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Speciation Analysis

Working Group 3.22

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The working group "Speciation Analysis" is responsible for the traceable quantification of elemental species.

For the assessment of the biological, chemical and toxicological influences of elements in nature, it is indispensible to know – in addition to the total content of the elements, which is mostly regulated by legislation – also the contents of the compounds (species) in which the elements occur. As these element species have completely different properties, the relevance of a specific element fraction in a sample can be reliably assessed only in connection with a species-analytical characterisation.


Different compounds of a metal may show

  • a different solubility and, thus, a different mobility: e.g. As2S3 0.0005 g/L versus Na2HAsO4 610 g/L
  • a different toxicity: e.g. Hg2Cl2 LD50 210 mg/kg, MeHgCl LD50 29.9 mg/kg
  • a different bioavailability

This means that – depending on the compound – the same total quantity of an element can have quite different effects. This is, for example, illustrated by the following copper compounds:

Copper in different compounds with different toxicity: copper sulfate as fungicides in viniculture, copper gluconate as dietary supplement and ceruloplasmin as copper storage protein

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Research/Development

As far as possible, we use isotope dilution analysis mass spectrometry (IDMS) for the quantitative determination of the elemental species. Thereby, two different approaches can be distinguished: Species-specific IDMS and post-column IDMS. In the case of species-specific IDMS, spike and analyte have the same chemical and structural composition – only the isotope signature is different. Therefore, it is the ideal internal standard, which behaves identically to the analyte during sample preparation and detection and allows a traceable quantification.

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Clinical Chemistry

Illustration of traceability from blood sampling via reference materials to SI

Different national and international directives require measurement results of clinical parameters to be traceable to higher-order reference materials and reference methods. These directives are, among others, the EU Directive on in vitro diagnostic medical devices (98/79/EC) and the "German Medical Association Directive on Quality Assurance of Quantitative Laboratory Tests for Medical Purposes" (RiLiBÄK). For many clinical analytes, in particular for proteins, reference methods or matrix-matched reference materials are, however, lacking. We consider the improvement of this situation and the development of measurement procedures which yield results that are traceable to the SI as one of the focal points of our work.

Our objective is to develop reference methods for important markers in clinical diagnosis which provide results traceable to the SI and, thus, comparable and reliable. A distinction can be made between:

Traceable quantification of small moleculesTraceable quantification of small molecules

In addition to proteins, cells and ribonucleic acids, also small molecules such as creatine, uric acid and the like play an important role in clinical diagnosis. In some cases in which it is difficult to determine the complete proteins, a detour via their smaller units such as peptides and amino acids is often made. An example is the determination of selenomethionine to estimate the selenium status of a patient instead of selenium proteins.

In addition to proteins, cells and ribonucleic acids, also small molecules such as creatine, uric acid and the like play an important role in clinical diagnosis. In some cases in which it is difficult to determine the complete proteins, a detour via their smaller units such as peptides and amino acids is often made. An example is the determination of selenomethionine to estimate the selenium status of a patient instead of selenium proteins.
Selenium is an essential element whose physiological range is, however, very narrow. In the case of a daily dose of < 30 µg for an adult, deficiency symptoms may occur, whereas a daily dose of > 700 µg will probably cause symptoms of poisoning. Selenium is a part of important proteins such as glutathione peroxidase which is involved in the defense against oxidative stress. It has also been found that the simultaneous administration of selenium during chemotherapy reduces side-effects. The prevention of poisoning requires an exact determination of the selenium content in the blood. Selenomethionine is a frequently used marker, although it is not a component of the selenoproteins themselves which contain selenocysteine instead. In a free state, selenocystein is very instable, which renders a reliable quantification difficult. Within the scope of the iMERA plus project T2.J10 TRACEBIOACVTIVITY, a method for the traceable quantification of selenomethionine in serum has been developed using 76Se-marked selenomethionine and isotope dilution analysis.

Traceable quantification of metalloproteinsTraceable quantification of metalloproteins

Haemoglobin (http://www.rcsb.org/pdb/)

With approx. 30 % of the total proteome, metalloproteins represent an important group of potential biomarkers for diseases. Examples of this are haemoglobin and transferrin as markers for anemia and inflammatory diseases, superoxide dismutase, which protects the cells against oxidative stress, and ceruloplasmin, the copper storage protein which is a marker for Wilson's or Menke's disease. For many proteins, however, reference measurement procedures are available so far. This means that each measurement kit of the different manufacturers has reference areas of its own; comparability of the results is, therefore, in many cases problematic.

We mainly use species-specific isotope dilution analysis for the quantitative determination of metalloproteins. For this purpose, the protein which contains the functional metal in an isotopically enriched form – the so-called “spike” – is added to the sample at the beginning of the sample preparation. In the ideal case, the spike and the investigated protein will behave identically during sample preparation, separation and detection, so that possible losses or conversions will have no effect on the result.

 

Superoxide dismutase: exchange of copper and zinc with natural isotopic composition with copper and zinc enriched in one isotope.

