Identification and characterization of new Fusarium masked mycotoxins,T2 and HT2 glycosyl derivatives,in naturally contaminated wheat and oats by liquid chromatography–high‐resolution mass spectrometry

The presence of glucoside derivatives of T‐2 and HT‐2 toxins (type A trichothecene mycotoxins) in naturally contaminated wheat and oats is reported for the first time. The use of advanced high‐resolution mass spectrometry based on Orbitrap technology allowed to obtain molecular structure details by measuring exact masses of main characteristic fragments, with mass accuracy lower than 2.8 ppm (absolute value). A monoglucoside derivative of T‐2 toxin and two monoglucoside derivatives of HT‐2 toxin were identified and characterized. The analysis of their fragmentation patterns provided evidence for glucosylation at C‐3 position for T‐2 toxin and at C‐3 or C‐4 position for HT‐2 toxin. A screening for the presence of these new masked forms of mycotoxins was carried out on a set of naturally contaminated wheat and oats samples. On the basis of peak area ratio between glucoside derivatives and free T‐2 and HT‐2 toxins, the presence of glucoside derivatives was more likely in wheat than in oats samples. The present work confirms the widespread occurrence of trichothecene glucosides in cereal grains naturally contaminated with the relevant unconjugated toxins, thus suggesting the importance of developing suitable analytical methods for their detection. Besides toxicity studies, tracking down these new masked forms of trichothecenes along the food/feed chain would enable to collect information on their relevance in human/animal exposure to mycotoxin risk. Copyright © 2012 John Wiley & Sons, Ltd.     

Natural toxins: risks,regulations and the analytical situation in Europe

Natural toxins in food and feed are considered important food safety issues of growing concern, in particular mycotoxins, phycotoxins and plant toxins. Most scientific developments have occurred in the past few decades in the area of mycotoxins. Formal health risk assessments have been carried out by the Joint Expert Committee on Food Additives of the World Health Organization and the Food and Agriculture Organization. Limits and regulations for mycotoxins in food and feed have been established in many countries, including practically all European countries. An array of (formally validated) analytical methods and (certified) reference materials have become available. Several European research projects, funded by the European Commission and supported by the European Standardization Committee, have significantly contributed to this development. Quantitative methods of analysis for mycotoxins often make use of immunoaffinity cleanup with liquid chromatographic or gas chromatographic separation techniques in combination with various types of detectors, including mass spectroscopy. For screening purposes (bio)sensor-based techniques are among the promising newcomers. For the phycotoxins the situation is less advanced. Formal risk assessments by authoritative international bodies have not been carried out. Methods of analysis, formally validated according to internationally harmonized protocols, are scarce and animal testing still plays a key role in official methodology. The development of the analytical methodology is partly hampered by the limited availability of certain reliable calibrants and reference materials, although this situation is gradually improving. New regulations in the European Union have increased the pressure to develop and validate chemical methods of analysis. Joint efforts in the European context are now directed towards significantly improving this situation, and techniques such as liquid chromatography–mass spectroscopy offer promise in this respect. Both the working group on biotoxins of the European Standardization Committee and the network of National Reference Laboratories for Marine Biotoxins have taken up responsibilities here. The plant toxins are a category of natural toxins, where the situation is the least developed with respect to regulations, validated methods of analysis and reference materials. Yet, their occurrence in a wide range of consumable plant species demands the attention of the analytical community.     

New advances in electrochemical biosensors for the detection of toxins: Nanomaterials,magnetic beads and microfluidics systems. A review

The use of nanotechnology in bioanalytical devices has special advantages in the detection of toxins of interest in food safety and environmental applications. The low levels to be detected and the small size of toxins justify the increasing number of publications dealing with electrochemical biosensors, due to their high sensitivity and design versatility. The incorporation of nanomaterials in their development has been exploited to further increase their sensitivity, providing simple and fast devices, with multiplexed capabilities. This paper gives an overview of the electrochemical biosensors that have incorporated carbon and metal nanomaterials in their configurations for the detection of toxins. Biosensing systems based on magnetic beads or integrated into microfluidics systems have also been considered because of their contribution to the development of compact analytical devices. The roles of these materials, the methods used for their incorporation in the biosensor configurations as well as the advantages they provide to the analyses are summarised.     

