Abstract
An efficient method for the determination of tetramine (tetramethylammonium, TM) in the salivary gland of gastropods (Neptunea intersculpta) was developed by using high performance liquid chromatography (HPLC) equipped with NH2-bonded silica gel column and refractive index (RI) detector. Isolation procedure is as follow. The homogenized salivary gland was refluxed in methanol for 30 min and the mixture was filtered after cooling. The methanol extract was then concentrated under reduced pressure and the residue was defatted with ether. The resulting aqueous layer was chromatographed on a column of DEAE-Sephadex A-25 and Sephadex LH-20. The tetramine chloride (TM-Cl) sample thus obtained was injected into a TSK-gel NH260 (4.6 mm i.d.×25 cm) column at 40°C by using methanol as a mobile phase. The retention times for TM-Cl and internal standard (ethyl p-hydroxybenzoate) were 3.6 min and 6.6 min, respectively. Recovery of TM-Cl from the salivary gland was 97.8%, and the determination limit was defined as 0.3 mg/g. The content (mg) of TM-Cl in the salivary gland of N. intersculpta was 9.8-5.7 mg/g.
