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Mycotoxins (toxic compounds formed by fungi) in food and feed have caused problems for mankind since the beginning of time. The group of ergot alkaloids plays a special role in human history. Several tens of thousands of deaths during the middle ages caused by to ergotism (the disease caused by continuous intake of ergot alkaloid contaminated food) underscore the importance of reliable analytical methods to ensure food safety.
More than 50 compounds belong to the group of ergot alkaloids. The 12 most found structures – the major ergot alkaloids – are typically measured, when it comes to ergot alkaloid quantification. High performance liquid chromatography (HPLC) with a fluorescence detector (FLD) is typically used to quantify the ergot alkaloid content. The main disadvantage of this method are the high costs for calibration standards (12 different calibration substances are required). But also, the time and effort required for the analysis of 12 peaks and overlapping signals that occur in complex food samples such as bread. As all ergot alkaloids share the ergoline structure and just differ in the substituents attached to this backbone, measurement of all ergot alkaloids in one sum parameter presents a time and cost saving alternative. The most important step for the development of such a sum parameter method is the reaction used to transfer all ergot alkaloids to one uniform structure. Two promising reactions, the acidic esterification to lysergic acid methyl ester and hydrazinolysis to lysergic acid hydrazide, were examined for possible use in a routine analysis method. In addition to yield and reaction rate, factors such as handling of the reaction and the possibility of parallel sample workup play a role.
Mycotoxins (toxins formed by fungi) in food and have caused problems for mankind since the beginning of time. The group of ergot alkaloids plays a special role in human history. Several tens of thousands of deaths during the middle ages caused by to ergotism (the disease caused by continuous intake of ergot alkaloid contaminated food) underscore the importance of reliable analytical methods to ensure food safety.
More than 50 compounds belong to the group of ergot alkaloids. The 12 most found structures – the major ergot alkaloids – are typically measured, when it comes to ergot alkaloid quantification. High performance liquid chromatography (HPLC) with a fluorescence detector (FLD) is typically used to quantify the ergot alkaloid content. The main disadvantage of this method are the high costs for calibration standards (12 different calibration substances are required). But also the time and effort required for the analysis of 12 peaks and overlapping signals that occur in complex food samples such as bread. As all ergot alkaloids share the ergoline structure and just differ in the substituents attached to this backbone, measurement of all ergot alkaloids in one sum parameter presents a time and cost saving alternative. The most important step for the development of such a sum parameter method is the reaction used to transfer all ergot alkaloids to one uniform structure. In the talk two promising reactions, the acidic esterification to lysergic acid methyl ester and hydrazinolysis to lysergic acid hydrazide, are examined for possible use in a routine analysis method. In addition to yield and reaction rate, factor such as the handling of the reaction and the possibility of parallelization play a role. Next to the current status of the ongoing research project, in this talk, current approaches to ergot alkaloid quantitation will be discussed.
Ergot alkaloids play a significant role in human history for more than 1000 years and are part of the most common contaminants of food and feed worldwide. Their high toxicity against humans and livestock even at low concentrations causes a high demand for quick and reliable analytics even though no European limits for ergot alkaloids have been determined yet. Currently the most common methods for the quantitation of the six major ergot alkaloids and their corresponding stereoisomers are HPLC based, using either fluorescence or mass spectrometric detection. Whereas these conventional detection methods measure each compound individually, a novel approach is to transfer all ergot alkaloids to one basic structure, which could be measured as a sum parameter.
Since all ergots contain a lysergic acid amide moiety and a differing peptide component, cleaving the molecule into a simple lysergic acid derivative, which could be quantified via either HPLC-FLD or MS/MS, is intended. To clean up the cleavage reaction mixture molecularly imprinted polymers (MIPs) are a simple and effective way to separate the desired structure. Due to the selectivity of MIPs, an improved matrix separation is expected, which results in fewer interferences in the FLD and the possibility to measure samples with more complex matrices. When fully developed, the novel method could overcome some major drawbacks of the conventional detection methods. Higher throughput and the need for less well-trained personnel are just two advantages, that should lead to a quick and cheap quantitation of ergot alkaloids. First results of this project will be presented, funded by the German ZIM program (Zentrales Innovationsprogramm Mittelstand) of the Federal Ministry for Economic Affairs and Energy.
Ergot alkaloids play a significant role in human history for more than 1000 years and are part of the most common contaminants of food and feed worldwide. Their high toxicity against humans and livestock even at low concentrations causes a high demand for quick and reliable analytics even though no European limits for ergot alkaloids have been determined yet. Currently the most common methods for the quantitation of the six major ergot alkaloids and their corresponding stereoisomers are HPLC based, using either fluorescence or mass spectrometric detection. Whereas these conventional detection methods measure each compound individually, a novel approach is to transfer all ergot alkaloids to one basic structure, which could be measured as a sum parameter.
