Optimization of the Method of Detection of Metabolites Produced by the Alternaria Genus: Alternariol, Alternariol Monomethyl Ether, Altenuene, Altertoxin I and Tentoxin

ABSTRACT: A simple method has been developed in this work for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), altertoxin I (ATX-I), tenuazonic acid (TA), and tentoxin by means of thin-layer chromatography from cultures of several strains of Alternaria. Strains were incubated in 2% malt extract broth for 7 d at 25 °C. For extraction of metabolites, chloroform was used. The solvent system benzene/methanol/acetic acid (92:6:2) was chosen. TLC plates were observed under ultraviolet light (254/366 nm). Results show that A. alternata IMI 354942 produced AOH, AME, ALT, ATX-I, tentoxin, and TA; A. alternata IMI 354943, AOH, AME, and TA; A. triticina IMI 289962 tentoxin; and A. triticina IMI 289680 AME, ATX-I, and TA.     

光照对交链孢毒素合成的调控

目的 明确光调控交链孢（Alternaria）产真菌毒素的作用。方法 在光/暗培养条件下,比较分析光照对2种交链孢菌株（ATCC 66981和Pear-3）的菌丝生长、孢子形成以及毒素合成的影响。结果 持续白光照射对交链孢菌丝生长影响不显著,而显著抑制其孢子形成。光照刺激交链孢菌ATCC 66981中交链孢酚（alternariol, AOH）、交链孢酚单甲醚（alternariol monomethyl ether, AME）和腾毒素（tentoxin, TEN）3种毒素的产生。菌株持续光照培养30天,AOH、AME和TEN毒素浓度分别为120、182和173 g/L;较之黑暗培养,分别增加了1.1、5.9和9.6倍。光照抑制交链孢菌Pear-3中细交链孢菌酮酸（tenuazonic acid, TeA）毒素的合成。菌株光照培养6天,TeA毒素浓度即已达142 g/L;而黑暗培养甚至高达325 g/L,毒素产率反而增加了1.3倍。结论 光照不影响交链孢菌丝生长,而对孢子形成影响显著;持续白光照射能够促进交链孢中AOH、AME和TEN毒素的产生,而抑制TeA毒素的产生。     

Alternaria toxins in wheat from the Autonomous Province of Vojvodina,Serbia: a preliminary survey

Although Fusarium species remain a main source of mycotoxin contamination of wheat, in recent years, due to the evident climatic changes, other mycotoxigenic fungi have been recognised as important wheat contaminants. Alternaria species, especially A. alternata, have been found as contaminants of wheat as well as wheat-based products. Under favourable conditions A. alternata very often produce alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA) and others Alternaria toxins. The aim of the present study was to examine the presence of three Alternaria toxins (AOH, AME and TeA) in wheat samples harvested during three years (2011–13). To this end, 92 samples were collected during wheat harvesting from different growing regions of the Autonomous Province of Vojvodina, which represents the most important wheat-growing area in Serbia. The presence of Alternaria toxins was analysed by HPLC with electrospray ionisation triple quadrupole mass spectrometry (LC-ESI-MS/MS). Among all the analysed wheat samples, 63 (68.5%) were contaminated with TeA, 11 (12.0%) with AOH and 6 (6.5%) with AME. Furthermore, the maximum and mean toxin concentrations were 2676 and 92.4 µg kg^?1, 48.9 and 18.6 µg kg^?1, and 70.2 and 39.0 µg kg^?1 for TeA, AOH and AME, respectively. Co-occurrence of three Alternaria toxins in wheat samples was detected in six samples; a combination of two toxins was found in two samples; and 64 samples contained one toxin. The results showed that among 92 analysed wheat samples, only 20 (21.7%) samples were without Alternaria toxins. The presence of Alternaria toxins was also investigated in terms of weather conditions recorded during the period of investigation, as well as with the sampling region. This study represents the first preliminary report of the natural occurrence of Alternaria toxins in wheat (Triticum aestivum) from Serbia.     

农产品及其制品中交链孢酚和交链孢酚单甲醚研究进展

交链孢菌(Alternaria spp.)是一类重要的植物病原体,能产生多种毒素,其中交链孢酚(alternariol,AOH)和交链孢酚单甲醚(alternariol monomethyl ether,AME)是目前从食物中检出的比较普遍和主要的2种交链孢毒素,可广泛污染蔬菜、水果及谷物等农产品及其制品。本文对AOH和AME的结构及理化性质、毒性、产生影响因素、生物及化学合成和污染现状等进行了综述,并对交链孢毒素限量标准的制定、快速检测方法等进行了展望。     

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection

ABSTRACT

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λ_exc = 258, λ_em = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD < 8%, n = 6) for AOH and 67–91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg^–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.     

