动物体液和组织中真菌毒素检测关键技术及应用研究
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摘要
致癌性真菌毒素对人类健康的危害十分严重,但同时监控致癌性真菌毒素的方法十分匮乏。根据国际癌症研究署(International Agency of Research on Cancer, IARC)对真菌毒素致癌性的评估,黄曲霉毒素B1被划定为Ⅰ类致癌物(Group1),其它黄曲霉毒素类、杂色曲霉素、桔霉素、伏马菌素B1、伏马菌素B2和赭曲霉毒素A定为2B类致癌物质(Group2B),棒曲霉素定为C类致癌物质(Group C),所以对这些致癌性真菌毒素进行同时监控十分必要。镰刀毒素中,极少文献报道检测所有的镰刀毒素及代谢产物。另外恩镰孢菌素A、恩镰孢菌素A1、恩镰孢菌素B、恩镰孢菌素B1和白僵菌素对人和动物同样造成很大的影响,在自然界广泛存在。但由于发现较晚,对它们的关注较少,到目前为止监控方法鲜有报道。胶霉毒素可以作为人类侵袭性曲霉病诊断的辅助指标,但是到目前为止,还没有建立起完善的定量确证方法。所以建立这些毒素的检测方法十分必要。目前报道的检测基质中,食品和饲料是关注的焦点,直接污染的体液和组织关注相对较少。而直接检测动物的体液和组织可以更直观的了解动物受真菌毒素的污染情况,不仅可以动态监控毒素在动物体内的代谢残留情况,避免了盲目宰杀食品动物造成阳性样品的浪费,从源头上降低人食用受污染动物性食品的风险。目前报道的前处理方法中,普遍采用的甲醇或乙腈为提取剂的液液萃取方法,同时结合传统的固相萃取技术如采用免疫亲和柱或常规的Oasis HLB柱进行萃取。而自动化程度高的先进的提取技术及简单实用的dilute, shoot","dilute, evaporate and shoot'和“QuEChERS'方法是样品前处理发展的方向。
因此,本研究的研究内容主要包括：
第一部分：致癌性真菌毒素的定量确证方法研究
方法：分别采用不同的前处理方法对不同的样品基质进行处理。首先采用自动化的PLE方法对猪,鸡的血液、尿液、肝脏和肾脏样品进行处理。经过优化前处理条件,最终采用的PLE萃取条件为：萃取温度140℃,萃取压力1500psi,静态萃取时间5min,冲洗体积设60%,吹扫时间120s,循环1次。萃取溶剂根据基质不同而不同,动物肝脏和肾脏采用乙腈/水/正己烷/乙酸(60/14/25/1,v/v/v/v)作为提取液,血液和尿液采用乙腈/乙酸(99/1,v/v)作为提取液。最终采用的适用于代谢工作者的提取方法为：小鼠和兔的血液采用dilute, evaporate and shoot方法进行提取,800μL乙腈/乙酸(99/1,v/v)为提取液。肝脏和肾脏样品采用QuEChERS方法提取,乙腈/水/正己烷/乙酸(60/14/25/1,v/v/v/v))为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。采用乙腈和0.1%乙酸水溶液作为流动相,在质谱优化的离子对条件下对12种致癌性真菌毒素进行检测。
结果：经过方法学考察,CCa和CCβ分别在0.028-0.402ug/kg (ug/L)和0.213-0.850ug/kg (ug/L)之间。在添加浓度0.25-3.2ug/kg (ug/L)的范围内,猪血浆、尿液、肝脏和肾脏中的平均回收率在64.6%～99.2%之间,RSDR≤15%；鸡血浆、肝脏和肾脏中的平均回收率在66.4%-101%之间,RSDR≤15%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在65.8%-101.6%之间,RSDR≤15.3%；兔血浆、尿液、肝脏和肾脏中的平均回收率在67.4%-98%之间,RSDR≤15.2%,可以满足分析方法的要求。
结论：最终建立了猪,鸡,小鼠,兔的血液、尿液、肝脏和肾脏样品中12种致癌性真菌毒素包括黄曲霉毒素B1(AFB1)、、黄曲霉毒素B2(AFB2)、黄曲霉毒素G1(AFG1)、黄曲霉毒素G2(AFG2)、黄曲霉毒素M1(AFM1)、黄曲霉毒素M2(AFM2)、杂色曲霉素(ST)、桔霉素(CIT)、伏马菌素B1(FB1)、伏马菌素B2(FB2)、赭曲霉毒素A(OTA)和棒曲霉素(PAT)的定量确证方法。此方法可以用来检测痕量的致癌性真菌毒素在食品动物和实验动物体内的含量,为残留监控和代谢工作者提供可靠的方法。
第二部分：恩镰孢菌素类和白僵菌素的定量确证方法研究
