Determination of ponazuril in pig excrement by high performance liquid chromatography
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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:ObjectiveTo 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.
MethodUrine 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.
ResultThe 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%.
ConclusionThis method has simple sample pretreatment and sensitive detection, and is suitable for the determination of ponazuril in pig excrement.
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Keywords:
- ponazuril /
- high performance liquid chromatography /
- pig /
- excrement /
- recovery rate /
- precision degree /
- stability
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表 1 帕托珠利在尿液中的回收率与相对标准偏差(RSD)
Table 1 Recovery rates and relative standard deviations (RSDs) of ponazuril in urine sample
n = 3 ρ(帕托珠利)/(µg·mL−1)
Ponazuril concentration回收率/% Recovery rate 批内相对标准偏差/% Intra-batch RSD 批间相对标准偏差/%
Inter-batch RSD1 2 3 1 2 3 0.05 93.54 97.56 93.49 7.62 5.95 5.78 6.43 1.00 98.26 98.78 98.75 1.34 0.97 1.29 1.17 5.00 99.16 97.43 98.86 6.15 6.09 5.87 5.72 
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表 2 帕托珠利在粪便样品中的回收率与相对标准偏差
Table 2 Recovery rates and relative standard deviations (RSDs) of ponazuril in feces samples
n = 3 w(帕托珠利)/(µg·g−1)
Ponazuril concentration回收率/% Recovery rate 批内相对标准偏差/% Intra-batch RSD 批间相对标准偏差/%
Inter-batch RSD1 2 3 1 2 3 0.25 95.88 89.55 90.65 1.76 2.30 2.00 3.63 25.00 91.95 93.57 94.00 2.29 2.46 2.34 2.40 100.00 93.87 95.24 94.50 2.77 2.97 3.17 2.82
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表 3 尿液样品中帕托珠利的冷冻放置稳定性试验结果
Table 3 Result of cryopreservation stability experiment of ponazuril in urine sample
n = 5 保存时间/d
Preservation timeρ(帕托珠利)起始/(µg·mL−1)
Initial ponazuril concentrationρ(帕托珠利)实际/(µg·mL−1) Actual ponazuril concentration RSD/% 1 2 3 4 5 $\bar X$±S 0 0.05 0.04 0.05 0.04 0.04 0.04 0.04±0.00 6.93 1.00 0.99 1.00 0.97 1.00 0.99 0.99±0.01 1.48 5.00 4.68 4.72 4.51 4.84 4.70 4.69±0.12 2.47 30 0.05 0.04 0.05 0.05 0.04 0.04 0.04±0.00 2.61 1.00 0.99 1.00 0.98 0.99 0.99 0.99±0.01 0.91 5.00 4.71 4.77 4.54 4.87 4.72 4.72±0.12 2.48 60 0.05 0.04 0.05 0.04 0.04 0.04 0.04±0.00 5.39 1.00 1.00 1.00 0.98 1.00 1.00 1.00±0.01 0.85 5.00 4.70 4.77 4.53 4.86 4.70 4.71±0.12 2.51
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表 4 粪便样品中帕托珠利的冷冻放置稳定性试验结果
Table 4 Result of cryopreservation stability experiment of ponazuril in feces sample
n = 5 保存时间/d
Preservation timew(帕托珠利)起始/(µg·g−1)
