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. GNE 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 ManNAc and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography (HILIC) 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 (LLOQ) 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.