AOAC 2018 Methods

Pipet 1 mL this 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 are 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 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. ( b )  High-protein matrices using Carrez extraction, >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 test solutions. 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). Transfer 25 mL filtrate to an additional cup using a serological pipet. Add 5 mL Carrez I solution, C(e) , along with a stir bar and allow to stir on a stir plate for 5 min. Add 5 mL Carrez II solution, C(f) , and allow to stir on a stir plate for 10 min. Note that the solution will begin to turn cloudy. Using a pH meter, bring the pH of the test solution to a value between 7.5 and 8.0 using 1 N NaOH. Note that a precipitate will begin to form. Quantitatively transfer the solution to a 50 mL volumetric flask using water, C(a) . Bring the flask to volume using water, C(a) . Cap and invert to mix. Filter

Table 2018.16B. Instrument settings Column temperature, °C

30±2 30±2

Detector temperature, °C Analytical flow, mL/min Postcolumn flow, mL/min

0.5 1.0

See Table 2018.16C

Waveform

Injection volume, µL

10 30

Run Time (min) Elution Gradient

See Table 2018.16D

the entire volume of the 50 mL flask through qualitative filter paper and a funnel. Collect the filtrate into a plasti c cup or other suitable container. Assemble the proper number of MW filter 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. 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 are completely dry, add 5 mL water, C(a) , to each. Cap and vortex 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 solutions using a disposable syringe and 0.45 μm GHP filter into an injection vial. ( c )  High-sugar matrices, >30% individual sugar compound or >50% total sugar content on as-is 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 250 mL water, C(a) , to the flask. 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 test solutions. Add a magnetic stir bar, cap with stopper, and mix vigorously on a stir plate for approximately 60 min. Add 250 mL ethanol, C(b) , to the flask. Cap again and shake vigorously to mix. 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.

Figure 2018.16. Instrument configuration.

© 2020 AOAC INTERNATIONAL