Stereoselective high-performance liquid chromatography determination of propranolol and 4-hydroxypropranolol in human plasma after pre-column derivatization

Abstract A stereoselective reversed-phase HPLC assay to quantify S -(−) and R -(+) enantiomers of propranolol and 4-hydroxypropranolol in human plasma was developed. The method involved liquid–liquid extraction for sample clean-up and employed 2,3,4,6-tetra- O -acetyl-β-glucopyranosyl isothiocyanate as a pre-column chiral derivatization reagent. The internal standard used was 4-methylpropranolol. The derivatized products were separated on an Altex C 18 column using a mixture of acetonitrile–water–phosphoric acid–triethylamine (58:42:0.1:0.06 and 50:50:0.15:0.06, v/v, for propranolol and 4-hydroxypropranolol, respectively) as mobile phase. The detection of propranolol derivatives was made at λ ex =280 nm and λ em =325 nm, and the corresponding 325 and 400 nm were used for 4-hydroxypropranolol derivatives. The assay was linear from 1 to 100 ng/ml and from 2 to 50 ng/ml using 0.5 ml of human plasma for propranolol and 4-hydroxypropranolol enantiomers, respectively. The present assay is used to quantify the enantiomers of propranolol and 4-hydroxypropranolol, respectively, in human plasma for pharmacokinetic studies.