Ultrasound assisted combined molecularly imprinted polymer for the selective micro-solid phase extraction and determination of aflatoxins in fish feed using liquid chromatography-tandem mass spectrometry.

A combined procedure based on using ultrasounds for target isolation followed by porous membrane-protected micro solid phase extraction using a molecularly imprinted polymer as an adsorbent has been developed as a highly selective extraction and clean-up procedure for isolating aflatoxins B1, B2, G1, and G2 from fish feed before ultra-high-performance liquid chromatography tandem mass spectrometry determination. Polymeric adsorbent beads have been synthesized by the precipitation polymerization method which guarantees a homogeneous particles size distribution and the integrity of the generated imprinted cavities. In addition, polymerization was performed using a higher proportion of organic solvent (toluene) in the porogen mixture, which generates MIP particles adequate for interacting with targets dissolved in organic (hydro-organic) mixtures (extracts from fish feed). These approaches led to a selective and high efficient pre-concentration method for AFs. Ultrasound-assisted extraction (10 mL of 60:40 acetonitrile/0.1 M KH₂PO₄ pH 6.0, 40% amplitude, continuous sonication for 7.0 min) allowed an efficient aflatoxins isolation from fish feed. In addition, the resulting pH of the extract (pH 7.0) has been found to be the optimum for performing clean-up/pre-concentration (enrichment factor of 33.3) by molecularly imprinted polymer based micro-solid phase extraction (orbital horizontal shaking speed at 150 rpm for 10 min for loading, and 5 mL of 95:5 acetonitrile/formic acid as eluting solution using ultrasounds 35 kHz for 15 min). The current proposal was shown to be an accurate and precise method through relative standard deviation of intraday and inter-day tests below 20% and analytical recoveries in the range of 80-100%. The limits of detection were within the 0.42-1.15 μg kg^-1 range, quite lower than those established by European Commission guidelines for aflatoxins in animal feeds.