Ergocornine

A Targeted UHPLC-MS/MS Method Validated for the Quantification of Ergot Alkaloids in Cereal-Based Baby Food from the Belgian Market.[Pubmed:34437402]

Toxins (Basel). 2021 Jul 29;13(8). pii: toxins13080531.

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, alpha-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).

Determination of the Main Ergot Alkaloids and Their Epimers in Oat-Based Functional Foods by Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry.[Pubmed:34207051]

Molecules. 2021 Jun 18;26(12). pii: molecules26123717.

An ultra-high performance liquid chromatography coupled to tandem mass spectrometry method is proposed for the determination of the major ergot alkaloids (ergometrine, ergosine, ergotamine, Ergocornine, ergokryptine, ergocristine) and their epimers (ergometrinine, ergosinine, ergotaminine, ergocorninine, ergokryptinine, and ergocristinine) in oat-based foods and food supplements. A modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) procedure was applied as sample treatment, reducing the consumption of organic solvent and increasing sensitivity. This method involved an extraction with acetonitrile and ammonium carbonate (85:15, v/v) and a clean-up step based on dispersive solid-phase extraction, employing a mixture of C18/Z-Sep+ as sorbents. Procedural calibration curves were established and limits of quantification were below 3.2 mug/kg for the studied compounds. Repeatability and intermediate precision (expressed as RSD%) were lower than 6.3% and 15%, respectively, with recoveries ranging between 89.7% and 109%. The method was applied to oat-based products (bran, flakes, flour, grass, hydroalcoholic extracts, juices, and tablets), finding a positive sample of oat bran contaminated with ergometrine, ergosine, ergometrinine, and ergosinine (total content of 10.7 mug/kg).

Unraveling the Ergot Alkaloid and Indole Diterpenoid Metabolome in the Claviceps purpurea Species Complex Using LC-HRMS/MS Diagnostic Fragmentation Filtering.[Pubmed:34148344]

J Agric Food Chem. 2021 Jun 30;69(25):7137-7148.

The plant parasitic fungus Claviceps purpurea sensu lato produces sclerotia containing toxic ergot alkaloids and uncharacterized indole diterpenoids in grasses including cereals. The aim of this study was to detect as many peptide ergot alkaloids and indole diterpenoids in ergot sclerotia as possible by using a liquid chromatography-high-resolution mass spectrometry (LC-HRMS/MS) approach and applying filtering of diagnostic fragment ions for data extraction. The sample set consisted of 66 Claviceps sclerotia from four different geographic locations in southeastern Norway as well as Saskatchewan, Canada. The host plants included both wild grasses and important cereal grains such as rye. DNA sequencing showed that the sclerotia were from three Claviceps species, i.e., Claviceps purpurea sensu stricto (s.s.), Claviceps humidiphila, and Claviceps arundinis (former C. purpurea genotypes G1, G2, and G2a, respectively). All sclerotia from cereal grains were from C. purpurea s.s. Diagnostic fragment filtering was based on detecting specific product ions in MS/MS data sets that are well-conserved across the different ergot alkaloid subgroups and indole diterpenoids of the paspaline/paxilline type. The approach extracted mass spectra from 67 peptide ergot alkaloids (including C-8 epimers and lactam variants) and five indole diterpenoids. In addition, three clavines were detected by using targeted analysis. The sum of the peak areas for ergot alkaloids, which have been assigned as "major" analogues by the European Food Safety Authority (ergometrine, ergosine, ergotamine, alpha-ergocryptine, Ergocornine, ergocristine, and their 8-S epimers), accounted for at least 50% of the extracted total ergot alkaloid metabolome. Univariate and multivariate statistical analyses showed that several of the alkaloids were specific for certain species within the C. purpurea species complex and could be used as chemotaxonomic markers for species assignment.

Occurrence of Ergot Alkaloids in Barley and Wheat from Algeria.[Pubmed:33925104]

Toxins (Basel). 2021 Apr 28;13(5). pii: toxins13050316.

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 microg/kg for barley and from 3.66 to 76.0 mug/kg for wheat samples. Ergosine, ergokryptine and ergocristine showed the highest incidences in wheat, while ergometrine was the most common ergot in barley.

Vasoactive Effects of Acute Ergot Exposure in Sheep.[Pubmed:33924041]

Toxins (Basel). 2021 Apr 20;13(4). pii: toxins13040291.

