HPLC-UV analysis of thymidine and deoxyuridine in plasma of patients with thymidine phosphorylase deficiency

We present a simple, fast and validated method for the determination of the two nucleosides thymidine (dThd) and deoxyuridine (dUrd) in plasma of patients with symptoms suggestive of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), using high performance liquid chromatography coupled with ultraviolet spectrophotometric detection (HPLC-UV). Plasma sample (100 μL) pretreatment was based on simple deproteinization by 1.2 M perchloric acid, using theophylline as internal standard (I.S.). HPLC-UV analysis was carried out on a Synergi 4 μm Hydro-RP, 150 × 4 mm I.D. column, at room temperature. The mobile phase was a mixture of potassium dihydrogen phosphate buffer (20 mM, pH 4.5) and acetonitrile (95:5, v/v), at an isocratic flow rate of 0.7 mL/min. The UV detector was set at 267 nm. The chromatographic run lasted 19 min. Similar pyrimidine nucleotides and nucleosides do not interfere with the assay. Calibration curves were linear for both dThd and dUrd over a range of 0.5 to 5.0 μg/mL. The limit of quantitation was 0.5 μg/mL for both nucleosides and the absolute recovery was >90% for dThd, dUrd and the I.S. Both intra- and inter-assay precision and accuracy were lower than 10% at all tested concentrations. The proposed method was successfully applied to measure plasma concentrations of dThd and dUrd in two MNGIE patients. This assay simplifies both plasma pretreatment and chromatographic conditions of previously reported procedures and describes the first validated method for the determination of the two nucleotides in human plasma.