Development and Validation of Analytical Method for Clenbuterol Chiral Determination in Animal Feed by Direct Liquid Chromatography

A direct chiral liquid chromatography with UV detection method was developed and validated for enantiomeric determination of clenbuterol beta(2)-agonist in feed samples for poultry. To obtain optimum chromatographic conditions for chiral separation, two commercially available columns packed with vancomycin and teicoplanin chiral stationary phase (150 x 2.1 mm i.d, 5 mu m) were tested. Experimental design and response surface analysis were used to model analytical responses and to get mobile phase composition for acceptable enantioresolution and performance in low analysis time. Different sample treatments based on HCX SPE and on the quick, easy, cheap, effective, rugged, and safe (QuEChERS) method were evaluated and compared to obtain the best extraction recoveries with negligible matrix effect. Special attention was paid to compatibility between sample preparation and chiral separation to get enantioresolution and low detection limits in the high-complex sample. QuEChERS-based extraction allowed obtaining cleaner feed extracts in low sample preparation time, providing a useful and reliable method capable to control the illegal use of clenbuterol enantiomers in feed for growth promotion in food-producing animals. Reproducible recoveries, from 64 to 101 % for clenbuterol enantiomers, with relative standard deviation values within the range 1-6 %, were achieved in feed samples of poultry spiked at low concentration levels of micrograms per gram.