Carbohydrate analysis using HPLC with PAD, FLD, CAD, and MS detectors

Analytical Session
David H Thomas, Thermo Fisher Scientific, Chelmsford, MA, USA
Co-author(s): Paul Ullucci and Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA, USA

ABSTRACT: Carbohydrates are a structurally diverse group of compounds that can be categorized as monosaccharides, disaccharides, oligosaccharides, glycoproteins, etc. Carbohydrates are difficult to analyze because they have similar physical and chemical characteristics and do not have a suitable chromophore for UV detection. Several different HPLC methods using various detector strategies (pulsed amperometric electrochemical, fluorescence following derivatization, charged aerosol detection, and mass spectrometry) were developed to help study carbohydrates, and examples for each approach are presented. Although fluorescent tags improve chromatographic resolution and detector sensitivity, they can lead to increased assay variability. Various HPLC modes can be used for carbohydrate separations, with ion exchange, hydrophilic interaction liquid chromatography (HILIC), and reversed phase (RP) on porous graphite column (PGC) being the most common. HPLC provides for simple chromatographic methods; direct detection using PAD or mass detectors such as ELSD, MS, and CAD are employed. CAD is an ideal detector when combined with HILIC or RP/PGC for measuring different carbohydrates. It is a mass-sensitive detector that can measure any non-volatile, and many semi-volatile compounds, typically with low ng sensitivity. Unlike ELSD, it shows high sensitivity, wide dynamic range, high precision, and more consistent inter-analyte response independent of chemical structure. For the analysis of glycans liberated from glycoproteins, the utility of the LC-MS-CAD platform is presented; CAD is used for quantitative analyses, while MS provides structural verification. The advantage of this approach over methods using fluorescent tags is discussed.

Dave Thomas received a Ph.D. degree in analytical chemistry from the University of Nebraska-Lincoln in 1994 for his work developing high-performance immunoaffinity chromatography. In post-doctoral appointments at Midwest Research Institute-California Operations and Sandia National Laboratories, he worked to implement HPIAC and HPLC approaches on miniaturized electrochromatographic separation and analysis platforms. Later, he spent several years developing a variety of IC, HPLC, and LC/MS applications at Dionex Corporation and Thermo Fisher Scientific, where he also served as manager of the HPLC and LCMS applications laboratory. After a few years in vaccine analytical development at Wyeth and Pfizer, Dave returned to Thermo Fisher Scientific, where he continues to develop applications for HPLC with charged aerosol and electrochemical detection.