AOAC SPIFAN Nutrients ERP (December 7, 2022)

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

Table 2: Example recipes to prepare 2AB labelling reagent

2AB [0.35 mol/L] – Picoline Borane [1.0 mol/L] in DMSO:acetic acid [70:30] solution

DMSO:Acetic Acid (70:30)

Maximum No. of Tests

DMSO:Acetic Acid (70:30) (mL)

Glacial Acetic Acid (mL)

Picoline Borane (mg)

DMSO (mL)

2AB (mg)

50

4.70

2.0

6.00

286 ± 5

625 ± 5

100

11.6

5.0

12.5

600 ± 10

1300 ± 15

250

23.3

10.0

30.0

1430 ± 15

3120 ± 15

F. Mobile Phase Preparation

Eluent A: Acetonitrile [D(g)] Eluent B: Ammonium formate (50 mM, pH 4.4): Add 2.3 g ± 0.1 g (1.89 mL) of formic acid (100 %) [D(e)] in a beaker containing 800 mL of water. Adjust the pH to 4.40 ± 0.05 with ammonium hydroxide solution (25-30 %) [D(i)]. Transfer quantitatively to a 1000-mL volumetric flask and dilute to the volume with water. This solution can be stored for up to 10 days at room temperature. G. Sample Preparation (a) For powder products, reconstitute powder or liquid concentrates according to instructions. For example, weigh 25 g of an infant formula powder into a bottle and add 200 g of water. Place the mixture in a water bath at 70 °C ± 2 °C for 20 - 25 min under constant stirring. Cool the solution to room temperature. (b) For reconstituted products (as prepared above), or products which are sold as ready to feed (RTF), weigh an amount of reconstituted product or RTF product in to a 50-mL volumetric flask, such that the concentration of HMO in the final volume is within the range of the calibration curve. Suggested dilutions are shown in Table 3. For viscous RTF products (such as adult nutritional products), high dilutions are recommended. Samples that do not contain GOS can be analysed using Preparation A [G(c)] only. Samples containing GOS, should be analysed using both Preparation A [G(c)] and Preparation B [G(d)]

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