Ochratest wb immunoaffinity column

OTA extraction followed the procedure described by Zhao et al. [51 (link)] with some modifications. Briefly, the other half of the irradiated samples (2.5 g) were extracted by stirring (25 °C at 150 rpm) with MeOH/1% NaHCO₃ solution (12.5 mL, 70:30, v/v) for 30 min and subsequently filtered through Whatman No. 4 filter paper. Afterwards, the extract (10 mL) was diluted with PBS-T (40 mL) and further filtered through a Whatman glass microfiber filter (934-AH). The filtered extract (20 mL) was purified through an Ochratest WB immunoaffinity column (VICAM, Watertown, MA, USA) and the column was washed first with PBS-T (10 mL) and then with ultra-pure water (10 mL). Afterwards OTA was eluted with methanol (2 mL), collected in a glass vial, filtered through 0.2 µm nylon filters (Whatman) and analysed by HPLC-FLD.
OTA samples were analysed using the HPLC system and column described above for AF analysis, but without the derivatization process. The fluorescence detector was set to λ_ex 330 nm and λem 463 nm, mobile phase consisted of a mixture with acetonitrile/water/acetic acid (70:29.5:0.5, v/v/v), with a flow rate of 0.8 mL/min, and the injection volume was 10 μL. OTA was identified by chromatographic comparison with the standard (OTA standard solutions, Sigma Aldrich Co.) and quantification was based on the fluorescence signal response.

Livers were collected from four broilers per pen. The livers were combined in pairs, resulting in two analytical samples per pen. The tissues were ground and stored at −20 °C until analysis. OTA analysis was performed by extracting 5.0 g of ground liver with 25 mL of 1% NaHCO₃: methanol (30:70, v/v). After centrifugation, 10.0 mL of the supernatant was mixed with an equal volume of PBS and applied onto an OchraTest WB immunoaffinity column (Vicam, Milford, MA, USA). The effluent was filtered through a 0.45 µm syringe filter and analysed by high pressure liquid chromatography equipped with a fluorescent detector (HPLC Prominence, Shimadzu, Kyoto, Japan). The HPLC separation was performed by means of a Phenomenex Luna C18(2) 3 µm, 150 × 4.60 mm column equipped with a Gemini C18, 4 × 3 mm SecurityGuard pre-column (Phenomenex Inc., Torrance, CA, USA) at a flow rate of 0.8 mL/min. Injection volume was set to 40 µL and column oven temperature 30 °C. Fluorescence detection was carried out at an excitation wavelength of 333 nm and emission wavelength of 460 nm. The limit of detection measured from standard deviation of background signal was 0.4 ng/kg and the limit of quantification was 2 ng/kg.