Fragmentation study on the phenolic alkaloid neferine and its analogues with anti-HIV activities by electrospray ionization tandem mass spectrometry with hydrogen/deuterium exchange and its application for rapid identification of in vitro microsomal metabolites of neferine

The application of mass spectrometry in drug discovery, especially in drug metabolites, is very important. This present paper is at first focused on the elucidation of fragmentation patterns of the phenolic bisbenzyltetrahydroisoquinoline alkaloid, neferine, together with its analogues isoliensinine and liensinine with anti-HIV activities using electrospray ionization tandem mass spectrometry (ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. All title compounds displayed major diagnostic fragments that formed by the cleavage of the C1'-C9' bond resulting in positive group CD, and the loss of 4-ethyl-1-phenol or 4-ethyl-1-methoxybenzene following rearrangements. Their ESI-MS/MS spectra also showed the relatively stable fragment ions formed by the elimination of H2O, CH3NH2, CH3OH, and CH3-N=CH2. Secondly, the metabolites of neferine from dog hepatic microsomal incubations were analyzed and characterized by high-performance liquid chromatography (HPLC) and data-dependent ESI-MS/MS. Based on fragmentation patterns and compared with their retention times in LC, molecular weights and ultraviolet (UV) absorbances with standard compounds, six metabolites were identified as isoliensinine, liensinine and four novel bisbenzyltetrahydroisoquinoline alkaloids named as 6-O-desmethylneferine, 2'-N-desmethylneferine, 2'-N-6-O-didesmethylneferine, and 6,13-O-didesmethylneferine. All metabolites were desmethyl or didesmethyl products of neferine. The possible metabolic pathways for neferine have been proposed. The results suggest that N-demethylation and O-demethylation are two important metabolic pathways of neferine in dog hepatic microsomal incubations. This is critical for screening and development of phenolic bisbenzyltetrahydroisoquinoline alkaloids with anti-HIV activities such as neferine and its analogues isoliensinine and liensinine. Copyright (C) 2007 John Wiley & Sons, Ltd.