超高效液相色谱检测奶及奶制品中的M族黄曲霉毒素

目的建立一种免疫亲和柱净化-超高效液相色谱法同时测定奶及奶制品中M族黄曲霉毒素的方法。方法以乙腈为提取剂和蛋白沉淀剂,采用涡旋混合及超声提取,免疫亲和柱净化。结果经Welch Ultimate XB-C18色谱柱(100 mm×2.1 mm,1.8μm)分离,应用大体积流通池荧光检测,流动相为水和乙腈/甲醇(1:1)。线性范围在0.01~5.00 ng/mL之间,线性系数均大于0.999,黄曲霉毒素M1、M2的检出限为0.003μg/kg。免疫亲和柱对黄曲霉毒素M1、M2的回收率均在80%以上,液态奶中添加0.05、0.2、1.0μg/kg的回收率在85.6%~106.4%之间,奶粉中添加0.5、2.0、10.0μg/kg的回收率在81.5%~93.4%之间。结论该方法可以适用于奶粉、液态奶中M族黄曲霉毒素检测。     

免疫亲和柱-高效液相色谱紫外法检测牛奶及奶粉中黄曲霉毒素M_1

建立免疫亲和柱富集净化、高效液相色谱-紫外法检测牛奶及奶粉中黄曲霉毒素M1的分析方法。该方法样品经离心处理、免疫亲和柱净化,即可应用液相色谱-紫外法检测。结果表明,不同加标水平下牛奶和奶粉添加黄曲霉毒素M1回收率分别为87.4%~108%、89.5%~103%,RSD分别为2.61%~7.83%和2.07%~4.15%。方法检测灵敏度为0.1 ng/mL牛奶、2.0 ng/g奶粉。该方法能够特异性的检测样品中的黄曲霉毒素M1,不需荧光检测器就能达到较高的灵敏度,前处理简便,实用性强。     

同位素内标-高效液相色谱-串联质谱法检测牛奶及奶粉中黄曲霉毒素M1

目的 建立一种高效液相色谱-串联质谱法(high performance liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)测定奶及奶粉中黄曲霉毒素M1的方法。方法 样品经甲醇溶液提取后, 经离心机离心, 上层清液用MycoSep?226 AflaZon+Multifunctional净化柱净化, 在线添加黄曲霉毒素M1同位素内标后采用LC-MS/MS进行检测。结果 黄曲霉毒素M1在0.05~5.0 μg/L范围内线性关系良好, 相关系数大于0.995; 在0.02、0.05和0.10 μg/L添加水平的回收率为89.02%~118.85%, 相对标准偏差小于7.06%(n=6), 定量限为0.06 μg/kg。结论 该方法快速、准确、灵敏, 适合用于测定奶及奶粉中黄曲霉毒素M1。     

超高效液相色谱串联质谱测定乳及乳制品中AFM1、AFM2

利用免疫亲和萃取结合超高效液相色谱-串联四极杆质谱技术(UPLC-MS/MS)建立了乳及乳制品中黄曲霉毒素M1和M2的提取、分离、确证与定量方法。乙腈和水提取样品中的黄曲霉毒素,乙腈二次去蛋白后用免疫亲和柱净化,采用Waters Acquity UPLC RHSS T3(2.1×100 mm,1.7μm)色谱柱实现分离,多反应监测(MRM)模式下以外标法定量。结果表明:黄曲霉毒素M1和M2在0.1μg/L~50μg/L范围内线性关系良好(r﹥0.998 5);检出限为0.029μg/L~0.047μg/L(S/N=3);2种黄曲霉毒素各添加水平在鲜牛奶、奶粉和奶油中的回收率为在69.56%~88.43%之间;相对标准偏差为3.69%~8.74%。     

高效液相色谱法测定牛奶中黄曲霉毒素M_1的含量

采用高效液相色谱荧光法测定牛奶中的黄曲霉毒素M1。牛奶中的黄曲霉毒素M1用乙腈超声提取,离心,上清液浓缩后用50mLPBS缓冲液稀释,过免疫亲和层析柱净化,10%甲醇水溶液10mL淋洗柱,2mL甲醇洗脱,洗脱液氮吹干后,用10%乙腈水溶液1mL蜗旋溶解残渣,过0.22μm滤膜,上机测定。对加入乙腈比例、淋洗液及洗脱液进行了优化,结果表明:m牛奶:V乙腈=1:2有效的沉淀蛋白避免了过柱堵塞问题,提高了过柱效率。用10%甲醇水溶液淋洗免疫亲和柱,能有效的去除掉杂质,避免了洗脱液成浑浊现象。该方法的线性范围宽,回收率为89.1%~93.5%,相对标准偏差在6.5%~9.7%之间,检出限为0.01μg/kg,低于1/10现行食品安全国家标准对食品中黄曲霉毒素M1的限量标准,符合检测要求,适用于对牛奶中黄曲霉素素M1的检测。     

