|Smith, Darren - EASTERN KENTUCKY UNIVERSI|
|Shafer, W - EASTERN KENTUCKY UNIVERSI|
Submitted to: American Society for Mass Spectrometry
Publication Type: Other
Publication Acceptance Date: April 15, 2006
Publication Date: May 31, 2006
Citation: Smith, D.L., Shafer, W.D., Smith, L.L., Klotz, J.L., Strickland, J.R. 2006. Monitoring the presence of ergot alkaloids in forage animal samples. American Society for Mass Spectrometry. Proceedings of the 54th ASMS Conference. Technical Abstract: Novel Aspect Initial liquid chromatography tandem mass spectrometry method development for metabolite profiling of ergot alkaloids consumed by forage animals Introduction The presence of ergot alkaloids in forages has been reported to produce acute toxicity when consumed by forage animals. A method was developed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to verify the presence of certain ergot alkaloids after in vitro dose-response constriction studies with bovine tissue. The developed LC-MS/MS method for the ergot alkaloids is then used for determining the presence of intact ergot alkaloids along with apparent metabolites in biological samples (i.e. urine, blood, fecal material) from forage animals. Methods For in vitro constriction studies, tissues were obtained from healthy mixed breed and gender cattle (cranial branch of the lateral saphenous vein) and treated. Detection of ergot alkaloids was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS) using a Varian 1200L (Varian Inc., Walnut Creek, CA). Data collection was done in positive ion and single ion monitoring was utilized for ergot alkaloid precursor ions. For in vitro studies, no sample clean-up or pre-concentration of the samples occurred before injection onto a reverse phase column (Synergi 4u Hydro-RP C18, Phenomenex, Torrance, CA). Elution solvents for the samples usually consisted of methanol/water gradient containing 0.1% formic acid. Preliminary results Methods for the determination of ergot alkaloids have been done previously with liquid chromatography with fluorescence detection. However, the lack of specificity and structure confirmation allows a margin of error with these compounds. An initial method for the separation and mass spectrometry detection of specific ergot alkaloids (lysergic acid and ergovaline) has been developed. This method was used to verify the relative concentrations of these ergot alkaloids from in vitro tissue studies. Each sample was first eluted with 10%/90% methanol/water (both solvents containing 0.1% formic acid) for 6.00 min. A linear gradient then increased the methanol to 95% by 16.3 minutes and then held at this solvent composition for three additional minutes. The solvent composition was then taken back to 10%/90% methanol/water for 10 min to equilibrate the column for the next sample run. Flow rate for each sample run was held at 0.1 mL/min. The elution from the column was coupled with the electrospray source (ESI; needle set at 5kV, shield 600V) to generate positive ions where mass spectrometry analysis could then occur. The retention time of the lysergic acid and ergovaline was on the order of 14.2 min + 0.1 min and 16.2 minutes + 0.1 min, respectively. Precursor ions were confirmed as the ergot alkaloids with tandem mass spectrometry utilizing collision-induced dissociation (CID).