AOAC SPIFAN Stakeholder Panel Meeting Book (September 7, 2019)

Analysis of HMO

Examples of techniques applied for HMO analysis • (RP)-HPLC • HPAEC • Hydrophilic Interaction chromatography (HILIC) • Capillary Electrophoresis (CE) • Lectin affinity chromatography • Porous graphitized carbon chromatography (PGC)

In combination with (examples) • UV • RI • FLD (fluorescence detection) / LIF (laser induced fluorescence detection) • PAD (pulsed amperometric detection) • MS • NMR

Depending on the technique derivatization/labeling may or may not be needed

Long sample preparation Typical analysis of HMO in milk

• Centrifugation • Washing with organic solvents (parallel precipitation of proteins) • Washing with organic solvents (EtOH, CHCl 3 /MeOH, Acetone, etc.) • Membrane filtration • Crystallization, • EtOH precipitation

Defatting

Protein removal

Diminishing lactose content

Fractionation (for simplified MO pools)

• Size-exclusion chromatography

The more complex the sample preparation the longer the preparation time and risk to induce material losses.