Determination of ponazuril in pig excrement by high performance liquid chromatography

Objective  To establish a method of high performance liquid chromatography (HPLC) with a photodiode array detector (PDA) for the determination of ponazuril in pig feces and urine.

Method  Urine samples were extracted twice with 0.2%(φ) acetic acetonitrile and dichloromethane. Feces samples were vortex-extracted by acetonitrile and purified by hydrophile-lipophile balance (HLB) solid phase extraction column. The mobile phase was 0.005 mol/L potassium dihydrogen phosphate solution (A)-acetonitrile (B), the mobile phase ratios of V(A)∶V(B) for urine and feces samples were 55∶45 and 56∶44 respectively. The detection wavelength was 255 nm, the column temperature was 35 ℃ and the injection volume was 30 µL.

Result  The detection limit and quantitative limit of ponazuril in urine were 0.02 and 0.05 µg/mL, respectively, which showed a good linear relationship in the range of 0.05−5.00 µg/mL, and the determination coefficients (R²) was 0.999 8. The average recovery rates ranged from 93.49% to 99.16% at three spiked levels of 0.05, 1.00 and 5.00 µg/mL, and the intra-batch and inter-batch relative standard deviations (RSDs) ranged from 0.97% to 7.62%. The detection limit and quantitative limit of ponazuril in feces were 0.10 and 0.25 µg/g, respectively, which showed a good linear relationship within the range of 0.25−100.00 µg/g, and R² was 0.999 5. The average recovery rates ranged from 89.55% to 95.88% at three spiked levels of 0.25, 25.00 and 100.00 µg/g, and the intra-batch and inter-batch RSDs ranged from 1.76% to 3.63%. The recovery rates of ponazuril in feces and urine were both higher than 89.50%, and the intra-batch and inter-batch RSDs were both lower than 8%.

Conclusion  This method has simple sample pretreatment and sensitive detection, and is suitable for the determination of ponazuril in pig excrement.