超高效液相色谱-串联质谱法测定全麦粉中的赭曲霉毒素A

目的：建立超高效液相色谱-串联质谱法测定全麦粉中赭曲霉毒素A的含量。方法：全麦粉样品经甲醇-水(60∶40,V∶V)提取,磷酸盐缓冲溶液稀释,赭曲霉毒素A免疫亲和柱净化,超高效液相色谱-串联质谱测定分析,内标法定量。结果：赭曲霉毒素A在0.475～9.500 ng·mL^-1具有良好的线性关系(R²=0.999 9),3个加标水平下的平均回收率为85.62%～99.42%,相对标准偏差为1.92%～4.66%,检出限为0.68μg·kg^-1、定量限为2.27μg·kg^-1。结论：该方法便捷、稳定、灵敏度高、准确度好,可用于快速分析全麦粉中赭曲霉毒素A的含量。     

高效液相色谱-串联质谱法测食品中的赭曲霉毒素A

研究建立高效液相色谱-串联质谱联用技术检测食品中赭曲霉毒素A的方法。根据不同样品,用甲醇-2%碳酸氢钠溶液(60:40,V/V)或甲醇-水(80:20,V/V)提取样品中的赭曲霉毒素A,经OchraTest亲和柱净化,以甲醇-5mmol/L(含0.1%甲酸)乙酸铵为流动相,采用正离子模式对赭曲霉毒素A进行检测。结果回收率在82.3%～98.5%之间,检出限为0.1μg/kg。该方法适用不同基质样品,准确性好、灵敏度高、抗干扰能力强,方法检出限可满足欧盟等最新限量要求。     

高效液相色谱-串联质谱法检测茶叶中的 赭曲霉毒素A

目的建立高效液相色谱-串联质谱联用技术检测茶叶中赭曲霉毒素A(OTA)的方法。方法用甲醇-2%Na HCO3溶液(60:40,V:V)提取样品中的赭曲霉毒素A,采用免疫亲和柱对茶叶中的赭曲霉毒素A净化,使用甲醇-5 mmol/L乙酸铵(含0.1%乙酸)为流动相,检测赭曲霉毒素A,采用正离子模式。结果本方法中赭曲霉毒素A在0.5~10 ng/m L质量浓度范围内具有良好的线性关系(r=0.996),回收率在83.6%~92.5%之间,相对标准偏差在6.5%~9.1%之间,检出限为0.1μg/kg。结论该方法准确性好、灵敏度高,适用于茶叶样品的检测。     

免疫亲和柱——高效液相色谱法测定肾脏中的赭曲霉毒素A

目的针对动物肾脏中可能污染的赭曲霉毒素A(OTA),建立了肾脏中赭曲霉毒素A的高效液相色谱方法。方法试样用磷酸酸化后经乙酸乙酯提取,免疫亲和柱净化,以乙腈-水-冰醋酸(450+525+25)为流动相,C18柱分离并通过荧光检测器定量。结果赭曲霉毒素A标准溶液浓度在0.10～20μg/L范围内呈线性相关(r=1.000 0),不同浓度水平的添加回收率为72.5%～87.4%,检出限为0.012μg/kg。结论使用免疫亲和柱净化能够达到良好的净化效果,是一种准确、方便的测定猪肾中赭曲霉毒素A的分析方法。     

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。     

免疫亲和层析-液相色谱-串联质谱法测定玉米和小麦中的赭曲霉毒素A

目的建立免疫亲和层析净化-液相色谱-串联质谱法测定食品中的赭曲霉毒素A的方法。方法玉米和小麦样品经50%甲醇水溶液(V:V)提取,免疫亲和柱净化后,采用液相色谱-串联质谱法进行测定,外标法定量。结果玉米、小麦添加浓度在1.0~10.0μg/kg时,回收率在70%~100%之间,变异系数小于10%,在2017年度法帕斯国际能力验证中(Food Analysis Performance Assessment Scheme),此方法测定样品中赭曲霉毒素A含量为2.8μg/kg,Z值为-0.4,统计结论为满意。结论该方法简单快速,灵敏度高,定量准确,适合于玉米和小麦中赭曲霉毒素A的检测。     

