CD BioGlyco provides serval HPLC-based analysis services for our clients' requirements. Because of the important roles of HMO in biology activity, it is a hot topic in scientific research today. We have experience and achievements in the field of HMO analysis, which provides high-quality services to our clients.

Introduction

The structures of human milk oligosaccharides (HMOs) have been examined using high-performance liquid chromatography (HPLC), high pH anion-exchange chromatography (HPAEC), capillary electrophoresis (CE), nuclear magnetic resonance (NMR), and mass spectrometry (MS).

Analysis Methods

Liquid chromatography (LC)

LC is a key component of the most widely used methods for quantifying HMOs. Due to the absence of chromophores and fluorophores in oligosaccharides, the chemical modification of HMOs is commonly used to improve chromatographic retention and detection, especially with fluorescence and UV detectors. MS can be linked to LC to obtain more precise molecular information.

Fig.1 A basic LC system. (LibreTexts)

HPLC

Reverse phase HPLC (RP-HPLC) is a well-liked extraction technique for HMO measurement. Because of their hydrophilic characteristics, native oligosaccharides cannot be maintained on RP material; derivatization is therefore necessary. The derivatization process has the biggest effect on the HMOs' preservation and separation on RP-LC. Their reducing extremities have been perbenzoylated for the purpose of studying HMOs, and chromophoric active markers like 1-phenyl-3-methyl-5-pyrazolone (PMP), 2-aminopyridine (PA), and 2-aminobenzoic acid have been used as labels (2-AA). They offer a chromophore for detection in addition to being hydrophobic identifiers and enabling chromatography separation on RP stationary phases.

Fig.2 The flow chart of HPLC. (Shen, 2019)

HPLC-RI

The addition of 2'-fucosyllactose (2'FL) and lacto-N-neotetraose (LNnT) to newborn formulae in recent years has made it crucial to manage both the quality and quantity of these substances. For the quantification of 2'FL and 3FL in various food samples, including infant formula, cereal bars, and milk-based goods, a reliable, simple-to-use HPLC approach with refractive index (RI) detection has been developed. HILIC column was used for the separation, and isocratic elution was used using an acetonitrile-water mobile phase that contained triethylamine. The technique was proven to be reliable, repeatable, and validated for the absolute measurement of 2'FL and 3FL in various food supplements using HPLC-RI.

Fig.3 Schematic diagram of a RI detector. (Moldoveanu & David, 2013)

HILIC

Hydrophilic interaction liquid chromatography (HILIC) is an additional technique to successfully separate small polar compounds on polar stationary phases. The advantages of HILIC over regular NP-LC and RP-LC are numerous. In the aqueous mobile phase of HILIC, polar materials usually have excellent solubility, which overcomes the drawbacks of the commonly observed weak solubility in NP-LC. Mass spectrometry (MS) can be simply coupled to HILIC since it doesn't call for pricey ion pair reagents, especially when employed in the electrospray ionization (ESI) mode.

Fig.4 HILIC combines the characteristics of the three major methods in liquid chromatography. (Buszewski & Noga, 2011)

HPAEC-PAD

One of the well-known methods for analyzing oligosaccharides without the need for derivatization is high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). This technique can be used to separate neutral or acidic HMOs, but it prolongs the analysis time since the neutral and acidic oligosaccharides must first be pre-fractionated.

Fig.5 HPAEC-PAD for oligosaccharide analysis. (Mechelke, et al., 2017)

SPE-HPLC

For the enrichment of HMOs, offline gel filtering and solid-phase extraction (SPE) with graphitized carbon have been utilized successfully. Offline pre-treatment, however, is not the best method for sensitive and high-throughput profiling. Food safety and drug metabolic analysis have both seen extensive usage of HPLC-MS with online SPE, which has the advantages of possible high throughput, automation, and robustness.

Fig.6 Schematic illustration of the online SPE-HILIC-MS system. (Yan, et al., 2018)

HPLC-based HMO Analysis Services at CD BioGlyco

Based on the current development of HPLC-based analysis methods (such as LC, HPLC, HPLC-RI, HILIC, HPAEC-PAD, SPE-HPLC, and so on), CD BioGlyco provides several HPLC-based analysis services. We not only use single methods but also combinate using of these analysis technologies according to customers' requirements.

Advantages of Us

• We have advanced lab equipment and a research scientists' team for HMO analysis services.
• We have custom analysis services by serval methods, such as LC, HPLC, HPLC-RI, HILIC, HPAEC-PAD, SPE-HPLC, and so on.
• According to client's needs, we have the ability to provide a combination of multiple analysis method services.
• We also have experienced researchers and well-trained technicians to handle these analysis projects.

CD BioGlyco aims to help customers to study the compounds, structure, and content of HMOs, we are a great choice for HMO analysis. If you are interested in our analysis services, please feel free to contact us for more detailed information. We look forward to inquiries from around the world and our research scientists will do their best to help our clients solve their problems and meet their needs in the field of HMO research.

References:

1. LaCourse, M.E.; and LaCourse, W.R. General instrumentation in HPLC *. Liquid Chromatography. 2017, 417–429.
2. Moldoveanu, S.C.; and David, V. Basic information about HPLC. Essentials in Modern HPLC Separations. 2013, 1–51.
3. Shen, C.H. Quantification and analysis of proteins. Diagnostic Molecular Biology. 2019, 187–214.
4. Buszewski, B.; and Noga, S. Hydrophilic interaction liquid chromatography (HILIC)—a powerful separation technique. Analytical and Bioanalytical Chemistry. 2011, 402(1): 231–247.
5. Yan, J.; et al. Profiling of sialylated oligosaccharides in mammalian milk using online solid phase extraction-hydrophilic interaction chromatography coupled with negative-ion electrospray mass spectrometry. Analytical Chemistry. 2018, 90(5): 3174–3182.