A new method for the determination of lolitrem B in plant materials

The endophyte fungus Neotyphodium lolii is associated with perennial ryegrass (Lolium perenne L) and can lead to the production of tremorgenic mycotoxins like lolitrem B, which is responsible for “Ryegrass staggers”, a disease in sheep, cattle and horses. We present a new simple, rapid, sensitive method to quantify lolitrem B in seeds and vegetative tissue. Lolitrem B is extracted using dichloromethane, purified on a solid-phase extraction (SPE) column and analysed by high performance liquid chromatography (HPLC) on a silicon column and a fluorimetric detector. This method is repeatable, reproducible, and linear at a concentration of 50–500 μg of lolitrem B/kg dry matter (DM) in seeds and hay. The limit of quantification was 50 μg of lolitrem B/kg DM. The mean recovery rates were 59.4 and 85.9% in seeds and hay, respectively. The method was applied to seeds and vegetative tissue collected in 2010 in France. Endophyte-infected and endophyte-free seeds and hay were mixed at varying percentage concentrations. The method was able to detect 1% of endophyte-infected material mixed with endophyte-free plant material.