Individual Ergot Alkaloids Research Articles

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).

Determination of principal ergot alkaloids in swine feeding.

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. 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 21.7μg 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 5.9μg kg-1 for ergosinine to 145.3μg kg-1 for ergometrine (the predominant ergot alkaloid), while the total ergot alkaloid content ranged from 5.9 to 158.7μg kg-1 . 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.

Proportions of predominant Ergot alkaloids (Claviceps purpurea) detected in Western Canadian grains from 2014 to 2016

Ergot alkaloids, produced by the fungusClaviceps purpurea, are contaminants of cereal crops. Depending on various factors, the relative composition of individual ergot alkaloids can differ among samples. The objective was to determine if the percentage of individual ergot alkaloids were similar across different cereal grains (barley n=39, rye n=7, triticale n=9, wheat n=94) collected in Western Canada over different years. Ergocristine was the predominant alkaloid accounting for half of the total alkaloids in all grain types. This study documented that barley, rye, triticale and wheat collected across Western Canada had similar percentages of ergocornine (6±1%,P=0.201), ergocristine (48±2%,P=0.939), ergocryptine (17±2%,P=0.302) and ergosine (5±0.5%,P=0.239). There were differences between grain types for ergometrine (P=0.027) and ergotamine (P=0.011), which ranged between 6 to 13% and 11 to 24%, respectively, of the total alkaloid content in different cereals. Both barley and wheat alkaloid percentages were similar between 2015 and 2016; ergocornine (7±1%,P=0.969), ergocristine (47±2%,P=0.680), ergocryptine (18±2%,P=0.572), ergometrine (8±1%,P=0.080), ergosine (15±1%,P=0.119) and ergotamine (P=0.189). The ergocornine percentage was higher in wheat (P=0.017) as compared to barley for 2015/2016 samples. Ergometrine was higher in barley (P=0.002) as compared to wheat for 2015/2016 samples. While two of the alkaloid proportions varied statistically, overall proportions of the six ergot alkaloids were comparable among the four grain types collected across Western Canada. If proportions of ergot alkaloids are similar across a region, then it may be deemed acceptable to recommend a maximum total ergot alkaloid concentration for that region. However, areas that exhibit variation among the ergot alkaloid proportions, individual ergot alkaloid guidelines based on a toxic equivalence factor, may be more appropriate. In contrast, since major differences were not seen between years or grain type, from a producer perspective there may be limited biological/toxicological significance for individual alkaloid guidelines.

In-house validation of a rapid and efficient procedure for simultaneous determination of ergot alkaloids and other mycotoxins in wheat and maize.

A fundamental step in addressing the global problem of mycotoxins is the development of highly sensitive, multi-class extraction and detection methods. This constitutes a field of research that has in recent years enjoyed a steady advance. Such methods, generally based on liquid chromatography coupled to mass spectrometry, are widely reported successfully detecting various mycotoxins in different food and feed samples. In this work, an innovative approach to multi-class mycotoxin control is proposed, offering specific advantages: a broader inclusion of more mycotoxin classes, robust and thorough extraction for all target compounds despite their varied chemical properties, and determination of all analytes from a single injection. The method involved the extraction and quantification of the main mycotoxins produced by Aspergillus, Fusarium, and Penicillium fungi, as well as their reported derivatives, together with 12 other compounds most commonly produced by Claviceps purpurea. The popularly reported QuEChERS technique has been reduced to a simple "salting-out liquid-liquid extraction" (SO-LLE) to obtain the most efficient extraction of the aforementioned mycotoxin classes in a very short time. This is in particular extremely important in ensuring correct determination of individual ergot alkaloids, for which short and robust sample preparation as well as short analytical sequences were key for minimizing the epimerization during analysis. The analyses of wheat and maize samples were performed using ultra-high performance liquid chromatography coupled with tandem mass spectrometry. Matrix-matched calibration curves were established and limits of quantification were below the maximum levels established by the EU regulation. The precision (repeatability and intermediate precision) was lower than 13% in all cases and recoveries ranged between 60 and 98% in maize and between 62 and 103% in wheat, fulfilling the current legislation. The method was applied to study the co-occurrence of these mycotoxins in wheat (n= 13) and maize (n= 15) samples from six European countries. A successful quantification of 23 different mycotoxins, from all major classes, in 85% of wheat and 93% of maize samples was achieved.