免疫亲和柱-高效液相色谱在白酒赭曲霉毒素A检测中的应用

将免疫亲和柱-高效液相色谱-荧光检测器相结合,测定白酒中赭曲霉毒素A的含量.建立了测定白酒中赭曲霉毒素A的方法.该方法用2%NaHCO3和15%NaCl的水溶液对白酒中赭曲霉毒素A进行提取,然后通过OchraTest免疫亲和柱净化,采用Agilent-C18柱(4.6 mm×300 mm,5μm),流动相为乙腈-水-乙酸(体积比为50:49:1),流速0.80 mL/min,激发波长335 nm,发射波长465 nm,柱温30℃,进样量20μL.结果表明,赭曲霉毒素A在1.0×10-9～10×10-9g/mL时.浓度与峰面积呈线性关系(r=0.9981),加标回收率为93.33%～97.40%,最低检出限为0.12×10-9g/mL,相对标准偏差分别为2.16%、3.13%和5.19%.     

免疫亲和柱净化-HPLC法同时测定玉米制品中的赭曲霉毒素A和玉米赤霉烯酮

建立了同时测定玉米制品中赭曲霉毒素A和玉米赤霉烯酮的免疫亲和柱净化高效液相色谱方法。试样经乙腈-水溶液(体积比80?20)提取,复合免疫亲和柱富集和净化后,采用Syncronis C18色谱柱(250 mm×4.6 mm,5μm)固定相,以0.5%冰乙酸-乙腈-甲醇(体积比10?45?45)为流动相,等度洗脱,荧光检测器检测(Ex:333 nm,Em:470 nm)。结果表明:赭曲霉毒素A和玉米赤霉烯酮检出限分别为5.65×10-2μg/kg和4.53×10-1-μg/kg;标准曲线的线性范围分别为1~50 ng/mL(r=0.9999)和10~500 ng/mL(r=0.9999);3个不同水平的加标平均回收率为95.81%~98.31%,RSD值为1.24%~1.65%。20批试样中赭曲霉毒素A和玉米赤霉烯酮测定值分别为0.59~0.69μg/kg和3.84~5.22μg/kg,均无过量残留。该方法具有操作简便、灵敏度高、重现性佳等优点,适用于同时检测玉米制品中赭曲霉毒素A和玉米赤霉烯酮。     

HPLC-MS/MS测定豆类及其制品中的赭曲霉毒素A含量

建立了基于高效液相色谱-串联质谱法检测豆类及其制品中赭曲霉毒素A含量的方法。该方法以有机溶剂提取结合免疫亲和柱净化作为前处理技术,以1%甲酸-乙腈为流动相,在正离子多反应监测模式下,采用高效液相色谱-三重四级杆串联质谱联用仪进行样品检测。结果表明:赭曲霉毒素A的质量浓度在0.5～20 μg/L范围内时,浓度与色谱峰面积的线性关系良好(R~20.99),方法检出限为0.3 μg/kg。样品的加标回收率为85.6%～106.2%,相对标准偏差介于0.96%～3.5%(n=6)。该方法具有分析时间短、操作简单、灵敏度高等优点,能够较好地满足豆类及其制品中赭曲霉毒素A含量检测的需要。     

小麦中真菌毒素的快速检测方法的研究

对小麦中黄曲霉毒素总量、赭曲霉毒素A和玉米赤霉烯酮的快速检测方法进行了研究.先采用胶体金快速检测卡法、免疫亲和柱层析净化荧光光度法进行快速检测,再用免疫亲和柱层析净化高效液相色谱法国标方法进行验证,从而根据实验室检测项目需要,快速对样品进行检测,既保证了准确度,又提高了检测效率.     

Determination of ochratoxin A in wine by immunoaffinity cleanup and liquid chromatography tandem mass spectrometry

A simple and accurate method has been developed for determining ochratoxin A (OTA), using an immunoaffinity column for cleanup and liquid chromatography-tandem mass spectrometry for identification and quantification. Wine samples were diluted with a solution containing polyethylene glycol 8000 and sodium hydrogen carbonate, filtered through a glass microfiber filter, and cleaned up on an immunoaffinity column. OTA was then eluted with methanol-acetic acid (98:2) and analyzed by liquid chromatography-tandem mass spectrometry. The average recoveries of OTA from red and white wines were 95 and 96.7% (spiked OTA level was 0.05 ng/ml) and repeatabilities (relative standard deviation) were 3.8 and 2.4%, respectively. The detection limit was 0.0003 ng/ml based on the signal-to-noise ratio in wine of 3:1. Analysis of 74 samples of domestic and imported wines showed OTA levels ranging from < 0.0003 to 0.82 ng/ml, with an incidence of contamination of 92.1% for red wines, and < 0.0003 to 0.51 ng/ml, with an incidence of contamination of 77.8% for white wines. These detection rates were higher than those rates of past reports of OTA contamination in wine, due to the high sensitivity of this method. However, all samples analyzed in this study complied with European Union regulations. It is concluded that this method is a useful tool for the quality assurance of wine.     

