Residues of melamine and cyanuric acid in milk and tissues of dairy cows fed different doses of melamine

Melamine (1,3,5-triazine-2,4,6-triamine) may be degraded into cyanuric acid and some other compounds by rumen microorganisms. This study was conducted to assess the transfer of melamine and cyanuric acid in to milk and tissues by dairy cows fed different doses of melamine. Forty mid-lactation dairy cows (157 ± 43 d in milk, 20.8 ± 1.4 kg of milk/d) were divided into 4 groups (n = 10/group) using a completely randomized design. The groups were fed the following amounts of melamine (purity ≥ 99.5%) at 0 (control), 300 [treatment (Trt) 1], 500 (Trt 2), and 1,000 (Trt 3) mg/d per cow, respectively. The trial lasted for 18 d (12-d feeding period, followed by a 6-d clearance period). Milk samples were collected from the 4 groups on d 1, 2, 3, 4, 8, 12, 13, 14, 15, and 18, and analyzed for melamine and cyanuric acid. On d 13, 3 cows from Trt 2 and Trt 3 were randomly selected and slaughtered; tissue samples including kidney, liver, mammary, bladder, gluteus medius, and longissimus dorsi were collected for melamine and cyanuric acid analyses. Milk and tissue samples were analyzed for melamine and cyanuric acid using a simultaneous liquid chromatography tandem mass spectrometry procedure. Neither melamine nor cyanuric acid was detected in concentrated feed that was being fed to the cows. In melamine-treated groups, milk melamine concentration increased quickly and reached a stable level by d 4 and was at similar levels on d 8 and 12 after the first administration of melamine. Milk melamine levels in treated groups were 0.18, 0.27, and 0.50 mg/L for Trt 1, Trt 2, and Trt 3, respectively, and were highly correlated (R² = 0.91) with melamine dosing levels. No cyanuric acid was detected in any of the milk collected from the various groups. Melamine residue levels in tissues of Trt 3 were about 2-fold higher than that in Trt 2, with the highest concentration being found in kidney. No differences in cyanuric acid levels in tissues were found between Trt 3 and Trt 2. Liver, kidney, and bladder tissues were found to contain the highest cyanuric acid levels. This study shows a relationship between dietary melamine levels and cyanuric acid levels found in tissues, which might be the result of melamine being converted to cyanuric acid by microorganisms in the rumen.     

液体乳中三聚氰胺的快速检测产品评价研究

目的 评价液体乳中三聚氰胺的快速检测产品。方法 采用空白基质加标的方式制备了三聚氰胺3个不同浓度水平的牛奶盲样,并验证盲样的均匀性和稳定性,对3个不同厂家的三聚氰胺胶体金试纸条、3台不同的便携式拉曼光谱仪进行评价测试,系统性地研究了快速检测产品评价方法。结果 评价结果表明,各快速检测产品灵敏度分别为100%（5个）和99.3%（1个）,特异性均为100%,假阴性率为0%（5个）和0.7%（1个）,假阳性率均为0%。结论 本研究用于液体乳中三聚氰胺检测的快速检测产品符合性能指标的要求,满足市场监管的需要。     

钛胶正相高效液相色谱法同时分析三聚氰胺和三聚氰酸

建立了同时分析液态奶、自来水和游泳池水中三聚氰胺和三聚氰酸的钛胶正相高效液相色谱法。色谱条件:分离柱为Titania Sachtopore-NP柱（250mm×4.6mm×5μm）,流动相为水∶甲醇=50∶50（V/V）、流动相中含醋酸盐1.0mmol/L、pH7.0、流速0.8mL/min、柱温60℃、UV检测波长210nm。三聚氰胺和三聚氰酸分别在0.1～10μg/mL和2～100μg/mL范围内与对应的峰面积呈良好的线性关系,检出限分别为0.02μg/mL和0.1μg/mL,本方法三聚氰胺的回收率为94.8%～111.7%,三聚氰酸回收率为103.9%～114.3%,三聚氰胺和三聚氰酸测定结果的相对标准偏差分别为0.62%和0.78%（n=8）。      