Correlation and variability between weighing, counting and analytical methods to determine ergot (Claviceps purpurea) contamination of grain

Ergot alkaloid mycotoxins produced by the fungusClaviceps purpurea, are contaminants of cereal crops and grasses. The objectives of this study were to determine the correlation between number of ergot sclerotia and weight compared to the total ergot alkaloid concentration, to evaluate the effect of grinding process (i.e. particle size (PS)) on ergot alkaloid analysis using high performance liquid chromatography – tandem mass spectrometry, and to determine the impact of sample volume on analytical variability. This study demonstrated that correlations exist between both ergot sclerotia count (R2=0.7242,P<0.001) and ergot sclerotia weight (R2=0.9618,P<0.001) compared to the total alkaloid concentration of 6 ergot alkaloids. However, at alkaloid ergot concentrations below 350 µg/kg grain, ergot sclerotia count (R2=0.0002,P=0.956) and ergot sclerotia weight (R2=0.0064,P=0.769) were not correlated to the total alkaloid concentration. A lower variability (P=0.041), defined by coefficient of variation (CV), was observed using a commercial UDY cyclone sample mill (PS=192 µm, CV=9 µg/kg) as compared to a household coffee grinder (PS=516 µm, CV=66 µg/kg). Total amount and concentration of individual ergot alkaloids varied (P<0.05) among sclerotia of similar weight. For the analytical method, CV was numerically reduced as sample volume increased (97% CV for 75 ml to 64% CV for 1000 ml; mean of all concentrations) but increased as sample concentration declined (17% CV for 81,678 µg/kg to 284% for 35 µg/kg; mean of all sample volumes). This implies that analysis of small sample volumes at low ergot alkaloid concentrations may result in highly variable and potentially misleading results. In conclusion, number of ergot sclerotia and weight are unreliable indicators of alkaloid content at ergot concentrations below 350 µg/kg and particle size influences the variability. An analytical approach with fine grinding (mean PS<200 µm, 85% particles <400 µm) of a large sample should be used to assess low-level ergot contamination.

Pattern and distribution of ergot alkaloids in cereals and cereal products from European countries

This paper reports on the occurrence of ergot alkaloids in cereals and cereal products in Europe. It includes occurrence data our group previously submitted to the European Food Safety Authority and new data we gathered afterwards. A total of 1,065 samples of cereals and cereal products intended for human consumption and animal feeding were analysed by liquid chromatography-tandem mass spectrometry for the presence of ergot alkaloids. The sample set included rye-, wheat- and multigrain-based food as well as rye-, wheat- and triticale-based feed. The study revealed that 59% of the analysed food and feed samples were contaminated with ergot alkaloids to some extent. In 55% of the samples, the levels of the -ine isomers were above the limit of quantification (LOQ), while contamination with the -inine isomers was found in 51% of the samples. The median values for the main ergot alkaloids (-ine forms) and the epimers (-inine forms) were 1 and 2 μg/kg, respectively. Ergot alkaloids were present in 84% of rye food, 67% of wheat food, 48% of multigrain food, 52% of rye feed, 27% of wheat feed, and 44% of triticale feed at total alkaloid levels ranging from ≤1 (LOQ) to 12,340 μg/kg. Though the highest frequencies of contamination were observed for food samples, the feed samples, in particular Swiss rye feed, accounted for the highest levels of ergot alkaloids. The frequencies and levels of contamination were significantly lower in organic samples compared to conventional samples. Maximum levels of individual ergot alkaloids up to 3,270 μg/kg (for ergotamine) were observed. Overall, ergosine, ergokryptine and ergocristine were the frequently occurring ergot alkaloids. The co-occurrence of all six ergot alkaloids was noted in 35% of the positive samples. Occurrence of a single ergot alkaloid was mainly observed for ergometrine.

Effects of Ergot Alkaloids on Liver Function of Piglets as Evaluated by the 13C-Methacetin and 13C-α-Ketoisocaproic Acid Breath Test

Ergot alkaloids (the sum of individual ergot alkaloids are termed as total alkaloids, TA) are produced by the fungus Claviceps purpurea, which infests cereal grains commonly used as feedstuffs. Ergot alkaloids potentially modulate microsomal and mitochondrial hepatic enzymes. Thus, the aim of the present experiment was to assess their effects on microsomal and mitochondrial liver function using the 13C-Methacetin (MC) and 13C-α-ketoisocaproic acid (KICA) breath test, respectively. Two ergot batches were mixed into piglet diets, resulting in 11 and 22 mg (Ergot 5-low and Ergot 5-high), 9 and 14 mg TA/kg (Ergot 15-low and Ergot 15-high) and compared to an ergot-free control group. Feed intake and live weight gain decreased significantly with the TA content (p < 0.001). Feeding the Ergot 5-high diet tended to decrease the 60-min-cumulative 13CO2 percentage of the dose recovery (cPDR60) by 26% and 28% in the MC and KICA breath test, respectively, compared to the control group (p = 0.065). Therefore, both microsomal and mitochondrial liver function was slightly affected by ergot alkaloids.

