AOAC OMB Final Action Recommendation (December 2019)-2016.14

2016.14 (October 2019) – FOS-03 METHOD

FOR ERP USE ONLY DO NOT DISTRIBUTE

(h) Hydrolysis of Fructans.— Transfer 700 µL of the eluate from the SPE column H(g) into a 1.5-mL

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microtube (marked “Sample”), add 200 µL sodium acetate buffer D(c) and 100 µL fructanase

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enzyme mixture D(k) . Into a second microtube (marked “Blank”) transfer 700 µL of the eluate

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H(g) and add 300 µL sodium acetate buffer D(c) . For all tubes (blank and sample), mix well

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(vortex) and incubate at 40 °C for 40 min. Note: The “Blank” is necessary only for some matrices

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and may be skipped if it has already been established in the laboratory that the blank measurement

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has a negligible impact on the result of a given sample. After cooling, centrifuge at 10 000 × g for

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5 min and then transfer 700 µL of the supernatant into a vial suitable for the instrument

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autosampler. NOTE: Adapt these conditions (buffer, time and temperature) according to the

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enzyme manufacturer recommendations.

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I.

Chromatographic Conditions (using PA20)

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If using a CarboPac PA1 column (or equivalent), skip this section and go to section J. 267

The HPAEC-PAD system is equipped with a CarboPac PA20 (3 × 150 mm, 6.5 µm) analytical column, or 268

equivalent. The column is held at 30 °C and the injection volume is 25 µL. NaOH (300 mM) is added post- 269

column (before the PAD detector) at a flow rate of 0.2 mL/min, using a T-piece and a 125-µL reaction coil 270 (or equivalent). Fructose and glucose are separated using the gradient described in Table 2016.14D . 271 Carbohydrates are detected by pulsed amperometry using a gold working electrode and an Ag/AgCl 272 reference electrode, and an appropriate waveform for the detection of carbohydrates. An example of a 273 suitable waveform is given in Table 2016.14E . 274

Table 2016.14D. HPAEC-PAD gradient for CarboPac PA20 analytical column (or equivalent) 275

Water,

Flow rate, mL/min

600 mM NaOH, % A

30 mM NaOH, % C

Time, min

% B

0.0

0.5

0

80

20

13