方法：首先采用自动化的PLE方法对各种基质进行处理。PLE萃取条件为：萃取温度120℃,萃取压力2000psi,静态萃取时间5mmin,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂根据基质不同而不同,动物肝脏和肾脏采用乙腈/水/正己烷(70/10/20,v/v/v)为提取液,血液采用乙腈作为提取液,尿液采用乙腈/甲酸(99/1,v/v)为提取液。然后血液样品采用dilute, evaporate and shoot方法进行提取,提取液为800μL乙腈。肝脏和肾脏样品采用QuEChERS方法提取,乙腈/水/正己烷(70/10/20,v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液样品采用"dilute and shoot'进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。采用乙腈和5mmol/L乙酸铵水溶液作为流动相,在质谱优化的离子对条件下对恩镰孢菌素类和白僵菌素进行检测。
结果：经过方法学考察,CCα和CCβ分别在0.110-0.220ug/kg (ug/L)和0.210-0.512ug/kg (μg/L)之间。在添加浓度0.25-4.8ug/kg (μg/L)的范围内,猪血浆、尿液、肝脏和肾脏中的平均回收率在76.6%-99%之间,RSDR≤15.7%；鸡血浆、肝脏和肾脏中的平均回收率在72%-98.2%之间,RSDR≤15.1%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在74.8%-97.4%之间,RSDR≤15%；兔血浆、尿液、肝脏和肾脏中的平均回收率在79.2%-99.2%之间,RSDR≤15%,可以满足分析方法的要求。
结论：最终建立了猪、鸡、小鼠、兔的血液、尿液、肝脏和肾脏样品中恩镰孢菌素A(ENA)、恩镰孢菌素A1(ENA1)、恩镰孢菌素B (ENB)、恩镰孢菌素B1(ENB1)和白僵菌素(BEA)的定量确证方法。方法不仅填补了恩镰孢菌素类和白僵菌素在食品动物和实验动物的体液和组织中检测方法空白,而且为代谢工作者提供可靠的方法。
第三部分：22种镰刀菌素类定量确证方法研究
本研究主要检测了实验动物的体液和组织以及食品动物的体液中22种镰刀菌素类的方法。
方法：首先采用自动化的PLE方法对各种基质进行处理。经过优化PLE条件,最终采用的PLE萃取条件为：萃取温度100℃,萃取压力1500psi,静态萃取时间7min,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂选择时,动物肝脏和肾脏采用甲醇/水/正己烷/甲酸(70/10/19/1,v/v/v/v)为提取液,血液采用甲醇/酸(99/1,v/v)作为提取液,尿液采用甲醇/甲酸(99.5/0.5,v/v)为提取液。然后血液采用dilute, evaporate and shoot方法进行提取,提取液为800μL甲醇/甲酸(99/1,v/v)。肝脏和肾脏样品采用QuEChERS方法提取,甲醇/水/正己烷/甲酸(70/10/19/1,v/v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot'进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。上机检测时,流动相采用乙腈和5mmol/1乙酸铵水溶液。
结果：经过方法学考察,方法的特异性和专一性良好,CCa和CCβ分别在0.040-0.680ug/kg (μg/L)和0.210-1.353ug/kg (μg/L)之间。在添加浓度0.25-4.8ug/kg (μg/L)的范围内,猪血浆和尿液中,鸡血浆中的平均回收率在69.8%-93%之间,RSDR≤15.5%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在72.2%-98.2%之间,RSDR≤14.9%；兔血浆、尿液、肝脏和肾脏中的平均回收率在73.4%-93.8%之间,RSDR≤15.1%,可以满足分析方法的要求。
结论：最终建立了小鼠和兔的血液、尿液、肝脏和肾脏样品,猪和鸡的血浆样品及猪的尿液样品中22种镰刀菌素的定量确证方法。不仅为测定镰刀菌素在动物体液和组织中的含量提供可靠方法,完善镰刀菌毒素的监控体系,而且为代谢工作者提供可靠的方法。
第四部分：胶霉毒素定量确证方法研究
方法：前处理方法一：采用PLE方法对各种基质进行处理。经过优化PLE条件,最终采用的PLE萃取条件为：萃取温度100℃,萃取压力2000psi,静态萃取时间6mmin,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂选择时,动物肝脏和肾脏采用甲醇/水/正己烷(70/10/20,v/v/v)为提取液,血液采用甲醇作为提取液,尿液采用甲醇/甲酸(99/1,v/v)为提取液。前处理方法二：血液采用dilute, evaporate and shoot方法进行提取,提取液为800μL甲醇。肝脏和肾脏样品采用QuEChERS方法提取,甲醇/水／正己烷/(70/10/20,v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。上机检测时,流动相采用甲醇和0.5%乙酸水溶液,质谱参数选择峰度强的离子对。
