O-Benzylhydroxylamine (BHA) as a Cleavable Tag for Isolation and Purification of Reducing Glycans

Glycoscience has been recognized as an important area in biomedical research. Currently, a major obstacle for glycoscience study is the lack of diverse, biomedically relevant, and complex glycans in quantities sufficient for exploring their structural and functional aspects. Complementary to chemoenzymatic synthesis, natural glycans could serve as a great source of biomedically relevant glycans if they are available in sufficient quantities. We have recently developed oxidative release of natural glycans (ORNG) for large-scale release of N-glycans as free reducing glycans. While free reducing glycans can be readily derivatized with ultraviolet or fluorescent tags for high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis, it is difficult to remove tags for the regeneration of free reducing glycans without affecting the structural integrity of glycans. To address this inconvenience, we explored the use of a cleavable tag, O-benzylhydroxylamine (BHA). Free reducing glycans are easily and efficiently labeled with BHA under mild conditions, enabling UV detection during HPLC purification. Individual glycan–BHA conjugates can then be separated using multidimensional HPLC and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and MS/MS. The BHA tag can then be easily removed by palladium-on-carbon (Pd/C)-catalyzed hydrogenation to efficiently regenerate free reducing glycans with little effect on glycan structures. This procedure provides a simple and straightforward way to tag free reducing glycans for purification at a preparative scale using multidimensional HPLC and subsequently recover purified free reducing glycans.

Chromatographic methods for determination of pork biochemical indicators

The study of the qualitative composition of the muscle tissue of animals is necessary in connection with the influence of the nutrient content on the technological, taste and dietary properties of meat. The major known changes in the biochemical parameters of the animal tissues are in the content of amino acids and fatty acids, etc. The aim of the work was to compare different chromatographic methods and to identify the most effective of them for the determination of fatty acids and amino acids in pig meat. The following HPLC methods are known for analyzing food products: ultra-HPLC, «hydrophilic» chromatography, two-dimensional and multidimensional HPLC methods, chiral chromatography, express chromatography, capillary HPLC, nano-HPLC, micellar HPLC, HPLC on monolithic columns, HPLC on surface porous sorbents, immunochromatographic methods, green chromatography, high-temperature HPLC. The most common methods for the determination of amino acids and fatty acids are reversed-phase and cation-exchange HPLC.