免疫亲和柱净化/高效液相色谱-串联质谱法检测婴幼儿配方乳粉中生物素含量

建立了免疫亲和柱净化/高效液相色谱串联质谱法测定婴幼儿配方乳粉中生物素的方法。样品用磷酸盐缓冲液溶解,并通过生物素免疫亲和柱净化后,采用Ultimate AQ-C18(2.1 mm×100 mm,3μm)色谱柱分离,以甲醇-0.1%甲酸(30∶70)为流动相,进样量为5.0μL,流速为0.3 mL/min,柱温为30℃,并经电喷雾电离串联质谱在多离子反应监测(MRM)模式下进行测定,定量子离子为227.2,定性子离子为227.2、96.9,碰撞能量分别为10、30 V。结果显示,生物素在0.1~1.0μg/mL范围内线性关系良好,方法检出限为20.3μg/kg。对空白试样进行3个浓度水平的加标回收实验,测得加标回收率为92.7%~98.5%,相对标准偏差为1.7%~1.9%。该方法具有样品处理简单、灵敏度高、重现性好、分析时间短等优点,可以满足婴幼儿配方乳粉中生物素含量的测定要求。     

多组分免疫亲和柱净化/超高效液相色谱-串联质谱检测水产饲料中的黄曲霉毒素

建立了多组分免疫亲和柱净化/超高效液相色谱-串联质谱(UPLC-MS/MS)测定水产饲料中黄曲霉毒素AFB_1、AFB_2、AFG_1及AFG_2含量的方法。饲料样品用80%乙腈水超声提取后,经多组分免疫亲和柱净化,采用UPLC-MS/MS测定,外标法定量。以0.1%甲酸-乙腈为流动相,梯度洗脱分离,电喷雾正离子多反应监测模式检测。结果表明,AFB_1、AFG_1和AFB_2、AFG_2分别在2.25～22.5 ng/mL和0.75～7.5 ng/mL质量浓度范围内呈良好线性,相关系数大于0.997;AFB_1、AFG_1的定量下限均为0.7μg/kg,AFB_2、AFG_2的定量下限均为0.2μg/kg。AFB_1、AFG_1在1.5μg/kg,AFB_2、AFG_2在0.5μg/kg加标水平下的回收率为78.7%～85.5%,日内相对标准偏差(RSD)为6.0%～6.5%,日间RSD为6.6%～7.0%。该法操作简单,耗时少,重复性好,灵敏度高,适用于水产饲料中黄曲霉毒素的测定。     

复合免疫亲和柱净化-液相色谱-串联质谱法测定动物源食品中6种黄曲霉毒素和6种玉米赤霉醇类真菌毒素残留量

建立了动物源食品(猪肉、鱼肉、猪肝)中6种黄曲霉毒素(AFB1、AFB2、AFG1、AFG2、AFM1和 AFM2)和6种玉米赤霉醇类真菌毒素(α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮)残留量的复合免疫亲和柱净化-高效液相色谱-串联质谱(HPLC-MS/ MS)检测方法。样品经β-葡萄糖苷酸/硫酸酯复合酶酶解后,用甲醇-乙腈(20∶80, V/ V)提取,提取液经玻璃纤维滤纸过滤,滤液用PBS 溶液稀释,复合免疫亲和柱富集和净化后,采用 HPLC-MS/ MS 法分析。12种目标分析物中 AFB2和 AFG2的线性范围为0.03~6.0μg/ L,其余目标分析物的线性范围为0.05~20μg/ L,线性相关系数均大于0.999,检出限在0.01~0.03μg/ kg 范围内,定量限在0.04~0.09μg/ kg 范围内。分别以0.5,1.0和5.0μg/ kg 添加浓度水平进行方法学验证,平均回收率为73.6%~98.4%,相对标准偏差(RSD)为1.9%~11.2%。本方法简便、灵敏,能够满足动物源食品中痕量黄曲霉毒素和玉米赤霉醇类真菌毒素残留的测定要求。     

New lysine-acetylated proteins screened by immunoaffinity and liquid chromatography-mass spectrometry

The lack of selective extraction specific for lysine-acetylated proteins has been a major problem in the field of acetylation biology,though acetylation plays a key role in many biological processes.In this paper,we report for the first time the proteomic screening of lysine-acetylated proteins from a mouse liver tissue,by a new approach of immunoaffinity purification of lysine-acetylated peptides combined with nano-HPLC/MS/MS analysis.We have found 20 lysine-acetylated proteins with 21 lysine-acetylated si...     

