免疫亲和净化-液相色谱-串联质谱法测定贝类中腹泻性贝类毒素

建立贝类中腹泻性贝类毒素的免疫亲和净化-液相色谱-串联质谱分析方法。样品采用80%甲醇溶液提取,选择磷酸盐缓冲液与提取液(4∶1,V/V)混合稀释后,免疫亲和选择专一净化,液相色谱-串联质谱分析。根据腹泻性免疫亲和柱的使用特性,对上样液、淋洗液、洗脱液等参数进行优化。质谱采用电喷雾负离子电离,多反应监测模式,外标法定量。3种分析物在1.0~100μg/L质量浓度范围内线性相关系数均大于0.996,对应的检出限和定量限均为0.3μg/kg和1.0μg/kg,平均回收率为82.7%~94.3%,相对标准偏差为0.70%~7.61%。本方法基质干扰小、净化效果强、灵敏度高,适合贝类中腹泻性贝类毒素的分析测定。     

不同净化柱处理对小麦粉中4种镰刀菌毒素的净化效果

比较MycoSep~?226多功能净化柱、DZT MS-PREP免疫亲和柱和实验室制备聚苯乙烯-二乙烯苯树脂免疫亲和柱在检测小麦粉中4种镰刀菌毒素(玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素、HT-2毒素)过程中的前处理效果。选择MycoSep~?226多功能净化柱、DZT MS-PREP免疫亲和柱和实验室制备聚苯乙烯-二乙烯苯树脂免疫亲和柱,通过不同的提取和前处理方法,以及建立高效液相色谱-串联质谱检测法,对小麦粉中的4种镰刀菌毒素进行测定。DZT MS-PREP免疫亲和柱处理小麦粉样品所得的回收率为88.01%~107.31%,相对标准偏差为1.09%~14.42%;聚苯乙烯-二乙烯苯树脂免疫亲和柱的回收率为70.05%~112.80%,相对标准偏差为3.42%~12.25%;MycoSep~?226多功能净化柱的回收率为69.42%~111.12%,相对标准偏差为1.46%~13.24%。免疫亲和柱具有重复利用,有机溶剂耗用量少、特异性强等显著优势,且聚苯乙烯-二乙烯苯树脂免疫亲和柱为新载体免疫亲和柱的制备提供了依据。     

免疫亲和柱净化-亲水液相色谱-串联质谱法测定水产食品中河鲀毒素

目的本文采用免疫亲和柱选择性吸附样品溶液中的河鲀毒素,建立了测定水产食品中河鲀毒素(TTX)的亲水液相色谱-串联质谱分析方法。方法样品以含1%乙酸的甲醇溶液提取,磷酸盐缓冲溶液稀释,再经免疫亲和柱富集和净化后进样分析。目标物以TSK-gel Amide-80亲水色谱柱(150 mm×2.0 mm,5μm)分离,乙腈-0.1%甲酸水溶液(含5 mmol/L乙酸铵)梯度洗脱,采用电喷雾离子源,选择反应监测(SRM)正离子模式检测,溶剂标准曲线校正,外标法定量。结果 TTX在1~1 000μg/L范围内线性良好,方法的检出限为1μg/kg,定量限为3μg/kg,在3~300μg/kg范围内加标回收率为73.6%~95.2%,相对标准偏差(RSD)为5.37%~10.7%。结论该方法可有效消除复杂基质样品中普遍存在的基质抑制效应,操作简便,色谱保留时间稳定,灵敏度高,准确度和重复性好,适用于烤鱼片、风味鱼干等水产食品中河鲀毒素的测定。     

柱前衍生-高效液相色谱法检测食品中的黄曲霉毒素B_1、B_2、G_1、G_2

目的:改进柱前衍生-高效液相色谱法测定食品中的黄曲霉毒素B1、B2、G1、G2。方法:分别用甲醇-水(8∶2,v∶v)或二氯甲烷提取食品中的黄曲霉毒素。提取液经免疫亲和柱净化后,采用三氟乙酸(或甲酸)进行衍生,并利用高效液相色谱仪进行测定。结果:黄曲霉毒素B1、B2、G1、G2的检出限分别为0.2、0.2、0.2、0.2μg/kg;在低、中、高加标浓度下的回收率分别为81.0%~94.1%、75.6%~92.0%、75.0%~92.4%、77.6%~91.3%。结论:改进后的柱前衍生-高效液相色谱法克服了样品基质的干扰,测定结果更准确。     

