Modified high-performance liquid chromatography assay for the measurement of 2 '-deoxyuridine in human plasma and its application to pharmacodynamic studies of antimetabolite drugs

A new method is presented for the HPLC determination of plasma 2'-deoxyurid ine (dUrd). Briefly, 1 ml of human plasma is deproteinised with perchloric acid followed by purification by solid-phase extraction using a non-polar h igh-capacity polymeric sorbent. The dUrd is separated on a C-18 reversed-ph ase column using a mobile-phase of 0.05% v/v trifluoroacetic acid in water, with a retention time of 8.5 min at a how-rate of 1.25 ml min(-1). Quantit ation is by UV detection at 261 nm using a photodiode array detector. The L imit of quantitation is 6 nM with a Linear response over the measured range 6-400 nM. Both intra- and inter-day RSD and bias are typically less than 1 3%. Chromatograms and pharmacodynamic data from a Phase 1 Clinical Trial of a new antifolate drug, ZD9331 are included to illustrate the utility of th e method. They show the increase in circulating dUrd as a result of drug in hibition of the target enzyme thymidylate synthase. The method has the sign ificant advantages of ease and simplicity over earlier methods and may be a pplied to the analysis of other nucleoside species. (C) 2000 Elsevier Scien ce B.V. All rights reserved.