Development of a liquid chromatography/tandem mass spectrometry method for the simultaneous determination of 16 mycotoxins on cellulose filters and in fungal cultures

The development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the simultaneous determination of 16 mycotoxins possibly related to the 'Sick Building Syndrome' on filters and in fungal cultures is described. Fungi-surface sampling as regards the 'Sick Building Syndrome' preferably happens by scraping off fungal material and vacuuming onto cellulose filters. Therefore, these two media were used as samples. They were spiked with nivalenol, deoxynivalenol, zearalenone, diacetoxyscirpenol, T-2 toxin, verrucarol, verrucarin A, neosolaniol, sterigmatocystin, roridin A, ochratoxin A, aflatoxin B1, aflatoxin B2, aflatoxin G1 and aflatoxin G2, which can be produced by isolates from fungi-damaged buildings. Deepoxy-deoxynivalenol was used as internal standard. Samples were extracted with organic solvents and the different mycotoxins were separated by high-performance liquid chromatography (HPLC) using a C18 reversed-phase SunFire analytical column and a mobile phase of variable mixtures of ammonium acetate (10 mM) and sodium acetate (20 microM) in water (solvent A) and in methanol (solvent B). The samples were run on-line with a Micromass Quattro Micro triple quadrupole mass spectrometer in positive electrospray ionisation mode using multiple reaction monitoring (MRM). The detection limits of the procedure varied from 50 to 0.009 pg/microL for filter samples and from 75 to 0.04 pg/microL for fungal culture samples. As the method includes few and non-labourious sample treatment steps, it should allow for a high throughput of samples.     

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.     

Determination of aflatoxins in peanuts by matrix solid-phase dispersion and liquid chromatography

A new method based on matrix solid-phase dispersion (MSPD) extraction was studied to determine aflatoxin B1, B2, G1 and G2 from peanuts. Optimization of different parameters, such as type of solid supports for matrix dispersion and elution solvents were carried out. The method used 2 g of peanut sample, 2 g of C18 bonded silica as MSPD sorbent and acetonitrile as eluting solvent. Recoveries of each aflatoxin spiked to peanut samples at 2.5 ng/g (5 ng/g for aflatoxin G2) level were between 78 and 86% with relative standard deviations ranging from 4 to 7%. The limits of quantification ranged from 0.125 to 2.5 ng/g for the four studied aflatoxins using liquid chromatography (LC) with fluorescence detection. In addition, LC coupled to mass spectrometry with an electrospray interface was used for confirmation of aflatoxins present in real samples. Eleven peanut samples from different countries were analyzed by the proposed method and by using an enzyme-linked immunosorbent assay (ELISA). ELISA test is a good screening method for investigation of these mycotoxins in peanut samples.     

Development and in-house validation of a robust and sensitive solid-phase extraction liquid chromatography/tandem mass spectrometry method for the quantitative determination of aflatoxins B1, B2, G1, G2, ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods

A sensitive and robust liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the simultaneous determination of aflatoxins (B(1), B(2), G(1), G(2)), ochratoxin A, deoxynivalenol, zearalenone, T-2 and HT-2 toxins in cereal-based foods. Samples were extracted with a mixture of acetonitrile/water (84:16, v/v) and cleaned up through a polymeric solid-phase extraction column. Detection and quantification of the nine mycotoxins were performed by reversed-phase liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (LC/ESI-MS/MS), using fully (13)C-isotope-labelled mycotoxins as internal standards. The method was validated in-house for five different cereal processed products, namely barley, oat and durum wheat flours, rye- and wheat-based crisp bread. Recoveries and repeatability of the whole analytical procedure were evaluated at contamination levels encompassing the EU maximum permitted levels for each tested mycotoxin. Recoveries ranged from 89 to 108% for deoxynivalenol, from 73 to 114% for aflatoxins, from 85 to 114% for T-2 and HT-2 toxins, from 64 to 97% for zearalenone, from 74 to 102% for ochratoxin A. Relative standard deviations were less than 16% for all tested mycotoxins and matrices. Limits of detection (signal-to-noise ratio 3:1) ranged from 0.1 to 59.2 μg/kg. The trueness of the results obtained by the proposed method was demonstrated by analysis of reference materials for aflatoxins, deoxynivalenol, zearalenone. The use of inexpensive clean-up cartridges and the increasing availability of less expensive LC/MS/MS instrumentation strengthen the potential of the proposed method for its effective application for reliable routine analysis to assess compliance of tested cereal products with current regulation.     

