Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy

Kipper, Karin; Hecht, Max; Antunes, Natalicia J.; Fairbanks, Lynette D.; Levene, Michelle; Kalkan Uçar, Sema; Schaefer, Andrew; Blakely, Emma L.; Bax, Bridget E.

doi:10.3390/jcm9030788

Open AccessArticle

Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy

by

Karin Kipper

^1,2

,

Max Hecht

^1,2,

Natalicia J. Antunes

^1,3,4,

Lynette D. Fairbanks

⁵,

Michelle Levene

⁶,

Sema Kalkan Uçar

⁷,

Andrew Schaefer

⁸,

Emma L. Blakely

⁸ and

Bridget E. Bax

^6,*

¹

Analytical Services International Ltd., St George’s University of London, Cranmer Terrace, London SW17 0RE, UK

²

University of Tartu, Institute of Chemistry, 14a Ravila Street, 50411 Tartu, Estonia

³

Department of Pharmacology, Faculty of Medical Sciences, State University of Campinas (UNICAMP), Campinas, SP 13083-881, Brazil

⁴

Clinical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK

⁵

The Purine Research Laboratory, St Thomas’ Hospital, London SE1 7EH, UK

⁶

Molecular and Clinical Sciences, St George’s University of London, London SW17 0RE, UK

⁷

Division of Inborn Error of Metabolism, Ege University Medical Faculty, 35100 Izmir, Turkey

⁸

The NHS Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne NE2 4HH, UK

^*

Author to whom correspondence should be addressed.

J. Clin. Med. 2020, 9(3), 788; https://doi.org/10.3390/jcm9030788

Submission received: 12 February 2020 / Revised: 5 March 2020 / Accepted: 11 March 2020 / Published: 13 March 2020

(This article belongs to the Section Clinical Laboratory Medicine)

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Abstract

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disorder caused by mutations in TYMP, 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 (r > 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.

Keywords: LC–MS/MS; method validation; thymidine; 2’-deoxyuridine; mitochondrial neurogastrointestinal encephalomyopathy; erythrocyte encapsulated thymidine phosphorylase; EE-TP

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MDPI and ACS Style

Kipper, K.; Hecht, M.; Antunes, N.J.; Fairbanks, L.D.; Levene, M.; Kalkan Uçar, S.; Schaefer, A.; Blakely, E.L.; Bax, B.E. Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy. J. Clin. Med. 2020, 9, 788. https://doi.org/10.3390/jcm9030788

AMA Style

Kipper K, Hecht M, Antunes NJ, Fairbanks LD, Levene M, Kalkan Uçar S, Schaefer A, Blakely EL, Bax BE. Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy. Journal of Clinical Medicine. 2020; 9(3):788. https://doi.org/10.3390/jcm9030788

Chicago/Turabian Style

Kipper, Karin, Max Hecht, Natalicia J. Antunes, Lynette D. Fairbanks, Michelle Levene, Sema Kalkan Uçar, Andrew Schaefer, Emma L. Blakely, and Bridget E. Bax. 2020. "Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy" Journal of Clinical Medicine 9, no. 3: 788. https://doi.org/10.3390/jcm9030788

APA Style

Kipper, K., Hecht, M., Antunes, N. J., Fairbanks, L. D., Levene, M., Kalkan Uçar, S., Schaefer, A., Blakely, E. L., & Bax, B. E. (2020). Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy. Journal of Clinical Medicine, 9(3), 788. https://doi.org/10.3390/jcm9030788

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Quantification of Plasma and Urine Thymidine and 2’-Deoxyuridine by LC-MS/MS for the Pharmacodynamic Evaluation of Erythrocyte Encapsulated Thymidine Phosphorylase in Patients with Mitochondrial Neurogastrointestinal Encephalomyopathy

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