Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC-MS/MS using isotope-labeled standard calibration

Dan Wang; Xiang Zhou; Lin Wang; Sihe Wang; Xue-Long Sun

doi:10.1016/j.jchromb.2013.11.016

Quantification of free sialic acid in human plasma through a robust quinoxalinone derivatization and LC-MS/MS using isotope-labeled standard calibration

J Chromatogr B Analyt Technol Biomed Life Sci. 2014 Jan 1:944:75-81. doi: 10.1016/j.jchromb.2013.11.016. Epub 2013 Nov 14.

Authors

Dan Wang¹, Xiang Zhou¹, Lin Wang², Sihe Wang³, Xue-Long Sun⁴

Affiliations

¹ Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, United States.
² School of Pharmaceutical Science, China Medical University, 92 Bei-er Road, Shenyang, Liaoning 110001, China.
³ Department of Clinical Pathology, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195, United States.
⁴ Department of Chemistry, Chemical and Biomedical Engineering and Center for Gene Regulation in Health and Disease (GRHD), Cleveland State University, 2121 Euclid Avenue, Cleveland, OH 44115, United States. Electronic address: x.sun55@csuohio.edu.

PMID: 24291723
DOI: 10.1016/j.jchromb.2013.11.016

Abstract

We report an accurate quantification of free sialic acid (SA) in human plasma using LC-MS/MS method with isotope-labeled standard calibration (ILSC) and robust derivatization. Specifically, derivatization of SA with a stable and inexpensive 3,4-diaminotoluene (DAT) provides a stable product of SA with high MS response, proving a convenient and cost-effective LC-MS/MS analysis of free SA. In addition, the use of (13)C3-SA as calibration standard ensured the accuracy for the measurement. This assay used ultra high performance liquid chromatography (UHPLC) for separation of native/labeled SA and IS from matrix interference, and employed mass spectrometry in multiple reaction monitoring (MRM) mode for sensitive and selective detection. We have achieved a lower limit of quantification (LLOQ) of 20ng/mL and a total running time of 4.2min, which is the most sensitive and quick measurement for free SA in biomatrices.

Keywords: 12-diamino-4,5-methylenedioxybenzene; 3,4-diaminotoluene; CV; DAT; DMB; ESI; Free sialic acid; Human plasma; ILSC; IS; Isotope-labeled standard calibration; KDN; LLOQ; Liquid chromatography–tandem mass spectrometry; MRM; PAD; PPT; QC; Quinoxaline derivatization; RE; RT; SA; UHPLC; coefficient of variation; electrospray ionization; internal standard; isotope-labeled standard calibration; ketodeoxynonulosonic acid; lower limit of quantification; multiple reaction monitoring; protein precipitation; pulsed amperometric detection; quality control; relative error; room temperature; sialic acid; ultra high performance liquid chromatography.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Calibration
Chromatography, Liquid / methods*
Humans
Linear Models
N-Acetylneuraminic Acid / blood*
N-Acetylneuraminic Acid / chemistry
Quinoxalines / chemistry*
Reproducibility of Results
Sensitivity and Specificity
Tandem Mass Spectrometry / methods*

Substances

Quinoxalines
N-Acetylneuraminic Acid