Within the scope of the EMRP project "Opens external link in new windowMetrology for Metalloproteins - HLT05", species-specific spikes and measurement procedures for the determination of haemoglobin, transferrin, superoxide dismutase, ceruloplasmin and selenoprotein P as well as glutathione peroxidase have been developed in cooperation with partner organisations from all over Europe.

The results are now applied within the EMPIR project Opens external link in new windowReMiND - 15HLT02 to the investigation of the role of these metalloproteins in the development of Alzheimer's disease.

Environmental analysis

Many substances which are contained in articles of daily use are ultimately found again in the environment. They are either already released during the lifetime of the products (such as the leaching of tributyltin from antifouling paints in shipbuilding) or afterwards from sewage treatment plants (e.g. pharmaceutical products and their metabolites which are excreted), waste disposal sites and waste combustion. From water and soil through the food chain , the toxic elements can finally also return to humans. 

A distinction must be made between polar substances, which are water-soluble, and non-polar substances, which are bound to particles or in the soil. The European Water Framework Directive (Directive 2000/60/EC  – amended by Directive 2013/39/EU) and the associated Directives 2008/105/EC and 2009/31/EC, therefore, require the determination of the content in whole water for organic analytes. In addition to the actual water phase, this also includes suspended particulate matter and sediments.

Examples are:

Flame retardants in waterFlame retardants in water

Polybrominated diphenyl ethers (PBDEs) are among the most frequently used flame retardants in polyurethane foams. These foams are used in car seats, furniture and packing material. In addition, these flame retardants can be found in electronic devices and in clothes. The so-called pentamix (PBDEs with an average of five bromine atoms per molecule) was prohibited in the EU in 1997. At that time, the worldwide production of this mix amounted to approx. 4000 tons per year. However, as PBDEs are additive (i.e. not covalently bound) flame retardants, and as the everyday objects, in which they were used, have a long lifetime, the PBDEs, which are contained in the mix, are still released. In spite of their low solubility in water (2  to 10 µg/L), they enriched in the food chain and are, in addition, biologically degradable only to a very small extent. The EU member states, therefore, concluded that PBDEs may pose a risk to public health and to the water ecosystems, especially since there are indications that the PBDEs contained in the pentamix could act like thyroid hormones.

For this reason, PBDEs from the pentamix were included in the list of priority substances of the European Water Framework Directive (Directive 2000/60/EC and Directives 2008/105/EC and 2009/90/EC associated with it). Until 2014, the established Environmental Quality Standards (EQS) were very low (0.5 ng/L for inland surface waters and 0.2 ng/L for all other surface waters). Therefore, very sensitive and selective measurement procedures are required in order to be able to  measure the PBDEs in water samples reliably and comparably throughout Europe. Within the scope of the European Metrology Research Programme (EMRP), the required methods were developed within the Opens external link in new windowENV08 project. The primary methods applied were species-specific isotope dilution and gas chromatography for the separation of the analytes from the matrix and from each other, and subsequent detection with mass spectrometry.

New toxicological findings required a further reduction of these EQS values. As it is very difficult to perform reliable measurements at such low concentrations due to blank values, it was agreed in 2014 to take the concentrations of PBDEs in biotas as an indicator of the concentrations of PBDEs in water. How reliable this relation is – and which biotas are best suited – is currently being investigated.

Mercury species in waterMercury species in water

Mercury (Hg) is an unusually mobile element in the environment. The quantities released by natural processes lead to a ubiquitous distribution, in which critical concentrations are reached only rarely. Anthropogenic releases may, however, lead to concentrations which are much higher at specific locations and which may be problematic in the environment due to high toxicity and different conversion processes.

Energiesparlampen mit QuecksilberIn industrialised countries, mercury is used, above all, in electronics, dentistry and for chemical and pharmaceutical products. In addition, mercury is released during the combustion of fossil fuels, in the production of cement and in the energy recovery of communal waste. For 2010, the Environmental Program of the United Nations (UNEP) estimated the global mercury emissions from anthropogenic sources to be 1960 tons. In 2013, it launched the "Minamata Convention on Mercury", with the aim of reducing worldwide mercury emissions and establishing comparable analysis and control mechanisms.

According to the European Water Framework Directive (2000/60/EC), mercury compounds are classified as priority hazardous substances (2013/39/EC). The currently valid Environmental Quality Standards (EQS) of 0.07 µg/L in surface waters and 20 µg/kg in biotas only relate exclusively to the total mercury content. A complete risk assessment, however, requires additional information about species distribution and possible conversion processes. For this reason, it is planned to develop and validate by 2017 sensitive and comparable measurement methods for the environmental compartments air, surface waters and biota within the scope of the European Metrology Research Programmes Opens external link in new windowENV51.

For water analysis, species-specific isotope dilution analysisi with gas chromatography for the separation of the analytes and subsequent detection with mass spectrometry are used. With the aid of asymmetrical flow-field-flow fractionation (AF4) of real water samples and subsequent quantitative analysis, it is intended to investigate, in addition, on which particle fraction or colloid fraction mercury mainly accumulates and to what extent sampling and storage conditions can be optimised.

natural water course
natural river course

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