Abstract

A sensitive and simple reverse-phase high performance liquid chromatographic (HPLC) assay has been validated for the determination of thymine as a measure of thymidine phosphorylase activity encapsulated in erythrocytes (EE-TP), a formulation which is under clinical development as an enzyme replacement therapy for the treatment of mitochondrial neurogastrointestinal encephalomyopathy (MNGIE). Diluted erythrocyte lysates were incubated in 100 mM sodium phosphate buffer and 10 mM thymidine at 37 °C for 10 min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C18 column (125 mm × 4.6 mm, 5 μm), with a mobile phase (40 mM ammonium acetate, 5 mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0 ml/min and run time of 8 min. Ultraviolet detection (UV) was employed at 254 nm. The method was linear in the range of 5–500 nmol/ml (r2 = 0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2 nmol/ml and 10 nmol/ml, respectively. The method was applied to quantify thymidine phosphorylase activity in samples of in-process controls and batches of EE-TP manufactured for clinical use