蛋白质、维生素、钙质及其它人类所需的营养素[2]，具有很高的营养价值。目前，饲养奶牛过程中存在滥用抗生素、激素的现象[3]，导致牛奶中含有一定量的雌三醇激素。雌三醇在雌性激素中占有很大比例，牛奶中雌性激素含量增加，人类与激素相关性疾病[4]易发生，如乳腺、卵巢、前列腺肿瘤、内分泌相关的肿瘤、出生缺陷和生育缺陷等[5]，还会对婴儿和青少年的生长发育造成严重影响[6]。国内外测定牛奶中的雌性激素采用气相色谱-质谱(GC-MS)、红外光谱法、酶免疫分析法、放射免疫法、荧光免疫分析法、高效液相色谱法[7]等方法。试验研究考察高效液相色谱检测牛奶中雌三醇含量的方法。1 材料与方法1.1 仪器与试剂Agilent 1200 高效液相色谱仪、紫外检测器、化学工作站；1810-B型石英自动双重纯水蒸馏器：常州国华电器有限公司；Laborta 4003型自动旋转蒸发仪：德国Heidolph公司；JY92-2D超声细胞粉碎机：宁波新芝生物科技股份有限公司；Minishaker MS1型漩涡混合器：德国IKA公司；TGL-16G台式离心机：上海安亭科学仪器厂；0.45 μm微孔滤膜；BS224S 精密电子天平；甲醇：HPLC，江苏汉邦科技有限公司；乙腈：纯度99.9%，天津市北辰方正试剂厂；雌三醇标准品：纯度≥99%，美国sigma公司；雌三醇标准储备液(100 μg/mL)，雌三醇标准使用液(10 μg/mL)。1.2 试验条件Agilent-C l8 (4.6 mm×300 mm，5 um)；流动相为乙腈+甲醇+四氢呋喃+醋酸钠溶液(0.01 moL/L) =20+20+10+50；流量为1.0 mL/min；检测波长为230 nm；柱温30℃；进样量为20 μL。1.3 样品处理准确量取牛奶200 mL加入200 mL乙腈，旋涡震荡混匀，超声提取40 min，静置30 min以沉淀蛋白质，以15 000 r/min速度离心15 min后吸出上清液，再加分析检测
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