Quantitation of cytidine-5′-monophospho-N-acetylneuraminic acid in human leukocytes using LC–MS/MS: method development and validation |
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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 |
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Institution: | 1. Alliance Pharma, 17 Lee Boulevard, Malvern, PA, USA;2. Therapeutic Development Branch, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD, USA;3. Medical Genetics Branch, National Human Genome Research Institute, Bethesda, MD, USA |
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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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Keywords: | cytidine-5′-monophospho-N-acetylneuraminic acid human leukocytes hydrophilic interaction chromatography LC–MS/MS |
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