HPLC-MS/MS同时检测大鼠体内三聚氰胺和三聚氰酸含量

目的建立HPLC-MS/MS同时检测大鼠血浆和组织中三聚氰胺和三聚氰酸含量的方法。方法以同位素标记的三聚氰胺和三聚氰酸作为内标物,大鼠血浆和组织经二氯甲烷-乙腈前处理后,经色谱柱分离,串联离子阱质谱采用ESI源按运行时间段切换正、负离子模式进行测定。结果在0.3～75μg/ml范围内,三聚氰酸和三聚氰胺具有良好的线性(r≥0.998)。在0.9、5.0、50μg/ml的给药大鼠的血浆样本中,三聚氰酸和三聚氰胺的回收率分别为97.4%～99.4%、96.8%～98.9%,两者的变异系数为2.43%～6.35%(日内)、1.70%～4.12%(日间)和0.30%～9.17%(日内)、0.75%～3.39%(日间);在0.9、5.0、50μg/ml的给药大鼠肝组织样本中,三聚氰酸和三聚氰胺的回收率分别为90.8%～104.6%、91.7%～106.0%,对应的变异系数为3.70%～5.14%(日内)、2.84%～4.50%(日间)和1.31%～2.21%(日内)、1.12%～3.36%(日间)。结论该方法样品前处理简单,基质干扰小,分析效率高,具有良好的精密度、准确度和特异性,是进行三聚氰酸、三聚氰胺在动物体内的组织代谢和毒性研究的理想分析方法。     

Aptamer-based rapid visual biosensing of melamine in whole milk

A rapid and facile visual method for the detection of melamine (MA) in whole milk based on aptamer-modified gold nanoparticles (AuNPs) was described in this paper. This strategy combined aptamer as a MA recognition element with AuNPs as the color indicator, providing a rapid and on-site detection of MA by naked eyes or UV–Vis spectrometer. The whole analytical process could finish in 30 min without any assistance of any instrument. Under the optimized condition, the proposed method could be used to detect MA in whole milk with a detection limit of 1.5 mg/L in naked eyes and 0.5 mg/L with UV–Vis spectrometer. The simple rapid technique provided a promising tool for on-site screening of MA adulterant in milk as well as in-house diagnosis.     

Detection of melamine and cyanuric acid in vegetable protein products used in food production

The multitude of food recalls in 2007 clearly demonstrated that total nitrogen-content (ΣN) determination by means of near-infrared spectroscopy (NIRS) and Kjeldahl-based measurements can be deceived, and should no longer be regarded as a complete quality assurance program for nutritive-protein evaluations. Furthermore, contemporary Canadian-employed analytical tools are precariously limited in their ability to effectively assure a product where there is no a priori knowledge of the environmental toxin(s) involved. In light of these challenges, this study explored a number of analytical techniques used to assess and furthermore assure the quality of vegetable protein products (VPPs). Using liquid chromatography with tandem mass spectrometry (LC/MS/MS) technologies, a combination of VPP-based samples was analyzed for the presence of nitrogen-bearing environmental toxicants. Of the 52 samples tested, involving an assortment of matrices, melamine and cyanuric acid were positively identified (>1 ng/mL) in 22 and 17 samples, respectively. Subsequent high pressure liquid chromatography with ultraviolet/visible (HPLC-UV) amino acid profiling further confirmed the adulteration of those materials contaminated with melamine and melamine-related compounds. Based on the evidence presented herein, LC/MS/MS in combination with HPLC-UV provides for a reliable food safety detection system as applied to VPPs. Moreover, HPLC-UV is indispensable as a stand-alone 1st level of screening to assess the integrity of a VPP or any nutritive protein-based sample.     

应用核酸层析技术快速检测奶粉中阪崎肠杆菌的研究

结合PCP与胶体金免疫层析试纸条技术建立了核酸层析检测技术用于奶粉中阪崎肠杆菌的快速检测.该技术根据寡-1,6-葡萄糖苷酶基因设计引物,并分别标记生物素和地高辛,通过试纸条上肉眼可见的红色条带对扩增结果进行判断.通过11株阪崎肠杆菌及27株常见致病菌的检测结果说明其特异性强,阪崎肠杆菌纯菌培养检测灵敏度达到103 mL-1,人工污染样品检测限达到0.08 g-1,检测奶粉样品时与传统方法(GB/T 4789.40-2010)相比,无显著性差异(x2=0,P＞0.05).该方法灵敏度高、快速、特异性强,可以很好地应用于奶粉以及其他食品中阪崎肠杆菌的检测与鉴定.     

A new approach to measure melamine, cyanuric acid, and melamine cyanurate using surface enhanced Raman spectroscopy coupled with gold nanosubstrates

Vibrational spectroscopic characteristics of melamine, cyanuric acid, and melamine cyanurate were measured using surface-enhanced Raman spectroscopy (SERS) coupled with gold nanosubstrates. Trace amounts of melamine and its analogues (cyanuric acid and melamine cyanurate) were characterized and quantified quickly and accurately by SERS in combination with partial least squares (PLS) analysis. Based on the relationship between the Raman intensity of the most prominent peak at around 676 cm⁻¹ and log values of melamine concentrations, the limit of detection (LOD) of SERS for melamine was estimated to be 2.6 × 10⁻⁷ mol L⁻¹ (∼33 ppb). An approximately 3 × 10⁴ fold of enhancement factor for SERS signals of melamine on gold nanosubstrates was obtained. This result was based upon the comparison of the peak at around 676 cm⁻¹ in the SERS spectra with that of normal Raman spectra of melamine in aqueous solutions. SERS spectra of cyanuric acid acquired from its solid form differ significantly from this compound in an aqueous solution, indicating a possible keto-enol isomerization reaction of cyanuric acid in water. When equal amounts of melamine and cyanuric acid were added together, spoke-like crystals of melamine cyanurate formed instantly, which could be measured and characterized by SERS. This study demonstrates that SERS could provide a fast and ultra-sensitive tool for detection and characterization of melamine and its derivative compounds in aqueous solutions.     

