HPLC-MS/MS Determination of Ractopamine in Internal Organs of Sheep

The homogenized sample of internal organ of sheep was extracted with 0.2 mol·L^-1 ammonium acetate buffer solution. After enzymatic hydrolysis for 6 h, the sample solution was cooled to room temperature and then 10 μg·L^-1 internal standard solution of ractopamine-D3 was added. The extract was removed protein with 0.1 mol·L^-1 perchloric acid solution and then extracted 2 times with ethyl acetate. The ethyl acetate layers were combined and concentrated to dryness. The residue was dissolved with 20% (φ) methanol solution and n-hexane saturated with acetonitrile was added. The lower level was taken and then filtered on 0.22 μm filtering membrane. HPLC-MS/MS was applied to the determination of ractopamine in the filtrate. Hypersil GOLD C₁₈ chromatographic column was used as stationary phase, and the mixture of 0.1% (φ) formic acid solution and methanol mixed in different ratios was used as mobile phase in gradient elution. ESI⁺ and multi-reactions monitor mode were adopted in MS/MS. Isotope was used as internal standard. Linear relationship between values of peak area and mass concentration of methanol was kept in the range of 0.25-5.0 μg·L^-1, with detection limit (3S/N) of 0.3 μg·kg^-1. On the base of blank sample, test for recovery was made by standard addition method; values of recovery found were in the range of 81.4%-90.2%, with RSDs (n= 6) of determined values in the range of 0.89%-1.4%.