Lateral-flow immunoassays for mycotoxins and phycotoxins: a review

Natural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters.

食品中主要霉菌毒素分析方法的研究进展

食品中霉菌毒素的检测面临基质复杂、污染浓度水平低的困扰,研发选择性高、富集能力强的样品前处理方法和高灵敏的分析方法对于提升食品中霉菌毒素的检测能力、保障食品安全具有重要意义。该文综述了近年来食品中霉菌毒素分析方法的研究进展,并对其发展方向作总结和展望。     

Current methods of analysis for the determination of trichothecene mycotoxins in food

This article describes the trends in analytical techniques for the determination of trichothecene mycotoxins, namely deoxynivalenol, and T-2 and HT-2 toxins in cereals and cereal products with particular emphasis on screening and rapid approaches. The driving force behind the changing methodologies is mainly attributed to legislative demands. However, for commercial and governmental testing laboratories, the need to use validated official methods is ever increasing to ensure quality assurance of results.Much research has been undertaken to improve screening assays, highlighted by the number of new methods using a variety of formats and platforms, including optical and electrochemical biosensors. Significant advances in the traditional reference methods have also been demonstrated in addition to the emergence of a variety of commercial immunoaffinity and solid-phase extraction columns for clean up. The use of liquid chromatography coupled to tandem mass spectrometry for mycotoxin detection is ever increasing, allowing simultaneous determination of many toxins in various sample matrices.     

食品中真菌毒素样品前处理方法的研究进展

真菌毒素是由真菌在一定环境条件下产生的一类具有毒性的小分子次级代谢产物。真菌毒素种类多,毒性强,污染范围广,可经食物链直接或间接进入人体,危及人体健康。食品基质形态多样,成分复杂,而实际样品中真菌毒素含量低,难以直接对目标物进行分析,故高效的样品前处理技术能实现待测物的分离和富集,在实际样品的分析中尤为重要。该文主要综述了基于磁性纳米材料、石墨烯类材料、分子印迹材料、免疫亲和材料、适配体功能材料等新型分离介质的液相萃取技术、固相萃取技术、场辅助提取技术(磁性固相萃取、超声辅助提取、微波辅助提取)、免疫亲和柱法、QuEChERS法等前处理技术在食品中真菌毒素分析中的应用,并对其分析的发展趋势进行了展望。     

Immunochemical methods for the determination of mycotoxins

The state-of-the-art immunochemical methods for the determination of mycotoxins are considered. Both instrumental (enzyme-linked immunosorbent assay, polarization fluoroimmunoassay, and sensor devices) and noninstrumental methods are presented. The principles of particular methods are considered, and the examples of the use of these methods for the determination of mycotoxins from various groups in food products and animal feeds are given; the main lines of development are discussed.     

Validation of analytical methods for determining mycotoxins in foodstuffs

The European Union (EU) has established demanding regulatory limits for controlling aflatoxins B₁, B₂, G₁ and G₂, in cereals, nuts, nut products and dried fruit, aflatoxin M₁ in milk, and ochratoxin A in cereals. These limits are likely to be extended in the future to additional commodities and other mycotoxins. For enforcement purposes and in particular for resolving any disputes between parties, it is essential that validated methods are available, with performance characteristics that meet certain minimum criteria. As such methods were not available and had not previously been validated either for matrices of interest in Europe or at the low European limits compared to the USA, the EU funded a method-validation project to fulfil this requirement. Immunoaffinity column clean-up methods with HPLC determination were established for aflatoxins B₁, B₂, G₁ and G₂ in peanut butter, pistachios, fig paste and paprika, aflatoxin B₁ in baby food, aflatoxin M₁ in liquid milk, and ochratoxin A in roasted coffee and baby food. For patulin in apple juice and apple puree, solvent extraction and solid-phase clean-up HPLC methods were developed. To undertake collaborative studies, particular care was taken in preparation of naturally-contaminated test materials containing the toxins at levels close to regulatory limits and in demonstrating the homogeneity of batches of material. To ensure that participants in the validation exercise could follow the procedures to be tested, videos of the methods were prepared showing, in particular, any critical steps. Prior to undertaking the method validation, participants were invited to collaborative study workshops to ensure that they fully understood the methods and their role in the study. This care in planning and executing the collaborative studies led to impressive performance characteristics and adoption of six procedures by AOAC International as First Action Methods and seven methods by CEN as European standards. The valuable lessons learned in undertaking these validation exercises are now being put to further use in studies aimed at validating methods for mycotoxins in foodstuffs, which are appropriate for developing countries based on TLC as the end determination but use more modern sample clean-up techniques.     

