AOAC 2018 Methods

( d )  Calibration standard working solutions (WS1–WS5) .— Prepare the calibration standard working solutions by combining appropriate volumes of the intermediate internal standard solution, E(b) , and the reference standard stock solution, E(c) , in separate 200 mL volumetric flasks as indicated in Table 2018.16A . Add approximately 20 mg sodium azide, C(h) , to each (mass not recorded). Bring to volume with water, C(a) , and mix each solution well. Note : Working standard solutions are stable for 6 months when stored refrigerated. F. Sample Preparation Note : All materials should undergo the appropriate processing to ensure the most representative test portion is taken for analysis. For certain drink mixes, this may require reconstitution, or for pet and animal feeds, this requires milling to achieve an appropriate particle size (e.g., 40 mesh or 0.42 mm). ( a )  Low-protein matrices, <35% total protein on dry matter basis .—Weigh 2.5±0.1 g test portion into a suitable container such as a 500 mL Erlenmeyer flask. Record mass to at least the nearest 0.001 g. Add 200 mL extraction solution, D(a) , with a bottle-top dispenser. Add 0.250 mL internal standard stock solution, E(a) , with a positive displacement pipet or electronic repeater pipet with equivalent accuracy. The same batch of internal standard stock solution, E(a) , must be used for the preparation of both standards and samples. Add a magnetic stir bar, cap with stopper, and mix vigorously on a stir plate for approximately 30 min. Add another 100 mL extraction solution, D(a) , using a bottle-top dispenser. Cap the flask and continue to stir on a stir plate for another 30 min. Add another 200 mL extraction solution, D(a) , to achieve a final volume of 500 mL. Cap again and mix well by shaking and inverting the flask. Allow sufficient time for the suspended particles to settle before filtering. Pour the solution through qualitative filter paper and a funnel and collect filtrate into a suitable container, such as a plastic cup. If needed, add Celite, C(d) , onto filter paper to aid filtration. Filtration may be further aided by transferring a portion of the test solution from the Erlenmeyer flask to an appropriately sized centrifuge tube and centrifuging until some of the solids have spun down (approximately 5 min). Assemble the proper number of MW filters for the analytical batch. Pipet 3 mL extraction solution, D(a) , into each MW filter. Cap and centrifuge at 1500× g for approximately 20 min. After 20 min, discard the filtered solution and any remaining liquid in the filter. Reassemble the MW filter and fill with approximately 4 mL filtrate. Centrifuge at 1500× g for at least 30 min or until more than 1 mL filtrate has been collected beneath the filter.

D. Reagent/Mobile Phase Preparation ( a )  Extraction solution, ethanol–water (1 + 1) .—Combine equal volumes of ethanol, C(b) , and water, C(a) , in an appropriate container. Mix thoroughly. Expiration is 6 months when stored in a sealed container at room temperature. ( b )  Mobile phase A (water) .—Measure 2000 mL water, C(a) , into a 2 L graduated cylinder. Vacuum degas or helium sparge for approximately 5 min. Place in appropriate mobile phase container and keep under inert gas while in use on the instrument. ( c )  Mobile phase B (100 mM sodium hydroxide) .—Measure 2000 mL water, C(a) , into a 2 L graduated cylinder. Transfer approximately 1900 mL to a 2 L polypropylene mobile phase container. Vacuum degas or helium sparge for approximately 5 min. Add 16±0.1 g 50% (w/w) sodium hydroxide, C(c) , to the container and add the remaining volume from the 2 L cylinder. Add a stir bar and stir slowly for 10 min. Keep under inert gas while in use on the instrument. ( d )  Mobile phase C and postcolumn eluent (200 mM sodium hydroxide) .—For each, measure 2000 mL water, C(a) , into a 2 L graduated cylinder. Transfer approximately 1900 mL to a 2 L polypropylene mobile phase container. Vacuum degas or helium sparge for approximately 5 min. Add 32.0±0.1 g 50% (w/w) sodium hydroxide, C(c) , to the container and add the remaining volume from the 2 L cylinder. Add a stir bar and stir slowly for 10 min. Keep under inert gas while in use on the instrument. ( e )  Mobile phase D (600 mM sodium hydroxide) .—Measure 2000 mL water, C(a) , into a 2 L graduated cylinder. Transfer approximately 1900 mL to a 2 L polypropylene mobile phase container. Vacuum degas or helium sparge for approximately 5 min. Add 96.0±0.1 g 50% (w/w) sodium hydroxide, C(c) , to the container and add the remaining volume from the 2 L cylinder. Add a stir bar and stir slowly for 10 min. Keep under inert gas while in use on the instrument. Note : All mobile phases expire after 1 week or earlier if removed from the blanked inert gas on the system and exposed to the atmosphere for an extended period. Sodium hydroxide can leach borate from borosilicate glass containers. Polypropylene bottles should be used for mobile phase preparation and storage. E. Preparation of Standard Solutions ( a )  Internal Standard Stock Solution (100000 µg/mL arabinose) .—Weigh 10.00±0.01 g arabinose, C(g) . Transfer to a 100 mL volumetric flask. Add approximately 10 mg sodium azide, C(h) , to the flask (mass not recorded). Add approximately 80 mL water, C(a) . Stir until completely dissolved and sonicate as needed. Bring to volume with water, C(a) . ( b )  Intermediate internal standard solution (1000 µg/mL arabinose) .—Pipet 1 mL internal standard stock solution, E(a) , into a 100 mL volumetric flask. Add approximately 10 mg sodium azide, C(h) , to the flask (mass not recorded). Dilute to volume with water, C(a) . Mix well. ( c )  Reference standard stock solution (approximately 100 µg/mL each compound) .—Weigh 0.10000±0.005 g each reference standard, C(g) , into a single 1000 mL volumetric flask. Add approximately 0.1 g sodium azide, C(h) , to the flask (mass not recorded). Fill flask about ¾ full with water, C(a) . Mix thoroughly until standard is dissolved. Sonicate the flask as necessary until all standards are dissolved. Allow flask to cool to room temperature and bring to volume with water, C(a) .

Table 2018.16A. Calibration standard preparation

Intermediate internal standard solution volume, mL

Calibration working standard

Ref. standard stock solution volume, mL

Final volume, mL

Target concn, µg/mL

Amt of NaN 3 , mg

WS1 WS2 WS3 WS4 WS5

1 1 1 1 1

1 4

20 20 20 20 20

200 0.5

200 200

2 5

10 20 30

200 10 200 15

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