结果：经过方法学考察,方法的特异性和专一性良好,CCa和CCββ分别在0.090-0.150ug/kg(ug/L)和0.170-0.310ug/kg(ug/L)之间。在添加浓度0.3-2.4ug/kg(ug/L)的范围内,胶霉毒素在猪,小鼠和兔血浆、尿液、肝脏和肾脏中的平均回收率在76.9%-99.6%之间,RSDR≤14.9%。胶霉毒素在鸡的不同样品中的平均回收率在83.3%-99.6%之间,RSDR≤10.8%。胶霉毒素在人不同样品中的平均回收率在80.8%-95.1%之间,RSDR≤13.5%。由以上数据可知GLI在不同动物的不同样品中的平均回收率均在76.9%-99.6%之间,RSDR≤14.9%。
结论：最终建立了人的血液和尿液,猪,鸡,小鼠和兔的血液、尿液、肝脏和肾脏中胶霉毒素的定量确证方法。不仅为测定胶霉毒素在人和动物体液和组织中的含量提供可靠方法,还可以作为人类侵袭性曲霉病诊断的潜在方法。
第五部分：致癌性真菌毒素在小鼠体内的血药浓度和组织浓度的的相关性研究
方法：通过小鼠经口灌胃致癌性真菌毒素后,在不同的时间点取血液与肝脏样品,利用所建立的致癌性真菌毒素检测方法,测定毒素的血药浓度,绘制药时曲线,分别比较血浆与组织浓度的相关性。
结果：小鼠经口灌胃黄曲霉毒素B1后血液和肝脏的相关系数为0.8524,通过小鼠口服桔霉素后,血液和肾脏的相关系数为0.9436,可见血液和组织之间存在相关性。
结论：血液中的浓度在一定程度上可以反映其在组织中的浓度,提示我们可以通过测定动物体液中的致癌性真菌毒素含量,间接估计动物组织器官受致癌性真菌毒素的侵害程度,评估人类和动物因致癌性真菌毒素引发癌症的潜在风险,间接推导食品动物中,可以通过对食品动物的血药浓度监控来评价组织中的浓度变化,从而制定食品动物被真菌毒素污染后的休药期,避免了盲目宰杀食品动物造成阳性样品的浪费。此外可以利用建立的方法测定人体液中的致癌性真菌毒素含量,间接估计组织器官受致癌性真菌毒素的侵害程度,为特殊人群健康普查提供方法。
Mycotoxins are secondary metabolites produced by different fungal species. They can contaminate many agricultural commodities during harvest or in storage. Currently, more than400mycotoxins have been identified in the world, although only a few of them present a significant source of food-borne illnesses and are of major concern worldwide. They can be categorized into Aflatoxins (AFs) family (produced by Aspergi//us spp.), Ochratoxin A (OTA), produced by Aspergillus and Penicillium spp., Zearalenones (ZENs), Fumonisins (FBs) and thricothecenes (TCT), produced by Fusarium spp. A group of carcinogenic mycotoxins is produced by some fungi under certain conditions. Therefore, monitoring carcinogenic mycotoxins in human and animal plasma and urine is important. Fusariums pose a significant potential threat to human and animal, so detecting fusariums in animal plasma and urine would be very useful. In addition, developing and validating methods to determine Enniatins(ENA), Enniatins A1(NA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON), Beauvericin(BEA) in biological matrices would be very useful because they could be tools during toxicokinetic and toxicology studies.
