Mar. 08, 2011

Dioxin Analysis - Optimized Workflows for Quick and Reliable Foodstuffs Inspections

Dioxin Analysis: The current dioxin scandal has created a great disturbance in Germany, but fortunately, specific improvement measures in the field of foodstuffs inspections have also followed on as the result. Accordingly, on 2nd February, the Federal cabinet adopted some changes to legislation concerning foodstuffs and fodder, including a disclosure obligation for private laboratories, and the setting-up of a dioxin early warning system. GIT spoke to Dr. Hans-Joachim Hübschmann and Dr. Kyle D´Silva from Thermo Fisher Scientific about the current requirements for the analysis of dioxin and other POPs (Persistent Organic Pollutants) and measures to improve workflows.

Dr. Hübschmann, the topic of dioxin is one that has been treated very sensitively in Germany, possibly as a reaction to the 1976 Seveso catastrophe. Why is it like this, and actually how great is the risk for consumers, in your estimation?

H.-J. Hübschmann: Of course the sensitivity springs from the fact that the toxicity of dioxin - for consumers - is a very difficult topic to approach. The way it operates is completely different from the normal perception of the toxicity of a substance. Whilst there are many substances that can be extremely poisonous for us, the damage caused by dioxins is long-term because it is cumulative. However, this long-term effect has hardly been taken into account in the discussion at the present time. Naturally, the concern about damage to health arises from the anticipation of a direct effect - except that there is no direct effect if I eat an egg in which the maximum threshold has been exceeded. Here, the maximum acceptable intake of dioxins in food comes up for discussion, although in everyday life and in practical terms it is very hard for us to measure it.

Consequently, the objective of foodstuffs & fodder inspections has to be the banning of accumulated substances from our environment. And this is also required by the UN Stockholm Convention. In the long-term, accumulation over a prolonged period will give rise to the health risks that have been discussed. The strict analytical monitoring of dioxins and other toxins over the last 20 years has demonstrably led to a marked reduction in such substances in the environment and hence also in our foods.

Lower levels of dioxin have prompted laboratories and apparatus manufacturers to require that apparatus should be able to measure these lower levels. Precisely what levels of concentration are we presented with at the present time?

H.-J. Hübschmann: Absolute concentrations in the range of between 10 and 100 femtograms are being measured. These are the lower levels which analysers have to indicate at the present time. Naturally, the sample can also be concentrated. But that would run counter to the requirement for a rapid reaction, in other words the more sensitive instrumentation provides better results thanks to improved quality and also precision. And it provides such results more rapidly, too, because it can deal with smaller sample quantities.

Accordingly, as in the present case, we are concerned with more than merely detecting amounts that exceed the limits of the threshold. The long-term prevention of dioxin in the environment also includes regularly monitoring the background levels. In this context, we measure concentrations that are markedly below the maximum level in order to detect and to eliminate sources of pollution. These concentrations are detected in terms of the EU "action level". When this level is exceeded, we are obligated to detect and eliminate the cause. Research into causes includes examining the range of congeners, the variously chlorinated individual compounds, because this sample is influenced by various processes and biological causes and consequently enables insight into the source of pollution.

In the course of implementing Minister Ilse Aigner's 14-point plan, goals include improved dioxin monitoring. Does this mean quicker and more sensitive instrumentation, and more efficient workflows?

H.-J. Hübschmann: Yes. In the present case, we have seen how meat has been sold within 1 to 2 days, in respect of which there has been a suspicion that it might have been contaminated. However, it has unfortunately taken several days before the results were available. That - of course - explains the concern for expediting the achievement of effective inspection of these analysis processes.

Does this mean that the challenges reside more in the preparation of specimens or in analysis itself?

K. D´Silva: The tricky point is the preparation of the samples. For two reasons, this is a limiting factor: firstly, the preparation of samples is necessary in all cases, and also I have to concentrate the sample and remove it from its usually fatty surrounding matrix. Accordingly we have to ask: up to what level should I apply purification? If we use instrumentation which needs good sample preparation in order to function reliably, then I may need 2 to 3 days for the purification of the sample, although the measurement itself will then take place relatively quickly. If the objective is rapidity, which is of course the case for dioxin screening, then we need a desktop system such as Thermo Fischer Scientific's TSQ Quantum XLS. This is capable of analysing relatively unprepared samples for purposes of monitoring the maximum level. Accordingly, the all-important time gage and the necessary productivity for screening is achieved.

For confirmation analysis, it is an option then to utilize the far more sensitive high-end instrumentation?

K. D´Silva: Yes. Here, the EU prescribes high resolution GC/MS as the standard method. The EU regulation refers in turn to the EPA 1613, which prescribes a sector field MS with resolution of 10,000. For this purpose, we recommend the Thermo Scientific DFS High Resolution GC/MS. For this procedure, we normally conduct high-quality sample preparation & cleaning because the results of confirmation analysis are usually significant for Court purposes. Such measurement normally takes 3 to 4 days to complete, because calibrations and control measurements also have to be conducted. Accordingly, additional expenditure has to be incurred for quality control on results. A screening method, by contrast, can be expected to provide results within one day.

No standard method is prescribed for screening. Are there any analysis methods other than GC/MS, and how are these assessed, by comparison?

H.-J. Hübschmann: The advantage of also using GC/MS for screening is that we are already very well acquainted with this well-established method based on dioxin confirmation analysis, and that such apparatus is also suitable for other analyses arising in the laboratory. A bioassay for dioxin can be used exclusively for dioxins. MS combined with GC or LC, on the other hand, is now used everywhere in trace analysis. The mass spectrometer is highly sensitive and selective, and - above all - it measures the substance directly, i.e., it provides data which can even be defended legally.

