Evaluation of matrix solid-phase dispersion (MSPD) extraction for multi-mycotoxin determination in different flours using LC-MS/MS

An existing matrix solid-phase dispersion (MSPD) method for aflatoxins (AFs) and ochratoxin A (OTA) extraction was extended by further 14 mycotoxins. After it careful optimization, this method was applied to determine the occurrence of these mycotoxins on commercial flour samples (with different cereals composition) collected from local markets. In a total of 49 samples investigated, 9 mycotoxins were identified. Nivalenol (NIV) and Beauvericin (BEA) were the mycotoxins found most frequently. The samples that presented major contamination were wheat flours and bakery preparations. Despite of the great number of positives finding, only one wheat flour sample exceeded the maximum limits (ML) for OTA established by the European Union (EU). However, it would be interesting to calculate the total ingest of these mycotoxins along the years.     

Evaluation of mycotoxins and their metabolites in human breast milk using liquid chromatography coupled to high resolution mass spectrometry

Humans can be exposed to mycotoxins through the food chain. Mycotoxins are mainly found as contaminants in food and could be subsequently excreted via biological fluids such as urine or human breast milk in native or metabolised form. Since breast milk is usually supposed as the only food for new-borns, the occurrence of mycotoxins in thirty-five human milk samples was evaluated by a newly developed method based on QuEChERS extraction and UHPLC–HRMS detection. The method described here allows the detection of target mycotoxins in order to determine the quality of this initial feeding. The method has been fully validated, with recoveries ranging from 64% to 93% and relative standard deviations (RSD, %) being lower than 20%. Using the method described, non-metabolised mycotoxins such as ZEA, NEO, NIV, ENA, ENA₁, ENB, ENB₁ and metabolites, such as ZEA metabolites, HT-2, DOM and T-2 triol were detected in human milk samples. Results obtained help to estimate the exposure of mothers and infants to mycotoxins. Moreover, to the best of our knowledge, this is the first work describing the simultaneous detection, quantification and screening of mycotoxins and their metabolites in human mature milk.     

应用超高效液相色谱-四极杆-飞行时间质谱法建立谷物中18种真菌毒素非靶向筛查数据库及确证方法

建立了基于超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF/MS)的18种真菌毒素非靶向筛查方法。真菌毒素标准物质用HSS T3色谱柱进行色谱分离后在UPLC-Q-TOF/MS MS^E模式下分别用正、负离子模式采集,获取MS和MS/MS的信息,记录对应保留时间、加合物离子、碎片离子精确质量数等信息,设置保留时间偏移为0.3 min,加合物离子和碎片离子的精确质量匹配容差为5×10^-6,在UNIFI中建立18种真菌毒素的数据库。在稻谷、小麦基质中,以筛查检出限(SDL)作为主要参数对筛查方法进行了验证。18种真菌毒素分为有最大限量和无最大限量两种类型,结果有最大限量的真菌毒素均能在其限量水平被准确筛查,无最大限量的真菌毒素其SDL的范围为2～800μg/kg。基质效应考察表明,稻谷中有14种真菌毒素有中等基质效应,小麦中有11种真菌毒素有中等基质效应。样品经乙腈提取后用QuEChERS萃取盐包和HLB净化柱净化,用建立的方法对25批稻谷、小麦进行筛查,结果2批稻谷中检出4种真菌毒素,2批小麦中检出2种真菌毒素。该方法能准确筛查SDL水...     