The aim of the work was to develop a high-throughput method based on LC-MS/MS for the detennination of mycotoxins and metabolites in animal biological matrices and tissues. Contents include the following:
Section one:Quantitative determination of carcinogenic mycotoxins in animal biological matrices and tissues using HPLC-MS/MS methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and 'QuEChERS'procedure have been developed. These procedures should be exhaustive with respect to the target compounds, as well as fast, simple and environmentally friendly for routine analysis. High-throughput was achieved by using PLE pre-treatment and without the need for any cleanup. The optimum extraction conditions were a static time of5min, the extraction pressure and the temperature was1500psi and140(?), respectively, the flush volume was60%. For the tissues samples, the extraction solvent was acetonitrile/water/hexane/acetic acid (60/14/25/1, v/v/v/v), for the plasma and urine samples, the extraction solvent was acetonitrile/acetic acid (99/1, v/v).
The limits of detection were defined as CCa varied from0.028ug/kg (ug/L) to0.402ug/kg (ug/L). The recoveries of spiked samples from0.25ug/kg (ug/L) to3.2ug/kg (ug/L) ranged from64.6%to101.6%with the relative standard deviations of less than15.3%. To our knowledge, this is the first method for large-scale testing of carcinogenic mycotoxins in all kinds animal of biological fluids and tissues using HPLC-MS/MS.
Section two:Quantitative determination of enniatins and beauvericin in animal biological matrices and tissues using HPLC-MS/MS Methods.
The method is to develop a simultaneous method for the determination of including Enniatins(ENA), Enniatins A1(ENA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON) and Beauvericin(BEA) in swine, chicken, rat and rabbit plasma, urine and tissues. The samples extracted with PLE and'dilute, evaporate and shoot'approach and 'QuEChERS' procedure.
CCa were0.110to0.220u.g/kg (ug/L) and CCB were0.210to0.512ug/kg (ug/L), respectively. The recoveries of enniatins and beauvericin, within the spiked level of quantitative limits, were from72.0%-99.2%. All the relative standard deviations of less than15.7%.
Section three:Quantitative determination of22fusariums in animal biological matrices and tissues using HPLC-MS/MS Methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and'QuEChERS'procedure have been developed. The optimum PLE extraction conditions were a static time of7min, the extraction pressure and the temperature was1500psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99.5/0.5, v/v/v), for the tissues samples, the extraction solvent was methanol/water/hexane/acetic acid (70/10/19/1, v/v/v), for the plasma samples, the extraction solvent was methanol/acetic acid (80/19/1, v/v/v). The limits of detection were defined as CCa varied from0.040ug/kg (ug/L) to0.680ug/kg (ug/L). The recoveries of spiked samples from0.210ug/kg (ug/L) to1.353ug/kg (ug/L) ranged from69.8%-98.2%with the relative standard deviations of less than15.5%.
Section four:Quantitative determination of gliotoxin in human and animal biological matrices and tissues using HPLC-MS/MS Methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and "QuEChERS" procedure have been developed. The optimum PLE extraction conditions were a static time of6min, the extraction pressure and the temperature was2000psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99/1, v/v), for the tissues samples, the extraction solvent was methanol/water/hexane (70/10/20, v/v/v). for the plasma samples, the extraction solvent was methanol.