Immunoaffinity column clean-up and thin layer chromatography for determination of ochratoxin A in green coffee.

An immunoaffinity clean-up-based method for determining ochratoxin A (OTA) in green coffee aiming at one-dimensional thin layer chromatography (TLC) analysis was established. OTA was extracted with a mixture of methanol and aqueous sodium hydrogen carbonate solution, purified through an immunoaffinity column, separated on normal or reversed-phase (RP) TLC plates and detected and quantified by visual and densitometric analysis. The linear equation of the standard calibration curve by densitometric analysis gave R(2) > 0.999 (0.04-84 ng). The mean recovery (R) of OTA from spiked samples (1.8-109 microg kg(-1)) by densitometric and visual analyses were 98.4 and 103.8%, respectively. The relative standard deviations (RSD) for densitometric and visual analysis varied from 1.1 to 24.9% and from 0.0 to 18.8%, respectively. The RSD for naturally contaminated samples by densitometry (three levels of contamination, n = 3) varied from 11.1 to 18.1%. The correlation (R(2)) between high-performance liquid chromatography (HPLC) and densitometry, and between visual and densitometric analysis for spiked samples were > 0.99. The limit of detection (LOD) of the method was 0.5 microg kg(-1) for normal TLC. Toluene-ethyl acetate-88% formic acid (6:3:1 v/v/v) and acetonitrile-methanol-water-glacial acetic acid (35:35:29:10 v/v/v/v) were regarded as the suitable TLC solvents for eluting both standards and samples on normal and RP TLC plates, respectively. Toluene-acetic acid (99:1 v/v) was chosen as the spotting solvent among several others for giving the best sensitivity and resolution of OTA on TLC plates as well as the best recovery of OTA from standard and sample extract residues. Preliminary studies were carried out to investigate the reuse of the immunoaffinity column and the interference of caffeine in the OTA recovery.     

A rapid high-performance liquid chromatography with fluorescence detection method developed to analyze ochratoxin A in wine

A rapid, sensitive, reproducible, and inexpensive method of high-performance liquid chromatography with fluorescence detection (HPLC-FLD) for the analysis of ochratoxin A (OTA) in wine was developed. It is characterized by direct injection of the wine into the HPLC apparatus, with no need of extraction or cleanup. The method uses acetonitrile, water, and acetic acid (49:49:2, vol/vol/vol, respectively) as the isocratic mobile phase and a 5-microm monolithic C18 column (100 by 3 mm inside diameter). The relative standard deviation obtained in the OTA determination varied between 0.22 and 1.76%, with a mean value of 0.89%, in samples with concentrations between 0.10 and 100 ng/ml. The recovery of OTA ranged from 102% in samples spiked with 1 ng/ml OTA to 120% in samples with 0.10 ng/ml OTA. The method compared favorably with a published method based on an immunoaffinity column cleanup and a chromatographic assay with a C18 conventional HPLC column.     

赭曲霉毒素A单克隆抗体免疫亲和柱的制备

制备了赭曲霉毒素A(OTA)单克隆抗体免疫亲和柱,并用间接竞争ELISA和HPLC法评价了免疫亲和柱的性能,其柱容量(结合OTA的能力)约为200ng,加标回收率为90.38%～100.1%,可反复使用3次。 建立了免疫亲和柱-HPLC联用分析谷物中OTA的方法,最低检出限为0.2μg/kg,线性范围为0.6～400μg/kg,OTA加标量为1～10μg/kg时谷物样品中的回收率为78.7%～87.1%,变异系数小于6.5%。用此法检测了大米、小麦、玉米和玉米饲料等15份市售样品,检出率为46.7%,其中OTA的最高含量为0.785μg/kg。     

Ochratoxin A in wine and grape juice sold in Canada.