番茄交链孢菌病斑及其外延组织中交链孢毒素的分布

目的 检测不同大小的交链孢菌病斑及其外延组织中交链孢毒素残留量, 明确交链孢毒素在番茄中的迁移规律。方法 样品经80%乙腈溶液提取后, 通过自制固相萃取柱排除杂质干扰, 流出液经氮吹至近干后, 采用超高效液相色谱-串联质谱仪(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)测定交链孢毒素残留量。结果 不同大小病斑组织中均检测到高浓度的4种交链孢毒素残留, 包括交链孢酚(alternariol, AOH), 交链孢酚单甲醚(alternariol monomethyl ether, AME), 细交链格孢酮酸(tenuazonic acid, TeA)和交链孢烯(altenuene, ALT); 外延组织中仅在病斑周围1 cm处检测到TeA, 含量为病斑组织的1/10左右。结论 番茄交链孢毒素能向病斑外延组织扩散, 但扩散情况与病斑大小无直接联系, 病斑周围2 cm处虽然用超高效液相色谱-串联质谱仪检测不到交链孢毒素, 但仍存在安全风险, 因此, 建议食用时或生产中将病斑及其外延2 cm范围内组织均剔除。     

Alternaria toxins in Argentinean wheat,bran, and flour

Alternaria species have been reported to infect a wide variety of vegetables, fruits, and cereal crops. Wheat is one of the most consumed cereal worldwide. A sensitive HPLC-DAD methodology was applied to quantify alternariol (AOH), alternariol methyl ether (AME) and tenuazonic acid (TeA) in 65 samples of whole wheat, bran, and flour. The extraction methodology allowed extracting the three toxins simultaneously. Limits of detection in wheat were 3.4, 4.5, and 0.5 µg kg^?1 for AOH, AME and TeA, respectively. For bran, these data were 3.1, 4.5, and 12 µg kg^?1 and for flour 50, 70, and 14 µg kg^?1, respectively. The studied recoveries were higher than 70% and RSD was below 10%. Wheat and bran samples showed low AOH and AME contamination compared to TeA. The averages levels found for TeA in wheat, bran and flour were 19,190, 16,760, and 7360 µg kg^?1, respectively.     

食品中链格孢霉毒素污染现状及检测技术研究进展

链格孢霉毒素是由链格孢霉属（Alternaria species）产生的次级代谢产物,具有致突变、致癌、致畸等多种毒性。文章概述了水果、蔬菜、谷物及其制品中链格孢霉毒素的污染现状,综述了链格孢霉毒素检测方法的研究现状,并展望了链格孢霉毒素检测技术今后的研究方向。     

分散固相萃取净化-超高效液相色谱-串联质谱法测定水果中7种链格孢霉毒素的含量

目的 建立分散固相萃取净化-超高效液相色谱-串联质谱检测法快速、准确地同时检测水果中7种链格孢霉毒素的方法。方法 试样用酸化乙腈提取,无水硫酸镁除水,氯化钠盐析,上层清液经C18与GCB分散固相萃取净化后,采用超高效液相色谱-串联质谱测定,基质匹配标准溶液外标法定量。结果 7种链格孢霉毒素在0.005～0.5 mg/L范围内线性关系好(r2＞0.99),检出限为0.005 mg/kg,定量限为0.01 mg/kg,回收率在77.30%~114.34%之间,批内相对标准偏差为0.65%~14.81%,批间相对标准偏差为0.03%~18.59%。结论 该方法简单、高效,精密度及准确度高,适用于水果中7种链格孢霉毒素的测定。     

超高效液相色谱-串联质谱法测定麦芯粉中四种交链孢毒素

探讨固相萃取方法,超高效液相色谱串联质谱仪(UPLC-MS/MS)测定麦芯粉中4种交链孢毒素。样品经0.05 mol/L Na H2PO4(pH 3.0)-乙腈-甲醇超声提取(450+450+100,V/V),上清液过HLB固相萃取柱净化,以1.0 mmol/L氨水溶液(pH 8.3)-甲醇为流动相,经Waters CORTECS C18(4.6×100 mm,2.7μm)柱分离梯度洗脱,采用电喷雾负离子(ESI-)、多反应监测(MRM)模式检测;以基质加标工作曲线定量。细交链孢菌酮酸(TeA)、交链孢酚(AOH)在2.0μg/L~100μg/L浓度范围内,腾毒素(TEN)、交链孢酚单甲醚(AME)在0.2μg/L~10.0μg/L浓度范围内线性关系良好,相关系数(R2)均大于0.998。回收率为76.3%~107.5%,TeA、AOH检出限和定量限分别为1.0μg/kg和3.0μg/kg、TEN、AME检出限和定量限分别为0.1μg/kg和0.3μg/kg。该方法灵敏度高,简便,准确。适于测定麦芯粉中的交链孢毒素。     

