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

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 µg/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 µg/kg).

Ruminal tryptophan-utilizing bacteria degrade ergovaline from tall fescue seed extract.

The objectives of this study were to evaluate degradation of ergovaline in a tall fescue [ (Schreb.) Darbysh.] seed extract by rumen microbiota ex vivo and to identify specific bacteria capable of ergovaline degradation in vitro. Rumen cell suspensions were prepared by harvesting rumen fluid from fistulated wether goats ( = 3), straining, and differential centrifugation. Suspensions were dispensed into anaerobic tubes with added Trypticase with or without extract (∼10 µg kg ergovaline). Suspensions were incubated for 48 h at 39°C. Samples were collected at 0, 24, and 48 h for ergovaline analysis and enumeration of hyper-ammonia producing (HAB) and tryptophan-utilizing bacteria. Ergovaline values were analyzed by repeated measures using the mixed procedure of SAS. Enumeration data were log transformed for statistical analysis. When suspensions were incubated with extract, 11 to 15% of ergovaline disappearance was observed over 48 h ( = 0.02). After 24 h, suspensions with added extract had 10-fold less HAB than controls ( = 0.04), but treatments were similar by 48 h ( = 1.00). However, after 24 h and 48 h, suspensions with extract had 10-fold more tryptophan-utilizing bacteria ( < 0.01) that were later isolated and identified by their 16S RNA gene sequence as . The isolates and other known rumen pure cultures ( JB1, B159, HD4, B, F, MD1, SR) were evaluated for the ability to degrade ergovaline in vitro. Pure culture cell suspensions were incubated as described above and samples were taken at 0 and 48 h for ergovaline analysis. Data were analyzed using the ANOVA procedure of SAS. All HAB, including the isolates, tested degraded ergovaline (54 to 75%; < 0.05). B14 was also able to degrade ergovaline but to a lesser capacity (12%; < 0.05), but all other bacteria tested did not degrade ergovaline. The results of this study indicate which rumen bacteria may play an important role in ergovaline degradation and that microbiological strategies for controlling their activity could have ramifications for fescue toxicosis and other forms of ergotism in ruminants.

Toxicity of endophyte-infected ryegrass hay containing high ergovaline level in lactating ewes.

The symbiotic association of var. (formerly named ) with perennial ryegrass () leads to the production of ergovaline (EV) and lolitrem B (LB) that are toxic for livestock. The objectives of this study were to determine the effects of feeding endophyte-infected ryegrass (SE+) hay on 16 lactating ewes (BW 80 ± 10 kg) in comparison with endophyte-free ryegrass (SE-) hay to investigate the putative mechanisms of action of EV and LB and to evaluate their persistence in milk and animal tissues. The mean EV and LB concentrations in SE+ hay were 851 and 884 µg/kg DM, respectively, whereas these alkaloids were below the limit of detection in SE- hay. No effect of SE+ was observed on animal health and skin temperature whereas prolactin decreased and significant differences between hays were observed from d 7 to 28 of the study ( < 0.03) but had no effect on milk production. Hematocrit and biochemical analyses of plasma revealed no significant difference between SE+ and SE-, whereas cortisol concentration differed significantly on d 28 ( = 0.001). Measurement of oxidative damage and antioxidant enzyme activities in plasma, liver, and kidneys revealed a slight increase in some enzyme activities involved in defense against oxidative damage in the SE+ fed ewes. Slight variations in the activities of hepatic and kidney flavin monooxygenase enzymes were observed, whereas in the kidney, glutathione -transferase activity decreased significantly ( = 0.002) in the SE+ fed ewes, whereas uridine diphosphate glucuronosyltransferase activity increased ( = 0.001). After 28 d of exposure of ewes to the SE+ hay, low EV and LB concentrations were measured in tissues. The highest concentration of EV was observed in the liver (0.68 µg/kg) whereas fat contained the highest concentration of LB (2.39 µg/kg). Both toxins were also identified at the trace level in milk.

Alteration of fasting heat production during fescue toxicosis in Holstein steers.

