Simultaneous determination of total ergot alkaloids in wheat flour by Orbitrap mass spectrometry.

The optimization screening methods for total ergot alkaloids in wheat extracts involve transforming them into a single compound, which is then analyzed via high-resolution Orbitrap mass spectrometry (Orbitrap MS). Orbitrap MS provides highly sensitive and accurate mass measurements, enhancing the selectivity and sensitivity of the analysis. Various hydrolysis and reduction methods have been investigated, and the use of superhydrides has emerged as the most effective method for transforming ergopeptine alkaloids. This study also focused on the epimerization of ergot alkaloids, particularly the differences between R- and S-epimers and their impact on the mass spectra. We validated our method by assessing the linearity, sensitivity, recovery, matrix effects, repeatability, and stability. The limits of detection and quantitation were set at 0.43 and 1.30 µg LSA/kg wheat, respectively. The proposed method offers a robust analytical approach for screening and quantifying total ergot alkaloids in wheat samples, addressing important concerns about their presence in food and feed.

Occurrence of Ergot Alkaloids in Major and Minor Cereals from Northern Italy: A Three Harvesting Years Scenario.

Ergot alkaloids (EAs), mycotoxins produced mainly by fungi of the Claviceps genus, have been frequently reported in rye, while their increasingly frequent occurrence in other cereals is likely related to weather conditions, with the incidence of ergot sclerotia in winter grains being related to heavy rainfall and moist soils at critical periods. However, compared to other regulated mycotoxins, data about the prevalence and occurrence of EAs in major and minor cereals harvested in the Mediterranean growing areas are still scant. In this regard, the current study reported the occurrence of EAs in 18 genotypes of winter cereals harvested over 3 years from an experimental field located in North Italy which were analyzed by HPLC-MS/MS. Results indicate a widespread occurrence of all the major EAs in all the considered cereal crops, especially under supportive meteorological conditions. EA contamination was dependent on the harvest year (p < 0.0001) which was particularly high in 2020 for all the considered species. The results also demonstrated a large co-occurrence of EAs with 98 cereal samples out of 162 contaminated with at least one of the 12 EAs (60% positive samples) in the range LOD: 15,389 µg/kg (median value: 2.32 µg/kg), expressed as the sum of the EAs. Rye was confirmed to be the crop more susceptible to the fungal infection (EAs content up to 4,302 µg/kg). To the best of our knowledge, we have reported the accumulation of EAs in tritordeum (LOD: 15,389 µg/kg) and in emmer (LOD: 1.9 µg/kg) for the first time.

Ergot alkaloids in sclerotia collected in Japan: synthetic profiles and induction of apoptosis by Clavine-type compounds.

The genus Claviceps (Clavicipitaceae) is famous for producing ergot alkaloids (EAs) in sclerotia. EAs can cause ergotism, resulting in convulsions and necrosis when ingested, making these compounds a serious concern for food safety. Agroclavine (2), a typical Clavine-type EA, is a causative agent of ergotism and is listed as a compound to be monitored by the European Food Safety Authority. Clavine-type EAs are known to cause cytotoxicity, but the mechanism has not been elucidated. We performed annexin V and PI double-staining followed by flow cytometric analysis to detect apoptosis in HepG2 and PANC-1 cells after exposure to Clavine-type EAs. Clavine-type EAs reduced cell viability and induced apoptosis in both cell lines. We then performed LC-MS analysis of EAs from 41 sclerotia samples of Claviceps collected in Japan. 24 out of 41 sclerotia extracts include peptide-type EAs (ergosine/inine: 4/4', ergotamine: 5, ergocornine/inine: 6/6', α-ergocryptine/inine: 8/8', and ergocristine/inine: 9/9') and 19 sclerotia extracts among 24 sclerotia detected peptide type EAs include Clavine-type EAs (pyroclavine: 1, agroclavine: 2, festuclavine: 3) by LC-MS. We then performed a metabolomic analysis of the EAs in the sclerotia using principal component analysis (PCA). The PCA score plots calculated for EAs suggested the existence of four groups with different EA production patterns. One of the groups was formed by the contribution of Clavine-type EAs. These results suggest that Clavine-type EAs are a family of compounds requiring attention in food safety and livestock production in Japan.

Ergometrine sensing in rye flour by a magnetic bead-based immunoassay followed by flow injection analysis with amperometric detection.