牛奶中掺入尿素的两种快速检测方法

建立了对二甲氨基苯甲醛法和亚硝酸盐——格里斯试剂法两种对牛奶中掺入的尿素的快速检测方法。通过对显色剂剂量和酸度等反应条件的研究和选择，确定了两种方法的最佳实验条件。     

牛奶中掺入尿素的两种快速检测方法

建立了对二甲氨基苯甲醛法和亚硝酸盐——格里斯试剂法两种对牛奶中掺入的尿素的快速检测方法。通过对显色剂剂量和酸度等反应条件的研究和选择,确定了两种方法的最佳实验条件。     

High-performance liquid chromatographic method for the simultaneous detection of the adulteration of cereal flours with melamine and related triazine by-products ammeline, ammelide, and cyanuric acid

Melamine has been used for the adulteration of cereal flours in order to increase their apparent protein content. Crude melamine may contain several by-products, i.e. ammeline, ammelide, and cyanuric acid. The simultaneous analysis of all four chemicals is difficult because of the formation of an insoluble salt between melamine and cyanuric acid. A simple and convenient high-performance liquid chromatography (HPLC) method for the detection of the adulteration of cereal flours with all four chemicals is proposed herein. The precipitate formation between melamine and cyanuric acid was prevented by using alkaline conditions (pH 11-12) for both standards preparation and sample extraction. The method uses matrix-matching, which involves the construction of a calibration curve on a blank (negative control) matrix, which is then used for the quantitation of melamine and by-products in adulterated (positive) samples. Matrix-matching compensates for analyte losses during sample preparation, and for matrix effects. The method was successfully applied to wheat, corn, and rice flours, and is expected to be applicable (with some modifications) to soy flour as well. The method allows for the detection of melamine, ammeline, and ammelide at approximately 5 µg g^-1, and cyanuric acid at approximately 90 µg g^-1 in wheat flour.     

利用不确定度比对乳粉中叶酸含量的快速检测方法

目的 利用不确定度比对乳粉中叶酸含量的快速检测方法。方法 分别用进口和国产的微生物法和酶联免疫法的试剂盒对代表性乳粉样品进行3 d 3平行检测, 对检测数据做正态分布分析及不确定度计算。结果 各方法检测结果正态分布检验值(anderson-darling, AD)均小于临界值0.725(95%置信区间), 服从正态分布; 微生物快速检测方法不确定度范围为13.3%~27.4%; 酶联免疫方法不确定度范围为9.6%~ 31.3%。结论 微生物试剂盒快速检测方法, 具有稳定性较好, 品牌差异较小的特点, 适合质控品的长期监测, 质控图的建立。酶联免疫检测方法, 具有简单、快速的特点, 适合样品中叶酸的快速检测。     

检测三聚氰胺胶体金免疫层析试验的建立

采用混合酸酐法制备三聚氰胺-OVA偶联抗原,胶体金标记三聚氰胺单克隆抗体,建立了检测三聚氰胺的胶体金免疫层析试验.该试验具有较好的敏感性,最低可检测50μg/L的三聚氰胺.胶体金免疫层析试验对原料奶和奶粉样品中三聚氰胺的最低检测量分别为250μg/L和1000μg/L,可初步应用于基层实验室快速筛查检测.     

牛奶中钙、钠快速检测方法的研究

目的为了更好地控制原料奶及成品的产品质量,形成一种简便的检测方法,更快更准确地鉴别掺假牛奶。方法实验选用传统的国标方法、直接进样法、标准加入-直接进样法三种不同的试验方法对几种产品进行检测,并比对各种方法的优劣,从而得到较理想的方法。结果各试验组的钙、钠检出限分别为0.42mg/100 g和3.75 mg/100 g、0.38 mg/100 g和0.96 mg/100 g、0.38 mg/100 g和0.96 mg/100 g。结论标准加入-直接进样法是一种新型的、快速且准确的检测方法。     

