AOAC SPIFAN Nutrients ERP (December 7, 2022)

HMO-02 (Analysis Paper) 10-2022 FOR ERP USE ONLY DO NOT DISTRIBUTE

H. Chromatographic Conditions

The UHPLC system is equipped with an Acquity UPLC BEH Glycan column (2.1 mm x 150 mm, 1.7 µm) or an Acquity UPLC BEH Premier Glycan column (2.1 mm x 150 mm, 1.7 µm). The column is held at 60 °C ± 2 °C and the injection volume is 2 µL. The analytes are separated using the gradient described in Table 4 and they are detected by means of a fluorescence detector tuned at the following wavelengths: excitation λ = 330 nm and emission λ = 420 nm. If samples based on goat or sheep milk or elemental formula are to be analysed for 6’SL, those samples should be injected a second time at a column temperature of 75 °C ± 2°C, for the determination of 6’SL. The calibration standards should also be injected a second time at 75 °C.

Table 4: Chromatographic Gradient Time (min) Flow (mL/min)

A (%)

B (%)

0.0

0.500 0.500 0.500 0.500 0.500 0.500 0.500

90.0 90.0 80.1 20.0 20.0 90.0 90.0

10.0 10.0 19.9 80.0 80.0 10.0 10.0

4.00

38.00 38.50 41.50 42.00 52.00

Eluent A = Acetonitrile; Eluent B = Ammonium Formate (50 mM, pH 4.4)

I. System Suitability Check

Run the LC system under the initial conditions and ensure the system pressure and detector baseline are stable. Inject the standard with the highest concentration. The height of the tallest peak should reach between 50 – 90% of full scale. Adjust the detector settings if this is not the case. Check that LNT, LNnT and 6’SL are well resolved from one another (resolution should be ≥ 1.6). If desired a sample of cow’s milk can be analysed with and without a spike of 2’FL, prepare the milk as a “ready to feed” sample. A small peak of lactose phosphate should be visible after the peak of 2’FL. The 2’FL and lactose phosphase should be separated with a resolution ≥ 1.6. If this is not the case, the column temperature can be adapted by ± 5°C. After changing temperature, check the resolution of the 2’FL and lactose phosphate as well as the resolution of LNT, LNnT and 6’SL (if these analytes are to be included in the analysis). Example chromatograms of the HMO standards spiked in to bovine milk, and of the seven HMOs in an infant fromula are shown in Figure 1. Inject 3-5 times one of the standards and check the peak areas are stable. Pay particular attention to 6’SL if it is being analysed. If the peak area is not stable, it may be nescessary to condition the column with fetuin. For fetuin conditioning, prepare a 10 mg/mL solution of fetuin, and make at least 3 injections (1 µL) of the fetuin solution running the method for HMO analysis. Repeat the injections of HMO standards to see if the peak areas are stable (repeat until stability is achieved).

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