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。     

全自动免疫亲和在线净化—高效液相色谱法快速测定粮食中赭曲霉毒素A

建立了全自动免疫亲和在线净化-高效液相色谱法快速测定粮食中赭曲霉毒素A(Ochtatoxin A,OTA)的方法。玉米、小麦样品经乙腈-水(60:40,V:V)提取,3%Tween-20(m/V)水溶液10倍稀释后,用自动进样器注入RIDA~? REST在线固相萃取系统,经赭曲霉毒素A免疫亲和小柱净化后,以乙腈-2%乙酸水(50:50,V:V)为流动相,流速为1.0 mL/min,C18色谱柱(150 mm×3.5 mm,3.5μm)分离,荧光检测器测定。根据3倍信噪比的峰响应值,确定赭曲霉毒素A的检出限为0.24μg/kg,在0.012 5～0.5μg/L范围内呈线性相关,R~2值为0.999 8;在玉米、小麦样品中加标回收率为80.1%～106.9%,变异系数为2.4%～8.2%。本方法一次装柱可检测60个样品,24 h可检测100个样品,满足谷物中赭曲霉毒素A快速准确定量检测的需要。     

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。     

药食两用类食品中赭曲霉毒素A的高效液相色谱-荧光检测方法

目的:采用免疫亲和净化和高效液相色谱技术,建立淀粉、糖类药食两用食品中赭曲霉毒素A的测定方法。方法:样品粉末经甲醇-水(8:2,V/V)涡旋、超声及振摇提取,提取液以磷酸盐缓冲液稀释后,用商品免疫亲和柱净化,含有赭曲霉毒素A的甲醇洗脱液用高效液相色谱技术分析,C18反相色谱柱分离,荧光检测器测定。结果:赭曲霉毒素A的最低检出浓度为1.0μg/kg(RSN=3);在0.5～100ng/mL范围内,峰面积与质量浓度呈线性关系(r=0.9995);以不含赭曲霉毒素A的太子参、莲子、薏苡、麦冬和龙眼肉为加标基质,加标水平为1～8μg/kg时,平均回收率在81.8%～107%之间,RSD为1.66%～15.0%(n=3)。结论:免疫亲和柱能和高效液相色谱-荧光检测相结合取得较为满意的结果,准确度高、精密度好,满足欧盟对食品饲料中OTA检测方法的要求,适合于淀粉、糖类药食两用食品中赭曲霉毒素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。     

高效液相色谱串联质谱法测定花生及制品中的五种真菌霉素

建立了高效液相色谱-串联质谱(HPLC-MS/MS)测定花生及制品中黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、玉米赤霉烯酮五种真菌霉素的快速分析方法。用甲醇-水(55:45,V/V)对样品进行提取,采用真菌毒素免疫亲和柱萃取,在ESI+模式下采用多反应监测(MRM)模式进行检测。目标物在C18色谱柱上实现了有效分离,在6 min内完成一个样品的分析,相关系数(r2,n=6)大于0.999,检测结果稳定、灵敏。黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇线性范围0.5~50.0μg/L,检出限为0.05μg/kg(LOD,S/N=3),赭曲霉毒素A、玉米赤霉烯酮线性范围5.0~100.0μg/L,检出限为0.5μg/kg(LOD,S/N=3),方法回收率为86.8~102.7%,精密度RSD为0.36~4.79%。该方法快速、灵敏,适用于花生及制品中黄曲霉毒素B1、黄曲霉毒素M1、脱氧雪腐镰刀菌烯醇、赭曲霉毒素A、玉米赤霉烯酮五种真菌霉素的检测与确证。     