Since all ergots contain a lysergic acid amide moiety and a differing peptide component, cleaving the molecule into a simple lysergic acid derivative, which could be quantified via either HPLC-FLD or MS/MS, is intended. To clean up the cleavage reaction mixture molecularly imprinted polymers (MIPs) are a simple and effective way to separate the desired structure. Due to the selectivity of MIPs, an improved matrix separation is expected, which results in fewer interferences in the FLD and the possibility to measure samples with more complex matrices. When fully developed, the novel method could overcome some major drawbacks of the conventional detection methods. Higher throughput and the need for less well-trained personnel are just two advantages, that should lead to a quick and cheap quantitation of ergot alkaloids. First results of this project will be presented, funded by the German ZIM program (Zentrales Innovationsprogramm Mittelstand) of the Federal Ministry for Economic Affairs and Energy.
Ergot alkaloids are a group of toxic compounds, formed by fungi on infested grasses. In 2022, the European Commission set into effect maximum levels for the sum of the twelve major ergot alkaloids in multiple foods. To facilitate the laborious and costly individual quantification of the twelve major ergot alkaloids by HPLC–MS/MS or -FLD, we recently reported a sum parameter method (SPM) for ergot alkaloid quantification. Here, derivatization to lysergic acid hydrazide—a derivative of the mutual ergoline backbone in all ergot alkaloids—allowed simplified determination of all ergot alkaloids in flour via HPLC-FLD. For the measurement of more complex matrices like processed foods, we now developed a MS/MS-based SPM. Two internal standards (IS), isotopically labelled at different positions of the molecule, were synthesized and employed in the MS/MS-measurements. Method performance using either the 13CD3-labelled or the 15N2-labelled IS was evaluated on naturally contaminated rye and wheat flour samples as well as on processed food matrices. Employing the 13CD3-labelled IS leads to lower variances and better consistency with the reference data (obtained by the FLD-based SPM) in flour samples compared to the 15N2-labelled IS. The novel method significantly improves the measurement of ergot alkaloids in complex food matrices, due to their increased selectivity and thus lower interferences. Furthermore, the application of isotope labelled IS obviates the need for time-consuming steps like the determination of recovery rate based, matrix specific correction factors as described in the MS/MS-based European standard method for ergot alkaloid quantification (EN 17425).
Ergot alkaloids are a group of mycotoxins occurring in products derived from various grasses (e.g., rye) and have been regulated in the EU recently. The new maximum levels refer to the sum of the six most common ergot alkaloids in their two stereoisomeric forms in different food matrices. Typically, these twelve compounds are individually quantified via HPLC-MS/MS or -FLD and subsequently summed up to evaluate food safety in a time-consuming process. Since all these structures share the same ergoline backbone, we developed a novel sum parameter method (SPM) targeting all ergot alkaloids simultaneously via lysergic acid hydrazide. After extraction and clean-up, in analogy to the current European standard method EN 17425 (ESM) for ergot alkaloid quantitation, the samples were derivatized by an optimized hydrazinolysis protocol, which allowed quantitative conversion after 20 min at 100 °C. The new SPM was evaluated against another established HPLC-FLD-based method (LFGB) and the HPLC-MS/MS-based ESM using six naturally contaminated rye and wheat matrix reference materials. While the SPM provided comparable values to the ESM, LFGB showed deviating results. Determined recovery rates, limits of detection and quantification of all three employed methods confirm that the new SPM is a promising alternative to the classical approaches for ergot alkaloid screening in food.
Ergot alkaloids are mycotoxins formed by fungi of the Claviceps genus, which are some of the most common contaminants of food and feed worldwide. These toxins are a structurally heterogeneous group of compounds, sharing an ergoline backbone. Six structures and their corresponding stereoisomers are typically quantified by either HPLC-FLD or HPLC-MS/MS and the values subsequently summed up to determine the total ergot alkaloid content. For the development of a screening method targeting all ergot alkaloids simultaneously, the alkaloids need to be transferred to one homogeneous structure: a lysergic acid derivative. In this study, two promising cleaving methods—acidic esterification and hydrazinolysis—are compared, using dihydroergocristine as a model compound. While the acidic esterification proved to be unsuitable, due to long reaction times and oxidation sensitivity, hydrazinolysis reached a quantitative yield in 40-60 min. Parallel workup of several samples is possible. An increasing effect on the reaction rate by the addition of ammonium iodide was demonstrated. Application of hydrazinolysis to a major ergot alkaloid mix solution showed that all ergopeptines were cleaved, but ergometrine/-inine was barely affected. Still, hydrazinolysis is a suitable tool for the development of a sum parameter screening method for ergot alkaloids in food and feed.