This application note demonstrates sensitive and reproducible HPLC analysis of N-linked glycans of human immunoglobulin G using the Agilent 1260 Infinity Bio-inert Quaternary LC System with fluorescence detection. The precision of retention times and areas was determined for analysis of a N-linked human IgG glycan library. In addition, good linearity in a range from 0.008 to 1 pmol was determined for a mix of five glycan standards (Man5, G2, G2S1, G2FS1, and G2S2) with low limits of detection and quantification. The Agilent 1260 Infinity Bio-inert Quaternary LC System with fluorescence detection is an optimal system for sensitive and reproducible analysis of 2-AB derivatized N-glycans released from human immunoglobulin G.
This Application Note demonstrates the use of the Agilent 1260 Infinity Bio-inert Quaternary LC System combined with fluorescence detection for the sensitive and reproducible analysis of N-linked glycans derived from human immunoglobulin G (IgG). The study focuses on the separation of 2-AB derivatized N-glycans released from human IgG using Hydrophilic Interaction Chromatography (HILIC)-HPLC.
The document details the experimental setup, including specific Agilent modules such as the Bio-inert Quaternary Pump, High Performance Bio-inert Autosampler, Thermostat, and Fluorescence Detector. It presents results on the precision of retention times and peak areas, the linearity of the method over a range of 0.008 to 1 pmol, and the limits of detection and quantification for a mix of five glycan standards.
| Excitation wavelength | 260 nm |
|---|---|
| Emission wavelength | 430 nm |
| Column temperature | 50 °C |
| Retention time precision | below 0.16% |
| Area precision range | 0.66% to 4.11% |
| Limit of detection (LOD) | 10 to 15 fmol |
| Limit of quantification (LOQ) | 34 to 51 fmol |
The optimized method uses an excitation wavelength of 260 nm and an emission wavelength of 430 nm.
Linearity was determined in a range from 0.008 to 1 pmol using a mix of five glycan standards.