高效液相色谱法测定猪排泄物中帕托珠利

    Determination of ponazuril in pig excrement by high performance liquid chromatography

    • 摘要:
      目的  建立高效液相色谱(HPLC)结合二极管阵列检测器(PDA)检测猪排泄物中帕托珠利的方法。
      方法  尿液样品用0.2%(φ)乙酸酸化乙腈和二氯甲烷进行2次提取;粪便样品经乙腈涡旋提取,亲水−亲油平衡(HLB)固相萃取柱净化。流动相为0.005 mol/L磷酸二氢钾溶液(A)−乙腈(B),尿液和粪便样品的流动相比例V(A)∶V(B)分别为55∶45和56∶44。检测波长为255 nm,柱温为35 ℃,进样量为30 µL。
      结果  猪尿液中帕托珠利的检测限和定量限分别为0.02和0.05 µg/mL,在0.05~5.00 µg/mL范围内呈良好的线性关系,决定系数(R2)为0.999 8;在3个添加剂量(0.05、1.00和5.00 µg/mL)下,帕托珠利在猪尿液中的平均回收率为93.49%~99.16%,批内和批间相对标准偏差(RSD)为0.97%~7.62%。猪粪便中帕托珠利的检测限和定量限分别为0.10和0.25 µg/g,在0.25~100.00 µg/g范围内呈良好的线性关系,R2为0.999 5;在3个添加剂量(0.25、25.00、100.00 µg/g)下,帕托珠利在猪粪便中的平均回收率为89.55%~95.88%,批内和批间RSD为1.76%~3.63%。帕托珠利在尿液和粪便样品中的提取回收率均大于89.50%,批内和批间RSD均小于8%。
      结论  本研究方法对样品的前处理操作简单、灵敏度高,可用于猪排泄物中帕托珠利的检测分析。

       

      Abstract:
      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.

       

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