title: Quantitative hydrophilic interaction chromatography-mass spectrometry analysis of N-acetylneuraminic acid and N-acetylmannosamine in human plasma.
abstract: N-acetylneuraminic acid (Neu5Ac or NANA) is the most predominant sialic acid in mammals. As a terminal component in many glycoproteins and glycolipids, sialic acid is believed to be an important biomarker related to various diseases. Its precursor, N-acetylmannosamine (ManNAc), is being investigated as a potential treatment for GNE myopathy. In this work, we developed two highly sensitive and selective liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for the quantitation of ManNAc and free Neu5Ac in human plasma. A fit-for-purpose approach was adopted during method validation and sample analysis. To measure the endogenous compounds and overcome the interference from plasma samples, a surrogate matrix that contained 5% bovine serum albumin (BSA) was used for the preparation of calibration standards and certain levels of quality control (QC) samples. QC samples at higher concentrations were prepared in the authentic matrix (human plasma) to best mimic incurred samples. For both methods, an Ostro 96-well phospholipid removal plate was used for sample extraction, which efficiently removed the phospholipids from the plasma samples prior to LC injection, eliminated matrix effect, and improved sensitivity. Chromatographic separation was achieved using hydrophilic interaction chromatography (HILIC) and gradient elution in order to retain the two polar compounds. The lower limit of quantitation (LLOQ) for ManNAc and Neu5Ac was 10.0 and 25.0ng/mL, respectively. The overall accuracy of the two assays was within 100%±8.3% based on three levels of QC samples. Inter- and intra-run precision (coefficient of variation (%CV)) across three analytical runs was less than 6.7% for ManNAc and less than 10.8% for Neu5Ac. These methods have been validated to support clinical studies.
authors.affiliation: Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA. Electronic address: shiyifan@gmail.com.
authors.forename: Yifan
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
authors.affiliation: Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA.
authors.forename: Meng
authors.initials: M
authors.lastname: Fang
authors.affiliation: Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA.
authors.forename: Michael
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA.
authors.forename: Yinghe
authors.initials: Y
authors.lastname: Li
authors.affiliation: Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA.
authors.forename: Brad
authors.initials: B
authors.lastname: Gillespie
authors.affiliation: New Zealand Pharmaceuticals, 68 Weld Street, RD2, Palmerston North 4472, New Zealand.
authors.forename: Selwyn
authors.initials: S
authors.lastname: Yorke
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors.forename: Nora
authors.initials: N
authors.lastname: Yang
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors.forename: John C
authors.initials: JC
authors.lastname: McKew
authors.affiliation: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20895, USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Gahl
authors.affiliation: Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20895, USA.
authors.forename: Marjan
authors.initials: M
authors.lastname: Huizing
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors.forename: Nuria
authors.initials: N
authors.lastname: Carrillo-Carrasco
authors.affiliation: Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
authors.forename: Amy Qiu
authors.initials: AQ
authors.lastname: Wang
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D004355:Drug Stability; D006595:Hexosamines / Q000097:blood*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D019158:N-Acetylneuraminic Acid / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D006595:Hexosamines; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine
keywords: HILIC; LC–MS/MS; N-acetylmannosamine; N-acetylneuraminic acid; Phospholipid removal; Validation