AOAC Final Action Methods in 2018

pH 9.0; with 1% SDS); 10 kDa protein internal standard (IS); acidic wash solution (high-purity 0.1 M HCl); basic wash solution (high- purity 0.1 M NaOH); and SDS-MW size standard (10–225 kDa, 16 mg/mL). Contents of the kit can be replaced by equivalent sources. (c)  Protein IS .—10 kDa, Part No. A26487 (Beckman Coulter, Inc.). (d)  Water .—LC grade. (e)  β-Mercaptoethanol .—Part No. M7154 or M6250 (Sigma- Aldrich). D. Preparation of System Buffer Trays (Beckman) and Standard and Sample Solutions (a)  Load reagents into system inlet (left) and outlet (right) using 6 × 6 buffer trays arranged according to configuration illustrated in Figure 2016.15A (buffer tray configuration). If using an equivalent system, follow instructions for that system. (b)  Weigh either 135 ± 5 mg skim milk powder (SMP; with protein content around 37%) or 500 ± 20 mg infant formula powder (with protein content around 11%) into a 10 mL centrifuge tube. (c)  Disperse sample to 5 mL final with deionized (DI) water. Mix each tube on a vortex mixer until solution appears homogeneous. Each final solution contains about 10–15 mg/mL protein. (d)  Prepare sample running presolution by mixing 1% SDS sample solution with 10 kDa IS peptide using an 84:1 ratio. Total volume of presolution is based on total number of samples to be analyzed in sample set (90 μL per sample). (e)  Pipet 10 μL of each sample solution into separate 2.0 mL microcentrifuge vials. (f)  Sequentially add 85 μL sample running presolution and 5 μL β-mercaptoethanol to each microcentrifuge vial. Mix well before heating vials in a water bath at 95 ± 5°C for 10 min. Cool down to

room temperature, then centrifuge at room temperature for 1 min at about 7000 rpm/5000 g . (g)  Mix on a vortex mixer before transferring each sample into corresponding injection vials. E. Sample Analysis (a)  Set up an optimized separation method for batch analysis, including one buffer blank (10 μL DI water), a molecular weight (MW) size standard, and an SMP sample. (b)  For each separation cycle (45 min), precondition the capillary first with basic wash solution for 3 min, followed by acidic wash solution for 1 min, DI water for 1 min, and SDS gel buffer for 10 min. Set up the separation run for 30 min. (c)  Introduce samples electrokinetically by applying voltage at –5 kV for 20 s. (d)  Perform electrophoresis at constant voltage with applied field strength of –497 V/cm and a capillary thermostatted to 25°C using recirculating liquid coolant. (e)  The generated current should be approximately 27 μA. (f)  To avoid reagent depletion, program the system to increment the vial position of all reagents every eight cycles. (g)  Test system suitability using the MW marker. Acceptance criteria for system suitability are as follows: ( 1 ) Migration time of the IS should be 12.3 ± 0.5 min using the instrument and capillary as per section B ( a ) and ( b ). If using an equivalent capillary or instrument, then define migration time of the IS accordingly. Migration pattern and migration times of the seven MW markers (10, 20, 35, 50, 100, 150, and 225 kDa) should be completely separated within 30 min using this method or when using an equivalent instrument (Figure 2016.15B ). ( 2 ) Acceptance criteria for the separation cycle are as follows: Migration time of the IS shouldbe 12.3±0.5min (if using an equivalent instrument, migration time should be as defined beforehand). Degree of baseline drop from migration time of IS to the peak valley

Figure 2016.15A. Buffer tray configuration (for Beckman instrument).

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