HPLC fluorescence determination of ochratoxin A utilizing a double internal standard and its application to poultry feed

Abstract

A validated liquid chromatography method employing a fluorescence detector for the determination of ochratoxin A (OTA) was developed with double internal standard and it was applied to ten different poultry feeds. The analysis was performed in an octadecyl silane column using a solvent system [ACN:water:formic acid (50:50:1.25, v/v/v)] by isocratic elution. The flow rate and injection volume were 1 mL min(-1) and 12 pi, respectively. Signals were detected at 278(lambda(ex))/315(lambda(em)) and 330(lambda(ex))/450(lambda(em)) nm between 0 and 8, and 8.01 and 20.0 min, respectively. The method was validated with precision, linearity, accuracy, limit of detection, limit of quantification, robustness, and stability. Good linearity (r(2) = 0.9998-0.9999) was achieved over a concentration range of 1.60 x 10(-8) M to 6.40 x 10(-6) M for OTA. LOD and LOQ values were 7.83 x 10(-1) M and 2.37 x 10(-9) M, and 2.01 x 10(-9) M and 6.10 x 10-9 M for internal standard 1 (IS1) and internal standard 2 (IS2), respectively, on an interday basis. The method was applied to poultry feed samples. Good recovery data ranged between 79.10% and 85.57%, and 71.98% and 76.66%, and the RSD% values were in the range of 1.36-11.70 and 2.07-2.34 for IS1 and IS2, respectively.