Chromatographic methods for the determination of mycotoxins in food products

Chromatographic methods for the determination of mycotoxins from different classes in food products of plant and animal origins are surveyed. The procedures of sample preparation and extract purification and the use of various chromatographic analysis techniques are considered.     

Volatilization of trichothecene mycotoxins for analysis

T-2 toxin and diacetoxyscirpenol (DAS), two trichothecene mycotoxins containing one hydroxy group, have been volatilized by induction heating, revolatilized, and analyzed by gas chromatography (GC) and/or GC mass spectroscopy. Seventy to eighty percent of DAS was recovered by this system; 60–70% T-2 toxin was recovered. When the hydroxy group is derivatized by acetate, 90–100% recovery is obtained. Other trichothecenes of the macrocyclic ester type (e.g., Roridan A) were also tried. Ten to twenty percent of the macrocyclic ester was obtained without derivatization.     

Chromatographic and electrophoretic methods used for analysis of milk proteins.

Current knowledge of milk proteins and their behavior in dairy foods is based on early applications of chromatography and electrophoresis. Electrophoretic identification of the number and genetic variety of milk proteins inaugurated a research effort in which chromatographic techniques were successfully applied to the isolation of each milk protein, thus facilitating the characterization and further study of milk and dairy products. This review focuses on recent applications of chromatography for separations and analysis and on analytical applications of electrophoresis.     

Screening methods for the detection of thirteen common mycotoxins

A study of screening methods for thirteen mycotoxins showed that they can be separated as neutral and acidic metabolites. RF values were determined in several solvent systems. The reactions of the mycotoxins with well known spray reagents were investigated, and their detection limits were established. A general procedure for the extraction of mycotoxins from contaminated samples is described.     

Fit-for-purpose shellfish reference materials for internal and external quality control in the analysis of phycotoxins

The need for reference materials for quality control of analysis of foodstuffs has been stressed frequently. This has been particularly true in the phycotoxins field, where there is a great shortage of both pure calibration standards and reference materials. Worldwide there are very few independent bodies that produce certified reference materials for phycotoxins, the main producers currently being the National Research Council Canada and the Japanese Food Research Laboratory. Limited availability of contaminated shellfish and algae, as well as the time and knowledge necessary for the production of adequate reference materials, continuously lead to limited editions of certified reference materials and even more limited production of in-house reference materials. The restricted availability of in-house quality control materials promotes the rapid use of the limited certified reference materials, which in turn hampers the production of the suite of materials required globally for complete protection of public health. This paper outlines the various options that analysts can pursue in the use of reference materials for internal and external quality control, with a view to optimising the efforts of both reference materials users and reference materials producers. For this purpose, the logical sequence is reviewed from the discovery of a new bioactive compound in shellfish, through initial method development up to regulation for food safety purposes including accepted reference methods. Subsequently, the requirements for and efforts typically spent in the production and characterisation of laboratory reference materials, certified reference materials and other test materials used in inter-laboratory studies or proficiency testing, in the area of marine biotoxins are evaluated. Particular emphasis is put on practical advice for the preparation of in-house reference materials. The intricate link between reference material characterisation and method performance is outlined to give guidance on the appropriate in-house method validation in the rapidly developing field of phycotoxins.      

Synthesis and characterization of colloidal gold particles as labels for antibodies as used in lateral flow devices

Based on well established citrate reduction protocols for the synthesis of colloidal gold particles, this work focuses on the characterization of these colloids for further use as color labels in lateral flow devices. A reproducible production method has been developed for the synthesis of well characterized colloidal gold particles to be employed in Lateral Flow Devices (LFDs). It has been demonstrated that when undertaking chemical reduction of gold salts with sodium citrate, the amount of reducing agent employed could be used to directly control the size of the resultant particles. A protocol was thereby developed for the synthesis of colloidal gold particles of pre-defined diameters in the range of 15 to 60 nm and of consistent size distribution. The absorption maxima (λ(max)) of the reaction solutions were analyzed by UV/VIS measurements to determine approximate particle sizes, which were confirmed with transmission electron microscopy (TEM) measurements. Colloidal gold particles of about 40 nm in diameter were synthesized and used for labeling monoclonal anti-mycotoxin antibodies (e.g. zearalenone). To deduce the extent of antibody coupling to these particles, smaller colloids with 15 nm diameter were labeled with anti-species specific antibodies. Both solutions were mixed and then scanned by TEM to obtain information about the success of coupling.     