This study was designed to examine alteration of fasting heat production (FHP) during fescue toxicosis. Six ruminally cannulated Holstein steers (BW = 348 ± 13 kg) were BW-matched into pairs and used in a 2 period crossover design experiment. Each period consisted of 2 temperature segments, one each at 22 and 30°C. During each period, 1 steer per pair was ruminally dosed twice daily with 0.5 kg of ground endophyte-infected fescue seed (E+) and the other with ground endophyte-free fescue seed (E-) for 7 d. Steers on E- treatment were pair-fed to E+ steers offered alfalfa cubes at 1.5 × NEm. On d 8 of each segment, steers were moved to individual metabolism stalls fitted with indirect calorimetry head boxes. Ruminal contents were removed, weighed, and subsampled for DM determinations. The reticulorumen was washed and filled with a buffer (NaCl = 96; NaHCO3 = 24; KHCO3 = 30; K2HPO4 = 2; CaCl2 = 1.5; MgCl2 = 1.5 mmol·kg buffer(-1)) that was gassed with a 75% N2 and 25% CO2 mixture before rumen incubation. During buffer incubation, an E+ or E- fescue seed extract was added at 12 h intervals to maintain treatment presentation to the animal. After a 12-h wait, heart rate, O2 consumption, CO2 production, and urinary output were recorded for 16 h. There was no difference (P = 0.931) in DMI/kg(0.75) between endophyte treatments by design; however, intake decreased (P = 0.004) at 30°C. Increased temperature had no effect (P > 0.10) on other measurements and there were no significant interactions (P > 0.11) of temperature and endophyte treatment. Heart rate was unaffected by fescue treatment or environmental temperature. Percent DM of ruminal contents as well as total rumen DM/kg(0.75) was increased (P < 0.0001) in E+ steers. Respiratory quotient was elevated (P = 0.02) in E+ steers. Oxygen consumption decreased (P = 0.04) and CO2 production tended to be reduced (P = 0.07) during E+ treatment. Calculated FHP (kcal/kg BW(0.75)) was also less (P = 0.006) in steers receiving E+ treatment. These data suggest that consumption of endophyte-infected tall fescue by cattle results in a reduction in basal metabolic rate.

Ergot alkaloid from the sea slug Pleurobranchus forskalii.

The sea slug Pleurobranchus forskalii is a carnivorous scavenger that is widely distributed in shallow subtidal areas. Very few investigations of the chemical components of this gastropod have been reported. In this study we performed a comprehensive analysis of an extract of the marine mollusc, P. forskalii, collected off Ishigaki Island, Japan. As a result, an alkaloid was isolated from the chloroform extract. Remarkably, the structure elucidation based on the spectral data revealed that it was an ergot alkaloid peptide, ergosinine. Various ergot alkaloids have previously been isolated mainly from terrestrial higher plants or fungi. This is the first report of the isolation of an ergopeptine from marine life, and thus the known geographical extent of ergot alkaloids now includes both terrestrial and aquatic organisms.

Evaluation of a ruminally dosed tall fescue seed extract as a model for fescue toxicosis in steers.

Tall fescue (Lolium arundinaceum) toxicosis research is often complicated by a reduction in intake of infected forage or seed, making treatment comparisons difficult. This study was conducted to develop a fescue toxicosis model that would allow for variations in DMI without altering the quantity of alkaloids consumed over the course of the experiment. Ground tall fescue seed and a tall fescue seed extract were used in two 2-period crossover experiments to determine the effectiveness of ruminal dosing of a tall fescue seed extract to induce fescue toxicosis. This experiment used 4 growing Holstein steers (BW = 337 ± 24 kg) surgically fitted with ruminal cannulas. Steers were maintained on a diet of endophyte-free fescue hay fed ad libitum throughout the experiment. Endophyte-infected (E+; 4.1 mg/kg of ergovaline) and uninfected (E-; 0.0 mg/kg of ergovaline) KY-31 tall fescue seed was ground and dosed or extracted with ethanol, concentrated, and lyophilized before ruminal dosing. Ergovaline concentration of the final extract was 102 mg/kg. Animals were given a minimum of a 3-wk washout period between treatments. Physiological indicators were measured over 7 d at 22°C (d 1 to 3) and 32°C (d 4 to 7) during both seed and extract dosing. Seed and extract E+ dosing reduced serum prolactin concentrations such that they were not different from zero (P < 0.10). Treatment with E+ reduced feed intake (P < 0.05) and heart rate (P < 0.001), and increased respiration rate (P < 0.01) and core temperature (P < 0.05) during both seed and extract dosing. Increasing environmental temperature from 22 to 32°C reduced total intake (P < 0.05) and increased core temperature (P < 0.001) and respiration rate (P < 0.001) during both seed and extract dosing. Diastolic blood pressure tended (P < 0.09) to be increased during E+ extract dosing and reduced during heat stress. These physiological alterations are consistent with those reported for cattle grazing or consuming seed from endophyte-infected tall fescue. These data indicate that a ruminally dosed ethanol extract of tall fescue seed is efficacious in inducing fescue toxicosis in cattle.

