AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (Jan. 2019) FOS-03 MLT Report FOR ERP USE ONLY DO NOT DISTRIBUTE

G. Preparation of Standards Using volumetric flasks, prepare a 6-level standard curve by diluting the glucose stock solution D(e) and the fructose stock solution D(f) to the final volume with water C(a), as described in Table 2016.14A . Treat each of the 6 standard solutions as follows: Into a microtube, transfer 200 µL of standard solution, add 200 µL of water C(a) and 100 µL of N,N’- diacetylchitobiose internal standard solution D(d) (use same N,N’-diacetylchitobiose solution for standards and samples) and mix well (vortex). Then transfer 400 µL of this solution to another microtube, add 1200 µL of SPE elute solution D(n) and mix well (vortex). To 700 µL of this solution, add 300 µL of sodium acetate buffer D(c) . Mix well (vortex) and centrifuge at 10 000 × g . Transfer 900 µL of the supernatant into a vial suitable for the instrument autosampler. Table 2016.14A. Dilution scheme for the preparation of the standard curve

Volume of fructose stock solution, µL

Volume of glucose stock solution, µL

Final volume,

Fructose concentration, µg/mL

Glucose concentration, µg/mL

Standard

mL 100

Level 1 Level 2 Level 3 Level 4 Level 5 Level 6

200 400 800

40

20

2

200 400 600 800

20 20 20 20 20

200 400 600 800

50

100 150 200 250

1200 1600 2000

1000

1000

H.

Sample Preparation (a) For analysis of products on a ready-to-feed basis (RTF) and for all powder products that are not homogeneous at the subgram level .—Reconstitute powder or liquid concentrates according to instructions. For example, accurately weigh 25 g ( m R ) of infant formula powder into an appropriate bottle, add 200 g ( V R ) water and mix well at room temperature to form a homogeneous suspension. Proceed as described in H(b) . (b) For analysis of reconstituted products (as prepared above), or products which are sold as RTF .—Mix well to ensure homogeneity of the test portion. Weigh 9 g ( m ) of sample into a 50-mL ( V ) volumetric flask. Add 30 mL water and confirm the pH is between 5 - 9 (adapt if needed, using 1 M hydrochloric acid or 1 M sodium hydroxide). Heat at 80 °C with constant agitation for 20 min. Cool to room temperature and make up to the mark with water (this is “Solution A”). Proceed as described in H(d) . (c) For analysis of homogeneous powder products without prior reconstitution .—Weigh 1 g ( m ) of powder into a 50-mL ( V ) volumetric flask. Add 30 mL water and confirm the pH is between 5 – 9 (adapt if needed, using 1 M hydrochloric acid or 1 M sodium hydroxide). Heat at 80 °C with constant agitation for 20 min. Cool to room temperature and complete to the mark with water (this is “Solution A”). Proceed as described in H(d) . (d) Dilution .—The solutions prepared above are further diluted depending on the expected fructan content (suggested dilutions are given in Table 2016.14B ) and the resulting solution is “Solution B”.