Challenges and trends in the determination of selected chemical contaminants and allergens in food

This article covers challenges and trends in the determination of some major food chemical contaminants and allergens, which-among others-are being monitored by Health Canada's Food Directorate and for which background levels in food and human exposure are being analyzed and calculated. Eleven different contaminants/contaminant groups and allergens have been selected for detailed discussion in this paper. They occur in foods as a result of: use as a food additive or ingredient; processing-induced reactions; food packaging migration; deliberate adulteration; and/or presence as a chemical contaminant or natural toxin in the environment. Examples include acrylamide as a food-processing-induced contaminant, bisphenol A as a food packaging-derived chemical, melamine and related compounds as food adulterants and persistent organic pollutants, and perchlorate as an environmental contaminant. Ochratoxin A, fumonisins, and paralytic shellfish poisoning toxins are examples of naturally occurring toxins whereas sulfites, peanuts, and milk exemplify common allergenic food additives/ingredients. To deal with the increasing number of sample matrices and analytes of interest, two analytical approaches have become increasingly prevalent. The first has been the development of rapid screening methods for a variety of analytes based on immunochemical techniques, utilizing ELISA or surface plasmon resonance technology. The second is the development of highly sophisticated multi-analyte methods based on liquid chromatography coupled with multiple-stage mass spectrometry for identification and simultaneous quantification of a wide range of contaminants, often with much less requirement for tedious cleanup procedures. Whereas rapid screening methods enable testing of large numbers of samples, the multi analyte mass spectrometric methods enable full quantification with confirmation of the analytes of interest. Both approaches are useful when gathering surveillance data to determine occurrence and background levels of both recognized and newly identified contaminants in foods in order to estimate human daily intake for health risk assessment.     

A portable automated multianalyte biosensor

The array biosensor employs an array of capture molecules on a planar optical waveguide to interrogate multiple samples simultaneously for multiple targets. In assay development and demonstration studies published previously, we have quantified this biosensor's capability for rapid identification of a wide variety of targets in complex sample media. This paper describes the miniaturization and automation of the array biosensor for portability and on-site use. The fluidics have been redesigned and constructed with reliability and commercial production of disposable components in mind. To demonstrate the automated operation, simultaneous assays were automatically conducted on samples containing both ovalbumin and staphylococcal enterotoxin B. Results demonstrate the capability of the biosensor for detection and quantification.     

Determination of marine biotoxins relevant for regulations: from the mouse bioassay to coupled LC-MS methods

The frequency of occurrence and intensity of harmful algal blooms (HABs) appear to be increasing on a global scale. Consequently, methods were established for the evaluation of possible hazards caused by the enrichment of algal toxins in the marine food chain. Different clinical types of algae-related poisoning have attracted scientific attention: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). In several countries fish specialties are consumed which may be contaminated with algal toxins typical for the respective region (e.g., ciguatera and tetrodotoxins). Bioassays are common methods for the determination of marine biotoxins. However, biological tests are not completely satisfactory, due to the low sensitivity and the absence of specialized variations. Moreover, there is growing resistance against the use of animal experiments. Therefore, many efforts have been made to determine algal toxins with chemical methods. In this context LC-MS methods replaced HPLC methods with optical detectors, allowing both effective seafood control and monitoring of phytoplankton in terms of the different groups of marine biotoxins.     

Optimization of two flow-injection spectrophotometric methods for the determination of indapamide in pharmaceutical dosage forms

Multivariate experimental design has been used to optimize 2 flow-injection spectrophotometric methods for the determination of indapamide in pharmaceutical dosage forms, both pure and commercial tablets. The methods are based on the oxidation of this drug with iron (III) in acidic medium and the subsequent formation of an intensive orange-red complex between the liberated iron (II) and 2,2'-bipyridyl or 1,10-phenanthroline reagents. Plackett-Burman designs were applied as a screening method to evaluate the most significant factors with few experiments. Central composite 2(3)+ star designs were performed to evaluate the response surfaces. The methods have been fully validated and were applied successfully to the determination of indapamide in pure and pharmaceutical forms with good accuracy and precision. Therefore, the 2 proposed procedures are simple, inexpensive, and rapid flow methods for the routine determination of indapamide in pharmaceutical preparations.     

