Isotopic separations of the drug N-0437 and its diastereoisomeric glucuronides by high-performance liquid chromatography

During the investigations of the metabolic pathways of the new dopaminergic drug N-0437 we encountered a substantial difference in HPLC-retention times between the metabolites, detected by a uv spectrophotometer, and their tritium-labeled markers, measured off-line by a scintillation counter. These distinct retention times can be ascribed to a phenomenon known as isotopic fractionation. In this article we quantified the isotopic separation by reversed-phase HPLC of the unlabeled N-0437, its deuterated and tritiated analogs, and their corresponding glucuronides, synthesized in vitro by rat liver microsomes. In the separation of the glucuronides we demonstrated that this isotope effect is dependent largely on the eluent pH.