超高效液相色谱-串联质谱法同时测定粮食中4种交链孢霉毒素

为同步测定大米、玉米和小麦中4种交链孢霉毒素,本研究建立了相应的超高效液相色谱-串联质谱法。试样中交链孢霉毒素用酸化乙腈水溶液提取,经HLB固相萃取柱净化后,以ACQUITY UPLC BEH C18柱(1.7μm, 2.1 mm×100 mm)为分析柱,以0.55 mmol/L氨水水溶液-乙腈为流动相进行梯度洗脱,以负离子电喷雾模式电离(ESI^-),多反应离子监测(MRM)方式进行定性和定量检测,外标法定量。在本方法条件下,4种交链孢霉毒素在3种基质中线性范围内均呈现良好的线性关系(R²>0.990 0),方法检出限0.4～4.0μg/kg,定量限1.0～10.0μg/kg。在3种加标水平下,4种毒素的回收率范围为62.50%～113.40%,相对标准偏差RSD≤7.89%。采用建立的方法检测了市售大米和小麦中的4种交链孢霉毒素,大米中4种毒素的污染水平为ND～12.18μg/kg,小麦中为ND～163.32μg/kg,本方法可满足实际样品的检测。     

人工接种温州蜜柑后链格孢霉毒素的产生及分布规律

采用超高效液相色谱-串联质谱法结合Qu ECh ERS(quick,easy,cheap,effective,rugged,safe)前处理方法,研究温州蜜柑人工接种链格孢菌(Alternaria alternate)后,病斑部位全果、非病斑部位果皮和果肉中腾毒素(tentoxin,Ten)、链格孢酚(alternariol,AOH)、交链格孢酚单甲醚(alternariol monomethyl ether,AME)、交链孢烯(altenuene,ALT)和细交链格孢菌酮酸(tenuazonic acid,Te A)5种链格孢霉毒素的产生和分布规律。结果表明,病斑部位全果、非病斑部位果皮和果肉中的链格孢霉毒素含量随着病斑直径的扩大,不同链格孢霉毒素的变化趋势不同。Te A、Ten、AME和AOH在所有部位中均有检出,其中Te A检出含量最高,在病斑部位的含量范围为3.05×10~3~55.88×10~3μg/kg,在非病斑部位的含量范围为65.35~40.68×10~3μg/kg;Ten、AME和AOH在病斑部位的含量范围分别为69.16~373.94、22.63~1 395.82μg/kg和8.18~689.19μg/kg;非病斑部位的含量范围分别为0~67.56、0~195.96μg/kg和0~301.91μg/kg;ALT在非病斑部位果肉中未检出,但在非病斑部位的果皮和病斑部位的全果中均有检出,含量最高达16.61μg/kg。研究表明,由于柑橘感染链格孢菌后产生的毒素会从发病部位扩散到健康部位累积,因此,在鲜食加工和风险评估中应引起关注和重视。     

QuEChERS-超高效液相色谱-串联质谱法测定小麦粉中4种交链孢霉菌素

目的建立QuEChERS-超高效液相色谱-串联质谱法测定小麦粉中4种交链孢霉菌素含量。方法样品以丙酮为提取剂,经QuEChERS法前处理,采用Endeavorsil C_(18)色谱柱(2.1 mm×100 mm,1.8μm),以甲醇-1.5 mmol/L碳酸氢铵为流动相,梯度洗脱,多反应监测模式检测,细交链孢菌酮酸含量采用内标法定量,交链孢酚、腾毒素和交链孢酚单甲醚含量采用外标法定量。结果该方法对4种交链孢霉菌素有很好的分离效果,检出限为0.06~0.50μg/kg,回收率为85.4%~104.3%,相对标准偏差为2.3%~9.8%,相关系数≥0.9990。结论本方法简便、快速,灵敏度、精密度、回收率均满足方法学要求,适用于小麦粉中4种交链孢霉菌素含量的同时测定。     

超高效液相色谱-串联质谱法同时测定小麦制品中4种交链孢霉毒素

目的建立测定4种小麦制品(挂面、馒头、面包和饼干)中4种交链孢霉毒素的超高效液相色谱-串联质谱法。方法 4种小麦制品加入NaCl后,经酸化乙腈提取,固相萃取柱(SPE)净化后,以HSS T3色谱柱(2.1 mm×100 mm,1.7μm)为分析柱,以0.15 mmol/L碳酸氢铵水溶液-甲醇为流动相进行梯度洗脱,以负离子电喷雾模式电离(ESI~-),多反应离子监测(MRM)方式进行定性及定量检测,外标法定量。结果在0.040～50μg/L的线性范围内,4种交链孢霉毒素的回归方程均呈良好的线性关系,相关系数(r)0.99。交链孢菌酮酸、交链孢酚、腾毒素和交链孢酚单甲醚的方法检出限分别为0.10、0.01、0.03和0.01μg/kg,3个加标水平的回收率为85.7%～102%,相对标准偏差(RSD)为0.8%～11.0%。应用该方法对流通环节的挂面、馒头、面包和饼干进行检测,4种交链孢霉毒素均有检出,其浓度为0.02～185.00μg/kg。结论该方法灵敏度高、简便,适用于小麦制品中多种交链孢霉毒素的同时测定。     

