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Direct determination of S ‐(−)‐ and R ‐(+)‐propranolol glucuronide in rat hepatic microsomes by RP‐HPLC
Author(s) -
Yu Lushan,
Luan Lianjun,
Shao Qing,
Zeng Su
Publication year - 2004
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.397
Subject(s) - chemistry , propranolol , chromatography , glucuronidation , glucuronide , microsome , high performance liquid chromatography , enantiomer , metabolism , in vitro , biochemistry , stereochemistry , endocrinology , medicine
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.