不同前处理方法对霉千张中赭曲霉毒素A检测效果对比

目的考察不同的固相萃取柱的净化效果,建立超高效液相色谱——串联质谱法检测霉千张中赭曲霉毒素A的检测方法。方法样品经甲醇+水(80:20,V:V)提取,萃取柱净化,目标化合物在多反应监测模式下进行检测,以基质匹配标准曲线法进行定量。优化色谱与质谱条件后,从回收率、基质效应、净化效率3个方面考察了HLB、C_(18)、MAX和免疫亲和柱,对霉千张中赭曲霉毒素A残留净化效果的影响。结果 HLB、C_(18)、免疫亲和柱、MAX固相萃取柱的基质效应分别为0.83、0.78、0.84、0.67;净化效率为75.6%、70.8%、81.5%、50.2%。赭曲霉毒素A在0.10～10.0 ng/mL范围内线性关系良好,相关系数为0.9998,加标回收率为86.0%～104.8%,相对标准偏差为1.2%～6.8%,方法的检出限为0.10μg/kg。结论HLB,C_(18)和免疫亲和柱均有较好的净化效果,免疫亲和柱和HLB价格昂贵,不太适合大批量样本的检测,因此选择C_(18)作为霉千张样品的前处理固相萃取柱。该方法前处理简单,选择性好,灵敏度高,适用于霉千张中赭曲霉毒素A的测定。     

免疫亲和柱净化-高效液相色谱法测定玉米油中F-2毒素含量的研究

建立免疫亲和柱净化-高效液相色谱法测定玉米油中F-2毒素的方法。样品经前处理后通过免疫亲和柱净化,采用高效液相色谱仪-荧光检测器进行检测。该方法最佳测定条件为:选用IAC-AZ型免疫亲和柱,柱吸附容量4μg;流动相乙腈-甲醇-水(体积比46∶46∶8),流速1.0m L/min;提取溶剂乙腈-水(体积比90∶10)。F-2毒素的质量浓度在0~250 ng/m L的范围内与峰面积呈良好的线性关系,方法检出限为0.06μg/kg,平均回收率为92.46%,RSD为0.942%~1.758%。该方法操作简便快捷、准确度高、重现性好,适用于玉米油中F-2毒素的测定。     

免疫亲和柱净化-柱后衍生高效液相色谱荧光法测定食用油中黄曲霉毒素B_1

样品经甲醇–水(7∶3,V/V)提取,提取液经过滤、稀释、免疫亲和柱净化,通过高效液相色谱柱后衍生法测定黄曲霉毒素B_1的含量。实验结果表明,黄曲霉毒素B_1在0.5~15.0 ng/m L范围内线性关系良好,相关系数为0.999。同一份样品加标后经三个不同品牌的免疫亲和柱纯化后,测定结果存在一定差异。不同样品中黄曲霉毒素B_1加标回收率在87.2%~95.8%之间,相对标准偏差(n=6)在2.12%~6.51%之间。     

免疫亲和柱净化-高效液相色谱-串联质谱法测定鱼肉和肝脏中河鲀毒素

目的采用免疫亲和柱净化鱼肉和肝脏中的河鲀毒素,建立高效液相色谱-三重四级杆质谱串联(LC-MS/MS)方法检测鱼肉和肝脏中的河鲀毒素,为水产品中的河鲀毒素检测提供方法依据。方法选用Zic-Hilic色谱柱(150 mm×2.1 mm,5μm),以10 mmol/L甲酸铵-0.1%甲酸-乙腈为流动相,采用梯度洗脱进行分离。样品用1%乙酸-甲醇沉淀蛋白提取,上清液加入PBS缓冲液后经免疫亲和柱净化,将洗脱液氮吹至干定容后上机测定。多重反应监测(MRM)方式检测。结果河鲀毒素的线性范围为1.0~1 000.0 ng/ml,鱼肉和肝脏中河鲀毒素的检出限分别为0.3和0.2μg/kg,回收率在52.4%~72.6%之间。结论本方法特异性强、提取效果好、无基质抑制效应,适用于鱼肉和肝脏中河鲀毒素的痕量检测。     

高效液相色谱法测定潮州花生糖中的黄曲霉毒素B_1

建立免疫亲和层析净化-高效液相色谱法测定潮州花生糖中的黄曲霉毒素B1的检测方法。样品用甲醇-水(7+3)提取,定性滤纸和玻璃纤维滤纸2次过滤,过免疫亲和柱净化;洗脱液氮气吹干后加正己烷和三氟乙酸衍生,以水-乙腈(85+15)溶解,用高效液相色谱仪带荧光检测器检测。方法在0.002 5 ng/μL~0.100 ng/μL的范围内呈良好的线性关系,加标平均回收率为85.3%~92.9%,试验的RSD为0.61%~1.08%。该方法通过改进柱前衍生化的条件和优化仪器分析条件,实验结果重现性好,回收率高,结果准确,为黄曲霉毒素B1的检测提供了准确可靠的方法。     

