超高效合相色谱法对克伦特罗对映体的拆分及其在猪尿中的残留分析

Chiral Separation of Clenbuterol Stereoisomer by Ultrahigh Performance Convergence Chromatography and Its Application in Residue Determination of Swine Urine

该文建立了一种超高效合相色谱法(UPC2)拆分克伦特罗对映体及测定猪尿中克伦特罗对映体含量的分析方法。试样经乙酸乙酯提取,阳离子交换固相萃取柱净化后,采用CHIRALPAK IA-3(4.6 mm×100 mm,3μm)分离,以超临界CO2与10 mol/L醋酸铵-甲醇溶液(0.5∶99.5,体积比)为流动相,流速为2.0 mL/min,梯度洗脱,检测波长为241 nm时,克伦特罗对映体的分离效果最佳,且在50~10000μg/L范围内呈良好线性,相关系数大于0.9998,方法的定量下限(S/N=10)均为1.0μg/L。猪尿中克伦特罗对映体在1.0、5.0、20.0μg/L加标水平的回收率为76.4%~94.5%,相对标准偏差(RSD)为3.6%~6.6%。方法用于克伦特罗外消旋体标准品及20份猪尿实际样品的测定,结果显示,克伦特罗外消旋体中(+)-克伦特罗和(-)-克伦特罗含量分别为5.6 mg/L和5.5 mg/L,该计算结果与文献报道基本相符。猪尿样品中均未检出两种克伦特罗对映体。该方法具有分析速度快、分离效果好、重现性好、有机溶剂消耗少等特点,可用于猪尿样品中克伦特罗对映体含量的测定,为手性药物开发、使用及相关法规的制定提供了技术支撑。

An ultrahigh performance convergence chromatography(UPC2)was established for the separation of two kinds of clenbuterol stereoisomers,which was applied to the residue analysis of swine urine samples.The experimental conditions,including back pressures,types of constant volume reagents,gradient elution conditions and types of purification columns were optimized.The best experimental conditions were as follows:the back pressure:13.8 MPa,constant volume reagent:heptane,gradient elution condition:gradient 3,purification column:PCX column.The swine urine samples were extracted with ethyl acetate,then purified on a cation exchange solid phase extraction column.The separation of two clenbuterol stereoisomers was performed on a CHIRALPAK IA-3(4.6 mm×100 mm,3μm)chiral column by gradient elution,with supercritical carbon dioxide and 10 mol/L ammonium acetate-methanol(0.5∶99.5,by volume)as mobile phases at a flow rate of 2.0 mL/min.The detection wavelenth for photo-diode array detector(PDAD)was set at 241 nm.Results showed that the linear ranges for two clenbuterol stereoisomers were between 50μg/Land 10000μg/L,with corelation coefficients greater than 0.9998.The limits of quantitation(S/N=10)for(+)-clenbuterol and(-)-clenbuterol were both 1.0μg/L.The recoveries at three spiked levels of 1.0,5.0,20.0μg/L ranged from 76.4%to 94.5%,with relative standard deviations(RSD)of 3.6%-6.6%.The method was used to detect standard clenbuterol racemates and 20 swine urine samples.Results showed that clenbuterol racemates contained two kinds of clenbuterol stereoisomers,i.e.(+)-clenbuterol and(-)-clenbuterol.The contents of(+)-clenbuterol and(-)-clenbuterol were 5.6 mg/L and 5.5 mg/L,respectively.The content ratio of(+)-clenbuterol and(-)-clenbuterol in standard clenbuterol raceme complied with the ratio reported in literature.Two kinds of clenbuterol stereoisomers were undetected in 20 swine urine samples.With the characteristics of rapid analysis,good separation effect,good reproducibility and less consumption of organic solvent,this method is applicable for the determination of clenbuterol stereoisomer content in swine urine samples.It provides a scientific support for the development and use of chiral drugs as well as the formulation of relevant regulations.