液相色谱-飞行时间质谱同时测定粮食中13种真菌毒素

建立了液相色谱-飞行时间质谱(LC-TOF MS)联用技术同时检测小麦和玉米中镰刀菌、曲霉菌和青霉菌产生的13种真菌毒素的分析方法。样品经乙腈-水-乙酸(84∶15∶1,体积比)混合溶剂提取,My-cosep 226多功能净化柱和强阴离子交换柱净化后,采用LC-TOF MS检测。在电喷雾正离子模式下,以保留时间和化合物精确分子离子质量对真菌毒素进行识别,以10 ppm为提取离子窗口进行定量。结果表明,13种真菌毒素在一定的线性范围内线性关系良好,相关系数均大于0.99,质量精确度均小于5 ppm,回收率为70%～113%,相对标准偏差为0.2%～14.5%。该方法可用于粮食中多种真菌毒素的同时测定。     

加速溶剂萃取-QuEChERS-超高效液相色谱-串联质谱法测定药食同源性食品中16种真菌毒素

建立了加速溶剂萃取-QuEChERS-超高效液相色谱-串联质谱测定药食同源性食品中16种真菌毒素的方法。样品经过加速溶剂萃取后用QuEChERS方法净化，液相色谱分离，在正、负离子同时扫描和多反应离子监测模式下检测，黄曲霉毒素B1和伏马毒素B1采用内标法定量，其余毒素采用基质外标法定量。在较宽的线性范围内，16种目标化合物的线性相关系数（r²）均大于0.99。该方法的检出限为0.008~0.3 μg/kg，定量限为0.03~1.0 μg/kg，在3个不同添加水平下的加标回收率为70.8%~118%，RSD为2.5%~10.2%。采用建立的方法分别对市面上销售的30个批次的山银花、葛根和沙棘产品进行检测，部分产品检出不同含量的真菌毒素。该方法快速、灵敏，适用于药食同源性食品中多种真菌毒素的同时检测。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速精准测定粮食中多种真菌毒素

采用直接提取稀释的快速前处理方法,结合稳定同位素稀释技术,利用超高效液相色谱-四极杆/静电场轨道阱高分辨质谱,建立了粮食中16种真菌毒素的快速精准分析方法。样品采用乙腈-水-乙酸溶液(70∶29∶1,体积比)提取,以C18色谱柱进行色谱分离,通过全扫描模式进行定量检测,并采用稳定同位素稀释以减少基质效应对定量分析的影响。结果表明,16种真菌毒素在一定浓度范围内均具有良好的线性关系,相关系数(r2)均大于0.999,4种常见粮食基质(小麦、玉米、大米、大麦)的限量浓度水平的加标回收率(n=6)为75.3%~123.5%,相对标准偏差为0.41%~14.7%。该方法简单、准确,适用于粮食中真菌毒素的检测,可满足日常监测工作的需要。     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱对粮谷产品中20种真菌毒素的快速筛查和确证

建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱快速筛查和确证粮谷产品中20种真菌毒素的方法。样品经乙腈（含2%（体积分数）甲酸）提取,用Captiva EMR-Lipid小柱净化,采用Thermo Hypersil Gold C₁₈柱（100 mm×2.1 mm,1.9 μm）分离,用四极杆/静电场轨道阱高分辨质谱进行分析。在全扫描模式下以分析物的保留时间和一级母离子信息实现快速筛查,以自动触发采集的二级碎片离子信息进行确证。结果显示,目标分析物在各自的质量浓度范围内线性关系良好（相关系数r²>0.99）,方法检出限为0.25~20 μg/kg,回收率为72.9%~117.8%,相对标准偏差为2.9%~15.2%（n=6）。该方法灵敏度高,结果准确、可靠,适用于粮谷产品中20种真菌毒素的快速筛查和确证。     

Screening of aflatoxins in feed samples using a flow system coupled to capillary electrophoresis

A method is proposed which presents a new approach to the joint use of capillary electrophoresis (CE) commercial equipment and a flow system. This flow system allows the total determination of several compounds by using a fluorimetric screening system. The individual determination for each analyte is performed by the CE proposed method. The screening procedure uses simple equipment and operations and provides a yes/no binary response that occasionally requires confirmation. A fast, simple, and reliable method has been developed in order to determine the most frequent mycotoxins in feed samples using micellar electrokinetic capillary chromatography (MECC). An extraction step followed by a purification step was carried out on the samples in order to remove interference substances before analysis. A C18 column was chosen to concentrate the mycotoxins, and the analytes were eluted from C18 using methanol. The MECC method allows the separation of six mycotoxins within 50 min with a reproducibility as RSD between 7.45 and 13.06%, and a limit of detection (LOD) between 0.02 and 0.06 mg l(-1) for all the mycotoxins. These LODs were clearly below legal limits (0.05 mg l(-1)).     

