AOAC SPIFAN WPC ERP-Final Action Review (Nov. 2017)

2016.15 (Nov. 2017) - WPC-31 Publications-Final Action Review

FOR ERP USE ONLY DO NOT DISTRIBUTE

D. Preparation of the system buffer trays (Beckman) as well as standard and sample solutions a) Load the reagents into the system inlet (left) and outlet (right) using 6x6 buffer trays arranged according to the configuration illustrated in Figure 2016.15A (buffer tray configuration). If using an equivalent system, follow the instructions for that system. b) Weigh either 135±5 mg skim milk powder (SMP, with protein content around 37%) or 500±20 mg of infant formula powder (with protein content around 11%) into a 1 0 -mL centrifuge tube. c) Disperse the sample to 5 mL final with DI water. Vortex each tube until the solution appears homogeneous. Each final solution contains about 10 to 15 mg/mL of protein. d) Prepare the sample running pre-solution: mix the 1% SDS sample solution with 10 kDa internal standard peptide using an 84:1 ratio. The total volume of the pre-solution is based on the total number of samples to be analyzed in the sample set (90 μL per sample). e) Pipette 10 μL of each sample solution into separate 2.0-mL microcentrifuge vials. f) Sequentially add 85 μL of the sample running pre-solution and 5 μL of β-mercaptoethanol to each micro-centrifuge vial. Mix well before heating the vials in a water bath at 100±5 °C for ten minutes. Cool down to room temperature, then centrifuge at room temperature for 1 minute at about 7,000 rpm / 5,000 g. g) Vortex 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 of DI water), a molecular weight size standard, (MW), and a skim milk powder (SMP) sample. b) For each separation cycle (45 minutes), precondition the capillary first with the basic wash solution for 3 minutes, followed by the acidic wash solution for 1 minute, deionized water for 1 minute, and SDS gel buffer for 10 minutes. Set up the separation run for 30 minutes. c) Introduce the samples electrokinetically by applying voltage at -5 kV for 20 seconds. d) Perform the 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 the system suitability using the MW marker. The acceptance criteria for the system suitability are as follows: • The migration time of the internal standard should be 12.3±0.5 minutes using the instrument and capillary as per section B (a&b). If using an equivalent capillary or instrument, then define the migration time of the internal standard accordingly. The migration pattern and the migration times of the seven MW markers (10, 20, 35, 50, 100, 150, and 225 kDa) should be completely separated within 30 minutes using this method or when using an equivalent instrument (refer to Figure 2016.15B).