HPLC Determination of Ochratoxin A in Roasted Coffee with Purification by Liquid-Liquid Extraction in Combination with Immunoaffinity Column Chromatography

Ochratoxin A in roasted coffee was extracted with chloroform,and purified by back-extraction with sodium bicarbonate solution and passing the aqueous extract after dilution with PBS of pH 7.0 through Ochra TestTM immunoaffinity column.Ochratoxin A was eluted from the column with methanol,and determined by HPLC.The KR100-10 C18 chromatographic column (4.6 mm×250 mm,5 μm) was used as the stationary phase,and a mixture of H2O,acetonitrile and acetic acid (mixed in the ratio of 51∶48∶1) was used as the mobile phase with a flow-rate of 1.0 mL·min-1,to elute the ochratoxin A from the column.Fluorescence detection at the wavelengths of (λex) 333 nm and (λem) 460 nm was used in the determination.Linear relationship between values of peak area (A) and concentration of ochratoxin A was obtained in the range of 1.0-50.0 μg·L-1.Determination limit (S/N=10) of the method was found to be 1.0 ng·g-1.Recovery and precision were tested by adding standard solutions of ochratoxin A at 3 different concentration levels of 1.0,10.0 and 50.0 ng·g-1,values of recovery found were in the range of 74.1%-78.0%,and values of RSD′s (n=8) obtained were in the range of 5.2%-9.6%.