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%.     

固相萃取-液相色谱-质谱法测定益母草中7种真菌毒素

建立液相色谱-质谱法测定益母草中黄曲霉毒素G2、G1、B2、B1、T-2毒素、赭曲霉毒素A、展青霉素7种真菌毒素的方法。样品经过70%甲醇溶液超声提取,用水稀释后用固相萃取柱富集净化,以C18色谱柱分离,多反应监测(MRM)模式进行检测,7种真菌毒素得到快速分离。7种真菌毒素的质量浓度与色谱峰面积成良好的线性关系,线性相关系数在0.9972~0.9992之间,检出限为0.047~4.724μg/kg。平均加标回收率为72.8%~95.5%,测定结果的相对标准偏差为3.1%~5.7%(n=6)。该检测方法可同时高效准确检测益母草中7种真菌毒素的含量,可用于赭曲霉毒素A的定量风险评估,为益母草的安全评价提供了科学依据。     

Determination of type B fumonisin mycotoxins in maize and maize-based products by liquid chromatography/tandem mass spectrometry using a QqQlinear ion trap mass spectrometer

A sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for determining the type B fumonisin mycotoxins in corn-based foodstuffs is described. Fumonisins FB1 and FB2 were extracted from a 1 g sample by homogenization with acetonitrile/water (75:25, v/v, 50 mmol/L formic acid, 25 mL final volume) and the extract was defatted on C18 phase. Volumes of 5 mL of crude extracts were cleaned up on Carbograph-4 cartridges. The final solution was analyzed by HPLC with electrospray ionization mass spectrometry in positive ion mode using multiple reaction monitoring with a QqQ linear ion trap mass spectrometer. Recoveries for spiked corn-based foodstuffs ranged from 91-105% (RSD% < or =8%), and method detection limits were < or =2 ng/g for FB1 and < or =1 ng/g for FB2. Two different spiking levels were tested (5000 and 100 ng/g for FB1, 1000 and 20 ng/g for FB2). Quantitation was achieved by an external calibration procedure using matrix-matched standards, with diclofenac added post-cleanup as internal standard for the LC/MS/MS analyses. Calibration curves showed linearity in the concentration range 0.005-5 ng/microL of final extract (0.992 < or = R2< or =0.995). Two other fumonisins, FB3 and FB4, were identified in naturally contaminated samples of corn meal using an information-dependent acquisition protocol that looped three experiments, including neutral loss scan, enhanced resolution scan, and enhanced product ion scan. FB3 and FB4 quantitation was estimated as peak area ratios relative to the FB2 response in view of the lack of both standards. This work also includes an application of the present LC/MS/MS method to some maize and maize-based product samples (corn meal, cornflakes and popcorn) collected from Italian stores. FB1 and FB2 contamination levels exceeding the European Union recommendation were found in 8 out of 15 corn meal samples.     

Thermospray high performance liquid chromatographic/mass spectrometric analysis of some fusarium mycotoxins

Thermospray high performance liquid chromatography/mass spectrometry (TSP HPLC/MS) was used to analyze five Fusarium mycotoxins in porcine plasma and urine. Four cytotoxic trichothecene mycotoxins, T-2 toxin, HT-2 toxin, diacetoxyscirpenol (DAS), deoxynivalenol (DON), T2 tetraol, and the fungal estrogen zearalenone (F-2 toxin) were analyzed. The thermospray mass spectrum contained molecular weight information with few, if any, fragment signals. Detection limits ranging from 1 to 10 ng of mycotoxin injected onto the HPLC column were obtained using selected ion monitoring (SIM) HPLC/MS. Neither the plasma nor the urine matrix interfered with TSP HPLC/MS analysis of these mycotoxins and no sample derivatization was necessary for the analysis. The TSP HPLC/MS technique appears to be ideal for very sensitive analysis of mycotoxins in biological samples.     

