Determination of clenbuterol in urine of calves by high-performance liquid chromatography with in series ultraviolet and electrochemical detection

Abstract

A method for the determination of clenbuterol in calf urine is described. After a simple two-step sample pretreatment, involving an Extrelut-3 column and a solid-phase extraction column (C2), the separation of clenbuterol from interfering compounds present in urine samples was performed with ion-pair chromatography on a LiChrospher RP-Select-B column with a mixture of acetonitrile and sodium dodecyl sulphate/acetate buffer (pH 3.5) as mobile phase. To obtain a higher specificity, two different physico-chemical detection techniques, i.e. UV-absorption (244 nm) and electrochemical detection (+1250 mV), were applied in series. The lowest limit of determination was 0.5 ng ml-1 and the mean recovery of clenbuterol spiked at 10 ng ml-1 level was 79.9% (RSD = 6.3%; n = 9). The analysis of one urine sample, including sample preparation, took less than 2 h. Results obtained with this method correlated well with GC-MS analysis. With the described method about 400 urine samples were analysed. In a pilot experiment, in which a calf received orally 4 micrograms clenbuterol.HCl per kilogram body weight twice a day (five times the therapeutic dose for oral application) for 5 days, the highest concentration of clenbuterol found in urine was 73 ng ml-1. In a second experiment, in which two calves received the therapeutic dose of clenbuterol.HCl twice a day over a period of 2 weeks, the highest concentration of clenbuterol was 75 ng ml-1 of urine. Eight days after the final application, concentrations of clenbuterol were lower than 0.5 ng ml-1. From this excretion study for clenbuterol a half-life value of approximately 1.5 days was calculated.