AOAC 2018 Methods

Pipet 1 mL filtrate into a 4-dram vial and evaporate the solution to complete dryness under a flow of nitrogen at 55±3°C. Typical time is less than 60 min to complete dryness. This may be achieved using an N-Evap 112 nitrogen evaporator or a heating block. Once vials a re completely dry, add 10 mL water, C(a) , to each. Cap and mix on a vortex mixer for at least 15 s. Allow vials to sit for at least 15 min and repeat the vortex mixing procedure. Dilute with water, C(a) , as necessary so that all analytes are within the calibration curve. It may be necessary to perform multiple dilutions. Filter the diluted test solution using a disposable syringe and 0.45 μm GHP filter into an injection vial. Note : Solutions stored in injection vials are stable for 1 week when stored at room temperature, and the final extracts in water are stable for 2 weeks when stored refrigerated (5±3°C). G. HPAEC–PAD Parameters ( a )  Instrument configuration.—See Figure 2018.16 . ( b )  Instrument settings.—See Table 2018.16B . ( c )  Waveform.—See Table 2018.16C . ( d )  Elution settings.—See Table 2018.16D . H. System Suitability/Analytical QC Each analytical batch must be analyzed with a validated control or matrix spike and a set of calibration standards covering the analytical range. An appropriate number of equilibration injections should be injected prior to three replicate injections of the check standard. These are injected immediately before the calibration standards. The response factor (RF) relative SD (RSD, %) of the three check standard injections must be ≤3% for all analytes. The RF is the ratio of the analyte area to the internal standard area divided by the analyte concentration. Failure to meet this criterion requires further equilibration of the system before the calibration standards are injected. A single set of calibrants is injected at the beginning of each injection sequence. The WS3 is injected as a check standard after every sixth vial containing unknown (or more frequent) and at the end of the injection sequence. The measured concentration of each analyte in the check standard must be ±5% of the analyte theoretical concentration. The obtained calibration curve should Table 2018.16C. Waveform, carbohydrate quadruple potential Time, s Potential, V Integration 0.00 +0.1 0.20 +0.1 Begin 0.40 +0.1 End 0.41 –2.0 0.42 –2.0 0.43 +0.6 0.44 –0.1 0.50 –0.1

Table 2018.16D. Elution settings

Time, min Flow, mL/min A, % B, % C, % D, %

Default gradient

0.00

0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5

90 10 0 0 90 10 0 0 0 0 100 0 0 0 100 0 90 10 0 0 90 10 0 0 0 0 0 100 0 0 0 100 90 10 0 0 90 10 0 0

13.00 25.00 27.00 27.01 30.00

System standby and shutdown

0.00

20.00 20.01

21.00 0.15, 0.01 a

a  0.15, Post-run system standby; 0.01, post-run system shutdown.

have acceptable linearity with a coefficient of determination r 2 ≥ 0.998. Calibration curve residuals (relative error) must be ≤10% for the lowest calibration level and ≤5% for the other calibration levels. I. Quantification Integrate the analyte and internal standard (arabinose) peak areas in each standard and unknown. Perform calibration using 1/ x weighted linear regression in which the abscissa ( x -axis) is the external working analyte standard concentration (micrograms per milliliter) and the ordinate ( y -axis) is an RF defined as:

= A A

RF

Istd

where A =analyte peak area and A Istd = internal standard peak area. Calculate the concentration of each analyte using the following calculation:

IS C V C W C × × × ID

IS

Analyte Concentration (micrograms per gram) =

where C (micrograms per milliliter) =concentration from the calibration curve; V IS (milliliters) =volume of the internal standard added; C IS (micrograms per milliliter) =concentration of the internal standard added; W (grams) = test portion mass; and C ID (micrograms per milliliter) =final concentration of the internal standard in the calibration working standards. References: AOAC SMPR 2018.001 (Sugars in Animal Feed, Pet Food, and Human Food) J. AOAC Int . 101 , 1280(2018) DOI: 10.5740/jaoacint.SMPR2018.001 J. AOAC Int . 103 , 89(2020) DOI: 10.5740/jaoacint.19-0193 Posted: August 2019 (pre-publication); January 2020 (First Action publication)

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