Final Activity Report Summary - SWORD (Se-speciation in real world food matrices: identification and the follow-up of the fate of Se-species in functional foods)
The objectives of the project SWORD were connected to the analysis of different forms of selenium (Se; also referred as Se-speciation), a microelement of paramount importance to human health. However Se-speciation has been a "hot" topic in science for about 15 years, some basic principles have not been established correctly and have therefore misled scientific research and decisions. One of the most important shortcomings is the incorrect identification and quantification of selenocysteine (Sec, or SeCys). SeCys is the 21st natural amino acid, a component of Se-enzymes that are responsible to, e.g. antioxidant defence of living cells. However this amino acid is not stable under usual analytical conditions, scientists have not always considered this fact important, which led to hundreds (!) of incorrect but peer-reviewed, impact factored publications.
The first and successfully achieved objective of the project was the synthesis of new SeCys standards that can be applied to ameliorate the quality control of this analyte. Two compounds were targeted: (i) carbamidomethyl-SeCys, a derivative of SeCys. We successfully synthesised the standard and characterised it by the means of ESI-MS. The technique and applications were published in a high quality journal (Journal of Analytical Atomic Spectrometry) in 2008 and it has already been independently cited several times, indicating its practical value. (ii) A SeCys2-containing oligopeptide. Up to now, the complete monitoring of all the steps of sample preparation in Se-amino acid (especially SeCys) oriented Se-speciation has been impossible, as neither certified reference materials, no quality control materials were available for SeCys. Therefore, we targeted and successfully synthesised a SeCys2-containing oligopeptide that can be used to validate the steps of reducing, alkylating, digesting, and quantifying. The value of this study is indicated by the fact that this new result could be presented in the frame of an oral presentation in the 3rd IUPAC conference on Trace Elements in Foods, Rome, Italy, 2009.
The second and successfully accomplished objective of the project was connected to the sample preparation of selected functional food samples. The difficulty of this task was that addressing real world food samples in Se-speciation is a demanding analytical job as usually very low extraction, recovery and identification ratios can only be achieved. In my case, I successfully developed enzymatic sample preparation based extraction techniques that were used for a Se-biofortified wheat flour sample and a Se-biofortified green bean sample. In the previous case, the results were validated in the frame of an interlaboratory comparison, organised by LGC (UK) where my results were rated "excellent". There is a relevant joint publication under preparation to be published in 2009, co-ordinated by Dr. H. Goenaga-Infante, LGC.
The third and successfully accomplished objective of the project was to identify new Se-species with the help of HPLC-ICP-MS and HPLC-ESI-MS instrumental set-ups. In both cases, multi-dimensional (also named orthogonal) separation and purification were applied, i.e. the selenium species extracted from the samples were purified by the means of consecutive chromatographic steps to achieve a certain level of purity. In my case, the extracted compounds from Se-biofortified yeast were first cleaned-up with size exclusion chromatography (SEC), then - depending on the chemical behavior of the species - strong anion exchange (SAX) or hydrophilic liquid chromatography (HILIC) HPLC columns were addressed on hyphenating to ICP-MS and ESI-MS. Altogether, more than 20 Se-species were identified, and partly quantified, from selenised yeast, including potential anti-cancer compounds. These results were published in parts in one of the highest ranked analytical journal, Analytical Chemistry, in 2008 (the value of this publication is mirrored by the fact that in 2008 only five articles were published in this journal from Hungarian authors), while another set of compounds is to be presented in a manuscript submitted to the newly emerged journal "Metallomics".
The first and successfully achieved objective of the project was the synthesis of new SeCys standards that can be applied to ameliorate the quality control of this analyte. Two compounds were targeted: (i) carbamidomethyl-SeCys, a derivative of SeCys. We successfully synthesised the standard and characterised it by the means of ESI-MS. The technique and applications were published in a high quality journal (Journal of Analytical Atomic Spectrometry) in 2008 and it has already been independently cited several times, indicating its practical value. (ii) A SeCys2-containing oligopeptide. Up to now, the complete monitoring of all the steps of sample preparation in Se-amino acid (especially SeCys) oriented Se-speciation has been impossible, as neither certified reference materials, no quality control materials were available for SeCys. Therefore, we targeted and successfully synthesised a SeCys2-containing oligopeptide that can be used to validate the steps of reducing, alkylating, digesting, and quantifying. The value of this study is indicated by the fact that this new result could be presented in the frame of an oral presentation in the 3rd IUPAC conference on Trace Elements in Foods, Rome, Italy, 2009.
The second and successfully accomplished objective of the project was connected to the sample preparation of selected functional food samples. The difficulty of this task was that addressing real world food samples in Se-speciation is a demanding analytical job as usually very low extraction, recovery and identification ratios can only be achieved. In my case, I successfully developed enzymatic sample preparation based extraction techniques that were used for a Se-biofortified wheat flour sample and a Se-biofortified green bean sample. In the previous case, the results were validated in the frame of an interlaboratory comparison, organised by LGC (UK) where my results were rated "excellent". There is a relevant joint publication under preparation to be published in 2009, co-ordinated by Dr. H. Goenaga-Infante, LGC.
The third and successfully accomplished objective of the project was to identify new Se-species with the help of HPLC-ICP-MS and HPLC-ESI-MS instrumental set-ups. In both cases, multi-dimensional (also named orthogonal) separation and purification were applied, i.e. the selenium species extracted from the samples were purified by the means of consecutive chromatographic steps to achieve a certain level of purity. In my case, the extracted compounds from Se-biofortified yeast were first cleaned-up with size exclusion chromatography (SEC), then - depending on the chemical behavior of the species - strong anion exchange (SAX) or hydrophilic liquid chromatography (HILIC) HPLC columns were addressed on hyphenating to ICP-MS and ESI-MS. Altogether, more than 20 Se-species were identified, and partly quantified, from selenised yeast, including potential anti-cancer compounds. These results were published in parts in one of the highest ranked analytical journal, Analytical Chemistry, in 2008 (the value of this publication is mirrored by the fact that in 2008 only five articles were published in this journal from Hungarian authors), while another set of compounds is to be presented in a manuscript submitted to the newly emerged journal "Metallomics".