Determination of 16 Mycotoxins in Maize by Ultrahigh-Performance Liquid Chromatography–Tandem Mass Spectrometry

An ultrahigh-performance liquid chromatography–tandem mass spectrometry method was developed and validated for the simultaneous determination of sterigmatocystin, verruculogen, enniatin A, fusarenon-X, fumonisins B1, B2, B3, aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, 3-acetyldeoxynivalenol, 5-acetyldeoxynivalenol, and zearalenone. The mycotoxins were extracted and cleaned up using a multitoxin column, separated on a C18 column, and then detected on a triple-quadrupole mass spectrometer. The limits of detection and quantification ranged within 0.2–2?µg/kg and 1–10?µg/kg, respectively. The recoveries ranged from 70.8 to 118.4%, with relative standard deviations below 15%. The method was used to analyze 80 samples obtained from Shandong Province in China. Fifty-eight samples were contaminated with 10 mycotoxins at concentrations ranging from 1.4 to 6566.1?µg/kg. Some samples exceeded the maximum limits in China and in European regulations for mycotoxins in unprocessed maize.     

Development of a liquid chromatography/time-of-flight mass spectrometric method for the simultaneous determination of trichothecenes, zearalenone and aflatoxins in foodstuffs

A liquid chromatography/atmospheric pressure chemical ionization mass spectrometry (LC/APCI-MS) method based on time-of-flight MS (TOFMS) with a real-time reference mass correction technique was developed for the simultaneous determination of Fusarium mycotoxins (nivalenol, deoxynivalenol, fusarenon X, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, HT-2 toxin, T-2 toxin, diacetoxyscirpenol, zearalenone) and Aspergillus mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2) in corn, wheat, cornflakes and biscuits. Samples were cleaned up with a MultiSep #226 column. Detection of the mycotoxins was carried out in exact mass chromatograms with a mass window of 0.03 Th. Calibration curves were linear from 2 to 200 ng x mL(-1) for trichothecenes and zearalenone, and 0.2 to 20 ng x mL(-1) for aflatoxins, by 20 microL injection. The limits of detection ranged from 0.1 to 6.1 ng x g(-1) in foodstuffs analyzed in this study. The LC/TOFMS method was found to be suitable for the screening of multiple mycotoxins in foodstuffs rapidly and with high sensitivity, and its performance was demonstrated for the confirmation for target mycotoxins.     

A quick,easy, cheap,effective, rugged,and safe sample pretreatment and liquid chromatography with tandem mass spectrometry method for the simultaneous quantification of 33 mycotoxins in Lentinula edodes

Lentinula edodes, one of the most cultivated edible fungi in the world, are usually neglected for mycotoxins contamination due to the initial thinking of its resistance to mycotoxingenic molds. In the present study, a sensitive and reliable liquid chromatography with tandem mass spectrometry method was developed for the simultaneous quantification of 33 mycotoxins in L. edodes. Targeted mycotoxins were extracted using a quick, easy, cheap, effective, rugged, and safe procedure without any further clean‐up step, and analyzed by liquid chromatography with tandem mass spectrometry on an Agilent Poroshell 120 EC‐C₁₈ column (100 × 3 mm, 2.7 μm) with a linear gradient elution program using water containing 5 mM ammonium acetate and methanol as the mobile phase. After validation by determining linearity (R² > 0.99), sensitivity (LOQ ≤ 20 ng/kg), recovery (73.6–117.9%), and precision (0.8–19.5%), the established method has been successfully applied to reveal the contamination states of various mycotoxins in L. edodes. Among the 30 tested samples, 22 were contaminated by various mycotoxins with the concentration levels ranging from 3.3–28 850.7 μg/kg, predicting that the edible fungus could be infected by the mycotoxins‐producing fungi. To the best of our knowledge, this is the first report about real mycotoxins contamination in L. edodes.     