Constriction of bovine vasculature caused by endophyte-infected tall fescue seed extract is similar to pure ergovaline.

Ergovaline has been extensively used to study vasoactive effects of endophyte- (Neotyphodium coenophialum) infected tall fescue (Lolium arundinaceum). However, initial results indicated that an extract of toxic tall fescue seed (E+EXT) is more potent than ergovaline alone in a right ruminal artery and vein bioassay. The E+EXT induced a greater contractile response than an equal concentration of ergovaline alone in the ruminal artery of heifers (P = 0.018). This led to a hypothesis that other compounds in the seed extract contribute to vasoconstriction. Thus, experiments were conducted to determine if vasoactivity of an E+EXT is different from a mixture of ergot alkaloids (ALK; ergovaline, ergotamine, ergocristine, ergocryptine, ergocornine, ergonovine, and lysergic acid) of similar concentrations and to determine if the vasoactivity of an E+EXT differs from an endophyte-free tall fescue seed extract (E-EXT). Segments of lateral saphenous vein and right ruminal artery and vein were collected from Holstein steers (n = 6) shortly after slaughter. Vessels were cleaned of excess connective tissue and fat and sliced into segments that were suspended in a multimyograph chamber with 5 mL of continually oxygenated Krebs-Henseleit buffer, equilibrated for 90 min, and exposed to a reference compound (120 mM KCl for ruminal vessels and 0.1 mM norepinephrine for saphenous vein). Increasing concentrations of each treatment (E+EXT, E-EXT, ALK, and ergovaline) were added to the respective chamber every 15 min after buffer replacement. Data were normalized as a percentage of maximal contractile response of the reference compound and fit to a sigmoidal concentration response curve. Ergovaline, ALK, and E+EXT induced similar responses in the saphenous vein, ruminal artery, and ruminal vein. The E+EXT displayed a smaller EC(50) (half maximal effective concentration) than ergovaline or ALK in the saphenous vein and ruminal vein (P < 0.008), but not the ruminal artery (P = 0.31). Extrapolated maximum response was greatest in the saphenous vein for ergovaline, least for E+EXT, and intermediate for ALK (P < 0.0001). The E-EXT did not induce a contractile response in any vessel tested (P > 0.1). Data from this study indicate that ergovaline is largely responsible for the locally induced vasoconstriction of bovine vasculature observed with endophyte-infected tall fescue.

Biotin ergopeptide probes for dopamine receptors.

The incorporation of chemical modifications into the structure of bioactive compounds is often difficult because the biological properties of the new molecules must be retained with respect to the native ligand. Ergopeptides, with their high affinities at D(1) and D(2) dopamine receptors, are particularly complex examples. Here, we report the systematic derivatization of two ergopeptides with different peptide-based spacers and their evaluation by radioligand binding assays. Selected spacer-containing ergopeptides with minimal biological alteration and a proper anchoring point were further derivatized with a biotin reporter. Detailed characterization studies identified 13 as a biotin ergopeptide maintaining high affinity and agonist behavior at dopamine receptors, being a useful tool for the study of heteromers involving D(1)R, D(2)R, or D(3)R.

Physiological and digestive effects of Neotyphodium coenophialum-infected tall fescue fed to lambs.