IAC-HPLC法检测牛奶中黄曲霉毒素和玉米赤霉醇及类似物质量分数

建立了IAC-HPLC(免疫亲和柱净化-高效液相色谱)法检测牛奶中6种黄曲霉毒素和玉米赤霉醇及类似物的方法。样品经免疫亲和柱净化后,黄曲霉毒素用高效液相色谱——荧光检测器柱后衍生检测,玉米赤霉醇及其类似物用高效液相色谱——紫外检测器检测。结果表明,牛奶中黄曲霉毒素(M2,M1,G2,G1,B2,B1)的检测限分别为0.004,0.004,0.004,0.003,0.002,0.002μg/L,6种黄曲霉毒素的平均回收率在91.20%~113.8%之间,变异系数小于8.79%;玉米赤霉醇及其类似物的检测限均为0.05μg/L,平均回收率在54.22%~90.76%之间,变异系数小于9.44%。     

Detection of DNA-bound advanced glycation end-products by immunoaffinity chromatography coupled to HPLC-diode array detection

Sugars and sugar degradation products are formed during food processing, but also endogenously in vivo. In vitro, nucleosides and DNA react readily with these carbonyl compounds during the formation of the two diastereomers of N(2)-carboxyethyl-2'-deoxyguanosine (CEdG(A,B)), leading to a loss of DNA integrity. Only little is known about DNA glycation in vivo and about the influence of nutrition on CEdG formation. In this study, we developed a sensitive method to analyze DNA glycation by HPLC. For this purpose, immunoaffinity chromatography (IAC) using a polyclonal antibody against N(2)-carboxyethylguanine (CEguanine) was coupled to HPLC-DAD. In some samples, peak identity was confirmed by LC-MS/MS. The recovery of CEguanine from the IAC columns was 52.5% +/- 3.6 (n = 4). Thus, it was possible for the first time to detect CEdG(A,B), N(2)-carboxyethylguanosine (CEG(A,B)), and CEguanine in 11 human urine samples. However, due to imprecision of IAC, valid quantification of the adducts could not be achieved. Furthermore, CEdG was also detected in the DNA of cultured human smooth muscle cells (SMCs) and bovine aorta endothelium cells (BAECs). In BAECs, CEdG(A,B) were found by HPLC-DAD and LC-MS/MS after immunoaffinity purification, whereas in SMCs DNA-advanced glycation end-products were only detected with the more sensitive LC-MS/MS method.     

探索用液质串联联用法测定牛肉中的瘦肉精

目的:探索用液质串联联用法测定抽查牛肉中的瘦肉精含量。方法:酶解后的样品溶液,加入氘代克伦特罗与沙丁胺醇作为混合内标,过C18固相萃取小柱,用OasisMCX小柱收集,以3%的氨水甲醇溶液对MCX小柱进行洗脱后吹干,残余物溶于0.1%甲酸水溶液-甲醇溶液(95:5),以0.1%甲酸乙腈溶液和0.1%甲酸溶液为梯度流动相,上液质串联联用仪测定。结果:各β受体激动剂在0.5～5.0μg/kg浓度范围良好线性关系,平均回收率90.47%～102.58%,仪器定量限0.0543～0.2978μg/kg。结论:此方法极大排除了其他成分干扰,重现性好,专属性强,灵敏准确,可作检测牛肉中的瘦肉精残留量参考。     

液相色谱-串联质谱法测定羊肉中的瘦肉精

利用液相-质谱串联法测定了市售羊肉中的瘦肉精残留量.氘代沙丁胺醇与克伦特罗混合内标溶液加入经酶解的样品中,用C18小柱净化后以3％的氨水甲醇溶液对Oasis MCX小柱进行洗脱,吹干,以0.1％甲酸水溶液-甲醇溶液(95:5)溶解残余物,用液相质谱串联连用法测定,以0.1％甲酸乙腈溶液和0.1％甲酸溶液为流动相梯度洗脱...     

