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J Chromatogr B Analyt Technol Biomed Life Sci. 2015 Sep 1;1000:105-11. doi: 10.1016/j.jchromb.2015.07.018. Epub 2015 Jul 17.

Quantitative hydrophilic interaction chromatography-mass spectrometry analysis of N-acetylneuraminic acid and N-acetylmannosamine in human plasma.

Author information

1
Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA. Electronic address: shiyifan@gmail.com.
2
Therapeutics for Rare and Neglected Diseases, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA.
3
Alliance Pharma, 17 Lee Boulevard, Malvern, PA 19355, USA.
4
Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21701, USA.
5
New Zealand Pharmaceuticals, 68 Weld Street, RD2, Palmerston North 4472, New Zealand.
6
Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20895, USA.

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.

KEYWORDS:

HILIC; LC–MS/MS; N-acetylmannosamine; N-acetylneuraminic acid; Phospholipid removal; Validation

PMID:
26218770
PMCID:
PMC4544686
DOI:
10.1016/j.jchromb.2015.07.018
[Indexed for MEDLINE]
Free PMC Article

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