A certain group of mycotoxins, the ergot alkaloids, has caused countless deaths throughout human history. They are found in rye and other cereals and ingesting contaminated foods can cause serious health problems. To identify contaminated food exceeding the legal limits for ergot alkaloids, a portable and cost-effective test system is of great interest to the food industry. Rapid analysis can be achieved by screening for a marker compound, for which we chose ergometrine. We developed a magnetic bead-based immunoassay for ergometrine with amperometric detection in a flow injection system using a handheld potentiostat and a smartphone. With this assay a limit of detection of 3 nM (1 µg L-1) was achieved. In spiked rye flour, ergometrine levels from 25 to 250 µg kg-1 could be quantified. All results could be verified by optical detection. The developed assay offers great promise to meet the demand for on-site ergometrine detection in the food industry.

Simultaneous Determination of Alternaria Toxins, Ergot Alkaloid Epimers, and Other Major Mycotoxins in Various Food Matrixes by LC-MS/MS.

BACKGROUND: Various food commodities are vulnerable to different types of fungal pathogens and could be contaminated with differential classes of mycotoxins as a result. It is ideal to implement a generic method for the simultaneous determination of multi-mycotoxins in different food matrixes or agricultural products. OBJECTIVE: In this study, a simplified sample preparation procedure and a reliable LC-MS/MS analytical method were developed for the comprehensive measurement of 37 regulated and emerging mycotoxins including five Alternaria toxins (ATs) and six major ergot alkaloids (EAs) and their corresponding epimers. Four different food commodities (baby wheat cereal, peanut, tomato puree, and blended flour) were chosen for method validation to demonstrate the applicability of this analytical method across a wide range of food types. METHODS: Sample extraction was performed using a formic acid-acidified acetonitrile-water (4 + 1, v/v) solution followed by extract dry-down and reconstitution in a water-methanol (1 + 1, v/v) solution for analysis on a biphenyl LC column. Chromatographic analysis was performed using regular acidic LC conditions for baseline separation of ergot alkaloid epimers and completed with a short 11 min cycle time. RESULTS: Accurate quantification was achieved using matrix-matched calibration standards in the range of 0.4 to 400 µg/kg. The recoveries of all mycotoxins (except citrinin) in fortified samples were from 70 to 120%, and the RSD was less than 20%. CONCLUSION: The established workflow was simple and fast for multi-mycotoxin determination in a wide variety of food commodities with LOQs needed to meet the regulatory levels. HIGHLIGHTS: The developed method provided the unique benefit of simultaneous analysis of Alternaria toxins (ATs) and ergot alkaloids (EAs) together with other major regulated mycotoxins.

Effects of Heating, Pelleting, and Feed Matrix on Apparent Concentrations of Cereal Ergot Alkaloids in Relation to Growth Performance and Welfare Parameters of Backgrounding Beef Steers.

As the contamination of cereal grains with ergot has been increasing in Western Canada, studies were undertaken to evaluate the impacts of heating (60, 80, 120, or 190 °C) alone or in combination with pelleting on concentrations of ergot alkaloids. Fifteen samples of ergot-contaminated grain from Alberta and Saskatchewan were assayed for R and S epimers of six alkaloids (ergocryptine, ergocristine, ergocornine, ergometrine, ergosine, and ergotamine) using HPLC MS/MS. Five samples with distinct alkaloid profiles were then selected for heating and pelleting studies. Heating resulted in a linear increase (p < 0.05) of total R and total S epimers with increasing temperature, although some individual R epimers were stable (ergometrine, ergosine, ergotamine). Pelleting also increased (p < 0.05) concentrations of total R and total S epimers detected, although ergometrine concentration decreased (p < 0.05) after pelleting. A feeding study arranged in a 2 × 2 factorial structure used 48 backgrounding Angus-cross steers fed four different diets: (1) Control Mash (CM, no added ergot), (2) Control Pellet (CP), (3) Ergot Mash (EM), or (4) Ergot Pellet (EP). Pelleting heated the ergot to 90−100 °C under 4 bars pressure, but the ergot used in the feeding study was not otherwise heated. Alkaloid concentrations of EM and EP varied by up to 1.1 mg/kg depending on the feed matrix assayed. No differences among treatments were noted for growth performance, feed intake, feed conversion, concentrations of serum prolactin and haptoglobin, hair cortisol, or in temperatures of extremities measured by infrared thermography. The only negative impacts of ergot alkaloids were on blood parameters indicative of reduced immune function or chronic inflammation. Pelleting did not heighten the negative clinical outcomes of ergot, although alkaloid concentrations of pelleted feed increased depending on the matrix assayed. It was hypothesized that the heat and pressure associated with pelleting may enhance the recovery of alkaloids from pelleted feed.