超高效液相色谱-四极杆串联离子阱复合质谱法筛查和定量玉米中5种链格孢菌毒素

目的 建立超高效液相色谱-四极杆串联离子阱复合质谱法(ultra performance liquid chromatography-Q-trap-tandem mass spectrometry, UPLC-Q-Trap-MS)对玉米中的5种链格孢菌毒素同时进行筛查和定量分析方法。方法 使用改进型QuEChERS法对玉米样品进行提取和净化,以多级反应监测(multi reaction monitoring, MRM)作为探针检测,并通过信息依赖性采集(information dependent acquisition, IDA)触发增强型子离子扫描(enhanced product ion scan, EPI)模式,采用外标法定量。结果 玉米中5种链格孢菌毒素的检出限为0.017~0.720 ?g/kg,定量限为0.057~2.400 ?g/kg,并且在1.0~200.0 ng/mL的线性范围内良好,相关系数r≥0.9929。在样品基质中分别添加20、40和200 ?g/kg三个水平的标准品进行加标回收试验,5种链格孢菌毒素的回收率为80.6%~104.7%,相对标准偏差（Relative standard deviations, RSDs）为1.57%~8.27% (n=6)。结论 该方法具有操作简单、灵敏度高、准确度高的特点,适合于玉米中5种链格孢菌毒素的同时快速筛查和准确定量。     

超高效液相色谱-串联质谱法快速检测麦类中典型链格孢霉毒素

建立一种超高效液相色谱-串联质谱结合分散液-液微萃取前处理方法测定麦类中7 种链格孢霉毒素（链格孢酚、交链格孢酚单甲醚、交链孢烯、细交链格孢菌酮酸、交链孢毒素I、腾毒素、细格菌素）的方法。样品经3 mL一级水、10 mL 1.5%甲酸乙腈-甲醇（4∶1,v/v）溶液提取,2 g无水MgSO4脱水和1 g NaCl盐析,振荡15 min、离心10 min后取1 mL上清液和100 μL三氯甲烷用于分散液-液微萃取。7 种链格孢霉毒素使用BEH C18色谱柱（50 mm×2.1 mm,1.7 μm）进行分离,采用电喷雾正负离子的多反应离子监测模式。结果表明：7 种链格孢霉毒素在5 min内完成色谱分离分析,在0.5～100 μg/L质量浓度范围内均呈现良好线性关系,R2＞0.987 277,检出限为0.11～0.16 μg/kg,定量限为0.42～0.49 μg/kg;以小麦为样品基质,在3 个不同加标水平下,7 种链格孢霉毒素回收率在70.7%～101.3%之间,相对标准偏差为1.22%～4.11%。将该方法用于分析实际麦类样品（黑麦、荞麦、莜麦、青稞、燕麦、小麦）中7 种链格孢霉毒素的污染状况,结果发现腾毒素和细交链格孢菌酮酸是6 种麦类中都检出的毒素,含量分别为0.6～10.7 μg/kg和未检出～31 μg/kg;细格菌素虽检出率低于腾毒素和细交链格孢菌酮酸,但最高含量37.3 μg/kg与细交链格孢菌酮酸相当。本方法稳定、准确、灵敏、快速,能够满足麦类样品中7 种链格孢霉毒素残留分析的需求。     

超高效液相色谱-串联质谱法测定食用植物油中典型链格孢霉毒素

建立超高效液相色谱-串联质谱法测定食用植物油中典型链格孢霉毒素残留。正交试验确定最优提取条件为4 mL甲醇-乙腈溶液（1∶1,V/V）涡旋萃取10 s。选取Waters BEH C18(100 mm×2.1 mm,1.7μm)色谱柱,乙腈和1 mmol/L碳酸铵溶液为流动相,梯度洗脱,采用电喷雾离子源负离子多反应监测模式测定。细交链格孢菌酮酸在0.02～0.2 mg/L质量浓度范围内线性关系良好,链格孢酚、交链孢烯、腾毒素、交链格孢酚单甲醚4种毒素在0.002～0.2 mg/L质量浓度范围内线性关系良好,R2≥0.998。验证了5种毒素在葵花籽油和大豆油基质下方法准确性和精密度,在0.002～0.2 mg/kg添加量下,5种毒素的平均回收率为98%～118%,相对标准偏差为2.9%～17.4%,定量限为0.002～0.02 mg/kg。该方法能够满足食用植物油中典型链格孢霉毒素分析要求。