Rapid and Simultaneous Extraction of Propranolol, Its Neutral and Basic Metabolites from Plasma and Assay by High-performance Liquid Chromatography

A high-performance liquid chromatographic method is described for the determination of propranolol, its neutral and basic metabolites from a single plasma sample. These analytes were extracted simply and efficiently by a solid-phase extraction column based on C18 modified silica (C18 Bond-Elut). Propranolol, the 4-hydroxy and N-desisopropyl metabolites were separated on a mu Bondapak C18 column with a mobile phase of acetonitrile--0.1% phosphoric acid. Propranolol glycol was selectively eluted from the C18 Bond-Elut column with acetonitrile and chromatographed separately with a mobile phase of acetonitrile--water. The recoveries of propranolol and all metabolites were greater than 78% with an intra-assay coefficient of variation between 4.9 and 7.3% at a concentration of 5-50 ng/ml. The minimum detectable levels in 1 ml of plasma were 1.0 ng/ml propranolol, 6.0 ng/ml 4-hydroxypropranolol, 1.0 ng/ml N-desisopropylpropranolol and 2.5 ng/ml propranolol glycol. Enzyme hydrolysis, Bond-Elut extraction and high-performance liquid chromatography revealed that propranolol, the neutral and basic metabolites were extensively conjugated in dog plasma (propranolol 67%, 4-hydroxypropranolol 98%, N-desisopropylpropranolol 55% and propranolol glycol 80%). With the use of pure enzymes and a selective inhibitor the nature of this conjugation appeared to involve both glucuronidation and sulfation. The conjugation of propranolol involved mainly glucuronidation (58-62%) compared to sulfation (7-12%), whilst that of 4-hydroxypropranolol mainly involved sulfation (55-65%) compared to glucuronidation (32-38%). The values for N-desisopropylpropranolol and propranolol glycol were 26-31% and 12% sulfation, 16-29% and 68-85% glucuronidation, respectively.