To ensure the health of humans and animals, the production of safe food and feed is indispensable. All over the world, food-borne diseases are among the most widespread health problems. These health problems are either of infectious origin, e.g. Salmonella, or they are associated with the consumption of toxic products, e.g. natural toxins such as mycotoxins or industrial chemical residues as for example polychlorinated biphenols (PCBs).
Especially for natural toxins the monitoring of possible contamination in food- and feedstuffs is an important and complex issue, causing a huge investment in time and effort by many regulatory and industrial laboratories. For more than 30 years, considerable research has also been devoted to develop methods for detecting and determining mycotoxins in foods, feeds and biological fluids.
But still, demands from consumers and regulators constantly grow to improve the quality and moreover the safety of food. To supply regulators, consumers and industry with long-term solutions to the complex problems associated with chemical contaminant monitoring, the European Commission has made several calls in its 4th, 5th, 6th and the current 7th Research Framework Program to improve methodologies for mycotoxin determinations. As a result of a recent call in the 6th framework program, the so called BioCop – Project was launched. The aim of BioCop is to develop fast and cost-effective technologies for the screening of food contaminants. One approach within the project is to identify specific transcriptional "alarm" responses to phytoestrogens, organochlorine pesticides and also mycotoxins.1 The Community Reference Laboratory (CRL) for Mycotoxins as a partner in BioCop focuses on novel determination techniques for mycotoxins
Table of Contents
1 Introduction and Scope of the work
2 Theoretical Part
2.1 Fungi, moulds and mycotoxins
2.2 Major Groups of mycotoxins occuring in food and feed
2.3 Food Safety Aspects and Regulations
2.4 Mycotoxin-Analysis
2.5 Immunoaffinity Clean-Up
2.5.1 Immunosorbents and Antibodies
2.5.2 Supports for Immunosorbents
2.5.3 Bonding density
2.5.4 Selective Extractions and Cross-Reactivity
2.5.5 Capacity
2.5.6 Immuno-based applications for Mycotoxins
2.6 Determination of Mycotoxins
2.6.1 Principles of reversed-phase high performance liquid chromatography (RP–HPLC)
2.6.2 Individual Mycotoxins
2.6.3 Liquid chromatography / mass spectrometry (LC-MS) as universal detector for multi-toxin extracts
3 Results and Discussion
3.1 Exploratory Experiments with Deoxynivalenol
3.1.1 Background
3.1.2 The Surveillance procedure
3.1.3 pH solutions
3.1.4 Heated elution procedures
3.1.5 Statistical analysis
3.2 Zearalenone
3.2.1 Mixtures of water with organic solvents
3.2.2 Discussions
3.2.2.1Unspecific bindings
3.2.2.2Recombination of antibodies
3.2.2.3Interfering Peaks
3.2.3 Statistical analysis
3.3 Aflatoxins
3.3.1 Statistical Analysis
3.3.1.1Aflatoxin B1
3.3.1.2Aflatoxin B2
3.3.1.3Aflatoxin G1
3.3.1.4Aflatoxin G2
3.4 Ochratoxin A
3.4.1 Statistical analysis
3.5 Fumonisins
3.5.1 Statistical analysis
3.6 T-2 and HT-2 toxin
4 Technical
4.1 Alternative heating procedures
4.1.1 Self-built heating Block
4.1.2 Microwave
4.1.3 Soldering Rod
4.1.4 Oscillating circuit
4.1.5 Electrical operated heating
4.2 (Semi-) Automation
5 Summary
Objectives and Topics
This thesis investigates the development and validation of alternative, solvent-free elution procedures for isolating mycotoxins from immunoaffinity columns (IAC). The primary research question addresses whether conditions like heat incubation can effectively release mycotoxins to replace current organic solvent-based methods, thereby facilitating automation and reducing costs while maintaining accurate detection limits.
- Investigation of water-based elution procedures using heat incubation.
- Evaluation of different heating techniques and their impact on antibody integrity and recovery.
- Statistical validation of new elution methods against established industrial standards for various mycotoxins (e.g., DON, Zearalenone, Aflatoxins).
- Assessment of the potential for automated sample preparation and miniaturization.
