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.

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

Fig. 1

Chemical structures of N acetylmannosamine (ManNAc), ManNAc-13C-d3, N acetylneuraminic acid (Neu5Ac), and Neu5Ac-d3.gr1

Fig. 2

Post-column infusion chromatograms of Neu5Ac-d3 after injection of (A) a plasma sample extracted with protein precipitation (PPT) and (B) a plasma sample extracted with phospholipid (PL) removal plate. A chromatogram of (C) Neu5Ac was overlaid to show the retention time.gr2

Fig. 3

Representative calibration curves of ManNAc and Neu5Ac in 5% BSA. A linear 1/x2 weighted regression model was used for ManNAc and a quadratic 1/x2 weighted regression model were used for Neu5Ac.gr3

Fig. 4

Representative chromatograms of ManNAc in (A) blank 5% BSA, (B) blank human plasma, (C) an LLOQ sample in 5% BSA, (D) an incurred sample, and (E) internal standard in human plasma.gr4

Fig. 5

Representative chromatograms of Neu5Ac in (A) blank 5% BSA, (B) blank human plasma, (C) an LLOQ in 5% BSA, (D) an incurred sample, and (E) internal standard in human plasma.gr5

Fig. 6

The incurred sample reanalysis results from the 30 samples tested demonstrated assay reproducibility (96.7% [29 of 30] for ManNAc and 100% [30 of 30] for Neu5Ac).gr6