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

KeyWord:ultrahigh performance convergence chromatography£¨UPC2£©  clenbuterol  swine urine  stereoisomer  chiral separation

Abstract:

      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£ºthe back pressure£º13.8 MPa£¬constant volume reagent£ºheptane£¬gradient elution condition£ºgradient 3£¬purification column£ºPCX 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 ¡Á 100 mm£¬3 ¦Ìm£© chiral column by gradient elution£¬with supercritical carbon dioxide and 10 mol/L ammonium acetate£­methanol£¨0.5¡Ã99.5£¬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 50 ¦Ìg/L and 10 000 ¦Ìg/L£¬with corelation coefficients greater than 0.999 8£®The limits of quantitation£¨S/N = 10£© for £¨+£©£­clenbuterol and £¨£­£©£­clenbuterol were both 1.0 ¦Ìg/L£®The recoveries at three spiked levels of 1.0£¬5.0£¬20.0 ¦Ìg/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.