Quantitation of cytidine-5′-monophospho-N-acetylneuraminic acid in human leukocytes using LC–MS/MS: method development and validation
| Autor: | Bradley Class, Yifan Shi, Xin Xu, Michael Zhang, Amy Wang, Wanjing Liu, William A. Gahl, Carla Ciccone, Meng Fang, Nuria Carrillo, Guodong Gu, Marjan Huizing |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
Glycan
Clinical Biochemistry 030226 pharmacology & pharmacy 01 natural sciences Biochemistry Article Analytical Chemistry 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine Glycolipid Drug Stability Limit of Detection Tandem Mass Spectrometry Drug Discovery medicine Cytidine Monophosphate Leukocytes Humans Myopathy Molecular Biology Pharmacology chemistry.chemical_classification Chromatography biology Hydrophilic interaction chromatography 010401 analytical chemistry Reproducibility of Results Cytidine General Medicine 0104 chemical sciences Sialic acid carbohydrates (lipids) chemistry biology.protein Linear Models Sialic Acids medicine.symptom Glycoprotein N-Acetylneuraminic acid Chromatography Liquid |
| Zdroj: | Biomed Chromatogr |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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