Smienk et al.: Journal of aoaC international Vol. 96, no. 1, 2013 77 Quantitative Determination of the Okadaic Acid Toxins Group by a Colorimetric Phosphatase Inhibition Assay: Interlaboratory Study Henry Smienk and elena Domínguez 1 ZEU-INMUNOTEC, C/Bari 25 Dpdo. 50197, Zaragoza, Spain maría l. roDríguez-V elaSco EURLMB-AESAN, Estación Marítima S/N, Muelle de Trasatlánticos, 36200 Vigo, Spain DaViD clarke Marine Institute, Rinville, Oranmore, Co. Galway, Ireland katrin kapp BFR—Federal Institute for Risk Assessment, Thielallee 88-92, 14195, Berlin, Germany panagiota katikou NRL Marine Biotoxins, Institute of Food Hygiene, 3A Limnou St, 54627 Thessaloniki, Greece ana g. cabaDo, alberto otero, and Juan m. VieiteS ANFACO CECOPESCA, Ctra. Colexio Universitario, 16, Lagoas, 36310, Vigo, Spain peDro razquin and luiS mata ZEU-INMUNOTEC, C/Bari 25 Dpdo., 50197, Zaragoza, Spain Received October 28, 2011. Accepted by AP May 23, 2012. 1 Corresponding author’s e-mail: edominguez@zeulab.com DOI: 10.5740/jaoacint.11-465 FOOD CHEMICAL CONTAMINANTS An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 µg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSD r ) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD R ) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group. O kadaic acid (OA) and its analogs dinophysistoxin-1 and -2 (DTX1, DTX2), together with their ester forms, are known as the OA toxins group. These lipophilic and heat stable toxins are produced by dinolagellates and can be found in various species of shellish, mainly in ilter-feeding bivalve molluscs. OA toxins causes diarrheic shellish poisoning, which is characterized by symptoms, such as diarrhea, nausea, vomiting, and abdominal pain. These symptoms may occur in humans shortly after consumption of contaminated bivalve molluscs, such as mussels, clams, scallops, or oysters. Inhibition of serine/ threonine phosphoprotein phosphatases (PPs) is assumed to be responsible for these toxic effects. These compounds are also involved in tumor promotion (1). Therefore, these toxins are regulated by European Union law. Regulation (EC) No. 853/2004 (2) states that live bivalve molluscs placed on the market for human consumption must not contain marine biotoxins in total quantities (measured in the whole body or any part edible separately) that exceed 160 µg of OA equivalents/kg for OA, dinophysistoxins, and pectenotoxins together. Commission Regulation (EC) No. 15/2011 (3) indicates that in the case of lipophilic toxins including OA toxins, LC/MS/MS is the reference method for routine testing of oficial controls or any checks done by food operators. This regulation has recently amended the Commission Regulation (EC) No. 2074/2005 (4), in which biological methods (mouse and rat bioassay) were considered the reference. From now on, they will only be used for a transitional period of time (until the end of 2014) or in special circumstances. Both regulations (No. 2074/2005 and No. 15/2011) contemplate other methods for routine testing of lipophilic toxins, providing they are intralaboratory-validated and successfully tested under a recognized proiciency test scheme. Those methods should detect, either alone or in combination with others, all of the lipophilic toxin analogs (OA, pectenotoxins, yesotoxins, and azaspiracids group toxins). The protein phosphatase inhibition assay (PPIA) is speciically mentioned in these regulations as an alternative or complementary method, considering that the PPs are known to be OA-toxins natural targets (5, 6). In-house PPIAs using different phosphatase sources and colorimetric or luorometric substrates have been previously developed (7–12). Later improvements to detect all OA derivatives by hydrolysis of samples were also suggested