多重PCR快速检测婴幼儿奶粉中的病原菌

为了建立同时检测婴幼儿奶粉中阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌的多重PCR方法,根据阪崎肠杆菌ompA基因、金黄色葡萄球菌nuc基因、蜡样芽孢杆菌hblA基因分别设计3对引物进行多重PCR扩增,并对反应条件进行优化。结果多重PCR扩增出长度为514、156、235bp的特异性目的条带。不增菌的情况下,多重PCR同时检测3种病原菌的灵敏度是103cfu/mL,3种病原菌在奶粉中的检出限是104cfu/g。建立的多重PCR反应准确、快速、高效,为同时检测婴幼儿奶粉中的阪崎肠杆菌、金黄色葡萄球菌、蜡样芽孢杆菌提供了新方法。     

SERS detection of urea and ammonium sulfate adulterants in milk with coffee ring effect

In the current work, surface-enhanced Raman scattering (SERS) based on gold nanoparticles (AuNPs) and silver-coated gold nanoparticles (Au@AgNPs) with coffee ring effect was employed to simultaneously ascertain urea and ammonium sulfate (AmS) in milk. A small drop (2 µL) of milk with adulterants was dried on a gold-coated slide to examine concentrations ranging from 5, 10, 20, 40 and 80 mg/dL based on spectra ranging from 400 to 1500 cm.^?1 A uniform distribution of analytes, with enhanced Raman signals was detected in a small region (maximum 1.9 mm) of coffee ring across the centre of coffee ring pattern. Nanoparticles with core (Au) diameter of 26 nm and shell thickness (Ag) of 6.5 nm were confirmed using transmission electron microscopy (TEM) images. A strong Raman peak at 980 cm^?1 was assigned to AmS, while that at 1001 cm^?1 was ascribed to urea. With AuNPs, coefficients of determination (R²) of 0.9873 and 0.9859 were achieved for urea and AmS, respectively, while for Au@AgNPs values of 0.9827 and 0.9855 were obtained for urea and AmS, respectively. This study revealed that SERS based on coffee ring effect has the potential to be further exploited for detecting other banned and hazardous adulterants in milk and milk products.     

牛奶中三聚氰胺的表面增强拉曼光谱检测法与 国标检测法的比较

目的 比较表面增强拉曼光谱法与国标法检测牛奶中三聚氰胺。方法 分别用表面增强拉曼光谱法和国标法(液相色谱-质谱法)检测牛奶中的三聚氰胺。结果 表面增强拉曼光谱快检法与国家标准方法GB/T 22388-2008第二法 液相色谱-质谱/质谱法在阳性样品的判定上结论一致;表面增强拉曼光谱法的平均回收率和精密度分别为117.3%和13.2%,国标法的平均回收率和精密度分别为106.2%和8.9%。结论 表面增强拉曼光谱法适用于牛奶中三聚氰胺的快速筛查;进一步的定量分析需要用国标法。     

Visual detection of melamine in raw milk using gold nanoparticles as colorimetric probe

We report the development of a simple and rapid colorimetric detection method for melamine in raw milk using gold nanoparticles as probe. This assay relies upon the distance-dependent optical properties of gold nanoparticles. In neutral media, melamine could rapidly induce the aggregation of gold nanoparticles, thereby resulting in red-to-blue (or purple) colour change. The concentration of melamine in raw milk can be determined by monitoring with the naked eye or a UV–vis spectrometer. The present limit of detection for melamine is 0.4 mg/L. The method is rather simple, and the whole process including sample pretreatment takes only 12 min at room temperature. The merits (such as simplicity, rapidity, low cost and visual colorimetry) make the proposed method specially useful for on-site screening melamine levels well below the current safety limit in raw milk.     

表面增强拉曼光谱法快速测定液态乳中三聚氰胺方法的研究和评价

目的 建立便携式拉曼光谱法定性检测液态乳中三聚氰胺的快速检验方法.方法 选择市场上有代表性的拉曼光谱仪,确定检测方法.探究增强试剂、常见的添加剂及原料对检测结果的影响以及三聚氰胺类似物对检测结果的影响,确定方法的适用范围并被验证.对指定的3家验证机构进行方法验证.结果 5家企业的快速检测产品中有3家在验证中完全满足方法...     

近红外光谱技术检测液态奶中微量三聚氰胺的可行性研究

为了快速检测液态奶中违法添加的三聚氰胺量,利用近红外光谱技术进行可行性研究分析。对采集的54组添加不同三聚氰胺含量的液态奶样本的近红外漫反射光谱进行小波变换(wavelet transform,WT)结合主成分分析(principal component analysis,PCA)后,提取得到特征信号,利用广义回归神经网络(general regression neural network,GRNN)建模进行三聚氰胺的定量分析,结果显示当三聚氰胺浓度低于0.05%时,模型预测能力较差,预测集模型R2仅为0.654。进一步利用支持向量机(support vector machine,SVM)建模对液态奶中是否添加三聚氰胺进行定性判别,结果显示测试集准确率为94.44%。因此,通过近红外光谱结合适当的化学计量学方法,对液态奶中添加三聚氰胺虽无法精确定量,但可准确对其进行定性判别。