Isolation, purification, and structure identification of glucuronic acid conjugates of propranolol and alprenolol and their ring-hydroxylated metabolites

The purpose of this study was to develop a procedure for the isolation, purification, and structure identification of glucuronic acid conjugates of propranolol and alprenolol and their active metabolites, 4-hydroxypropranolol and 4-hydroxyalprenolol. As both aliphatic and aromatic glucuronides may be formed from 4-hydroxypropranolol and 4-hydroxyalprenolol, the structure identification of these conjugates has to be based on the intact conjugates. Using DEAE-Sephadex anion-exchange chromatography, milligram quantities of these glucuronides were isolated from urine of dogs pretreated with propranolol and alprenolol, respectively. A high degree of purification was achieved by reversed-phase HPLC. Structure identification of the methyl ester-trimethylsilyl derivatives was accomplished by electron impact GC/MS. Only one 4-hydroxypropranolol glucuronide was found, having the glucuronic acid linked to the aromatic hydroxyl group. For 4-hydroxyalprenolol glucuronide, however, two structures were identified, one with the glucuronic acid linked to the aromatic hydroxyl group, and the other with the glucuronic acid linked to the aliphatic hydroxyl group of the side chain. Using this analytical approach, 4-hydroxypropranolol glucuronide was identified in the urine of patients on propranolol therapy. In man, as in the dog, only the aromatic glucuronide was found, i.e. containing an unsubstituted beta-blocking side chain.