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Development of HPLC-MS/MS methods for the detection of protein based binding systems in meat and meat products

  • Jira, Wolfgang
Publication Date
Dec 01, 2017
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In this work, analytical methods based on a coupling of high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS) for the detection of the market relevant protein-based binding systems microbial transglutaminase (TG) and fibrinogen/thrombin (FT) from beef and pork in meat and raw meat products have been developed. For the development of an HPLC-MS/MS method for the detection of TG in restructured meat (pork, beef, chicken and turkey), meat binding experiments with two technical TG mixtures with and without caseinate were performed. After the determination of six characteristic TG marker peptides (8 – 11 amino acids), establishing a methodology for defatting of samples using pressurized liquid extraction and optimizing the conditions of protein extraction and tryptic digestion, samples of restructured meat and corresponding blank values were analyzed in a raw and heated state. Using four selected TG marker peptides with three mass transitions each, no false-positive or –negative results were obtained. By investigation of samples pre-treated with oil marinade, emulsion marinade, seasoning salt and breadcrumbs, only very little effects of the type of pre-treatment on the detectability of TG were found. The limits of detection of the developed method were about a factor of 10 below the recommended amount of TG for raw as well as heated restructured meat and thus in a scale, in which no binding efficiency was achieved any more. By the additional measurement of casein marker peptides, the simultaneous detection of these important milk allergens was possible. On the basis of investigations of restructured hams, which had been produced with different TG concentrations it was verified, whether the proteolytic activity during the ripening process leads to an enzymatic degradation of TG and consequently to a decrease of the detectability of the TG marker peptides. It was shown that a decrease of TG detectability during the storage time for 43 days of dry-cured formed ham was not observed. The contents of two selected TG marker peptides were calculated using isotope-labeled peptides as internal standards, obtained by microwave-assisted solid-phase synthesis. These were characterized by good recoveries and high correlations to the applied TG concentration. The limits of detection of the TG marker peptides were about a factor of 20 below the recommended addition of TG. Based on the HPLC-MS/MS method established for TG analysis and the conditions of protein extraction and tryptic digestion used therein, an analytical method for the simultaneous detection of TG and FT was developed by the integration of six characteristic marker peptides for beef and pork each (8 – 16 amino acids). It was shown that the simultaneous detection of TG and FT is possible. Using the recommended amounts of FT for restructuring of meat of the same animal species, significantly increased peak areas of the fibrinogen marker peptides (about a factor of 100) in restructured meat compared to the control samples due to residual blood were observed. These substantial increases were confirmed by calculating the contents of selected fibrinogen marker peptides using isotope-labelled internal standards. A differentiation between the use of blood plasma powder and FT using the ratios of fibrinogen to serotransferrin peptide peak areas seems to be possible.

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