Direct determination of S ‐(−)‐ and R ‐(+)‐propranolol glucuronide in rat hepatic microsomes by RP‐HPLC

Propranolol, available commercially as a racemic mixture, is a non‐selective β ‐adrenergic blocking agent used in the treatment of hypertension, angina pectoris and cardic arrhythmias. We have developed and validated an RP‐HPLC assay method for direct determination of R ‐(+)‐ and S ‐(−)‐propranolol glucuronide in rat hepatic microsomes to investigate the enantioselectivity of propranolol glucuronidation metabolism. A baseline separation of propranolol glucuronide enantiomers was achieved on a 5 µm reversed‐phase ODS column, with a mixture of phosphate buffer (pH 3.5, 0.067 mol/L) and methanol (55:45, v/v) as mobile phase. Ultraviolet detection was set at 220 nm, and p ‐nitrobenzoic acid was used as internal standard. The standard curve of assay for R ‐(+)‐ and S ‐(−)‐propranolol glucuronide in spiked microsomal incubate showed good linearity throughout the concentration range from 0.50 to 20.0 µmol/L. The analytical method affords average recovery of 99.8 and 100.1% for R ‐(+)‐ and S ‐(−)‐propranolol glucuronide, respectively. The method provides a high sensitivity and good precision for R ‐(+)‐ and S ‐(−)‐propranolol glucuronide (RSD < 10%). The LOD was 0.15 µmol/L and the LOQ was 0.5 µmol/L (RSD < 8%, n = 5) for both R ‐(+)‐ and S ‐(−)‐propranolol glucuronide. The method is simple, precise and accurate, and is suitable for quantifying the propranolol glucuronides enantiomers in rat hepatic microsomes. Copyright © 2004 John Wiley & Sons, Ltd.