Inter-laboratory validation of the fluorescent protein phosphatase inhibition assay to determine diarrhetic shellfish toxins: intercomparison with liquid chromatography and mouse bioassay

Abstract

Toxic episodes of diarrhetic shellfish toxins (DSP) in shellfish harvesting areas have serious economic and public health implications, where fluorescent protein phosphatase inhibition assay (FPPIA) may be a highly useful tool for monitoring purposes. This paper presents results from the first inter-laboratory study to validate the assay. Three laboratories participated in the design and development of the inter-laboratory work. Standard solutions and spiked samples of the main toxin, okadaic acid, were used at the beginning of the validation exercise to avoid cross-inhibition of other toxins that would otherwise deteriorate the quantitative significance of the data. HPLC with fluorimetric detection of okadaic acid was also submitted to inter-laboratory validation to be subsequently used as a quantitative reference method. FPPIA results from spiked samples were free of systematic bias in any laboratory and determinations repeated over 3 days showed that the classic “repeatability” was the main within-laboratory source of variability (15–26% R.S.D. depending on the sample). After the inter-laboratory validation of both HPLC and FPPIA methods, 83 samples of mussel hepatopancreas collected during a toxic DSP episode were analyzed over 9 weeks. Toxic levels determined with FPPIA were in line with mouse bioassay results, highlighting the lack of false negative results of the FPPIA test: 98.7% of samples whose concentration of okadaic acid equivalents was over 0.8 μg/g hep., provided positive bioassay results within 24 h of observation time. The reliability and the quantitativeness of the FPPIA method in naturally contaminated samples was demonstrated by intercomparison with mouse bioassay and HPLC.