CD BioGlyco's special HMO Profiling services include HPAEC-PAD, RP-HPLC, HILIC, CE, LC-MS, and IM-MS. We have developed systematic solutions to provide clients with One-stop Solution for HMO Production.
The study of HMO is one of the most active research fields at present. Despite the well-known diversity and complexity of HMOs, manufactured infant formulas remain poor in terms of HMO complexity, mainly because their chemical synthesis is often a tedious and laborious process. Therefore, understanding the complexity and variability of HMO structures is not only highly relevant for the development of better food supplements, but also increases biochemical knowledge of these oligosaccharides.
With very few exceptions, HMO are formed from a reduced lactose core. Not only are HMOs structurally complex, but multiple positional and linkage isomers also be found for the same monosaccharide composition (and thus mass), further complicating their analysis.
Fig.1 Structure of HMOs. (Figueroa‐Lozano & de Vos, 2019)
CD BioGlyco provides various effective methods for structural characterization, including high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), reverse-phase high-performance liquid chromatography (RP-HPLC), hydrophilic interaction chromatography HPLC (HILIC), capillary electrophoresis (CE), liquid chromatography (LC) - mass spectrometry (MS), and ion mobility mass spectrometry (IM-MS).
Given the ability to separate isomers, HPAEC-PAD is used to separate HMO. Notably, prior isolation of neutral and acidic glycans is required.
RP-HPLC requires sample derivatization because natural HMO are usually polar and not retained by the column.
HMOs are separated by HILIC, which provides good separation of the isomers. This method requires oligosaccharide labeling, usually using 2-aminobenzamide for reductive amination.
CE is applied to HMO analysis, often requiring end-labeling.
Besides derivatization, the main disadvantage of separation-based methods is the need for standards against which retention times can be compared. Depending on the exact structure of the HMO, standards are very expensive or not available, making quality identification almost a must-have tool for analysis. Mass-based methods are used independently, often using MALDI-FTICR-MS or in combination with HPLC separations. While offline methods may be faster because no separation is required, this is a major disadvantage in itself if the sample is not completely pure, and isomeric separation is not possible without prior column separation. Therefore, the most efficient methods rely on LC separation and MS ionization and fragmentation of HMO molecular species to characterize the chemical structure.
IM-MS analysis is capable of distinguishing almost all HMO studied, especially due to the single ion mobility monitoring acquisition using a capture ion mobility spectrometer, which provides high ion mobility resolution and enhanced ion mobility separation. Combining IM-MS separations with MS/MS experiments improve performance in identifying HMO, especially isomers that are poorly separated by ion mobility alone.
CD BioGlyco provides an integrated solution for HMO structural characterization service. For decades, we have won the trust of global scientists with high-quality services, and technical support. Our company has unique and advanced technology platforms. Excellent researchers and sophisticated instruments have become our powerful assistance. If you are interested in our services, please contact us for more details without any hesitation.
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