A European perspective on progress in moving away from the mouse bioassay for marine-toxin analysis

This review considers the ethical and technical problems currently associated with employing mouse bioassays for marine-toxin analysis and the challenges and the difficulties that alternative methods must overcome before being deemed applicable for implementation into a regulatory monitoring regime. We discuss proposed alternative methods, classified as functional, immunological and analytical, for well-established European toxins as well as emerging toxins in European waters, highlighting their advantages and disadvantages. We also consider emerging tools and technologies for future toxin analysis.Even though regulatory bodies have recently recommended analytical methods for a number of toxins, there is still scope for functional and immunological methods in rapid screening and detecting emerging toxins. Future developments foreseen in the analysis of marine biotoxins are multiplex-based analysis, miniaturization and portability for on-site testing. However, the longstanding lack of reference materials and standards continues to pose a severe limitation on progress in development, validation and therefore implementation of any alternative method based on the criteria stipulated by European Union legislation.     

LC-MS analysis in the aquatic environment and in water treatment technology – a critical review

Environmental contaminants of recent concern are pharmaceuticals, estrogens and other endocrine disrupting chemicals (EDC) such as degradation products of surfactants, algal and cyanobacterial toxins, disinfection by-products (DBPs) and metalloids. In addition, pesticides (especially their transformation products), microorganisms, and humic substances (HS), in their function as vehicles for contaminants and as precursors for by-products in water treatment, traditionally play an important role. The present status of the application of LC-MS techniques for these water constituents are discussed and examples of application are given. Solid-phase extraction with various non-selective materials in combination with liquid chromatography (LC) on reversed-phase columns have been the most widely used methods for sample preconcentration and separation for different compound classes like pesticides, pharmaceuticals or estrogens. Electrospray ionization (ESI) and atmospheric pressure ionization (APCI) are the most frequently used ionization techniques for polar and ionic compounds, as well as for less polar non-ionic ones. The facilities of LC-MS have been successfully demonstrated for different compound classes. Polar compounds from pharmaceuticals used as betablockers, iodinated X-ray contrast media, or estrogens have been determined without derivatization down to ultratrace concentrations. LC-MS can be viewed as a prerequisite for the determination of algal and cyanobacterial toxins and the homologues and oligomers of alkylphenol ethoxylates and their metabolites. Tandem mass spectrometric techniques and the use of diagnostic ions reveal their usefulness for compound-class specific screening and unknown identification, and are also valid for the analysis of pesticides and especially for their transformation products. Structural information has been gained by the application of LC-MS methods to organometallic species. New insights into the structural variety of humic substances have been made possible by FT-ICR-MS due to its ultrahigh mass resolution. Finally, exciting possibilities for rapid detection and identification of microorganisms have been made possible by MALDI and LC-MS methods.     

Immunoassay methods for paralytic shellfish poisoning toxins

The current status of immunochemical techniques for analysis of paralytic shellfish poisoning (PSP) toxins is summarized. Important aspects regarding production of the biological reagents necessary for immunochemical methods, the characteristics of polyclonal and monoclonal antibodies against saxitoxin and neosaxitoxin, and the importance of test sensitivity and specificity are discussed. Applications of immunochemical techniques for PSP toxins include microtiter plate enzyme immunoasays and enzyme-linked immunofiltration assays for toxin detection, and immunoaffinity chromatography (IAC) for sample extract cleanup. A major advantage of enzyme immunoassay (EIA) is simplicity and rapidity of the test procedure, and higher sensitivity than other methods. However, quantitative agreement between EIA and mouse bioassay is dependent on antibody specificity and the toxin profile in the shellfish; thus, both over- and underestimation of total toxicity may occur. For screening purposes, however, EIAs offer major advantages over the mouse bioassay, which is criticized in Europe because of animal welfare. A major application of antibodies against PSP toxins is their use for extract cleanup by IAC, which gives highly purified extracts, thereby enhancing determination of PSP toxins by conventional physicochemical methods such as liquid chromatography. IAC can also be used to isolate PSP toxins for preparation of analytical standard solutions.     

