Development and validation of solvent-free elution
procedures for the isolation of mycotoxins by immunoaffinity

A Thesis Presented to the Faculty of Chemistry at the
Aalen University of Applied Sciences
in Fulfilment of the Requirements for the Degree
"Diplom-Ingenieur (FH) - Chemie"

Jörg Seidler

August 2007
Aalen University of Applied Sciences
Aalen, Germany
European Commission
Joint Research Centre
Institute for Reference Materials & Measurements (IRMM)
Geel, Belgium

Table of Contents

1 Introduction and Scope of the work ...  4

2 Theoretical Part ...  7
2.1 Fungi, moulds and mycotoxins  ...  7
2.2 Major Groups of mycotoxins occuring in food and feed ...  8
2.3 Food Safety Aspects and Regulations ...  12
2.4 Mycotoxin-Analysis ...  13
2.5 Immunoaffinity Clean-Up  ...  14
2.5.1 Immunosorbents and Antibodies ...  14
2.5.2 Supports for Immunosorbents  ...  17
2.5.3 Bonding density ...  18
2.5.4 Selective Extractions and Cross-Reactivity ...  19
2.5.5 Capacity  ...  19
2.5.6 Immuno-based applications for Mycotoxins  ...  20
2.6 Determination of Mycotoxins  ...  21
2.6.1 Principles of reversed-phase high performance liquid chromatography (RP–HPLC)  ...  21
2.6.2 Individual Mycotoxins  ...  23
2.6.3 Liquid chromatography / mass spectrometry (LC-MS) as universal detector for multi-toxin extracts ...  23

3 Results and Discussion  ...  25
3.1 Exploratory Experiments with Deoxynivalenol ...  25
3.1.1 Background  ...  25
3.1.2 The Surveillance procedure  ...  27
3.1.3 pH solutions ...  27
3.1.4 Heated elution procedures  ...  28
3.1.5 Statistical analysis  ...  32
3.2 Zearalenone  ...  36
3.2.1 Mixtures of water with organic solvents ...  37
3.2.2 Discussions  ...  39
3.2.2.1Unspecific bindings  ...  39
3.2.2.2Recombination of antibodies ...  41
3.2.2.3Interfering Peaks ...  41
3.2.3 Statistical analysis  ...  44
3.3 Aflatoxins  ...  46
3.3.1 Statistical Analysis ...  51
3.3.1.1Aflatoxin B1 ...  51
3.3.1.2Aflatoxin B2 ...  52
3.3.1.3Aflatoxin G1  ...  54
3.3.1.4Aflatoxin G2  ...  55
3.4 Ochratoxin A ...  56
3.4.1 Statistical analysis  ...  57
3.5 Fumonisins  ...  59
3.5.1 Statistical analysis  ...  60
3.6 T-2 and HT-2 toxin ...  

4 Technical  ...  65
4.1 Alternative heating procedures  ...  65
4.1.1 Self-built heating Block ...  65
4.1.2 Microwave  ...  67
4.1.3 Soldering Rod ...  68
4.1.4 Oscillating circuit  ...  69
4.1.5 Electrical operated heating ...  70
4.2 (Semi-) Automation ...  67

5 Summary  ...  72

1 Introduction and Scope of the work

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.

Until today, various methods for the determination of mycotoxins exist, whereunder chromatographic methods are the most widespread used for final separation of matrix components and detection of the analyte of interest. High performance liquid chromatography (HPLC) methods have been developed for almost all major mycotoxins in cereals and other agricultural commodities, and are nowadays widely used because of their good performance and reliability.2 Thin layer chromatography (TLC) is mostly the method of choice for rapid screening purposes and for situations where advanced HPLC equipment is not available, but can also be used for quantitative analyses with densitometric detection. The use of gas chromatography (GC) is restricted to a limited number of mycotoxins, especially the trichothecenes.

All these methods depend on a suitable extraction and isolation procedure prior to the measurement. This clean-up procedure is often equal for TLC, HPLC and GC, depending on the separation and the specificity of the detector.

Most methods are reliable and stable, so the main challenge today is to provide comparable results: Several projects of the European Commission deal with the production of calibrants and (certified) reference materials as well as the organisation of intercomparison studies between different laboratories, a prerequisite for the establishment and implementation of EU guidelines.

Inside the BioCop - Project a range of new technologies such as transcriptomics, proteomics and biosensors will be utilised. These new approaches are based on measuring effect rather than on measuring single target compound concentrations. For these new methods, rather pure and if possible organic solvent free extracts were aimed, as it was noticed that biosensors and proteomics can obtain more reliable results using aqueous solutions.

Nowadays, a key component for the sample extract purification in modern mycotoxin analysis is immunoaffinity column chromatography. Immunoaffinity columns (IACs) can be self made, but are also commercial available. Different suppliers provide different IACs using several immunosorbents containing different antibodies. Currently the only used and recommended procedure by manufacturers of IACs is to elute the purified mycotoxins from the column with neat organic solvents to break the antibody-antigen bond.

One limitation of immunoaffinity clean ups is, that the purified mycotoxins - eluted with pure organic solvents such as methanol or acetonitrile - cannot be directly injected in larger aliquots for chromatographic separation systems, as this would result in insufficient separation in commonly used reversed-phase systems.3

Hence, only a rather small fraction of the eluate is injected after dilution with water or the IAC eluate is evaporated and re-dissolved in mobile phase, which is essential for rather polar analytes that require mobile phases with water contents higher than 85%.4 In the first case, this means that the capacity of the IAC must be sufficient to allow the analysis of the fraction injected and that larger amounts of sample extracts must be applied. In the latter case, the evaporation limits the simple automation of the clean-up procedure.

Therefore, this study aimed to investigate alternative, preferably solvent free procedures for the elution of mycotoxins from IACs. New elution procedures may contain the use of more dramatically conditions such as heat or protein denaturating agents such as glycin HCl or urea. Goal should be a procedure which offers several advantages, such as automation, the need of less antibodies in an IAC and as result of that, better limits of detection.

[....]