Ergotism is a common and increasing problem in Saskatchewan's livestock. Chronic exposure to low concentrations of ergot alkaloids is known to cause severe arterial vasoconstriction and gangrene through the activation of adrenergic and serotonergic receptors on vascular smooth muscles. The acute vascular effects of a single oral dose with high-level exposure to ergot alkaloids remain unknown and are examined in this study. This study had two main objectives; the first was to evaluate the role of alpha1-adrenergic receptors in mediating the acute vasocontractile response after single-dose exposure in sheep. The second was to examine whether terazosin (TE) could abolish the vascular contractile effects of ergot alkaloids. Twelve adult female sheep were randomly placed into control and exposure groups (n = 6/group). Ergot sclerotia were collected and finely ground. The concentrations of six ergot alkaloids (Ergocornine, ergocristine, ergocryptine, ergometrine, ergosine, and ergotamine) were determined using HPLC/MS at Prairie Diagnostic Services Inc., (Saskatoon, SK, Canada). Each ewe within the treatment group received a single oral treatment of ground ergot sclerotia at a dose of 600 microg/kg BW (total ergot) while each ewe in the control group received water. Animals were euthanized 12 h after the treatment, and the pedal artery (dorsal metatarsal III artery) from the left hind limb from each animal was carefully dissected and mounted in an isolated tissue bath. The vascular contractile response to phenylephrine (PE) (alpha1-adrenergic agonist) was compared between the two groups before and after TE (alpha1-adrenergic antagonist) treatment. Acute exposure to ergot alkaloids resulted in a 38% increase in vascular sensitivity to PE compared to control (Ctl EC50 = 1.74 x 10(-6) M; Exp EC50 = 1.079 x 10(-6) M, p = 0.046). TE treatment resulted in a significant dose-dependent increase in EC50 in both exposure and control groups (p < 0.05 for all treatments). Surprisingly, TE effect was significantly more pronounced in the ergot exposed group compared to the control group at two of the three concentrations of TE (TE 30 nM, p = 0.36; TE 100 nM, p < 0.001; TE 300 nM, p < 0.001). Similar to chronic exposure, acute exposure to ergot alkaloids results in increased vascular sensitivity to PE. TE is a more potent dose-dependent antagonist for the PE contractile response in sheep exposed to ergot compared to the control group. This study may indicate that the dry gangrene seen in sheep, and likely other species, might be related to the activation of alpha1-adrenergic receptor. This effect may be reversed using TE, especially at early stages of the disease before cell death occurs. This study may also indicate that acute-single dose exposure scenario may be useful in the study of vascular effects of ergot alkaloids.

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.[Pubmed:33784227]

Food Addit Contam Part A Chem Anal Control Expo Risk Assess. 2021 Jun;38(6):997-1012.

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 alpha- and beta-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.

Molecular and Alkaloid Characterization of Claviceps purpurea Sensu Lato From Grass Seed Production Areas of the U.S. Pacific Northwest.[Pubmed:33141647]

Phytopathology. 2021 May;111(5):831-841.

Ergot, caused by Claviceps purpurea sensu lato, is an economically important seed replacement disease of Kentucky bluegrass (Poa pratensis) and perennial ryegrass (Lolium perenne) seed crops. C. purpurea sensu stricto is considered the primary Claviceps species responsible, but genetic diversity and cryptic species within C. purpurea sensu lato have previously been reported. Fifty-six C. purpurea sensu lato isolates collected from P. pratensis (n = 21) and L. perenne (n = 35) in Oregon and Washington between 2010 and 2014 were characterized via random amplified polymorphic DNA (RAPD), partial internal transcribed spacer (ITS), beta-tubulin and elongation factor-1alpha (EF-1alpha) sequences, conidial size, and ergot alkaloid chemotype. Based on RAPD analysis, seven isolates from P. pratensis and 33 isolates from L. perenne collected in Oregon corresponded to C. purpurea sensu stricto, and 13 isolates collected from P. pratensis in Washington and Oregon were identified as C. humidiphila. Partial ITS, beta-tubulin, and EF-1alpha sequences identified 10 isolates from P. pratensis as C. humidiphila, and seven isolates from P. pratensis and 33 isolates from L. perenne were identified as C. purpurea sensu stricto. Several isolates generated ambiguous RAPD bands or sequences that prevented identification. Ergot alkaloid chemotype profiling found that Ergocornine and its epimer were predominant in sclerotia from P. pratensis, whereas ergotamine and its epimer were most abundant in sclerotia from L. perenne. This study confirms the presence of the C. purpurea sensu lato species complex in the U.S. Pacific Northwest and suggests that more research is needed to characterize and mitigate Claviceps spp. infection of grass seed crops in North America.

