AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (October 2019) – FOS-03 MANUSCRIPT

FOR ERP USE ONLY DO NOT DISTRIBUTE

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Then perform the following steps under gravity if possible, or up to an elution

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flow rate of 2 mL/min with positive pressure:

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c. Apply 400 µL of the enzyme treated solution H(e) or H(f) .

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d. Wash with 2 × 1 mL NaCl solution D(m) .

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e. Wash with 4 × 1 mL water.

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f. Elute the fructans into a 2-mL microtube using 4 × 400 µL SPE elute solution

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D(m) .

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g. Apply a little positive pressure to eliminate all solution from the column.

h. Mix eluates from the SPE column well (vortex). 9 (h) Hydrolysis of Fructans.— Transfer 700 µL of the eluate from the SPE column H(g) into a 10 1.5-mL microtube (marked “Sample”), add 200 µL sodium acetate buffer D(c) and 100 µL 11 fructanase enzyme mixture D(k) . Into a second microtube (marked “Blank”) transfer 12 700 µL of the eluate H(g) and add 300 µL sodium acetate buffer D(c) . For all tubes (blank 13 and sample), mix well (vortex) and incubate at 40 °C for 40 min. Note: The “Blank” is 14 necessary only for some matrices and may be skipped if it has already been established in 15 the laboratory that the blank measurement has a negligible impact on the result of a given 16 sample. After cooling, centrifuge at 10 000 × g for 5 min and then transfer 700 µL of the 17 supernatant into a vial suitable for the instrument autosampler. NOTE: Adapt these 18 conditions (buffer, time and temperature) according to the enzyme manufacturer 19 recommendations. 20 I. Chromatographic Conditions (using PA20) 21 If using a CarboPac PA1 column (or equivalent), skip this section and go to section J. 22 The HPAEC-PAD system is equipped with a CarboPac PA20 (3 × 150 mm, 6.5 µm) analytical 23 column, or equivalent. The column is held at 30 °C and the injection volume is 25 µL. NaOH 24 (300 mM) is added post-column (before the PAD detector) at a flow rate of 0.2 mL/min, using a T- 25 piece and a 125-µL reaction coil (or equivalent). Fructose and glucose are separated using the 26

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