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.

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.

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.

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

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.

Alkaloid variation among epichloid endophytes of sleepygrass (Achnatherum robustum) and consequences for resistance to insect herbivores.

Epichloid endophytes are well known symbionts of many cool-season grasses that may alleviate environmental stresses for their hosts. For example, endophytes produce alkaloid compounds that may be toxic to invertebrate or vertebrate herbivores. Achnatherum robustum, commonly called sleepygrass, was aptly named due to the presence of an endophyte that causes toxic effects to livestock and wildlife. Variation in alkaloid production observed in two A. robustum populations located near Weed and Cloudcroft in the Lincoln National Forest, New Mexico, suggests two different endophyte species are present in these populations. Genetic analyses of endophyte-infected samples revealed major differences in the endophyte alkaloid genetic profiles from the two populations, which were supported with chemical analyses. The endophyte present in the Weed population was shown to produce chanoclavine I, paspaline, and terpendoles, so thus resembles the previously described Epichloë funkii. The endophyte present in the Cloudcroft population produces chanoclavineI, ergonovine, lysergic acid amide, and paspaline, and is an undescribed endophyte species. We observed very low survival rates for aphids feeding on plants infected with the Cloudcroft endophyte, while aphid survival was better on endophyte infected plants in the Weed population. This observation led to the hypothesis that the alkaloid ergonovine is responsible for aphid mortality. Direct testing of aphid survival on oat leaves supplemented with ergonovine provided supporting evidence for this hypothesis. The results of this study suggest that alkaloids produced by the Cloudcroft endophyte, specifically ergonovine, have insecticidal properties.

A systematic assessment of the variability of matrix effects in LC-MS/MS analysis of ergot alkaloids in cereals and evaluation of method robustness.

The study presents for the first time a systematic investigation of matrix effects in the LC-MS/MS analysis of ergot alkaloids in cereals. In order to assure the accuracy of the results, several approaches to minimize/eliminate matrix effects were investigated including variation of ionization techniques, chromatography and sample preparation on different grain types and grain varieties. It was revealed that the use of UPLC and careful choice of sample preparation might reduce signal suppression/enhancement. In general, ergometrine was found to be the most susceptible among the ergot alkaloids studied, but none of the used approaches suggested a total elimination of matrix effects; only less than half of its MS signal could be recovered. The late-eluting compounds were less affected by matrix components in all conditions tested. Further, the robustness of the applied LC-MS method was checked by means of a fractional factorial design. The results indicate that small changes to the sample preparation parameters, namely pH and concentration of extraction buffer, shaking time, drying temperature and extraction volumes, did not significantly (α = 0.05) affect the recoveries of ergot alkaloids.

Ergot alkaloids in rye flour determined by solid-phase cation-exchange and high-pressure liquid chromatography with fluorescence detection.

Ergot alkaloids are mycotoxins that are undesirable contaminants of cereal products, particularly rye. A method was developed employing clean-up by cation-exchange solid-phase extraction, separation by high-performance liquid chromatography under alkaline conditions and fluorescence detection. It is capable of separating and quantifying both C8-isomers of ergocornine, alpha-ergocryptine, ergocristine, ergonovine, and ergotamine. The average recovery was 61% +/- 10% with limits of detection from 0.2 to 1.1 microg kg(-1). Twenty-four unknown rye flour samples from Danish mills contained on average 46 microg kg(-1) with a maximum content of 234 microg kg(-1). The most common ergot alkaloids were ergotamine and alpha-ergocryptine including their C8-isomers. A total of 54% of the ergot alkaloids were detected as C(8)-S isomers.

Determination of ergometrine maleate by fluorescence detection.

A simple, accurate, sensitive and selective fluorescence analysis method for rapid determination of trace amounts of ergometrine maleate has been developed. The method is based on the fluorescence of ergometrine maleate. The calibration graph was linear in the range of 1 x 10(-4) to 0.2 microg/mL and the detection limit was 4 x 10(-5) microg/mL, with a relative standard deviation of 0.32% at 0.05 microg/mL (n = 11) ergometrine maleate. The influence of foreign compounds was tested. The proposed method was applied successfully to the determination of ergometrine maleate in pharmaceutical preparations and human plasma.

Fragmentation patterns of selected ergot alkaloids by electrospray ionization tandem quadrupole mass spectrometry.

