Determination of domoic acid in mussels by HPLC with post-column derivatization using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and fluorescence detection.

A new, sensitive method was developed for the determination of the neurotoxin domoic acid (DA) using a reversed phase separation followed by post-column derivatization (PCD) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and subsequent fluorescence detection. The PCD conditions which involves a two-step reaction was fully optimized for the lowest detection limit. The first reaction occurs between DA and NBD-Cl while the second makes possible the detection of the derivative causing the destruction of the interfering fluorescent 4-hydroxy-7-nitrobenzo-2-oxa-1,3-diazole (NBD-OH) which is the hydrolysis product of NBD-Cl. Kainic acid a similar base structure compound with DA was used as an internal standard. The developed post-column method provides the ability for a fully automated analysis, low detection limits (LOD 25 ppb in real samples of mussel extracts), it requires less sample preparation, and it gives clean simple chromatograms without chromatographic interferences from coeluting compounds such as tryptophan. The method was successfully applied to for the quantitative determination of DA in mussel tissues at quantities as low as 75 microg/kg tissue.