Isolation of an unknown metabolite of capecitabine, an oral 5-fluorouracil prodrug, and its identification by nuclear magnetic resonance and liquid chromatography–tandem mass spectrometry as a glucuroconjugate of 5′-deoxy-5-fluorocytidine, namely 2′-(β- d-glucuronic acid)–5′-deoxy-5-fluorocytidine

Abstract

A new metabolite of capecitabine, a prodrug of 5-fluorouracil, was detected by 19F NMR in bile and liver of rats treated with this anticancer drug. Crude bile and perchloric acid extract of liver was subjected to liquid–liquid separation followed by a pre-purification step on a preparative octadecyl silane column (C 18). The compound was purified by HPLC optimised to allow the detection of the unknown metabolite and its assumed precursor 5′-deoxy-5-fluorocytidine (5′-DFCR). Treatment with β-glucuronidase from three sources showed that it was a glucuroconjugate of 5′-DFCR. HPLC–TIS-MS–MS and 1H NMR allowed identification of the unknown metabolite as 2′-(β- d-glucuronic acid)–5′-deoxy-5-fluorocytidine.