单克隆抗体亲和柱分离荧光法测定花生和玉米中黄曲霉毒素B1的研究

本文建立了用单克隆抗体亲和柱分离荧光法测定花生和玉米中黄曲霉毒Ｂ１的方法，用６０％甲醇提取样品中的ＡＦＴ，经氢氧化铁沉淀除去大部分杂质，通过亲和柱将ＡＴＦＢ１进一步与杂质分离，甲醇洗脱后加溴处理，测定荧光强度，与标准比较定量。     

免疫亲和柱在兽药残留检测应用中的研究进展

免疫亲和柱(IAC)是一种有效的兽药残留检测净化技术,可以简化样品净化过程并且提高待测物的提取效率,近几年在兽药残留检测中得到广泛应用,并表现出良好的发展前景。本文简要叙述了IAC的原理、制备过程以及在各种抗微生物类兽药检测中的应用,就IAC对目前已研制出的抗微生物药的残留检测及净化效果进行综述,并展望了IAC在未来兽药残留检测应用中的发展趋势,为研究者提供参考和研发思路。     

Molecular diagnostics using analytical immuno high performance liquid affinity chromatography

Analytical immuno high performance liquid affinity chromatography (analytical immuno HPLAC) was evaluated as a molecular diagnostic tool. Antibodies raised in rabbits against bovine neurophysin II were immobilized through Protein A crosslinking onto coated silica. Interaction of immobilized antibody with mobile antigen was characterized by zonal and frontal elutions of¹⁴C-labeled bovine neurophysin II under isocratic, nondenaturing conditions. The Chromatographic behavior shows that analytical immuno HPLAC with immobilized antibodies can be used to detect the number and functional nature of matrix-interacting antigens in mixtures, thus providing a quantitative Chromatographic technology for “antigen mapping”.     

免疫亲和固相萃取-超高效合相色谱-串联质谱法同时测定牛奶中6种玉米赤霉醇类化合物残留

建立了免疫亲和固相萃取（IAC-SPE）-超高效合相色谱-串联质谱（UPC²-MS/MS）同时测定牛奶中α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉烯酮和玉米赤霉酮残留的分析方法。样品用去离子水稀释,经IAC-SPE富集净化后,采用Waters ACQUITY UPC² Torus 2-PIC色谱柱（50 mm×3.0 mm,1.7 μm）分离,以超临界CO₂和0.1%（v/v）甲酸甲醇溶液为流动相,经梯度洗脱后在ESI^-模式下检测。经过稀释离心的牛奶样品采用免疫亲和柱净化后没有明显的基质效应,6种目标化合物在1~200 ng/mL范围内线性关系良好,相关系数（r²）≥0.9957;6种目标化合物在3个加标水平下的平均回收率为75.9%~106.5%,日内和日间精密度均≤11.4%。该法专属性好,操作简便,有机溶剂使用量小,与已有的样品测定方法比较更绿色环保,可用于牛奶中α-玉米赤霉醇、β-玉米赤霉醇、α-玉米赤霉烯醇、β-玉米赤霉烯醇、玉米赤霉酮和玉米赤霉烯酮的残留检测。     

Evaluation of Fumonitest Immunoaffinity Columns

Abstract

A method for the determination of fumonisins B1 and B2 in corn was developed. The method involves sample extraction with methanol:water (80:20) and the use of a commercially available Fumonitest column for sample cleanup. The capacity, selectivity, column-to-column and lot-to-lot reproducibility of the Fumonitest columns were evaluated. The total capacity of the column was found to be 1.2 μg fumonisin. Both fumonisins B1 and B2 had an equal affinity toward the Fumonitest column, with the sample matrix demonstrating little effect on the column performance. The maximum sample size was 0.5 g for samples containing total fumonisins of less than 2 ppm. After elution from the immunoaffinity column, fumonisins B1 and B2 were reacted with naphthalene-2, 3-dicarboxaldehyde (NDA) to produce a highly fluorescent derivative, 1-cyano-2-alkyl-benz[f]isoindole (CBI). The derivatives were then separated from the sample matrix on a reverse phase C-18 column with a mobile phase consisting of acetonitrile:water:acetic acid (55:45:1) Average recoveries of fumonisins B1 and B2 from corn samples spiked at a level of 1000 ng (500ng B1 + 500ng B2)/g were 85.4 and 87.1%, respectively. The detection limit for B1 and B2 was estimated to be 10 and 4 ppb, respectively. The coefficient of variations for fumonisins B1 and B2 were determined to be 10.2% and 10.6%, respectively.     

Development of a liquid–chromatography tandem mass spectrometry and ultra-high-performance liquid chromatography high-resolution mass spectrometry method for the quantitative determination of zearalenone and its major metabolites in chicken and pig plasma

A sensitive and specific method for the quantitative determination of zearalenone (ZEN) and its major metabolites (α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), α-zearalanol (α-ZAL), β-zearalanol (β-ZAL) and zearalanone (ZAN)) in animal plasma using liquid chromatography combined with heated electrospray ionization (h-ESI) tandem mass spectrometry (LC–MS/MS) and high-resolution Orbitrap^® mass spectrometry ((U)HPLC–HR–MS) is presented. The sample preparation was straightforward, and consisted of a deproteinization step using acetonitrile. Chromatography was performed on a Hypersil Gold column (50 mm × 2.1 mm i.d., dp: 1.9 μm, run-time: 10 min) using 0.01% acetic acid in water (A) and acetonitrile (B) as mobile phases.