P175 Purification of Biosynthesized Drug Metabolites by Supercritical Fluid Chromatography

Pingrong Liu , Boehringer Ingelheim, Ridgefield, CT
Zaid Temrikar , Boehringer Ingelheim, Ridgefield, CT
Heewon Lee , Boehringer Ingelheim, Ridgefield, CT
Amy Gao , Boehringer Ingelheim, Ridgefield, CT
Nina Gonnella , Boehringer Ingelheim, Ridgefield, CT
Dongyue Xin , Boehringer Ingelheim, Ridgefield, CT
Mitchell E Taub , Boehringer Ingelheim, Ridgefield, CT
Aaron Teitelbaum , Boehringer Ingelheim, Ridgefield, CT
Drug metabolite structure elucidation and synthesis is a key component of drug development for investigations regarding bioanalytical quantification of metabolites, qualification of metabolites from preclinical toxicological species, and in vitro drug metabolism studies. Traditionally, metabolite identification is performed utilizing high resolution LC-MS/MS and metabolite structures are tentatively assigned based on MS/MS fragmentation. However, even with the most advanced and sophisticated mass spectrometry instrumentation, some metabolite structures can only be partially elucidated. The complete structural elucidation of a metabolite can be achieved only when an authentic standard is available either through chemical synthesis or biosynthesis. One bottleneck of the traditional biosynthesis approach is the isolation of desired metabolites from a biological matrix in sufficient quantity and purity suitable for structure elucidation by NMR spectroscopy. Current strategies utilize liquid chromatographic based purification systems which ultimately require the complete removal of water from a sample prior to NMR analysis, a nontrivial matter when isolating only micrograms of material. Following the production of metabolites via classical liver microsomal incubations, we developed a strategy utilizing supercritical fluid chromatography, which does not require any aqueous solvent for purification and improves the overall efficiency of the biosynthesis process. We have applied this strategy during the biosynthesis of 4-hydroxyestrone ether glucuronides and a piperidine oxidative metabolite from BI-P. Subsequently, the isolated metabolites were unequivocally characterized and quantified by NMR spectroscopy. The biosynthetic process to generate, isolate, purify, elucidate, and quantify the aforementioned metabolites will be evaluated and the scope and limits of supercritical fluid purification of the biosynthesized metabolites will be discussed.