Task 4: Improved Methodology to Measure Mycotoxin Contamination
Biosensor methodology
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Intercomparison of Trichothecene Analysis and Feasibility to Produce Certified Calibrants and Reference Material - H. Pettersson
Annual Report
Period: 1998-10-01 - 1999-09-30
Project No: SMT4-CT96-2047
Reporter and Coordinator: H. Pettersson, Swedish University of Agricultural Sciences
Main Partner: Wenche Langseth, Central Veterinary Laboratory, Norway
Introduction
The first Intercomparison study, on trichothecenes in solution, in the project showed a relatively high variation, both in-laboratory and between-laboratories. The variations were, however, similar to those obtained in previous collaborative studies using gas chromatographic methods.
A special study on method performance and discussions with the participating laboratories revealed several serious method problems. All laboratories got more or less matrix induced response enhancement for the studied trichothecenes. Trichothecenes added to an uncontaminated wheat sample give higher response than when pure
calibrants are analyzed. It will give an overestimation of the trichothecene content in the samples, when external calibration curves based on pure trichothecene
calibrants are used for quantification. Many laboratories have solved the problem by applying matrix assisted
calibrants or a derivatized internal standard. These approaches have, however, not been accepted for certification of reference material. Most laboratories had also problems to obtain linear calibration curves for the trichothecenes, and some reported carry-over or memory effects from previous sample, matrix interferences or drifting detector response for the trichothecenes.
Such method problems as described above can't be tolerated in methods to be used in certification of reference material. Investigations of the method problems in order to eliminate them were therefore included as a new part in the project. Nearly all project time during the year has been used for such studies.
WP 2B1. Method studies to eliminate identified problems
Studies have been done in both the coordinator and the main partners laboratories using quite different gas chromatographic equipment and procedures. A preliminary and detailed report on the work done during the first six months has been produced and presented at the mid-term meeting of the project in Oslo in May 1999. A summary of the main results is given below.
The main problem, the matrix enhancement of the trichothecene response may be up to 120%, but is normally between 10 and 40 %. It has been found after all types of
derivatization. Internal GC standards, which are derivatized in the same way as the trichothecenes, like (-chloralose and trichothecene like compounds show similar matrix response enhancement, while the response of
underivatized GC standards like methoxychlor and DDE are less enhanced. The use of relative response factors to
derivatized internal GC standards and matrix assisted calibration curves compensate for most of the difference in response. If such methods are going to be used for certification of reference materials, it has to be shown that the response is the same for all samples of the same grain species, which may be difficult to prove.
The more matrix the injected solution contains, the more the response of the trichothecenes will increase up to a certain but varying level. The response of the trichothecenes were found to be the same for different samples of the same grain species, while small variation were sometimes observes between wheat , barley and oats. The matrix response enhancement seem to be more pronounced on GC's with MS-detector compared to those with EC-detectors, probably because more sample matrix is used for MS-detection. This matrix response enhancement seems to be partly due to adsorption of the
derivatized trichothecenes to active sites in the splitless injector and the first part of the column. Other compounds in the matrix are believed to compete with or block these active sites.
By using a new deactivated liner/insert in the splitless injector and cutting away the first part of the analytical column it was shown that the adsorption could be reduced significantly. Still, however, some matrix enhancement may be observed, and injection of new samples resulted in a rapid accumulation of impurities and active sites accompanied with lower response. Water or buffer treatment/washing of the extract after
derivatization, used in most methods, destroy and remove most of the excess of
derivatization reagent. Some of the trichothecene derivatives and other
derivatized compounds may, however, also be destroyed, which may cause accumulation of
underivatized impurities and formation of active sites in the injector and first part of column. Injection of BSTFA, a silylating reagent, reduces the actives sites and partly clean the column and injector from impurities and thereby increases the response. The matrix response enhancement was more or less eliminated for fluoro-derivated trichothecenes by regular injections of BSTFA in a clean deactivated system with EC-detection, but it was difficult to obtain reproducible results.
Different splitless injection conditions have also been studied. Normal injector temperatures or exchange of hexane with heptane or isooctane in the injection solution had no effect on the response, while variables like the internal volume of the liner, the volume injected, air gap in the syringe, and how fast the sample is injected may influence the response significantly. Too short «hold»-time (the time after injection before the split is opened and the injector purged) caused low reproducibility and reduced response. But even when the «hold»-time was increased so that no difference in response was observed compared to shorter «hold»-time, the amount of trichothecenes that reached the column seemed to increase with the amount of matrix present in the solution injected.
The use of a cool on-column injector will eliminate the problems appearing from the splitless injector, and matrix response enhancement observed as a consequence of adsorption to the column can more or less be eliminated by frequent injections of BSTFA. However, many laboratories do not have access to an on-column injector, and many on-column injectors can only be operated manually.
