Effects of initial and extended exposure to an endophyte-infected tall fescue seed diet on faecal and urinary excretion of ergovaline and lysergic acid in mature geldings.

AIM: To determine the amount of ergovaline and lysergic acid retained or excreted by geldings fed endophyte-infected seed containing known concentrations of these alkaloids, and the effects of exposure time on clinical expression of toxicosis. METHODS: Mature geldings (n=10) received diets containing either endophyte-free (E-) or endophyte-infected (E+) tall fescue seed during three experimental phases. The first phase (Days -14 to -1) was an adaptation phase, to allow all horses to adapt to a diet containing E- tall fescue seed. The second (Days 0 to 3) was the initial exposure phase to E+ tall fescue seed, used for the delivery of ergovaline and lysergic acid at 0.5 and 0.3 mg/kg of diet, respectively, to test the initial effects of exposure on routes and amounts of elimination of alkaloid. During this phase, half the geldings were exposed to an E+ diet while the rest served as controls by remaining on the E- diet. Once assigned to treatments, geldings remained on the same diet through the third phase (Days 4 to 21), which served as the extended exposure phase. Total outputs of faeces and urine were collected within each phase, to determine retention of ergovaline and lysergic acid and nutrient digestibility. Serum was collected weekly and analysed for activities of enzymes and concentrations of prolactin. Bodyweights (BW) and rectal temperatures were recorded weekly. RESULTS: BW, rectal temperature, enzyme activities and concentrations of prolactin in serum, and nutrient digestibility were not affected by treatment. Total intake of ergovaline by geldings on the E+ diet was 3.5 and 3.6 (SE 0.20) mg/day, and 2.1 and 2.3 (SE 0.11) mg/day were not accounted for in initial and extended phases, respectively. Lysergic acid was excreted in the urine (4.0 and 4.9 (SE 0.97) mg/day) and faeces (2.5 and 2.7 (SE 0.35) mg/day) at greater amounts than that consumed (2.0 and 1.9 (SE 0.09) mg/day) during the initial and extended exposure phases, respectively. Animals exposed to E+ seed for a period of 20 days appeared to excrete more (1.5 vs 1.2 mg/day; SE 0.08; p=0.03) ergovaline in the faeces than those exposed for only 4 days. CONCLUSIONS: Exposure time to the ergot alkaloids had a limited effect on the route of elimination or the amounts of ergovaline or lysergic acid excreted by horses. The primary alkaloid excreted was lysergic acid, and urine was the major route of elimination. These data will aid future research to improve animals' tolerance to toxic endophyte-infected tall fescue.

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.

Determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite, in human urine by high performance liquid chromatography.

A specific method for the determination of 10 alpha-methoxy-9,10-dihydrolysergol, a nicergoline metabolite (metabolite 2), in urine is described. Metabolite 2 was well separated from the urine components on a reversed phase column, Hypersil ODS 5 microns, using an acetonitrile:pH 3.5 phosphate buffer (40:60, v/v) as the mobile phase at a flow rate of 1 mL/min. UV detection was set up at 220 nm. After addition of a known amount of lysergamide as the internal standard, the compounds were extracted from alkalysed urine on a pre-packed glass column (Extrelut 1) with dichloromethane. With 0.5 mL urine, concentrations down to 0.56 mumol/L could be determined.

Liquid chromatography-mass spectrometry in the pharmaceutical industry: objectives and needs.

The role of liquid chromatography-mass spectrometry (LC-MS) in the analysis of drugs is discussed. The main fields of application are thermally labile compounds, compounds with low volatility and compounds with rather high molecular weights, all of which are not generally suitable for analysis by combined gas chromatography-mass spectrometry. The objectives, needs, limitations and abilities of LC-MS for the analysis of by-products, degradation products, traces of drug substances for pharmacokinetic studies and metabolites in complex matrices are presented. The LC-MS coupling is discussed as a sophisticated LC detector for sensitive and selective quantitative determinations or as an on-line sample-introduction system for the mass spectrometer to obtain structural information for identification or structural elucidation. LC-MS combined with a flow-switching technique can be used for the analysis of mixtures containing large amounts of components which otherwise would be detrimental to the LC-MS technique. With flow-injection techniques the LC-MS interface is used as a sample-introduction system with possibilities for sample preparation, sample clean-up and chemical derivatization.