Effects of the Ergot Alkaloids Dihydroergotamine, Ergonovine, and Ergotamine on Growth ofEscherichia coliO157:H7In Vitro

A series of experiments were conducted to evaluate the effects of ergot alkaloids (dihydroergotamine, ergonovine, and ergotamine) on E. coli O157:H7 in both pure and mixed ruminal fluid culture. Alkaloids were added to solutions of E. coli O157:H7 strains 933 (pure and ruminal cultures) and 6058 (ruminal culture only), and growth rates and colony-forming units (CFU) of E. coli O157:H7 were measured. Two mixtures of all three alkaloids at either 2 or 500 microM for each alkaloid decreased (p < 0.001) the growth rate of E. coli O157:H7 in pure culture compared to the individual alkaloids. Dihydroergotamine tended (p = 0.07) to reduce growth rate of E. coli O157:H7 in pure culture compared with ergonovine or ergotamine alone. Increased concentrations of dihydroergotamine and ergotamine decreased (p < 0.003) growth rate of E. coli O157:H7 but increasing concentrations of ergonovine did not influence (p > 0.10) E. coli O157:H7 growth rate. Similar to results in pure culture, a mixture of all three alkaloids at various concentrations for each alkaloid decreased (p < 0.001) the CFU of E. coli O157:H7 strain 6058 in mixed ruminal culture compared to the individual ergot alkaloids. Dihydroergotamine decreased (p = 0.04) CFU of E. coli O157:H7 strain 6058 when compared to ergonovine but CFU were similar (p > 0.10) between dihydroergotamine and ergotamine. Ruminal and (or) intestinal populations of E. coli O157:H7 may be influenced in livestock consuming endophyte-infected tall fescue, and these alterations could be due to the presence of ergot alkaloids in fescue plants.

Effects of different levels of ergot in concentrate on the health and performance of male calves

A number of studies dealing with the effects of ergot and ergot alkaloids on the health and performance of poultry and pigs were reported in the past, but only a few studies and field reports are available for ruminants. Therefore, a dose-response study was carried out with calves since young animals are considered to be especially sensitive to ergot.A total of 35 male Holstein calves were randomly assigned to three feeding groups after one month of feeding milk replacer. The mean initial live weight of the calves was 49.4±5.7 kg. One control group was fed an ergot-free concentrate (n=12), one group an ergot proportion of 1000 mg/kg in the concentrate (n=ll), and another group was fed a concentrate containing 5000 mg/kg ergot (n=12). Hay, grass silage and water were available forad libitum consumption, whereas the daily concentrate portion was restricted to 2 kg. Live weight, health parameters and feed intake were monitored over the experimental period of 84 days. In addition, blood samples were taken from theVena jugularis at the beginning and at the end of the experiment and analysed for ergot alkaloids and liver parameters.Total dry matter intake, live weight gain and feed-to-gain ratio were not significantly influenced by increasing ergot proportions when the whole experimental period was considered, although there was a trend for an ergot-related decrease in concentrate intake during the first 6 weeks of the experiment. After this period of time, it seemed that calves got used to the presence of ergot in the concentrate and were able to adjust their intake to the level of the control group. Moreover, health and liver parameters, such as total bilirubin, aspartate aminotransferase, glutamate dehydrogenase, gamma-glutamyl transpeptidase and creatine kinase in the serum were not significantly influenced by dietary treatments. Concentrations of the individual ergot alkaloids in serum were lower than the detection limits of the applied HPLC-method.In conclusion, it can be assumed that an ergot contamination of the concentrate up to 5000 mg/kg resulted in a transient depression of concentrate intake by the calves. However, no significant effects on health and performance could be detected when the entire test period of 84 days was considered.

Effect of ergotamine and ergonovine on thermal regulation and cardiovascular function in cattle.

Research was conducted to determine whether individual ergot alkaloids could induce signs of fescue toxicosis. Nine Angus heifers received single i.v. injections of ergotamine tartrate, ergonovine maleate, and saline vehicle in a simple cross-over design. Each heifer received a different compound each week and all treatments during the study. Physiological traits measured 15 min before and 30, 60, and 90 min after treatment were respiration rate, rectal and skin temperatures, systolic and diastolic pressures, and heart rate. Blood samples were collected 5 min before and 105 min after treatments to assess plasma prolactin concentrations. Heifers were on a fescue-free diet in drylot. Ambient temperature averaged 35 degrees C during data collection. A treatment x time interaction existed (P < .05) for respiration rate and prolactin concentrations. Ergot alkaloids altered (P < .05) all traits across time, except rectal temperature. Heifers under the influence of ergot alkaloids exhibited significantly lower skin temperature, heart rate, and prolactin and had higher respiration rate and blood pressure. Results indicated that individual ergot alkaloids administered i.v. induced signs of fescue toxicosis in cattle.