Excerpt from the Book
3.1.4 Heated Elution Procedures
It is widely known that temperatures above 45°C lead to a denaturation of most proteins like antibodies. As a result the specific affinity of antibodies to antigens can disappear due to heat incubation. Hence, a water elution of the mycotoxins following heat incubation of the IAC promised to be useful and lead to acceptable recoveries. Therefore aqueous standards of DON were percolated through the IAC. The mycotoxins link to the antibodies resulting in "DON-containing IACs". These were placed into a water bath as respective heating device. The IACs including the mycotoxins bonded to the antibodies are filled with the eluate before heating, while the bottom outlet of the IAC is closed with the cap during the heating procedure. After heating up the IAC to the desired temperature and incubation for a certain time, the cap is taken off and the mycotoxins are eluted hot, immediately after removing the columns from the heating system.
Alternatively the bottom closed columns including the mycotoxins were heated up without eluate, removed from the heating device and allowed to cool down to room temperature. The IAC reverted down to room temperature, the eluate (also at room temperature) is applied to the column and the mycotoxins are eluted.
Figure 3-3 shows the scheme and the experimental setup of the water bath used in this investigation to treat IACs with defined temperatures. Once the water bath has reached the designated temperature, the mycotoxin containing IACs were placed into the water bath, which is continuously stirred while incubation to ensure a constant temperature.
Summary of Chapters
1 Introduction and Scope of the work: Describes the global necessity for food safety and the role of the European Commission in funding research for rapid mycotoxin detection technologies.
2 Theoretical Part: Provides a scientific background on mycotoxins, their toxicological impact, current regulatory limits, and the fundamental principles of immunoaffinity chromatography.
3 Results and Discussion: Details the experimental results of water-based, heat-induced elution procedures for specific mycotoxins like DON, Zearalenone, and Aflatoxins, including comprehensive statistical analysis.
4 Technical: Evaluates various heating hardware configurations, ranging from self-built heating blocks to microwave and electrical heating, to assess their viability for laboratory automation.
5 Summary: Concludes that heat-assisted water elution is a viable, eco-friendly alternative to solvent-based elution, offering competitive recovery rates and improved potential for automated analysis.
Keywords
Mycotoxins, Immunoaffinity columns, Solvent-free elution, Deoxynivalenol, Zearalenone, Aflatoxins, Food safety, Thermal elution, Antibodies, Chromatography, Sample preparation, Automation, Immunosorbents, Ochratoxin A, Fumonisins
Frequently Asked Questions
What is the primary objective of this research?
The study aims to develop and validate solvent-free elution procedures for mycotoxins from immunoaffinity columns, using heat incubation to overcome the need for organic solvents, which currently limit automation.
Which mycotoxins are the focus of this analysis?
The study covers a range of mycotoxins including deoxynivalenol (DON), zearalenone (ZON), aflatoxins (B1, B2, G1, G2), ochratoxin A (OTA), fumonisins, and T-2/HT-2 toxins.
What analytical methods are utilized for detection?
The research relies on high-performance liquid chromatography (HPLC) with fluorescence detection and gas chromatography-mass spectrometry (GC-MS) for quantification.
What is the central scientific challenge addressed?
The main challenge is that current elution methods require organic solvents, which cannot be directly injected into reversed-phase systems without an evaporation step, which in turn hinders automation.
What heating methods were investigated for the new procedure?
The author tested several systems, including water baths, a self-built heating block, microwave-based heating, soldering rods, and electrical conductive heating.
What are the key keywords characterizing this work?
The primary keywords include mycotoxin analysis, immunoaffinity chromatography, thermal elution, food safety, and solvent-free extraction techniques.
Why is 85°C identified as a critical threshold for some applications?
Incubation at 85°C was found to be sufficient for the full release of certain mycotoxins, though the study notes that for sensitive mycotoxins like Aflatoxin G2, temperature control must be extremely precise to avoid degradation.
Does the water-based elution method affect the precision of the results?
Statistical analyses across multiple samples demonstrate that the proposed water-based methods provide precision (RSD values) and recovery rates comparable to the traditional organic solvent methods.
- Quote paper
- Dipl.-Ing. (FH) Joerg Seidler (Author), 2007, Development and validation of solvent free elution procedures for the isolation of mycotoxins by immunoaffinity, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/88603
