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


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.


Fang, Meng; Xu, Xin; Zhang, Michael; Shi, Yifan; Gu, Guodong; Liu, Wanjing; Class, Bradley; Ciccone, Carla; Gahl, William A; Huizing, Marjan; Carrillo, Nuria; Wang, Amy;


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

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