Determination of aflatoxins B1, G1, B2 and G2 in medicinal herbs by liquid chromatography-tandem mass spectrometry

An easy method for the determination of aflatoxins B1, G1, B2 and G2 in Rhammus purshiana by LC coupled to mass spectrometry has been developed. Aflatoxins were extracted with a mixture of methanol and water and then it was purified by solid-phase clean-up using a polymeric sorbent, not described previously, for the determination of these toxins. The eluted extract was injected into the chromatographic system using a reversed-phase C18 short column with an isocratic mobile phase composed of methanol-water (30:70). A single-quadruple mass spectrometry using an electrospray ionization source operating in the positive ion mode was used to detect aflatoxins due to derivatization presenting several disadvantages. Recoveries of the full analytical procedure were 110% for aflatoxin B1, 89% for aflatoxin B2, 81% for aflatoxin G1 and 77% for aflatoxin G2. Detection limit (S/N = 3) was 10 ng and quantification limit (S/N = 10) was 25 ng, calculated as amount in medicinal herb.     

进出口食品中常见霉菌毒素的污染及其检测技术探讨

阐述了进出口食品中常见霉菌毒素的种类,包括黄曲霉毒素、赭曲霉毒素、脱氧雪腐镰刀菌烯醇、玉米赤霉烯酮、伏马毒素、T-2毒素以及蛇形毒素等;分析了它们在进出口食品中污染的产生、来源和危害性;同时对霉菌毒素的检测技术及其研究进展进行了简要介绍。     

花生及其制品中多种霉菌毒素残留同时测定方法

利用液相色谱-质谱联用（LC-MS／MS）建立了花生及其制品中多种霉菌毒素包括黄曲霉毒素（B1,B2,G1,G2）、赭曲霉毒素A、伏马毒素B1、脱氧雪腐镰刀菌烯醇、T-2毒素、HT-2毒素及玉米赤霉烯酮的同时测定方法。样品经PBS溶液和甲醇-水溶液提取,提取液经稀释、过滤后,用免疫亲和柱净化,通过淋洗去除免疫亲和柱上的杂质,随后用洗脱液过柱,将目标物分离下来,氮吹干后定容。以液相色谱-质谱／质谱测定,外标法定量。方法的检出限黄曲霉毒素B1为0．0005mg／kg,黄曲霉毒素B2,G1,G2为0．001mg／kg,赭曲霉毒素A为0．002mg／kg,伏马毒素B1为0．020mg／kg,脱氧雪腐镰刀菌烯醇为0．050mg／kg,T-2毒素为0．010mg／kg,HT-2毒素为0．010mg／kg,玉米赤霉烯酮为0．002mg／kg。在样品中添加检出限水平的毒素混标溶液,加标回收率为72．35％-97．82％,测定结果的相对标准偏差为8．95％～18．41％（n=10）.     

免疫亲和柱净化-柱后光化学衍生-高效液相色谱法同时检测粮谷中的黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

建立了同时检测粮谷中黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法。样品经过甲醇-水(体积比为80∶20)提取,通过免疫亲和柱富集和净化,采用Waters Nova-Pak色谱柱(3.9 mm i.d.×150 mm,4 μm),以甲醇、乙腈和1%的磷酸溶液为流动相,梯度洗脱,柱后光化学衍生、改变波长荧光检测。黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A检出限分别为0.24,4.0和0.5 μg/kg,标准曲线的线性范围分别为0.24~6.0,4.0~100.0和0.5~40.0 μg/L;在小麦、玉米、黑麦样品中,平均加标回收率为70.8% ~94.0%,相对标准偏差为2.79% ~9.38%。     

Determination of pantothenic acid in foods by optical biosensor immunoassay

An optical biosensor inhibition immunoassay was developed using a specific pantothenic acid-binding protein for the quantitation of free pantothenic acid (vitamin B5) in foodstuffs. Samples were prepared by a simple extraction procedure in buffer, and vitamin content was estimated against authentic calibrants in the same buffer. Performance parameters included a working range of 10-5000 ng/mL, a limit of detection of 4.4 ng/mL, precision relative standard deviation of 5.4-7.1% over a range of concentrations, and recoveries > 95% in the matrixes tested. A wide range of foodstuffs, including National Institute of Standards and Technology reference samples, were tested in 3 independent laboratories and the results were compared with microbiological assay and liquid chromatography/mass spectrometry (LC/MS) methods. The results indicate that the biosensor technique is appropriate for the estimation of pantothenic acid in a wide range of foodstuffs.     

