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1、液相色谱-串联质谱法测定奶和奶制品中三聚氰胺残留Determination of melamine residues in milk and dairy products by liquid chromatography tandem mass spectrometryLiu Han 1, Sun Ningning 1, Yang Wenquan 1, Liu Fei 2, Ding Tao 1, Xu chin Chung 11. 、 animal and vegetable and food inspection center of Jiangsu entry exit inspectio
2、n and Quarantine Bureau, Nanjing, 210001,2. thermofisher technology Shanghai Co. Ltd. Shanghai 201206, GC-MS,A method for determination of melamine residues in milk and dairy products by high performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC/MS/MS) was reported
3、. The protein in the sample was precipitated in three acetic acid solution, and the target analyte was extracted. The strong cation solid phase extraction column was enriched and purified by liquid chromatography electrospray ionization tandem mass spectrometry. Methods the low limit was 0.05 mg/kg,
4、 and the recovery rate of HPLC/MS/MS was between 72 and 87% (matrix matching curve correction). The relative standard deviation (RSD) ranged from 4.2% to 8.2%.Keywords melamine; milk and milk products; liquid chromatography tandem mass spectrometry;Determination, of, melamine, residues, in, and, dai
5、ry, milk, products, by, liquid, chromatography, tandem, mass, spectrometryHan, Liu1, Fei, Liu2, Ningning, Sun1, Wenquan, Yang1, Tao, Ding1, Jinzhong, Xu1,Animal, Plant, and, Food, Inspection, Center (APFIC), of, Jiangsu, Entry-Exit, Inspection, and, Quarantine, Bureau, Nanjing, 210001,2, Thermo, Fis
6、her, Scientific, Shanghai, 201206Abstracts: A method for analysis of melamine residues in milk and dairy products was developed by liquid chromatography tandem mass spectrometry (LC/MS/MS) Trichloroacetic solutions were used. Acid to precipitate proteins and dissociate the target analytes from sampl
7、e matrix. After matrix purified by strong cation exchange solid phase extraction cartridge, analyses can be performed using HPLC/MS/MS in the selected reaction monitoring (SRM) mode. The limit of dection is mg/kg when using HPLC/MS/MS method. The 0.05 mean recovery was 72 108% (matrix match calibrat
8、ion curve and the standard deviation was) 4.2 12.5% by HPLC/MS/MS method.KEYWORDS: melamine; milk, and, dairy, products; HPLC-MS/MSIn early September, Gansu and other places reported a number of cases of urinary tract stones in infants and young children, and the survey showed that the main reason f
9、or many children suffering from urinary lithiasis was melamine in the milk powder taken by children. Melamine is a kind of non food chemical raw material. It is forbidden to be used as food additive according to the regulation of the state. Some infant formula contains melamine, which is artificiall
10、y added to increase the amount of protein detected in raw milk or milk powder.Melamine (melamine) referred to the three amines, is a kind of important heterocyclic organic chemical raw material (structure shown in Figure 1), mainly for the production of melamine formaldehyde resin, widely used in wo
11、od, plastic, paint, paper, textile, leather, electrical, pharmaceutical and other industries, is currently the important urea processing products. In addition, melamine can also be used as flame retardant, water reducing agent, formaldehyde cleaner and so on. In some Asian countries, it is also used
12、 to make fertilizers. At the same time its also called a pesticide cyromazine metabolite 1. Melamine is a non food chemical substance, which is strictly prohibited in food. In order to increase the content of protein in milk and milk products and add a large amount of melamine, the consumption can l
13、ead to urinary system diseases 1.Figure 1 chemical formula of melamineFig.1三聚氰胺的化学结构目前测定三聚氰胺的检测方法包括气相色谱法 2 、气质联用法3-4、高效液相色谱紫外或二极管阵列和串联质谱检测方法5-8。气质联用方法需要衍生化，过程比较繁琐。高效液相色谱配合紫外或二极管阵列由于其无需衍生化是目前最为常用的检测方法，但是抗干扰能力差，定量限难以满足要求高效液相色谱串联质谱方法灵敏度高、抗干扰能力强，在三聚氰胺检测中具有优势。本文介绍了利用高效液相色谱电喷雾串联质谱联用（HPLCMSMS）测定奶和奶制品中三聚氰胺残
