title: Validation of a HPLC method for the measurement of erythrocyte encapsulated thymidine phosphorylase (EE-TP) activity.
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 100mM sodium phosphate buffer and 10mM thymidine at 37°C for 10min and the reaction stopped with 40% trichloroacetic acid. Following centrifugation, the supernatant was washed with water saturated diethyl ether, and injected onto a Spherisorb C(18) column (125mm×4.6mm, 5μm), with a mobile phase (40mM ammonium acetate, 5mM tetrabutyl ammonium hydrogen sulphate, pH 2.70) delivered at a flow rate of 1.0ml/min and run time of 8min. Ultraviolet detection (UV) was employed at 254nm. The method was linear in the range of 5-500nmol/ml (r(2)=0.992), specific with intra- and inter-day precisions of <9.6 and accuracies within ±20%. Limits of detection and quantification were 1.2nmol/ml and 10nmol/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.
authors.affiliation: Purine Research Laboratory, GSTS Pathology, London SE1 7EH, United Kingdom.
authors.forename: Lynette D
authors.initials: LD
authors.lastname: Fairbanks
authors.forename: Michelle
authors.initials: M
authors.lastname: Levene
authors.forename: Bridget E
authors.initials: BE
authors.lastname: Bax
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D057230:Limit of Detection; D012680:Sensitivity and Specificity; D013936:Thymidine / Q000032:analysis* / Q000378:metabolism; D013939:Thymidine Phosphorylase / Q000008:administration & dosage / Q000032:analysis*
chemical_list: D013939:Thymidine Phosphorylase; D013936:Thymidine