Carbohydrates are carbon sources essential for cell growth and product synthesis and are commonly analyzed in the food and beverage and biopharmaceutical/pharmaceutical industries. Fast and accurate determination of carbohydrates is vital for food and beverage quality control, nutritional labeling, and identification of possible adulteration. In the biopharmaceutical industry, characterization of glycoproteins routinely involves carbohydrate analysis, and changes in protein glycosylation are often studied in cancer research to identify potential biomarkers. There are over 30 approved glycoprotein-based biodrugs on the market, and the number is increasing rapidly.

The U.S. FDA and the European Medicines Agency have increased pressure on biopharmaceutical manufacturers to demonstrate satisfactory programs for understanding, measuring, and controlling glycosylation in glycoprotein-based drugs. Carbohydrates are difficult to analyze due to their highly polar nature, similar structural characteristics, and lack of a suitable chromophore. By using HPAE-PAD, manufacturers can easily and effectively separate and quantify underivatized carbohydrates.

Carbohydrate analysis
Block diagram of a capillary Reagent-Free HPAE-PAD system with electrochemical detection
Block diagram of a capillary Reagent-Free HPAE-PAD system with electrochemical detection.

HPAE-PAD – High-Performance Anion Exchange chromatography with Pulsed Amperometric Detection – is a technique that was developed specifically for the separation and detection of carbohydrates. Unlike other carbohydrate analysis techniques, HPAE-PAD allows for highly selective separation and quantification of non-derivatized carbohydrates.

High-Performance Anion Exchange (HPAE) chromatography is used to separate analytes that are ionized under high pH conditions, such as carbohydrates (pKas are usually between 12-13). This is accomplished through hydroxide-based eluents and a strong anion-exchange stationary phase. The columns’ nonporous resins have small anion-exchange microbeads carrying the anion-exchange functional groups. These small anion-exchange microbeads are permanently attached electrostatically to a larger cation-exchange resin particle. The nonporous nature of the resin minimizes band-broadening and allows for highly effective separation of a wide variety of carbohydrates, including branched oligosaccharides.

Pulsed Amperometric Detection (PAD) allows for the detection of underivatized analytes. A series of potentials known as a waveform results in oxidizing and reducing conditions on the electrode surface. This causes the analytes to be oxidized at the working electrode surface, which automatically cleans and prepares the electrode for detection. Detection is sensitive and highly selective for electroactive species, as pulse amperometry only detects compounds which contain the functional groups that are oxidized at the detection voltage utilized. With the optimized pulse settings, PAD can be made to be highly specific for carbohydrates. Many potentially interfering species cannot be oxidized or reduced, so they are not detected. In addition, neutral or cationic sample components in the matrix elute in, or close to, the void volume of the column. As a result, the carbohydrate components of interest are not impacted even if the neutral or cationic sample components are oxidized.

Determination of glycoprotein sialic acid composition using HPAE-PAD

Determination of sialic acid content of a glycoprotein is crucial when assaying for therapeutic efficacy and safety. HPAE-PAD allows for direct detection of sialic acids when they are released from the glycoprotein.

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Structural characterization of mucin O-linked glycans using HPAE-PAD-MS

There is an increasing demand for reproducible, fast, and simple methods for glycan profiling in the biologics and food industries. HPAE-PAD can be leveraged for structrual analysis of various native glycans.

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Improved carbohydrate analysis of agave syrup using HPAE-PAD

Knowledge of the chemical composition of foods is important for both human health and authenticity. HPAE-PAD can be used for accurate carbohydrate determination in products such as agave syrup.

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