The limits of detection were defined as CCa varied from0.090to0.150ug/kg (ug/L). The recoveries of spiked samples from0.30ug/kg (ug/L) to2.4ug/kg (ug/L) ranged from76.9%~99.6%with the relative standard deviations of less than14.9%.
Section five:Application of carcinogenic mycotoxins method in rat plasma and tissues.
The method was applied in real samples obtained from rats. It could be used to check the efficacy of detoxification strategies (adsorbents, detoxification microorganisms, and enzymes) in vivo, and reduce mycotoxin toxicity in farm animals. In the future, this method can be widely applied in mycotoxins exposure assessment of human and animals.
因此,本研究的研究内容主要包括：
第一部分：致癌性真菌毒素的定量确证方法研究
方法：分别采用不同的前处理方法对不同的样品基质进行处理。首先采用自动化的PLE方法对猪,鸡的血液、尿液、肝脏和肾脏样品进行处理。经过优化前处理条件,最终采用的PLE萃取条件为：萃取温度140℃,萃取压力1500psi,静态萃取时间5min,冲洗体积设60%,吹扫时间120s,循环1次。萃取溶剂根据基质不同而不同,动物肝脏和肾脏采用乙腈/水/正己烷/乙酸(60/14/25/1,v/v/v/v)作为提取液,血液和尿液采用乙腈/乙酸(99/1,v/v)作为提取液。最终采用的适用于代谢工作者的提取方法为：小鼠和兔的血液采用dilute, evaporate and shoot方法进行提取,800μL乙腈/乙酸(99/1,v/v)为提取液。肝脏和肾脏样品采用QuEChERS方法提取,乙腈/水/正己烷/乙酸(60/14/25/1,v/v/v/v))为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。采用乙腈和0.1%乙酸水溶液作为流动相,在质谱优化的离子对条件下对12种致癌性真菌毒素进行检测。
结果：经过方法学考察,CCa和CCβ分别在0.028-0.402ug/kg (ug/L)和0.213-0.850ug/kg (ug/L)之间。在添加浓度0.25-3.2ug/kg (ug/L)的范围内,猪血浆、尿液、肝脏和肾脏中的平均回收率在64.6%～99.2%之间,RSDR≤15%；鸡血浆、肝脏和肾脏中的平均回收率在66.4%-101%之间,RSDR≤15%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在65.8%-101.6%之间,RSDR≤15.3%；兔血浆、尿液、肝脏和肾脏中的平均回收率在67.4%-98%之间,RSDR≤15.2%,可以满足分析方法的要求。
结论：最终建立了猪,鸡,小鼠,兔的血液、尿液、肝脏和肾脏样品中12种致癌性真菌毒素包括黄曲霉毒素B1(AFB1)、、黄曲霉毒素B2(AFB2)、黄曲霉毒素G1(AFG1)、黄曲霉毒素G2(AFG2)、黄曲霉毒素M1(AFM1)、黄曲霉毒素M2(AFM2)、杂色曲霉素(ST)、桔霉素(CIT)、伏马菌素B1(FB1)、伏马菌素B2(FB2)、赭曲霉毒素A(OTA)和棒曲霉素(PAT)的定量确证方法。此方法可以用来检测痕量的致癌性真菌毒素在食品动物和实验动物体内的含量,为残留监控和代谢工作者提供可靠的方法。
第二部分：恩镰孢菌素类和白僵菌素的定量确证方法研究
方法：首先采用自动化的PLE方法对各种基质进行处理。PLE萃取条件为：萃取温度120℃,萃取压力2000psi,静态萃取时间5mmin,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂根据基质不同而不同,动物肝脏和肾脏采用乙腈/水/正己烷(70/10/20,v/v/v)为提取液,血液采用乙腈作为提取液,尿液采用乙腈/甲酸(99/1,v/v)为提取液。然后血液样品采用dilute, evaporate and shoot方法进行提取,提取液为800μL乙腈。肝脏和肾脏样品采用QuEChERS方法提取,乙腈/水/正己烷(70/10/20,v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液样品采用"dilute and shoot'进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。采用乙腈和5mmol/L乙酸铵水溶液作为流动相,在质谱优化的离子对条件下对恩镰孢菌素类和白僵菌素进行检测。