No drift or increase in response was observed for the trichothecenes with an EC-detector, when repeatedly injected together with wheat matrix. Repeated injection of silylated trichothecene
calibrants show on the other hand a trend of increasing response. A less pronounced increase was observed with fluorinated trichothecenes. Injections of BSTFA increased the response of the trichothecene
calibrants, but it decreased rapidly during 5-8 injections of calibrants, followed by a
continuous increase in the response with further injections.
Further studies on Clean-up and Implementation
At the mid-term meeting it was decided to further study and improve the extraction and clean-up methods. Most of the participating laboratories promised to try different approaches and some results have been reported, and the most important are
summarized below.
Adding somewhat more water at extraction of naturally nivalenol contaminated samples with ethylacetate will increase the recovery of nivalenol (Möller). Extraction of deoxynivalenol with 10% methanol in water for 3 min from naturally deoxynivalenol contaminated samples is very effective and similar to 2 hours with acetonitrile-water (84+16) (Usleber). Methanol-water (10+90) extract seems on the other hand to give higher response enhancements than acetonitrile-water (84+16) (Lew). The more polar extraction solvent also showed a lower recovery of HT-2 and T-2 toxins from naturally contaminated samples compared to acetonitrile-water. Water-polyethylene glycol extraction of deoxynivalenol from BCR - reference samples for immuno affinity columns and HPLC gives only about 50 % of the certified value although spiked samples gave >80% recovery (Solfrizzo).
The volume of extract passing the Mycosep column is of importance (Josephs/Krska). The trichothecene response and recovery increase with the volume passing the Mycosep column and 2 mL gave normally around 100% recovery and higher volumes higher
response, up to 158% recovery for HT-2 and T-2 toxin when passing 4 ml (1 gram). Analysing 0.5 ml portions of 3mL extracts passing the column showed that the nivalenol and deoxynivalenol response and the presumed recovery increased from 0 to 140%. Nivalenol was most retarded on the Mycosep column. Toxins in spiked extracts were more retarded than if added only to pure extraction solvent. The higher response when passing high volume through the column is probably due to more matrix present in the injected sample.
Additional C18 column/disc clean-up after the Romer type column has been tested (Eskola). Problems with low recovery of the polar trichothecenes nivalenol and deoxynivalenol was obtained. An extra ethylacetate extraction using an Extrelute column after the Mycosep column was tested by Lepschy. The additional clean-up step reduced or eliminated the response enhancement for nivalenol and deoxynivalenol and the slope of the calibration curves were similar to those without matrix. No response enhancement was seen for HT-2 and T-2 toxins with either clean-up procedures. Improvement of methods by implementation of some of the recommendations made at the mid-term meeting has also been reported by some laboratories.
Measure fumonisins - A. Visconti
Abstract of progress report: SMT-CT97-2193 - A. Visconti
The report describes the results of a method development/optimization study for fumonisin B1 (FB1) and fumonisin B2 (FB2) in five maize based matrices (maize flour, cornflakes, extruded product, muffins and infant formula). Fifteen materials, naturally contaminated at 1.5 µg/g FB1 + FB2 and spiked at 0.5 and 1.5 µg/g FB1 + FB2 were prepared. Their homogeneity was checked and found to be adequate to be used in the ruggedness test. The influence on method performances of the following five possible critical factors were examined by four laboratories by using a ruggedness test: extraction solvent (MeOH+water, 3+1, v+v and ACN+water, 1+1, v+v), volume of the extraction solvent (100 ml and 50 ml), test sample size (10 g and 25 g), extraction mode (shaking and blending) and clean-up (SAX and immunoaffinity).
The results of the ruggedness-test permitted to identify two critical steps in the method for the analysis of fumonisin in maize, cornflakes, extruded maize, muffins and infant formula. The extraction step of fumonisins from the materials and the clean-up step of the crude extracts were demonstrated to be critical, whereas test portion size, extraction solvent volume and extraction mode were demonstrated to have minor or no effect on method performances. The use of immunoaffinity columns proved to be essential for the purification of cornflakes, muffins and infant formula extracts showing that the SAX clean-up is insufficient for this purpose. The use of ACN+water, 1+1, v+v seemed to give higher fumonisins recoveries, as compared to MeOH+water, 3+1, v+v for all but infant formula matrices, however phase separation occurring during the extraction step showed that this solvent mixture is inappropriate.
These results can be usefully employed in order to optimize the mixture of ACN+MeOH+water and the use of immunoaffinity column to produce a robust method for fumonsin analysis in the five matrices considered in the present study.
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