Use of immunoaffinity columns for clean-up of diarrhetic toxins (okadaic acid and dinophysistoxins) extracts from shellfish prior to their analysis by HPLC fluorimetry

Diarrhetic Shellfish Poisoning (DSP) is a severe gastro-intestinal disease caused by consumption of seafood contaminated by microalgal toxins, mainly okadaic acid (OA) and structurally related toxins, dinophysistoxins (DTXs). Regulatory monitoring is generally based on rodent bioassays which, howeve r, present some technical and ethical disadvantages. The most promising tec hnique of analysis of these toxins involves an HPLC separation with spectro fluorimetric detection after derivatization of the toxins with a fluorescen t reagent. The lack of specificity of the extraction procedure (liquid-liqu id partition), and the presence of interfering compounds in the matrix, doe s nor allow the determination and the quantification of low amounts of toxi ns in seafood. In this paper, the authors report the development and the ch aracterization of immunoaffinity columns (IAC), which were elaborated using anti-okadaic acid monoclonal antibodies, for a specific retention of the O A group of toxins. The coupling yield and the stability of these columns we re investigated as well as their capacity to remove interfering compounds. Cross-reactivity was observed between the antibodies and the DTX-1 and the DTX-2, allowing the detection of the different toxins in a single analysis. Different spiked (1 mu g OA/ g) or naturally-contaminated (mussel digestiv e gland 2 mu g OA/g, algae. 165 mu g OA/g) matrices were tested. The recove ry for OA varied from 55 to 95% according to the matrices. The IAC purifica tion was then included as a step of a global [IAC/HPLC/spectrofluorimetric detection] method and the performance of the method was evaluated. Estimati ons of the linearity and the accuracy (percentages of the presumptive respo nse for OA in the range +101% to + 114%) were satisfactory in accordance wi th the method validation criteria.