|SMITH, DARRIN - Eastern Kentucky University|
|SMITH, LORI - University Of Kentucky|
|SHAFER, WILSON - University Of Kentucky|
Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2009
Publication Date: 7/31/2009
Citation: Smith, D.L., Smith, L.L., Shafer, W.D., Klotz, J.L., Strickland, J.R. 2009. Development and validation of a LC-MS method for quantitation of ergot alkaloids in lateral saphenous vein tissue. Journal of Agricultural and Food Chemistry. 57:7213-7220.
Interpretive Summary: “Fescue toxicosis” is a costly animal syndrome with nearly one billion dollars in annual production losses for forage-animal enterprises within the USA. The syndrome has been linked to consumption of tall fescue containing ergot alkaloids produced by a symbiotic endophytic fungus located within the plant. A major hindrance to finding a complete solution to the fescue toxicosis problem has been our inability to study the distribution and metabolism of the alkaloids with the target tissues of the intoxicated animal. This has been due to a lack of sensitive and selective analytical methods suitable for the identification and quantification of the alkaloids and their metabolites. LC/MS is a tool that offers the required combination of sensitivity and selectivity to conduct these types of studies. This manuscript reports on the development of a new LC/MS method for the quantitation of ergot alkaloids in animal tissues (specifically vascular tissue). Although there are a number of LC/MS methods for detecting these alkaloids in plant based materials, to our knowledge a LC/MS method does not exist for animal tissues/fluids (for the ergot alkaloids of concern in endophyte-infected tall fescue). The few published reports for quantitatively analyzing for these compounds in animals tissues and fluids have primarily been based on HPLC/fluorescence or ELISA. None of these methods, however, offer the necessary combination of selectivity and sensitivity needed for the study of the alkaloids in animal tissues as reported in this manuscript. The method reported in this manuscript exhibits good sensitivity and selectivity and will allow for new studies in the distribution and metabolism of these compounds in the vasculature of grazing animals. Further, the method forms a solid platform from which additional extraction and sample cleanup and separation protocols for other body tissues/fluids may be developed for future study of these alkaloids in other tissues/cells.
Technical Abstract: A liquid chromatography-mass spectrometry (LC/MS) method for simultaneous quantitation of seven ergot alkaloids (lysergic acid, ergonovine, ergovaline, ergocornine, ergotamine, ergocryptine and ergocrystine) in vascular tissue was developed and validated. Reverse-phase chromatography, coupled to an electrospray ionization source, was used to separate and detect alkaloids. Singly-protonated molecular ions for each alkaloid and methysergide (internal standard), were detected by single-ion monitoring (SIM). Calibration curves were obtained over a linear range of 0.1 to 40 pmol on column with correlation coefficients better than 0.994. Method recoveries were 68.4% to 111.0%. Intra-assay precision was 3.4% to 16.1%. Matrix effects were observed and overcome by introducing matrix components into calibrant solutions to create matrix-diluted standards. Limits of detection and quantitation were 0.05 pmol and 0.1 pmol, respectively. Method ruggedness tests resulted in recoveries of 86.1 to 122% with an inter-assay precision of 7.9% to 22.8%. These results indicate that this method is suitable for quantitation of alkaloids extracted from in vitro – exposed vascular tissue.