取鸡肉样品约2.0 g,加入100 μg·L^-1金刚烷胺-d₆和利巴韦林-¹³C₅的混合内标溶液100 μL和体积比为7∶3的20 g·L^-1三氯乙酸溶液-乙腈的混合溶液15 mL,涡旋30 s,振荡15 min,离心5 min,上层有机相转移至50 mL离心管中。重复上述操作一次,合并上层有机相,过普通滤纸,滤液用约100 μL氨水调节酸度至pH 8.5,过活化好的Bond Elut PBA固相萃取柱。固相萃取柱先以体积比1∶9的乙腈-0.25 mol·L^-1乙酸铵混合溶液3 mL和含5%(体积分数)氨水的甲醇溶液3 mL淋洗,然后以含2%(体积分数)甲酸的甲醇溶液4 mL洗脱。收集洗脱液,氮吹至干,残渣用体积比1∶9乙腈-水混合溶液1 mL复溶后,过0.22 μm滤膜。滤液进入高效液相色谱-串联质谱仪,在Eclipse XDB-C₈色谱柱上用不同体积比的含5 mmol·L^-1乙酸铵的1%(体积分数)甲酸溶液-甲醇-水的混合溶液梯度洗脱分离其中的金刚烷胺和利巴韦林,在多反应监测模式和电喷雾离子源正离子模式下检测,以内标法定量。结果显示:金刚烷胺和利巴韦林在Eclipse XDB-C₈色谱柱上的保留较强,分别在4.30,9.13 min出峰;标准曲线的线性范围均为2~40 μg·L^-1,检出限(3S/N)分别为0.2,0.3 μg·kg^-1;对空白鸡肉样品进行3个浓度水平的加标回收试验,回收率分别为70.5~84.4%和98.5%~99.6%,测定值的相对标准偏差(n=6)均小于5.0%。

The chicken sample of about 2.0 g was taken, and 100 μL of 100 μg·L^-1 mixed internal standard (amantadine-d₆ and ribavirin-¹³C₅) solution together with 15 mL of 20 g·L^-1 trichloroacetic acid solution-acetonitrile mixed solution at volume ratio of 7∶3 were added. The mixture was vortexed for 30 s, shaken for 15 min, and centrifuged for 5 min. The upper organic phase was transferred into a 50 mL-centrifuge tube. The above extraction process was repeated once, and the upper organic phase was combined and filtered through ordinary filter paper. Then about 100 μL of aqueous ammonia was added into the above solution for adjusting the acidity to pH 8.5. The solution obtained was passed through the Bond Elut PBA solid phase extraction column activated beforehand. The solid phase extraction column was washed with 3 mL of acetonitrile-0.25 mol·L^-1 ammonium acetate mixed solution at volume ratio of 1∶9 and 3 mL of methanol solution containing 5% (volume fraction) aqueous ammonia in turn, and eluted with 4 mL of methanol solution containing 2% (volume fraction) formic acid. The eluent was collected, and blown to dry ness by nitrogen. The residue was redissolved with 1 mL of acetonitrile-water mixed solution at volume ratio of 1∶9, and then passed through a 0.22 μm filter membrane. The filtrate was introduced into the high performance liquid chromatography-tandem mass spectrometer. Amantadine and ribavirin in the filtrate were separated on Eclipse XDB-C₈ column by gradient elution with mixed solutions composed of 1% (volume fraction) formic acid containing 5 mmol·L^-1 ammonium acetate, methanol and water at different volume ratios, detected by multiple reaction monitoring mode and positive ion mode of electrospray ion source, and quantified by internal standard method. It was shown that the retention of amantadine and ribavirin on the Eclipse XDB-C₈ column was strong, and the peaks appeared at 4.30 min and 9.13 min, respectively. The linear ranges of standard curves were found in the same range of 2-40 μg·L^-1, with detection limits (3S/N) of 0.2, 0.3 μg·kg^-1. Test for the spiked recovery was made on the blank chiken at 3 cencentration levels, giving results of recovery in the ranges of 70.5%-84.4% and 98.5%-99.6%, and RSDs (n=6) of the determined values were less than 5.0%.