The digestive responses and degradation of ergovaline and production of lysergic acid in the rumen of sheep offered Neotyphodium coenophialum-infected tall fescue straw at 2 ergovaline levels were investigated. Six crossbred wethers (56 +/- 3.0 kg of BW) were used in a randomized crossover design involving 2 treatments, for a total of 6 observations per treatment. The experiment consisted of two 28-d feeding periods with a 14-d washout period between them. The treatments were 1) tall fescue straw containing <0.010 mg of ergovaline/kg (E-), and 2) tall fescue straw containing 0.610 mg of ergovaline/kg (E+). Feed, orts, and feces were measured and analyzed for DM, ADF, and CP, and used to determine digestibilities. Feed and water intake were monitored throughout the feeding periods. Body weight and serum prolactin levels were measured at the beginning and end of each feeding period. Ruminal fluid was sampled 3 times (d 0, 3, and 28) during each 28-d feeding period for determination of ergovaline, lysergic acid, ammonia, and pH. Samples were collected before feeding (0 h) and at 6 and 12 h after feeding. Total fecal and urine collection commenced on d 21 and continued until d 25 of each feeding period. Ruminal ammonia, ruminal pH, and rectal temperature were not influenced by ergovaline concentration (P > 0.10). Digestion of DM, ADF, and CP was not different between treatments (P > 0.10). Daily water intake was less for the E+ diet (2.95 vs. 2.77 L/d; P < 0.05) as was serum prolactin (22.9 vs. 6.4 ng/mL; P < 0.05). Ergovaline concentration in ruminal fluid increased over sampling days at each sampling time (P < 0.05). Lysergic acid concentration in ruminal fluid increased over time from d 0 to 3 (P < 0.05) but was not different between d 3 and 28 (P > 0.10). In the E+ treatment, ergovaline was not detectable in the urine, whereas the concentration in the feces was 0.480 mg/kg. Lysergic acid was detected in the diet of the E+ treatment at 0.041 g/kg, lysergic acid in the urine was 0.067 mg/kg and in the feces was 0.102 mg/kg. The apparent digestibility of the alkaloids was 64.2% for ergovaline and -12.5% for lysergic acid. Approximately 35% of dietary ergovaline and 248% of dietary lysergic acid were recovered in the feces and urine. The appearance of lysergic acid in the feces, urine, and ruminal fluid is likely due to microbial degradation of ergovaline in the rumen and further breakdown in the lower digestive tract.

Detection of lysergic acid in ruminal fluid, urine, and in endophyte-infected tall fescue using high-performance liquid chromatography.

Ergot alkaloids present in endophyte-infected tall fescue induce fescue toxicosis in livestock consuming the plant. The lysergic acid (LA) ring structure is a common moiety among the ergot alkaloids. Little is known about the bioavailability of LA because of limitations in available analytical protocols. Thus, a high-performance liquid chromatography procedure was developed to analyze biological matrices for LA. The biological matrices of interest were tall fescue straw and seed, and ruminant feces, urine, and ruminal fluid. Lysergic acid was added to each matrix at a high (150 ng/ml) or low (30 ng/ml) level. Using the high-level addition, the greatest recovery of LA was obtained from ruminal fluid, feces, and urine (P < 0.05), with an average 85.1% recovered. At the low level, a greater recovery of added LA was observed in the ruminal fluid, urine, and feces (82.1%; P < 0.05) than that in the other 2 matrices (62.6%). The limit of quantitation (LOQ) in ruminal fluid and urine was 5.5 and 18.4 ng/ml, respectively. Seed, straw, and feces had higher LOQ (24.2, 14.5, and 36.0 ng/g, respectively). Limit of detection (LOD) was 1.64, 10.80, 4.35, 5.52, and 7.26 ng/g for ruminal fluid, feces, urine, seed, and straw, respectively. To test the assay in vivo, samples of ruminal fluid and urine were collected from steers consuming a diet containing 400 ng of ergovaline/g and 30 ng of LA/g. All matrices sampled resulted in levels above the LOD and LOQ for the assay, indicating that this assay is sufficiently sensitive for use in assessing the bioavailability of LA.

Norleucine, a natural occurrence in a novel ergot alkaloid gamma-ergokryptinine.