Analysis of sterigmatocystin in cereals,animal feed,seeds, beer and cheese by immunoaffinity column clean-up and HPLC and LC-MS/MS quantification

A method is reported for the analysis of sterigmatocystin in various food and feed matrices using a commercial sterigmatocystin immunoaffinity column (IAC) for sample clean-up prior to HPLC analysis by UV with mass spectrometric detection (LC-MS/MS). Cereals (wheat, oats, rye, maize and rice), sunflower seeds and animal feed were spiked with sterigmatocystin at levels from 0.75 to 50 µg kg^?1 to establish method performance. Using acetonitrile/water extraction followed by IAC clean-up, and analysis by HPLC with detection at 325 nm, recoveries ranged from 68% to 106%, with repeatability from 4.2% to 17.5%. The limit of quantification with UV detection in these matrices was 1.5 µg kg^?1. For the analysis of beer and cheese the sample preparation prior to IAC clean-up was changed to accommodate the different properties of the matrix, prior to analysis by LC-MS/MS. For beer and cheese spiked at 5.0 µg kg^?1 the recoveries were 94% and 104%, and precision (RSDs) were 1.9% and 2.9% respectively. The limits of quantification by LC-MS/MS in beer and cheese were 0.02 and 0.6 µg kg^?1 respectively. The sterigmatocystin IAC was demonstrated to provide an efficient clean-up of various matrices to enable this mycotoxin to be determined by either HPLC with UV detection or LC-MS/MS.     

高压消解—石墨炉原子吸收光谱法测定贝类中镉含量的条件优化

建立了高压消解—石墨炉原子吸收光谱法测定贝类中镉含量的方法,研究对比几种基体改进剂对海水贝肉中镉原子吸光值的影响,优化了程序升温中灰化及原子化温度。实验结果:采用2 g/L的硝酸钯溶液作为基体改进剂,选择灰化温度650℃,原子化温度1 400℃,方法检出限0.091μg/kg,样品加标回收率90.3%~104.0%,相对标准偏差小于3.5%。试验结果表明该方法线性关系好,检出限、准确度和精密度均满足贝类样品的测定要求。     

以聚苯乙烯-二乙烯苯为载体的多目标免疫亲和柱的制备及应用

选取3 种不同聚苯乙烯-二乙烯苯基材,通过硝基化、氨基化、活化、偶联抗体等步骤制备免疫亲和柱,用于4 种镰刀菌毒素（玉米赤霉烯酮、脱氧雪腐镰刀菌烯醇、T-2毒素、HT-2毒素）的同时纯化,并将3 种基材制备的免疫亲和柱进行比较。以Cleanert PS型聚苯乙烯-二乙烯苯为基材制备的免疫亲和柱与抗体偶联的偶联率为97.64%,且其空白柱对样品的吸附作用最小,3 个加标水平下的回收率为66.16%～112.80%,相对标准偏差为3.09%～12.25%,可应用于实际样品的纯化。     

Screening for the presence of lipophilic marine biotoxins in shellfish samples using the neuro-2a bioassay

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out.     

Immunoaffinity chromatography purification and ultra-high-performance liquid chromatography-tandem mass spectrometry determination of four β-agonists in beef

A highly selective and sensitive method was developed for the simultaneous determination of four β-agonists (clenbuterol, salbutamol, ractopamine and terbutaline) in beef by immunoaffinity chromatography purification coupled to ultra-high-performance LC-MS/MS. The MS/MS conditions, ultra-high-performance LC mobile phase, injection solution, sample purification process and matrix effect were studied to optimise the operation conditions. The limits of detection (LODs) of the instrument for the studied β-agonists ranged from 0.20 to 0.25 μg l(-1), and the LODs of the method for the studied β-agonists ranged from 0.20 to 3.00 μg kg(-1) for beef. Calibration curves were constructed using a standard solution diluted with blank beef matrix. The linear ranges of the calibration curves ranged from 5 to 100 μg kg(-1) and the coefficients of determination were >0.9942 (n = 10) for all four β-agonists. Samples spiked at 5, 10 and 50 μg kg(-1) showed recoveries >72% and RSDs <6.6%. The method is suitable for the simultaneous detection of four β-agonists at trace levels in beef.     

液相色谱-串联质谱基质效应及其消除方法

综合近年来国内外进行液相色谱-串联质谱方法开发中关于基质效应的相关报道,重点介绍了液相色谱-串联质谱基质效应的消除方法,主要包括:选择合适的样品前处理方法;选择最佳的色谱分离条件;优化的质谱分析参数;使用恰当的内标;采用基质标准溶液校正。这些方法的应用,可有效改善基质效应的影响,优化液相色谱-串联质谱检测方法。