液相色谱-串联质谱法同时测定动物血浆中21种霉菌毒素或其代谢物残留

建立了同时检测动物血浆中黄曲霉毒素B1等21种霉菌毒素或其代谢物残留的液相色谱-串联质谱方法.动物血浆样品中加入0.1％甲酸-乙腈溶液、NaCl和无水MgSO4进行萃取,无水MgSO4和C18,PSA,A-AL对提取液进行脱水净化,经浓缩、复溶和离心后,再进行测定.采用反相C18色谱柱分离,以0.1％甲酸-0.5 mmol/L乙酸铵溶液和0.1％甲酸-甲醇溶液作为流动相进行梯度洗脱,采用电喷雾离子源(ESI)多反应监测离子模式(MRM)进行检测,基质标准曲线外标法进行定量分析,线性范围在0.05 ～ 100 ng/mL之间,方法的定量限为0.05 ～0.5 ng/mL.在高、中、低3个添加浓度水平下,21种霉菌毒素的平均回收率为62.0％ ～ 116.4％,相对标准偏差小于19％.     

Analytical method for the determination of mycotoxins in indoor/outdoor airborne particulate matter by HPLC-MS-MS

An effective analytical method for the screening of mycotoxins, in indoor/outdoor airborne particulate matter, was developed and method performance data are presented. Mycotoxins are natural compounds produced, in particular conditions, as secondary metabolites by filamentous fungi and moulds, and, after their production, they can be transported far from their source. To simulate real samples, an urban dust (reference material 1649a) free from mycotoxins was used as matrix and spiked by the most common mycotoxins, chosen on the basis of studies carried out previously in other real matrices: deoxynivalenol, aflatoxin B1, ochratoxin A, T-2 toxin, zearalenone and sterigmatocystin. The analytical method was optimised and structured in four successive steps: (1) accelerated solvent extraction of the (spiked) analytes from matrix, (2) solid-phase purification (SPE) of the previous extract, (3) pre-concentration of the eluates from SPE and (4) analysis of the concentrated eluates by high performance liquid chromatography tandem mass spectrometry in multiple reaction monitoring mode. After a proper sampling campaign, the method was applied to real indoor and outdoor particulate matter samples, where the clean-up step showed to be very effective and fundamental to avoid misleading analytical results.     

Determination of Fusarium mycotoxins by liquid chromatography/tandem mass spectrometry coupled with immunoaffinity extraction

A method for the simultaneous quantitative determination of deoxynivalenol (DON), T‐2 toxin (T‐2), HT‐2 toxin (HT‐2) and zearalenone (ZEN) in wheat and biscuit by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS) coupled with immunoaffinity extraction is described. A clean‐up was carried out using a DZT MS‐PREP® immunoaffinity column (IAC), and the effect of the sample dilution rate and sample loading was investigated. Furthermore, the effects of ion suppression of a multifunctional column (MFC) and the IAC in the clean‐up were compared. The results with the DZT MS‐PREP® IAC showed that it is possible to make the sample dilution rate low, and indicated a higher solvent‐tolerance than usual with an IAC. Sample loading was optimized at 0.25 g. Ion suppression was lowered by purification of the toxins using the DZT MS‐PREP® IAC. Recoveries of each mycotoxin from wheat and biscuit samples spiked at two levels ranged from 78 to 109%. The limits of detection in wheat and biscuit was in the range of 0.03–0.33 ng·g^?1. From these studies, it is suggested that use of an IAC is effective in the clean‐up of each mycotoxin, and, when combined with LC/ESI‐MS/MS, it is good for the determination of mycotoxins in foodstuffs due to its rapidity and high sensitivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Determination of aflatoxins in food by overpressured-layer chromatography

Legislative measures for monitoring and control of aflatoxin levels in foods and foodstuffs have been introduced in many countries. The aim of the present work was to make developments in the field of aflatoxin analysis, focusing upon the use of overpressured-layer chromatography (OPLC) for quantitative determination. OPLC methods have been developed for the determination of aflatoxins B1, B2, G1, G2 in different foods. These methods are suitable for sample clean-up and separation as well. Using OPLC we could analyze 10 samples simultaneously. The methods were investigated with fish, corn and wheat samples spiked with 2-10 ng/g aflatoxins. Quantitative evaluation of aflatoxins was accomplished by densitometry. Average recoveries from each food were greater than 73%. The OPLC technique seems to be a rapid, reproducible and cost-effective analysis for quantitative determination of aflatoxins in foods.     