基于QuEChERS提取的超高效液相色谱-串联质谱法测定小麦粉中5种镰刀菌毒素

建立了小麦粉中恩镰孢菌素A、恩镰孢菌素A1、恩镰孢菌素B、恩镰孢菌素B1、白僵菌毒素5种镰刀菌毒素的超高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。小麦粉样品采用改良的Qu ECh ERS方法进行提取,无需进一步净化,以甲醇-2 mmol/L乙酸铵为流动相梯度洗脱,经Agilent Eclipse Plus C_(18)色谱柱(2.1 mm×50 mm,1.8μm)分离,在电喷雾电离(ESI)正离子模式下采用多反应监测(MRM)进行测定,基质外标法定量。在较宽的线性范围内,5种镰刀菌毒素的相关系数(r2)均不小于0.993,方法检出限为0.3~0.8μg/kg,定量下限为0.8~2.4μg/kg。样品在1倍、2倍、10倍定量下限3个加标浓度下的平均回收率为74.0%~85.4%,相对标准偏差(RSDs)为5.6%~13.1%。采用建立的方法对市售的30批次小麦粉中5种镰刀菌毒素进行筛查,数批产品检出不同含量的镰刀菌毒素。该方法简单快速、准确、灵敏,可用于小麦粉中多种镰刀菌毒素的同时分析。     

复合免疫亲和柱-高效液相色谱法同时测定谷物及其制品中9种真菌毒素

建立了复合免疫亲和柱-在线光化学衍生-高效液相色谱同时测定谷物及其制品中9种真菌毒素的检测方法。以乙腈-水（80：20,v/v）混合溶液提取样品中9种真菌毒素,提取液经自制真菌毒素复合免疫亲和柱净化,采用高效液相色谱进行分离,在线光化学衍生后进入荧光检测器测定,外标法定量。结果表明,9种真菌毒素在相应浓度范围内线性关系良好,相关系数均大于0.999;在低、中、高3个不同加标浓度下,9种真菌毒素的回收率均大于80%,相对标准偏差（RSD）为1.0%~5.6%;方法的检出限（LOD）为0.02~5.00 μg/kg,定量限（LOQ）为0.07~16.70 μg/kg。该方法具有重现性好、灵敏度高、结果准确的特点,适用于谷物及其制品中9种真菌毒素残留的分析检测。     

Minimization of carryover for high‐throughput liquid chromatography with tandem mass spectrometry analysis of 14 mycotoxins in corn grits

A method for the simultaneous analysis of 14 mycotoxins with the minimization of carryover was developed. Our verification experiments suggested that the carryover occurred due to the chelation of fumonisins with the metal. To wash the fumonisins from the metal, the inner surface of the injection needle was rinsed with 10 mM trisodium citrate and 1% formic acid in water/methanol/acetonitrile/isopropanol after each injection, and the analysis was performed on a metal‐free Mastro C18 column. This approach remarkably minimized the carryover of fumonisins. Fourteen mycotoxins in samples were extracted with 2% acetic acid in water/acetonitrile and a quick, easy, cheap, effective, rugged, and safe extraction kit, purified on a MultiSep 229 Ochra, and then quantified by liquid chromatography with tandem mass spectrometry. Determinations performed using this method produced a linearity greater than 0.99 and recoveries ranging from 72.6 to 117.4%, with good intraday precision from 4.0 to 12.4%, and interday precision from 6.5 to 17.0%. The limits of detection ranged from 0.01 to 0.71 μg/kg, demonstrating that a highly sensitive method for the simultaneous analysis of mycotoxins over a wide range of concentrations was achieved with minimal carryover. When 12 samples of commercially available corn grits were analyzed with this method, deoxynivalenol, fumonisin B1, fumonisin B2, fumonisin B3, and zearalenone were present most frequently.     

