Simultaneous Determination of Amantadine and Ribavirin Residues in Chicken by High Performance Liquid Chromatography- Tandem Mass Spectrometry with Bond Elut PBA Solid Phase Extraction Column Purification

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%.