Multi-mycotoxin analysis in eggs using a QuEChERS-based extraction procedure and ultra-high-pressure liquid chromatography coupled to triple quadrupole mass spectrometry

A reliable and rapid method has been developed for the determination of 10 mycotoxins (beauvericin, enniatin A, A1, B1, citrinin, aflatoxin B1, B2, G1, G2 and ochratoxin A) in eggs at trace levels. Ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) has been used for the analysis of these compounds in less than 7 min. Mycotoxins have been extracted from egg samples using a QuEChERS-based extraction procedure (Quick, Easy, Cheap, Effective, Rugged and Safe) without applying any further clean-up step. Extraction, chromatographic and detection conditions were optimised in order to increase sample throughput and sensitivity. Matrix-matched calibration was used for quantification. Blank samples were fortified at 10, 25, 50 and 100 μg kg(-1), and recoveries ranged from 70% to 110%, except for ochratoxin A and aflatoxin G1 at 10 μg kg(-1), and aflatoxin G2 at 50 μg kg(-1). Relative standard deviations were lower than 25% in all the cases. Limits of detection ranged from 0.5 μg kg(-1) (for aflatoxins B1, B2 and G1) to 5 μg kg(-1) (for enniatin A, citrinin and ochratoxin A) and limits of quantification ranged from 1 μg kg(-1) (for aflatoxins B1, B2 and G1) to 10 μg kg(-1) (for enniatin A, citrinin and ochratoxin A). Seven samples were analyzed and aflatoxins B1, B2, G1, G2, and beauvericin were detected at trace levels.     

Standardized high-performance liquid chromatography of 182 mycotoxins and other fungal metabolites based on alkylphenone retention indices and UV-VIS spectra (diode array detection)

A general standardized method for the analysis of mycotoxins and other fungal secondary metabolites has been developed, based on high-performance liquid chromatography (HPLC) with an alkylphenone retention index and photodiode-array detection combined with thin-layer chromatography (TLC) in two different eluents. Each fungal secondary metabolite is characterized by its bracketed alkylphenone retention time index, its UV-VIS absorption maxima and its retardation factors relative to griseofulvin in two TLC eluents. This system is effective for the comparison of chemotaxonomic data in different laboratories and for a precise identification of fungi based on organic solvent extracts of fungal cultures. All important groups of mycotoxins and other fungal secondary metabolites could be detected in the HPLC system described and data are listed for 182 metabolites. The fungal secondary metabolites separated and characterized include aflatoxin B1, B2, G1 and G2, ochratoxin A, citrinin, penicillin acid, viomellein, penitrem A, patulin, sterigmatocystin, alternariol, tenuazonic acid, trichothecenes, roquefortines, fusarin C, zearalenone, PR-toxin, citreoviridin, viridicatumtoxin, verruculogen, rugulosin, cyclopiazonic acid, penicillin G and many other alkaloids, polyketides and terpenes.     

Liquid Chromatography with mass spectrometry analysis of mycotoxins in food samples using silica hydride based stationary phases

Liquid chromatography with mass spectrometry analysis of selected food samples using silica hydride stationary phases allowed for the identification and quantification of common mycotoxins including aflatoxin B1, B2, G1, G2, ochratoxin A, and fumosinin B1. Phenyl and C₁₈ columns showed relatively similar selectivity based on hydrophobicity but the phenyl phase provides an additional mechanism, π–π interaction. The most hydrophobic of the analyzed compounds was more strongly retained on the C₁₈ column and also has fewer unsaturated sites, which limited the interaction with the phenyl phase. Bean, maize, rice, and wheat samples were harvested and stored under conditions conducive to fungal development, and all samples presented toxin contamination exceeding the maximum tolerable limits.