高效液相色谱法同时检测粮谷中的黄曲霉毒素和赭曲霉毒素

建立粮谷类食品中黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 的同时检测方法。样品经过甲醇- 水(80:20,V/V)提取,液液萃取净化和富集后,三氟乙酸衍生,采用Agilent ZORBAX SB-C18 色谱柱(4.6mm ×250mm),以乙腈和体积分数2% 冰醋酸溶液为流动相梯度洗脱,在线变换波长荧光检测。根据3 倍信噪比的峰 响应值,确定黄曲霉毒素(B1、B2、G1 和G2)和赭曲霉毒素A 检出限分别为0.06、0.03、0.18、0.05μg/kg 和0.51μg/kg,上述5 种毒素分别在质量浓度0.05～100、0.125～25.00、0.05～100、0.125～25.00μg/L 和0.05～50.00μg/L 范围内呈线性相关,相关系数r 分别为0.9998、0.9998、0.9998、0.9996 和0.9998;在玉米、大米、小麦3 类样品中加标回收率平均为71.73%～115.37%,相对标准偏差为3.00%～9.88%,方法学验证结果表明,黄曲霉毒素和赭曲霉毒素A 5 种毒素同时检测结果与现行国标的单独检测方法检测结果无显著性差异(P ＞ 0.05)。     

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。     

同位素内标高效液相色谱-串联质谱法测定粮食及其制品中赭曲霉毒素A、B和C

建立一种同位素内标高效液相色谱—串联质谱法同时测定粮食及其制品中赭曲霉毒素A、B和C的方法。样品用乙腈-水(84:16,V/V)提取,振荡离心后取上清液过赭曲霉毒素免疫亲和柱富集净化,经Waters ACQUITY BEH C₁₈(50 mm×2.1 mm,1.7 μm)进行液相色谱-串联质谱分析,采用多反应监测(MRM)、正离子模式和内标法定量,同时对线性范围、准确度、精密度和加标回收率等进行方法学验证。结果表明,在优化的条件下,赭曲霉毒素A、B和C在0.25~2.5 ng/mL线性范围内线性良好,决定系数均在0.999以上,本方法对赭曲霉毒素的检出限(LODs,S/N=3)和定量限(LOQs,S/N=10)分别为0.003~0.018、0.011~0.059 μg/kg;在2.0~10.0 μg/kg 三个加标水平下,方法的平均回收率为80.50%~107.08%,相对偏差(RSDs)介于0.14%~4.94%(n=6)。利用此方法对10个批次的粮食及其制品进行检测,发现1个批次的样品检出赭曲霉毒素A,含量为(0.35±0.01) μg/kg。此方法操作简便、灵敏度高,适用于粮食及其制品中赭曲霉毒素A、B和C的检测。     

超高效液相色谱-串联质谱法检测中草药中赭曲霉毒素A

本研究通过优化液相色谱条件和质谱条件,并结合碳酸氢钠溶液提取稀释的方法法有效克服了基质效应的干扰,建立了中草药中赭曲霉毒素A的超高效液相色谱-串联质谱法快速检方法。试样经碳酸氢钠溶液提取,超声波提取,再经过免疫亲和柱净化后,用C18液相色谱柱分离,多级反应选择离子正离子模式检测。经方法学验证,赭曲霉毒素A质量浓度在0.1~50.0μg/L范围内呈现良好的线性关系,r0.99;样品在1.0、2.0和10.0μg/kg三个添加水平下的回收率为78.5%~98.0%;相对标准偏差为2.6%~11.8%;方法检出限为1.0μg/kg。将该方法应用于实际8批样品的检测,结果显示8批样品中检出1批的赭曲霉毒素A检测结果呈阳性(7.3μg/kg)。实际样品检测结果表明,本方法可实现中草药中赭曲霉毒素A灵敏、准确的定性定量分析。     

Validation of a high-performance liquid chromatography analytical method for ochratoxin A quantification in cocoa beans

A validated high-performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in cocoa beans is described. OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified with immunoaffinity columns before its analysis by HPLC. The validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within- and between-day variability) and recovery, robustness and uncertainty. Detection and quantification limits were 0.04 and 0.1 μg kg^-1, respectively. Recovery was 88.9% (relative standard deviation = 4.0%). This method was successfully applied to the measurement of 46 cocoa bean samples of different origins. A total of 63% of cocoa bean samples was contaminated with a level greater than the limit of detection. The means and medians obtained for cocoa bean were 1.71 and 1.12 μg kg^-1, respectively. Surveillance controls should be set up in both crops and factories involved in transformation processes to avoid this mycotoxin in final products.     

