High-performance liquid chromatography with amperometric detection applied to the screening of beta-blockers in human urine.

A high-performance liquid chromatographic method with electrochemical detection has been developed for the determination of six beta-blockers: atenolol, nadolol, timolol, metoprolol, oxprenolol, and alprenolol. The chromatographic separation was performed using a mu Bondapack C18 column, a mobile phase of acetonitrile-water (40:60), containing 5 mM KH2PO4/K2HPO4 proved to be optimal at a 1.3 ml/min flow-rate, and a pH of 6.5. The temperature was optimized at 30 +/- 0.2 degrees C. The amperometric detector, equipped with a glassy carbon electrode, was operated at 1300 mV versus Ag/AgCl in the direct current mode. The method was applied to the determination of these compounds at two concentration levels: ppm and ppb (ng/ml), obtaining relative standard deviations lower than 5% at ppm levels and lower than 10% at ppb levels, and quantitation limits ranging from 15 ppb to 500 ppb. The method was applied to the screening of beta-blockers in spiked urine samples, with a total elution time lower than 12 min, obtaining the best recoveries for timolol and metoprolol (never greater than 93%). These recoveries together with the low limits of quantitation achieved, allows its application to doping analysis in human urine.