Initial ponazuril concentrationw(帕托珠利)实际/(µg·g−1) Actual ponazuril concentration RSD/% 1 2 3 4 5 $\bar X$±S 0 0.25 0.21 0.24 0.22 0.22 0.22 0.22±0.01 6.29 25.00 22.87 23.47 24.28 24.56 23.54 23.74±0.68 2.85 100.00 93.50 89.04 97.27 99.24 95.00 94.81±3.89 4.11 30 0.25 0.21 0.22 0.23 0.21 0.23 0.22±0.01 3.44 25.00 24.03 23.83 23.40 23.44 22.70 23.48±0.51 2.17 100.00 94.23 87.96 94.32 84.57 94.84 91.18±4.65 5.10 60 0.25 0.21 0.22 0.23 0.24 0.21 0.23±0.01 4.94 25.00 22.39 21.76 21.96 21.70 21.47 21.86±0.35 1.59 100.00 97.38 97.03 83.50 83.98 94.73 91.33±7.00 7.66
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表 5 不同时间段粪便和尿液中帕托珠利的平均排出量1)
Table 5 Average elimination amounts of ponazuril in feces and urine samples at different time intervals
采样时间/d
Sampling time粪便 Feces 尿液 Urine 平均排出量/mg
Average elimination amount累积占比/%
Accumulative ratio平均排出量/mg
Average elimination amount累积占比/%
Accumulative ratio0~0.5 3.76±0.46 7.75 0.026±0.020 16.48 0.5~1.0 6.72±1.68 21.62 0.033±0.023 37.60 1.0~1.5 11.14±0.54 44.63 0.022±0.008 51.75 1.5~2.5 7.55±2.16 60.22 0.008±0.003 56.64 2.5~3.5 3.91±1.16 68.30 0.006±0.000 60.75 3.5~4.5 2.13±0.44 72.71 0.007±0.003 64.98 4.5~5.5 1.62±0.45 76.04 0.007±0.002 69.42 5.5~6.5 1.72±0.46 79.59 0.010±0.006 75.74 6.5~7.5 1.15±0.57 81.97 0.008±0.006 80.48 7.5~8.5 0.76±0.42 83.54 0.006±0.006 84.37 8.5~9.5 0.98±0.96 85.55 0.016±0.016 94.43 9.5~10.5 1.08±0.42 87.77 0.005±0.003 97.40 10.5~11.5 0.56±0.38 88.93 0.002±0.003 98.89 11.5~12.5 0.82±0.35 90.63 0.001±0.002 99.70 12.5~13.5 0.49±0.47 91.65 — 99.70 13.5~14.5 0.44±0.09 92.56 — 99.70 14.5~15.5 0.43±0.18 93.44 — 99.70 15.5~16.5 0.47±0.27 94.41 — 99.70 16.5~17.5 0.37±0.40 95.17 — 99.70 17.5~18.5 0.24±0.14 95.67 — 99.70 18.5~19.5 0.20±0.07 96.08 — 99.70 19.5~20.5 0.28±0.16 96.67 — 99.70 20.5~21.5 0.23±0.11 97.14 — 99.70 21.5~22.5 0.21±0.16 97.58 — 99.70 22.5~23.5 0.09±0.05 97.78 — 99.70 23.5~24.5 0.15±0.10 98.09 — 99.70 24.5~25.5 0.05±0.01 98.19 — 99.70 25.5~26.5 0.16±0.18 98.52 — 99.70 26.5~27.5 0.07±0.03 98.67 — 99.70 27.5~28.5 0.12±0.08 98.92 — 99.70 28.5~29.5 0.06±0.01 99.05 — 99.70 29.5~30.5 0.09±0.11 99.23 — 99.70 30.5~31.5 0.08±0.07 99.40 — 99.70 31.5~32.5 0.07±0.05 99.55 — 99.70 32.5~33.5 0.08±0.09 99.72 — 99.70 33.5~34.5 0.03±0.00 99.78 — 99.70 34.5~35.5 0.03±0.02 99.85 — 99.70 35.5~36.5 0.02±0.02 99.89 — 99.70 36.5~37.5 0.02±0.03 99.93 — 99.70 37.5~38.5 0.01±0.02 99.95 — 99.70 38.5~39.5 0.01±0.02 99.98 — 99.70 39.5~40.5 0.01±0.01 99.99 — 99.70 40.5~41.5 — 99.99 — 99.70 1)采样时间指给药后天数；“—”表示未检出
1) Sampling time indicates days after administration; “—” represents not detectable
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