14、留的方法。利用三氯乙酸溶液沉淀蛋白，阳离子交换固相萃取柱净化，进行高效液相色谱串联质谱分析。经过大量样品分析结果表明，该方法快速、简单、抗干扰能力强，可以作为奶和奶制品中三聚氰胺残留的测定方法。1实验部分1.1仪器、试剂与材料赛默飞世尔TSQ量子液相色谱-三重四极杆质谱联用仪，配有电喷雾电离源。甲醇（色谱纯，德国默克），乙腈（色谱纯，德国默克），三氯乙酸、氨水（28%）、氢氧化钠（分析纯，南京化学试剂厂）、强阳离子交换柱（PCX，60mg 3ml艾杰尔公司）；水为重蒸蒸馏水。三聚氰胺标准品购买自西格玛奥德里奇公司，纯度98%。液态奶、奶粉、奶油等来自送检样品。1.2测定条件色谱条件：色谱柱Va
15、rian C18（150 mm内径2.1毫米，5m）；流动相：10mmol/L醋酸铵水溶液甲醇（90:10），等度洗脱；流速0.20毫升/分钟。柱温：室温。进样体积：5L.分析时间：9分钟。质谱条件：电喷雾离子化，正离子方式检测；毛细管温度350；喷雾电压4500 V；三聚氰胺的质谱条件见表1。1三聚氰胺质谱条件表表1三聚氰胺的电喷雾质谱化合物母离子（mz）子离子（mz）碰撞能量/电动车三聚氰胺一百二十七八十五六十八二十一十五1.3标准工作溶液的配制将三聚氰胺标准用甲醇水（2:8）配制成1 g/L的单标储备液，避光冷藏保存。按实际检测需求，用阴性样品提取后吹干，添加一定量的标准溶液和1%三氯乙
16、酸溶液配制相应浓度的标准工作溶液。1.4样品前处理称取（10.02）G样品于50毫升具塞离心管中，加入1%三氯乙酸溶液10毫升，于液体混匀器上快速混合30 s，超声波仪超声30min。离心5 min（8000 r/min），将上层清液过滤纸到干净离心管中，待净化。用3毫升3毫升甲醇、水分别活化强阳离子交换柱，将滤液上柱，3毫升水和3ml甲醇洗涤SPE小柱，3毫升氨水甲醇（5% V：洗脱后V），40水浴旋转蒸发至干。用1毫升三氯乙酸溶液（1%）定容后，过滤膜到进样瓶中，供液质联用仪测定。2结果与讨论2.1提取和净化条件的选择和优化三聚氰胺极性较强，因此提取溶剂一般都使用极性较强的体系：缓冲水溶液
17、、极性较强的有机溶剂或有机溶剂和水的混合溶剂。实验中比较了甲醇、甲醇水混合溶剂（1:1）、乙腈、乙腈水混合溶剂（1:1）、水、1%三氯乙酸水溶液和5%三氯乙酸水溶液作为提取溶剂，在阴性液态奶、奶粉、奶油中添加0.5毫克/公斤浓度的三聚氰胺标准，The extraction effects of different extraction solvents were compared.The results showed that the direct extraction rate of pure organic solvent was low, and the extraction effic
18、iency of other solvents was close. Found in a large number of samples, there are some milk samples in the use of water or a mixture of water and organic solvent extraction, very sticky, after high speed centrifugation or filtration filtration also cannot be difficult, and some constant volume sample
19、 solution in place 2hr after precipitation, extremely easy to cause the column chromatographic analysis or analysis of pre column blockage. 1%, three trichloroacetic acid solution can precipitate the protein in the sample, avoid this kind of phenomenon appearance. In experiment three, 5% and 10% of
20、the chloroacetic acid was used as the extraction solvent, increase the content of three chloroacetic acid protein precipitation effect is more obvious, but with the increase of the content of three chloroacetic acid in the extraction solvent, has a great impact on the chromatographic analysis of pea
21、k or non peak phenomenon. Finally, 1%, three chloroacetic acid solution was used as the extraction solvent.Due to the export of milk and milk products components are complex, directly after extracting three chloroacetic acid high performance liquid chromatography tandem mass spectrometry, prone to i
22、on source vents pollution leads to decreased sensitivity of the phenomenon, the need for purification of samples. Melamine contains three primary amine groups, which belongs to the basic compound, form positive ions in acidic conditions, using strong cation solid-phase extraction column to purify sa
23、mples, remove samples of water-soluble impurities, inorganic salt and non ionized lipophilic impurity 9 by washing water and methanol can. Experiments show that the exchange column used to purify the sample by using a strong cation, can remove impurities effectively, through the analysis of a large
24、number of samples, still can effectively prevent the occurrence of dirty samples resulted in pollution of nozzle.2.2 optimization of chromatographic and mass spectrometric conditionsMelamine is a strong polar compound, almost no reserve on the C18 chromatographic column, resulting in melamine and in
25、terfering substances can not be separated, causing interference to the detection. In order to increase the melamine retained in the C18 column, HPLC DAD analysis in general in the mobile phase adding ion pair reagent, such as sodium heptanesulfonate, sodium 1-octanesulfonate 8 and twelve sodium dode