Ion mobility-mass spectrometry to extend analytical performance in the determination of ergot alkaloids in cereal samples.

This work evaluates the potential of ion mobility spectrometry (IMS) to improve the analytical performance of current liquid chromatography-mass spectrometry (LC-MS) workflows applied to the determination of ergot alkaloids (EAs) in cereal samples. Collision cross section (CCS) values for EA epimers are reported for the first time to contribute to their unambiguous identification. Additionally, CCS values have been inter-laboratory cross-validated and compared with CCS values predicted by machine-learning models. Slight differences were observed in terms of CCS values for ergotamine, ergosine and ergocristine and their corresponding epimers (from 3.3 to 4%), being sufficient to achieve a satisfactory peak-to-peak resolution for their unequivocal identification. A LC-travelling wave ion mobility (TWIM)-MS method has been developed for the analysis of EAs in barley and wheat samples. Signal-to-noise ratio (S/N) was improved between 2.5 and 4-fold compared to the analog LC-TOF-MS method. The quality of the extracted ion chromatograms was also improved by using IMS.

In-House Validation of an Efficient and Rapid Procedure for the Simultaneous Determination and Monitoring of 23 Mycotoxins in Grains in Korea.

A high-performance liquid chromatography tandem mass spectrometry method is described for the simultaneous determination of mycotoxins, including Ergot alkaloids (EAs), in 3 types of grains. The extraction of 23 mycotoxins was evaluated and performed by using a modified QuEChERS-based sample preparation procedure. The proposed method was fully validated on spiked grain samples (barley, wheat and oat) to assess the linearity, limit of detection (LOD) and limit of quantitation (LOQ), matrix effects, precision and recovery. After validation, this method was applied to 143 samples of various types of 3 grains from the Republic of Korea to survey the level of mycotoxin contamination in Republic of Korean grains. A total of 42 grain samples (29%) were contaminated with at least one of these mycotoxins at levels higher than the LOQ. The results demonstrated that the procedure was suitable for simultaneously determining these mycotoxins in cereals and could be performed for their routine analysis in mycotoxin laboratories.

Ammonization of the R- and S-Epimers of Ergot Alkaloids to Assess Detoxification Potential.

Detoxification of ergot-contaminated feed by ammonia would be a practical application, given that ammonia is routinely used in the agriculture industry. To assess the effects of ammonia on ergot alkaloids, natural ergot-contaminated wheat was ammoniated. The total concentration of ergot alkaloids (R- and S-epimers) decreased after exposure to ammonia (8-29%). Separately, the total R-epimers decreased in concentration (40-66%), whereas the total S-epimers increased (21-81%). Specific ergot alkaloids demonstrated degradation and/or epimerization after exposure to ammonia, potentially associated with structural differences, and influenced the total concentrations observed. Ammonization of ergot standards resulted in potential degradation products and epimerization, supporting the above results. The use of ultrahigh-performance liquid chromatography-tandem mass spectrometry provides an updated assessment of the detoxification potential of ammonia for ergot alkaloids and the quantification of the S-epimers. Ammonia alters the R- and S-epimers of ergot alkaloids, which may lead to a potential practical detoxification process of ergot-contaminated feed.

Correlation patterns prevalence, and co-occurrence of ergot alkaloids in cool-season adapted cereal grains revealed with molecular spectroscopy and LC-MS/MS equipped HPLC system.

It is necessary to obtain more recent data on the prevalence and co-occurrence of mycotoxins in feed and food to minimize risks. This study examined the recent presence, co-occurrence, and correlation patterns of six major ergot alkaloids (EAs; i.e. ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) in cool-season adapted barley (n = 57) and wheat (n = 80) submitted by livestock producers and industries for testing ergot alkaloids/mycotoxins by liquid chromatography - tandem mass spectrometry method. Overall, 91% industry-submitted barley samples and 84% industry-submitted wheat samples tested positive for at least one ergot alkaloid and 33% industry-submitted barley and 38% industry-submitted wheat samples were found to be co-contaminated with all six major EAs. The content of total EAs in 9 industry-submitted barley (16%) and 18 industry-submitted wheat (23%) samples exceeded the recommended maximum allowable level for lactating or pregnant animals (250 ppb). All the barley and wheat samples that contained detectable ergosine were found to co-occur with other EAs. Overall, the content of individual EAs was positively correlated with each other and strong correlations (r > 0.8, P < 0.01) were detected between the content of individual EAs and total EAs. These results implied that the industry and producers submitted cool-season adapted barley and wheat samples contaminated with a single EA is likely to contain high levels of other major EAs. The patterns of individual EAs in this study were distinct from previous studies that focus on samples from European countries. Ergocristine was remained as the predominant EAs in the industry-submitted cool-season adapted barley and wheat samples at levels up to 9438.8 and 12416.2 ppb, respectively. While the mean contents of ergosine were the lowest (68.5 and 50.6 ppb for the industry-submitted cool-season adapted barley and wheat samples, respectively). The high prevalence and co-occurrence of EAs indicated that ergot contamination is still posing a significant threat to food and feed industry and more research is expected to reduce the contamination level and explore the toxicological significance of various co-occurrence profiles. A full scale of investigation for all barley and wheat samples is needed to obtain a full picture of mycotoxin contamination.