Effect of Selected Microorganisms on Fusarium Toxins Production

The aim of this study was to examine the effect of selected microorganisms on mycotoxins production by molds of the genus Fusarium, namely HT-2 and T-2 toxins. Appropriate nutritive media were inoculated with test microorganisms (Rhodotorula spp., Leuconostoc spp., Pantoea agglomerans), subsequently inoculated with Fusarium molds, then incubated under various conditions. Content of Fusarium mycotoxins in individual samples was determined using HPLC/MS/MS. Separation of mycotoxins was performed on a C₁₈ stationary phase column using gradient elution. Total analysis time was less than 20 minutes. In examining the effect of accompanying microflora on the production of HT-2 and T-2 toxins, a decrease in production of both mycotoxins was observed under various experimental conditions. Greatest inhibitory effect was observed in the presence of Pantoea agglomerans CCM 298 bacteria. It was found that the amount of HT-2 and T-2 toxins produced by the examined mold strains also depends on cultivation conditions and the nutritive medium used.     

The array biosensor: portable, automated systems.

With recent advances in surface chemistry, microfluidics, and data analysis, there are ever increasing reports of array-based methods for detecting and quantifying multiple targets. However, only a few systems have been described that require minimal preparation of complex samples and possess a means of quantitatively assessing matrix effects. The NRL Array Biosensor has been developed with the goal of rapid and sensitive detection of multiple targets from multiple samples analyzed simultaneously. A key characteristic of this system is its two-dimensional configuration, which allows controls and standards to be analyzed in parallel with unknowns. Although the majority of our work has focused on instrument automation and immunoassay development, we have recently initiated efforts to utilize alternative recognition molecules, such as peptides and sugars, for detection of a wider variety of targets. The array biosensor has demonstrated utility for a variety of applications, including food safety, disease diagnosis, monitoring immune response, and homeland security, and is presently being transitioned to the commercial sector for manufacturing.     

Determination of phenolic acids using Trametes versicolor laccase

Two biosensors based on Trametes versicolor laccase (TvL) were developed for the determination of phenolic compounds. Commercial oxygen electrode and ferrocene-modified screen-printed graphite electrodes were used for preparation of laccase biosensors. The systems were calibrated for three phenolic acids. Linearity was obtained in the concentration range 0.1-1.0 μM caffeic acid, 0.05-0.2 μM ferulic acid, 2.0-14.0 μM syringic acid for laccase immobilised on a commercial oxygen electrode and 2.0-30.0 μM caffeic acid, 2.0-10.0 μM ferulic acid, 4.0-30.0 μM syringic acid for laccase immobilised on ferrocene-modified screen-printed electrodes. Furthermore, optimal pH, temperature and thermal stability studies were performed with the commercial oxygen electrode. Both electrodes were used for determination of a class of phenolic acids, achieving a cheap and fast tool and an easy to be used procedure for screening real samples of human plasma.     

A mussel tissue certified reference material for multiple phycotoxins. Part 1: design and preparation

The development of multi-analyte methods for lipophilic shellfish toxins based on liquid chromatography–mass spectrometry permits rapid screening and analysis of samples for a wide variety of toxins in a single run. Validated methods and appropriate certified reference materials (CRMs) are required to ensure accuracy of results. CRMs are essential for accurate instrument calibration, for assessing the complete analytical method from sample extraction to data analysis and for verifying trueness. However, CRMs have hitherto only been available for single toxin groups. Production of a CRM containing six major toxin groups was achieved through an international collaboration. Preparation of this material, CRM-FDMT1, drew on information from earlier studies as well as improved methods for isolation of toxins, handling bulk tissues and production of reference materials. Previous investigations of stabilisation techniques indicated freeze-drying to be a suitable procedure for preparation of shellfish toxin reference materials and applicable to a wide range of toxins. CRM-FDMT1 was initially prepared as a bulk wet tissue homogenate containing domoic acid, okadaic acid, dinophysistoxins, azaspiracids, pectenotoxin-2, yessotoxin and 13-desmethylspirolide C. The homogenate was then freeze-dried, milled and bottled in aliquots suitable for distribution and analysis. The moisture content and particle size distribution were measured, and determined to be appropriate. A preliminary toxin analysis of the final material showed a comprehensive toxin profile.     

