Wednesday, November 15, 2006

A method for analyzing alkaloid composition in hardinggrass.

Andrew Hopkins1, Lili Zhou2, David Huhman3, and Lloyd Sumner3. (1) The Samuel Roberts Noble Foundation, Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401, (2) North Carolina State University, 4219 Gardner Hall, Raleigh, NC 27695, (3) Noble Foundation, 2510 Sam Noble Parkway, Ardmore, OK 73401

Hardinggrass (Phalaris aquatica L.) is a cool season perennial that has potential for forage production in the south central USA.  However, a number of alkaloids present in hardinggrass have been implicated in animal health disorders.  Our objective was to develop a sensitive, selective and stable method for analysis of alkaloids in hardinggrass. An HPTLC (High Performance Thin Layer Chromatography) method was developed utilizing glass plates pre-coated with silica gel 60F-254 as the stationary phase. The solvent system consisted of ethyl acetate/chloroform/methanol with ammonia (7N NH3 in methanol) (8/2/1, v/v/v). Using uni-dimensional, double development, bands were well separated for 10 alkaloid standards as well as alkaloids observed in hardinggrass plant extracts. The identities of compounds observed using HPTLC were validated by HPLC/MS (High Performance Liquid Chromatography / Mass Spectrometry).  Software was used to quantify individual alkaloids in plant samples based on HPTLC retention factors and intensities relative to standards of known concentration.  Correlation coefficients of 0.99 were obtained between estimated and actual concentrations for four standards (methyltyramine, hordenine, gramine, 5-methoxy dimethyltryptamine).  The HPTLC method is repeatable and specific for β-carboline, tryptamine, gramine and tyramine type alkaloids in mixed standard and plant extracts.  Initial results indicate substantial variation in alkaloid composition among and within hardinggrass populations. Application of this HPTLC method will be helpful in gaining a better understanding of genotype and genotype by environment effects on alkaloid composition in hardinggrass, and for developing hardinggrass cultivars with reduced risk of alkaloid toxicity.