Covariation of Ergot Severity and Alkaloid Content Measured by HPLC and One ELISA Method in Inoculated Winter Rye across Three Isolates and Three European Countries.[Pubmed:33114663]

Toxins (Basel). 2020 Oct 26;12(11). pii: toxins12110676.

Ergot caused by Claviceps purpurea is a problem for food and feed security in rye due to the occurrence of toxic ergot alkaloids (EAs). For grain elevators and breeders, a quick, easy-to-handle, and cheap screening assay would have a high economic impact. The study was performed to reveal (1) the covariation of ergot severity (= percentage of sclerotia in harvested grain) and the content of 12 EAs determined by high performance liquid chromatography (HPLC) and (2) the covariation between these traits and results of one commercial enzyme linked immunosorbent assays (ELISA). In total, 372 winter rye samples consisting of a diverse set of genotypes, locations from Germany, Austria, and Poland over two years, and three isolates were analyzed. Ergocornine and alpha-ergocryptine were detected as major EAs. Ergocristinine occurred as a minor component. Claviceps isolates from different countries showed a similar EA spectrum, but different quantities of individual EAs. A moderate, positive covariation between ergot severity and EA content determined by HPLC was observed across two years (r = 0.53, p < 0.01), but large deviation from the regression was detected. ELISA values did neither correlate with the HPLC results nor with ergot severity. In conclusion, a reliable prediction of the EA content based on ergot severity is, at present, not possible.

Epimerization of ergot alkaloids in feed.[Pubmed:32637710]

Heliyon. 2020 Jun 30;6(6):e04336.

Chronic intake of cereals contaminated with ergot alkaloids can cause ergotism and result in the loss of toes and fingers or even death. Today, due to common risk management practices, ergotism is rare as a human disease but remains a problem in livestock husbandry. Each alkaloid coexists under two forms (R and S), though only the R-form presents toxic effects. The epimerization occurs spontaneously but the mechanisms remain globally unknown. Therefore, different processing methods were evaluated for their respective influences on the epimerization. The results suggest that ergotamine and ergosine are very stable ergot alkaloids, and neither their concentrations, nor their respective R/S ratios, are significantly influenced by heating, protic solvents or UV light. In contrast, for ergocristine, ergokryptine, Ergocornine and ergometrine, heating can decrease the concentrations of these alkaloids and heat, protic solvents and UV light influence the R/S ratio towards the S-form, though the respective influence on the epimerization of these compounds is variable. In addition, the total concentration of all ergot alkaloids is reduced through heating. However, all these effects are not strong enough to change the composition of ergot alkaloids in feed substantially and to transform toxic feed into non-toxic feed.

Ergot Alkaloids in Wheat and Rye Derived Products in Italy.[Pubmed:31052444]

Foods. 2019 May 1;8(5). pii: foods8050150.

Genus Claviceps is a plant pathogen able to produce a group of toxins, ergot alkaloids (EAs), whose effects have been known since the Middle Ages (ergotism). Claviceps purpurea is the most important representative specie, known to infect more than 400 monocotyledonous plants including economically important cereal grains (e.g., rye, wheat, triticale). EAs are not regulated as such. Maximum limits are in the pipeline of the EU Commission while at present ergot sclerotia content is set by the Regulation (EC) No. 1881/2006 in unprocessed cereals (0.05% as a maximum). This study aimed to investigate the presence of the six principal EAs (ergometrine, ergosine, Ergocornine, alpha-ergocryptine, ergotamine and ergocristine) and their relative epimers (-inine forms) in rye- and wheat-based products. Of the samples, 85% resulted positive for at least one of the EAs. Wheat bread was the product with the highest number of positivity (56%), followed by wheat flour (26%). Rye and wheat bread samples showed the highest values when the sum of the EAs was considered, and durum wheat bread was the more contaminated sample (1142.6 mug/kg). These results suggest that ongoing monitoring of EAs in food products is critical until maximum limits are set.

Links Between Genetic Groups, Host Specificity, and Ergot-Alkaloid Profiles within Claviceps purpurea (Fr.) Tul. on Slovenian Grasses.[Pubmed:30673578]

Plant Dis. 2018 Jul;102(7):1334-1340.

