LC/MS/MS VALIDATED METHOD FOR QUANTITATION OF ERGOPEPTINES AND -ININES IN GRAINS AND GRASSES

PROGRAM SUMMARYThe current Official US Standards for Grains regulatory guidelines for ergot contamination ingrains is a crude measurement and determines the percentage of ergot sclerotia in a sample,with ergoty grain defined as > 0.05% sclerotia in wheat, > 0.1% sclerotia in barley, oats, andtriticale, and > 0.3% sclerotia in rye (CFR Title 7). The European Union Scientific Panel onContaminants in the Food Chain concluded that a consistent relationship between the alkaloidconcentration in the sclerotia and total ergot alkaloid concentration could not be established(EFSA 2005). No country has limits for individual ergot alkaloids in food or feed. Several recentfactors have increased ergot invasion in crops, including no-till farming practices leaving thevegetation and ergot sclerotia on the soil surface for increased infection rates, the move toorganic farming and avoiding fungicides, and the dramatic weather patterns with huge rainfallsthat enhance increased Claviceps purpurea invasion of cereals and grasses in thespring/summer.No US veterinary diagnostic laboratory is providing a validated ergot alkaloid analysis of cerealgrains and grasses by LC/MS/MS for the main Claviceps ergot alkaloids, the ergopeptines (-ines, biologically active) include ergotamine, ergosine, ergocristine, ergocryptine, andergocornine, and their diastereomers the ergopeptinines (-inines, biologically inactive). Innature, ergopeptinines always accompany ergopeptines, and during storage of raw materialsover time or improper storage, more ergopeptinines may form. The conversion of -ines to -ininescan be rapid under certain laboratory conditions and -inines can be reactivated to -ines undercertain conditions. Therefore, both epimers have to be determined for ergot alkaloidcontamination of foods and feeds (Krska and Crews).This proposal is to develop and validate a sensitive LC/MS/MS method for ergpopeptines,ergotamine, ergosine, ergocristine, ergocryptine and ergocornine, and their -inines in cerealgrains of wheat, barley, rye and grasses. The inclusion of a grass matrix for ergot alkaloidanalysis by LC/MS/MS is not commonly addressed in any method but is extremely important forveterinary livestock exposures. The analytical range of detection chosen for the assay, 20 to8000 ug/kg, represents veterinary toxicological important data for interpretation of animal feedand food contamination.RELEVANCEThe LC/MS/MS analytical method for ergopeptines and ergopeptinines in small cereals andgrasses will provide chemical analysis of potentially contaminated feed to be used for foodanimals or sold into the food chain. The current US Official Grain Standards of determiningpercentage contamination by ergot sclerotia in cereal grains is often inaccurate and ignores thegreat variation of alkaloid content in individual sclerotia. The increased use of no-till farmingand leaving vegetation and sclerotia on the soil surface for the successive crops, consumerdesire to utilize organic foods without chemicals, and dramatic weather events will tend toincrease the incidence of Claviceps purpurea in cereal crops and grasses and exposure to ergotalkaloids.