Analysis of domoic acid in shellfish by thin-layer chromatography

Abstract

A thin-layer chromatography (TLC) method has been developed for the semi-quantitative analysis of domoic acid (DA) in shellfish tissues. Tissues were extracted in a single-step homogenization of tissue with 50 % aqueous methanol and then taken through a selective strong anion exchange cleanup. Cleaned extracts were applied directly to silica gel TLC plates and developed with a butanol-acetic acid-water mixture (3:1:1, Rf = 0.45 for DA). As little as 10 microg DA per gram of tissue could be detected after chromatography using a hand-held short-wave UV lamp to detect fluorescence quenching. Confirmation was provided by spraying the plate with ninhydrin, which reacts with the secondary amine of DA to give a distinctive yellow colored product. The extraction, cleanup and TLC procedures are fast and simple, and do not require the use of expensive equipment. This method should prove useful for the routine screening of shellfish tissues in those laboratories not equipped with an LC system. It should also be useful as a chemical confirmation method for DA in samples tested positive by assay methods such as immunoassay.