Prolonged absorption and susceptibility to enterohepatic circulation after oral administration of ergot alkaloids in ewes.

The objective of this study was to evaluate the pharmacokinetics profile of ergot alkaloids when administered to sheep orally. Although ergot alkaloids frequently contaminate animal feed, current understanding of their pharmacokinetics in animals cannot adequately predict toxicity. Blood samples were collected from ewes at 0.5, 1, 3, 5, and 12 h after oral exposure to 4 ergot alkaloids: ergocornine, ergocristine, ergocryptine, and ergosine, followed by serum analysis of these alkaloids using high performance liquid chromatography and tandem mass spectrometry. The alkaloids showed extended absorption time, in addition to clear signs of enterohepatic circulation. This pharmacokinetic profile suggests potential enhanced toxicity in animals with disorders related to secretion of bile acid. It may also explain the high susceptibility of sheep to ergot poisoning compared to other species. An extended sampling protocol (> 12 h) is necessary, however, to identify the pharmacokinetic properties of ergot alkaloids in ewes. In conclusion, ewes exposed to ergot alkaloids showed a prolonged absorption phase and enterohepatic circulation, which is in contrast with human ergot pharmacokinetics.

L'objectif de cette étude était d'évaluer le profil pharmacocinétique des alcaloïdes de l'ergot lorsqu'ils sont administrés à des moutons par voie orale. Bien que les alcaloïdes de l'ergot contaminent fréquemment les aliments pour animaux, la compréhension actuelle de leur pharmacocinétique chez les animaux ne permet pas de prédire de manière adéquate la toxicité. Des échantillons de sang ont été prélevés chez les brebis à 0,5, 1, 3, 5 et 12 h après exposition orale à quatre alcaloïdes de l'ergot : ergocornine, ergocristine, ergocryptine et ergosine, suivi d'une analyse sérique de ces alcaloïdes par chromatographie liquide à haute performance et spectrométrie de masse en tandem. Les alcaloïdes ont montré un temps d'absorption prolongé, en plus de signes évidents de circulation entérohépatique. Ce profil pharmacocinétique suggère une toxicité potentiellement accrue chez les animaux présentant des troubles liés à la sécrétion d'acide biliaire. Cela peut également expliquer la forte sensibilité des moutons à l'empoisonnement par l'ergot par rapport aux autres espèces. Un protocole de prélèvement étendu (> 12 h) est cependant nécessaire pour identifier les propriétés pharmacocinétiques des alcaloïdes de l'ergot chez les brebis. En conclusion, les brebis exposées aux alcaloïdes ont montré une phase d'absorption prolongée et une circulation entérohépatique, ce qui contraste avec la pharmacocinétique de l'ergot chez l'humain.(Traduit par Docteur Serge Messier).

Undertaking a New Regulatory Challenge: Monitoring of Ergot Alkaloids in Italian Food Commodities.

The present manuscript reports on monitoring data of 12 ergot alkaloids (EAs) in cereal and cereal-derived products, collected in Italy over the period 2017-2020, for official control purposes under the edge of the Commission Recommendation 2012/154/EU on the monitoring of the presence of EAs in feed and food. To these purposes, an LC-MS/MS method was set up and applied, after in-house verification of its analytical performance. Besides satisfactory recoveries and precision, the method's quantification limits proved suitable to assess the compliance of cereals and cereal-based foods with the recently issued EU maximum permitted levels (Commission Regulation 2021/1399/EU). The validity of the generated data was also evaluated through the adoption of four proficiency tests, from which acceptable z-score values (-2 ≤ z ≤ 2) were obtained. The method was then applied to analyse a total of 67 samples, collected in Italy over the period 2017-2020. The samples consisted of 18 cereal grains, 16 flours (14 of wheat and 2 of spelt) and 31 other types of cereals derivatives (including 9 for infants). Overall, the EAs analysis returned a high percentage of left-censored data (>86%). Among the positive samples, the highest contamination levels, up to 94.2 µg/kg, were found for ergocristine (12% incidence), followed by ergocristinine (7% incidence) with levels of up to 48.3 µg/kg.

