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′-deoxyuridine (dUrd). Briefly, 1 ml of human plasma is deproteinised with perchloric acid followed by purification by solid-phase extraction using a non-polar high-capacity polymeric sorbent. The dUrd is separated on a C18 reversed-phase column using a mobile-phase of 0.05% v/v trifluoroacetic acid in water, with a retention time of 8.5 min at a flow-rate of 1.25 ml min−1. Quantitation is by UV detection at 261 nm using a photodiode array detector. The limit 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 13%. Chromatograms and pharmacodynamic data from a Phase 1 Clinical Trial of a new antifolate drug, ZD9331 are included to illustrate the utility of the method. They show the increase in circulating dUrd as a result of drug inhibition of the target enzyme thymidylate synthase. The method has the significant advantages of ease and simplicity over earlier methods and may be applied to the analysis of other nucleoside species.