2.2. HMO Profiling

Frozen milk samples were thawed by storage at 4 °C overnight. The thawed samples were mixed and two 0.5 mL aliquots were taken. Each aliquot was diluted with the same volume of water. The diluted samples were centrifuged (20,000× g, 4 °C, 20 min) to separate the fat from the serum.

The extraction of the milk oligosaccharides was performed by solid phase extraction (SPE) using a graphitised carbon solid phase extraction cartridge (Supelclean ENVI-Carb, Supelco, Darmstadt, Germany, 3 mL). The SPE cartridges were first conditioned by passing 1.5 mL of 80% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid (TFA), followed by washing with 1.5 mL of water. An aliquot of 100 µL milk serum was loaded onto the column, followed by 1.5 mL water for washing. Lactose and 3′-fucosyllactose (3′-FL) were eluted using 3 mL 3% (v/v) acetonitrile (Fraction A). The rest of the oligosaccharides were eluted in 1.5 mL 40% (v/v) acetonitrile containing 0.05% (v/v) TFA (Fraction B). Both fractions were dried under a stream of nitrogen and redissolved in 500 µL water.

Analysis of 3′-FL was performed on the supernatant of fraction A after ten times dilution, using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD; Dionex ICS5000, Thermo Scientific, Landsmeer, The Netherlands) equipped with CarboPac PA1 column (2 × 250 mm) and a guard column (2 × 50 mm) (Thermo Scientific). The flow rate was kept at 0.3 mL/min. The 3′-FL was eluted in a 4 min isocratic elution of 20 mM sodium acetate in 0.1 M sodium hydroxide. After every analysis, the column was cleaned using 1 M sodium acetate in 0.1 M sodium hydroxide for 5 min, followed by 11 min re-equilibration under the starting condition. Quantification of 3′-FL was performed based on a standard range of 3′-FL from 1 to 20 µg/mL.

Reduction step: the oligosaccharides in Fraction B were reduced by mixing 200 µL Fraction B with 200 µL 0.5 M sodium borohydride, followed by overnight incubation at room temperature. The reduced oligosaccharides were then extracted from the mixture using a graphitised carbon solid phase extraction cartridge (Supelclean ENVI-Carb, Supelco, 3 mL). The SPE cartridges were conditioned and washed as described above. After loading the reduced sample, excess reagent was eluted using 6 mL of water. The reduced oligosaccharides were then eluted in 40% (v/v) acetonitrile containing 0.05% (v/v) TFA, dried and redissolved in 400 µL water. Mixtures of oligosaccharide standards (200 µL) containing 20 µg/mL of each standard compound were treated in the same way as the samples.

UHPLC-MS analysis: the samples and standards after reduction were diluted two times, followed by analysis of the oligosaccharides on a Vanquish UHPLC system (Thermo Scientific) equipped with a Hypercarb column (100 × 2.1 mm, Thermo Scientific) maintained at 25 °C. The eluents were UHPLC grade water (Biosolve, Dieuze, France) containing 1% (v/v) acetonitrile and 0.1% (v/v) formic acid (Eluent A), and UHPLC grade acetonitrile containing 0.1% (v/v) formic acid (Eluent B). The elution was started by an isocratic elution of 3% (v/v) B for 5 min, followed by a 1%/min gradient to 20% (v/v) B and sequentially 2%/min gradient to 40% (v/v) B. The flow rate was 0.2 mL/min. Afterwards, the column was cleaned in 100% B for 10 min at 0.3 mL/min, followed by re-equilibration at the starting condition for 10 min at 0.3 mL/min and 11 min at 0.2 mL/min.

Detection of the oligosaccharides was performed using an LTQ VelosPro Mass Spectrometer (Thermo Scientific) set to negative mode, with an m/z range of 300–2000. Capillary temperature was set at 250 °C, and the source heater temperature was 50 °C. The MS was tuned using maltotetraose. Quantification was performed based on the average peak area from standards injected in the beginning, middle and end of the sample analysis.