免疫亲和层析净化-高效液相色谱法同时测定牛奶和奶粉中黄曲霉毒素B1、B2、G1、G2、M1、M2

建立牛奶和奶粉中黄曲霉毒素B1、B2、G1、G2、M1、M2的免疫亲和层析净化和柱后衍生高效液相色谱测定方法。样品经溶解、离心、过滤后,通过免疫亲和柱,黄曲霉毒素特异性抗体选择性地与存在的黄曲霉毒素抗原键合,形成抗体-抗原复合体。甲醇-乙腈混合溶液(4:5,v:v)洗脱,带荧光检测器的高效液相色谱仪经柱后衍生测定,外标法定量。标准曲线线性良好,添加回收率在57.0%～88.7%,相对标准偏差在3.37%～16.9%,牛奶中各黄曲霉毒素检出限:B1为2ng/kg,B2为1ng/kg,G1、G2为3ng/kg,M1、M2为5ng/kg;奶粉中B1为20ng/kg,B2为10ng/kg,G1、G2为30ng/kg,M1、M2为50ng/kg,检测低限能够满足各国对牛奶和奶粉中黄曲霉毒素的限量要求。该方法准确、快速、灵敏度高,适用于牛奶和奶粉中黄曲霉毒素的测定。     

HPLC法测定乳制品中的黄曲霉毒素M1

目的 建立孔制品中黄曲霉毒素M1的HPLC的检测方法.方法 样品经提取、过免疫亲和柱净化后,用高效液相色谱-荧光检测器进行分析.结果 黄曲霉毒素M1在0.1～1 μg/L范围内线性关系良好,γ＞0.999.回收率在90%～110%之间,定量限为5 pg,检测限为2 pg.结论 该方法快速、简便、准确,改善了现有国标GB/T18980-2003操作繁琐,测定结果易受操作影响的缺点.可作为乳制品中黄曲霉毒素M1的定量测定.     

免疫亲和柱净化-大体积流通池高效液相色谱法同时检测食品中6种黄曲霉毒素

建立了免疫亲和柱净化-大体积流通池无需衍生同时测定食品中6种黄曲霉毒素(Aflatoxins)的检测方法。样品采用乙腈-水(84:16,V/V)超声辅助提取,用免疫亲和柱净化,经过XBridge TM C18柱(150 mm×4.6 mm,5μm)分离。采用乙腈-甲醇-水(15:25:60,V/V)为流动相,大体积流通池荧光检测器检测,无需衍生,外标法定量。6种黄曲霉毒素在9.5 min内有效分离,从样品前处理到结果分析整个过程小于50 min。黄曲霉毒素B1、B2、G1、G2、M1、M2的检出限(LOD,S/N=3)分别为0.05μg/kg、0.02μg/kg、0.05μg/kg、0.02μg/kg、0.04μg/kg、0.03μg/kg,能够满足我国对食品中黄曲霉毒素限量的要求。6种黄曲霉毒素标准曲线线性良好,线性相关系数r2大于0.999;样品加标回收率为77.6%~90.5%,精密度RSD为2.42%~6.08%。本方法简便、准确、灵敏度高,无需衍生即可同时检测食品中6种黄曲霉毒素,适用于食品中6种黄曲霉毒素的快速定量测定。     

单克隆免疫亲和柱-高效液相色谱法测定牛乳中黄曲霉毒素M1

研究了单克隆免疫亲和柱－高效液相色谱法测定牛乳中黄曲霉毒素M1的方法。样品通过离心脱脂,脱脂乳用免疫亲和柱净化,SpherisorM C18色谱柱分离,荧光检测器检测,外标法定量。对添加不同含量水平的黄曲霉毒素M1 5次重复实验的平均回收率为80．4％－88．7％。变异系数（CV）为7．7％－8．9％。检测低限为0．05μg/kg。     

