Determination Of Phenobarbital Sodium In Animal Feeds

Phenobarbital Sodium is forbidden to be used in animal feeds and drinking water according to the 176 announcement of ministry of agriculture. At present, there is no method and criterion to use for determining Phenobarbital Sodium. There are hidden risks for quality supervision and healthful eating. Establishing the detection method of Phenobarbital Sodium allows of no delay.Phenobarbital Sodium is weakly acidic salt which would be hydrolyzed to Phenobarbital Sodium in the acidic environment. After pretreatment by n-hexane, Phenobarbital Sodium in the feed sample was transformed to Phenobarbital which was extracted by(HCl+CH3OH)solution. The extracting solution was purified by Waters Oasis HLB SPE, then, leached by CH3OH+H2O(2+98), and eluted by Ammonia+ CH3OH(5+95). The eluent was dried by nitrogen. The liquid sample was detected by HPLC after residue dissovled in CH3OH+H2O(2+98). A C8 chromatographic column of 5 μm,250 mm×4.6 mm was selected for analyzation. The target object was detected by VWD at 215 nm on the condition of 30 ℃. Gradient elution was used at the flow rate of 1 mL/min. The content of Phenobarbital Sodium was obtained through formula calculating and transforming. It was indicated that the LOD of HPLC was 0.5mg/kg and the LOQ of HPLC was 1mg/kg. The method had good degree of accuracy and precision. It was also showed that an extensive applicability of mixed feed, premixed feed, concentrated feed as well as concentrate supplement with recovery of 80%-120% and RSD of 10% below. The points of standard curve with a related coefficient uo to 0.999 was 0.5μg/mL、1.0μg/mL、2.0μg/mL、5.0μg/mL、10.0μg/mL 、 20.0μg/mL and 50.0μg/m L. At these conditions, drugs such as diazepam,secobarbital etc. had no effect on the determination of Phenobarbital Sodium.The LC-MS-MS method was studied on the basis of the research of HPLC. The conditions of extraction, purification and concentration were further optimized. A C18(100mm×2.1 mm) column was selected for the seperation of Phenobarbital. At a flow rate of0.2 mL/min and 40 ℃, Phenobarbital was seperated to be detected on MS-MS. The Qualitative and quantitative monitoring ions of Phenobarbital were 231.0>84.9 and 231.0>188.1. The capillary outlet voltage was 90 V and the collision energy was 5eV �'� 3eV. The standard curve has a good linear correlation with a related coefficient of 0.997 while the standard series were 0.05μg/mL,0.1μg/mL,0.2μg/mL,0.5μg/mL,1.0μg/mL,2.0μg/m L.The final content of Phenobarbital Sodium was obtained by calculation and transformation. It was shown that the LOD and LOQ of LC-MS-MS were 0.1mg/kg and 0.2mg/kg, respectively.The method applied to mixed feed, premixed feed, concentrated feed and concentrate supplement while the recovery was 60%-120% and RSD was 20% below. This LC-MS-MS method provides basis for the the qualitative and quantitative detection of Phenobarbital Sodium.