In relation to bioassays, we should also recall that these require special biological laboratories capable of cultivating the cells needed for the assays and preserving them for a prolonged period, and the corresponding quality-control has to be developed for the purpose. Moreover, it is required - as discussed at the 2010 dioxin conference in San Antonio, Texas, - that these bioassays should be calibrated by means of mass spectrometry, and for various matrixes. Accordingly, they also require the use of instrumentation capable of achieving adequate quality. And of course we should not forget that a bioassay requires the same laborious sample preparation as GC/MS. We have seen how the topic of dioxin bioassay far transcends the level of a rapid test kit or a form of litmus paper with a "positive" or "negative" result.

Exactly what processes and apparatus will be entailed in the preparation of samples?

K. D'Silva: For sample preparation, I will initially require extraction, i.e., the food is ground or freeze-dried; the proportion of fat has to be determined, because limit values are mostly defined in pg TEQ per gram of fat, and a conventional Soxhlet extraction process is performed. This is the established method, although, as it takes up approximately 24 hours, it is very time-consuming and uses a large quantity of solvents. Alternatively, recourse can also be had to the more rapid "Pressurized Liquid Extraction" (PLE ) or "Accelerated Solvent Extraction" (ASE, as supplied, for example, by Dionex). Both Soxhlet and ASE yield extracts which have to be purified. This is conducted in three stages of column chromatography: firstly, separation of matrix constituents; secondly the oxidation of the proportion of fat by means of concentrated sulphuric acid; thirdly the separation of congeners into various fractions. In order to simplify and accelerate these three stages of purification, FMS, for example, supplies a fully automated system.

Simplified, more rapid sample preparation processes for screening would dispense with the need for thorough purification, i.e. stages 1 and 3 of column chromatography could be obviated and a high level of selectivity could be set on the mass spectrometer. Naturally, we believe that we can claim for our mass spectrometers that thanks to the special technology that we employ in the field of the quadrupole, we can achieve higher selectivity by virtue of higher mass resolution. The TSQ Quantum XLS is used in many well-known laboratories for screening with a high sample throughput rate. The results achieved by means of this method have been presented at numerous dioxin conferences, such that now we have a very solid footing.

Accordingly, then, there are best-practice methods for mass spectrometry; what is the situation with workflow in other respects?

H.-J. Hübschmann: For this purpose, we have developed the "POPs Integrated Workflow", an analytical solution for laboratories dealing with POPs analysis. This takes in consultation and the know-how that we share with our customers. For example: in the field of extraction, GC/MS measurement and data analysis, which is also subject to an EU Directive. In laboratories, furthermore, samples have to be prepared and data has to be managed. We cover the entire workflow by collaborating in certain areas with other companies, for example with standards, some of which are 13C marked standards for investigation of recovery in extraction. And on clean-up, in which context we collaborate with the specialist firm of FMS, already mentioned, from Boston. This places us at a stage at which we can also hand over these products more quickly. Overall, then, the objective in the compilation of workflows is to ensure that we achieve productivity, to provide reports and of course to assure the quality of results.

How did this workflow come about, and what service do you offer your customers with regard to POP analysis?

H.-J. Hübschmann: The integrated workflow is achieved thanks to worldwide collaboration between various divisions of Thermo, and accordingly, for example, our GC/MS systems come from Austin, the Triple-Quads for the TSQ Quantum are made in San Jose and the consumables are covered in the UK, and the LIM system comes from Madison. The know-how from all of these areas is combined in the new "POPs Centre of Excellence" initiative. This is a group of people here in Bremen, taking in colleagues from the Food Safety Response Center in Dreieich, which in addition to functioning as the knowledge center, funds and represent the core of the POPs centers. . We plan to manage all activities as projects and to combine and bundle the necessary consultation services and information from the various divisions, so that we can also provide our customers with recommendations as to which types of apparatus are most suitable for the customer's own workflow.

K. D'Silva: We also configure user meetings on the topic of POPs analysis (refer info box) and at this stage I should emphasise that we are not dependent on any particular manufacturer. Naturally, in our range of customers, we have laboratories operating with various products, but the topic of POPs analysis is so important and so dynamic and has such a considerable financial repercussions - bearing in mind that 3,500 farms have been closed in Germany - that we are making every effort to keep contact with all of the players in this field of analysis and to continue to develop the best-practice solution by virtue of a global exchange of information.

Dioxins and PCBs are topics with a very high emotional charge. What other urgent analytical challenges, perhaps ones which are not at the centre of public attention at the present time, can we also consider?

H.-J. Hübschmann: Major topics certainly include plant pesticides and insecticides and flame retardants as frequently employed in electronics components, and also in textiles such as theatre curtains or commissioning materials in various items of equipment. These halogen compounds have a similar threat to health as dioxins, and in the final analysis they also follow the same path through the food chain. Although substances such as PCBs (polychlorinated biphenyls) and DDT have already been banned for many years, DDT is nevertheless still permissible (exclusively) in the combating of malaria and so there may be some concerns with historic pollution; the analysis of this compound with regard to background loading is an up-to-date topic.



Thermo Fisher Scientific GmbH
Hanna-Kunath-Str. 11
28199 Bremen
Phone: +49 421 5493 0
Telefax: +49 421 5493 200

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