In the present study, the genetic relationships and ergot-alkaloid production of the fungus Claviceps purpurea on grasses were investigated, to determine any associations between grass host specificity, ergot-alkaloid production, and geographic origin. C. purpurea sclerotia were obtained from wild and cultivated grasses along a 300-km climatic gradient, from sub-Mediterranean to continental climates. Twenty-one infected grass samples provided 39 sclerotia for analysis of the ergot alkaloids ergometrine, ergosine, ergotamine, Ergocornine, ergocryptine, and ergocristine, and their "-inine" epimers, using liquid chromatography-tandem mass spectrometry. C. purpurea ribosomal DNA underwent molecular classification to determine any grass host or geographic specificity of ergot-alkaloid composition for the different operational taxonomic units. Molecular analysis of sclerotia ribosomal DNA showed three genetic groups, with some associations with specific grass host taxonomic groups. The ergot-alkaloid composition data were in agreement with the data obtained by molecular methods. The most frequent ergot-alkaloid epimers were ergocristine, and ergosine. The total ergot-alkaloid concentrations in sclerotia varied from 59 to 4,200 mg kg(-1), which corresponds to 0.059 to 4.2 mg kg(-1) in animal feed (assuming ergot alkaloids at 1,000 mg kg(-1) sclerotia). Therefore, grasses can be associated with significant levels of ergot alkaloids. In addition, the ergot-alkaloid compositions of C. purpurea sclerotia can be different for infections with different C. purpurea genetic groups, because these show different ergot-alkaloid compositions.

Tall fescue ergot alkaloids are vasoactive in equine vasculature.[Pubmed:29293720]

J Anim Sci. 2017 Nov;95(11):5151-5160.

Mares grazing endophyte-infected () tall fescue () typically exhibit reproductive dysfunction rather than problems associated with peripheral vasoconstriction as a primary sign of the fescue toxicosis syndrome. Research using Doppler ultrasonography demonstrated that consumption of endophyte-infected tall fescue seed causes measurable vasoconstriction in the medial palmar artery. The objective of this study was to evaluate contractile responses of medial palmar artery and vein to increasing concentrations of various tall fescue alkaloids. Medial palmar arteries and veins were collected immediately following euthanasia from 23 horses of mixed breed, age, and gender from both forelimbs, and uterine arteries were collected from females ( = 12). Vessels were separated, cleaned of excess connective and adipose tissue, divided into 2- to 3-mm cross-sections, and suspended in a multimyograph chamber with continuously oxygenated Krebs-Henseleit buffer (95% O/5% CO; pH 7.4; 37 degrees C). Following a 90-min equilibration and recovery from reference compound exposure, increasing concentrations of norepinephrine, 5-hydroxytryptamine, ergotamine, and ergonovine for the palmar artery and vein and uterine artery and ergovaline, ergocryptine, ergocristine, Ergocornine, and lysergic acid for the palmar artery and vein were added to assess vasoactivity. Data were normalized as a percentage of contractile response induced by the reference compound addition and analyzed as a completely randomized design. Both norepinephrine and serotonin were vasoactive in all 3 types of blood vessels. Neither ergotamine nor ergonovine were vasoactive in the uterine artery. All alkaloids tested with the palmar artery and vein produced a contractile response, except that neither the palmar artery nor the palmar vein responded to lysergic acid ( > 0.05). Ergovaline was the most vasoactive ergot alkaloid in both the palmar artery and the palmar vein ( < 0.05) followed by ergonovine, whereas out of the 4 remaining ergopeptine alkaloids tested, ergocristine induced the lowest contractile response. Although horses do not outwardly appear to be affected by peripheral vasoconstriction as observed in cattle, these data indicate that tall fescue alkaloids are vasoactive and suggest that potential exists for peripheral vascular effects of tall fescue alkaloids in horses. This does not appear to be the case for the uterine artery, and future research should be directed at understanding how ergot alkaloids cause equine reproductive dysfunction.

Effects of Continuously Feeding Diets Containing Cereal Ergot Alkaloids on Nutrient Digestibility, Alkaloid Recovery in Feces, and Performance Traits of Ram Lambs.[Pubmed:29257065]

Toxins (Basel). 2017 Dec 19;9(12). pii: toxins9120405.

