MEASUREMENT OF AROMATIC CHOLINE ESTERS IN RAPESEED BY HPLC

Aromatic choline esters are natural compounds occurring in Brassicaceae especially in rapeseed among whom the most important is called sinapine (choline ester of the 3,5-dimethoxy-4 hydroxycinnamic acid). Choline esters are easily hydrolysed by entero bacteria in digestive tractus giving the correspondent acid and a choline molecule. Choline could then be broken down, by a deaminase, into trimethylamine, compound inducing a negative flavour ("fishy odour") in the milk, the meat or in yolk. So, the method improved in our laboratory is a power tool for breeders and other scientists who would like to evaluate individual aromatic choline esters in seeds or meals. Seeds (10 g) are first ground in a coffee mill (20 sec). 200 mg of ground seeds are weighed in a test tube and then placed in a water bath (75°C). Then 10 ml of boiling methanol/acetic acid (0.05M) mixture (70/30) are added plus 0.5 ml of internal standard solution (3,5-dimethoxybenzoic choline ester, 10 µmol/ml).The heterogeneous content is stirred with a magnetic stirrer for 10 min. Only one extraction step is necessary to obtain good results. Then, 1 ml of crude extract is purified with a cation exchange column (CM Sephadex C25-120). Aromatic choline esters are eluted with acetic acid 1N. Separation and quantification of individual choline esters are further realised by high performance liquid chromatography with an Inertsil 5 ODS-2 (3 x 250 mm, 5 µm) using a ternary solvent gradient (water-acetonitrile-phosphate buffer: NaH2PO4, 20 mM at pH 2 with o-phosphoric acid). In these optimised chromatographic conditions, the choline esters are separated and quantified. The retention times and the response factors have been determinated for the 36 different choline esters (benzoic or cinnamic structures with hydroxy or methoxy groups in ortho, para or meta position) synthesised in our laboratory.