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ARS Home » Midwest Area » Lexington, Kentucky » Forage-animal Production Research » Research » Publications at this Location » Publication #210389

Title: Separation and Quantitation of Seven Ergot Alkaloids in Bovine Vein Tissue

item Klotz, James

Submitted to: Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics
Publication Type: Abstract Only
Publication Acceptance Date: 3/21/2007
Publication Date: 6/3/2007
Citation: Shafer, W.D., Smith, D.L., Smith, L.L., Klotz, J.L., Strickland, J.R. 2007. Separation and Quantitation of Seven Ergot Alkaloids in Bovine Vein Tissue. Proceedings of the ASMS Conference on Mass Spectrometry and Allied Topics, June 3-7, 2007. #ThPh-118.

Interpretive Summary:

Technical Abstract: Ergot alkaloid related toxicosis has been documented in forage animals for over thirty years, but few mass spectrometry analytical methods for the detection and quantitation of these compounds exist. A primary ergot alkaloid of interest was ergovaline, the predominant toxicant in endophyte-infected fescue. Therefore, extraction protocols and LC-MS quantitative methods were developed for ergovaline in vein tissue. The vein tissue was previously exposed to known concentrations of ergovaline in vitro. An additional preliminary method for the simultaneous separation, detection, and quantitation of six other naturally occurring ergot alkaloids (and associated epimers) in solution was developed using LC-MS. The development of these methods is vital to screen for ergot alkaloids levels and determine thresholds associated with the onset of toxicosis. Vein tissues obtained from healthy mixed breed and gender cattle (cranial branch of the lateral saphenous vein) ten minutes post mortem were exposed to a known concentration of ergovaline in vitro and washed. Subsequent methanolic extraction of vein segments was performed using 2-bromo-ergocryptine as an internal standard. Reverse-phase LC-MS (Varian 1200L) and selected ion monitoring of protonated precursor ions were used to generate standard addition calibration curves for ergovaline quantitation. A solution of seven ergot alkaloids (and 2-bromo-ergocryptine) was also prepared at known concentrations in 5% acetonitrile / 95% water with 0.1% formic acid and analyzed using SIM conditions. Calibration curves for each component in the mixture were then produced. Two different methods were developed: i) quantify ergovaline from forage animal veins using standard addition and ii) separation, identification, and quantification of multiple ergots and their epimers from a solution. i) Vein tissue originating from seven different animals was exposed to the same concentration of ergovaline (10-4 M) in vitro, washed with buffer, spiked with internal standard, and then extracted. The extract was separated into equal volumes and subjected to standard addition analysis (one blank sample followed by a series of known concentrations of ergovaline). A standard addition calibration curve was generated and the quantity of the ergovaline sequestered in vein samples was determined by extrapolation. Linear correlation coefficients for standard addition curves were 0.99. The average amount of ergovaline in the exposed veins was 3.98 + 1.40 ng / mg vein (on a dry matter basis). Additional sample clean up is required to eliminate matrix effects (ion suppression in the electrospray ionization process) and improve precision. Also, use of a calibration curve obtained daily might then be used to reduce analysis time. ii) Since ergovaline may not be the only ergot alkaloid involved in toxicosis, the separation and quantitation of ergonovine ([M+H]+ 326), lysergic acid ([M+H]+ 269), ergotamine ([M+H]+ 583), ergocryptine ([M+H]+ 576), ergocrystine ([M+H]+ 610), ergocornine ([M+H]+ 561) along with associated epimers in solution in addition to ergovaline ([M+H]+ 534) was achieved. Using the sample / internal standard peak area ratio versus concentration, calibration curves for all the ergot alkaloids in solution gave linear correlation coefficients greater than 0.990 (excluding lysergic acid). A wider range of correlation coefficients for lysergic acid calibration curves (0.979 – 0.990 over time) was observed and suggests chemical instability in solution. Tandem analysis was performed to verify the identity of each ergot alkaloid and its associated epimer.