Effect of ergot alkaloids from fungal endophyte-infected grasses on fall armyworm (Spodoptera frugiperda)

Ergot alkaloids produced by endophytic fungi in the tribe Balansiae (Clavicipitaceae, Ascomycetes), which infect grasses, may provide plant defense against herbivores. This study examined the effects of six ergot alkaloids on survivorship, feeding, and growth of larvae of the fall armyworm (Spodoptera frugiperda, Lepidoptera: Noctuidae), a generalist herbivore of grasses. Corn leaf disks were soaked in solutions of individual ergot alkaloids at different concentrations and presented to neonate larvae. At the highest concentrations (77-100 mg/liter) of ergonovine, ergotamine, ergocryptine, agroclavine, and elymoclavine, larval weights and/or leaf area consumed after eight days were reduced relative to controls. Lysergol had no effect on larval weights and leaf consumption at any concentration. Although active concentrations were higher than those reported from two host grasses, in vivo levels of ergot alkaloids have not been quantified for most endophyte-infected grasses. The detrimental effects on fall armyworm observed in this study suggest that ergot alkaloids could be responsible, at least in part, for the greater insect resistance of endophyte-infected grasses.

Chemical and toxicologic studies of native corn in flour and bread

During the bread-baking process, total alkaloid content is reduced by about 50%, the pharmacologically active alkaloids (the '-in' types) being reduced to a much greater extent than the less active type ('-inin' type). Using ergotamin and ergokryptin as a basis, total alkaloid levels were calculated from the HPLC data; these results were in a good agreement with the total alkaloid content calculated from 13 individual ergot alkaloids. The changes in the alkaloid spectrum caused by pelletin are shown. In feeding experiments with growing, pregnant, and lactating mice, diets with 0%, 1% and 2% unbaked (MM) or baked (MB) rye ergot were compared for possible toxic effects on reproduction, using animals fed ad libitum or pair-fed controls. A mean ingestion of 3.6 mg of alkaloids/kg body weight per day (MM 2%) was maternally toxic and caused inhibition of conception and implantation. Feeding with 1.3 or 1.6 mg of alkaloids/kg body weight per day (MM 1% and MB 2%) during pregnancy had no significant toxic effect on the embryo or fetus, but resulted in gestation time being prolonged to about 10 h. During lactation, an intake of 2.9 and 3.0 mg of alkaloids/kg body weight per day (MM 1% and MB 2%) led to a considerable reduction in growth (up to 50%) in the young mice compared to the control animals fed ad libitum. Both the determination of total ergot alkaloid content and the feeding experiment indicate that the bread-baking process causes a reduction and consequently a detoxification of ergot alkaloids of about 50%.

Liquid Chromatographic Determination of Ergot Alkaloids in Wheat

A method is described for the determination of individual ergot alkaloids in wheat. The sample is extracted with ethyl acetate-4% ammonium hydroxide (100 + 10), and the extract is cleaned up by liquid-liquid partition. The ergot alkaloids are resolved by liquid chromatography (LC), using a porous cross-linked polystyrene-divinylbenzene resin column and a mobile phase consisting of acetonitrile-0.05 M dibasic ammonium phosphate (55 + 45) buffered at pH 10.0. The ergot alkaloids ergonovine, ergonovinine, ergotamine, ergotaminine, alpha-ergocryptine, alpha-ergocryptinine, ergocristine, and ergocristinine are separated by LC and detected with a fluorescence detector. Recovery of ergot alkaloids added to wheat at levels of 16-760 ng/g averaged 85.6% with a coefficient of variation of 11.1%.

Étude par chromatographie liquide à haute pression des alcaloïdes de l'ergot

High-performance liquid chromatography of ergot alkaloid. A method for the analysis of ergot alkaloids has been developed by high-performance liquid chromatography. A silica column was used for preparation of ergotinine and ergotoxine from ergot. Separation of the individual ergot alkaloids was successfully accomplished on a column of Hitachi Gel No. 3011-O (porous polystyrene modified by hydroxymethyl) with an eluent of n-hexane—ethanol—triethylamine (70:30:0.5, v/v). The method was applied to the analysis of α-ergocryptinine, ergocorninine, ergocristinine, α-ergocryptine, ergocornine and ergocristine in ergot and dihydroergocryptine (α + β), dihydroergocornine and dihydroergocristine in pharmaceutical tablets.