结果：经过方法学考察,CCα和CCβ分别在0.110-0.220ug/kg (ug/L)和0.210-0.512ug/kg (μg/L)之间。在添加浓度0.25-4.8ug/kg (μg/L)的范围内,猪血浆、尿液、肝脏和肾脏中的平均回收率在76.6%-99%之间,RSDR≤15.7%；鸡血浆、肝脏和肾脏中的平均回收率在72%-98.2%之间,RSDR≤15.1%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在74.8%-97.4%之间,RSDR≤15%；兔血浆、尿液、肝脏和肾脏中的平均回收率在79.2%-99.2%之间,RSDR≤15%,可以满足分析方法的要求。
结论：最终建立了猪、鸡、小鼠、兔的血液、尿液、肝脏和肾脏样品中恩镰孢菌素A(ENA)、恩镰孢菌素A1(ENA1)、恩镰孢菌素B (ENB)、恩镰孢菌素B1(ENB1)和白僵菌素(BEA)的定量确证方法。方法不仅填补了恩镰孢菌素类和白僵菌素在食品动物和实验动物的体液和组织中检测方法空白,而且为代谢工作者提供可靠的方法。
第三部分：22种镰刀菌素类定量确证方法研究
本研究主要检测了实验动物的体液和组织以及食品动物的体液中22种镰刀菌素类的方法。
方法：首先采用自动化的PLE方法对各种基质进行处理。经过优化PLE条件,最终采用的PLE萃取条件为：萃取温度100℃,萃取压力1500psi,静态萃取时间7min,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂选择时,动物肝脏和肾脏采用甲醇/水/正己烷/甲酸(70/10/19/1,v/v/v/v)为提取液,血液采用甲醇/酸(99/1,v/v)作为提取液,尿液采用甲醇/甲酸(99.5/0.5,v/v)为提取液。然后血液采用dilute, evaporate and shoot方法进行提取,提取液为800μL甲醇/甲酸(99/1,v/v)。肝脏和肾脏样品采用QuEChERS方法提取,甲醇/水/正己烷/甲酸(70/10/19/1,v/v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot'进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。上机检测时,流动相采用乙腈和5mmol/1乙酸铵水溶液。
结果：经过方法学考察,方法的特异性和专一性良好,CCa和CCβ分别在0.040-0.680ug/kg (μg/L)和0.210-1.353ug/kg (μg/L)之间。在添加浓度0.25-4.8ug/kg (μg/L)的范围内,猪血浆和尿液中,鸡血浆中的平均回收率在69.8%-93%之间,RSDR≤15.5%；鼠血浆、尿液、肝脏和肾脏中的平均回收率在72.2%-98.2%之间,RSDR≤14.9%；兔血浆、尿液、肝脏和肾脏中的平均回收率在73.4%-93.8%之间,RSDR≤15.1%,可以满足分析方法的要求。
结论：最终建立了小鼠和兔的血液、尿液、肝脏和肾脏样品,猪和鸡的血浆样品及猪的尿液样品中22种镰刀菌素的定量确证方法。不仅为测定镰刀菌素在动物体液和组织中的含量提供可靠方法,完善镰刀菌毒素的监控体系,而且为代谢工作者提供可靠的方法。
第四部分：胶霉毒素定量确证方法研究
方法：前处理方法一：采用PLE方法对各种基质进行处理。经过优化PLE条件,最终采用的PLE萃取条件为：萃取温度100℃,萃取压力2000psi,静态萃取时间6mmin,冲洗体积设60%,吹扫时间120s,循环1次,萃取溶剂选择时,动物肝脏和肾脏采用甲醇/水/正己烷(70/10/20,v/v/v)为提取液,血液采用甲醇作为提取液,尿液采用甲醇/甲酸(99/1,v/v)为提取液。前处理方法二：血液采用dilute, evaporate and shoot方法进行提取,提取液为800μL甲醇。肝脏和肾脏样品采用QuEChERS方法提取,甲醇/水／正己烷/(70/10/20,v/v/v)为提取液,2.0g无水硫酸钠和0.5g无水醋酸钠为去除水分和杂质的试剂。尿液采用'dilute and shoot进行提取,酶解后直接用750μL乙腈/水(10/90,v/v)的混合溶液稀释,过滤膜后待测。上机检测时,流动相采用甲醇和0.5%乙酸水溶液,质谱参数选择峰度强的离子对。