Rapid analysis of food products by means of high speed gas chromatography

Since the invention of GC, there has been an ever increasing interest within the chromatographic community for faster GC methods. This is obviously related to the fact that the number of samples subjected to GC analysis has risen greatly. Nowadays, in routine analytical applications, sample throughput is often the most important aspect considered when choosing an analytical method. Gas chromatographic instrumentation, especially in the last decade, has been subjected to continuous and considerable improvement. High-speed injection systems, electronic gas pressure control, rapid oven heating/cooling and fast detection are currently available in a variety of commercial gas chromatographs. The main consequence of this favourable aspect is that high-speed GC is being increasingly employed for routine analysis in different fields. Furthermore, the employment of dedicated software makes the passage from a conventional to a fast GC method a rather simple step. The present review provides an overview of the employment of fast GC techniques for the analysis of food constituents and contaminants. A brief historical and theoretical background is also provided for the approaches described.     

剧毒性Ⅱ型核糖体失活蛋白蓖麻毒素和相思子毒素的检测鉴定方法研究进展

Ⅱ型核糖体失活蛋白(RIPs)是一类重要的蛋白毒素,该类毒素大都具有一对二硫键连接的A-B链结构特征,B链具有半乳糖结合特性,能够与真核细胞膜表面受体特异性结合,将具有N-糖苷酶活性的A链导入细胞,与核糖体特定位点发生脱嘌呤作用使核糖体失活,最终通过抑制蛋白质合成而展现出细胞毒性。Ⅱ型RIPs毒素毒性极强,来源于植物的蓖麻毒素(ricin)和相思子毒素(abrin)的毒性分别是神经性毒剂维埃克斯(Vx)的385倍和2885倍。同时,该类毒素来源广泛、易于制备、稳定性好,成为一类潜在化生恐怖战剂,受到国内外广泛关注,其中蓖麻毒素作为唯一的蛋白毒素被收录于禁止化学武器公约禁控清单。近年来发生的多次蓖麻毒素邮件恐怖事件,进一步促进了有关Ⅱ型RIPs毒素的准确、灵敏、快速的检测鉴定技术的发展。剧毒性Ⅱ型RIPs毒素的检测鉴定方法主要涉及免疫分析法为代表的特异性识别和生物质谱分析为主的定性定量检测方法,以及基于脱嘌呤反应活性和细胞毒性的毒素活性检测方法。基于抗原-抗体作用的免疫检测法及基于寡核苷酸适配体的特异性识别检测法具有速度快、灵敏度高的优势,但对于复杂样品中高度同源蛋白的检测,易产生假阳性结果。随着生物质谱技术的快速发展,电喷雾离化(ESI)或基质辅助激光解吸离化(MALDI)等技术广泛应用于蛋白质的准确鉴定,不仅能够提供蛋白毒素的准确分子量和结构序列信息,而且能够实现准确定量。酶解质谱法是应用最为广泛的检测鉴定方法,通过酶解肽指纹谱分析,实现蛋白毒素的准确鉴定;对于复杂样品中蛋白毒素的分析,通过多种蛋白酶酶解策略获得丰富的特异性肽段标志物,然后进行肽段标志物的靶向质谱分析从而获得准确的定性及定量信息,方法有效提升了Ⅱ型RIPs毒素鉴定的准确度和灵敏度。免疫分析法和生物质谱法能够准确鉴定Ⅱ型RIPs毒素,但无法识别毒素是否还保持毒性。对于Ⅱ型RIPs毒素的活性分析,主要包括基于N-糖苷酶活性的脱嘌呤反应测定法和细胞毒性测定法,两种方法均可实现毒素毒性的简便、快速、灵敏的分析检测,是Ⅱ型RIPs毒素检测方法的有效补充。由于该类毒素的高度敏感性,国际禁止化学武器组织(OPCW)对相关样品中Ⅱ型RIPs毒素的分析提出了唯一性鉴定的技术要求。该文引用了Ⅱ型RIPs毒素及其检测方法相关的70篇文献,综述了以上Ⅱ型RIPs毒素的结构性质、中毒机理及典型剧毒性Ⅱ型RIPs毒素检测方法的研究进展,对不同检测方法的特点和应用潜力进行了总结,并结合OPCW对Ⅱ型RIPs毒素唯一性鉴定的技术需求,展望了未来Ⅱ型RIPs毒素检测技术研究的发展趋势。     