A novel natural peptide ergot alkaloid gamma-ergokryptinine containing norleucine has been isolated from ergot sclerotia of the field-growing parasitic fungus Claviceps purpurea CCM 8059. Its structure was deduced from the NMR and mass spectral data. The final structural proof was provided by the crystal structure determination, which is the first X-ray structure of a natural Nle-containing secondary metabolite. The conformations of three ergopeptinines: gamma-ergokryptinine, ergoladinine, and alpha-ergokryptinine were compared.

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.

Ergovaline toxicity on Caco-2 cells as assessed by MTT, alamarBlue, and DNA assays.

The exact mechanisms of fescue toxicity in animals have yet to be established, but it has been associated with an inability to thrive. Ergovaline is the major ergopeptine alkaloid associated with fungal infections of tall fescue. Gastrointestinal (GI) toxicity of ergovaline (10(-11) to 10(-4) M) was evaluated in Caco-2 cells (mimicking the GI epithelium) beginning on days 1, 8, and 18 of culture. Acute and chronic toxicity was assessed after 24 and 72 h of exposure. Treatment periods were chosen to study undifferentiated, semidifferentiated, and completely differentiated cells. Cell loss and metabolic activity were assessed by thiazolyl blue reduction (3-(4,5-dimethylthiozole-2-yl)-2,5,-biphenyl tetrazolium bromide [MTT], mitochondrial succinate dehyrdogenase activity), alamarBlue assay (cytochrome oxidase activity), and deoxyribonucleic acid (DNA) quantitation. Undifferentiated cells were sensitive to 1 x 10(-4) M ergovaline after acute exposure (from 52 to 74% of control values depending on assay). After 72 h of exposure to 1 x 10(-4) M ergovaline, in all three assays, treatment means were reduced to approximately 10% of the control means. By day 11 in culture, ergovaline toxicity to cells had decreased. With 24 h exposure, an apparent paradoxical increase in MTT was seen at some concentrations. This increase in MTT was also found in fully differentiated cells (day 21), whereas alamarBlue activity decreased. No change in DNA was found until 72 h of exposure, when DNA was reduced approximately 12% over most concentrations. These findings indicate differentiation state-dependent sensitivity of Caco-2 cells to ergovaline, potential problems of the MTT assay as an indicator of cellular toxicity, and usefulness of alamarBlue assay over DNA assay for toxicity assessment.

Antagonistic effects of simultaneous exposure of ergot alkaloids on kidney adenosine triphosphatase system.

Much of the research on fescue toxicosis has concentrated on evaluating animal response to grazing endophyte-infected (E+) versus endophyte-free tall fescue or the effects of single toxins such as ergonovine (EN), ergovaline (EV), or ergotamine (ET) on animal performance. Such approaches have eliminated the opportunity to test the possible additive, synergistic, or antagonistic interactions of one or more ergot alkaloids with the other ergot alkaloids found in E+ tall fescue. This study was conducted to determine the effects of simultaneous exposure of pairs of EN, EV, and ET on the kidney adenosine triphosphatase (ATPase) system in vitro. Tests were performed using three separate rat kidney homogenates and were repeated four times at concentrations of 0, 75, and 200 microM. Individually, EN, EV, and ET induced dose-dependent inhibitions of kidney Na(+)/K(+) ATPase, with EN being most potent, followed by purified EV, and then by ET. The ergot alkaloids inhibited Mg(2+) ATPase to a lesser degree than Na(+)/K(+) ATPase, with EN again being the most potent toxin. Simultaneous exposure to any combination of the ergot alkaloid pairs tested (EV + ET, EV + EN, and ET + EN) resulted in significant interactions (P < 0.05), indicating antagonistic effects on the inhibition of Na(+)/K(+) ATPase and Mg(2+) ATPase for most concentration combinations. These interactions suggest that in studies of the effects of any ergot alkaloid on animal performance, effects of other ergot alkaloids may also be present. Effects may not be additive, as was the case in this study, and the presence of one toxin may enhance or hinder the effectiveness of others.

Technical note: epimerization of ergopeptine alkaloids in organic and aqueous solvents.

