A method for the simultaneous determination of R- and S-propranolol and R- and S-hyoscyamine in human plasma was developed, validated and applied to the analysis of samples from a clinical study. Sample preparation was performed by solid-phase extraction of 2 ml of human plasma using Oasis MCX cartridges and the enantioselective separations were achieved using a Chirobiotic V chiral stationary phase. The chromatography was carried out using gradient elution with a mobile phase composed of methanol:acetic acid:triethylamine which was varied from 100:0.05:0.04 to 100:0.05:0.1 (v/v/v) over 30 min and delivered at a flow rate 1 ml/min. The internal standard was R,S-propranolol-d7 and the analytes were quantified using a single quadrupole mass spectrometer employing APCI interface operated in the positive ion mode with single ion monitoring. The enantioselective separation factors, alpha, were 1.15 and 1.07 for S- and R-propranolol and R- and S-hyoscyamine, respectively. The standard curves were linear for all target compounds with coefficients of determination (r2) ranging from 0.9977 to 0.9999. The intra- and inter-day precision and accuracy were <or=13.2% and <or=10.2%, respectively. The assay was used to analyze plasma samples from seven subjects who had received i.v. infusions of R,S-propranolol and R,S-hyoscyamine. The initial data indicate that R-propranolol was eliminated faster than S-propranolol (CL/f=2.34+/-0.13 L/kg min vs. 2.07+/-0.22 L/kg min) and that R-propranolol had a larger volume of distribution at steady-state (Vss/f=705+/-165 L/kg vs. 589+/-130 L/kg). In the case of R,S-hyoscyamine, S-hyoscyamine was eliminated faster than R-hyoscyamine (CL/f=0.0537+/-0.0073 L/kg min vs. 0.0439+/-0.0086 L/kg min), while the volumes of distribution at steady-state were similar for the hyoscyamine enantiomers (Vss/f=7.82+/-2.66 L/kg vs. 7.73+/-1.39 L/kg).