Occurrence of ochratoxin A in cocoa beans: Effect of shelling

The aim of this study was to investigate the influence of the shelling process on the presence of ochratoxin A (OTA) in cocoa samples. Twenty-two cocoa samples were analysed for the determination of OTA before (cocoa bean) and after undergoing manual shelling process (cocoa nib). In order to determine OTA contamination in cocoa samples, a validated high-performance liquid chromatography (HPLC) method with fluorescence detection was used for the quantitative analysis of ochratoxin A (OTA). In both types of samples, OTA was extracted with methanol-3% sodium hydrogen carbonate solution and then purified using immunoaffinity columns prior to HPLC analysis. Due to the fact that different recovery values were obtained for OTA from both types of samples, a revalidation of the method in the case of cocoa nibs was needed. Revalidation was based on the following criteria: Selectivity, limits of detection and quantification (0.03 and 0.1 µg kg^-1, respectively), precision (within-day and between-day variability) and recovery 84.2% (RSD = 7.1%), and uncertainty (30%). Fourteen of the twenty-two cocoa bean samples (64%) suffered a loss of OTA of more than 95% due to shelling, six samples suffered a loss of OTA in the range 65-95%, and only one sample presented a reduction of less than 50%. The principal conclusion derived from this study is that OTA contamination in cocoa beans is concentrated in the shell; therefore, improvements of the industrial shelling process could prevent OTA occurrence in cocoa final products.     

降解赭曲霉毒素A菌株的筛选及其鉴定

为了提供筛选生物降解赭曲霉毒素A（ochratoxin A,OTA）的便利条件,优化水系中OTA的测定方法。通过在水系中添加甲醇或乙醇的方法,使得水系中OTA回收率超过了90%,此外通过对比,当甲醇或乙醇与样液体积比为1∶1时,足以准确测定水系中OTA含量。利用以上方法对降解OTA的菌株进行筛选,发现细菌B-1和酵母Y-2均能够降解OTA,且细菌B-1在2 d时能降解87%的OTA,3 d将OTA全部降解,而酵母Y-2在2 d时就能够降解84%的OTA,在后续的培养中维持降解率不变。此外,利用液质联用法进一步确定了菌株B-1和Y-2的降解作用,并分析了降解产物。最后,通过分别分析细菌B-1和酵母Y-2的16S rDNA和ITS rDNA序列,菌株B-1鉴定为泡囊短波单胞菌,菌株Y-2鉴定为耶罗维亚酵母。     

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.     

ELISA法检测赭曲霉毒素A的改进研究

针对ELISA法检测赭曲霉毒素A(OTA),使用微振荡酶标板可以缩短免疫反应检测时间约30min。采用ELISA方法检测小麦样品中的OTA时,使用空白样品提取液来配制OTA标准溶液可以消除样品基质造成的干扰。同时对提取溶剂加以改进,采用NaCl-水-甲醇(10%:70:30)作为提取溶剂的提取效率最高。0.1、1、10μg/kg三个浓度水平的加标回收率为95%～110%,方法的重现性很好,变异系数小于10%。     

赭曲霉毒素A生成转化及致毒机制的研究进展

赭曲霉毒素A(Ochratoxin A,OTA)是由曲霉属(Aspergillus.sp)和青霉属(Penicillium.sp)真菌产生的一种次级代谢产物,它的生成受温度、水活度等的影响。检测食品及饲料中OTA含量的基本方法有薄层层析法、高效液相色谱法和酶联免疫吸附法。OTA因被认为与巴尔干半岛肾病有关而引起全球的关注,研究发现,OTA具有肾毒性、肝毒性、免疫毒性、基因毒性等,并且主要是通过促进膜的过氧化反应,抑制线粒体的呼吸作用和影响细胞信号传导通路中蛋白及关键因子的转录表达等来达到致毒效应。吸附、转化、降解是OTA脱毒的主要方式。本文就OTA的检测方法、生物合成、致毒机制和脱毒转化的相关研究进展进行了综述。     

Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters

The occurrence of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA) in the winter wheat of 1997 and 1998 grown under organic farming conditions was investigated using ELISAs (R-Biopharm®) for quantification. The influence of delayed drying of the grain after harvest on the development of DON and OTA was determined in storage trials (moisture: 17% and 20%; temperature: 20°C; duration: four and six weeks). The Tox5 PCR assay was used both to detect Fusarium species with the potential to produce trichothecenes and as a measure of their relative DNA content during the storage trials. The intensity of the PCR signals was correlated with the DON concentration. Fusarium species were identified microscopically by standard methods. All the freshly harvested grain samples were contaminated with DON and showed further increases in the DON concentration during storage. OTA contamination was found in 14.3% of the 1997 samples and in 24.1% of the 1998 samples. OTA increased during storage trials of the 1997 samples but not in the 1998 samples.     

A simple chemical method reduces ochratoxin A in contaminated cocoa shells

Ochratoxin A (OTA) is a mycotoxin produced by Aspergillus and Penicillium species, which contaminates cocoa among other food commodities. It has been previously demonstrated that the toxin is concentrated in cocoa shells. The aim of this study was to assay a simple chemical method for ochratoxin A reduction from naturally contaminated cocoa shells. In order to determine the efficiency of the method, a high-performance liquid chromatography method with fluorescence detection was set up beforehand and validated. Ochratoxin A was extracted from cocoa shells with methanol-3% sodium bicarbonate solution and then purified with immunoaffinity columns. The recovery attained was 88.7% (relative standard deviation = 6.36%) and the limits of detection and quantification were 0.06 and 0.2 kg/kg, respectively. For decontamination experiments, the solvent extractor ASE 200 was used. First, aqueous solutions of 2% sodium bicarbonate and potassium carbonate were compared under the same conditions (1,500 lb/in2 at 40 degrees C for 10 min). Higher ochratoxin A reduction was obtained with potassium carbonate (83 versus 27%). Then, this salt was used under different conditions of pressure, temperature, and time. The greatest ochratoxin A reduction was achieved with an aqueous potassium carbonate solution (2%), at 1,000 lb/in2 at 90 degrees C for 10 min. This method could probably be applicable to the cocoa industry because it is fast and relatively economic. From the point of view of human health, the use of potassium carbonate, partially eliminated by rinsing the sample with water, does not likely represent a risk for human health.     

High levels of ochratoxin A in licorice and derived products

The ochratoxin A (OTA) content of 30 samples of licorice root and derived products (licorice-confectionery, licorice block, and licorice extract) was analyzed by a standard HPLC-fluorescence technique and confirmed by methyl-ester formation. All analyzed samples of licorice and derived products were found to contain ochratoxin A, and some of them showed extremely high concentrations up to 252.8 ng/g of OTA. Highest levels of ochratoxin A were found in dry licorice root, averaging 63.6 ng/g, while mean contents in fresh licorice root were 9.2 ng/g. Licorice-confectionery (sweets) contained 3.8 ng/g of OTA. Ochratoxin A was also abundant in two licorice derivatives, liquid licorice extract (16.0 ng/g) and solid licorice block (39.5 ng/g). The ochratoxin levels found in licorice and derived products are higher than those reported in the literature for other food commodities. The experiments of OTA transfer into the tea beverages showed that almost 5% of the OTA present in dry licorice root is transferred to the corresponding decoction tea, whereas only 1% of OTA remains in infusion tea. The significance of the levels of ochratoxin A in licorice and its derivatives is discussed in the context of existing data on ochratoxin contamination in foods.