Purified ergopeptine alkaloids are often used in studies related to tall fescue toxicosis without regard to epimerization that occurs when ergopeptines are solvated. The objectives of this study were to measure the rates of alpha-ergocryptine epimerization to alpha-ergocryptinine at room temperature and at -40 degrees C, and to measure the rate of ergovaline epimerization to ergovalinine at 37 degrees C. Alpha-ergocryptine tartrate was stable (< 0.5% epimerization) in protic or aprotic solvents when stored at -40 degrees C for 20 to 52 d. At room temperature, alpha-ergocryptine epimerization in chloroform did not occur; epimerization was modest in acetone and acetonitrile (< 5%) but was substantial in methanol (78% by 38 d) and in a 70:30 water methanol mix (47% by 42 d). Ergovaline epimerization to ergovalinine occurred at 37 degrees C in 0.1 M phosphate buffers (pH 3, 7.5, and 9) in 9% aqueous solutions of fetal bovine serum (FBS), and in water, methanol, and acetonitrile. The degree of epimerization at 37 degrees C was solvent-dependent. Epimerization rates with respect to time were roughly linear in phosphate buffer (pH 3 only), water, methanol, and acetonitrile; epimerization rates resembled first-order kinetics in phosphate buffers (pH 7.5 and 9) and in the presence of FBS (pH 3, 7.5 and in Dulbecco's culture media). Epimerization equilibria (48 to 63% ergovaline) were reached within approximately 1 to 19 h. Results from this study indicate that researchers conducting studies with purified ergopeptines should carefully control the storage conditions of solvated ergopeptines and measure isomeric composition under the actual experimental conditions used in experiments.

Analysis of endophyte toxins: fescue and other grasses toxic to livestock.

Research on livestock toxicoses caused by Acremonium (endophyte)-infected grasses strongly implicate the ergopeptine alkaloids with A. coenophialum-infected fescue and paxilline and the lolitrem alkaloids with A. lolii-infected perennial ryegrass as the causative agents. Isolation, identification, and detection of these toxins involves extraction with appropriate solvents, clean-up procedures, and chromatographic methods with known standards. Thin-layer, high-performance liquid and gas chromatography along with ultraviolet and mass spectrometric (i.e., electron impact, chemical ionization, tandem mass) characterizations have been reported. These methods have varying degrees of success depending on the matrix from which the alkaloids have been extracted. Ergovaline is the primary ergopeptine alkaloid isolated from cultures of A. coenophialum and also from infected fescue grass and seeds toxic to livestock. Other compounds isolated from the endophyte-infected fescue include: lysergic acid amide (ergine), the clavine class of ergot alkaloids (chanoclavine I, agroclavine, elymoclavine, penniclavine), the pyrrolizidine alkaloids (N-formylloline, N-acetylloline, N-methyloline, N-acetylnorloline), and the unique pyrrolopyrazine alkaloid peramine. The loline alkaloids and peramine have been more associated with the insect-deterrent properties of the endophyte-infected fescue than with livestock toxicoses. Also, both peramine and the ergopeptine alkaloids (ergovaline, ergotamine) have been isolated from A. lolii-infected perennial ryegrass. More recently, paxilline and lolitrem B have been detected in laboratory cultures of A. coenophialum isolated from tall fescue. The ergot alkaloids in endophyte-infected perennial ryegrass may be more related to decreased animal productivity (weight gains, reproduction problems), whereas the lolitrems cause the staggers syndrome. The detection, isolation, identification, and analyses of these compounds from Acremonium-infected grasses is presented.

Mass spectrometry as an aid to the identification of ergots and dihydroergots: comparison of hard and soft ionization techniques.

An analysis of the 70 eV electron impact (EI) and fast atom bombardment (FAB) mass spectral features of a variety of ergoline and dihydroergoline derivatives of therapeutic importance is presented with emphasis upon analytical utility. Derivatives which carry non-peptide based C-8 substituents are fully characterized by EI-MS through provision of molecular wieght evidence and fragment ions diagnostic of both the ergoline skeleton and the C-8 substituent. Peptidic ergolines and dihydroergolines are poorly characterized by EI-MS, but their FAB-MS clearly reveal [M + 1]+ (high intensity) and [M - 1]- (high to low intensity) ions in positive and negative ion spectra, respectively. Negative FAB spectra of salts also display diagnostic anion-base conjugate ions.