冷冻-离心净化-高效液相色谱法测定植物油中黄曲霉毒素B1

通过冷冻-离心净化,建立了高效液相色谱定量测定植物油中黄曲霉毒素B1的方法,并将其用于测定市场上30批植物油产品中黄曲霉毒素B1的含量。以水/乙腈溶液为提取剂对植物油中黄曲霉毒素B1进行萃取,低温冷冻固化植物油,冷冻离心使植物油和提取液分离、净化提取液,经衍生后上液相色谱进行定量分析。优化的乙腈/水提取液配比为90:10,低温冰箱冷冻温度为-12 ℃,冷冻离心转速为12000 r/min、离心力约为1.4万 × g,以水浴加热的方式进行衍生。方法的定量检出限为0.02 μg/kg,校准曲线回归方程为y=2.321987x＋0.001377,相关系数（R2）为0.9997,在0.10～5.0 μg/L范围内线性关系良好。当样品中黄曲霉毒素B1添加量为0.5、1.0、5.0 μg/kg时,平均回收率为80.2%～93.2%,相对标准偏差为2.9%～4.7%（n=6）,均优于免疫亲和柱净化处理的结果。市售植物油产品中黄曲霉毒素B1的浓度范围为< LOD至5.72 μg/kg,均低于GB 2761—2017中对该指标的限值,花生油和玉米油中黄曲霉毒素B1均有检出,油茶籽油和核桃油中黄曲霉毒素B1均低于检出限。该方法样品前处理简便、稳定性好、检出限低,适合植物油中黄曲霉毒素B1的批量检测。     

牛奶及奶粉中黄曲霉毒素M1的快速测定

采用免疫亲和柱－荧光光度法快速测定了牛乳及乳制品中黄曲霉毒素M1。试样经过离心、脱脂、过滤后滤液经过键合有黄曲霉毒素M1特殊抗体的免疫亲和柱净化，此抗体对黄曲霉毒素M1具有专一的识别能力，黄曲霉毒素M1键合在分离柱中的抗体上，用甲醇与水之比为10：90的混合液将免疫亲和柱上杂质除去；以甲醇与水之比为80：20的混合液通过分离柱洗脱；加入溴溶液衍生，以提高测定灵敏度，衍生化后的洗脱液于荧光光度计中测定黄曲霉毒素M1，检测低限为0．1μg/kg；在0．1－1．0μg/kg范围内，回收率为89．6％－96．1％，变异系数为0．52％－5．8％，分析一个样品的时间小于30min，分析过程中不使用黄曲霉毒素M1标准物质，结果表明，本方法具有准确、简单、快速、安全等优点，可以满足少量和批量样品的检测需要。     

酶联免疫法测定婴幼儿配方乳粉中黄曲霉毒素M1的方法研究

采用酶联免疫法建立婴幼儿配方奶粉中黄曲霉毒素M1的测定方法。样品经冷冻离心、脱脂棉去脂等简便的前处理操作,采用黄曲霉毒素M1的酶联免疫试剂盒进行分析。结果显示,黄曲霉毒素M1在0～0.08 μg/L范围内线性关系良好,方法检出限为0.05 μg/kg,回收率为90.0%～105.0%,精密度相对标准偏差（RSD）为8.1%。该方法快速、简便、特异、可靠,适于现代批量检测的需求,为保证乳制品的质量安全提供了一可靠的筛选方式,将更好地配合液相色谱-质谱法作定性和定量分析。     

基于群青蓝纳米粒子的免疫层析法快速检测牛奶中的黄曲霉毒素M1

目的 建立一种基于群青蓝纳米粒子的免疫层析法(immunochromatographic assay, ICA),用于快速检测黄曲霉毒素M1(aflatoxin M1,AFM1)。方法 通过离心筛选出合适粒径的群青蓝纳米粒子,经羧基化修饰后与AFM1单克隆抗体通过碳二亚胺法共价偶联,制备免疫探针,用于AFM1检测。实验结果通过肉眼观察并结合Image J 软件进行定性定量分析,对所建立方法的灵敏度、特异性、准确性等进行评价。结果 经条件优化后,该方法定性视觉检测限为0.30 ng/mL,定量检测限(limit of detection, LOD)为0.051 ng/mL,与其他真菌毒素无交叉反应。结论 该方法可应用于牛奶样品中AFM1检测,检测结果符合国家限量标准,同时操作简单易于判断、特异性好,适用于现场快速初步筛查。     

ELISA法测定牛奶中黄曲霉毒素M1不确定度评定

目的本实验对酶联免疫法检测牛奶中黄曲霉毒素M1含量的测定结果进行不确定度的评定,确保检测结果的准确性及可靠性。方法实验分析了酶联免疫法测定牛奶中黄曲霉毒素M1的不确定度的分量及其来源,并通过计算各分量的不确定度得出检测结果的合成标准不确定度。结果酶联免疫法测定牛奶中黄曲霉毒素M1的浓度为45.741 pg/m L,扩展不确定度为11.704 pg/m L,置信水平P=95%,k=2。结论不确定度的主要来源为测量的重复性、试剂盒的灵敏度、标准曲线拟合,而酶标仪测定OD值、ELISA法操作过程中导致的不确定度可以忽略不计。选用酶联免疫法检测黄曲霉毒素M1时增加平行样的测定、注意试剂盒的灵敏度、保持标准曲线的稳定性对于提高酶联免疫法检测的准确性和可靠性具有较强的实用价值。     

