Quantitation of plasma thymidine by high-performance liquid chromatography—atmospheric pressure chemical ionization mass spectrometry and its application to pharmacodynamic studies in cancer patients

Abstract

A novel method employing high-performance liquid chromatographic–mass spectrometry analysis (LC–MS) has been developed and validated for the quantitation of plasma thymidine (TdR). It involves a plasma clean-up step with strong anion-exchange solid-phase extraction (SAX-SPE) followed by HPLC separation and atmospheric pressure chemical ionization mass spectrometry detection (APCI–MS) in a selected-ion monitoring (SIM) mode. The ionization conditions were optimised in negative ion mode to give the best intensity of the dominant formate adduct [ M+HCOO] − at m/ z 287. Relative retention times were 6.5 and 8.5 min for thymidine and 5-iodo-deoxyuridine, an iodinated analogue internal standard (IS), respectively. Peak area ratios of thymidine to IS were used for regression analysis of the calibration curve. The latter was linear from 1.0 to 100 ng/ml using 1 ml sample volume of plasma. The average recovery was 80.0 and 62.8% for thymidine and 5-iodo-deoxyuridine, respectively. The method has sufficient sensitivity, precision, accuracy and selectivity for routine analysis of human plasma thymidine concentration with a lower limit of quantitation (LOQ) of 1 ng/ml. The method was used to measure plasma thymidine in healthy volunteers and cancer patients as well as laboratory rodents. Preliminary studies in cancer patients treated with the new fluoropyrimidine analogue capecitabine ( N 4-pentoxycarbonyl-5′-5-fluorocytidine) found that plasma thymidine was reduced after 1 week of treatment, consistent with inhibition of thymidylate synthase (TS). Marked differences between plasma thymidine in humans as compared to mice and rats confirms the observation that studies of anticancer drugs targeting TS may be confounded by differences in thymidine salvage between these species. In conclusion, the method developed is highly selective and sensitive and enables the study of thymidine pharmacodynamics in several species.