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1、36-奶粉中三聚氰胺的测定（Determination of melamine in 36- milk powder）奶粉中三聚氰胺的测定奶粉中三聚氰胺的测定1原理三聚氰胺是一种三嗪类含氮杂环有机化合物，微溶于水，可溶于甲醇、甲醛、乙酸、热乙二醇、甘油、吡啶等。本方法采用20%甲醇溶解样品，5%三氯乙酸沉淀蛋白，提取样品中的三聚氰胺，经液相色谱仪C18柱分离，检测波长235nm条件下进行检测。2试剂使用优级纯、色谱纯试剂，色谱用水符合GB / t6682一级水的规定。2.1 20%甲醇溶液：甲醇+水（2 + 8）2.2 5%三氯乙酸溶液：称取三氯乙酸5g溶解于100ml水中。2.3乙腈2.4混
2、合液：称取2.10g一水合柠檬酸和1.88克己烷磺酸钠，溶于水中，稀释至1000ml混匀。2.5流动相：混合液（2.4）+乙腈（2.3）= 92 + 8。2.6标准储备液配制：准确称取三聚氰胺标准品0.1000g，用20%甲醇溶液（2.1）定容至100ml。三聚氰胺含量为1.00mg/毫升，于4冰箱内避光保存。2.7三聚氰胺标准系列的配制：准确吸取标准储备液（2.6）0.100ml；1.00ml；2.50ml；5.00ml；10.00ml至100ml容量瓶中，并用20%甲醇溶液（2.1）定容至刻度。配制成三聚氰胺浓度分别为1.00mg/L；10.0mg/L；25.0mgL；50.0mgL；10
3、0mg/L的标准系列。3仪器和设备3.1液相色谱仪：附二级管阵列检测器（爸爸）或紫外检测器（UV）3.2超声波清洗器3.3超速离心机3.4涡旋混合器3.5滤膜：0.45M4分析步骤4.1试样处理：将奶粉样品混匀，准确称取样品15g于50ml比色管中，加入20%甲醇溶液（2.1）约35ml，震荡混匀，超声提取5min，加入5%三氯乙酸（2.2）5ml，并用20%甲醇溶液（2.1）定容至刻度，摇匀。以为3000rpm/min时超速离心5min。取上清液经0.45M滤膜过滤，备用。4.2色谱参考条件4.2.1流动相：10mmol/L己烷磺酸钠+ 10mmol/L柠檬酸乙腈= 928；4.2.2流速：
4、1.0ml/min；4.2.3柱温：35；4.2.4检测器：二极管阵列检测器（爸爸）或紫外检测器（UV），波长235nm；4.2.5色谱柱：C18柱子250mm4.6毫米，5M；4.2.6进样量：1020L4.3色谱分析：进样1020L标准使用液及样品处理液于高效液相色谱仪中，以保留时间及光谱定性，以样品峰面积与标准系列比较定量。5结果计算样品中三聚氰胺含量按下列公式进行计算。式中：X样品中三聚氰胺的含量，单位为毫克每千克（毫克/公斤）；C从曲线上查出样品中三聚氰胺浓度，单位为毫克每升（毫克/升）；V样品中总体积，单位为毫升（ML）；M样品质量，单位为克（G）。6色谱图图1三聚氰胺标准高效色谱
5、图（1三聚氰胺）2三聚氰胺标准品紫外吸收光谱图图图3奶粉样品三聚氰胺液相色谱图（1三聚氰胺）4奶粉样品中三聚氰胺的紫外吸收光谱图图7精密度在重复性条件下获得的两个独立测定结果的绝对差值不得超过算术平均值的10%。8其它8.1标准曲线及线性范围在上述实验条件下，分别取三聚氰胺标准使用液0.911、9.11、22.8、45.6、91.1mgL，以峰面积对比浓度绘制标准曲线，结果如下：线性方程及相关系数：a = 81。7c+56.3 r=0.9999Type: A- peak area; the concentration of c-, units of milligrams per liter (
6、mg/L)8.2 detection limitThe reference sample volume, capacity factors and instrument sample size, the method of the minimum detection limit was 5 mg/kg.8.3 precision experiment (n=7)According to the method of determination of melamine in milk powder, the parallel determination of 7 copies, the data
7、in table 1.Table 1 results of precision determination of melamine in milk powderDetermination of the number ofThe content of (mg/kg)OneForty-five point eightTwoForty-six point eightThreeForty-six point sevenFourForty-six point twoFiveForty-six point twoSixForty-five point nineSevenForty-sixaverage v
8、alueForty-six point twoRSD (%)Zero point nine8.4 recovery testBy adding the recovery experiment to examine the accuracy of the method, determination of melamine in milk powder recovery experiment results are shown in table 2.Table 2 melamine in milk spiked recovery experimentsSample nameThe backgrou
9、nd value of mg/kgAdd scalarMg/kgmeasured valueMg/kgThe recovery rate (%)The average recovery rate (%)Milk powder 1Six hundred and sixteenEighty-nine point twoSeven hundred and twoNinety-six point fourNinety-eight point fiveSeven hundred and oneNinety-five point threeSeven hundred and fourNinety-eigh
10、t point sevenThree hundred and sixty-fourNine hundred and seventy-eightNinety-nine point fiveNine hundred and sixty-sevenNinety-six point fourNine hundred and sixty-twoNinety-five point one1.46 x 1032.10 x 103One hundred and two2.10 x 103One hundred and two2.09 x 103One hundred and oneMilk powder 2S
11、eventy-three point nineEighty-nine point twoOne hundred and sixty-twoNinety-eight point eightNinety-nine point fiveOne hundred and sixtyNinety-six point fiveOne hundred and fifty-eightNinety-four point threeThree hundred and sixty-fourFour hundred and twenty-nineNinety-seven point sixFour hundred an
12、d thirty-eightOne hundredFour hundred and fiftyOne hundred and three1.46 x 1031.55 x 103One hundred and one1.57 x 103One hundred and two1.57 x 103One hundred and two8.5 interference experimentThe determination results of 8.5.1 reagent blank display reagent in the retention time of melamine were no i
13、nterference peaks.8.5.2 add nutrients in vitamin B1, vitamin B2, vitamin B6, vitamin C, biotin, folic acid, calcium pantothenate, nicotinic acid and nicotinamide on formula in accordance with GB10766-1997 provisions of 5 times the amount of interference test showed no interference.8.5.3 by UV detect
14、or (UV) determination results as positive, suggested using diode array detector (DAD) or mass spectrometry (MS) to further confirm.Reference：Determination of 1 Chinese, peoples Republic of China standard NY/T agriculture industry 1372-2007 melamine in feed2, Wang Hui, Cao Xiaoyan, Peng Xinkai, Chen
15、Liguo and other high performance liquid chromatography diode array method for the determination of melamine in high protein food and J food machinery, October 2007 twenty-third, volume fifth3, Zhang Meijin, Lin Haidan, Raymond Lam, Li Xun and other high performance liquid chromatography for the dete
16、rmination of melamine in feed and pet food hygiene inspection Chinese J magazine in December 2007 seventeenth volume twelfth4, Wendy C Anderson, Sherri B. Turnipseed, Christine M. Karbiwnyk, and Mark R. Madson Determination of Melamine Residues in Catfish Tissue by Triple Quadrupole LC-MS-MS with HILIC chromatography Laboratory Information Bulletin LIB No.4396 Volume 23, May 2007One