结果：经过方法学考察,方法的特异性和专一性良好,CCa和CCββ分别在0.090-0.150ug/kg(ug/L)和0.170-0.310ug/kg(ug/L)之间。在添加浓度0.3-2.4ug/kg(ug/L)的范围内,胶霉毒素在猪,小鼠和兔血浆、尿液、肝脏和肾脏中的平均回收率在76.9%-99.6%之间,RSDR≤14.9%。胶霉毒素在鸡的不同样品中的平均回收率在83.3%-99.6%之间,RSDR≤10.8%。胶霉毒素在人不同样品中的平均回收率在80.8%-95.1%之间,RSDR≤13.5%。由以上数据可知GLI在不同动物的不同样品中的平均回收率均在76.9%-99.6%之间,RSDR≤14.9%。
结论：最终建立了人的血液和尿液,猪,鸡,小鼠和兔的血液、尿液、肝脏和肾脏中胶霉毒素的定量确证方法。不仅为测定胶霉毒素在人和动物体液和组织中的含量提供可靠方法,还可以作为人类侵袭性曲霉病诊断的潜在方法。
第五部分：致癌性真菌毒素在小鼠体内的血药浓度和组织浓度的的相关性研究
方法：通过小鼠经口灌胃致癌性真菌毒素后,在不同的时间点取血液与肝脏样品,利用所建立的致癌性真菌毒素检测方法,测定毒素的血药浓度,绘制药时曲线,分别比较血浆与组织浓度的相关性。
结果：小鼠经口灌胃黄曲霉毒素B1后血液和肝脏的相关系数为0.8524,通过小鼠口服桔霉素后,血液和肾脏的相关系数为0.9436,可见血液和组织之间存在相关性。
结论：血液中的浓度在一定程度上可以反映其在组织中的浓度,提示我们可以通过测定动物体液中的致癌性真菌毒素含量,间接估计动物组织器官受致癌性真菌毒素的侵害程度,评估人类和动物因致癌性真菌毒素引发癌症的潜在风险,间接推导食品动物中,可以通过对食品动物的血药浓度监控来评价组织中的浓度变化,从而制定食品动物被真菌毒素污染后的休药期,避免了盲目宰杀食品动物造成阳性样品的浪费。此外可以利用建立的方法测定人体液中的致癌性真菌毒素含量,间接估计组织器官受致癌性真菌毒素的侵害程度,为特殊人群健康普查提供方法。
Mycotoxins are secondary metabolites produced by different fungal species. They can contaminate many agricultural commodities during harvest or in storage. Currently, more than400mycotoxins have been identified in the world, although only a few of them present a significant source of food-borne illnesses and are of major concern worldwide. They can be categorized into Aflatoxins (AFs) family (produced by Aspergi//us spp.), Ochratoxin A (OTA), produced by Aspergillus and Penicillium spp., Zearalenones (ZENs), Fumonisins (FBs) and thricothecenes (TCT), produced by Fusarium spp. A group of carcinogenic mycotoxins is produced by some fungi under certain conditions. Therefore, monitoring carcinogenic mycotoxins in human and animal plasma and urine is important. Fusariums pose a significant potential threat to human and animal, so detecting fusariums in animal plasma and urine would be very useful. In addition, developing and validating methods to determine Enniatins(ENA), Enniatins A1(NA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON), Beauvericin(BEA) in biological matrices would be very useful because they could be tools during toxicokinetic and toxicology studies.
