In vitro formation of phase I and II metabolites of propranolol and determination of their structures using chemical derivatization and liquid chromatography-tandem mass spectrometry

Derivatization with 1,2-dimethylimidazole-4-sulfonyl chloride (DMISC) has been successfully used as a tool to differentiate between aromatic and aliphatic O-glucuronides of hydroxypropranolol. The analyses were performed with liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) with both a triple quadrupole and an ion trap instrument. Hydroxylated forms of propranolol can be glucuronidated in aliphatic as well as aromatic positions. These isoforms are not distinguishable by tandem MS alone, as they both initially lose 176 Da, i.e. monodehydrated glucuronic acid, giving back the aglycone. Two in vitro systems were set up for the production of propranolol metabolites. The obtained isomers of 4'-hydroxypropranolol glucuronide were determined to correspond to one aliphatic and one aromatic form, using chemical derivatization with DMISC and LC-MS(n). DMISC was shown to react with the secondary amine in the case where the naphtol was occupied by the glucuronyl moiety, resulting in a different fragmentation pattern compared with that of the aliphatic glucuronide, where the naphtol group was accessible to derivatization. Copyright (C) 2009 John Wiley & Sons, Ltd.