Molecular approaches for monitoring potentially toxic marine and freshwater phytoplankton species

Harmful phytoplankton species are a growing problem in freshwater and marine ecosystems, because of their ability to synthesize toxins that threaten both animal and human health. The monitoring of these microorganisms has so far been based on conventional methods, mainly involving the microscopic counting and identification of cells, and using analytical and bioanalytical methods to identify and quantify the toxins. However, the increasing number of microbial sequences in the GeneBank database and the development of new tools in the last 15 years nowadays enables the use of molecular methods for detection and quantification of harmful phytoplankton species and their toxins. These methods provide species-level identification of the microorganisms of interest, and their early detection in the environment by PCR techniques. Moreover, real time PCR can be used to quantify the cells of interest, and in some cases to evaluate the proportion of toxin-producing and non-toxin-producing genotypes in a population. Recently, microarray technologies have also been used to achieve simultaneous detection and semi-quantification of harmful species in environmental samples. These methods look very promising, but so far their use remains limited to research. The need for validation for routine use and the cost of these methods still hamper their use in monitoring programs.     

Screening of lipophilic marine toxins in shellfish and algae: development of a library using liquid chromatography coupled to orbitrap mass spectrometry

Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the analysis of the 13 toxins that are stated in European Union legislation. However, to date over 200 lipophilic marine toxins have been described in the literature. To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100 000) for marine lipophilic toxins has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin (PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were obtained from Norway, Ireland, United Kingdom, Portugal and Italy. Each sample extract was analyzed with and without collision induced dissociation fragmentation. Based on their mass and specific fragmentation pattern, 85 different toxins were identified comprising 33 OA, 26 YTX, 18 AZA and 8 PTX group toxins. A major complication of full scan HRMS is the huge amount of data generated (file size), which restricts the possibility of a fast search. A software program called metAlign was used to reduce the orbitrap MS data files. The 200-fold reduced data files were screened using an additional software tool for metAlign: ‘Search_LCMS’. A search library was constructed for the 85 identified toxins. The library contains information about compound name, accurate mass, mass deviation (<5 ppm), retention time (min) and retention time deviation (<0.2 min). An important feature is that the library can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested by analyzing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. A toxin profile was determined for each of the contaminated samples. No toxins were found in the blank sample, which is in line with the results obtained for this sample in the routine monitoring program (rat bioassay and tandem LC–MS).     

发铅检测法的临床应用价值评估

从铅中毒发铅诊断标准、发铅诊断试验及发铅诊断临床应用3个方面论述了发铅检测法在铅中毒诊断、筛查和监督中的实际应用问题。根据临床经验和发铅-血铅比值确定了居民发铅正常值上限及铅中毒发铅诊断分级标准;即使以血铅测定值为"金标准",诊断试验表明,发铅测定在铅中毒诊断中仍有实际应用价值;发铅检测法自上世纪60年代以来一直沿用至今,绝大多数研究者认为,发铅测定是诊断、筛查铅中毒和监督环境铅污染最简单、有效的工具。     

Development of an Electrochemical Biosensor for the Rapid Detection of Cholera Toxin Based on Air Stable Lipid Films with Incorporated Ganglioside GM1 Using Graphene Electrodes

The present work describes a miniaturized potentiometric cholera toxin sensor on graphene nanosheets with incorporated lipid films. Ganglioside GM1, the natural cholera toxin receptor, immobilized on the stabilized lipid films, provided adequate selectivity for detection over a wide range of toxin concentrations, fast response time of ca. 5 min, and detection limit of 1 nM. The proposed sensor is easy to construct and exhibits good reproducibility, reusability, selectivity, long shelf life and high sensitivity of ca. 60 mV/decade of toxin concentration. The method was implemented and validated in lake water samples. This novel ultrathin film technology is currently adapted to the rapid detection of other toxins that could be used in bioterrorism.