Allowable limits for cereal ergot alkaloids in livestock feeds are being re-examined, and the objective of this study was to compare nutrient digestibility, growth performance and carcass characteristics of ram lambs fed a range of alkaloid concentrations, including the maximum currently allowed in Canada (2 to 3 ppm). Four pelleted diets were fed: control, with no added alkaloids; 930; 1402; and 2447 ppb alkaloids based on total R and S epimers. Eight ram lambs (30.0 +/- 3.1 kg) were used to examine the impacts of dietary treatments on nutrient digestibility and alkaloid recovery from feces. Concentrations of dietary alkaloids evaluated did not affect nutrient digestibility or N metabolism. Excepting Ergocornine and ergocryptine, recovery of alkaloids in feces varied among periods, suggesting that individual lambs may differ in their ability to metabolize ergocristine, ergometrine, ergosine, ergotamine and their S epimers. In a second experiment, ram lambs (n = 47, 30 +/- 8 kg) were randomly assigned to a diet and weighed weekly until they achieved a slaughter weight of >/= 45 kg (average 9 weeks; range 6 to 13 weeks). Intake of DM did not differ (p = 0.91) among diets, although lambs fed 2447 ppb alkaloids had a lower (p < 0.01) ADG than did lambs receiving other treatments. The concentration of serum prolactin linearly declined (p < 0.01) with increasing alkaloids. Feeding 2447 ppb total alkaloids negatively impacted growth, while feeding 1402 ppb did not harm growth performance, but reduced carcass dressing percentage. Due to different concentrations of alkaloids affecting growth and carcass characteristics in the present study, determining allowable limits for total dietary alkaloids will require a better understanding of impacts of alkaloid profiles and interactions among individual alkaloids.

Detection of Total Ergot Alkaloids in Cereal Flour and in Bread by a Generic Enzyme Immunoassay Method.[Pubmed:28964275]

J AOAC Int. 2018 May 1;101(3):618-626.

Four sets of polyclonal antibodies against ergot alkaloids ergometrine, ergotamine, alpha-ergocryptine, and Ergocornine were produced and characterized in a competitive direct or indirect enzyme immunoassay (EIA). Standard curve LODs were 0.03 ng/mL (ergometrine EIA) to 2.0 ng/mL (Ergocornine EIA). Three EIAs were highly specific, whereas the ergometrine EIA had a broad specificity pattern and reacted, albeit weakly, with all seven major ergot alkaloids and their epimeric forms. Using the ergometrine EIA, a generic test system was established in which total ergot alkaloids are quantified by a standard curve for a toxin mixture composed of three alkaloids that matched the ergot alkaloid composition in naturally contaminated rye and wheat products. Sample extraction with acetonitrile-phosphate-buffered saline at pH 6.0 without further cleanup was sufficient for EIA analysis. The LODs for total ergot alkaloids were 20 ng/g in rye and wheat flour and 14 ng/g in bread. Recoveries were 85-110% (RSDs of 0.1-11.7%) at a concentration range of 50-1000 ng/g. The total ergot alkaloid EIA was validated by comparison with HPLC-fluorescence detection. Although some under- and overestimation by the total ergot alkaloid EIA was observed, it was suitable for the reliable identification of positive samples at 10-20 ng/g and for the determination of total ergot alkaloids in a concentration range between 100 and 1000 ng/g.

Human and animal dietary exposure to ergot alkaloids.[Pubmed:32625563]

EFSA J. 2017 Jul 6;15(7):e04902.

The ergot alkaloids (EAs) are mycotoxins produced by several species of fungi in the genus Claviceps. In Europe, Claviceps purpurea is the most widespread species and it commonly affects cereals such as rye, wheat, triticale, barley, millets and oats. Food and feed samples used to estimate human and animal dietary exposure were analysed for the 12 main C. purpurea EAs: ergometrine, ergosine, Ergocornine, ergotamine, ergocristine, ergocryptine (alpha- and beta-isomers) and their corresponding -inine (S)-epimers. The highest levels of EAs were reported in rye and rye-containing commodities. In humans, mean chronic dietary exposure was highest in 'Toddlers' and 'Other children' with maximum UB estimates of 0.47 and 0.46 mug/kg bw per day, respectively. The 95th percentile exposure was highest in 'Toddlers' with a maximum UB estimate of 0.86 mug/kg bw per day. UB estimations were on average fourfold higher than LB estimations. Average acute exposure (MB estimations) ranged from 0.02 mug/kg bw per day in 'Infants' up to 0.32 mug/kg bw per day estimated in 'Other children'. For the 95th percentile acute exposure, the highest estimate was for a dietary survey within the age class 'Other children' (0.98 mug/kg bw per day). Dietary exposure estimates for animals, assuming a mean concentration scenario, varied between 0.31-0.46 mug/kg bw per day in beef cattle and 6.82-8.07 mug/kg bw per day (LB-UB) in piglets, while exposure estimates assuming a high concentration scenario (95th percentile) varied between 1.43-1.45 mug/kg bw per day and 16.38-16.61 mug/kg bw per day (LB-UB) in the same species. A statistically significant linear relationship between the content of sclerotia and the levels of EAs quantified was observed in different crops (barley, oats, rye, triticale and wheat grains). However, the absence of sclerotia cannot exclude the presence of EAs as samples with no sclerotia identified showed measurable levels of EAs ('false negatives').