超高效液相色谱-四极杆/静电场轨道阱高分辨质谱法测定焙烤食品及其原料中11种真菌毒素

建立了超高效液相色谱-四极杆/静电场轨道阱高分辨质谱(UPLC-HRMS)测定焙烤食品及其原料中11种真菌毒素的检测分析方法。样品经20 mL含1%(体积分数)甲酸的乙腈-水(9∶1,v/v)溶液提取,经2.0 g无水硫酸、0.5 g氯化钠和300 mg C18盐析、净化后进行检测。采用CORTECS C18色谱柱(100 mm×2.1 mm,1.6μm),以含0.1%(体积分数)甲酸的2 mmol/L乙酸铵溶液和含0.1%(体积分数)甲酸的2 mmol/L乙酸铵甲醇溶液为流动相进行梯度洗脱。结果显示,11种真菌毒素在各自的质量浓度范围内线性关系良好(相关系数r2≥0.996 0),方法的定量限为0.15~20.00μg/kg,样品加标回收率为64.38%~122.61%,相对标准偏差为1.52%~12.99%(n=6)。该方法简单快速、灵敏度高、结果准确、可靠,利用该方法可有效测定焙烤食品及其原料中常见真菌毒素的含量。     

Fast determination of 14 mycotoxins in chestnut by dispersive solid‐phase extraction coupled with ultra high performance liquid chromatography‐tandem mass spectrometry

A dispersive solid‐phase extraction coupled with ultra high performance liquid chromatography with tandem mass spectrometry method was developed and validated for the simultaneous determination of T‐2 toxin, penicillic acid, fumonisins B₁, B₂, and B₃, aflatoxins B₁, B₂, G₁, and G₂, ochratoxin A, deoxynivalenol, 3‐acetyldeoxynivalenol, 15‐acetyldeoxynivalenol, and zearalenone in chestnut samples. The method was used to analyze 136 samples obtained from Shandong province in China. The mycotoxins were extracted using a dispersive solid‐phase extraction method and cleaned using an improved quick, easy, cheap, effective, rugged, and safe approach. The mycotoxins were then detected using a triple‐quadrupole mass spectrometer. The limits of detection and quantification ranged from 0.02 to 1 and 0.1 to 2 μg/kg, respectively. The recovery rates ranged from 74.2 to 109.5%, with relative standard deviations below 15%. A total of 71 samples were contaminated with seven mycotoxins at concentrations ranging from 1.2 to 105.5 μg/kg, with a number of samples exceeding the maximum limits set in the European regulations for mycotoxins in unprocessed chestnuts.     

Determination of alpha- and beta-zearalenol and zearalenone in cereals by gas chromatography with ion-trap detection.

A method for determination of estrogenic mycotoxins, alpha- and beta-zearalenol and zearalenone, in cereals (wheat, barley, oats, corn) is described. After extraction with ethylacetate, clean-up involved a base treatment and partition with water; derivatization was by trimethylsilylation. For quantitation and confirmation a capillary gas chromatograph combined with a selective mass detector (ion trap), working in the electron impact-mode was used. The detection limit for the complete method is 1 microgram/kg for each of the three mycotoxins in full scan. Recoveries from spiked cereals were 82-86%.     