Ochratoxin A (OTA) was determined in 251 samples of wines and grape juice collected over 3 years in Canada. In total, 25/84 samples of red wine, 22/96 samples of white wine, 3/46 red grape juices and 1/25 white grape juices contained OTA levels above the limit of quantitation (LOQ). Canadian wines, when compared with imported products, showed both a lower OTA occurrence, noted as positive (19 versus 48% above the limit of detection (LOD) for wines), and a lower level of OTA contamination (upper bound mean of 17.5 versus 163pg ml(-1) for wines). Wines from the USA contained no quantifiable levels of ochratoxin A. OTA was found in Canadian and US grape juice samples, with 12.9% above the LOD and an upper bound mean of 13.3pg ml(-1). It was extracted from a wine or grape juice sample by passing it through an immunoaffinity column. The sample matrix was washed off the column with water. OTA was eluted from the column with methanol and quantitatively determined by liquid chromatography using a fluorescence detector. The presence of OTA was confirmed by esterification with boron trifluoride-methanol. The LOQ of OTA was estimated as 20 pg ml(-1) in white wine (S/N 10:1) and 40 pg ml(-1) in red wine, white grape juice and red grape juice (S/N 20.1). The LOD was estimated as 4pgml(-1) for white wine and 8pgml(-1) for red wine and white and red grape juices (S/N 3:1).     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol-5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g(-1) and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65-100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50-75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.     

Ochratoxin A exposure assessment of the inhabitants of Lisbon during winter 2007/2008 through bread and urine analysis

A survey on the occurrence of ochratoxin A (OTA) in 41 bread samples was carried out in the Portuguese capital, Lisbon. Maize (5) and wheat bread (36) and 43 representative urine samples from the Lisbon region were assayed for OTA levels using immunoaffinity column cleanup (IAC) and HPLC with fluorimetric detection (LC–FD). The percentage of OTA-positive samples was slightly higher for maize bread (80%) than wheat bread (70.8%), although, due to its higher consumption, the latter contributes more to OTA exposure, featuring a higher estimated daily intake (EDI). In the urine samples analyzed, both female and male residents displayed similarly high levels of OTA frequency and average contamination. In summary, OTA is a food contaminant of concern and may constitute a hazard for public health through consumption of cereal-based products.     

Ochratoxin A exposure assessment of the inhabitants of Lisbon during winter 2007/2008 through bread and urine analysis

A survey on the occurrence of ochratoxin A (OTA) in 41 bread samples was carried out in the Portuguese capital, Lisbon. Maize (5) and wheat bread (36) and 43 representative urine samples from the Lisbon region were assayed for OTA levels using immunoaffinity column cleanup (IAC) and HPLC with fluorimetric detection (LC–FD). The percentage of OTA-positive samples was slightly higher for maize bread (80%) than wheat bread (70.8%), although, due to its higher consumption, the latter contributes more to OTA exposure, featuring a higher estimated daily intake (EDI). In the urine samples analyzed, both female and male residents displayed similarly high levels of OTA frequency and average contamination. In summary, OTA is a food contaminant of concern and may constitute a hazard for public health through consumption of cereal-based products.     

Influence of roasting and different brewing processes on the ochratoxin A content in coffee determined by high-performance liquid chromatography-fluorescence detection (HPLC-FLD)

A rapid and reliable procedure has been developed for the determination of ochratoxin A (OTA) in green and roasted coffee. The method consists of extraction of the sample with methanol–5% aqueous sodium hydrogen carbonate/1% PEG8000 (20:80), followed by immunoaffinity column (IAC) clean-up and, finally, high-performance liquid chromatography (HPLC) determination with fluorimetric detection. Mean recoveries for green and roasted coffee spiked at different levels ranging from 94 and 105% were obtained. The limit of determination (S/N = 3) was 0.032 ng g^?1 and the precision (within-laboratory relative standard deviation) was 6%. The method described has been used to assess the influence of roasting and different brewing processes on OTA content in commercial lots of green and roasted coffee. The results provided evidence that roasting led to a significant drop on OTA levels (65–100%). Also, the way coffee is prepared affects the OTA content: brewing using a Moka Express (Italian coffee) led to a significant reduction of OTA concentration (50–75%) since hot water stays in contact with coffee for a short time. On the contrary, Turkish coffee-making (infusion for about 10 min) cause poor reduction in OTA.