Kipper Karin, Hecht Max, Antunes Natalicia J, Fairbanks Lynette D, Levene Michelle, Kalkan Uçar Sema, Schaefer Andrew, Blakely Emma L, Bax Bridget E
Analytical Services International Ltd., St George's University of London, Cranmer Terrace, London SW17 0RE, UK.
University of Tartu, Institute of Chemistry, 14a Ravila Street, 50411 Tartu, Estonia.
J Clin Med. 2020 Mar 13;9(3):788. doi: 10.3390/jcm9030788.
Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in , leading to a deficiency in thymidine phosphorylase and a subsequent systemic accumulation of thymidine and 2'-deoxyuridine. Erythrocyte-encapsulated thymidine phosphorylase (EE-TP) is under clinical development as an enzyme replacement therapy for MNGIE. Bioanalytical methods were developed according to regulatory guidelines for the quantification of thymidine and 2'-deoxyuridine in plasma and urine using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for supporting the pharmacodynamic evaluation of EE-TP. Samples were deproteinized with 5% perchloric acid (v/v) and the supernatants analyzed using a Hypercarb column (30 × 2.1 mm, 3 µm), with mobile phases of 0.1% formic acid in methanol and 0.1% formic acid in deionized water. Detection was conducted using an ion-spray interface running in positive mode. Isotopically labelled thymidine and 2'-deoxyuridine were used as internal standards. Calibration curves for both metabolites showed linearity ( > 0.99) in the concentration ranges of 10-10,000 ng/mL for plasma, and 1-50 µg/mL for urine, with method analytical performances within the acceptable criteria for quality control samples. The plasma method was successfully applied to the diagnosis of two patients with MNGIE and the quantification of plasma metabolites in three patients treated with EE-TP.
线粒体神经胃肠性脑肌病（MNGIE）是一种由特定基因突变引起的极为罕见的疾病，该突变导致胸苷磷酸化酶缺乏，进而使胸苷和2'-脱氧尿苷在全身蓄积。红细胞包封的胸苷磷酸化酶（EE-TP）作为MNGIE的一种酶替代疗法正在进行临床开发。根据监管指南开发了生物分析方法，用于使用液相色谱-串联质谱法（LC-MS/MS）定量血浆和尿液中的胸苷和2'-脱氧尿苷，以支持EE-TP的药效学评估。样品用5%高氯酸（v/v）进行脱蛋白处理，上清液使用Hypercarb柱（30×2.1 mm，3 µm）进行分析，流动相为甲醇中含0.1%甲酸和去离子水中含0.1%甲酸。使用以正模式运行的离子喷雾接口进行检测。同位素标记的胸苷和2'-脱氧尿苷用作内标。两种代谢物的校准曲线在血浆浓度范围为10-10,000 ng/mL和尿液浓度范围为1-50 µg/mL时显示出线性（>0.99），方法分析性能在质量控制样品的可接受标准范围内。血浆方法成功应用于两名MNGIE患者的诊断以及三名接受EE-TP治疗患者的血浆代谢物定量。
