Quantitation of cytidine-5'-monophospho-N-acetylneuraminic acid in human leukocytes using LC-MS/MS: method development and validation.

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.

• 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
• Journal: Biomedical chromatography : BMC (Biomed Chromatogr) Vol. 34 Issue 2 Pg. e4735 (Feb 2020) ISSN: 1099-0801 [Electronic] England
• PMID: 31691999 (Publication Type: Journal Article)
• Copyright: © 2019 John Wiley & Sons, Ltd.

Chemical References

• Sialic Acids
• cytidine-5'-monophosphosialic acid
• Cytidine Monophosphate

Topics

• Chromatography, Liquid (methods)
• Cytidine Monophosphate (analogs & derivatives, blood)
• Drug Stability
• Humans
• Leukocytes (chemistry)
• Limit of Detection
• Linear Models
• Reproducibility of Results
• Sialic Acids (blood)
• Tandem Mass Spectrometry (methods)