Simple Separation and Detection Techniques for the Analysis of Carbohydrates

Carbohydrates are diffi cult to analyze because they are very polar compounds, exhibit similar structural characteristics, and do not have a suitable chromophore. Their analysis is often performed directly using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD) with a high pH mobile phase. This technique has been approved for use in a variety of offi cial methods for the analysis of carbohydrates in foods. Another common technique is to derivatize the sample with a suitable fl uorescent tag and use HPLC with fl uorescence detection for the analysis. This approach can assist with the separation of sugar isomers and provide sensitive detection, although derivatization can contribute to increased assay variability. HPLC column technology for the separation of carbohydrates has advanced during the past several years. New hydrophilic interaction liquid chromatography (HILIC) mode separations are providing a unique way to separate polar carbohydrates using simple organic/water mobile phases. This chromatographic approach not only provides suitable separation, but also enables the use of sensitive nebulizer-based detectors (i.e., mass spectrometry [MS] or charged aerosol detection). The work presented here describes the use of the Thermo Scientifi c Dionex CoronaTM Charged Aerosol Detector (CADTM) for the measurement of simple carbohydrates. The method has a limit of detection of <10 ng on column and a wide dynamic range that covers ng to μg levels with high reproducibility. The determination of more complex carbohydrates—for example, glycans liberated from glycoproteins—can be achieved using a combination of MS and charged aerosol detection. Because the Dionex charged aerosol detector is a universal detector, there is no need to form fl uorescent tagged derivatives, thus simplifying their analysis. The Dionex charged aerosol detector is used for quantitative analyses while the MS is used for structural verifi cation. Data showing the utility of the LC-MS-CAD platform for analysis of glycoproteins is presented.