Development and validation of a gas chromatography mass spectrometry method for the analysis of phytoestrogens,phytosterols and mycotoxins in estuarine water samples

Many pollutants currently released into the environment may cause adverse effects on exposed organisms leading to disruption of the endocrine system. Among them are natural compounds such as phytoestrogens and phytosterols, found in various plants and mycotoxins produced by a wide range of fungal species. The presence of some classes of phytoestrogens and phytosterols has been demonstrated in environmental samples while little information can be found about the presence of mycotoxins. Due to the complexity of environmental matrices and the low concentrations normally found, sensitive and selective methods are required for an unequivocal quantification of this type of compounds. The aim of this study was to develop and validate an analytical method for the simultaneous quantification of seven phytoestrogens, two phytosterols and three mycotoxins in estuarine water samples. The method consisted of the preconcentration of estuarine water samples (1 L) on 200 mg OASIS HLB cartridges, followed by a clean-up step in 1 g silica cartridges and quantification by gas chromatography with mass spectrometry ion trap analyser. Validation parameters were evaluated and the method demonstrated to be selective and linear with correlations higher than 0.99. Method detection limits were in the ng L^?1 levels ranging from 18.0 to 90.0 ng L^?1, recovery rates ranged from 59.8% to 99.5% and precision (RSD) from 5.5% and 9.7%. The feasibility of the method was demonstrated in surface water samples from Douro River estuary, a polluted estuary, with reported incidence of endocrine-disrupting phenomena.     

Simultaneous determination of aflatoxins, ochratoxin A and Fusarium toxins in maize by liquid chromatography/tandem mass spectrometry after multitoxin immunoaffinity cleanup

A liquid chromatography/tandem mass spectrometry method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, fumonisins (B(1), B(2)), deoxynivalenol, zearalenone, T-2 and HT-2 toxins in maize. A double extraction approach, using a phosphate-buffered solution followed by methanol, was applied to achieve effective co-extraction of the 11 mycotoxins under investigation having quite different polarities and chemical structures. A new multitoxin immunoaffinity column containing antibodies for all these mycotoxins was used to clean up the extract. Detection and quantification of the 11 mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS) using, as chromatographic mobile phase, a linear gradient of methanol/water containing 0.5% acetic acid and 1 mM ammonium acetate. Method performances were quite satisfactory for all tested mycotoxins at contamination levels close to or below the relevant EU maximum permitted or recommended levels. Limits of detection in maize ranged from 0.3 to 4.2 microg/kg. Recoveries higher than 79% were obtained for all tested mycotoxins with relative standard deviations less than 13%.     

Development of a sensitive and reliable high performance liquid chromatography method with fluorescence detection for high-throughput analysis of multi-class mycotoxins in Coix seed

As an edible and medicinal plant, Coix seed is readily contaminated by more than one group of mycotoxins resulting in potential risk to human health. A reliable and sensitive method has been developed to determine seven mycotoxins (aflatoxins B₁, B₂, G₁, G₂, zearalenone, α-zearalenol, and β-zearalenol) simultaneously in 10 batches of Coix seed marketed in China. The method is based on a rapid ultrasound-assisted solid–liquid extraction (USLE) using methanol/water (80/20) followed by immunoaffinity column (IAC) clean-up, on-line photochemical derivatization (PCD), and high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). Careful optimization of extraction, clean-up, separation and detection conditions was accomplished to increase sample throughput and to attain rapid separation and sensitive detection. Method validation was performed by analyzing samples spiked at three different concentrations for the seven mycotoxins. Recoveries were from 73.5% to 107.3%, with relative standard deviations (RSDs) lower than 7.7%. The intra- and inter-day precisions, expressed as RSDs, were lower than 4% for all studied analytes. Limits of detection and quantification ranged from 0.01 to 50.2 μg kg⁻¹, and from 0.04 to 125.5 μg kg⁻¹, respectively, which were below the tolerance levels for mycotoxins set by the European Union. Samples that tested positive were further analyzed by HPLC tandem electrospray ionization mass spectrometry for confirmatory purposes. This is the first application of USLE-IAC-HPLC-PCD-FLD for detecting the occurrence of multi-class mycotoxins in Coix seed.