Chromatographic methods as tools in the field of mycotoxins

Achievements in the applications of chromatographic techniques in mycotoxicology are reviewed. Historically, column chromatography (CC) and paper chromatography (PC) were applied first, followed by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) and gas chromatography (GC). Although PC techniques are no longer used in the analysis of mycotoxins, selected applications of PC are included to underline historical continuity. The most important achievements published from 1980 onwards are described. They include clean-up methods, TLC, CC, HPLC and GC of mycotoxins in environmental samples, foods, feeds, body fluids and in studies on biosynthesis and biotransformations of mycotoxins. Advantages and disadvantages of chromatographic techniques used in mycotoxicology are also evaluated.     

Rapid test strips for analysis of mycotoxins in food and feed

An overview is given on recent trends and applications of rapid immunodiagnostic tests for screening of food and feed for mycotoxins. Different test formats are discussed, and challenges in the development of lateral-flow devices for on-site determination of mycotoxins, with requirements such as being robust, fast, and cost-effective, are briefly elucidated.     

Development and evaluation of C18 and immunosorbent solid-phase extraction methods prior immunochemical analysis of chlorophenols in human urine

Two solid-phase extraction (SPE) methods, based on hydrophobic and selective (antibody-antigen) interactions, have been established and evaluated as clean-up methods prior the immunochemical analysis of 2,4,6-trichlorophenol (2,4,6-TCP) in urine samples. Without a clean-up method the extent of interferences caused by the urine matrix in the ELISA [R. Galve, M. Nichkova, F. Camps, F. Sanchez-Baeza, M.-P. Marco, Anal. Chem. 74 (2002) 468] varies depending on individual urine samples and accurate measurements are only possible when 2,4,6-TCP concentration levels are higher than 40 μg L⁻¹. Both sample preparation methods improve detectability of the immunochemical method getting rid of the variability due to the intrinsic individual differences within the urine samples. Even though, the immunosorbent (IS)-SPE method developed has proven to be a superior sample preparation method eliminating completely matrix effects caused by both, non-hydrolyzed (NH) and hydrolyzed urine samples. The LOD reached by the C18-SPE-ELISA method (∼4 μg L⁻¹ for free and total chlorophenols) is sufficient for exposure assessment of the occupationally exposed population. However, the detectability (0.66 and 0.83 μg L⁻¹ in NH and hydrolyzed urine samples, respectively) accomplished by the IS-SPE-ELISA allows also biomonitoring potential exposure of non-occupationally exposed groups. Moreover, the specificity of the IS-SPE procedure can be modulated to provide a group-specific (9 chlorophenols and 2 bromophenols are extracted with an efficacy superior to 85%) or a more selective protocol (only 2,3,4,6-TtCP, 2,4,6-TCP are extracted with a recovery superior to 80% and 2,4,6-tribromophenol with a 70% recovery). On the other hand, the IS-SPE extracts produce cleaner chromatograms allowing quantitation by GC-ECD (or GC-MS) after toluene extraction and derivatization with a LOD near 0.1 μg L⁻¹ in NH and hydrolyzed urine samples. The IS-SPE-ELISA method has been validated with GC-ECD using spiked and real urine samples. This study also provides evidences of the general exposure of the population to organochlorinated and organobrominated substances. Measurable levels of 2,4,6-TCP, 2,4,5-TCP, 2,3,4,6-TtCP, 2,4,6-TBP and 2,4-DBP have been detected in some of the samples used in this study.     

Statistical validation of sulfate quantification methods used for analysis of acid mine drainage

Turbidimetric method (TM), ion chromatography (IC) and inductively coupled plasma atomic emission spectrometry (ICP-AES) with and without acid digestion have been compared and validated for the determination of sulfate in mining wastewater. Analytical methods were chosen to compare the performance of a portable field turbidimetric instrument and to validate the underlying assumption utilized in conversion of total sulfur to sulfate during ICP-AES analysis. Accuracy and precision of analytical techniques were compared to one another using control and field samples collected from a mine site using the Bonferroni multiple comparison test. Effects of sample dilution, filter pore size and acidification on sulfate quantification were also studied. The results showed that IC and ICP-AES with and without acid digestion provided excellent recoveries in the case of control samples (within 90-110%). These analytical methods also showed lower relative standard deviation for both control and field samples. On the other hand, performance of the turbidimetric method was severely affected by sample dilution and acidification, and also revealed poor sulfate recoveries for control samples ranging from 0 to 83.5%. Analysis of variance (ANOVA) was used to evaluate the response (sulfate concentration) obtained from factorial design. Analytical method had significant effect (P < 0.0001) on the sulfate quantification. The interaction between determination method and sample dilution was more significant than other two-way interactions.     

Chromatographic analysis of mycotoxins on thin-layers of rice starch

Summary Chromatographic separation and UV detection of twelve mycotoxins on thin-layers of rice starch are presented. The effect of solvent systems on the fluorescence of mycotoxins and the mechanism of the chromatographic separation are described.