取均质完毕的烟叶样品10.00 g,加入含1%(体积分数)乙酸的乙腈溶液10 mL和2~3 g氯化钠,振荡提取10 min,离心。取上清液5 mL置于预先加有200 mg N-丙基乙二胺(PSA)和900 mg无水硫酸镁(MgSO₄)的离心管中,涡旋,离心。取上清液2.0 mL于35℃氮吹至近干,用酸化水溶液(pH 3)500 μL、邻苯二甲醛(OPA)/2-巯基乙醇溶液250 μL和0.05 mol·L^-1氢氧化钠溶液250 μL涡旋溶解残渣,经0.2 μm微孔膜过滤于进样小瓶中,用酸化水溶液(pH 3)定容至1 mL,于80℃避光加热衍生30 min。所得溶液以EXTEND-C₁₈色谱柱为固定相,不同体积比的甲醇-乙腈-水的混合液为流动相进行梯度洗脱分离,采用附荧光检测器的高效液相色谱法(HPLC)测定其中抗蚜威的含量。结果表明,抗蚜威标准曲线的线性范围为0.01~1.00 mg·L^-1,检出限(3S/N)为0.01 mg·kg^-1。按标准加入法进行回收试验,回收率为84.4%~85.5%,测定值的相对标准偏差(n=6)为1.4%~2.4%。用此法分析30份烟叶样品,检出率为20%,最高检出量为0.06 mg·kg^-1。

The sample of homogenized tobacco leaf (10.00 g) was taken, 10 mL of acetonitrile solution containing 1% (φ) acetic acid and 2-3 g of sodium chloride were added, and the mixture was extracted for 10 min by shaking. After centrifugation, 5 mL of the supernatant was taken, and put into a centrifuge tube pre-filled with 200 mg of PSA and 900 mg of MgSO₄. After vortex and centrifugation, 2.0 mL of the supernatant was taken, and blow to nearly dryness at 35℃ by nitrogen. The residue was dissolved by vortex in 500 μL of acidified aqueous solution (pH 3), 250 μL of OPA/2-mercaptoethanol solution and 250 μL of 0.05 mol·L^-1 sodium hydroxide solution, which was filtered into the injection vial though 0.2 μm microporous membrane. The solution was made its volume up to 1 mL with acidified aqueous solution (pH 3), and heated at 80℃ for 30 min for derivatization without light. The obtained solution was separated on EXTEND-C₁₈ column as the stationary phase and a mixture of methanol, acetonitrile and water with different volume ratios as the mobile phase for gradient elution, and pirimicarb was determined by high performance liquid chromatography (HPLC) with fluorescence detector. As shown by the results, the linear range of the standard curve of pirimicarb was 0.01-1.00 mg·L^-1, with detection limit (3S/N) of 0.01 mg·kg^-1. Test for recovery was made by standard addition method, giving results in the range of 84.4%-85.5%, with RSDs (n=6) of the determined values in the range of 1.4%-2.4%. This method was applied to the analysis of 30 tobacco leaf samples, the detection rate was 20% and the maximum detection amount was 0.06 mg·kg^-1.