Diversification of ergot alkaloids and heritable fungal symbionts in morning glories.

Heritable microorganisms play critical roles in life cycles of many macro-organisms but their prevalence and functional roles are unknown for most plants. Bioactive ergot alkaloids produced by heritable Periglandula fungi occur in some morning glories (Convolvulaceae), similar to ergot alkaloids in grasses infected with related fungi. Ergot alkaloids have been of longstanding interest given their toxic effects, psychoactive properties, and medical applications. Here we show that ergot alkaloids are concentrated in four morning glory clades exhibiting differences in alkaloid profiles and are more prevalent in species with larger seeds than those with smaller seeds. Further, we found a phylogenetically-independent, positive correlation between seed mass and alkaloid concentrations in symbiotic species. Our findings suggest that heritable symbiosis has diversified among particular clades by vertical transmission through seeds combined with host speciation, and that ergot alkaloids are particularly beneficial to species with larger seeds. Our results are consistent with the defensive symbiosis hypothesis where bioactive ergot alkaloids from Periglandula symbionts protect seeds and seedlings from natural enemies, and provide a framework for exploring microbial chemistry in other plant-microbe interactions.

Simultaneous Determination of Ergot Alkaloids in Swine and Dairy Feeds Using Ultra High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Ergot alkaloids (EAs) are mycotoxins mainly produced by the fungus Claviceps purpurea. EAs are known to affect the nervous system and to be vasoconstrictors in humans and animals. This work presents recent advances in swine and dairy feeds regarding 11 major EAs, namely ergometrine, ergosine, ergotamine, ergocornine, ergocryptine, ergocristine, ergosinine, ergotaminine, ergocorninine, ergocryptinine, and ergocristinine. A reliable, sensitive, and accurate multiple mycotoxin method, based on extraction with a Mycosep 150 multifunctional column prior to analysis using UHPLC-MS/MS, was validated using samples of swine feed (100) and dairy feed (100) for the 11 targeted EAs. Based on the obtained validation results, this method showed good performance recovery and inter-day and intra-day precision that are in accordance with standard criteria to ensure reliable occurrence data on EA contaminants. More than 49% of the swine feed samples were contaminated with EAs, especially ergocryptine(-ine) (40%) and ergosine (-ine) and ergotamine (-ine) (37%). However, many of the 11 EAs were not detectable in any swine feed samples. In addition, there were contaminated (positive) dairy feed samples, especially for ergocryptine (-ine) (50%), ergosine (-ine) (48%), ergotamine (-ine), and ergocristine (-ine) (49%). The mycotoxin levels in the feed samples in this study almost complied with the European Union regulations.

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market.

Following pending new legislation in the European Union setting a maximum of 20 ng g-1 for the total sum of ergot alkaloids in dry cereal-based baby food, a new UHPLC-MS/MS method was developed. It is suitable for the quantification of six ergot alkaloids: Ergocornine, ergocristine, ergometrine, ergosine, ergotamine, α-ergocryptine, and their corresponding epimers. The method is able to reliably detect individual ergot alkaloids at a level as low as 0.5 ng g-1. The method uses a modified QuEChERS extraction approach before UHPLC-MS/MS analysis. The method showed good sensitivity, accuracy, and precision. It has been applied to 49 samples from the Belgian market. In 26 samples, not a single ergot alkaloid was detected while in 23 out of 49 samples at least one ergot alkaloid was detected with 2 samples containing 12 ergot alkaloids. Ergometrine was the alkaloid most frequently detected i.e., 16 out of 49 samples. Only one sample, testing positive for all 12 ergot alkaloids, would be non-conforming to the newly proposed Maximum Residue Level (MRL).

Cleaving Ergot Alkaloids by Hydrazinolysis-A Promising Approach for a Sum Parameter Screening Method.

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.

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.

