Synthesis Of Patulin-Selective Molecular Imprinting Polymers Grafted On Silica Bead And Their Applications To SPE-HPLC Determination

Patulin, 4-hydroxy-4H-furo[3,2-c]pyran-2(6H)-one, is a mycotoxin produced by mainly Penicillium and Aspergillus species. Recently, determination of patulin in apple juice has become the focus of concern because of its adverse effects.As opposed to other mycotoxins, until now, no commercial ELISA-kits are available for patulin assay. Conventional methods for the determination of patulin include thin layer chromatography (TLC), gas chromatography (GC), and high-performance liquid chromatography (HPLC). Reversed-phase HPLC, coupled with UV detection has become the most commonly used method for patulin analysis. However, sample pretreatments for the HPLC method usually include liquid-liquid extraction(LLE) with ethylacetate accompanied with purification by sodium carbonate solution, which may cause degradation of patulin due to the basic condition. On the other hand, patulin was difficult to be separated from the co-extracted 5-hydroxymethylfurfural (HMF) in LLE sample. Analytical methods with high accuracy, low detection limits and high specificity are needed.In this study, a patulin-selective polymer was successfully synthesized by molecular imprinting technology (MIT) and applied to solid phase extraction (SPE) as a pretreatment method.First, APTS-silica was prepared by silanization on silica bead surface after activation. After then, 4,4'-Azobis(4-cyanopentanoic acid) was grafted on the silica bead by two methods alternatively. The synthesis of molecularly imprinted polymers was carried on at 70℃with 2-Hydroxynicotinic acid as template substitude, acrylamide, methacrylic acid, 4-vinyl pyridine and 2,2,2-trifluoroethyl methacrylateas as functional monomers, ethylene glycol dimethacrylate as crosslinker and 4,4'-Azobis(4-cyanopentanoic acid) grafted silica (ACPA-Silica) as azo-initiator. The optimum SPE conditions was determined to be using acetic acid solution (pH 2.95) as loading solution and phosphate buffer (0.05mol/L,pH 4.14) as eluting solution. Under these conditions, a separation degree of 13.42 of patulin and 5-hydroxymethylfurfural was achieved and a recovery of about 93.73-97.62% for Pat was obtained.A HPLC-UV method with LOD of 10μg/L and LOQ of 40μg/L for Pat analysis was also developed. The RSD for repeatability is about 0.16% and the linear range of this analysis was between 0.5 ng and 250 ng with a correlation coefficient of 0.9998.The results could be used as a fast and accurate method for patulin analysis and relating products.