Tall fescue toxicosis and other maladies in livestock result from the ingestion of vasoconstrictive ergot alkaloids produced by fungal endophytes associated symbiotically with the grass. In order to facilitate future analyses of grass extracts considered responsible for outbreak of related livestock diseases, we examined the electrospray ionization mass spectra of specific ergot alkaloids under conditions that permit protonation. Our purposes were both to record the spectra with interpretation of mechanisms of fragmentation and to derive commonalities that would allow the prediction of mass spectra of related compounds for which standards were not readily available. With [M + H](+) values in parentheses, water-insoluble lysergic acid peptide ergot derivatives ergovaline (m/z 534), ergotamine (m/z 582), ergocornine (m/z 562), ergocryptine (m/z 576) and ergocrystine (m/z 610) exhibited a consistent loss of water (-18 u) from the C-12' alpha-hydroxy functionality. Of this group, ergovaline and ergotamine generated an m/z 320 fragment deriving from cleavage of ring E amide and ether functions with retention of the peptide ring system methyl group. Ergocornine, ergocryptine and ergocrystine similarly formed an m/z 348 fragment with retention of isopropyl. These assignments were supported by the lack of similar fragments from the water-soluble ergot ergonovine, which lacks a peptide ring system. Clavine-type ergot alkaloids lysergic acid and lysergol lack any substituents beyond simple ones directly on the C-8 position and, similarly to ergonovine, lack significant fragments at m/z 268, 251 and 225 shared by the peptide ergot alkaloids.

A comparison of high performance liquid chromatographic assays with the current pharmacopoeial assays for the combined formulation of ergometrine and oxytocin.

This paper reports the comparison of recently-developed chromatographic assays (using high performance liquid chromatography) with the present British Pharmacopoeial methods for the combined formulation of ergometrine and oxytocin. The estimates using high performance liquid chromatography were in good agreement with both the Pharmacopoeial determinations and nominal contents. The variability of estimates for ergometrine was similar for both procedures, whereas the variability of estimates of oxytocin content by chromatographic assay was significantly less than the variability of estimates by the Pharmacopoeial bioassay.

Variability in the content and composition of alkaloids found in Canadian ergot. II. Wheat.

The total alkaloid content and individual alkaloid composition were determined by colorimetry and high performance liquid chromatography, respectively, for Canadian wheat ergot sclerotia. The total alkaloid content was highly variable between individual sclerotia from the same or different sources and ranged from 0.013 to 0.307%; the overall average for bulked samples from 75 sites was 0.163%. Ergocristine and its isomer ergocristinine were the major constituents (approximately 46%). Other alkaloid pairs observed were ergotamine (approximately 17%), ergocryptine (approximately 12%), ergocornine (approximately 11%), ergometrine (approximately 7%), and ergosine (approximately 5%), plus about 2% unidentified alkaloids.

Fluoremetric determination of methylergonovine maleate and ergonovine maleate in tablets and injections.

A column cleanup, followed by a shakeout and fluorometric determination, has been developed to determine methylergonovine maleate and ergonovine maleate in tablets and injections. The ether eluate is extracted with tartaric acid solution. An aliquot is diluted with sodium tartrate solution, and this solution is excited at 325 nm. The resulting fluorescence is measured at 432 nm. Results obtained using this method compare favorably with those from the official NF XIV procedure and a semiautomated procedure. Recoveries of 100 and 99% were obtained from one sample each of spiked excipient tablets of methylergonovine maleate and ergonovine maleate, respectively.

Semiautomated analysis of ergonovine maleate or methylergonovine maleate tablets and injections by colorimetric or fluorometric systems.

Two semiautomated analytical systems have been developed to determine ergonovine and methylergonovine maleate in single tablets and injections. The active ingredient is dissolved in a tartrate buffer. In the fluorometric method the stream is automatically diluted with tartrate buffer and excited with ultraviolet light; the resulting fluorescence is measured and recorded. In the colorimetric method, the stream is automatically made basic with sodium hydroxide and extracted with n-butanol. The extract is mixed with p-dimethylaminobenzaldehyde reagent. After reaction, the solution is mixed with sodium nitrite solution, and the developed color is measured at 550 nm. Recoveries of 100% were obtained from spiked placebos. Standard deviations for powdered tablet and injection samples ranged from 0.63 to 1.24%. Comparisons with the USP XVIII methods are presented.