The aim of the work was to develop a high-throughput method based on LC-MS/MS for the detennination of mycotoxins and metabolites in animal biological matrices and tissues. Contents include the following:
Section one:Quantitative determination of carcinogenic mycotoxins in animal biological matrices and tissues using HPLC-MS/MS methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and 'QuEChERS'procedure have been developed. These procedures should be exhaustive with respect to the target compounds, as well as fast, simple and environmentally friendly for routine analysis. High-throughput was achieved by using PLE pre-treatment and without the need for any cleanup. The optimum extraction conditions were a static time of5min, the extraction pressure and the temperature was1500psi and140(?), respectively, the flush volume was60%. For the tissues samples, the extraction solvent was acetonitrile/water/hexane/acetic acid (60/14/25/1, v/v/v/v), for the plasma and urine samples, the extraction solvent was acetonitrile/acetic acid (99/1, v/v).
The limits of detection were defined as CCa varied from0.028ug/kg (ug/L) to0.402ug/kg (ug/L). The recoveries of spiked samples from0.25ug/kg (ug/L) to3.2ug/kg (ug/L) ranged from64.6%to101.6%with the relative standard deviations of less than15.3%. To our knowledge, this is the first method for large-scale testing of carcinogenic mycotoxins in all kinds animal of biological fluids and tissues using HPLC-MS/MS.
Section two:Quantitative determination of enniatins and beauvericin in animal biological matrices and tissues using HPLC-MS/MS Methods.
The method is to develop a simultaneous method for the determination of including Enniatins(ENA), Enniatins A1(ENA1), Enniatins B(ENB), Enniatins B1(ENB1), Moniliformin (MON) and Beauvericin(BEA) in swine, chicken, rat and rabbit plasma, urine and tissues. The samples extracted with PLE and'dilute, evaporate and shoot'approach and 'QuEChERS' procedure.
CCa were0.110to0.220u.g/kg (ug/L) and CCB were0.210to0.512ug/kg (ug/L), respectively. The recoveries of enniatins and beauvericin, within the spiked level of quantitative limits, were from72.0%-99.2%. All the relative standard deviations of less than15.7%.
Section three:Quantitative determination of22fusariums in animal biological matrices and tissues using HPLC-MS/MS Methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and'QuEChERS'procedure have been developed. The optimum PLE extraction conditions were a static time of7min, the extraction pressure and the temperature was1500psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99.5/0.5, v/v/v), for the tissues samples, the extraction solvent was methanol/water/hexane/acetic acid (70/10/19/1, v/v/v), for the plasma samples, the extraction solvent was methanol/acetic acid (80/19/1, v/v/v). The limits of detection were defined as CCa varied from0.040ug/kg (ug/L) to0.680ug/kg (ug/L). The recoveries of spiked samples from0.210ug/kg (ug/L) to1.353ug/kg (ug/L) ranged from69.8%-98.2%with the relative standard deviations of less than15.5%.
Section four:Quantitative determination of gliotoxin in human and animal biological matrices and tissues using HPLC-MS/MS Methods.
The analytes were extracted using PLE first, then,'dilute, and shoot'approach,'dilute, evaporate and shoot'approach and "QuEChERS" procedure have been developed. The optimum PLE extraction conditions were a static time of6min, the extraction pressure and the temperature was2000psi and100(?), respectively, the flush volume was60%. For the urine samples, the extraction solvent was methanol/acetic acid (99/1, v/v), for the tissues samples, the extraction solvent was methanol/water/hexane (70/10/20, v/v/v). for the plasma samples, the extraction solvent was methanol.
The limits of detection were defined as CCa varied from0.090to0.150ug/kg (ug/L). The recoveries of spiked samples from0.30ug/kg (ug/L) to2.4ug/kg (ug/L) ranged from76.9%~99.6%with the relative standard deviations of less than14.9%.
Section five:Application of carcinogenic mycotoxins method in rat plasma and tissues.
The method was applied in real samples obtained from rats. It could be used to check the efficacy of detoxification strategies (adsorbents, detoxification microorganisms, and enzymes) in vivo, and reduce mycotoxin toxicity in farm animals. In the future, this method can be widely applied in mycotoxins exposure assessment of human and animals.
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