Enantiomers of salbutamol, extracted from human urine, were successfully separated and quantitated by high-performance liquid chromatography with electrochemical detection. This direct resolution was accomplished using a chiral alpha 1-acid glycoprotein column (EnantioPac) maintained at 0 degrees C and a mobile phase consisting of a 0.1% (v/v) triethylamine in 5.3 mM citrate buffer, pH 7.2. An amperometric detector incorporating a glassy carbon electrode was employed for detection. The between-day coefficients of variation for the determination of R(-)- and S(+)-salbutamol in human urine were 4.1 and 4.7%, respectively, at a drug level of 1.0 microgram/ml. The urinary excretion ratio of the biologically active (-)-isomer to (+)-isomer in one healthy subject who received an intravenous dose of racemic salbutamol (1.0 mg) decreased continuously over a 12-h period. A similar excretion pattern exhibiting a far more extensive distortion in the enantiomeric ratio was found in three subjects dosed with a single 4-mg tablet of racemic salbutamol.