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

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 \mu L, 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 \times 10^{-8} M to 6.40 \times 10^{-6} M for OTA. LOD and LOQ values were 7.83 \times 10^{-10} M and 2.37 \times 10^{-9} M, and 2.01 \times 10^{-9} M and 6.10 \times 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.