高效液相色谱法测定猪尿中克伦特罗对映异构体残留量

Determination of Residual Clenbuterol Enantiomers in Swine Urine by High Performance Liquid Chromatography

建立了测定猪尿中克伦特罗对映异构体残留量的高效液相色谱分析方法。在碱化的条件下,用乙酸乙酯提取10mL猪尿样品,提取液经稀HCl反萃取,萃取液直接过SCX固相萃取小柱,再用5%氨化甲醇洗脱,洗脱液经氮气吹干后用200μL甲醇定容。采用Astec CHIROBIOTICTM V手性色谱柱,以V(甲醇)∶V(冰乙酸)∶V(三乙胺)=99.94∶0.02∶0.04为流动相进行HPLC分析,检测波长301nm,外标法定量。克伦特罗单-对映体的峰面积与其浓度在70～5000μg/L范围内呈良好的线性;线性相关系数均大于0.9996;猪尿样品中检出限为0.30μg/L。猪尿中克伦特罗对映体在1.0～20.0μg/L范围内的添加回收率为76.3%～91.5%;相对标准偏差RSD均小于7%(n=5)。

A method was developed for the determination of residual clenbuterol enantiomers in swine urine by high performance liquid chromatography. A 10-mL sample was extracted with ethyl acetate under basic condition. Then, clenbuterol enantiomers were stripped from the extract using diluted hydrochloric acid to remove the fat. The hydrochloric acid extract was purified by SCX solid phase extraction（SPE） cartridge. The eluent was dried by nitrogen and then dissolved in 200 μL methanol. The samples were analyzed on a chiral column（Astec CHIOBIOTICTM V） with a mixture of methanol-acetic acid-triethylamine as the mobile phase and quantified with the external standard calibration curve method. The detection wavelength was set at 301 nm. Good linearities were obtained for the two enantiomers at the concentration of 70-5000 μg/L with the linear relative coefficient more than 0.9996. The recoveries for the two enantiomers in urine were 76.3%-91.5% at the fortified levels of 1.0-20.0 μg/L with the limit of detection of 0.30 μg/L. The relative standard deviations were less than 7.0%.