A simple steady-state model for carry-over of aflatoxins from feed to cow's milk

A simple steady-state model is derived from two kinetic one-compartment models for the disposition of aflatoxin B₁ (AFB₁₎ and aflatoxin M₁ (AFM₁) in the lactating cow. The model relates daily intake of AFB₁ in feed of dairy cattle and the cow's lactation status to resulting concentrations of AFM₁ in milk. Moreover, assuming a linear relationship between the cow's lactation status and feed intake, the model relates daily milk production and AFB₁ concentration in total feed to AFM₁ levels in milk. The model explains similar experimental outcomes from different investigations into carry-over of aflatoxins from feed to milk. Although it is difficult to set a permanent limit for AFB₁ in feed, the European Union (EU) limit of 5 µg AFB₁ kg^-1 concentrate has proved, thus far, to be an appropriate level in preventing the EU limit of 0.05 µg AFM₁ kg^-1 milk being exceeded.     

Occurrence of aflatoxin M1 in domestic milk in Japan during the winter season

A total of 208 samples of commercial pasteurized milk gathered from retail outlets across Japan during the winter season were analysed for aflatoxin M₁ (AFM₁). Japan was divided into 11 regions from north to south, and nine to 45 milk samples from each region were randomly purchased between December 2001 and February 2002. Each milk sample was cleaned up by an immunoaffinity column, and AFM₁ was quantified by liquid chromatography with fluorescence detection in four independent laboratories. The limit of detection of the method was 0.001 μg kg^-1. The identity of the putative AFM₁ in milk sample was confirmed by the formation of AFM₁ hemi-acetal with trifluoroacetic acid. Based on the results obtained with spiked samples (0.05 μg AFM₁ kg^-1), the mean recovery was 91.4%, the relative standard deviation for repeatability was 4.6%, and the relative standard deviation for reproducibility was 8.0% among four independent laboratories. AFM₁ was detected in 207 (99.5%) of 208 milk samples at 0.001-0.029 μg kg^-1, with a mean of 0.009 μg kg^-1 and a 90th percentile of 0.014 μg kg^-1. No significant difference of the level of AFM₁ contamination was observed among the regions.     

Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples

Forty-nine samples of raw cow's milk and 20 samples of fresh white soft cheese were collected directly from 20 local dairy factories in the north-west of Libya and analysed for the presence of aflatoxin M₁ (AFM₁). The samples were analysed using a high-performance liquid chromatography technique for toxin detection and quantification. Thirty-five of the 49 milk samples (71.4%) showed AFM₁ levels between 0.03 and 3.13 ng ml^-1 milk. Multiple analyses of five milk samples free of AFM₁ artificially contaminated with concentrations of AFM₁ at 0.01, 0.05, 0.1, 1.0 and 3.0 ng ml^-1 showed average recoveries of 66.85, 72.41, 83.29, 97.94 and 98.25%, with coefficients of variations of 3.77, 4.11, 1.57, 1.29 and 0.54%, respectively. Fifteen of 20 white soft cheese samples (75.0%) showed the presence of AFM₁ in concentrations between 0.11 and 0.52 ng g^-1 of cheese. Multiple assays of five cheese samples free of AFM₁ spiked with different concentration of AFM₁ (0.1, 0.5, 1.0 and 3.0 ng g^-1) showed average recoveries of 63.23, 78.14, 83.29 and 88.68%, with coefficients of variation of 1.53, 9.90, 4.87 and 3.79%, respectively. The concentrations of AFM₁ were lower in the cheese products than in the raw milk samples.     

Determination of aflatoxins by enzyme-linked immunosorbent assay with special reference to aflatoxin M1 in raw milk

There is an increasing requirement for the screening of animal feedstuffs, plant products and edible animal tissues for human consumption, for the presence of contamination by aflatoxins. An enzyme-linked immunosorbent assay is described which may be used to determine aflatoxins in raw milk at 0.1μg litre^?1 and in extracted feedstuffs at the level of 5μgkg^?1. The method includes a novel conjugation procedure allowing heterologous conjugates to be prepared resulting in increased sensitivity without interference by sample matrix effects. Suitably cross-reactive antisera were produced using the cyclopentanone ring of the aflatoxin molecule as the principal immunogenic determinant.