26、cyl sulfate, and melamine to form neutral ion pair. In HPLC/MS/MS determination, the mobile phase can only use a volatile ion pair reagent, such as seven fluoro butyric acid 6. But the use of seven fluorine butyric acid, although retention can improve the melamine in the ordinary C18 column, but it
27、will lead to negative ions can not be normal determination, is generally not recommended, so the direct use of ammonium acetate aqueous solution and methanol as mobile phase 90:10.In the experiment, we found that using 1%, three chloroacetic acid solution, constant volume sample and preparing standa
28、rd solution can not only make the melamine play a good role in retention of common C18 analysis column, but also increase the sensitivity. May be due to three chloroacetic acid also has certain ion pair reagent function, can make the analysis of melamine in the ordinary C18 column which is left, whi
29、le three of chloroacetic acid added to melamine in the mobile phase is pre ionized form, leading to increased sensitivity.It was found that there was a matrix inhibitory effect in the determination of HPLC/MS/MS. With negative liquid milk, milk and cream after extraction, purification of SPE standar
30、d solution volume after the preparation compared with the same concentration of standard solution preparation directly, signal decreased by 30 60% or so, which directly leads to the low recovery rate. In order to compensate for the numerical deviation due to the matrix inhibition effect, the Matrix
31、matched calibration (curve) was used to correct the final result.2.3 linear range and quantitative limitAfter the negative matrix was extracted and dried, a series of standard solutions of corresponding mass concentration were prepared by adding a certain amount of standard solution and 1%, three ch
32、loroacetic acid. The linear regression of the peak area was carried out for the mass concentration of the analytes. The results show that when the concentration is 0.02 g/mL to 2 g/mL, the correlation coefficient (R) is 0.9981, and the linearity is good. The quantitative limit of this method is 0.05
33、mg/kg.2.4 recovery and precision testsMelamine standard solution was added to the negative liquid milk, milk powder and cream. The added level was 0.05 mg/kg, 0.10mg/kg and 0.20mg/kg, and 7 times were repeated, and the recovery and precision were calculated. The results are shown in table 2.Table 2
34、recovery and precision test results (n = 7)Table 2, Results, of, Recovery, and, precision, test (n=7)Dosage / (mg/kg)Liquid milkPowdered MilkDairyDetectable quantity (mg/kg)Recovery rate (%)RSD (%)Detectable quantity (mg/kg)Recovery rate (%)RSD (%)Detectable quantity (mg/kg)Recovery rate (%)RSD (%)Z
35、ero Point Zero FiveZero point zero three sevenSeventy-fourSix point eightZero point zero three sixSeventy-twoEight point twoZero point zero three nineSeventy-eightSeven point oneZero point oneZero point zero eight threeEighty-threeFour point fiveZero point zero seven sevenSeventy-sevenSix point sixZ
36、ero point zero eight twoEighty-twoFour point sevenZero point twoZero point one seven fourEighty-sevenFive point oneZero point one six fourEighty-twoFive point oneZero point one five eightSeventy-nineFour point twoFig. 2 Chromatogram and mass spectrum of melamine (0.1 mg/kg) added in milk powderFig.
37、2, Chromatography, and, mass, spectrum, of, melamine, spiked, in, milk, powder (, mg/kg)3 conclusionA HPLC/MS/MS method was established for the determination of melamine residues in milk and dairy products. Using three acetic acid solution to precipitate protein and extract melamine, cation exchange
38、 column purification, simple, fast, anti-interference ability. A large number of samples have been tested and proved to be suitable for monitoring and testing at home and abroad.Reference1.http:/www.cfsan.fda.gov/dms/melamra.html, May 25, 20072.Bardalaye, P., C., Wheeler, W., B., Meister, C., W., J.
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