Quantitation of Cytidine-5'-monophospho-N-acetylneuraminic Acid in Human Leukocytes Using LC-MS/MS: Method Development and Validation

Overview

Journal Biomed Chromatogr

Publisher Wiley

Specialties Biology
Chemistry

Date 2019 Nov 7

PMID 31691999

Citations 5

Authors

Meng Fang

Xin Xu

Michael Zhang

Yifan Shi

Guodong Gu

Wanjing Liu

Bradley Class

Carla Ciccone

William A Gahl

Marjan Huizing

Nuria Carrillo

Amy Q Wang

Affiliations

Soon will be listed here.

Abstract

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.

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