The natural occurrence of six major ergot alkaloids, ergometrine, ergosine, ergotamine, ergocornine, ergokryptine and ergocristine, as well as their corresponding epimers, were investigated in 60 cereal samples (barley and wheat) from Algeria. Ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and a QuEChERS extraction method were used for sample analysis. The results revealed that 12 out of 60 samples (20%) were contaminated with ergot alkaloids. Wheat was the most contaminated matrix, with an incidence of 26.7% (8 out of 30 samples). The concentration of total ergot alkaloids ranged from 17.8 to 53.9 µg/kg for barley and from 3.66 to 76.0 µg/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

A critical review of analytical methods for ergot alkaloids in cereals and feed and in particular suitability of method performance for regulatory monitoring and epimer-specific quantification.

Cereals and feed contaminated with ergot alkaloids (EAs) have been of concern for several decades. Nowadays, analysis of EAs is focused on ergometrine, ergotamine, ergosine, ergocristine, ergocryptine (a mixture of α- and ß-isomers) and ergocornine and their related -inine epimers as listed in the European Commission Recommendation 2012/154/EU. Liquid chromatography with fluorescence detection has been used for quantification of EAs for decades whilst LC-MS has become the work-horse for quantification of EAs in the last decade. However, in LC-MS analysis matrix effects of different magnitudes exist for each EA epimer, especially ergometrine/ergometrinine, even after different clean-up procedures. This leads to an underestimation or overestimation of EAs levels. Moreover, isotopic labelled standards for EAs are still not available in the market. This review aims to provide background information on different analytical methods, discuss their advantages and disadvantages and possible advancement. Moreover, the method performance requirements to support forthcoming regulations are also discussed.

Determination of principal ergot alkaloids in swine feeding.

BACKGROUND: Ergot alkaloids are secondary metabolites produced by fungi in the genus Claviceps. They contaminate a large variety of cereals, such as rye, triticale, wheat and barley. The ingestion of contaminated cereals might cause adverse health effects in humans and animals. In fact, pigs, cattle, sheep, and poultry are involved in sporadic outbreaks and, although there are several studies about occurrence of ergot alkaloids in grain cereals, there are scarce studies focused on compound feed. RESULTS: Twelve ergot alkaloids have been quantified in 228 feed samples intended for swine. The analytes were extracted using QuEChERS with Z-Sep+ as sorbent in the clean-up step, which reduced the matrix effect, allowing limits of quantification between 2.1 and 21.7 µg kg-1 . The analytes were subsequently quantified by ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS). A total of 29 samples (12.7%) revealed contamination by at least one ergot alkaloid, and among contaminated samples, 65% were contaminated by more than one. Only 6 of 12 target ergot alkaloids showed concentrations above the limit of quantification. The concentrations for individual ergot alkaloids ranged between 5.9 µg kg-1 for ergosinine to 145.3 µg kg-1 for ergometrine (the predominant ergot alkaloid), while the total ergot alkaloid content ranged from 5.9 to 158.7 µg kg-1 . CONCLUSIONS: The occurrence of ergot alkaloids in feed samples in Spain seems to be lower than in other regions of Europe. All the samples fulfilled current recommendations of the feed industry about practical limits for ergot alkaloids in pig feeds. This suggests that the feeds are safe for pig consumption, regarding the presence of ergot alkaloids. © 2021 Society of Chemical Industry.

Validation of a New Sensitive Method for the Detection and Quantification of R and S-Epimers of Ergot Alkaloids in Canadian Spring Wheat Utilizing Deuterated Lysergic Acid Diethylamide as an Internal Standard.

Ergot sclerotia effect cereal crops intended for consumption. Ergot alkaloids within ergot sclerotia are assessed to ensure contamination is below safety standards established for human and animal health. Ergot alkaloids exist in two configurations, the R and S-epimers. It is important to quantify both configurations. The objective of this study was to validate a new ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for quantification of six R and six S-epimers of ergot alkaloids in hard red spring wheat utilizing deuterated lysergic acid diethylamide (LSD-D3) as an internal standard. Validation parameters such as linearity, limit of detection (LOD), limit of quantification (LOQ), matrix effects, recovery and precision were investigated. For the 12 epimers analyzed, low LOD and LOQ values were observed, allowing for the sensitive detection of ergot epimers. Matrix effects ranged between 101-113% in a representative wheat matrix. Recovery was 68.3-119.1% with an inter-day precision of <24% relative standard deviation (RSD). The validation parameters conform with previous studies and exhibit differences between the R and S-epimers which has been rarely documented. This new sensitive method allows for the use of a new internal standard and can be incorporated and applied to research or diagnostic laboratories.