Determination of Citrinin in Red Yeast Rice by High Performance Liquid Chromatography with Immuno-Affinity Column

Abstract

To prepare immune-affinity column (IAC) of citrinin, CNBr activated Sepharose 4FF was used as a carrier to couple with monoclonal antibody against citrinin. A reversed-phase high performance liquid chromatography (HPLC) with fluorescence detection method was used to determine the samples elution after the IAC cleaning up. The fluorescence excitation and emission maxima of citrinin were 331 and 500 nm, respectively. Citrinin is determined by HPLC with a retention time of 6.5 min (flow rate of 1 mL/min) with acetonitrile-H2O (45:55, v/v, pH 2.0). The linear range is 1-500 ng/mL. The column capacity is 0.35 μg when using 0.5 mL CNBr activated Sepharose 4FF and 500 μg monoclonal antibody against citrinin. Average recoveries of citrinin from red yeast rice spiked at levels of 0.1-0.6 mg/kg range from 74.2% to 87.12%. 16 samples of red yeast rice were determined by HPLC with immune-affinity column. The results show 12 samples contain citrinin, and concentrations of citrinin of red yeast rice are 100.6~443.6 μg/kg. The IAC method is found to be highly effective, sensitive and convenient for isolating the target analyte from samples.